# Phosphate (P i )-regulated heterodimerization of the high-affinity sodium-dependent P i transporters PiT1/Slc20a1 and PiT2/Slc20a2 underlies extracellular P i sensing independently of P i uptake

Nina Bon, Greig Couasnay, Annabelle Bourgine, Sophie Source, Sarah

Beck-Cormier, Jérôme Guicheux, Laurent Beck

► To cite this version:

Nina Bon, Greig Couasnay, Annabelle Bourgine, Sophie Source, Sarah Beck-Cormier, et al.. Phosphate (P i )-regulated heterodimerization of the high-affinity sodium-dependent P i transporters PiT1/Slc20a1 and PiT2/Slc20a2 underlies extracellular P i sensing independently of P i uptake. Journal of Biological Chemistry, 2018, 293 (6), pp.2102-2114. 10.1074/jbc.M117.807339. hal-02333862

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# Phosphate (Pᵢ)-regulated heterodimerization of the high-affinity sodium-dependent Pᵢ transporters PiT1/Slc20a1 and PiT2/Slc20a2 underlies extracellular Pᵢ sensing independently of Pᵢ uptake

Received for publication, July 19, 2017, and in revised form, November 16, 2017 Published, Papers in Press, December 12, 2017, DOI 10.1074/jbc.M117.807339

Nina Bon<sup>‡§1</sup>, Greig Couasnay<sup>‡§2</sup>, Annabelle Bourgine<sup>‡§3</sup>, Sophie Sourice<sup>‡§</sup>, Sarah Beck-Cormier<sup>‡§</sup>,
Jérôme Guicheux<sup>‡§¶</sup>, and Laurent Beck<sup>‡§4</sup>

From ‡INSERM, U1229, RMeS "Regenerative Medicine and Skeleton," STEP team "Skeletal Physiopathology and Joint Regenerative Medicine," Nantes F-44042, France, the §Université de Nantes, UMR-S 1229, RMeS, UFR Odontologie, Nantes F-44042, France, and ¶CHU Nantes, PHU 4 OTONN, Nantes F-44042, France

Edited by Amanda J. Fosang

Extracellular phosphate (Pᵢ) can act as a signaling molecule that directly alters gene expression and cellular physiology. The ability of cells or organisms to detect changes in extracellular Pᵢ levels implies the existence of a Pᵢ-sensing mechanism that signals to the body or individual cell. However, unlike in prokaryotes, yeasts, and plants, the molecular players involved in Pᵢ sensing in mammals remain unknown. In this study, we investigated the involvement of the high-affinity, sodium-dependent Pᵢ transporters PiT1 and PiT2 in mediating Pᵢ signaling in skeletal cells. We found that deletion of PiT1 or PiT2 blunted the Pᵢ-dependent ERK1/2-mediated phosphorylation and subsequent gene up-regulation of the mineralization inhibitors matrix Gla protein and osteopontin. This result suggested that both PiTs are necessary for Pᵢ signaling. Moreover, the ERK1/2 phosphorylation could be rescued by overexpressing Pᵢ transport-deficient PiT mutants. Using cross-linking and bioluminescence resonance energy transfer approaches, we found that PiT1 and PiT2 form high-abundance homodimers and Pᵢ-regulated low-abundance heterodimers. Interestingly, in the absence of sodium-dependent Pᵢ transport activity, the PiT1-PiT2 heterodimerization was still regulated by extracellular Pᵢ levels. Of note, when two putative Pᵢ-binding residues, Ser-128 (in PiT1) and Ser-113 (in PiT2), were substituted with alanine, the PiT1-PiT2 heterodimerization was no longer regulated by extracellular Pᵢ. These observations suggested that Pᵢ binding rather than Pᵢ uptake may be the key factor in mediating Pᵢ signaling through the PiT proteins. Taken together, these results

demonstrate that Pᵢ-regulated PiT1-PiT2 heterodimerization mediates Pᵢ sensing independently of Pᵢ uptake.

Phosphorus is the sixth most abundant element in the human body, constituting ~1% of total body weight (1). About 85% of total phosphate can be found in the skeleton, where it is a major constituent of hydroxyapatite crystals deposited on the extracellular organic matrix during the mineralization process. The remaining 15% is found mainly in cells from soft tissues and in extracellular volume, where it represents <1% of total phosphate (2–4). In plasma, ~16% of circulating phosphate is found as organic phosphate bound to proteins and lipids, whereas the major part (84%) is orthophosphate, or free inorganic phosphate (Pᵢ),<fnref n="5" /> that can be filtered by the kidney (1). At physiological pH, the monovalent H₂PO₄⁻ and the divalent HPO₄<fnref n="2" />⁻ forms are present at a 1:4 molar ratio (5). Although this plasma Pᵢ represents a small fraction of total body phosphorus, it serves as an exchange pool between the various Pᵢ-containing and -regulating organs, and disturbances in Pᵢ homeostasis can affect almost all organ systems.

In addition to the widespread structural and metabolic functions of Pᵢ, it has become increasingly apparent during the past 15 years that extracellular Pᵢ can act as a signaling molecule directly altering gene expression and cell phenotype (6–9). The abundance of Pᵢ in the skeleton has led to early studies describing the effects of extracellular Pᵢ in this organ. Exposing cultured chondrocytes to a high level of extracellular Pᵢ leads to their terminal maturation and subsequent matrix mineralization (10–13). Similarly, the apoptosis of terminally differentiated hypertrophic chondrocytes was shown to be dependent upon the circulating plasma Pᵢ levels *in vivo* (14). In both of these *in vitro* and *in vivo* approaches, the Pᵢ-mediated apoptosis of chondrocytes was dependent upon the activation of the MAPK ERK1/2 pathway (15–17), but not of other mitogen-

<footnote n="5">⁵ The abbreviations used are: Pᵢ, inorganic phosphate; iLoop, large intracellular loop; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; eYFP, enhanced yellow fluorescent protein; BRET, bioluminescence resonance energy transfer; qPCR, quantitative PCR; F1–F3, fractions 1–3, respectively.</footnote>

<footnote>This work was supported by in part grants from INSERM, Région des Pays de la Loire (CIMATH 2, Nouvelle Equipe/Nouvelle Thématique and SENSEO). The authors declare that they have no conflicts of interest with the contents of this article.</footnote>

<footnote>This article contains Figs. S1–S5.</footnote>

<footnote n="1">¹ Recipient of a fellowship from Région des Pays de la Loire (SENSEO) and University of Nantes.</footnote>

<footnote n="2">² Recipient of a fellowship from Région des Pays de la Loire (Nouvelle Equipe/ Nouvelle Thématique) and University of Nantes.</footnote>

<footnote n="3">³ Recipient of a fellowship from Région des Pays de la Loire (BIOREGOS) and INSERM.</footnote>

<footnote n="4">⁴ To whom correspondence should be addressed: INSERM U1229-RMeS, Faculté de Chirurgie Dentaire, 1 Place Alexis Ricordeau, 44042 Nantes, France. Tel.: 332-40-41-29-20; Fax: 332-40-08-37-12; E-mail: laurent.beck@inserm.fr.</footnote>
# Phosphate (Pᵢ)-regulated heterodimerization of the high-affinity sodium-dependent Pᵢ transporters PiT1/Slc20a1 and PiT2/Slc20a2 underlies extracellular Pᵢ sensing independently of Pᵢ uptake

Received for publication, July 19, 2017, and in revised form, November 16, 2017 Published, Papers in Press, December 12, 2017, DOI 10.1074/jbc.M117.807339

Nina Bon<sup>‡§1</sup>, Greig Couasnay<sup>‡§2</sup>, Annabelle Bourgine<sup>‡§3</sup>, Sophie Sourice<sup>‡§</sup>, Sarah Beck-Cormier<sup>‡§</sup>,
Jérôme Guicheux<sup>‡§¶</sup>, and Laurent Beck<sup>‡§4</sup>

From ‡INSERM, U1229, RMeS "Regenerative Medicine and Skeleton," STEP team "Skeletal Physiopathology and Joint Regenerative Medicine," Nantes F-44042, France, the §Université de Nantes, UMR-S 1229, RMeS, UFR Odontologie, Nantes F-44042, France, and ¶CHU Nantes, PHU 4 OTONN, Nantes F-44042, France

Edited by Amanda J. Fosang

Extracellular phosphate (Pᵢ) can act as a signaling molecule that directly alters gene expression and cellular physiology. The ability of cells or organisms to detect changes in extracellular Pᵢ levels implies the existence of a Pᵢ-sensing mechanism that signals to the body or individual cell. However, unlike in prokaryotes, yeasts, and plants, the molecular players involved in Pᵢ sensing in mammals remain unknown. In this study, we investigated the involvement of the high-affinity, sodium-dependent Pᵢ transporters PiT1 and PiT2 in mediating Pᵢ signaling in skeletal cells. We found that deletion of PiT1 or PiT2 blunted the Pᵢ-dependent ERK1/2-mediated phosphorylation and subsequent gene up-regulation of the mineralization inhibitors matrix Gla protein and osteopontin. This result suggested that both PiTs are necessary for Pᵢ signaling. Moreover, the ERK1/2 phosphorylation could be rescued by overexpressing Pᵢ transport-deficient PiT mutants. Using cross-linking and bioluminescence resonance energy transfer approaches, we found that PiT1 and PiT2 form high-abundance homodimers and Pᵢ-regulated low-abundance heterodimers. Interestingly, in the absence of sodium-dependent Pᵢ transport activity, the PiT1-PiT2 heterodimerization was still regulated by extracellular Pᵢ levels. Of note, when two putative Pᵢ-binding residues, Ser-128 (in PiT1) and Ser-113 (in PiT2), were substituted with alanine, the PiT1-PiT2 heterodimerization was no longer regulated by extracellular Pᵢ. These observations suggested that Pᵢ binding rather than Pᵢ uptake may be the key factor in mediating Pᵢ signaling through the PiT proteins. Taken together, these results

demonstrate that Pᵢ-regulated PiT1-PiT2 heterodimerization mediates Pᵢ sensing independently of Pᵢ uptake.

Phosphorus is the sixth most abundant element in the human body, constituting ~1% of total body weight (1). About 85% of total phosphate can be found in the skeleton, where it is a major constituent of hydroxyapatite crystals deposited on the extracellular organic matrix during the mineralization process. The remaining 15% is found mainly in cells from soft tissues and in extracellular volume, where it represents <1% of total phosphate (2–4). In plasma, ~16% of circulating phosphate is found as organic phosphate bound to proteins and lipids, whereas the major part (84%) is orthophosphate, or free inorganic phosphate (Pᵢ),<fnref n="5" /> that can be filtered by the kidney (1). At physiological pH, the monovalent H₂PO₄⁻ and the divalent HPO₄<fnref n="2" />⁻ forms are present at a 1:4 molar ratio (5). Although this plasma Pᵢ represents a small fraction of total body phosphorus, it serves as an exchange pool between the various Pᵢ-containing and -regulating organs, and disturbances in Pᵢ homeostasis can affect almost all organ systems.

In addition to the widespread structural and metabolic functions of Pᵢ, it has become increasingly apparent during the past 15 years that extracellular Pᵢ can act as a signaling molecule directly altering gene expression and cell phenotype (6–9). The abundance of Pᵢ in the skeleton has led to early studies describing the effects of extracellular Pᵢ in this organ. Exposing cultured chondrocytes to a high level of extracellular Pᵢ leads to their terminal maturation and subsequent matrix mineralization (10–13). Similarly, the apoptosis of terminally differentiated hypertrophic chondrocytes was shown to be dependent upon the circulating plasma Pᵢ levels *in vivo* (14). In both of these *in vitro* and *in vivo* approaches, the Pᵢ-mediated apoptosis of chondrocytes was dependent upon the activation of the MAPK ERK1/2 pathway (15–17), but not of other mitogen-

<footnote n="5">⁵ The abbreviations used are: Pᵢ, inorganic phosphate; iLoop, large intracellular loop; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; eYFP, enhanced yellow fluorescent protein; BRET, bioluminescence resonance energy transfer; qPCR, quantitative PCR; F1–F3, fractions 1–3, respectively.</footnote>

<footnote>This work was supported by in part grants from INSERM, Région des Pays de la Loire (CIMATH 2, Nouvelle Equipe/Nouvelle Thématique and SENSEO). The authors declare that they have no conflicts of interest with the contents of this article.</footnote>

<footnote>This article contains Figs. S1–S5.</footnote>

<footnote n="1">¹ Recipient of a fellowship from Région des Pays de la Loire (SENSEO) and University of Nantes.</footnote>

<footnote n="2">² Recipient of a fellowship from Région des Pays de la Loire (Nouvelle Equipe/ Nouvelle Thématique) and University of Nantes.</footnote>

<footnote n="3">³ Recipient of a fellowship from Région des Pays de la Loire (BIOREGOS) and INSERM.</footnote>

<footnote n="4">⁴ To whom correspondence should be addressed: INSERM U1229-RMeS, Faculté de Chirurgie Dentaire, 1 Place Alexis Ricordeau, 44042 Nantes, France. Tel.: 332-40-41-29-20; Fax: 332-40-08-37-12; E-mail: laurent.beck@inserm.fr.</footnote>
Pivotal role of PiT1 and PiT2 in bone phosphate sensing

activated protein kinases, such as p38 or c-Jun N-terminal
kinase. Interestingly, the Pᵢ-dependent activation of the ERK1/2
pathway up-regulated the gene expression of the mineraliza-
tion inhibitors matrix Gla protein (Mgp) and osteopontin
(Opn), most likely setting off a feedback mechanism to control
Pᵢ-induced mineralization (7, 16, 18, 19). The elevated extracel-
lular Pᵢ level was also shown to be important in osteoblast pro-
liferation and differentiation (16, 20–24), cementoblast forma-
tion (25), odontoblast differentiation (26, 27), and osteoclast
differentiation (28–30).

The Pᵢ-mediated signaling underlies the notion that cells
must possess a Pᵢ-sensing mechanism on the surface of or
within the cell that is able to detect and respond to the variation
of extracellular Pᵢ levels. The ability of organisms to detect
changes in extracellular levels of other metabolites (such as
Ca²⁺, glucose, or amino acids) has already been described (31–
33), and emerging evidence suggests that similar events are at
work to mediate the cellular effects of Pᵢ (8, 34–36). Although
the identity of the molecules involved in these mechanisms is
still unknown in mammals, Pᵢ-sensing machineries have been
characterized in prokaryotic and eukaryotic unicellular organ-
isms (37). In *Escherichia coli*, the phosphate transporter PstS
and other periplasmic proteins (PstC, Pst, and PstB/PhoU)
detect the variation of external Pᵢ concentrations. In case of a
low extracellular Pᵢ level, this system increases the efficiency of
Pᵢ retention in the bacteria (38). In *Saccharomyces cerevisiae*, a
low extracellular Pᵢ level resulted in induction of the Pᵢ trans-
porter Pho84, now identified as the essential component of the
Pᵢ-sensing system (38, 39). Interestingly, following Pᵢ restric-
tion, it was demonstrated that Pho84 could trigger the rapid
activation of protein kinase A without transporting Pᵢ (40).

In mammals, the Slc20a1/PiT1 and Slc20a2/PiT2 proteins
are expressed at the plasma membrane and have been described
as mediating the intracellular uptake of P<sub>i</sub> with a high affinity
(41–43). PiT1 and PiT2 have a wide tissue distribution, being
the only P<sub>i</sub> transporters expressed in bone (44, 45). Interest-
ingly, their expression can be modulated by extracellular P<sub>i</sub> (21,
43, 44, 46, 47), and previous studies have suggested that they
can mediate downstream effects of extracellular P<sub>i</sub>. In bone, the
elegant study of Kimata *et al*. (48) suggests that the chondrocyte
response to extracellular P<sub>i</sub> is mediated by a PiT1-dependent
up-regulation of cyclin D1 through ERK1/2 pathway activa-
tion. The authors hypothesize that P<sub>i</sub>-driven conformational
changes of PiT1 could be involved in the P<sub>i</sub>-sensing mechanism.
In parathyroid cells, PiT1 was suggested to act as a P<sub>i</sub> sensor to
modulate the secretion of the phosphaturic parathyroid hor-
mone (49). On the other hand, based on its property of oli-
gomerizing upon extracellular P<sub>i</sub> variation, PiT2 was also pro-
posed to serve as a P<sub>i</sub> sensor (50). Although these data support a
possible role for PiT1 or PiT2 as P<sub>i</sub> sensors, little is known about
the underlying mechanisms. Because PiT1 and PiT2 have very
close P<sub>i</sub> transport characteristics (51), they may also share P<sub>i</sub>-
sensing properties and thus have interconnected roles in P<sub>i</sub>
sensing. Moreover, because P<sub>i</sub>-independent functions have
been highlighted recently for PiT1 (52–56), the involvement of
P<sub>i</sub> transport in the P<sub>i</sub> sensing by PiT1 or PiT2 remains to be
investigated.

In this report, we investigated the role of PiT1 and PiT2 as Pᵢ
sensors in osteoblastic and chondrocytic cell lines. We show
that both PiT1 and PiT2 are required for mediating Pᵢ-depen-
dent signaling. We demonstrate that PiT1 and PiT2 could
interact together and that extracellular Pᵢ modulates this inter-
action. Finally, we show that cellular Pᵢ uptake is not required to
mediate Pᵢ signaling through the PiT proteins.

