# Capacitative calcium entry and transient receptor potential canonical 6 expression control human hepatoma cell proliferation

Charbel El Boustany, Gabriel Bidaux, Antoine Enfissi, Philippe Delcourt, Natalia Prevarskaya, Thierry Capiod

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Charbel El Boustany, Gabriel Bidaux, Antoine Enfissi, Philippe Delcourt, Natalia Prevarskaya, et al. Capacitative calcium entry and transient receptor potential canonical 6 expression control human hepatoma cell proliferation. Hepatology, 2008, 47 (6), pp.2068-2077. 10.1002/hep.22263. hal-03060260

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Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
Capacitive Calcium Entry and Transient Receptor
Potential Canonical 6 Expression Control Human
Hepatoma Cell Proliferation

Charbel El Boustany,¹* Gabriel Bidaux,¹* Antoine Enfissi,¹* Philippe Delcourt,¹ Natalia Prevarskaya,¹ and
Thierry Capiod¹¹

Store-operated calcium entry (SOCE) is the main Ca²⁺ influx pathway involved in controlling proliferation of the human hepatoma cell lines Huh-7 and HepG2. However, the molecular nature of the calcium channels involved in this process remains unknown. Huh-7 and HepG2 cells express transient receptor potential canonical 1 (TRPC1) and TRPC6, as well as STIM1 and Orai1, and these 4 channels are the most likely candidates to account for the SOCE in these cells. We generated stable TRPC6-overexpressing or TRPC6-knockdown Huh-7 clones, in which we investigated correlations between the presence of the protein, the rate of cell proliferation, and SOCE amplitude. TRPC6-overexpressing Huh-7 cells proliferated 80% faster than did untransfected cells and their SOCE amplitude was 160% higher. By contrast, proliferation rate was 50% lower and SOCE amplitude 85% lower in TRPC6-knockdown clones than in untransfected cells. OAG (olyl acetyl glycerol)-induced calcium entry was similar in all cells, and small interfering RNA (siRNA) against TRPC1 had no effect on SOCE amplitude, highlighting the relationship among SOCE, TRPC6 and cell proliferation in Huh-7 cells. SOCE amplitude was reduced by STIM1 and Orai1 knockdowns, suggesting possible cooperation between these proteins and TRPC6 in these cells. Endothelial growth factor and hepatocyte growth factor increased TRPC6 expression and SOCE amplitude in Huh-7 cells, and cyclin D1 expression was decreased by STIM1, Orai1, and TRPC6 knockdowns. Conclusion: TRPC6 was very weakly expressed in isolated hepatocytes from healthy patients and expressed more strongly in tumoral samples from the liver of a cancer patient, strongly supporting a role for these calcium channels in liver oncogenesis. (HEPATOLOGY 2008;47:2068-2077.)

The processes involved in the transformation of normal cells into tumorigenic cells and tumor progression are complex and only partially understood.¹,² Many proteins in cancer cells are produced in

larger or smaller amounts than in normal cells. Some of
these proteins associated with cancer progression are in-
volved in calcium homeostasis. Several types of cancer
cells require an influx of calcium to proliferate, and it has
been known for more than 30 years that the proliferation
and growth of normal vertebrate cells depend on the phys-
iological concentration of extracellular calcium.³,⁴ Cal-
cium influx is required at various stages of the cell cycle,⁴
but little is known about the nature of the calcium chan-
nels involved in this process in nonexcitable cells such as
liver cells. Two separate calcium entry pathways are si-
multaneously activated in liver cells. Phospholipase C
stimulation results in InsP³ and diacylglycerol (DAG)
generation, leading to capacitative or store-operated cal-
cium entry (SOCE) because of a decrease in the calcium
concentration of the endoplasmic reticulum (ER) lumen.
A second type of calcium channel, activated indepen-
dently of store depletion by the membrane-permeant an-
logue of DAG, 1-oleoyl-2-acetyl-glycerol (OAG),⁵ has
also been described and is involved in non-capacitative

Abbreviations used: SOCE, store-operated calcium entry; STIM1, stromal inter-
acting molecule 1; NCCE, noncapacitative calcium entry; TRPC6, transient recep-
tor potential canonical 6; OAG, 1-oleoyl-2-acetyl-glycerol.

