Bone 35 (2004) 1077 – 1085
www.elsevier.com/locate/boneSite-specific localization of two distinct phosphatases along the osteoblast
plasma membrane: tissue non-specific alkaline phosphatase andplasma membrane calcium ATPase
Yukiko Nakanoa,*, Wouter Beertsenb, Theo VanDenBosb, Tadafumi Kawamotoc,
Kimimistsu Odad, Yoshiro TakanoabaBiostructural Science, Graduate School of Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, 113-8549, Japan
Department of Periodontology, Academic Centre for Dentistry Amsterdam (ACTA), Universiteit van Amsterdam and Vrije Universiteit,
Amsterdam, The Netherlandsc
RI Research Center, School of Dental Medicine, Tsurumi University, Yokohama, Japand
Biochemistry, Niigata University, Graduate School of Medical and Dental Science, Niigata, Japan

Received 7 March 2004; revised 30 June 2004; accepted 20 July 2004
Available online 23 September 2004

Abstract
In osteoblasts, alkaline phosphatase has been reported to be restricted to the basolateral domains. In recent studies, we have demonstrated
phosphatase activities different from those of tissue non-specific alkaline phosphatase (TNSALP) along the osteoidal aspect of osteoblast
membrane at alkaline and neutral pH on undecalcified freshly frozen sections of rat bones. In the present study, we sought to further
characterize and define the nature of membrane-associated phosphatases along the osteoidal aspect of osteoblasts. Histochemical propertiesof the enzymes and their localization in vivo were examined in long bones of normal Wistar rats and TNSALP null mutant mice and theirwild type littermates. Molecular profiles of the enzymes in the osteoblast extracts were also examined. The enzymatic activity of thephosphatase along the osteoidal surface of osteoblasts proved to be activated by both Mg2+ and Ca2+. Unlike TNSALP, the activity was
inhibited by vanadate but resistant to levamisole, implicating a similarity between this enzyme and plasma membrane Ca2+ transport ATPase(PMCA). Immunohistochemistry showed that PMCA immunoreactions were restricted to the osteoidal domain of the plasma membrane.
Native-PAGE analysis of osteoblast extracts suggested the presence of two phosphatases corresponding, respectively, to TNSALP and
PMCA. Western blot analysis after SDS-PAGE of osteoblast extracts confirmed the existence of PMCA (140 kDa) and TNSALP (80 kDa).
Gel-chemical analysis of the osteoblast extract from TNSALP null mutant mice depicted phosphatase activity, which was resistant tolevamisole.
These data suggest the presence of a phosphatase different from TNSALP, most plausibly PMCA, on the osteoidal surface of osteoblasts.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Osteoblasts; Alkaline phosphatase; Ca2+-ATPase; Bone histology

Introduction
Alkaline phosphatase (ALPase) [EC 3.1.3.1] is amember of the phosphoric monoester hydrolases [EC* Corresponding author. Biostructural Science, Graduate School ofTokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo
113-8549, Japan. Fax: +81 3 5803 5442.
E-mail address: yuki.bss@tmd.ac.jp (Y. Nakano).
8756-3282/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.bone.2004.07.009

3.1.3]. Human ALPase is classified as four isoenzymes(intestinal/tissue-nonspecific/germ cell/placental) [1]. Tissue-nonspecific alkaline phosphatase (TNSALP) has three
isoforms: bone, kidney, and liver types, attributed to thevariety of post-transcriptional N- and O-glycosylation [2].
These enzymes have in common that their pH optimumin the test tube is in the alkaline range. Natural
substrate(s) and actual working pH for these enzymes
have not sufficiently been clarified. In bone tissue,