Results

Requirement of both PiT1 and PiT2 for P<sub>i</sub>-mediated signaling

We first investigated whether PiT1 and/or PiT2 were
involved in the P<sub>i</sub>-dependent up-regulation of *Mgp* and *Opn*
expression. To this aim, using RNA interference, we established
stably transfected osteoblastic MC3T3-E1 clones in which PiT1
or PiT2 expression was knocked down. In MC3T3-E1 *shPiT1*
clones, *PiT1* gene expression showed a 63% reduction, together
with a significant up-regulation of *PiT2* (Fig. 1A). Similarly, the
MC3T3-E1 *shPiT2* clones displayed a 62% decrease in *PiT2*
mRNA level, together with a significant up-regulation of *PiT1*
(Fig. 1A). Comparable results were observed when cells were
incubated with 10 mM extracellular P<sub>i</sub> concentration (Fig. S1A).
Interestingly, the sodium-dependent P<sub>i</sub> uptake was similar in
MC3T3-E1 *shPiT1* and *shPiT2* clones and control MC3T3-E1
cells (Fig. 1B), suggesting that a depletion of either PiT may be
compensated by the remaining PiT, as was previously suggested
(56). Consistent with this possibility, MC3T3-E1 clones stably
transfected with both *shPiT1* and *shPiT2* resulted in a 52%
reduction of both PiTs (Fig. 1A), resulting in a similar decrease
in sodium-dependent P<sub>i</sub> uptake (Fig. 1B). In contrast to wild-
type differentiated MC3T3-E1 cells in which *Mgp* and *Opn*
expression was up-regulated following stimulation with 10 mM
extracellular P<sub>i</sub> for 24 h, the up-regulation of *Mgp* and *Opn*
expression in PiT-depleted MC3T3-E1 clones was blunted (Fig.
1C). The defect in P<sub>i</sub>-dependent *Mgp* and *Opn* up-regulation
arose despite a normal P<sub>i</sub> transport in the *shPiT1* or *shPiT2*
MC3T3-E1 clones, suggesting that a variation in intracellular
P<sub>i</sub> content is unlikely to account for defects in P<sub>i</sub>-dependent
signaling in the absence of either PiTs. Because the ERK1/2
signaling pathway was shown to be required for P<sub>i</sub>-depen-
dent regulation of *Mgp* and *Opn* expression (16, 19), we
investigated the P<sub>i</sub>-dependent ERK1/2 activation in differen-
tiated PiT-depleted MC3T3-E1 clones. We showed that fol-
lowing a 30-min (Fig. S1B) or 24-h (Fig. 1D) stimulation with 10
mM extracellular P<sub>i</sub>, the activation of ERK1/2 pathway was
blunted in *shPiT1*, *shPiT2*, or *shPiT1/shPiT2* clones, as com-
pared with untransfected and *shScramble*-transfected cells.
Similar data were obtained in three separate PiT-depleted
MC3T3-E1 clones (Fig. S1, B–D) and in transiently transfected
MC3T3-E1 cells (Fig. S2). Interestingly, the effect of PiT deple-
tion on the activation of ERK1/2 pathway following stimulation
with 10% fetal bovine serum (FBS) was not as pronounced as P<sub>i</sub>
stimulation, arguing for a specificity of PiT1 and PiT2 in the
P<sub>i</sub>-dependent activation of the ERK1/2 pathway.

Moreover, we performed similar experiments in the MC615
chondrogenic cell line. We used a transient transfection
approach leading to a 50 and 56% deletion of *PiT1* and *PiT2*
mRNA levels, respectively (Fig. 2A). A similar extent of PiT
Pivotal role of PiT1 and PiT2 in bone phosphate sensing

activated protein kinases, such as p38 or c-Jun N-terminal
kinase. Interestingly, the Pᵢ-dependent activation of the ERK1/2
pathway up-regulated the gene expression of the mineraliza-
tion inhibitors matrix Gla protein (Mgp) and osteopontin
(Opn), most likely setting off a feedback mechanism to control
Pᵢ-induced mineralization (7, 16, 18, 19). The elevated extracel-
lular Pᵢ level was also shown to be important in osteoblast pro-
liferation and differentiation (16, 20–24), cementoblast forma-
tion (25), odontoblast differentiation (26, 27), and osteoclast
differentiation (28–30).

The Pᵢ-mediated signaling underlies the notion that cells
must possess a Pᵢ-sensing mechanism on the surface of or
within the cell that is able to detect and respond to the variation
of extracellular Pᵢ levels. The ability of organisms to detect
changes in extracellular levels of other metabolites (such as
Ca²⁺, glucose, or amino acids) has already been described (31–
33), and emerging evidence suggests that similar events are at
work to mediate the cellular effects of Pᵢ (8, 34–36). Although
the identity of the molecules involved in these mechanisms is
still unknown in mammals, Pᵢ-sensing machineries have been
characterized in prokaryotic and eukaryotic unicellular organ-
isms (37). In *Escherichia coli*, the phosphate transporter PstS
and other periplasmic proteins (PstC, Pst, and PstB/PhoU)
detect the variation of external Pᵢ concentrations. In case of a
low extracellular Pᵢ level, this system increases the efficiency of
Pᵢ retention in the bacteria (38). In *Saccharomyces cerevisiae*, a
low extracellular Pᵢ level resulted in induction of the Pᵢ trans-
porter Pho84, now identified as the essential component of the
Pᵢ-sensing system (38, 39). Interestingly, following Pᵢ restric-
tion, it was demonstrated that Pho84 could trigger the rapid
activation of protein kinase A without transporting Pᵢ (40).

In mammals, the Slc20a1/PiT1 and Slc20a2/PiT2 proteins
are expressed at the plasma membrane and have been described
as mediating the intracellular uptake of P<sub>i</sub> with a high affinity
(41–43). PiT1 and PiT2 have a wide tissue distribution, being
the only P<sub>i</sub> transporters expressed in bone (44, 45). Interest-
ingly, their expression can be modulated by extracellular P<sub>i</sub> (21,
43, 44, 46, 47), and previous studies have suggested that they
can mediate downstream effects of extracellular P<sub>i</sub>. In bone, the
elegant study of Kimata *et al*. (48) suggests that the chondrocyte
response to extracellular P<sub>i</sub> is mediated by a PiT1-dependent
up-regulation of cyclin D1 through ERK1/2 pathway activa-
tion. The authors hypothesize that P<sub>i</sub>-driven conformational
changes of PiT1 could be involved in the P<sub>i</sub>-sensing mechanism.
In parathyroid cells, PiT1 was suggested to act as a P<sub>i</sub> sensor to
modulate the secretion of the phosphaturic parathyroid hor-
mone (49). On the other hand, based on its property of oli-
gomerizing upon extracellular P<sub>i</sub> variation, PiT2 was also pro-
posed to serve as a P<sub>i</sub> sensor (50). Although these data support a
possible role for PiT1 or PiT2 as P<sub>i</sub> sensors, little is known about
the underlying mechanisms. Because PiT1 and PiT2 have very
close P<sub>i</sub> transport characteristics (51), they may also share P<sub>i</sub>-
sensing properties and thus have interconnected roles in P<sub>i</sub>
sensing. Moreover, because P<sub>i</sub>-independent functions have
been highlighted recently for PiT1 (52–56), the involvement of
P<sub>i</sub> transport in the P<sub>i</sub> sensing by PiT1 or PiT2 remains to be
investigated.

In this report, we investigated the role of PiT1 and PiT2 as Pᵢ sensors in osteoblastic and chondrocytic cell lines. We show that both PiT1 and PiT2 are required for mediating Pᵢ-dependent signaling. We demonstrate that PiT1 and PiT2 could interact together and that extracellular Pᵢ modulates this interaction. Finally, we show that cellular Pᵢ uptake is not required to mediate Pᵢ signaling through the PiT proteins.

Results

Requirement of both PiT1 and PiT2 for P<sub>i</sub>-mediated signaling

We first investigated whether PiT1 and/or PiT2 were
involved in the P<sub>i</sub>-dependent up-regulation of *Mgp* and *Opn*
expression. To this aim, using RNA interference, we established
stably transfected osteoblastic MC3T3-E1 clones in which PiT1
or PiT2 expression was knocked down. In MC3T3-E1 *shPiT1*
clones, *PiT1* gene expression showed a 63% reduction, together
with a significant up-regulation of *PiT2* (Fig. 1A). Similarly, the
MC3T3-E1 *shPiT2* clones displayed a 62% decrease in *PiT2*
mRNA level, together with a significant up-regulation of *PiT1*
(Fig. 1A). Comparable results were observed when cells were
incubated with 10 mM extracellular P<sub>i</sub> concentration (Fig. S1A).
Interestingly, the sodium-dependent P<sub>i</sub> uptake was similar in
MC3T3-E1 *shPiT1* and *shPiT2* clones and control MC3T3-E1
cells (Fig. 1B), suggesting that a depletion of either PiT may be
compensated by the remaining PiT, as was previously suggested
(56). Consistent with this possibility, MC3T3-E1 clones stably
transfected with both *shPiT1* and *shPiT2* resulted in a 52%
reduction of both PiTs (Fig. 1A), resulting in a similar decrease
in sodium-dependent P<sub>i</sub> uptake (Fig. 1B). In contrast to wild-
type differentiated MC3T3-E1 cells in which *Mgp* and *Opn*
expression was up-regulated following stimulation with 10 mM
extracellular P<sub>i</sub> for 24 h, the up-regulation of *Mgp* and *Opn*
expression in PiT-depleted MC3T3-E1 clones was blunted (Fig.
1C). The defect in P<sub>i</sub>-dependent *Mgp* and *Opn* up-regulation
arose despite a normal P<sub>i</sub> transport in the *shPiT1* or *shPiT2*
MC3T3-E1 clones, suggesting that a variation in intracellular
P<sub>i</sub> content is unlikely to account for defects in P<sub>i</sub>-dependent
signaling in the absence of either PiTs. Because the ERK1/2
signaling pathway was shown to be required for P<sub>i</sub>-depen-
dent regulation of *Mgp* and *Opn* expression (16, 19), we
investigated the P<sub>i</sub>-dependent ERK1/2 activation in differen-
tiated PiT-depleted MC3T3-E1 clones. We showed that fol-
lowing a 30-min (Fig. S1B) or 24-h (Fig. 1D) stimulation with 10
mM extracellular P<sub>i</sub>, the activation of ERK1/2 pathway was
blunted in *shPiT1*, *shPiT2*, or *shPiT1/shPiT2* clones, as com-
pared with untransfected and *shScramble*-transfected cells.
Similar data were obtained in three separate PiT-depleted
MC3T3-E1 clones (Fig. S1, B–D) and in transiently transfected
MC3T3-E1 cells (Fig. S2). Interestingly, the effect of PiT deple-
tion on the activation of ERK1/2 pathway following stimulation
with 10% fetal bovine serum (FBS) was not as pronounced as P<sub>i</sub>
stimulation, arguing for a specificity of PiT1 and PiT2 in the
P<sub>i</sub>-dependent activation of the ERK1/2 pathway.

Moreover, we performed similar experiments in the MC615
chondrogenic cell line. We used a transient transfection
approach leading to a 50 and 56% deletion of *PiT1* and *PiT2*
mRNA levels, respectively (Fig. 2A). A similar extent of PiT
Pivotal role of PiT1 and PiT2 in bone phosphate sensing

<figure><img src="image_1.png" /></figure>

<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>**Figure 1.** Pᵢ-dependent *Mgp* and *Opn* gene regulation and ERK1/2 signaling require both PiT1 and PiT2 in MC3T3-E1 cells. A, RT-qPCR analysis of *PiT1* (*white bars*) and *PiT2* (*black bars*) mRNA levels in untransfected (UT) or stably transfected MC3T3-E1 cells, as indicated. Data are means ± S.E. (*, versus shScramb, *p* < 0.05, *n* = 3). B, sodium-dependent Pᵢ uptake was measured in untransfected or stably transfected MC3T3-E1 cells, as indicated. Data are means ± S.E. (*n* = 3). C, RT-qPCR analysis of *Mgp* and *Opn* mRNA levels in untransfected or stably transfected MC3T3-E1 cells, as indicated. Cells were incubated in low-serum (0.5%) medium for 24 h and stimulated with 1 mM (*white bars*) or 10 mM (*black bars*) extracellular Pᵢ concentration for 24 h. Data are means ± S.E. (*error bars*) (#, *p* < 0.05; ##, *p* < 0.01 versus 1 mM Pᵢ control; and *, *p* < 0.05; **, *p* < 0.01 versus shScramb; *n* = 3) D, Western blot analysis of ERK1/2 phosphorylation (P-ERK 1/2) in untransfected or stably transfected MC3T3-E1 cells, as indicated. Cells were incubated in low-serum (0.5%) medium for 24 h and stimulated for another 24 h with 1 mM or 10 mM extracellular Pᵢ concentration or with 10% FBS used as a positive control for ERK1/2 phosphorylation. Total ERK1/2 proteins were used as a loading control.</figcaption></figure>

deletion was obtained at the protein level, as shown by immu-
nofluorescence (Fig. S3). MC615 cells were also used to rescue
PiT deletion by overexpressing human PiT1 and PiT2 in PiT1-
and PiT2-depleted cells, respectively (Fig. 2B). Similar to what
was observed in MC3T3-E1 cells, depletion of either PiT1 or
PiT2 in MC615 cells blunted the activation of the ERK1/2 path-
way by 10 mM extracellular Pᵢ, despite the up-regulation of the
remaining PiT (Fig. 2C). When human *PiT1* was overexpressed
in PiT1-depleted MC615 cells, we could rescue the Pᵢ-depen-
dent ERK1/2 phosphorylation (Fig. 2C). Similar results were
obtained when human *PiT2* was overexpressed in PiT2-de-
pleted MC615 cells or when both human PiT1 and PiT2 were
overexpressed in PiT1-PiT2-depleted MC615 cells (Fig. 2C).
This further illustrated the requirement of both PiT1 and PiT2
for the Pᵢ-dependent ERK signaling in cell lines of skeletal
origin.

PiT1 and PiT2 form hetero-oligomers upon variation of
extracellular Pᵢ concentrations

The requirement of both PiT1 and PiT2 for Pᵢ-dependent ERK1/2 signaling may indicate the existence of a functional protein complex comprising both PiTs. This possibility is also reinforced by the presence in PiT1 and PiT2 protein sequences of a conserved and highly hydrophobic 127-amino acid domain that was suggested to be important for determining the quaternary structure of the protein (57). To investigate the formation of hetero- and homo-oligomers, we used HEK293T cells that are easy to transfect, allow a robust expression of PiTs at the plasma membrane, and have been shown to have similar Pᵢ-mediated ERK1/2-specific activation (58). In addition, because PiT proteins are often difficult to detect using total cell extracts in Western blot experiments, we used a crude cell fractionation
Pivotal role of PiT1 and PiT2 in bone phosphate sensing

<figure><img src="image_2.png" /><figcaption>**Figure 1.** P<sub>i</sub>-dependent *Mgp* and *Opn* gene regulation and ERK1/2 signaling require both PiT1 and PiT2 in MC3T3-E1 cells. A, RT-qPCR analysis of *PiT1* (*white bars*) and *PiT2* (*black bars*) mRNA levels in untransfected (UT) or stably transfected MC3T3-E1 cells, as indicated. Data are means ± S.E. (*, versus shScramb, *p* < 0.05, *n* = 3). B, sodium-dependent P<sub>i</sub> uptake was measured in untransfected or stably transfected MC3T3-E1 cells, as indicated. Data are means ± S.E. (*n* = 3). C, RT-qPCR analysis of *Mgp* and *Opn* mRNA levels in untransfected or stably transfected MC3T3-E1 cells, as indicated. Cells were incubated in low-serum (0.5%) medium for 24 h and stimulated with 1 mM (*white bars*) or 10 mM (*black bars*) extracellular P<sub>i</sub> concentration for 24 h. Data are means ± S.E. (error bars) (#, *p* < 0.05; ##, *p* < 0.01 versus 1 mM P<sub>i</sub> control; and *, *p* < 0.05; **, *p* < 0.01 versus shScramb; *n* = 3) D, Western blot analysis of ERK1/2 phosphorylation (P-ERK 1/2) in untransfected or stably transfected MC3T3-E1 cells, as indicated. Cells were incubated in low-serum (0.5%) medium for 24 h and stimulated for another 24 h with 1 mM or 10 mM extracellular P<sub>i</sub> concentration or with 10% FBS used as a positive control for ERK1/2 phosphorylation. Total ERK1/2 proteins were used as a loading control.</figcaption></figure>

deletion was obtained at the protein level, as shown by immu-
nofluorescence (Fig. S3). MC615 cells were also used to rescue
PiT deletion by overexpressing human PiT1 and PiT2 in PiT1-
and PiT2-depleted cells, respectively (Fig. 2B). Similar to what
was observed in MC3T3-E1 cells, depletion of either PiT1 or
PiT2 in MC615 cells blunted the activation of the ERK1/2 path-
way by 10 mM extracellular Pᵢ, despite the up-regulation of the
remaining PiT (Fig. 2C). When human *PiT1* was overexpressed
in PiT1-depleted MC615 cells, we could rescue the Pᵢ-depen-
dent ERK1/2 phosphorylation (Fig. 2C). Similar results were
obtained when human *PiT2* was overexpressed in PiT2-de-
pleted MC615 cells or when both human PiT1 and PiT2 were
overexpressed in PiT1-PiT2-depleted MC615 cells (Fig. 2C).
This further illustrated the requirement of both PiT1 and PiT2
for the Pᵢ-dependent ERK signaling in cell lines of skeletal
origin.