¹From the Institut National de la Santé et la recherche Médicale (INSERM), U800, IFR143, Université Sciences et Technologies Lille 1, F-59655, Villeneuve d'Ascq, France.

Received October 2, 2007; accepted January 24, 2008

Supported by Association pour la Recherche contre le Cancer grant 4615 to T.C.

*These authors contributed equally to this work.

Present address of Antoine Enfissi: Institut de Pharmacologie Moléculaire et Cellulaire, 660 Route des Lucioles Sophia Antipolis, Valbonne, F-06560 France.

Address reprint requests to: Dr. Thierry Capiod, INSERM U800, Laboratoire de Physiologie Cellulaire, Bâtiment SN3, Université Sciences et Technologies Lille 1, 59655 Villeneuve d'Ascq cedex, France. E-mail: thierry.capiod@univ-lille1.fr; fax: (33) 0-320434066.

Copyright © 2008 by the American Association for the Study of Liver Diseases.
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI 10.1002/hep.22263

Potential conflict of interest: Nothing to report.
calcium entry (NCCE).⁶ Several reports have shown that TRPC (transient receptor potential canonical) channels are probably involved in SOCE and NCCE.⁷ However, little is known about the role of these channels in hepatocytes, and 1 previous study has reported the involvement of TRPC1 in SOCE activation.⁸ Both transient receptor potential canonical 6 (TRPC6) and TRPC1 have been associated with cell proliferation and SOCE,⁹ and we recently showed that SOCE is essential for human hepatoma cell line proliferation.¹⁰ We focused on TRPC6, as it has recently been suggested that this receptor is associated with cell proliferation in vascular smooth muscle cells⁹,¹¹ and prostate cancer epithelial cells.¹² Two other proteins, stromal interacting molecule 1 (STIM1) and Orai1, have also been shown to be involved in controlling SOCE.¹³,¹⁴ We therefore first established expression profiles for TRPC, STIM1, and Orai1 in human hepatoma cells in order to identify the candidates most likely to account for SOCE activation in these cells. We modified the expression of the genes concerned and monitored the effects of these changes on the amplitude of SOCE and rates of cell proliferation in human hepatoma cells. We found that TRPC6 plays an essential role in proliferation and SOCE activity in human hepatoma cells.

Close associations between several TRP channels and cancer have been demonstrated,¹⁵ but the role of these calcium channels in liver cancer has yet to be determined. We assessed whether our observations in two model cell lines were relevant to human hepatocarcinoma development by monitoring the pattern of expression of these channels in normal quiescent adult human hepatocytes, in nontumoral and tumoral zones of human livers infected with hepatitis B (HBV+) or hepatitis C (HCV+), and in HBV-/HCV- tumoral livers. The changes in expression observed and the results obtained with human hepatoma cells suggest that TRPC6 plays an important role in the development of hepatocellular carcinomas.

## Materials and Methods

**Cell Culture and Human Liver Samples.** Huh-7 and HepG2 human hepatoma cells were provided by Dr. Doris Cassio (INSERM U757, Orsay, France). Cells were cultured as previously described.¹⁰ Freshly dissociated human hepatocytes from healthy patients were obtained from Biopredic (Rennes, France), and human liver samples were obtained from Dr. Jean-Marc Pascussi (INSERM U632, Montpellier, France). Liver tumors from patients infected with hepatitis B or C were obtained from Dr. Mounia Chami-Maggiora (INSERM U807, Paris, France). Liver slices were a gift from Professor Valérie Paradis (CNRS UMR 8149, Paris, France).

**Intracellular [Ca²⁺]ᵢ Measurements.** Huh-7 cells were plated on glass delta T culture dishes (Bioptechs, Butler, PA) coated with collagen. Calcium imaging was performed as previously described.¹⁶ Images were collected at 2-second intervals. Background fluorescence signals were collected at the same rate for the same wavelengths (340 and 380 nm) and were subtracted from the corresponding fluorescence images. The results (ΔR/R) are expressed as ratios of fluorescence signals measured at 340 nm to fluorescence signals measured at 380 nm during a response divided by the ratio obtained in resting conditions (that is, before the addition of an agent). ΔR/R was used to assess the amplitude of SOCE- and OAG-evoked Ca²⁺ responses in these cells.