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TNSALP is localized mainly at the plasma membrane ofosteoblasts [3,4] and is bound via a glycosylphosphatidylinositol (GPI) anchor [5–7]. This enzyme has beenused as a marker for phenotyping osteoblastic cells in
vitro [8].
Hypophosphatasia is a clinically heterogeneous heritable
disorder characterized by defective bone mineralization,
which is caused by an impaired TNSALP activity due tomutation of the TNSALP gene [9,10]. Data from studies on
hypophosphatasia patients and TNSALP mutant mice
showing symptoms of hypomineralization suggest thatTNSALP is one of the factors involved in bone mineralization [9–18].
Two types of adenosine triphosphatase (ATPase) [EC
3.6.1.3], Na, K-ATPase and plasma membrane Ca2+transport ATPase (PMCA) have been reported to belocated on the osteoblast plasma membrane [19–22]. The
natural substrate of these ATPases, ATP, is also hydrolyzed
by TNSALP in vitro [23] and is therefore supposed to be
one of the candidates of the natural substrate of TNSALP.
Despite an abundance of data on TNSALP and ATPases inosteoblasts, the physiological roles and kinetics of these
phosphatases on the osteoblast plasma membrane have yet
to be determined [19–22,24–32].
In initial mineralization, it is well known that matrix
vesicles (MV) present TNSALP and ATPase activities on
the outer surface of their membrane and contribute toinduction of mineral deposition [33–35]. Though thepresence or absence of MV in the osteoid undergoing
appositional mineralization had long been uncertain, Babet al. [36] demonstrated by biochemical methods theexistence of MV in the osteoid as well as the presenceof both ATPase and TNSALP in these MV fractions. Coexistence of TNSALP and ATPase on the osteoblastderived MV is therefore feasible. Interestingly, however, inosteoblasts, enzyme reactions of TNSALP have beenreported to be associated with the basolateral aspects ofplasma membrane, facing away from the bone matrix,
when examined in sections of routinely fixed and decalcified specimens [3,4]. Immunoreactivity of TNSALP was
also shown to be restricted to these sites [37,38]. On theother hand, Morris et al. [39,40] reported the presence of
TNSALP along the entire surface of the osteoblast
membrane. Furthermore, Stains et al. [22] recently reported
a polarized localization of PMCA on the cultured
osteoblasts and claimed an association of PMCA on thebasolateral domains of these cells. In our previous studies,
however, we used undecalcified bone sections and could
successfully demonstrate phosphatase activities along theosteoidal surface of osteoblasts at both alkaline and near
neutral pH, and suggested the presence of enzyme(s)different from TNSALP [37]. Therefore, it appears
necessary to further examine the precise spatial localizationand enzymatic properties of these phosphatases in theosteoblasts to elucidate their roles in the mineralization of
osteoid matrix.

Materials and methods
Tissue preparation
Protocols for animal experiments had been approved by
the Institutional Animal Care and Use Committee of Tokyo
Medical and Dental University and the local institutional
review board of the Academic Medical Centre in Amsterdam, The Netherlands. All experiments were properly
carried out under the Guideline for Animal Experimentation
at Tokyo Medical and Dental University.
Whole body sectioning of freshly frozen animals
One- or 2-week-old Wistar rats (Charles River Japan. Inc,
Kanagawa, Japan) were anesthetized by ether inhalation
followed by an intraperitoneal injection of 8% chloral
hydrate solution (400 mg/kg B.W.), and subjected to wholebody freeze sectioning according to Kawamoto’s procedures[41,42]. In brief, the deeply anesthetized animals werefrozen by quenching in cold hexane kept at 948C in thecooling bath of a freeze dryer (Neocool Bath, Yamato
Kagaku, Tokyo, Japan). The frozen animals were rapidly
embedded in 5% carboxymethylcellulose (CMC) gel andfurther frozen in cold hexane. The frozen CMC blocks ofanimals were set in a cryomicrotome (CM 3500, Leica
Microsystems, Wetzlar, Germany) kept at 208C and cut
with a disposable type carbide tungsten steel blade (Leica
Microsystems, Nugloch, Germany). When the appropriate
plane of CMC-embedded frozen animal was exposed, anadhesive tape made of polyvinylidene chloride film (Asahikasei Kogyo, Tokyo, Japan) was glued onto the cut
surface. In this way 5-Am-thick frozen sections supported bya polyvinylidene chloride film were collected one by one.
The whole body frozen sections were freeze-dried in thecold chamber of the cryomicrotome and stored at 808C
until use. Anesthetized 8- to 12-day-old TNSALP nullmutant mice and their wild type littermates (JacksonLaboratory, Animal Resources, Bar Habor, ME, USA) were
decapitated and immediately frozen in liquid nitrogen. The
frozen bodies were processed for whole body sectioning inthe same manner.
Paraffin or cryosectioning of chemically fixed specimens
A group of Wistar rats (1–5 week old) were similarly
anesthetized and perfused via the left cardiac ventricle
with 4% paraformaldehyde in 0.1 M cacodylate buffer(pH 7.4). Upper and lower jaws and hind limbs ofperfused animals were excised and further immersed in
the fixative for 1 day at 48C. They were processed either
for paraffin sectioning or for cryosectioning with orwithout prior EDTA decalcification. Before cryosectioning, specimens were immersed overnight in 30% sucrose/
PBS solution for cryoprotection. A support film cryosectioning method [41,42], as already described for wholebody sectioning, was applied for the undecalcified
specimens.