PiT1 and PiT2 form hetero-oligomers upon variation of
extracellular Pᵢ concentrations

The requirement of both PiT1 and PiT2 for P<sub>i</sub>-dependent ERK1/2 signaling may indicate the existence of a functional protein complex comprising both PiTs. This possibility is also reinforced by the presence in PiT1 and PiT2 protein sequences of a conserved and highly hydrophobic 127-amino acid domain that was suggested to be important for determining the quaternary structure of the protein (57). To investigate the formation of hetero- and homo-oligomers, we used HEK293T cells that are easy to transfect, allow a robust expression of PiTs at the plasma membrane, and have been shown to have similar P<sub>i</sub>-mediated ERK1/2-specific activation (58). In addition, because PiT proteins are often difficult to detect using total cell extracts in Western blot experiments, we used a crude cell fractionation
Pivotal role of PiT1 and PiT2 in bone phosphate sensing

<figure><img src="image_6.png" /><figcaption>**Figure 2.** Pᵢ-dependent ERK1/2 signaling requires both PiT1 and PiT2 in MC615 cells. A, RT-qPCR analysis of *mPiT1* (white bars) and *mPiT2* (black bars) mRNA levels in transiently transfected MC615 cells, as indicated. Data are means ± S.E. (error bars) (**, *p* < 0.01; **, *p* < 0.001 versus *shScramb*; *n* = 3). B, RT-qPCR analysis of *hPiT1* (white bars) and *hPiT2* (black bars) mRNA levels in transiently transfected MC615 cells, as indicated. The endogenous murine *PiT1* or *PiT2* genes were used as reference genes to evaluate the overexpression of the transfected human *PiT* genes. Data are expressed as mean ± S.E. (***, *p* < 0.001 versus *shScramb+pcDNA*, *n* = 3). N/A, not applicable. C, Western blot analysis of ERK1/2 phosphorylation (*P*-ERK 1/2) in transiently transfected MC615 cells, as indicated. Cells were incubated in low-serum (0.5%) medium for 24 h and stimulated for 30 min with 1 mM or 10 mM extracellular Pᵢ concentration as indicated. Total ERK1/2 proteins were used as a loading control.</figcaption></figure>

approach to analyze an enriched plasma membrane protein
fraction revealed by the specific expression of the Na/K-AT-
Pase, as shown in Fig. 3A. When analyzing the enriched plasma
membrane fraction from PiT1- and/or PiT2-transfected cells
by Western blotting, we could observe protein complexes after
cell surface cross-linking using BS³, a membrane-impermeable
cross-linker (Fig. 3B). The apparent molecular mass of 142–165
kDa that we detected from cells transfected with hPiT2 alone
recapitulated the results obtained by Salaün *et al.* (50) demon-
strating the formation of PiT2 homodimers. Similarly, the 151–
188 kDa apparent molecular mass band detected from cells
transfected with *hPiT1* is consistent with the association of two
PiT1 molecules. When cells were co-transfected with both
*hPiT1* and *hPiT2*, no change was observed in the band profile,
apart from a less intense signal due to transfection with 50%
fewer *hPiT1* or *hPiT2* plasmids. In this condition, we could not
detect a distinct band at the theoretical PiT1-PiT2 heterodimer
molecular mass, most likely due to the expected similar molec-
ular weights of PiT homo- and heterodimers.

To study further the PiT oligomerization, we then used a
BRET approach. To this aim, we constructed hPiT1 and hPiT2
chimeric proteins expressing the eYFP acceptor or Rluc donor.
Because structure-function studies have excluded a role of the
large intracellular loop (iLoop) in Pᵢ transport and retrovirus
binding (59–62) and showed no overlapping between iLoop
and the highly hydrophobic domain (57), we substituted the
iLoop with eYFP and Rluc sequences (Fig. 3C). When expressed
in HEK293T, the chimeric hPiT1-eYFP or -Rluc and hPiT2-

eYFP or -Rluc proteins could be visualized at the plasma mem-
brane, as shown by confocal microscopy (Fig. 3D), enabling us
to study their role in detecting the variation of extracellular Pᵢ
levels. We performed saturation BRET experiments in living
cells to investigate their potential hetero- and homo-oligomer-
ization. As shown in Fig. 3E, we obtained typical BRET-satura-
ble curves when using hPiT1-eYFP and hPiT2-Rluc proteins,
together with a high BRET ratio. In contrast, when the BTN3A2
protein was used instead of either PiT, no saturation could be
achieved, together with a weak BRET ratio (Fig. S4, A and B).
These data support the notion that PiTs can form hetero- and
homo-oligomers specifically. We confirmed the specificity of
the hetero-oligomers by a competition assay (Fig. S4C) whereby
overexpression of untagged PiT1 or PiT2 reduces the BRET
ratio, whereas unrelated BTN3A2 expression does not. We next
determined whether the interaction of PiTs could be modu-
lated by the variation of extracellular Pᵢ concentration. We
therefore performed the same saturation BRET experiments
after a 10-min stimulation with 1, 3, or 10 mM extracellular Pᵢ.
Results reported in Fig. 3 (F and G) and Fig. S5A showed that
saturation curves were different upon extracellular Pᵢ concen-
tration only for hPiT1-hPiT2 hetero-oligomers. Indeed, calcu-
lating the BRET 50 values from these curves, we showed a
significant decrease at 10 mM extracellular Pᵢ, suggesting a
stronger interaction between PiT1 and PiT2 in this condition,
whereas no variation was observed for homo-oligomers. In all
conditions, the BRET max values did not vary significantly
(Fig. S5B).
Pivotal role of PiT1 and PiT2 in bone phosphate sensing

<figure><img src="image_3.png" /></figure>

**Figure 2.** Pᵢ-dependent ERK1/2 signaling requires both PiT1 and PiT2 in MC615 cells. A, RT-qPCR analysis of *mPiT1* (white bars) and *mPiT2* (black bars) mRNA levels in transiently transfected MC615 cells, as indicated. Data are means ± S.E. (error bars) (**, *p* < 0.01; **, *p* < 0.001 versus *shScramb*; *n* = 3). B, RT-qPCR analysis of *hPiT1* (white bars) and *hPiT2* (black bars) mRNA levels in transiently transfected MC615 cells, as indicated. The endogenous murine *PiT1* or *PiT2* genes were used as reference genes to evaluate the overexpression of the transfected human *PiT* genes. Data are expressed as mean ± S.E. (***, *p* < 0.001 versus *shScramb+pcDNA*, *n* = 3). N/A, not applicable. C, Western blot analysis of ERK1/2 phosphorylation (*P*-ERK 1/2) in transiently transfected MC615 cells, as indicated. Cells were incubated in low-serum (0.5%) medium for 24 h and stimulated for 30 min with 1 mM or 10 mM extracellular Pᵢ concentration as indicated. Total ERK1/2 proteins were used as a loading control.

approach to analyze an enriched plasma membrane protein
fraction revealed by the specific expression of the Na/K-AT-
Pase, as shown in Fig. 3A. When analyzing the enriched plasma
membrane fraction from PiT1- and/or PiT2-transfected cells
by Western blotting, we could observe protein complexes after
cell surface cross-linking using BS³, a membrane-impermeable
cross-linker (Fig. 3B). The apparent molecular mass of 142–165
kDa that we detected from cells transfected with hPiT2 alone
recapitulated the results obtained by Salaün *et al.* (50) demon-
strating the formation of PiT2 homodimers. Similarly, the 151–
188 kDa apparent molecular mass band detected from cells
transfected with *hPiT1* is consistent with the association of two
PiT1 molecules. When cells were co-transfected with both
*hPiT1* and *hPiT2*, no change was observed in the band profile,
apart from a less intense signal due to transfection with 50%
fewer *hPiT1* or *hPiT2* plasmids. In this condition, we could not
detect a distinct band at the theoretical PiT1-PiT2 heterodimer
molecular mass, most likely due to the expected similar molec-
ular weights of PiT homo- and heterodimers.

To study further the PiT oligomerization, we then used a
BRET approach. To this aim, we constructed hPiT1 and hPiT2
chimeric proteins expressing the eYFP acceptor or Rluc donor.
Because structure-function studies have excluded a role of the
large intracellular loop (iLoop) in Pᵢ transport and retrovirus
binding (59–62) and showed no overlapping between iLoop
and the highly hydrophobic domain (57), we substituted the
iLoop with eYFP and Rluc sequences (Fig. 3C). When expressed
in HEK293T, the chimeric hPiT1-eYFP or -Rluc and hPiT2-

eYFP or -Rluc proteins could be visualized at the plasma mem-
brane, as shown by confocal microscopy (Fig. 3D), enabling us
to study their role in detecting the variation of extracellular Pᵢ
levels. We performed saturation BRET experiments in living
cells to investigate their potential hetero- and homo-oligomer-
ization. As shown in Fig. 3E, we obtained typical BRET-satura-
ble curves when using hPiT1-eYFP and hPiT2-Rluc proteins,
together with a high BRET ratio. In contrast, when the BTN3A2
protein was used instead of either PiT, no saturation could be
achieved, together with a weak BRET ratio (Fig. S4, A and B).
These data support the notion that PiTs can form hetero- and
homo-oligomers specifically. We confirmed the specificity of
the hetero-oligomers by a competition assay (Fig. S4C) whereby
overexpression of untagged PiT1 or PiT2 reduces the BRET
ratio, whereas unrelated BTN3A2 expression does not. We next
determined whether the interaction of PiTs could be modu-
lated by the variation of extracellular Pᵢ concentration. We
therefore performed the same saturation BRET experiments
after a 10-min stimulation with 1, 3, or 10 mM extracellular Pᵢ.
Results reported in Fig. 3 (F and G) and Fig. S5A showed that
saturation curves were different upon extracellular Pᵢ concen-
tration only for hPiT1-hPiT2 hetero-oligomers. Indeed, calcu-
lating the BRET 50 values from these curves, we showed a
significant decrease at 10 mM extracellular Pᵢ, suggesting a
stronger interaction between PiT1 and PiT2 in this condition,
whereas no variation was observed for homo-oligomers. In all
conditions, the BRET max values did not vary significantly
(Fig. S5B).
Pivotal role of PiT1 and PiT2 in bone phosphate sensing

<figure><img src="image_7.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>
Pivotal role of PiT1 and PiT2 in bone phosphate sensing

<figure><img src="image_4.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_11.png" /></figure>

BRET 50

<figure><img src="image_12.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_13.png" /></figure>

BRET 50

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

BRET 50

<figure><img src="image_16.png" /></figure>
Pivotal role of PiT1 and PiT2 in bone phosphate sensing

PiT1-PiT2-mediated ERK1/2 activation by extracellular Pᵢ and regulation of PiT hetero-oligomerization are Pᵢ transport-independent

Our results illustrated that both PiT proteins are important for Pᵢ-dependent ERK signaling and that Pᵢ can modulate PiT hetero-oligomerization. To elucidate whether the Pᵢ transport function of PiTs was important to mediate Pᵢ-dependent ERK1/2 signaling, we used the previously reported hPiT1S¹²⁸A and hPiT2S¹¹³A Pᵢ transport-deficient mutants (56, 63, 64). When hPiT1S¹²⁸A was overexpressed in PiT1-depleted cells in which Pᵢ-dependent ERK1/2 activation was lost, we could rescue the activation of ERK1/2 signaling (Fig. 4A). Similarly, we could rescue the Pᵢ-dependent ERK1/2 activation in PiT2-depleted cells by overexpressing hPiT2S¹¹³A mutant. This was also true when cells depleted from both PiTs were transfected by both hPiT1S¹²⁸A and hPiT2S¹¹³A (Fig. 4A). These data demonstrated that the Pᵢ transport function of PiTs was dispensable for the Pᵢ-dependent ERK1/2 activation. To further study whether the sodium-dependent Pᵢ transport function of PiTs was important to mediate Pᵢ-dependent PiT hetero-oligomerization, we performed BRET experiments in the absence of Na⁺. In this condition where Pᵢ transport was blunted, the variation of extracellular Pᵢ concentrations was still able to modulate PiT interaction (Fig. 4 (B and C) and Fig. S5C). Because Pᵢ was able to modulate PiT interaction without being transported, this suggests that the binding of Pᵢ to PiT proteins rather than its actual uptake into the cell may be involved in the modulation of PiT interaction. We next generated mutated versions of the chimeric hPiT1-eYFP and hPiT2-Rluc proteins in which Ser¹²⁸ or Ser¹¹³ was replaced by an alanine residue. As expected, over-expression of chimeric hPiT1S¹²⁸A-eYFP and hPiT2S¹¹³A-Rluc resulted in decreased sodium-dependent Pᵢ transport compared with transporting chimeric PiT proteins (Fig. 4D). Interestingly, using a BRET approach, we showed that although hPiT1S¹²⁸A-eYFP and hPiT2S¹¹³A-Rluc were still able to interact together, this interaction was not modulated anymore by extracellular Pᵢ variations (Fig. 4E (**left**) and Fig. S5C), consistent with a role for Ser¹²⁸ or Ser¹¹³ in Pᵢ binding. When BRET experiments were performed with a transport-deficient mutant (hPiT1S¹²⁸A-eYFP or hPiT2S¹¹³A-Rluc) and a transporting chimeric PiT protein (hPiT1-eYFP and hPiT2-Rluc), we could recover the modulation of PiT interaction by Pᵢ (Fig. 4E (**middle and left**) and Fig. S5C), further supporting a role of Ser¹²⁸ or Ser¹¹³ in the Pᵢ-dependent interaction of PiTs.

Discussion

The ability of a cell to detect changes in extracellular Pᵢ levels is paramount for its adequate response to environmental fluctuations and critical for the appropriate modulation of Pᵢ homeostasis and skeletal mineralization. In this work, we provided mechanistic insights into the molecular events leading to the detection of changes in extracellular Pᵢ concentrations using skeletal cell lines as a model.

Skeletal cells are constantly exposed to high local extracellular Pᵢ levels, mainly due to the continual resorption and formation of the mineralized extracellular matrix of bone and the need for tremendous quantities of Pᵢ for mineralization purposes (65). This has made bone a model of choice to study Pᵢ signaling, where it has been shown in early studies to regulate the programmed cell death of hypertrophic chondrocytes (10, 11, 13). In subsequent studies, we and others have shown that Pᵢ could regulate skeletal mineralization by controlling the expression of the mineralization inhibitors *Mgp* and *Opn* through the activation of the ERK1/2 pathway (7, 15, 16, 18, 19). Our present study brings evidence for a role of PiT1 and PiT2 in transmitting the Pᵢ signal to the cell by showing that the Pᵢ-dependent up-regulation of *Mgp* and *Opn* and ERK1/2 phosphorylation were blunted in PiT1- or PiT2-depleted cells. A role for PiT1 in mediating ERK1/2 signaling has been reported earlier (48, 55); however, a similar role for PiT2 has never been illustrated before.

A critical aspect in studying the molecular events involved in Pᵢ signaling is whether or not Pᵢ needs to be transported within the cell to fulfill its role. This question is particularly relevant in view of the role of PiT1 and PiT2 as mediators of Pᵢ signaling because these two proteins have well-described Pᵢ transport functions (41–43). By using Pᵢ transport-deficient mutants of PiT proteins, we could rescue the ERK1/2 signaling, demonstrating that PiT1 and PiT2 can mediate a Pᵢ signal without transporting the ion. Furthermore, the loss of ERK1/2 signaling in the absence of PiT1 or PiT2 was not associated with a change in cellular Pᵢ uptake, further indicating that the transport of Pᵢ into the cell is not necessary for Pᵢ signaling.