**RT-PCR.** A classical reverse transcription polymerase chain reaction (RT-PCR) protocol was used, as described elsewhere.¹⁷ The various sets of primers used to identify human TRPC, TRPV, and TRPM channels are shown in Table 1.

**Proliferation Assays.** Cells were seeded at a density of 1000 cells/cm² in a final volume of 1 mL of culture medium. Proliferation rate was determined daily over 3 or 4 days, using an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) reagent kit (Promega).

**Cell Transfection.** Ready-to-use sihTRPC6, siTRPC1, siSTIM1, and siORAI1 cells were generated by transiently transfecting the Huh-7 cell line overnight with 100 nM siRNA, using the JetSI ENDO transfection reagent (Polyplus transfection). Control siRNA experiments were carried out in which Huh-7 cells were transfected with an siRNA against mTRPC6 (mouse transient receptor potential canonical 6).

**Generation of a TRPC6-Knockdown Cell Line and TRPC6-Overexpressing Cells.** Stable cell lines were obtained by transfecting Huh-7 cells with a vector encoding the human TRPC6 protein or short hairpin RNA (shRNA) against TRPC6, using Nucleofector, as recommended by the manufacturer (Amaxa GmbH, Köln, Germany). We transected 2 × 10⁶ trypsin-treated Huh-7 cells with 3 µg of vector and then used the transfected cells to seed a 24-well plate (Nunc) at very low density. Clones overexpressing or underexpressing TRPC6 were selected on the basis of their antibiotic resistance.

**Immunofluorescence.** Huh-7 cells and frozen sections of human liver were subjected to immunofluorescence analysis as previously described,¹⁷ using a rabbit polyclonal anti-TRPC6 antibody produced in our laboratory and diluted 1:500.