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Semi-thin sectioning of freshly frozen, freeze-substituted
specimens
Three-week-old Wistar rats were anesthetized (asdescribed above) and killed by phlebotomy. Maxillae,
mandible, tibiae, and femora were immediately excised,
cut into small pieces, and quenched in liquid propane cooledwith liquid nitrogen for rapid freezing. The frozen pieces
were freeze-substituted with absolute ethanol at 808C for 4days, gradually brought to 48C, and embedded in Technovit
7100 (Heraeus Kulzer GmbH and Co. KG., Werrhein,
Germany) at 08C. Two-micron-thick sections were cut by
glass knives on a Histoknife (Diatome, Bienne, Switzerland)
attached to the ultramicrotome (Leica ULTRA CUT, Leica
Aktiengesellschaft, Vienna, Austria) and were adhered to
the glass slide without heating.

periosteum of femora, tibiae, humeri, and radii was carefully
removed, leaving the osteoblast layer remaining on the bone
surface [48]. The osteoblasts were then collected byscraping the bone surface by a scalpel under the dissection
microscope at a magnification of 10. Osteoblasts weresimilarly collected from skeletal tissue of 8- to 12-day-old
TNSALP wild type or null mutant mice, which had been
kept frozen at 808C following decapitation. Collected
osteoblasts were then transferred to 0.1 M Tris–HCl buffer(pH 7.3) supplemented with or without 1% Triton X-100.
Subsequently, the samples in the Triton-free buffer werebriefly homogenized using an ultrasonic homogenizer(Sonics and Materials, Inc., Newtown, CT, USA). After
10,000  g centrifugation at 48C for 30 min, the supernatant
was obtained as the osteoblast extract.

Enzyme histochemistry of phosphatases

Electrophoresis of osteoblast extract

To detect phosphatase reactions, the Technovit sectionsof freshly frozen, freeze-substituted specimens were incubated either by the Azo-dye method or the lead salt method.
The Azo-dye method was performed basically according to
the Burstone’s method [43]. Briefly, the sections wereincubated in the medium comprising 1.5 mM Naphthol ASMX phosphate as substrate and 0.5 mM Fast Red Violet LB
salt as capture agent, in 0.1 M Tris–HCl buffer (pH 9.2),
containing 3 mM MgSO4 at 378C for 60 min. For the leadsalt method, sections were incubated in the medium
containing 3 mM ATP as substrate, 8% glucose, 3 mM
MgSO4, and 3 mM PbNO3 in 0.1 M Tris–maleate buffer(pH 9.2). After completion of the incubation for 60 min at
378C, the sections were thoroughly rinsed in distilled waterand treated with 1% ammonium sulfide solution to visualize
sites of lead precipitation.

Native-polyacrylamide gel disc electrophoresis
The osteoblast extract prepared after a brief ultrasonichomogenization in Triton-free buffer was diluted with 60%
sucrose solution (3.5 Ag protein per tube) and applied on a
7.5% polyacrylamide gel (pH 8.9). Electrophoresis was
carried out at a constant current of 2.0 mA per gel tube in
Tris–glycine buffer (pH 8.3) in a cold chamber [49,50].
Subsequently, the gels were removed from the glass tubes
and incubated in medium for the detection of phosphataseactivity either by the Azo-dye or the lead-salt method
containing ATP as substrate at pH 9.2 for 60 min at 378C.
To determine the effect of inhibitors, either 0.5 mMlevamisole or 0.35 mM vanadate was added to the
incubation media.