These data have major implications for the understanding of the Pᵢ-signaling mechanism. The absence of functional compensation by PiT1 in PiT2-depleted cells, and vice versa, together with the requirement of both PiTs for Pᵢ signaling, suggested that they could interact functionally and/or physically to mediate the Pᵢ signal. Consistent with this hypothesis, using a BRET approach, we showed that PiT proteins could

**Figure 3. Specific interaction between PiT1 and PiT2 varies upon extracellular Pᵢ concentration.** A, Western blotting analysis of Na/K-ATPase, hPiT1, and hPiT2 expression in *pmaxGFP*-transfected (Ctrl) or *hPiT1* and *hPiT2* co-transfected HEK293T cells after crude cellular fractionation, as indicated (see "Experimental procedures" for details). Overexpression of hPiT1 and PiT2 allowed a better signal than Ctrl. B, Western blotting analysis (*IB*) of hPiT1 (*left*) and hPiT2 (*right*) expression in HEK293T cells untransfected (*UT*) or transfected with hPiT1, hPiT2, or both after cell surface cross-linking using bis(sulfosuccinimidyl)suberate and cellular fractionation. Enriched plasma membrane fraction (*F1*) was analyzed. C, schematic representation of chimeric hPiT1-eYFP or -Rluc and hPiT2-eYFP or -Rluc proteins used for BRET assays. The DNA region encoding for the large internal loop of PiT1 and PiT2 (iLoop1 and iLoop2, respectively) was replaced by coding sequences of eYFP or Rluc. Transmembrane domains (*black rectangles*) are shown. D, confocal images obtained from transfected HEK293T cells, as indicated. Rluc was detected after immunofluorescence and nuclei were stained using Hoechst (*blue*). Scale bar, 10 µm. *E*, representative BRET saturation curves of co-transfected HEK293T cells, as indicated. *F*, representative BRET saturation curves (*left panel*) and BRET 50 index (*right panel*) of HEK293T co-transfected with hPiT1-eYFP and hPiT2-Rluc obtained following a 10-min incubation with 1, 3, or 10 mM extracellular Pᵢ concentration, as indicated. BRET 50 data are means ± S.E. (*error bars*) (**, *p* < 0.01 versus 1 mM Pᵢ condition, *n* = 8). *G*, the BRET 50 index was measured from BRET saturation curves obtained from HEK293T co-transfected with hPiT1-eYFP and hPiT1-Rluc (*left*) or hPiT2-eYFP and hPiT2-Rluc (*right*) following a 10-min incubation with 1, 3, or 10 mM extracellular Pᵢ concentration. BRET 50 data are means ± S.E., and no statistically difference was observed (*n* = 4–8).
Pivotal role of PiT1 and PiT2 in bone phosphate sensing

PiT1-PiT2-mediated ERK1/2 activation by extracellular Pᵢ and
regulation of PiT hetero-oligomerization are Pᵢ
transport-independent

Our results illustrated that both PiT proteins are important
for Pᵢ-dependent ERK signaling and that Pᵢ can modulate PiT
hetero-oligomerization. To elucidate whether the Pᵢ trans-
port function of PiTs was important to mediate Pᵢ-dependent
ERK1/2 signaling, we used the previously reported hPiT1S¹²⁸A
and hPiT2S¹¹³A Pᵢ transport-deficient mutants (56, 63, 64).
When hPiT1S¹²⁸A was overexpressed in PiT1-depleted cells in
which Pᵢ-dependent ERK1/2 activation was lost, we could res-
cue the activation of ERK1/2 signaling (Fig. 4A). Similarly, we
could rescue the Pᵢ-dependent ERK1/2 activation in PiT2-de-
pleted cells by overexpressing hPiT2S¹¹³A mutant. This was also
true when cells depleted from both PiTs were transfected by
both hPiT1S¹²⁸A and hPiT2S¹¹³A (Fig. 4A). These data demon-
strated that the Pᵢ transport function of PiTs was dispensable
for the Pᵢ-dependent ERK1/2 activation. To further study
whether the sodium-dependent Pᵢ transport function of PiTs
was important to mediate Pᵢ-dependent PiT hetero-oligomeri-
zation, we performed BRET experiments in the absence of Na⁺.
In this condition where Pᵢ transport was blunted, the variation
of extracellular Pᵢ concentrations was still able to modulate PiT
interaction (Fig. 4 (B and C) and Fig. S5C). Because Pᵢ was able
to modulate PiT interaction without being transported, this
suggests that the binding of Pᵢ to PiT proteins rather than its
actual uptake into the cell may be involved in the modulation of
PiT interaction. We next generated mutated versions of the
chimeric hPiT1-eYFP and hPiT2-Rlac proteins in which Ser¹²⁸
or Ser¹¹³ was replaced by an alanine residue. As expected, over-
expression of chimeric hPiT1S¹²⁸A-eYFP and hPiT2S¹¹³A-Rlac
resulted in decreased sodium-dependent Pᵢ transport com-
pared with transporting chimeric PiT proteins (Fig. 4D). Inter-
estingly, using a BRET approach, we showed that although
hPiT1S¹²⁸A-eYFP and hPiT2S¹¹³A-Rlac were still able to inter-
act together, this interaction was not modulated anymore by
extracellular Pᵢ variations (Fig. 4E (**left**) and Fig. S5C), consistent
with a role for Ser¹²⁸ or Ser¹¹³ in Pᵢ binding. When BRET exper-
iments were performed with a transport-deficient mutant
(hPiT1S¹²⁸A-eYFP or hPiT2S¹¹³A-Rlac) and a transporting chi-
meric PiT protein (hPiT1-eYFP and hPiT2-Rlac), we could
recover the modulation of PiT interaction by Pᵢ (Fig. 4E (**middle
and left**) and Fig. S5C), further supporting a role of Ser¹²⁸ or
Ser¹¹³ in the Pᵢ-dependent interaction of PiTs.

Discussion

The ability of a cell to detect changes in extracellular Pᵢ levels is paramount for its adequate response to environmental fluctuations and critical for the appropriate modulation of Pᵢ homeostasis and skeletal mineralization. In this work, we provided mechanistic insights into the molecular events leading to the detection of changes in extracellular Pᵢ concentrations using skeletal cell lines as a model.

Skeletal cells are constantly exposed to high local extracellular Pᵢ levels, mainly due to the continual resorption and formation of the mineralized extracellular matrix of bone and the need for tremendous quantities of Pᵢ for mineralization purposes (65). This has made bone a model of choice to study Pᵢ signaling, where it has been shown in early studies to regulate the programmed cell death of hypertrophic chondrocytes (10, 11, 13). In subsequent studies, we and others have shown that Pᵢ could regulate skeletal mineralization by controlling the expression of the mineralization inhibitors *Mgp* and *Opn* through the activation of the ERK1/2 pathway (7, 15, 16, 18, 19). Our present study brings evidence for a role of PiT1 and PiT2 in transmitting the Pᵢ signal to the cell by showing that the Pᵢ-dependent up-regulation of *Mgp* and *Opn* and ERK1/2 phosphorylation were blunted in PiT1- or PiT2-depleted cells. A role for PiT1 in mediating ERK1/2 signaling has been reported earlier (48, 55); however, a similar role for PiT2 has never been illustrated before.

A critical aspect in studying the molecular events involved in Pᵢ signaling is whether or not Pᵢ needs to be transported within the cell to fulfill its role. This question is particularly relevant in view of the role of PiT1 and PiT2 as mediators of Pᵢ signaling because these two proteins have well-described Pᵢ transport functions (41–43). By using Pᵢ transport–deficient mutants of PiT proteins, we could rescue the ERK1/2 signaling, demonstrating that PiT1 and PiT2 can mediate a Pᵢ signal without transporting the ion. Furthermore, the loss of ERK1/2 signaling in the absence of PiT1 or PiT2 was not associated with a change in cellular Pᵢ uptake, further indicating that the transport of Pᵢ into the cell is not necessary for Pᵢ signaling.

These data have major implications for the understanding of
the Pᵢ-signaling mechanism. The absence of functional com-
pensation by PiT1 in PiT2-depleted cells, and vice versa,
together with the requirement of both PiTs for Pᵢ signaling,
suggested that they could interact functionally and/or physi-
cally to mediate the Pᵢ signal. Consistent with this hypothesis,
using a BRET approach, we showed that PiT proteins could

**Figure 3. Specific interaction between PiT1 and PiT2 varies upon extracellular Pᵢ concentration.** A, Western blotting analysis of Na/K-ATPase, hPiT1, and hPiT2 expression in *pmaxGFP*-transfected (Ctrl) or *hPiT1* and *hPiT2* co-transfected HEK293T cells after crude cellular fractionation, as indicated (see "Experimental procedures" for details). Overexpression of hPiT1 and PiT2 allowed a better signal than Ctrl. B, Western blotting analysis (*IB*) of hPiT1 (*left*) and hPiT2 (*right*) expression in HEK293T cells untransfected (*UT*) or transfected with hPiT1, hPiT2, or both after cell surface cross-linking using bis(sulfosuccinimidyl)suberate and cellular fractionation. Enriched plasma membrane fraction (*F1*) was analyzed. C, schematic representation of chimeric hPiT1-eYFP or -Rlac and hPiT2-eYFP or -Rlac proteins used for BRET assays. The DNA region encoding for the large internal loop of PiT1 and PiT2 (iLoop1 and iLoop2, respectively) was replaced by coding sequences of eYFP or Rlac. Transmembrane domains (*black rectangles*) are shown. D, confocal images obtained from transfected HEK293T cells, as indicated. Rlac was detected after immunofluorescence and nuclei were stained using Hoechst (*blue*). Scale bar, 10 µm. *E*, representative BRET saturation curves of co-transfected HEK293T cells, as indicated. *F*, representative BRET saturation curves (*left panel*) and BRET 50 index (*right panel*) of HEK293T co-transfected with hPiT1-eYFP and hPiT2-Rlac obtained following a 10-min incubation with 1, 3, or 10 mm extracellular Pᵢ concentration, as indicated. BRET 50 data are means ± S.E. (*error bars*) (**, *p* < 0.01 versus 1 mm Pᵢ condition, *n* = 8). *G*, the BRET 50 index was measured from BRET saturation curves obtained from HEK293T co-transfected with hPiT1-eYFP and hPiT1-Rlac (*left*) or hPiT2-eYFP and hPiT2-Rlac (*right*) following a 10-min incubation with 1, 3, or 10 mm extracellular Pᵢ concentration. BRET 50 data are means ± S.E., and no statistically difference was observed (*n* = 4–8).
Pivotal role of PiT1 and PiT2 in bone phosphate sensing

<figure><img src="image_19.png" /><figcaption>**Figure 4.** P<sub>i</sub>-dependent ERK1/2 signaling and PiT1-PiT2 hetero-oligomerization are independent of P<sub>i</sub> transport. A, Western blot analysis of ERK1/2 phosphorylation (P-ERK 1/2) in transiently transfected MC615 cells, as indicated. Overexpression of P<sub>i</sub> transport-deficient hPiT1 (PiT1<sup>S128A</sup>) and/or hPiT2 (PiT2<sup>S113A</sup>) was performed in PiT1-, PiT2-, or PiT1-PiT2-depleted cells, respectively. Cells were incubated in low-serum (0.5%) medium for 24 h and stimulated for 30 min with 1 or 10 mm extracellular P<sub>i</sub> concentration. Total ERK1/2 proteins were used as a loading control. B, sodium-dependent and -independent P<sub>i</sub> uptake was measured in HEK293T cells transfected as indicated. Data are means ± S.E. (error bars) (*, *p* < 0.05 *versus* sodium-dependent, *n* = 4). C, BRET 50 index was measured from BRET saturation curves obtained after a 10-min stimulation with 1, 3, or 10 mm extracellular P<sub>i</sub> concentration from HEK293T co-transfected with *hPiT1-eYFP* and *hPiT2-Rluc* in an Na<sup>+</sup>-free medium. Data are means ± S.E. (*, *p* < 0.05 *versus* 1 mm P<sub>i</sub> condition, *n* = 4). D, sodium-dependent P<sub>i</sub> uptake was measured in HEK293T cells transfected with pcDNA6A plasmid (Ctrl) or with plasmids containing normal (hPiT1 and hPiT2) or P<sub>i</sub> transport-deficient mutants (hPiT1<sup>S128A</sup> and hPiT2<sup>S113A</sup>) with or without BRET chimeric acceptor or donor, as indicated. Data are means ± S.E. (*, *p* < 0.05 *versus* P<sub>i</sub>-transporting PiTs, *n* = 4). E, BRET 50 index was measured from BRET saturation curves obtained after 1, 3, or 10 mm extracellular P<sub>i</sub> concentration stimulation for 10 min from HEK293T co-transfected with the indicated plasmids. Data are means ± S.E. (*, *p* < 0.05 *versus* 1 mm P<sub>i</sub> condition, *n* = 4–5).</figcaption></figure>

form homo- and heterodimers. More strikingly, we could illus-
trate that the formation of PiT1-PiT2 heterodimers only was
affected by the variation of extracellular Pᵢ levels. We could not
quantify the relative importance of hetero- *versus* homodimers,
but the cross-linking data supported the idea that the Pᵢ-sensi-
tive PiT1-PiT2 heterodimers were present at much lower quan-
tities. It is possible that a low-abundance Pᵢ-sensitive PiT1-PiT2
heterodimer may be more effectively tunable than a highly
abundant Pᵢ sensor. This abundance may also be consistent
with the on/off Pᵢ effect on ERK1/2 signaling that we have
observed when deleting the PiT proteins.

Although a Pᵢ-sensitive PiT1-PiT2 heterodimer is likely to
represent an important component of the Pᵢ-sensing machin-
ery, deciphering the detailed functioning of such a sensor
requires additional work. Nevertheless, our data provide sev-
eral important mechanistic insights that may give clues to the

understanding of the Pᵢ-signaling cascade. We showed that in
the absence of Na⁺, which blunts the Pᵢ transport activity of PiT
proteins, the formation of PiT1-PiT2 heterodimers was still
sensitive to extracellular Pᵢ variations. This is consistent with
the idea that binding of Pᵢ, rather than transport itself, is impor-
tant for PiT1-PiT2 heterodimerization. This also suggests that
Ser¹²⁸ and Ser¹¹³ residues that were demonstrated to be critical
for Pᵢ transport by PiT1 and PiT2 (56, 63) may indeed be
involved in Pᵢ binding, a prerequisite step for Pᵢ transport (51).
In line with this possibility, when BRET experiments were per-
formed using PiT1 and PiT2 chimeric proteins in which Ser¹²⁸
and Ser¹¹³ were substituted with alanine residues, the PiT1-
PiT2 heterodimer was not responsive anymore at extracellular
Pᵢ variations. The substitution of only one serine, however, res-
cued Pᵢ sensitivity of the PiT1-PiT2 heteroduplex, illustrating
the complex relationship between the structural arrangement
Pivotal role of PiT1 and PiT2 in bone phosphate sensing

<figure><img src="image_17.png" /><figcaption>**Figure 4.** P<sub>i</sub>-dependent ERK1/2 signaling and PiT1-PiT2 hetero-oligomerization are independent of P<sub>i</sub> transport. A, Western blot analysis of ERK1/2 phosphorylation (P-ERK 1/2) in transiently transfected MC615 cells, as indicated. Overexpression of P<sub>i</sub> transport-deficient hPiT1 (PiT1<sup>S128A</sup>) and/or hPiT2 (PiT2<sup>S113A</sup>) was performed in PiT1-, PiT2-, or PiT1-PiT2-depleted cells, respectively. Cells were incubated in low-serum (0.5%) medium for 24 h and stimulated for 30 min with 1 or 10 mm extracellular P<sub>i</sub> concentration. Total ERK1/2 proteins were used as a loading control. B, sodium-dependent and -independent P<sub>i</sub> uptake was measured in HEK293T cells transfected as indicated. Data are means ± S.E. (error bars) (*, *p* < 0.05 *versus* sodium-dependent, *n* = 4). C, BRET 50 index was measured from BRET saturation curves obtained after a 10-min stimulation with 1, 3, or 10 mm extracellular P<sub>i</sub> concentration from HEK293T co-transfected with *hPiT1-eYFP* and *hPiT2-Rluc* in an Na<sup>+</sup>-free medium. Data are means ± S.E. (*, *p* < 0.05 *versus* 1 mm P<sub>i</sub> condition, *n* = 4). D, sodium-dependent P<sub>i</sub> uptake was measured in HEK293T cells transfected with pcDNA6A plasmid (Ctrl) or with plasmids containing normal (hPiT1 and hPiT2) or P<sub>i</sub> transport-deficient mutants (hPiT1<sup>S128A</sup> and hPiT2<sup>S113A</sup>) with or without BRET chimeric acceptor or donor, as indicated. Data are means ± S.E. (*, *p* < 0.05 *versus* P<sub>i</sub>-transporting PiTs, *n* = 4). E, BRET 50 index was measured from BRET saturation curves obtained after 1, 3, or 10 mm extracellular P<sub>i</sub> concentration stimulation for 10 min from HEK293T co-transfected with the indicated plasmids. Data are means ± S.E. (*, *p* < 0.05 *versus* 1 mm P<sub>i</sub> condition, *n* = 4–5).</figcaption></figure>

form homo- and heterodimers. More strikingly, we could illus-
trate that the formation of PiT1-PiT2 heterodimers only was
affected by the variation of extracellular Pᵢ levels. We could not
quantify the relative importance of hetero- *versus* homodimers,
but the cross-linking data supported the idea that the Pᵢ-sensi-
tive PiT1-PiT2 heterodimers were present at much lower quan-
tities. It is possible that a low-abundance Pᵢ-sensitive PiT1-PiT2
heterodimer may be more effectively tunable than a highly
abundant Pᵢ sensor. This abundance may also be consistent
with the on/off Pᵢ effect on ERK1/2 signaling that we have
observed when deleting the PiT proteins.