**Western Blotting.** Western blotting was carried out as previously described.¹⁷ The primary antibodies used were: rabbit anti-TRPC6, rabbit anti-TRPC1 (Alomone Labs, Israel), mouse anti-STIM1 (Chemicon International).
<table><caption>Table 1. List of Primers Used for RT-PCR Assays</caption><thead><tr><th></th><th>5' Forward 3'</th><th>5' Reverse 3'</th><th>Expected Size (bp)</th><th>Accession Number</th></tr></thead><tbody><tr><td>TRPC1</td><td>GATTTTGGAAAATTCTTGGGATGT</td><td>TTTGCTTTCATGATTTGCTATCA</td><td>369</td><td>NM_003304</td></tr><tr><td>TRPC3</td><td>GACATATTCAAGTTCATGGTCCTC</td><td>ACATCACTGTCATCCTCAATTTC</td><td>323</td><td>NM_003305</td></tr><tr><td>TRPC4</td><td>CTGCAAATATCTCTGGGAAGA</td><td>GCTTTGTTCGTGCAAATTTC</td><td>412</td><td>NM_016179</td></tr><tr><td>TRPC5</td><td>ACTACTAGCTTTTGCCAATGGAC</td><td>ATCAGCGTCATCCTCAATTTC</td><td>301</td><td>NM_012471</td></tr><tr><td>TRPC6</td><td>GACATCTTCAAGTTCATGGTCATA</td><td>ATCAGCGTCATCCTCAATTTC</td><td>321</td><td>NM_004621</td></tr><tr><td>TRPC7</td><td>CTTCAACTGTGTGGACTACATG</td><td>GCACTTCAGAAGTAGTCGTG</td><td>391</td><td>NM_020389</td></tr><tr><td>TRPV1</td><td>GACAACGAGTTCAAAGACCCT</td><td>CAGTTCACCGAAGTAGAATCC</td><td>318</td><td>NM_080704</td></tr><tr><td>TRPV2</td><td>GACAACGAGTTCAAAGACCCT</td><td>CAGTTCACCGAAGTAGAATCC</td><td>352</td><td>NM_016113</td></tr><tr><td>TRPV3</td><td>CCTCTACGACCTCACCAACG</td><td>CAGCAGGAAGATGGCAATG</td><td>361</td><td>NM_145068</td></tr><tr><td>TRPV4</td><td>ATCCTCTTTGACATCGTGTCC</td><td>GGGCCACGAGAAGTTCCACGT</td><td>322</td><td>NM_021625</td></tr><tr><td>TRPV5</td><td>GGCCTATGAGACACGTGAAGATATC</td><td>ATAGAATTGCCCCAGACTGGTTG</td><td>448</td><td>NM_019841</td></tr><tr><td>TRPV6</td><td>TGGTGCAACGTCATGTACTTC</td><td>CATGGGGTAGTCGTAGAAGT</td><td>201</td><td>AF365927</td></tr><tr><td>TRPM1</td><td>AGCAGGACATCGAGATGGCAAT</td><td>GTGTTGCATGTTCACTCCGTTT</td><td>413</td><td>NM_002420</td></tr><tr><td>TRPM2</td><td>TCTCAGAATGGAGCCCTCAGC</td><td>AAGACGATGTCGCCAAAGGCAT</td><td>402</td><td>NM_002420</td></tr><tr><td>TRPM3</td><td>CGCAGCTGGAAGACCTTATC</td><td>AAGCTGCTCTGACGGACAAT</td><td>142</td><td>NM_020952</td></tr><tr><td>TRPM4</td><td>AGCATGGTGGTGCCGGAGAA</td><td>GGTGTCTCTATTCCGGACCACA</td><td>244 (a), 600 (b, c)</td><td>a, AY297044; b, AY297046; c, AY297046</td></tr><tr><td>TRPM5</td><td>CAGAACATCACCTCACACCAG</td><td>GGTTCTCGCTCTTCTGGTTC</td><td>640</td><td>NM_014555</td></tr><tr><td>TRPM6</td><td>AGATGAAGGGATGCTGCGAC</td><td>GCAAGAGTGTATTAGTAGGTCC</td><td>470</td><td>NM_017662</td></tr><tr><td>TRPM7</td><td>TGGACAGCAGTGGCTGGTTG</td><td>TATTTCCGCCAGACCTCCGA</td><td>362</td><td>NM_017672</td></tr><tr><td>TRPM8</td><td>TGCTGCAGAGGATGCTGATCG</td><td>GGTGCCCAACCGTGTAGCCAA</td><td>419</td><td>NM_024080</td></tr><tr><td>hOrai1</td><td>CTTCAGTGCCCTGCACCACAG</td><td>CCTGGAACTGTCGGTCAGTC</td><td>450</td><td>(NM_032790)</td></tr><tr><td>hSTIM1</td><td>GCGGGAGGGTACTGAG</td><td>TCCATGTCATCCACGTCGTCA</td><td>533</td><td>(NM_003156)</td></tr><tr><td>Real-time PCR</td><td></td><td></td><td></td><td></td></tr><tr><td>TRPM8</td><td>CGGTCATCTACGAGCCCTAC</td><td>CACACACAGTGGCTTGGACT</td><td></td><td>AY328400</td></tr><tr><td>TRPC6</td><td>TGGTTACGTTCTTTATGGAGTC</td><td>CACATCAGCGTCATCCTCA</td><td>112</td><td>NM_004621</td></tr><tr><td>18S mRNA</td><td>CAGCTTCCGGGAAACCAAAGTC</td><td>AATTAAGCCGCAGGCTCCACTC</td><td>113</td><td>NR_003286.1</td></tr></tbody></table>

tional), mouse anti-β-actin (Sigma, St. Louis, MO), and mouse anticalnexin (Chemicon International).

The intensity of the signals obtained was evaluated by densitometry and semiquantified using the ratio between the intensity of the protein of interest divided by the intensity of calnexin or actin in each experiment. Each experiment presented was repeated at least three times.

**Quantitative Real-Time PCR Analysis.** PCR product level was quantified by real-time quantitative PCR on an ABI-Prism 5700 Sequence Detection System. For each reaction, 10 ng of complementary DNA was added to 20 µL of reaction mixture containing 12.5 µL of 2× QuantiTect SYBR Green PCR Master Mix (Applied Biosystems) and 300 nM primers (Table 1). The primers used for the quantification of TRPC6 messenger RNA (mRNA) are shown in Table 1. We used 18S mRNA as an endogenous control to normalize variations in RNA extraction, the degree of RNA degradation, and variability in RT efficiency. We quantified the results with the comparative Ct method.¹⁸

**Materials.** All culture media were obtained from Invitrogen (Cergy-Pontoise, France). Thapsigargin was obtained from Alomone Laboratories (Jerusalem, Israel), and fura-2/AM was obtained from Molecular Probes Europe (Leiden, the Netherlands). All other reagents were from Sigma.