Specificity tests of enzyme reactions
To test metal ion dependency of the phosphatase, someTechnovit sections of freshly frozen, freeze-substituted
specimens adhering to the glass side were immersed in 5%
EDTA for 1 day at 48C to eliminate metal ions. Demineralized
sections were then reactivated by immersion in 0.1 M Tris–
maleate buffer (pH 7.3) supplemented either with 50 mM
MgSO4 or 50 mM CaCl2, for 3 days at 48C [44], followed byan incubation in Azo-dye medium containing 3 mM MgSO4
or 3 mM CaCl2. The influence of phosphatase inhibitors was
also tested on undecalcified freshly frozen, freeze-substituted
Technovit sections by adding levamisole (5 mM), a specificinhibitor of TNSALP [45] or vanadate (0.2 mM), a potent
inhibitor of Mg2+/Ca2+-ATPase [46,47] to the incubation
medium by the lead salt method, using ATP as substrate.
Preparation of osteoblast extracts
Anesthetized 5-week-old Wistar rats were decapitated
and their limbs immediately excised. Subsequently, the

SDS-PAGE
To determine the molecular mass of target proteins, theosteoblast extract prepared in Triton-supplemented buffer
was dissolved in 0.32 M Tris containing 10% glycerol 2%
SDS and 2 h-mercaptoethanol (7.0 Ag protein per well),boiled for 5 min, and run on a 12% SDS-polyacrylamide gelat 200 V.
Western blot analysis
After electrophoresis (native PAGE or SDS-PAGE),
proteins were transferred to nitrocellulose or PVDF membrane using a semi-dry transfer system. The membrane wasincubated in blocking reagent (Hoffmann-La Roche Inc.,
Nutley, NJ, USA) and primary/secondary antibody treatment was performed. The primary antibodies used wererabbit anti-human TNSALP (1:500) [51] and mouse antihuman PMCA (1:1000) (Affinity Biologicals Inc., Golden,
Colorado, USA). HRP-conjugated goat anti-rabbit IgG(WAKO, Tokyo, Japan) (1:1000) and AP-conjugated goatanti-mouse IgG (KPL, Inc., Gaithersburg, ML, USA)
(1:5000) were employed as secondary antibodies, respectively. Reactive protein bands were visualized by treating

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the membranes with 2% 3V 3-diaminobenzidine (DAB) or
NBT/BCIP reagent.
Immunohistochemistry
Paraffin sections or film-supported undecalcified frozen
sections were immersed in a solution of 0.3% H2O2 inabsolute methanol to inhibit endogenous peroxidase activity.
Following a blocking treatment with a mixture of normalswine (10%) and goat (5%) sera, rabbit anti-human
TNSALP antibody (1:100) and mouse anti-human PMCA
antibody (1:2000) were applied as primary antibodies for 12
h. A biotin-conjugated swine anti-rabbit antibody (1:200) or
HISTOFINE simple stain mouse MAX-PO (NICHIREI
Corporation, Tokyo, Japan) was used as the secondary
antibody and incubated for 1 h. The site of immunoreaction
was visualized by treating sections with a 2% DAB solution
supplemented with 0.002% H2O2. Normal rabbit or mouse
immunoglobulins were used as negative control of primary
antibodies. For TNSALP immunostaining, HRP conjugatedstreptavidin treatment (Dako Cytomation, Glostrup, Denmark) was performed for 30 min before DAB treatment.
For PMCA immunostaining on TNSALP null mutant or
wild type mice, immunocomplex of primary antibody (4000times diluted mouse anti-human PMCA) and secondary
antibody (HISTOFINE biotin conjugated rabbit anti-mouse
Igs (NICHIREI Corporation)) supplemented with 5%
normal mouse IgG was prepared before antibody incubation.
The sections treated by 0.3% H2O2 in absolute methanol and
10% normal rabbit serum were immersed in the immunocomplex solution overnight, after which HRP conjugatedstreptavidin treatment was performed for 30 min, and the siteof immunoreaction was visualized by 2% DAB treatment.