Although a Pᵢ-sensitive PiT1-PiT2 heterodimer is likely to
represent an important component of the Pᵢ-sensing machin-
ery, deciphering the detailed functioning of such a sensor
requires additional work. Nevertheless, our data provide sev-
eral important mechanistic insights that may give clues to the

understanding of the Pᵢ-signaling cascade. We showed that in
the absence of Na⁺, which blunts the Pᵢ transport activity of PiT
proteins, the formation of PiT1-PiT2 heterodimers was still
sensitive to extracellular Pᵢ variations. This is consistent with
the idea that binding of Pᵢ, rather than transport itself, is impor-
tant for PiT1-PiT2 heterodimerization. This also suggests that
Ser¹²⁸ and Ser¹¹³ residues that were demonstrated to be critical
for Pᵢ transport by PiT1 and PiT2 (56, 63) may indeed be
involved in Pᵢ binding, a prerequisite step for Pᵢ transport (51).
In line with this possibility, when BRET experiments were per-
formed using PiT1 and PiT2 chimeric proteins in which Ser¹²⁸
and Ser¹¹³ were substituted with alanine residues, the PiT1-
PiT2 heterodimer was not responsive anymore at extracellular
Pᵢ variations. The substitution of only one serine, however, res-
cued Pᵢ sensitivity of the PiT1-PiT2 heteroduplex, illustrating
the complex relationship between the structural arrangement
Pivotal role of PiT1 and PiT2 in bone phosphate sensing

of the Pᵢ binding site and Pᵢ sensitivity. A determination of the crystal structure of PiT proteins is therefore necessary to help determine the identity of the Pᵢ binding site and its role in Pᵢ transport and sensing.

An interesting consequence of our work is that PiT proteins should not be considered anymore as Pᵢ transporters solely but also as Pᵢ receptors able to mediate Pᵢ signaling by activating specific downstream pathways. Such a hybrid transporter-receptor behavior may indicate that the PiT1-PiT2 heteroduplex could behave as a Pᵢ transceptor, whereby conformational changes during the transport cycle (including the Pᵢ-binding step) affect a signal transduction component that triggers a downstream signaling pathway (66). The difference between a transceptor and a pure receptor is that the transceptor can also transport the ligand into the cell. In recent years, evidence for transporters functioning as transceivers has been obtained in several eukaryotic systems (67). Interestingly, in yeast, the Pho84 phosphate carrier that is considered as an essential component of the Pᵢ-sensing system was characterized as a Pᵢ transceptor (40). In prokaryotic or lower eukaryotic organisms, true transceivers could be functionally characterized by specific mutants that either lack the signaling capacity and retain normal transport or have lost transport but retain signaling. In the case of PiT proteins, the signaling capacity is lost when either PiT1 or PiT2 is deleted, indicating that the possible transceptor function may only be revealed by heterodimerization and may not be associated with structural changes of either PiT. The gain of function provided through PiT heterodimerization implies that a unique protein complex mediates Pᵢ signaling. This underlies the idea that specific PiT1 and PiT2 protein partners may be involved in this process. If this is the case, the identification of PiT-specific partners in the future may provide unique targets to modulate Pᵢ signaling in specific organs or specific physiological conditions.

In summary, this study provided mechanistic insights into the Pᵢ-signaling cascade in skeletal cell line models by unraveling PiT1-PiT2 heterodimers as essential components of a Pᵢ-sensing machinery. Although *in vivo* studies are required to strengthen the physiological relevance of these findings, our work may help in deciphering the mechanisms underlying the ability of the organism to respond to the serum Pᵢ level variations, which is the necessary first step in the Pᵢ homeostasis-regulating cascade.

Experimental procedures

Cells and culture conditions

Murine preosteoblastic MC3T3-E1 cells were seeded at 10⁴ cells/cm² and cultured for 10 days in α-minimum essential medium GlutaMAX™ (catalogue no. 32751, Thermo Fisher Scientific, Saint-Aubin, France) supplemented with 10% FBS and 1% penicillin/streptomycin. Murine chondrogenic MC615 cells were seeded at 10⁴ cells/cm² and maintained in a medium consisting of Dulbecco's modified Eagle's medium (DMEM) high glucose GlutaMAX™/Ham's F-12 (1:1) (catalogue no. 31966 and 31765, respectively, Thermo Fisher Scientific) supplemented with 10% FBS and 1% penicillin/streptomycin. HEK293T cells were maintained in DMEM

<caption>Table 1</caption>

Primer sequences used for RNA interference, site-directed mutagenesis and RT-qPCR

m, mouse; h, human; F, Forward primer; R, Reverse primer. N/A, not applicable.

<table><thead><tr><th>Applications</th><th>Target genes</th><th>Primer sequences (5' to 3')</th><th>Accession Number</th></tr></thead><tbody><tr><td>RNA interference*</td><td>mPiT2</td><td>F: ACTAGATCTCCCGATGAAAGCCTCAGGAAATTCAA<br>GAGATTTCCTGAGGCTTTTCATCGTTTTTAAGCTTCA<br>R: TGAAGCTTAAAAACGATGAAAGCCTCAGGAAATCT<br>CTTGAATTTCCTGAGGCTTTCATCGGGAGATCTAGT</td><td>N/A</td></tr><tr><td rowspan="4">Site-directed mutagenesis⁵</td><td>hPiT1ΔV5-6xHis</td><td>F: GTGCTGGAATTCGGCTTCTACATTCTGAGGATGAC<br>AT<br>R: ATGTCATCCTCAGAATGTAGAAGCCGAATTCCAGC<br>AC</td><td rowspan="4">N/A</td></tr><tr><td>hPiT2ΔV5-6xHis</td><td>F: GATCCTTCCATATGTGTGATCTAGAGGGCCCTTCG<br>R: CGAAGGGCCCTCTAGATCACACATATGGAAGGATC</td></tr><tr><td>hPiT1-eYFP<sup>S1284</sup></td><td>F: CAATGGGTTCCAGCAATAGGGAGCTTCAAAACGA<br>AGC<br>R: GCTTCGTTTTTGAAGCTCCCTATTGCTGGAACCCAT<br>TG</td></tr><tr><td>hPiT2-Rluc<sup>S134</sup></td><td>F: TGCAGTGCGTTTCCTGCGATTGGAAGCCTCAG<br>R: CTGAGGCTTCCAATCGCAGGAACGCACTGCA</td></tr><tr><td rowspan="7">RT-qPCR</td><td>Gapdh</td><td>F: GAGCCAAACGGGTCATCA<br>R: CATATTTCTCGTGGTTCACACC</td><td>M32599</td></tr><tr><td>Mgp</td><td>F: GTCCTATGAAATCAGTCCCTTCA<br>R: TTGTTGCGTTCCCTGGACTCT</td><td>NM_008597</td></tr><tr><td>Opn</td><td>F: CCCGGTGAAAGTGACTGATT<br>R: TTCTTCAGAGGACACAGCATTC</td><td>NM_009263</td></tr><tr><td>mPiT1</td><td>F: TGTGGCAAATGGGCAGAAG<br>R: AGAAAGCAGCGGAGAGACGA</td><td>NM_015747</td></tr><tr><td>mPiT2</td><td>F: CCATCGGCTTCTCACTCGT<br>R: AAACCAGGAGGCGACAATCT</td><td>NM_011394</td></tr><tr><td>hPiT1</td><td>F: GTTCGTGCATTCATCCTCCAT<br>R: TGGTACCCACAGAGGAAGTTT</td><td>NM_005415</td></tr><tr><td>hPiT2</td><td>F: TCTCATGGCTGGGGGAAGTTAGT<br>R: TTGCGACCAGTGAGAATCCTAT</td><td>NM_006749</td></tr></tbody></table>

*, siRNA sequences are underlined.

#, desired mutation is underlined.

high glucose GlutaMAX™ supplemented with 10% FBS, 10 mM HEPES, and 50 µg/ml gentamicin. Cells were cultured at 37 °C in a humidified atmosphere of 5% CO₂ in air, and media were renewed every 2–3 days. When indicated, cells were incubated in low-serum (0.5%) medium for 24 h and stimulated with various concentrations of Pᵢ for 30 min or 24 h. Pᵢ was added as a mixture of NaH₂PO₄ and Na₂HPO₄ (pH 7.4), as described previously (10).

RNA interference

Inactivation of PiT1 or PiT2 were performed by cloning an *shPiT1* (56) or *shPiT2* (see sequences in Table 1) into pSUPER vector (68) expressing a puromycin resistance gene. A scramble sequence cloned into pSUPER was used as a negative control (56). Stable knockdown of PiT1 or PiT2 was obtained by transfecting MC3T3-E1 cells with 5 µg of the pSUPER-*shRNAs* using the T-20 program of the Amaxa nucleofector system (Cell Line Nucleofector™ Kit V VCA-1003, Lonza, Bâle, Switzerland). Cells were plated at limiting density, and puromycin-resistant clones were picked, expanded, and tested for PiT expression. Experiments were performed with three independent stable transfectants, and the data presented illustrate a representative clone. Transient inactivation of PiT was also performed in MC3T3-E1 and MC615 cells using transfection as described above. These transient experiments were stopped 72 h after transfection.
Pivotal role of PiT1 and PiT2 in bone phosphate sensing

of the Pᵢ binding site and Pᵢ sensitivity. A determination of the crystal structure of PiT proteins is therefore necessary to help determine the identity of the Pᵢ binding site and its role in Pᵢ transport and sensing.

An interesting consequence of our work is that PiT proteins should not be considered anymore as Pᵢ transporters solely but also as Pᵢ receptors able to mediate Pᵢ signaling by activating specific downstream pathways. Such a hybrid transporter-receptor behavior may indicate that the PiT1-PiT2 heteroduplex could behave as a Pᵢ transceptor, whereby conformational changes during the transport cycle (including the Pᵢ-binding step) affect a signal transduction component that triggers a downstream signaling pathway (66). The difference between a transceptor and a pure receptor is that the transceptor can also transport the ligand into the cell. In recent years, evidence for transporters functioning as transceptrons has been obtained in several eukaryotic systems (67). Interestingly, in yeast, the Pho84 phosphate carrier that is considered as an essential component of the Pᵢ-sensing system was characterized as a Pᵢ transceptor (40). In prokaryotic or lower eukaryotic organisms, true transceptrons could be functionally characterized by specific mutants that either lack the signaling capacity and retain normal transport or have lost transport but retain signaling. In the case of PiT proteins, the signaling capacity is lost when either PiT1 or PiT2 is deleted, indicating that the possible transceptor function may only be revealed by heterodimerization and may not be associated with structural changes of either PiT. The gain of function provided through PiT heterodimerization implies that a unique protein complex mediates Pᵢ signaling. This underlies the idea that specific PiT1 and PiT2 protein partners may be involved in this process. If this is the case, the identification of PiT-specific partners in the future may provide unique targets to modulate Pᵢ signaling in specific organs or specific physiological conditions.

In summary, this study provided mechanistic insights into the Pᵢ-signaling cascade in skeletal cell line models by unraveling PiT1-PiT2 heterodimers as essential components of a Pᵢ-sensing machinery. Although *in vivo* studies are required to strengthen the physiological relevance of these findings, our work may help in deciphering the mechanisms underlying the ability of the organism to respond to the serum Pᵢ level variations, which is the necessary first step in the Pᵢ homeostasis-regulating cascade.

Experimental procedures

Cells and culture conditions

Murine preosteoblastic MC3T3-E1 cells were seeded at 10⁴ cells/cm² and cultured for 10 days in α-minimum essential medium GlutaMAX™ (catalogue no. 32751, Thermo Fisher Scientific, Saint-Aubin, France) supplemented with 10% FBS and 1% penicillin/streptomycin. Murine chondrogenic MC615 cells were seeded at 10⁴ cells/cm² and maintained in a medium consisting of Dulbecco's modified Eagle's medium (DMEM) high glucose GlutaMAX™/Ham's F-12 (1:1) (catalogue no. 31966 and 31765, respectively, Thermo Fisher Scientific) supplemented with 10% FBS and 1% penicillin/streptomycin. HEK293T cells were maintained in DMEM

<caption>Table 1</caption>

Primer sequences used for RNA interference, site-directed mutagenesis and RT-qPCR

m, mouse; h, human; F, Forward primer; R, Reverse primer. N/A, not applicable.

<table><thead><tr><th>Applications</th><th>Target genes</th><th>Primer sequences (5' to 3')</th><th>Accession Number</th></tr></thead><tbody><tr><td>RNA interference*</td><td>mPiT2</td><td>F: ACTAGATCTCCCGATGAAAGCCTCAGGAAATTCAA<br>GAGATTTCCTGAGGCTTTTCATCGTTTTTAAGCTTCA<br>R: TGAAGCTTAAAAACGATGAAAGCCTCAGGAAATCT<br>CTTGAATTTCCTGAGGCTTTCATCGGGAGATCTAGT</td><td>N/A</td></tr><tr><td rowspan="4">Site-directed mutagenesis⁹</td><td>hPiT1ΔV5-6xHis</td><td>F: GTGCTGGAATTCGGCTTCTACATTCTGAGGATGAC<br>AT<br>R: ATGTCATCCTCAGAATGTAGAAGCCGAATTCCAGC<br>AC</td><td rowspan="4">N/A</td></tr><tr><td>hPiT2ΔV5-6xHis</td><td>F: GATCCTTCCATATGTGTGATCTAGAGGGCCCTTCG<br>R: CGAAGGGCCCTCTAGATCACACATATGGAAGGATC</td></tr><tr><td>hPiT1-eYFP<sup>S1284</sup></td><td>F: CAATGGGTTCCAGCAATAGGGAGCTTCAAAACGA<br>AGC<br>R: GCTTCGTTTTTGAAGCTCCCTATTGCTGGAACCCAT<br>TG</td></tr><tr><td>hPiT2-Rluc<sup>S134</sup></td><td>F: TGCAGTGCGTTTCCTGCGATTGGAAGCCTCAG<br>R: CTGAGGCTTCCAATCGCAGGAACGCACTGCA</td></tr><tr><td rowspan="7">RT-qPCR</td><td>Gapdh</td><td>F: GAGCCAAACGGGTCATCA<br>R: CATATTTCTCGTGGTTCACACC</td><td>M32599</td></tr><tr><td>Mgp</td><td>F: GTCCTATGAAATCAGTCCCTTCA<br>R: TTGTTGCGTTCCCTGGACTCT</td><td>NM_008597</td></tr><tr><td>Opn</td><td>F: CCCGGTGAAAGTGACTGATT<br>R: TTCTTCAGAGGACACAGCATTC</td><td>NM_009263</td></tr><tr><td>mPiT1</td><td>F: TGTGGCAAATGGGCAGAAG<br>R: AGAAAGCAGCGGAGAGACGA</td><td>NM_015747</td></tr><tr><td>mPiT2</td><td>F: CCATCGGCTTCTCACTCGT<br>R: AAACCAGGAGGCGACAATCT</td><td>NM_011394</td></tr><tr><td>hPiT1</td><td>F: GTTCGTGCATTCATCCTCCAT<br>R: TGGTACCCACAGAGGAAGTTT</td><td>NM_005415</td></tr><tr><td>hPiT2</td><td>F: TCTCATGGCTGGGGAAGTTAGT<br>R: TTGCGACCAGTGAGAATCCTAT</td><td>NM_006749</td></tr></tbody></table>

*, siRNA sequences are underlined.

#, desired mutation is underlined.

high glucose GlutaMAX™ supplemented with 10% FBS, 10 mM HEPES, and 50 µg/ml gentamicin. Cells were cultured at 37 °C in a humidified atmosphere of 5% CO₂ in air, and media were renewed every 2–3 days. When indicated, cells were incubated in low-serum (0.5%) medium for 24 h and stimulated with various concentrations of Pᵢ for 30 min or 24 h. Pᵢ was added as a mixture of NaH₂PO₄ and Na₂HPO₄ (pH 7.4), as described previously (10).

RNA interference

Inactivation of PiT1 or PiT2 were performed by cloning an *shPiT1* (56) or *shPiT2* (see sequences in Table 1) into pSUPER vector (68) expressing a puromycin resistance gene. A scramble sequence cloned into pSUPER was used as a negative control (56). Stable knockdown of PiT1 or PiT2 was obtained by transfecting MC3T3-E1 cells with 5 µg of the pSUPER-*shRNAs* using the T-20 program of the Amaxa nucleofector system (Cell Line Nucleofector™ Kit V VCA-1003, Lonza, Bâle, Switzerland). Cells were plated at limiting density, and puromycin-resistant clones were picked, expanded, and tested for PiT expression. Experiments were performed with three independent stable transfectants, and the data presented illustrate a representative clone. Transient inactivation of PiT was also performed in MC3T3-E1 and MC615 cells using transfection as described above. These transient experiments were stopped 72 h after transfection.
# Pivotal role of PiT1 and PiT2 in bone phosphate sensing

## Gene expression analysis

Total RNA was isolated from cells using the NucleoSpin RNA II kit (Macherey-Nagel, Hoerdt, France) according to the manufacturer's instructions. RNA was reverse transcribed using Affinity Script (Agilent Genomics, Santa Clara, CA) as per the manufacturer's recommendations. Real-time PCR was performed on a Mx3000P system (Stratagene, San Diego, CA) using Brilliant III Ultra-Fast SYBR QPCR Master Mix (Agilent Genomics). The following temperature profile was used: denaturation at 95 °C for 3 min, amplification during 40 cycles of 5 s at 95 °C and 20 s at 60 °C, followed by a step at 95 °C for 1 min and 65 °C for 30 s. Expression of target genes were normalized to GAPDH expression levels and were calculated as described previously (69). Primer sequences are listed in Table 1.