**Statistics.** Data were analyzed and curves fitted with Microcal Origin software (Microcal Software Inc., Northampton, MA). Video imaging experiments were repeated at least three times in triplicate, and between 140 and 250 cells were examined in all conditions. An unpaired t test was used to compare [Ca²⁺]; values. Results are expressed as means ± SEMs, with statistically significant at *P* < 0.5, **P* < 0.1, and ***P* < 0.01.

## Results

**TRPC, STIM1 and Orai1 Expression in Normal and Tumoral Human Liver and in Primary Cultures of Human Hepatocytes.** We used a TRPC6-enhanced green fluorescent protein (TRPC6-eGFP) chimera to validate our TRPC6 antibody. We detected a 106-kDa protein and a glycosylated form eliminated by peptide N-glycosidase F-mediated deglycosylation on western blots (Fig. 1A,B). Immunocytochemical staining with the same construct showed TRPC6 to be localized at the plasma membrane of human hepatoma cells (Fig. 1C). We then monitored TRPC expression in livers collected
<figure><img src="image_3.png" /><figcaption>Fig. 1. TRPC6 expression and localization. (A) Immunoblotting showing detection of a TRPC6-eGFP chimera produced in Huh-7 cells and detected with either anti-GFP antibody (left) or anti-TRPC6 antibody (right). The expected chimera was detected at about 125 kDa, together with another band at around 150 kDa. (B) Reduction of the glycosylated form of the wild-type TRPC6 protein overexpressed in HEK-293 cells by PNGase treatment. (C) Validation of the anti-TRPC6 antibody in an immunofluorescence experiment showing the codetection of a TRPC6-eGFP chimera overexpressed (green) in Huh-7 cells with anti-TRPC6 antibody (red).</figcaption></figure>

from healthy patients, tumoral and nontumoral tissue
from HBV+, HCV+, and HBV-/HCV- patients, and
dissociated human hepatocytes from the livers of healthy
donors. RNA analyses showed that TRPC1 and TRPC4
were absent in liver tissues (six of six samples) and freshly
dissociated human hepatocytes (nine of ten samples) from
healthy donors, but were present in all tested tumors from
HBV+ (seven of seven samples), HCV+ (seven of seven
samples), and HBV-/HCV- (six of six samples) pa-
tients. TRPC5, STIM1, and Orai1 were expressed in all
conditions. Quantitative PCR was used to compare

TRPC6 mRNA expression in isolated human hepatocytes
from healthy patients and nontumoral and tumoral sam-
ples taken from the same liver of HBV+, HCV+, and
HBV-/HCV- patients. TRPC6 was very weakly ex-
pressed in isolated hepatocytes from healthy patients. It
was expressed 100 times and 1,000 times more strongly in
nontumoral and tumoral samples, respectively, from the
liver of a cancer patient (Fig. 2A). Immunocytochemical
staining indicated that TRPC6 was expressed on hepato-
cytes solely in the tumoral zones (Fig. 2B) of slices of
HBV-/HCV- livers, indicating that the increase in
<figure><img src="image_4.png" /><figcaption>Fig. 2. Pattern of TRPC expression in normal and tumoral human liver and in primary cultures of human hepatocytes. (A) Histogram summarizing mRNA expression for human TRPC6 in isolated human hepatocytes (n = 9), and nontumoral (NT) and tumoral (T) specimens from the same liver (n = 6 for each condition). Values are presented as means ± SDs. (B) TRPC6 was overexpressed in cancerous hepatocytes in liver tumors (right) with respect to normal liver (left).</figcaption></figure>

TRPC6 expression in tumors was independent of hepatitis viruses. The expression observed in nontumoral zones may have been a result of other cell types such as endothelial cells or Kupffer cells (not shown).