Results

Fig. 1. Phosphatase reactions on the osteoblast layer and its metal ion
dependency by the Azo-dye method. (A) Frozen section of a chemically
fixed and EDTA-decalcified specimen demonstrates reactions only along
the basolateral aspects of osteoblasts after Mg2+ reactivation. (B) Technovit
section of a freshly frozen, freeze-substituted specimen shows intense
reactions along the whole perimeter of osteoblasts (ob). (C) A total loss ofreactions in Technovit section of a freshly frozen, freeze-substituted
specimen after EDTA-chelation before incubation. (D, E) Recovery of
enzyme reactions in EDTA-chelated sections after reactivation with Mg2+(D) or Ca2+ (E) ions. Osteoid: ost. Scale bars = 25 Am.

frozen, freeze-substituted specimens. The phosphatase
reactions along the perimeter of osteoblasts (Fig. 1B) weretotally abolished by EDTA chelation before histochemical
incubation according to the Azo-dye method (Fig. 1C).
Unlike conventional aldehyde fixed and decalcified sections, the phosphatase reaction on the freshly frozen sections
was restored along the entire perimeter of the osteoblasts by
pre-treatment of chelated sections with Mg2+ (Fig. 1D) andless efficiently with Ca2+ (Fig. 1E).

Metal ion dependency
Inhibition experiment
In sections of conventional aldehyde fixed and decalcified specimens, phosphatase reactions in osteoblasts wereundetectable without reactivation with metal ions, such as
Mg2+ or Ca2+. In reactivated sections, distinct phosphatase
reactions appeared along the plasma membrane of osteoblasts but only along the basolateral aspects (Fig. 1A). On
the freshly frozen and freeze-substituted Technovit sections,on the other hand, an intense phosphatase reaction emerged
along the entire perimeter of osteoblasts including theosteoidal surface (Fig. 1B). In whole body cryo-sections offreshly frozen rats, osteoblasts in all types of bones
displayed identical localization of phosphatase reactions(data not shown).
To characterize the phosphatase(s) responsible for theintense reactions along the osteoidal aspect of osteoblast
plasma membrane, the dependency for metal ions (Mg2+
and Ca2+) was tested on Technovit sections of the freshly

When the lead salt method was applied on freshly frozen,
freeze-substituted Technovit sections, for histochemical
localization of phosphatase activities, intense reactions werealso detected along the entire perimeter of the osteoblast
plasma membrane, using ATP as substrate (Fig. 2A). The
reaction was abolished from the basolateral domains of theosteoblast plasma membrane in the presence of levamisole,while the osteoidal aspect of the same cells retained thereaction (Fig. 2B). Inversely, the phosphatase reaction on
the osteoidal aspect was inhibited by vanadate, whereas theactivity remained on the basolateral membrane (Fig. 2C).
Immunolocalization of TNSALP and PMCATNSALP immunoreaction along the osteoblast plasma
membrane was restricted to the basolateral domains and was

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1081

Fig. 2. Phosphatase reactions on Technovit sections of undecalcified freshly frozen and freeze-substituted specimens using ATP as substrate. (A) Control
without inhibitor. (B) Levamisole (5 mM) abolishes the reaction on the basolateral domains but causes no effect on the phosphatase along the osteoidal surface(arrows). (C) Vanadate (0.2 mM) inhibits phosphatase activity along the osteoidal surface (arrowheads) but much less on the basolateral aspects of osteoblasts.
osteoblast, ob; osteoid, ost. Scale bar = 25 Am.