## Immunofluorescence and confocal microscopy

One day after transient transfection, MC615 cells were fixed/permeabilized in methanol at -20 °C for 5 min and blocked in 1% bovine serum albumin for 1 h at room temperature. Immunodetection of PiT1 and PiT2 was performed using rabbit anti-mouse PiT1 or PiT2 antibody, respectively (generously provided by Dr. G. Friedlander), at a 1:200 dilution overnight at 4 °C, and goat anti-rabbit Alexa488 secondary antibody (catalogue no. A11008, Thermo Fisher Scientific) at a 1:1,000 dilution for 1 h at room temperature. The nuclei were counterstained using 1 µg/ml TO-PRO 3 iodide solution (Thermo Fisher Scientific). The stained cells were mounted in Prolong Antifade mounting medium (Thermo Fisher Scientific). Images were acquired with a Nikon Eclipse TE2000E confocal microscope (Nikon, Badhoevedorp, The Netherlands) equipped with a ×60 oil immersion objective. The averaged intensity for PiT1 and PiT2 staining was determined using Metamorph version 7.5 software.

HEK293T cells were seeded at $3 \times 10^4$ cells/cm$^2$ in µ-slide 8-well ibiTreat (catalogue no. 80826, Ibidi, Madison, WI) pre-coated with poly-L-lysine (Sigma-Aldrich). Cells were transfected with 0.125 µg/well of plasmid using JetPrime (Polyplus transfection, Illkirch, France) according to the manufacturer's instructions. Twenty-four hours post-transfection, HEK293T cells were fixed in 4% paraformaldehyde, permeabilized in 0.2% Triton X-100, and blocked in 1% bovine serum albumin for 1 h at room temperature. Immunodetection of hPiT1-Rluc, hPiT2-Rluc, and BTN3A2-Rluc was performed using anti-Rluc antibody (catalogue no. GTX47953, GeneTex, Irvine, CA) at a 1:200 dilution overnight at 4 °C and goat anti-rabbit Alexa488 secondary antibody (catalogue no. A11008, Thermo Fisher Scientific) at a 1:1,000 dilution for 1 h at room temperature. Nucleus staining was performed using 2 µg/ml Hoescht solution (catalogue no. H3569, Thermo Fisher Scientific), and immunofluorescence was preserved in Citifluor AF1 (Biovalley, Nanterre, France). Images were acquired with a Nikon A1Rsi confocal microscope (Nikon, Minato-ku, Tokyo, Japan) equipped with a ×60 oil immersion objective.

## Phosphate uptake measurements

HEK293T cells were seeded at $10^5$ cells/cm$^2$ in poly-L-lysine (Sigma-Aldrich)-precoated 24-well plates. Cells were transfected with 0.5 µg/well of plasmid using JetPrime (Polyplus

transfection) according to the manufacturer's instructions. $P_i$ uptake in HEK293T cells was measured 24 h after transfection. To perform uptake experiments in MC3T3-E1 cells, PiT-depleted MC3T3-E1 clones were seeded at $1.5 \times 10^5$ cells/cm$^2$, and uptake was performed 24 h later. $P_i$ uptake was performed as described previously (70). Briefly, after three washing steps, cells were incubated in an uptake medium containing 100 µM $[^{32}P]KH_2PO_4$ (0.5 µCi/ml) in the presence of 137 mM NaCl (total $P_i$ transport) or N-methyl-D-glucamine (sodium-independent transport) at 37 °C for 10 min. sodium-dependent $P_i$ transport was calculated as the difference between total and sodium-independent $P_i$ transports. Cells were washed three times and lysed with 0.1 M NaOH solution, and aliquots of cell lysates were taken for the determination of protein content (Pierce BCA protein assay kit, Thermo Fisher Scientific) and the radioactivity by liquid scintillation counting (Ultima Gold LLT, PerkinElmer Life Sciences) in a Hidex 300 SL β counter.

## Immunoblot analysis

Cells were lysed for 30 min in ice-cold lysis buffer (20 mM Tris-HCl, pH 7.5, 100 mM potassium chloride, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 20 mM β-glycerophosphate, 2 mM Na$_3$VO$_4$, 1 mM phenylmethylsulfonyl fluoride, and 1 mM NaF). After centrifugation at $12,000 \times g$ for 10 min at 4 °C, the protein extracts (supernatants) were boiled in Laemml loading buffer before SDS-PAGE. For cross-linking experiments, proteins were not boiled, and electrophoresis was done on NuPAGE™ 3–8% Tris acetate gel (catalogue no. EA0378BOX, Thermo Fisher Scientific). Proteins were transferred to PVDF membrane, and blocking was performed with 5% nonfat dry milk/TBST (10 mM Tris, 154 mM NaCl, 0.15% Tween 20) for 1 h at room temperature. Blots were probed with primary antibodies in 5% nonfat dry milk/TBST overnight at 4 °C followed by secondary antibodies for 1 h at room temperature. Anti-ERK1/2 (catalogue no. 9102) and anti-phospho-ERK1/2 (catalogue no. 4370) were from Cell Signaling (Danvers, MA) and used at a 1:2,000 dilution. Anti-PiT1 (catalogue no. 12423-1-AP) and anti-PiT2 (catalogue no. 12820-1-AP) were from ProteinTech (Rosemont, IL) and used at a 1:1,000 dilution. Anti-Na/K-ATPase (catalogue no. ab7671) was from Abcam (Cambridge, UK) and used at a 1:5,000 dilution. Monoclonal anti-β-actin clone AC-74 (Sigma-Aldrich) was used at a 1:5,000 dilution as a loading control. Anti-rabbit (catalogue no. 7074, Cell Signaling) and anti-mouse (catalogue no. A9917, Sigma-Aldrich) secondary antibodies were used at 1:2,000 and 1:80,000 dilutions, respectively. Signal detection was performed using ECL Western blotting detection reagent and ECL hyperfilm (GE Healthcare) or ChemiDoc Imaging System™ (Bio-Rad).

## Crude cellular fractionation and cross-linking

HEK293T cells were seeded at $10^5$ cells/cm$^2$ in 6-well plates and transfected with 1 µg/well each of *hPiT1* and *hPiT2* plasmids using JetPrime (Polyplus transfection) according to the manufacturer's instructions. Twenty-four hours post-transfection, cells were scraped and lysed in 1 ml of buffer A/well (10 mM Hepes, pH 7, 10% sucrose, 5 mM EDTA, Phosphatase Inhibitor Mixture 3 (catalogue no. P0044), and Protease Inhibitor
# Pivotal role of PiT1 and PiT2 in bone phosphate sensing

## Gene expression analysis

Total RNA was isolated from cells using the NucleoSpin RNA II kit (Macherey-Nagel, Hoerdt, France) according to the manufacturer's instructions. RNA was reverse transcribed using Affinity Script (Agilent Genomics, Santa Clara, CA) as per the manufacturer's recommendations. Real-time PCR was performed on a Mx3000P system (Stratagene, San Diego, CA) using Brilliant III Ultra-Fast SYBR QPCR Master Mix (Agilent Genomics). The following temperature profile was used: denaturation at 95 °C for 3 min, amplification during 40 cycles of 5 s at 95 °C and 20 s at 60 °C, followed by a step at 95 °C for 1 min and 65 °C for 30 s. Expression of target genes were normalized to GAPDH expression levels and were calculated as described previously (69). Primer sequences are listed in Table 1.

## Immunofluorescence and confocal microscopy

One day after transient transfection, MC615 cells were fixed/permeabilized in methanol at -20 °C for 5 min and blocked in 1% bovine serum albumin for 1 h at room temperature. Immunodetection of PiT1 and PiT2 was performed using rabbit anti-rabbit PiT1 or PiT2 antibody, respectively (generously provided by Dr. G. Friedlander), at a 1:200 dilution overnight at 4 °C, and goat anti-rabbit Alexa488 secondary antibody (catalogue no. A11008, Thermo Fisher Scientific) at a 1:1,000 dilution for 1 h at room temperature. The nuclei were counterstained using 1 µg/ml TO-PRO 3 iodide solution (Thermo Fisher Scientific). The stained cells were mounted in Prolong Antifade mounting medium (Thermo Fisher Scientific). Images were acquired with a Nikon Eclipse TE2000E confocal microscope (Nikon, Badhoevedorp, The Netherlands) equipped with a ×60 oil immersion objective. The averaged intensity for PiT1 and PiT2 staining was determined using Metamorph version 7.5 software.

HEK293T cells were seeded at $3 \times 10^4$ cells/cm$^2$ in µ-slide 8-well ibiTreat (catalogue no. 80826, Ibidi, Madison, WI) pre-coated with poly-L-lysine (Sigma-Aldrich). Cells were transfected with 0.125 µg/well of plasmid using JetPrime (Polyplus transfection, Illkirch, France) according to the manufacturer's instructions. Twenty-four hours post-transfection, HEK293T cells were fixed in 4% paraformaldehyde, permeabilized in 0.2% Triton X-100, and blocked in 1% bovine serum albumin for 1 h at room temperature. Immunodetection of hPiT1-Rluc, hPiT2-Rluc, and BTN3A2-Rluc was performed using anti-Rluc antibody (catalogue no. GTX47953, GeneTex, Irvine, CA) at a 1:200 dilution overnight at 4 °C and goat anti-rabbit Alexa488 secondary antibody (catalogue no. A11008, Thermo Fisher Scientific) at a 1:1,000 dilution for 1 h at room temperature. Nucleus staining was performed using 2 µg/ml Hoescht solution (catalogue no. H3569, Thermo Fisher Scientific), and immunofluorescence was preserved in Citifluor AF1 (Biovalley, Nanterre, France). Images were acquired with a Nikon A1Rsi confocal microscope (Nikon, Minato-ku, Tokyo, Japan) equipped with a ×60 oil immersion objective.

## Phosphate uptake measurements

HEK293T cells were seeded at $10^5$ cells/cm$^2$ in poly-L-lysine (Sigma-Aldrich)-precoated 24-well plates. Cells were transfected with 0.5 µg/well of plasmid using JetPrime (Polyplus

transfection) according to the manufacturer's instructions. $P_i$ uptake in HEK293T cells was measured 24 h after transfection. To perform uptake experiments in MC3T3-E1 cells, PiT-depleted MC3T3-E1 clones were seeded at $1.5 \times 10^5$ cells/cm$^2$, and uptake was performed 24 h later. $P_i$ uptake was performed as described previously (70). Briefly, after three washing steps, cells were incubated in an uptake medium containing 100 µM $[^{32}P]KH_2PO_4$ (0.5 µCi/ml) in the presence of 137 mM NaCl (total $P_i$ transport) or N-methyl-D-glucamine (sodium-independent transport) at 37 °C for 10 min. sodium-dependent $P_i$ transport was calculated as the difference between total and sodium-independent $P_i$ transports. Cells were washed three times and lysed with 0.1 M NaOH solution, and aliquots of cell lysates were taken for the determination of protein content (Pierce BCA protein assay kit, Thermo Fisher Scientific) and the radioactivity by liquid scintillation counting (Ultima Gold LLT, PerkinElmer Life Sciences) in a Hidex 300 SL β counter.

## Immunoblot analysis

Cells were lysed for 30 min in ice-cold lysis buffer (20 mM Tris-HCl, pH 7.5, 100 mM potassium chloride, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 20 mM β-glycerophosphate, 2 mM Na$_3$VO$_4$, 1 mM phenylmethylsulfonyl fluoride, and 1 mM NaF). After centrifugation at $12,000 \times g$ for 10 min at 4 °C, the protein extracts (supernatants) were boiled in Laemml loading buffer before SDS-PAGE. For cross-linking experiments, proteins were not boiled, and electrophoresis was done on NuPAGE™ 3–8% Tris acetate gel (catalogue no. EA0378BOX, Thermo Fisher Scientific). Proteins were transferred to PVDF membrane, and blocking was performed with 5% nonfat dry milk/TBST (10 mM Tris, 154 mM NaCl, 0.15% Tween 20) for 1 h at room temperature. Blots were probed with primary antibodies in 5% nonfat dry milk/TBST overnight at 4 °C followed by secondary antibodies for 1 h at room temperature. Anti-ERK1/2 (catalogue no. 9102) and anti-phospho-ERK1/2 (catalogue no. 4370) were from Cell Signaling (Danvers, MA) and used at a 1:2,000 dilution. Anti-PiT1 (catalogue no. 12423-1-AP) and anti-PiT2 (catalogue no. 12820-1-AP) were from ProteinTech (Rosemont, IL) and used at a 1:1,000 dilution. Anti-Na/K-ATPase (catalogue no. ab7671) was from Abcam (Cambridge, UK) and used at a 1:5,000 dilution. Monoclonal anti-β-actin clone AC-74 (Sigma-Aldrich) was used at a 1:5,000 dilution as a loading control. Anti-rabbit (catalogue no. 7074, Cell Signaling) and anti-mouse (catalogue no. A9917, Sigma-Aldrich) secondary antibodies were used at 1:2,000 and 1:80,000 dilutions, respectively. Signal detection was performed using ECL Western blotting detection reagent and ECL hyperfilm (GE Healthcare) or ChemiDoc Imaging System™ (Bio-Rad).

## Crude cellular fractionation and cross-linking

HEK293T cells were seeded at $10^5$ cells/cm$^2$ in 6-well plates and transfected with 1 µg/well each of *hPiT1* and *hPiT2* plasmids using JetPrime (Polyplus transfection) according to the manufacturer's instructions. Twenty-four hours post-transfection, cells were scraped and lysed in 1 ml of buffer A/well (10 mM Hepes, pH 7, 10% sucrose, 5 mM EDTA, Phosphatase Inhibitor Mixture 3 (catalogue no. P0044), and Protease Inhibitor
Pivotal role of PiT1 and PiT2 in bone phosphate sensing

<table><caption>**Table 2**

Primer sequences used for FastCloning</caption><thead><tr><th>Chimeric constructs*</th><th>Primer for</th><th>Primer sequences (5' to 3')</th></tr></thead><tbody><tr><td rowspan="3">eYFP insert in hPiT1 plasmid</td><td>Plasmid</td><td>F: GACAGAAAAGGAAGTAATGGC<br>R: AGGACTACACTTTATTTCCTG</td></tr><tr><td>Insert</td><td>F: ATAAAGTGTAGTCCTATGGTGAGCAAGGGCGAG<br>R: ACTTCCTTTTCTGTTCCTTGTACAGCTCGTCCAT</td></tr><tr><td>Plasmid</td><td>F: GACAGAAAAGGAAGTAATGGC<br>R: AGGACTACACTTTATTTCCTG</td></tr><tr><td rowspan="3">Rluc insert in hPiT1 plasmid</td><td>Insert</td><td>F: ATAAAGTGTAGTCCTATGACCAGCAAGGTGTACGA<br>R: ACTTCCTTTTCTGTCATCCCCCTGCTCGTTCTTCA</td></tr><tr><td>Plasmid</td><td>F: GGGCGGCGTGGAGATGAA<br>R: CTACTCGTGATAAAGCACCTTCTTT</td></tr><tr><td>Insert</td><td>F: CTTTATCACGAGTAGCCATGGTGAGCAAGGGCGA<br>R: ATCTCCACGCCGCCGCCGCTTGTACAGCTCGTCCAT</td></tr><tr><td rowspan="3">eYFP insert in hPiT2 plasmid</td><td>Plasmid</td><td>F: GGGCGGCGTGGAGATGAA<br>R: CTACTCGTGATAAAGCACCTTCTTT</td></tr><tr><td>Insert</td><td>F: CTTTATCACGAGTAGCCATGACCAGCAAGGTGTACGA<br>R: ATCTCCACGCCGCCGCCGCTTCTTCAGCACTC</td></tr><tr><td>Plasmid</td><td>F: ATGACCAGCAAGGTGTACGAC<br>R: GGTGGCTCTAGCGGATCTG</td></tr><tr><td rowspan="3">BTN3A2 in hRlucC1 plasmid</td><td>Insert</td><td>F: TCCGCTAGAGCCACCATGAAAATGGCAAGTTCCCTGGCT<br>R: CACCTTGCTGGTCATGGCTGACTTATTGGTATCGGACGA</td></tr><tr><td>Plasmid</td><td>F: ATGGTGAGCAAGGGCGAGGA<br>R: ATCTGAGTCCGGTAGCGCTA</td></tr><tr><td>Insert</td><td>F: CTACCGGACTCAGATATGAAAATGGCAAGTTCCCTGGCT<br>R: GCCCTTGCTCACCATGGCTGACTTATTGGTATCGGACGA</td></tr></tbody></table>

Mixture (catalogue no. P8340) from Sigma-Aldrich. Lysates were passed 10 times through a 22-gauge 1-ml syringe and centrifuged at 800 × g for 5 min at 4 °C. Pellet (fraction 1; F1) was resuspended in 20 µl of radioimmune precipitation assay buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 20 mM EDTA, Phosphatase Inhibitor Mixture 3, and Protease Inhibitor Mixture). The supernatant was centrifuged at 20,000 × g for 45 min at 4 °C, and the resulting supernatant (Fraction 2; F2) was reserved. The pellet (Fraction 3; F3) was resuspended in 20 µl of radioimmune precipitation assay buffer and submitted to two freeze/thaw cycles, a 10-min incubation at 100 °C, and a 30-s sonication. All fractions were incubated 1 h at 4 °C with gentle mix. F3 was centrifuged at 12,000 × g for 10 min at 4 °C, and supernatant was used for analysis.