**Transient Overexpression and Underexpression of TRPC6.** We investigated the involvement of TRPC6 in cell proliferation and cancer development in the liver using two human hepatoma cell lines, Huh-7 and Hep G2. We first demonstrated (Table 2) that these two cell lines produced mRNA for STIM1 and Orai1 and the same set

of TRPs, with only TRPC1 and TRPC6 associated with SOCE.⁷,⁹ We investigated the role of TRPC6 in SOCE, using Huh-7 cells transiently transfected with a plasmid encoding the channel (increasing expression by 150% 72 hours after transfection) or with an siRNA against TRPC6 (decreasing expression by 95% 72 hours after transfection; Fig. 3A). Cells were then challenged by incubation with 1 µM thapsigargin for 3 to 4 minutes in the absence of external calcium for ER depletion and full activation of SOCE. Once fluorescence traces had re-

<table><caption>Table 2. Pattern of TRP mRNA Expression in Human Hepatoma Cell Lines Huh-7 and Hep G2</caption><thead><tr><th rowspan="2"></th><th colspan="6">hTRPC</th><th colspan="6">hTRPV</th><th colspan="6">hTRPM</th></tr><tr><th>1</th><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th><th>8</th></tr></thead><tbody><tr><td>Huh-7</td><td>✓</td><td>-</td><td>-</td><td>-</td><td>✓</td><td>-</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>Hep G2</td><td>✓</td><td>-</td><td>-</td><td>-</td><td>✓</td><td>-</td><td>✓</td><td>✓</td><td>-</td><td>✓</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>✓</td><td>-</td><td>✓</td><td>✓</td><td>✓</td></tr></tbody></table>
<figure><img src="image_5.png" /><figcaption>Fig. 3. Transient overexpression and underexpression of TRPC6 and SOCE amplitude (A) Fura-2 loaded Huh-7 cells were challenged by incubation with thapsigargin (TG, 1 µM) in a saline medium containing no calcium and 100 µM EGTA. SOCE was monitored in simTRPC6 cells (ctrl; Huh-7 cells transiently transfected with the plasmid encoding hTRPC6) or Huh-7 cells transiently transfected with the siRNA against hTRPC6 (sihTRPC6 cells). The addition of 1.8 mM Ca²⁺ to the external solution triggered SOCE in ctrl (thick line) hTRPC6 (thin line), and sihTRPC6 (dashed line) Huh-7 cells. CCE was estimated as the difference between [Ca²⁺], increase in the presence and the absence of TG (basal). Increases in [Ca²⁺], are expressed as ratios of 340:380 nm fluorescence signals (ΔR/R). (B) Means ± SEMs from a series of experiments similar to A for control (black), hTRPC6 (gray), and sihTRPC6 (white) Huh-7 cells.</figcaption></figure>

turned to baseline, we added 1.8 mM Ca²⁺ for the assessment of SOCE amplitude (Fig. 3B). ER content was assessed by determining peak TG-evoked responses and SOCE amplitudes as the peak ratio after the addition of

Ca²⁺. Huh-7 cells overexpressing TRPC6 displayed a 110% increase in SOCE amplitude, and Huh-7 cells underexpressing this receptor displayed an 85% decrease in SOCE amplitude (Fig. 3C), whereas ER content was similar in all conditions.

**Stable TRPC6 Overexpression.** We developed two stable clones giving similar results for the evaluation of TRPC6 involvement in cell proliferation. TRPC6 over-expression was stable for at least 20 passages, as shown by western blots (see Materials and Methods section). A 106-kDa band corresponding to the protein was significantly more intense (+ 70%) for transfected Huh-7 cells than for controls (Fig. 4A). This increase in TRPC6 level resulted in a 145% increase in SOCE amplitude in transfected Huh-7 cells, with no significant difference in TG-sensitive Ca²⁺ pool contents between control and transfected Huh-7 cells (Fig. 4B). We monitored the rate of cell proliferation at 24-hour intervals in control cells and cells overexpressing TRPC6 plated at the same density. The time required for doubling of the cell population was determined from absorbance measurements. This doubling time was shorter for TRPC6-overexpressing cells (19 hours) than for control cells (about 21 hours; Fig. 4C), confirming the intimate relationship between TRPC6 expression, SOCE amplitude, and cell proliferation rate.