lacking or negligible along the osteoidal surface (Figs. 3A,
E). With anti-PMCA antibody, on the other hand, theimmunoreaction was observed only along the osteoidal
surface of osteoblasts and negligible along the basolateral
aspects (Figs. 3C, F). No reaction products were depicted
when the primary antibodies were replaced with normal
immunoglobulin (Figs. 3B, D).
Gel electrophoresis and Western blot analysis of osteoblast
extract
To further characterize and distinguish the phosphatase
reactions on the osteoblast plasma membrane, the osteoblast
extracts from long bones of normal rats (Fig. 4B) were
subjected to micro-disc Native-PAGE. When the micro-discgel thus prepared was incubated for phosphatase reactions,
two intensely reactive bands were detected (Fig. 4A).
Similar bands were also confirmed when the gel was
incubated by the Azo-dye method (data not shown). The
upper band was inhibited by levamisole dose dependently,

Fig. 3. Immunolocalization of TNSALP (A, E) and PMCA (C, F,) in
osteoblasts of rat bone. (A, E) TNSALP is located along basolateraldomains of osteoblasts and is lacking or negligible on osteoidal aspect(arrows in E). Decalcified paraffin section. Osteoblast: ob. (C, F): PMCA isdetected mainly on osteoidal aspect (arrowheads in F). Undecalcified frozen
section. (B, D): negative control of TNSALP (B) and PMCA (D). Scalebars = 25 Am.

but resistant to vanadate, whereas the lower band wasresistant to levamisole but inhibited by vanadate. Western
blot analysis of micro-disc gel revealed single bands
immunopositive either for PMCA or TNSALP. Since themolecular weight of these bands cannot be determined bythis system, we then examined these bands after SDSPAGE. As the result, we depicted single bands positive
either for PMCA or TNSALP antibodies and confirmed themolecular mass of these bands to be 140 and 80 kDa,
respectively (Fig. 5).
Analysis of osteoblast extract of TNSALP mutant mice
To examine the phosphatase reaction on the osteoblast
plasma membrane of TNSALP null mutant mice, a protocol
similar to that for normal rats was employed and data werecompared with those of wild type animals.
Native-PAGE gel analysis of the osteoblast extract of
TNSALP wild type mice revealed two distinct bands eachshowing phosphatase activities, using ATP as substrate (Fig.
6A, +/+). As predicted, the osteoblast extracts fromTNSALP null mutant mice only displayed a single reactive
band (Fig. 6A, / ), which was resistant to levamisole(data not shown). As shown in Figs. 6B and C, only one ofFig. 4. Gel analysis of osteoblast extracts of normal rat. (A) Native PAGE
gel incubated for phosphatase activity using ATP as substrate by the leadsalt method with or without inhibitors (0.5 mM levamisole or 0.35 mM
vanadate). (B) Photomicrograph of the osteoblast layer (ob) remaining onbone after removal of periosteum. These osteoblasts were scraped off andcollected to obtain osteoblast extracts for Gel analysis. Technovit section ofundecalcified rat femur. Scale bar = 25 Am.

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Discussion
In the present study, we could successfully obtainhistochemical and immunohistochemical evidence showing
the presence of two distinct phosphatases, plasma membrane Ca2+ transport ATPase (PMCA) and tissue nonspecific alkaline phosphatase (TNSALP), located on theosteoidal and basolateral domains of the plasma membraneof osteoblasts, respectively.
Phosphatase along the osteoidal surface

the two phosphatase reactive bands in TNSALP wild typelane was immunopositive either for TNSALP or PMCA,
whereas TNSALP null mutant lane was lacking a positive
band for TNSALP and showed a single immunopositive
band for PMCA after Western blotting.
Immunostaining of PMCA on the whole body fresh
frozen sections of TNSALP null mutant and wild type micedemonstrated the immunoreactivity for this protein associated with the osteoidal domain of osteoblasts (Fig. 7).

In our previous study, we reported the presence of intense
phosphatase activity along the osteoidal aspect of theplasma membrane of osteoblasts, and suggested that thisenzyme is different from TNSALP, which is known to belocated along the basolateral domains of the same cells [37].
The scarcity of information regarding the phosphataseactivity associated with the osteoidal domain of theosteoblast plasma membrane is attributable at least in partto the difficulty to demonstrate histochemically the enzyme
in routinely processed specimens, most notably chemically
fixed and EDTA-decalcified specimens. Our data from themetal ion dependency test (Fig. 1) indicate that thephosphatase reactions along the osteoidal aspect arereactivated and become detectable by supplementation ofboth Mg2+ and Ca2+ in the chelated (EDTA treated)
specimens if the specimens had not been treated withchemical fixatives. It is now clear that a combined treatmentof the specimens with chemical (aldehyde) fixation and
EDTA-chelation irreversibly abolish the phosphatase reactions on the osteoidal domain of the osteoblasts, but notfrom basolateral domains. A reactivation treatment of suchspecimens with divalent cations before histochemical
staining [44] is not efficient for the enzyme located along
the osteoidal surface for unknown reasons.