For cross-linking experiments, transfected HEK293T cells were washed three times in cold PBS and cross-linked 30 min at 4 °C with 1 mM bis(sulfosuccinimidyl)suberate (catalogue no. 21580, Thermo Fisher Scientific). This cell surface cross-linker was blunted with ice-cold 50 mM Tris-HCl, pH 8, for 15 min and washed twice with cold PBS. Cells were then lysed in Buffer A and processed for cell fractionation as described above.

Construction of chimeric and transport-deficient PiT plasmids

The *hPiT1* and *hPiT2* sequences were previously cloned into pcDNA6A plasmid (56). The V5-His₆ fusion tag sequence present in pcDNA6A was excluded by introducing a stop codon at the end of the *hPiT* coding sequence using site-directed mutagenesis (QuikChange, Agilent Genomics) (see primers in Table 1). The iLoop of hPiT1 and hPiT2 was then replaced by eYFP or RLuc proteins using FastCloning (71). Briefly, using Phusion® high-fidelity DNA polymerase (New England Bio-labs) and overlapping specific primers (Table 2), we PCR-amplified the *pcDNA6A-hPiT1* and *pcDNA6A-hPiT2* vectors with the exception of the iLoop regions and the coding regions of *eYFP* and *Rluc* from *pEYFP-N1* (Clontech, Mountain View, CA) and *phRluc-C1* (PerkinElmer Life Sciences) plasmids, respectively. After digestion of the DNA templates by DpnI, PCR-amplified overlapping sequences were reassembled by *E. coli*-mediated recombination-ligation following transformation in high-efficiency NEB® 10-β competent cells (New England Bio-labs, Ipswich, MA). In the final hPiT1-eYFP or -Rluc chimeric constructs, the eYFP or Rluc coding sequences were inserted in place of iLoop1 (amino acids 268–492). Similarly, the *eYFP* or *Rluc* coding sequence was inserted in place of iLoop2 (amino acids 256–450) in the *hPiT2-eYFP* or *-Rluc* constructs. The integrity of the constructs was verified by sequencing. To serve as a control for BRET experiments, the *BTN3A2* DNA sequence (generously provided by Dr. Scotet, INSERM UMR1232, Centre de Recherche en Cancérologie et Immunologie, Nantes-Angers, France), encoding a small cell surface-expressed protein, was fused to the N terminus of eYFP or Rluc coding sequences using the same strategy (primers used are reported in Table 2).

To generate transport-deficient mutant versions of *hPiT1-eYFP* and *hPiT2-Rluc* chimeric constructs, we replaced the Ser¹²⁸ of hPiT1 or Ser¹¹³ of hPiT2 by an alanine using site-directed mutagenesis (QuikChange, Agilent Genomics), as reported previously (56, 63). The sequences of the primers used are listed in Table 1.

BRET saturation assays

HEK293T cells were seeded at 5 × 10⁴ cells/cm² in 12-well plates. The next day, cells were co-transfected using JetPrime (Polyplus transfection) with a fixed amount of *hPiT1-Rluc* (50 ng/well), *hPiT2-Rluc* (100 ng/well), or *BTN3A2-Rluc* (10 ng/well) plasmids (encoding BRET donors) and variable amounts of *hPiT1-eYFP* or *hPiT2-eYFP* (from 12.5 to 1,500 ng/well) or *BTN3A2-eYFP* (from 1.56 to 50 ng/well) plasmids (encoding BRET acceptors). The *pcDNA6A* empty vector was used to compensate for the variable amounts of transfected DNA and to ensure equivalent transfection conditions in each well. Twenty-four hours later, transfected cells were detached using 0.5 mM EDTA solution and seeded at 1.5 × 10⁵ cells/cm² in white flat bottom 96-well plates in duplicate. BRET experiments were performed 48 h post-transfection. Cells were washed once with 0.9% NaCl solution and stimulated with various concentrations of Pᵢ for 10 min. Pᵢ was added as a mixture of NaH₂PO₄ and Na₂HPO₄ (pH 7.4). When indicated, cells were previously starved of Pᵢ by incubating cells overnight with DMEM high glucose no phosphates (catalogue no. 11971, Thermo Fisher Scientific) supplemented with 10% FBS, 10 mM HEPES, and 50 µg/ml gentamicin before Pᵢ stimulation. The coelenterazine h substrate (UPR3078, Interchim Uptima, Montluçon, France) was added at a final concentration of 5 µM by automated injection in the Mithras LB940 plate reader (Berthold Technologies, Versailles, France), and 485- and 530-nm light emissions were measured consecutively several times. The BRET ratio was calculated as the ratio of light emitted by the acceptor fusion protein at 530 nm over the light emitted by the donor fusion protein at 485 nm. Values were

<footnote>*, overlapping sequences underlined.</footnote>
Pivotal role of PiT1 and PiT2 in bone phosphate sensing

<table><caption>**Table 2**

Primer sequences used for FastCloning</caption><thead><tr><th>Chimeric constructs*</th><th>Primer for</th><th>Primer sequences (5' to 3')</th></tr></thead><tbody><tr><td rowspan="3">eYFP insert in hPiT1 plasmid</td><td>Plasmid</td><td>F: GACAGAAAAGGAAGTAATGGC<br>R: AGGACTACACTTTATTTCCTG</td></tr><tr><td>Insert</td><td>F: ATAAAGTGTAGTCCTATGGTGAGCAAGGGCGAG<br>R: ACTTCCTTTTCTGTTCCTTGTACAGCTCGTCCAT</td></tr><tr><td>Plasmid</td><td>F: GACAGAAAAGGAAGTAATGGC<br>R: AGGACTACACTTTATTTCCTG</td></tr><tr><td rowspan="3">Rluc insert in hPiT1 plasmid</td><td>Insert</td><td>F: ATAAAGTGTAGTCCTATGACCAGCAAGGTGTACGA<br>R: ACTTCCTTTTCTGTCATCCCCCTGCTCGTTCTTCA</td></tr><tr><td>Plasmid</td><td>F: GGGCGGCGTGGAGATGAA<br>R: CTACTCGTGATAAAGCACCTTCTTT</td></tr><tr><td>Insert</td><td>F: CTTTATCACGAGTAGCCATGGTGAGCAAGGGCGA<br>R: ATCTCCACGCCGCCGCCGCTTGTACAGCTCGTCCAT</td></tr><tr><td rowspan="3">eYFP insert in hPiT2 plasmid</td><td>Plasmid</td><td>F: GGGCGGCGTGGAGATGAA<br>R: CTACTCGTGATAAAGCACCTTCTTT</td></tr><tr><td>Insert</td><td>F: CTTTATCACGAGTAGCCATGACCAGCAAGGTGTACGA<br>R: ATCTCCACGCCGCCGCCGCTTCTTCAGCACTC</td></tr><tr><td>Plasmid</td><td>F: ATGACCAGCAAGGTGTACGAC<br>R: GGTGGCTCTAGCGGATCTG</td></tr><tr><td rowspan="3">BTN3A2 in hRlucC1 plasmid</td><td>Insert</td><td>F: TCCGCTAGAGCCACCATGAAAATGGCAAGTTCCCTGGCT<br>R: CACCTTGCTGGTCATGGCTGACTTATTGGTATCGGACGA</td></tr><tr><td>Plasmid</td><td>F: ATGGTGAGCAAGGGCGAGGA<br>R: ATCTGAGTCCGGTAGCGCTA</td></tr><tr><td>Insert</td><td>F: CTACCGGACTCAGATATGAAAATGGCAAGTTCCCTGGCT<br>R: GCCCTTGCTCACCATGGCTGACTTATTGGTATCGGACGA</td></tr></tbody></table>

Mixture (catalogue no. P8340) from Sigma-Aldrich. Lysates were passed 10 times through a 22-gauge 1-ml syringe and centrifuged at 800 × g for 5 min at 4 °C. Pellet (fraction 1; F1) was resuspended in 20 µl of radioimmune precipitation assay buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 20 mM EDTA, Phosphatase Inhibitor Mixture 3, and Protease Inhibitor Mixture). The supernatant was centrifuged at 20,000 × g for 45 min at 4 °C, and the resulting supernatant (Fraction 2; F2) was reserved. The pellet (Fraction 3; F3) was resuspended in 20 µl of radioimmune precipitation assay buffer and submitted to two freeze/thaw cycles, a 10-min incubation at 100 °C, and a 30-s sonication. All fractions were incubated 1 h at 4 °C with gentle mix. F3 was centrifuged at 12,000 × g for 10 min at 4 °C, and supernatant was used for analysis.

For cross-linking experiments, transfected HEK293T cells were washed three times in cold PBS and cross-linked 30 min at 4 °C with 1 mM bis(sulfosuccinimidyl)suberate (catalogue no. 21580, Thermo Fisher Scientific). This cell surface cross-linker was blunted with ice-cold 50 mM Tris-HCl, pH 8, for 15 min and washed twice with cold PBS. Cells were then lysed in Buffer A and processed for cell fractionation as described above.

Construction of chimeric and transport-deficient PiT plasmids

The *hPiT1* and *hPiT2* sequences were previously cloned into pcDNA6A plasmid (56). The V5-His₆ fusion tag sequence present in pcDNA6A was excluded by introducing a stop codon at the end of the *hPiT* coding sequence using site-directed mutagenesis (QuikChange, Agilent Genomics) (see primers in Table 1). The iLoop of hPiT1 and hPiT2 was then replaced by eYFP or RLuc proteins using FastCloning (71). Briefly, using Phusion® high-fidelity DNA polymerase (New England Bio- labs) and overlapping specific primers (Table 2), we PCR-am

plified the *pcDNA6A-hPiT1* and *pcDNA6A-hPiT2* vectors with the exception of the iLoop regions and the coding regions of *eYFP* and *Rluc* from *pEYFP-N1* (Clontech, Mountain View, CA) and *phRluc-C1* (PerkinElmer Life Sciences) plasmids, respectively. After digestion of the DNA templates by DpnI, PCR-amplified overlapping sequences were reassembled by *E. coli*-mediated recombination-ligation following transformation in high-efficiency NEB® 10-β competent cells (New England Bio- labs, Ipswich, MA). In the final hPiT1-eYFP or -Rluc chimeric constructs, the eYFP or Rluc coding sequences were inserted in place of iLoop1 (amino acids 268–492). Similarly, the *eYFP* or *Rluc* coding sequence was inserted in place of iLoop2 (amino acids 256–450) in the *hPiT2-eYFP* or *-Rluc* constructs. The integrity of the constructs was verified by sequencing. To serve as a control for BRET experiments, the BTN3A2 DNA sequence (generously provided by Dr. Scotet, INSERM UMR1232, Centre de Recherche en Cancérologie et Immunologie, Nantes-Angers, France), encoding a small cell surface-expressed protein, was fused to the N terminus of eYFP or Rluc coding sequences using the same strategy (primers used are reported in Table 2).

To generate transport-deficient mutant versions of *hPiT1-eYFP* and *hPiT2-Rluc* chimeric constructs, we replaced the Ser¹²⁸ of hPiT1 or Ser¹¹³ of hPiT2 by an alanine using site-directed mutagenesis (QuikChange, Agilent Genomics), as reported previously (56, 63). The sequences of the primers used are listed in Table 1.

BRET saturation assays

HEK293T cells were seeded at 5 × 10⁴ cells/cm² in 12-well plates. The next day, cells were co-transfected using JetPrime (Polyplus transfection) with a fixed amount of *hPiT1-Rluc* (50 ng/well), *hPiT2-Rluc* (100 ng/well), or *BTN3A2-Rluc* (10 ng/well) plasmids (encoding BRET donors) and variable amounts of *hPiT1-eYFP* or *hPiT2-eYFP* (from 12.5 to 1,500 ng/well) or *BTN3A2-eYFP* (from 1.56 to 50 ng/well) plasmids (encoding BRET acceptors). The *pcDNA6A* empty vector was used to compensate for the variable amounts of transfected DNA and to ensure equivalent transfection conditions in each well. Twenty-four hours later, transfected cells were detached using 0.5 mM EDTA solution and seeded at 1.5 × 10⁵ cells/cm² in white flat bottom 96-well plates in duplicate. BRET experiments were performed 48 h post-transfection. Cells were washed once with 0.9% NaCl solution and stimulated with various concentrations of Pᵢ for 10 min. Pᵢ was added as a mixture of NaH₂PO₄ and Na₂HPO₄ (pH 7.4). When indicated, cells were previously starved of Pᵢ by incubating cells overnight with DMEM high glucose no phosphates (catalogue no. 11971, Thermo Fisher Scientific) supplemented with 10% FBS, 10 mM HEPES, and 50 µg/ml gentamicin before Pᵢ stimulation. The coelenterazine h substrate (UPR3078, Interchim Uptima, Montluçon, France) was added at a final concentration of 5 µM by automated injection in the Mithras LB940 plate reader (Berthold Technologies, Versailles, France), and 485- and 530-nm light emissions were measured consecutively several times. The BRET ratio was calculated as the ratio of light emitted by the acceptor fusion protein at 530 nm over the light emitted by the donor fusion protein at 485 nm. Values were

<footnote>*, overlapping sequences underlined.</footnote>
# Pivotal role of PiT1 and PiT2 in bone phosphate sensing

corrected with the background signal calculated from a well without donor fusion protein. The BRET 50 was calculated as the eYFP/Rluc value at which the BRET ratio is half of the maximum BRET ratio achieved at saturating substrate concentration.

## Statistical analysis

Data are expressed as mean ± S.E. GraphPad version 5.0 software was used to perform Mann-Whitney tests. A *p* value of <0.05 was considered statistically significant. Unless otherwise stated, experiments were repeated at least three times.

Author contributions—N.B., G.C., and A.B. conducted most of the experiments, from conception and design to acquisition of data or analysis and interpretation of data. S.S. provided technical assistance. L.B., J.G., and S.B.C. conceived the idea and supported the coordination of the project. N.B. and L.B. wrote the paper. N.B., G.C., A.B., S.B.C., J.G., and L.B. made adjustments to the final paper version. All authors reviewed the results and approved the final version of the manuscript.

Acknowledgments—We thank the IMPACT platform of the Federal Research Structure François Bonamy (Nantes, France) for technical support and expertise to carry out the BRET assays. Specifically, we gratefully acknowledge Dr. Fabien Gautier for help with this technique. We also thank Philippe Hulin and Steven Nedellec of the Cellular and Tissular Imaging Core Facility (MicroPCell) of the Federal Research Structure François Bonamy (Nantes, France) for assistance with confocal microscopy.

## References

1. Berner, Y. N., and Shike, M. (1988) Consequences of phosphate imbalance. *Annu. Rev. Nutr.* **8**, 121–148 *CrossRef Medline*

2. Walser, M. (1961) Ion association. VI. Interactions between calcium, magnesium, inorganic phosphate, citrate and protein in normal human plasma. *J. Clin. Invest.* **40**, 723–730 *CrossRef Medline*

3. Marshall, W. (1976) Plasma fractions. In *Calcium, Phosphate, and Magnesium Metabolism* (Nordin, B. E. C., ed) pp. 162–185, Churchill Livingstone, London

4. Knochel, J. P. (1977) The pathophysiology and clinical characteristics of severe hypophosphatemia. *Arch. Intern. Med.* **137**, 203–220 *CrossRef Medline*

5. Klahr, S., and Peck, W. A. (1980) Cyclic nucleotides in bone and mineral metabolism. II. Cyclic nucleotides and the renal regulation of mineral metabolism. *Adv. Cyclic Nucleotide Res.* **13**, 133–180 *Medline*

6. Camalier, C. E., Yi, M., Yu, L. R., Hood, B. L., Conrads, K. A., Lee, Y. J., Lin, Y., Garneys, L. M., Bouloux, G. F., Young, M. R., Veenstra, T. D., Stephens, R. M., Colburn, N. H., Conrads, T. P., and Beck, G. R. (2013) An integrated understanding of the physiological response to elevated extracellular phosphate. *J. Cell Physiol.* **228**, 1536–1550 *CrossRef Medline*

7. Beck, G. R., Jr., Zerler, B., and Moran, E. (2000) Phosphate is a specific signal for induction of osteopontin gene expression. *Proc. Natl. Acad. Sci. U.S.A.* **97**, 8352–8357 *CrossRef Medline*

8. Khoshniat, S., Bourgine, A., Julien, M., Weiss, P., Guicheux, J., and Beck, L. (2011) The emergence of phosphate as a specific signaling molecule in bone and other cell types in mammals. *Cell Mol. Life Sci.* **68**, 205–218 *CrossRef Medline*

9. Michigami, T. (2013) Extracellular phosphate as a signaling molecule. *Contrib. Nephrol.* **180**, 14–24 *CrossRef Medline*

10. Magne, D., Bluteau, G., Faucheux, C., Palmer, G., Vignes-Colombeix, C., Pilet, P., Rouillon, T., Caverzasio, J., Weiss, P., Daculsi, G., and Guicheux, J. (2003) Phosphate is a specific signal for ATDC5 chondrocyte maturation and apoptosis-associated mineralization: possible implication of apo-

ptosis in the regulation of endochondral ossification. *J. Bone Miner. Res.* **18**, 1430–1442 *CrossRef Medline*