**Stable TRPC6 Silencing.** The relationship between SOCE and TRPC6 was also investigated in Huh-7 cells transfected with a shRNA against TRPC6, resulting in a stable decrease in TRPC6 protein level. Western blots showed that the intensity of the 106-kDa band corresponding to TRPC6 was 60% lower in transfected cells than in controls (Fig. 4A). The 50% smaller SOCE amplitude and greater cell population doubling time (32 hours, 30 minutes versus 19 hours, 45 minutes for control cells) observed for transfected cells may therefore be directly related to the lower level of expression of TRPC6 in the transfected Huh-7 cells (Fig. 4B,C). The low level of TRPC6 expression had no significant effect on TG-sensitive Ca²⁺ pool content (Fig. 4B). TRPC6 expression was not totally abolished in the two clones, so SOCE was not entirely prevented. Cells continued to grow and divide, but with a doubling time about 40% higher than that of control cells. This close relationship confirms our previous data showing that the abolition of SOCE results in the total inhibition of proliferation.¹⁰,¹⁹

**OAG-Activated Calcium Entry.** Noncapacitative calcium entry is an alternative pathway to SOCE,²⁰ and has often been associated with TRPC6 expression.²¹,²² Control human hepatoma cells and stable clones over- or underexpressing TRPC6 were challenged by incubation for 3 minutes with 100 µM OAG in the absence of ex-
<figure><img src="image_6.png" /></figure>

Fig. 4. SOCE amplitude, cell proliferation, and TRPC6 level in stably transfected Huh-7 clones. (A) Western blots showed the overproduction (left) or underproduction (right) of a 106-kDa protein corresponding to hTRPC6 in transfected Huh-7 cells. The numbers above the gel indicate expression with respect to control conditions. (B) Mean results from a series of experiments similar to those shown in Fig. 2A. Values are means ± SEMs for ER Ca²⁺ content and SOCE amplitude in control (black) and TRPC6-overexpressing (hTRPC6, gray) Huh-7 cells (left) and control (black) and TRPC6 knockdown (KD hTRPC6, white) Huh-7 cells (the right). (C) Cell number determinations from DO absorbance in hTRPC6 (left) and KD hTRPC6 (right) Huh-7 cells plated at a cell density of 1000 cells/cm² in 24-well plates. Absorbance was measured every day for 3 days, and doubling time was estimated from the slopes of the curves.

ternal calcium. NCCE was then measured after adding external calcium. The histogram obtained showed that OAG-evoked calcium influx did not differ significantly in control cells and in cells over- and underexpressing TRPC6. Thus, OAG-activated calcium influx was clearly independent of TRPC6 level (Fig. 5). No increase in [Ca²⁺]ᵢ was observed after the addition of 100 µM OAG in the absence of external calcium.

**TRPC6 Expression and Growth Factors.** The transition from a normal to a tumoral liver is associated with an increase in the expression of growth factor receptors.²³ We therefore investigated the possible regulation of TRPC6 expression by endothelial growth factor (EGF) or hepatocyte growth factor (HGF). Huh-7 cells were

seeded in 2% fetal bovine serum and treated for 24 hours with 20 ng/mL EGF or 10 ng/mL HGF. These growth factors increased TRPC6 levels by a factor of 3 (Fig. 6A) and induced a large increase in SOCE amplitude (Fig. 6B).

**TRPC1, STIM1, and Orai1 Expression, SOCE Amplitude, and Cyclin Activity.** TRPC1—which is expressed in Huh-7 cells, together with STIM1 and Orai1—is known to be associated with SOCE in several cell types.²¹,²² We therefore investigated the effects of siRNA against these proteins on SOCE amplitude and protein level (Fig. 5). Western blots showed that TRPC1 and STIM1 levels decreased in the presence of the corresponding siRNA (Fig. 7A,B). Control experiments
<figure><img src="image_7.png" /><figcaption>Fig. 5. OAG-activated calcium entry. (A) Fura-2 loaded Huh-7 cells were challenged by 100 µM OAG in a saline medium containing no calcium and 100 µM EGTA. NCCE was monitored after the addition of 1.8 mM Ca²⁺ in the external solution in simTRPC6 (ctrl), stable Huh-7 clones overexpressing hTRPC6 or expressing shRNA hTRPC6 (KD hTRPC6). (B) Means ± SEMs from a series of three experiments for control (black), hTRPC6 (gray), and KD hTRPC6 (white).</figcaption></figure>