Fig. 6. (A) Native PAGE gel of the osteoblast extracts of TNSALP wild
type (+/+) and null mutant ( / ) mice incubated for phosphatase activities
using ATP as substrate. (B and C) Immunodetection of TNSALP (B) and
PMCA (C) on the TNSALP +/+ and / lane. TNSALP / lane shows aband immunopositive for PMCA but not for TNSALP.

Fig. 7. Immunolocalization of PMCA in the osteoblast layer of TNSALP
null mutant (A) and wild type (B) mice showing significant reactions
associated with the osteoidal aspect of osteoblasts (arrowheads). Undecalcified frozen sections of bone. Osteoblast: ob. Scale bar = 25 Am.

Fig. 5. Western blot analysis of osteoblast extract from normal rats for
PMCA and TNSALP following SDS-PAGE. The molecular mass of PMCA
and TNSALP positive bands are near 140 and 80 kDa, respectively.

Y. Nakano et al. / Bone 35 (2004) 1077–1085

It is well known that PMCA is resistant to levamisole [52]but sensitive to vanadate [46,47], whereas TNSALP is
sensitive to levamisole [45] but is reported to be lesssensitive to vanadate than PMCA [53]. In our histochemical
observations (Fig. 2), the phosphatase activity located on theosteoidal surface was shown to be inhibited by vanadate but
resistant to levamisole in an inverse manner to thephosphatase (TNSALP) associated with the basolateraldomains of osteoblasts. Taking substrate specificity, metal
ion dependency, and sensitivity to inhibitors together, PMCA
appears to be the most plausible candidate for the phosphatase associated with the osteoidal aspect of the osteoblast
plasma membrane. In fact, our Western blot analysis of
Native-PAGE of osteoblast extracts confirmed the presenceof PMCA in the osteoblast layer (Fig. 4). Immunoreactivity
for PMCA did indeed reveal the presence of the enzyme on
the osteoidal domain of osteoblasts (Figs. 3,7).
A careful comparison between the histochemical data
and the two phosphatase-reactive protein bands which
activity could be inhibited either by vanadate or levamisoleon the Native-PAGE gel (Fig. 4) has clearly shown that thelevamisole-sensitive but vanadate-resistant band on the gelcorresponds to TNSALP and the other band to PMCA. The
molecular weights of the PMCA-positive band (140 kDa)
and the TNSALP-positive band (80 kDa) as shown by the
SDS-PAGE of the osteoblast extract correspond to thosereported previously [2,51,54,55]. The lower concentrationof inhibitor needed to abolish phosphatase activity on thegel relative to that in histochemical sections may be due to adifference in the state and/or microenvironment of the
enzyme between the in situ and laboratory conditions.
A polarized protein localization of PMCA in osteoblasts
had already been reported in previous in vitro [22] studies.
Interestingly, however, the distribution patterns of PMCA
demonstrated were opposite to those found in our current
experiment. This discrepancy may be attributed at least inpart to possible alteration in the polarization of functional
domains of the osteoblastic cells in cell culture system. Data
in a previous cytochemical study of Ca2+-ATPase in1083