11. Mansfield, K., Rajpurohit, R., and Shapiro, I. M. (1999) Extracellular phosphate ions cause apoptosis of terminally differentiated epiphyseal chondrocytes. *J. Cell Physiol.* **179**, 276–286 *CrossRef Medline*

12. Teixeira, C. C., Mansfield, K., Hertkorn, C., Ischiropoulos, H., and Shapiro, I. M. (2001) Phosphate-induced chondrocyte apoptosis is linked to nitric oxide generation. *Am. J. Physiol. Cell Physiol.* **281**, C833–C839 *CrossRef Medline*

13. Mansfield, K., Teixeira, C. C., Adams, C. S., and Shapiro, I. M. (2001) Phosphate ions mediate chondrocyte apoptosis through a plasma membrane transporter mechanism. *Bone* **28**, 1–8 *CrossRef Medline*

14. Sabbagh, Y., Carpenter, T. O., and Demay, M. B. (2005) Hypophosphatemia leads to rickets by impairing caspase-mediated apoptosis of hypertrophic chondrocytes. *Proc. Natl. Acad. Sci. U.S.A.* **102**, 9637–9642 *CrossRef Medline*

15. Khoshniat, S., Bourgine, A., Julien, M., Petit, M., Pilet, P., Rouillon, T., Masson, M., Gatius, M., Weiss, P., Guicheux, J., and Beck, L. (2011) Phosphate-dependent stimulation of MGP and OPN expression in osteoblasts via the ERK1/2 pathway is modulated by calcium. *Bone* **48**, 894–902 *CrossRef Medline*

16. Julien, M., Khoshniat, S., Lacreusette, A., Gatius, M., Bozec, A., Wagner, E. F., Wittrant, Y., Masson, M., Weiss, P., Beck, L., Magne, D., and Guicheux, J. (2009) Phosphate-dependent regulation of MGP in osteoblasts: role of ERK1/2 and Fra-1. *J. Bone Miner. Res.* **24**, 1856–1868 *CrossRef Medline*

17. Miedlich, S. U., Zalutskaya, A., Zhu, E. D., and Demay, M. B. (2010) Phosphate-induced apoptosis of hypertrophic chondrocytes is associated with a decrease in mitochondrial membrane potential and is dependent upon Erk1/2 phosphorylation. *J. Biol. Chem.* **285**, 18270–18275 *CrossRef Medline*

18. Julien, M., Magne, D., Masson, M., Rolli-Derkinderen, M., Chassande, O., Cario-Toumaniantz, C., Cherel, Y., Weiss, P., and Guicheux, J. (2007) Phosphate stimulates matrix Gla protein expression in chondrocytes through the extracellular signal regulated kinase signaling pathway. *Endocrinology* **148**, 530–537 *CrossRef Medline*

19. Beck, G. R., Jr., and Knecht, N. (2003) Osteopontin regulation by inorganic phosphate is ERK1/2-, protein kinase C-, and proteasome-dependent. *J. Biol. Chem.* **278**, 41921–41929 *CrossRef Medline*

20. Adams, C. S., Mansfield, K., Perlot, R. L., and Shapiro, I. M. (2001) Matrix regulation of skeletal cell apoptosis: role of calcium and phosphate ions. *J. Biol. Chem.* **276**, 20316–20322 *CrossRef Medline*

21. Beck, G. R., Jr., Moran, E., and Knecht, N. (2003) Inorganic phosphate regulates multiple genes during osteoblast differentiation, including Nrf2. *Exp. Cell Res.* **288**, 288–300 *CrossRef Medline*

22. Conrads, K. A., Yi, M., Simpson, K. A., Lucas, D. A., Camalier, C. E., Yu, L. R., Veenstra, T. D., Stephens, R. M., Conrads, T. P., and Beck, G. R. (2005) A combined proteome and microarray investigation of inorganic phosphate-induced pre-osteoblast cells. *Mol. Cell Proteomics* **4**, 1284–1296 *CrossRef Medline*

23. Naviglio, S., Spina, A., Chiosi, E., Fusco, A., Illiano, F., Pagano, M., Romano, M., Senatore, G., Sorrentino, A., Sorvillo, L., and Illiano, G. (2006) Inorganic phosphate inhibits growth of human osteosarcoma U2OS cells via adenylate cyclase/cAMP pathway. *J. Cell Biochem.* **98**, 1584–1596 *CrossRef Medline*

24. Yoshiko, Y., Candeliere, G. A., Maeda, N., and Aubin, J. E. (2007) Osteoblast autonomous $P_1$ regulation via Pit1 plays a role in bone mineralization. *Mol. Cell Biol.* **27**, 4465–4474 *CrossRef Medline*

25. Foster, B. L., Nociti, F. H., Jr., Swanson, E. C., Matsa-Dunn, D., Berry, J. E., Cupp, C. J., Zhang, P., and Somerman, M. J. (2006) Regulation of cementoblast gene expression by inorganic phosphate *in vitro*. *Calcif. Tissue Int.* **78**, 103–112 *CrossRef Medline*

26. Lundquist, P. (2002) Odontoblast phosphate and calcium transport in dentinogenesis. *Swed. Dent. J. Suppl.*, 1–52 *Medline*

27. Bourgine, A., Beck, L., Khoshniat, S., Wauquier, F., Oliver, L., Hue, E., Alliot-Licht, B., Weiss, P., Guicheux, J., and Wittrant, Y. (2011) Inorganic phosphate stimulates apoptosis in murine MO6-G3 odontoblast-like cells. *Arch. Oral Biol.* **56**, 977–983 *CrossRef Medline*

<figure><img src="image_20.png" /></figure>
# Pivotal role of PiT1 and PiT2 in bone phosphate sensing

corrected with the background signal calculated from a well without donor fusion protein. The BRET 50 was calculated as the eYFP/Rluc value at which the BRET ratio is half of the maximum BRET ratio achieved at saturating substrate concentration.

## Statistical analysis

Data are expressed as mean ± S.E. GraphPad version 5.0 software was used to perform Mann-Whitney tests. A *p* value of <0.05 was considered statistically significant. Unless otherwise stated, experiments were repeated at least three times.

Author contributions—N.B., G.C., and A.B. conducted most of the experiments, from conception and design to acquisition of data or analysis and interpretation of data. S.S. provided technical assistance. L.B., J.G., and S.B.C. conceived the idea and supported the coordination of the project. N.B. and L.B. wrote the paper. N.B., G.C., A.B., S.B.C., J.G., and L.B. made adjustments to the final paper version. All authors reviewed the results and approved the final version of the manuscript.

Acknowledgments—We thank the IMPACT platform of the Federal Research Structure François Bonamy (Nantes, France) for technical support and expertise to carry out the BRET assays. Specifically, we gratefully acknowledge Dr. Fabien Gautier for help with this technique. We also thank Philippe Hulin and Steven Nedellec of the Cellular and Tissular Imaging Core Facility (MicroPCell) of the Federal Research Structure François Bonamy (Nantes, France) for assistance with confocal microscopy.

## References

1. Berner, Y. N., and Shike, M. (1988) Consequences of phosphate imbalance. *Annu. Rev. Nutr.* **8**, 121–148 *CrossRef Medline*

2. Walser, M. (1961) Ion association. VI. Interactions between calcium, magnesium, inorganic phosphate, citrate and protein in normal human plasma. *J. Clin. Invest.* **40**, 723–730 *CrossRef Medline*

3. Marshall, W. (1976) Plasma fractions. In *Calcium, Phosphate, and Magnesium Metabolism* (Nordin, B. E. C., ed) pp. 162–185, Churchill Livingstone, London

4. Knochel, J. P. (1977) The pathophysiology and clinical characteristics of severe hypophosphatemia. *Arch. Intern. Med.* **137**, 203–220 *CrossRef Medline*

5. Klahr, S., and Peck, W. A. (1980) Cyclic nucleotides in bone and mineral metabolism. II. Cyclic nucleotides and the renal regulation of mineral metabolism. *Adv. Cyclic Nucleotide Res.* **13**, 133–180 *Medline*

6. Camalier, C. E., Yi, M., Yu, L. R., Hood, B. L., Conrads, K. A., Lee, Y. J., Lin, Y., Garneys, L. M., Bouloux, G. F., Young, M. R., Veenstra, T. D., Stephens, R. M., Colburn, N. H., Conrads, T. P., and Beck, G. R. (2013) An integrated understanding of the physiological response to elevated extracellular phosphate. *J. Cell Physiol.* **228**, 1536–1550 *CrossRef Medline*

7. Beck, G. R., Jr., Zerler, B., and Moran, E. (2000) Phosphate is a specific signal for induction of osteopontin gene expression. *Proc. Natl. Acad. Sci. U.S.A.* **97**, 8352–8357 *CrossRef Medline*

8. Khoshniat, S., Bourgine, A., Julien, M., Weiss, P., Guicheux, J., and Beck, L. (2011) The emergence of phosphate as a specific signaling molecule in bone and other cell types in mammals. *Cell Mol. Life Sci.* **68**, 205–218 *CrossRef Medline*

9. Michigami, T. (2013) Extracellular phosphate as a signaling molecule. *Contrib. Nephrol.* **180**, 14–24 *CrossRef Medline*

10. Magne, D., Bluteau, G., Faucheux, C., Palmer, G., Vignes-Colombeix, C., Pilet, P., Rouillon, T., Caverzasio, J., Weiss, P., Daculsi, G., and Guicheux, J. (2003) Phosphate is a specific signal for ATDC5 chondrocyte maturation and apoptosis-associated mineralization: possible implication of apo-

ptosis in the regulation of endochondral ossification. *J. Bone Miner. Res.* **18**, 1430–1442 *CrossRef Medline*

11. Mansfield, K., Rajpurohit, R., and Shapiro, I. M. (1999) Extracellular phosphate ions cause apoptosis of terminally differentiated epiphyseal chondrocytes. *J. Cell Physiol.* **179**, 276–286 *CrossRef Medline*

12. Teixeira, C. C., Mansfield, K., Hertkorn, C., Ischiropoulos, H., and Shapiro, I. M. (2001) Phosphate-induced chondrocyte apoptosis is linked to nitric oxide generation. *Am. J. Physiol. Cell Physiol.* **281**, C833–C839 *CrossRef Medline*

13. Mansfield, K., Teixeira, C. C., Adams, C. S., and Shapiro, I. M. (2001) Phosphate ions mediate chondrocyte apoptosis through a plasma membrane transporter mechanism. *Bone* **28**, 1–8 *CrossRef Medline*

14. Sabbagh, Y., Carpenter, T. O., and Demay, M. B. (2005) Hypophosphatemia leads to rickets by impairing caspase-mediated apoptosis of hypertrophic chondrocytes. *Proc. Natl. Acad. Sci. U.S.A.* **102**, 9637–9642 *CrossRef Medline*

15. Khoshniat, S., Bourgine, A., Julien, M., Petit, M., Pilet, P., Rouillon, T., Masson, M., Gatius, M., Weiss, P., Guicheux, J., and Beck, L. (2011) Phosphate-dependent stimulation of MGP and OPN expression in osteoblasts via the ERK1/2 pathway is modulated by calcium. *Bone* **48**, 894–902 *CrossRef Medline*

16. Julien, M., Khoshniat, S., Lacreusette, A., Gatius, M., Bozec, A., Wagner, E. F., Wittrant, Y., Masson, M., Weiss, P., Beck, L., Magne, D., and Guicheux, J. (2009) Phosphate-dependent regulation of MGP in osteoblasts: role of ERK1/2 and Fra-1. *J. Bone Miner. Res.* **24**, 1856–1868 *CrossRef Medline*

17. Miedlich, S. U., Zalutskaya, A., Zhu, E. D., and Demay, M. B. (2010) Phosphate-induced apoptosis of hypertrophic chondrocytes is associated with a decrease in mitochondrial membrane potential and is dependent upon Erk1/2 phosphorylation. *J. Biol. Chem.* **285**, 18270–18275 *CrossRef Medline*

18. Julien, M., Magne, D., Masson, M., Rolli-Derkinderen, M., Chassande, O., Cario-Toumaniantz, C., Cherel, Y., Weiss, P., and Guicheux, J. (2007) Phosphate stimulates matrix Gla protein expression in chondrocytes through the extracellular signal regulated kinase signaling pathway. *Endocrinology* **148**, 530–537 *CrossRef Medline*

19. Beck, G. R., Jr., and Knecht, N. (2003) Osteopontin regulation by inorganic phosphate is ERK1/2-, protein kinase C-, and proteasome-dependent. *J. Biol. Chem.* **278**, 41921–41929 *CrossRef Medline*

20. Adams, C. S., Mansfield, K., Perlot, R. L., and Shapiro, I. M. (2001) Matrix regulation of skeletal cell apoptosis: role of calcium and phosphate ions. *J. Biol. Chem.* **276**, 20316–20322 *CrossRef Medline*

21. Beck, G. R., Jr., Moran, E., and Knecht, N. (2003) Inorganic phosphate regulates multiple genes during osteoblast differentiation, including Nrf2. *Exp. Cell Res.* **288**, 288–300 *CrossRef Medline*

22. Conrads, K. A., Yi, M., Simpson, K. A., Lucas, D. A., Camalier, C. E., Yu, L. R., Veenstra, T. D., Stephens, R. M., Conrads, T. P., and Beck, G. R. (2005) A combined proteome and microarray investigation of inorganic phosphate-induced pre-osteoblast cells. *Mol. Cell Proteomics* **4**, 1284–1296 *CrossRef Medline*

23. Naviglio, S., Spina, A., Chiosi, E., Fusco, A., Illiano, F., Pagano, M., Romano, M., Senatore, G., Sorrentino, A., Sorvillo, L., and Illiano, G. (2006) Inorganic phosphate inhibits growth of human osteosarcoma U2OS cells via adenylate cyclase/cAMP pathway. *J. Cell Biochem.* **98**, 1584–1596 *CrossRef Medline*

24. Yoshiko, Y., Candeliere, G. A., Maeda, N., and Aubin, J. E. (2007) Osteoblast autonomous $P_1$ regulation via Pit1 plays a role in bone mineralization. *Mol. Cell Biol.* **27**, 4465–4474 *CrossRef Medline*

25. Foster, B. L., Nociti, F. H., Jr., Swanson, E. C., Matsa-Dunn, D., Berry, J. E., Cupp, C. J., Zhang, P., and Somerman, M. J. (2006) Regulation of cementoblast gene expression by inorganic phosphate *in vitro*. *Calcif. Tissue Int.* **78**, 103–112 *CrossRef Medline*

26. Lundquist, P. (2002) Odontoblast phosphate and calcium transport in dentinogenesis. *Swed. Dent. J. Suppl.*, 1–52 *Medline*

27. Bourgine, A., Beck, L., Khoshniat, S., Wauquier, F., Oliver, L., Hue, E., Alliot-Licht, B., Weiss, P., Guicheux, J., and Wittrant, Y. (2011) Inorganic phosphate stimulates apoptosis in murine MO6-G3 odontoblast-like cells. *Arch. Oral Biol.* **56**, 977–983 *CrossRef Medline*
Pivotal role of PiT1 and PiT2 in bone phosphate sensing

28. Kanatani, M., Sugimoto, T., Kano, J., Kanzawa, M., and Chihara, K. (2003) Effect of high phosphate concentration on osteoclast differentiation as well as bone-resorbing activity. *J. Cell Physiol.* **196**, 180–189 *CrossRef Medline*

29. Takeyama, S., Yoshimura, Y., Deyama, Y., Sugawara, Y., Fukuda, H., and Matsumoto, A. (2001) Phosphate decreases osteoclastogenesis in coculture of osteoblast and bone marrow. *Biochem. Biophys. Res. Commun.* **282**, 798–802 *CrossRef Medline*

30. Mozart, A., Haren, N., Chasseraud, M., Louvet, L., Mazière, C., Wattel, A., Mentaverri, R., Morlière, P., Kamel, S., Brazier, M., Mazière, J. C., and Massy, Z. A. (2008) High extracellular inorganic phosphate concentration inhibits RANK-RANKL signaling in osteoclast-like cells. *J. Cell Physiol.* **215**, 47–54 *CrossRef Medline*

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Pivotal role of PiT1 and PiT2 in bone phosphate sensing

28. Kanatani, M., Sugimoto, T., Kano, J., Kanzawa, M., and Chihara, K. (2003) Effect of high phosphate concentration on osteoclast differentiation as well as bone-resorbing activity. *J. Cell Physiol.* **196**, 180–189 *CrossRef Medline*

29. Takeyama, S., Yoshimura, Y., Deyama, Y., Sugawara, Y., Fukuda, H., and Matsumoto, A. (2001) Phosphate decreases osteoclastogenesis in coculture of osteoblast and bone marrow. *Biochem. Biophys. Res. Commun.* **282**, 798–802 *CrossRef Medline*

30. Mozart, A., Haren, N., Chasseraud, M., Louvet, L., Mazière, C., Wattel, A., Mentaverri, R., Morlière, P., Kamel, S., Brazier, M., Mazière, J. C., and Massy, Z. A. (2008) High extracellular inorganic phosphate concentration inhibits RANK-RANKL signaling in osteoclast-like cells. *J. Cell Physiol.* **215**, 47–54 *CrossRef Medline*

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