osteoblasts [56] also appear to be different from ourobservations. This contradiction may be due at least in partto the fact that the authors of the latter study employed en
bloc incubation for the lead salt method, which might nothave allowed enough penetration of the substrate and/or thecapture agent in the incubation medium to the site of
enzyme reaction (osteoidal surface).
TNSALP mutant mice
In this study, we could also confirm the presence of aphosphatase different from TNSALP in the osteoblast
extract of TNSALP null mutant mice. The gel electrophoretic profiles of the osteoblast extract by Native-PAGE
treated for the expression of phosphatase activities, and thedata from the Western blot analysis, all indicate characteristics of an enzyme different from TNSALP. Since thephosphatase detected in the osteoblast extract of TNSALP
mutant mice is capable of hydrolyzing ATP and is resistantto levamisole, this enzyme is most likely PMCA. In fact,
PMCA immunoreactivity was in close association with theosteoidal surface of the osteoblasts in TNSALP null mutant
mice (Fig. 7A).
Role of PMCA
Under physiological conditions, osteoblast TNSALP
hydrolyzes inorganic pyrophosphate (PPi) [57], a natural
inhibitor of crystallization, and promotes mineralization ofbone matrix [10]. It is noteworthy that, in TNSALP nullmutant mice, bone mineralization proceeds to some extent
despite severe hypophosphatasia [11–16], which accompanies elevation of PPi [57]. Nevertheless, a slow but steadyprogression of mineralization of osteoid under these
conditions may implicate the existence of enzymes otherthan TNSALP that might at least partially hydrolyze andlower the local level of PPi, and promote mineralization of
osteoid matrix. Though data are not available yet as towhether PMCA can hydrolyze PPi, the enzyme associatedFig. 8. Summary diagram of localization of TNSALP (red) and PMCA (green) on the osteoblast plasma membrane and its possible diffusion into the intercellularspaces and also into the osteoidal matrix. Arrows in PMCA positive osteoidal plasma membrane indicate the direction of putative Ca2+-extrusion pump.

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with the osteoidal aspect of osteoblast plasma membrane
and/or that released into osteoid matrix may contribute to
the regulation of PPi concentration. Under severe hypophosphatemic conditions like in TNSALP null mutant
animal, this system may perhaps remain functional.
Aside from the possible role as a regulator of pyrophosphate concentration, the known physiological role of PMCA
is to maintain intracellular Ca2+ concentration by regulating
intra/extra-cellular Ca2+ homeostasis [54,55,58–60]. PMCA
is an outwardly directed Ca2+ pump that extrudes intracellular Ca2+ fuelled by hydrolysis of ATP [54,55,58–60].
As illustrated in the summary diagram of plausible localization patterns of TNSALP and PMCA based on ourcurrent observations (Fig. 8), the exclusive, polarized
localization of PMCA on the osteoidal aspect of osteoblast
plasma membrane may also imply a role of this enzyme as apotential calcium pump that provides Ca2+ ions toward theosteoid. This calcium pump may act synergistically with the
Na+/Ca2+ exchanger, which is also reported to be present on
the osteoidal aspects of osteoblasts [22,61].
In case of the periodontal ligament, 10% of TNSALP is
associated with its extracellular matrix and 90% of the
enzyme is firmly bound to cell membranes [62]. Thus,
partial release of PMCA from the osteoidal aspect ofosteoblast membrane into the osteoid matrix may also be
possible. Rather broad band-like immunoreactivity for
PMCA along the osteoidal surface (Figs. 3,7) support thishypothesis. Liberated PMCA in the osteoid matrix may
function as a regulator of local pyrophosphate concentrations, whereas the membrane-bound PMCA may serve as aCa2+-exclusion pump. Takano et al. [63] provided cytochemical evidence for such a liberation of PMCA from theplasma membrane of presecretory ameloblasts into the
osteoid-like predentin matrix at the early stage of toothformation.

Conclusion
In conclusion, the phosphatase associated with theosteoidal aspect of the osteoblast plasma membrane isdifferent from TNSALP, which is restricted to the basolateral domains of osteoblasts. Current histochemical andimmunochemical data indicate that PMCA is the most likely
candidate enzyme for the phosphatase located on theosteoidal aspects of the osteoblast plasma membrane thoughpossible co-localization of minor activities of phosphatase(s) such as inorganic pyrophosphatase should not bedisregarded.

Acknowledgment
This study was supported in part by Grants-in-Aid for
Scientific Research [15791036], [(B) 14370577] from Japan
Society for the Promotion of Science.

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