showed that siRNA against TRPC6 had no effect on TRPC1 or STIM1 levels (data not shown). Huh-7 cells were transiently transfected with siRNA against TRPC1, STIM1, or Orai1, and TG-induced Ca²⁺ content or SOCE amplitude was assessed, as previously described. Control experiments showed that the transfection of cells with an siRNA against mouse TRPC6 had no effect on SOCE amplitude. Transfection with an siRNA against TRPC1 had no effect on SOCE amplitude, whereas transfection with an siRNA against STIM1 or Orai1 resulted in a decrease in SOCE amplitude (about 40% for STIM1 and 30% for Orai1; Fig. 7C). The decrease in SOCE amplitude was smaller than that observed for TRPC6 knockdown, probably because of the lower transient transfection efficiency, as TRPC6 expression was inhibited by 95%, whereas STIM1 expression was inhibited by only 50%. ER Ca²⁺ content was similar in all these

conditions, clearly indicating that the observed decrease in Ca²⁺ influx was not a result of a decrease in ER calcium content.

Calcium channels form signaling complexes in the plasma membrane for the activation of specific transduction cascades.²⁴ STIM1, Orai1, and TRPC6 are therefore probably directly linked to the pathway controlling the cell cycle in cancerous Huh-7 cells, making it possible to activate cell proliferation specifically in these cells. SOCE is involved in the activation and expression of cyclins involved in G1 phase progression such as cyclin D1.²⁵ We therefore investigated whether STIM1, Orai1, and TRPC6 interacted with these proteins to control the rate of cell proliferation. Knocking down the expression of these SOCE components in Huh-7 cells greatly decreased cyclin D1 levels (Fig. 7D), strongly suggesting that as part as a molecular complex with STIM1 and Orai1, TRPC6 directly controls cell proliferation in these cells. As TRPC6 is expressed only in tumoral livers, it seems likely

<figure><img src="image_8.png" /><figcaption>Fig. 6. TRPC6 expression and growth factors. (A) Western blots showing an increase in TRPC6 levels in Huh-7 cells incubated in the presence of EGF or HGF for 24 hours. The numbers above the gels indicate expression with respect to control conditions. (B) Mean results from a series of experiments similar to those shown in Fig. 2A. Values are means ± SEMs for ER Ca²⁺ content and SOCE amplitude for control (black), EGF (light gray), and HGF (gray).</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>Fig. 7. TRPC1, STIM1, and Orai1 levels and SOCE amplitude. (A, B) Western blots showing the decrease in TRPC1 and STIM1 levels in siTRPC1- and siSTIM1-transfected Huh-7 cells, respectively. (C) Mean results from a series of experiments similar to those shown in Fig. 1. Values are means ± SEMs for ER $Ca^{2+}$ content and SOCE amplitude in control (black), siTRPC1 (white), siSTIM1 (light gray), and siOrai1 (gray) Huh-7 cells. (D) Blots showing the decrease in cyclin D1 levels in sihTRPC6-transfected, siSTIM1-transfected, and siOrai1-transfected Huh-7 cells. The numbers above the gels indicate relative expression with respect to control conditions.</figcaption></figure>

that this protein plays a major role in the development of human hepatocarcinomas.

Discussion

The nature of the proteins involved in SOCE may differ between cell types. TRPC6 is often described as a channel involved in NCCE,²¹,²² but there is evidence to suggest it is also involved in SOCE in some tissues.⁹,¹¹ Human hepatoma cells express only TRPC6 and TRPC1, and TRPC1 silencing had no effect on SOCE amplitude; therefore, our results clearly demonstrate that SOCE activity is closely related to TRPC6 expression in these cells. STIM1 and Orai1 are alternative calcium channels dis-

playing functional interactions that have recently been implicated in SOCE.¹³,¹⁴ The silencing of STIM1 or Orai1 in Huh-7 cells reduced SOCE amplitude. As SOCE amplitude is closely correlated with STIM1 and TRPC6 levels, these two proteins seem to be equally important in this process. Poor efficiency of the siRNA against Orai1 was probably also involved in the 40% reduction of SOCE amplitude in Huh-7 cells. It has been shown that STIM1 binds to TRPC1, TRPC4, or TRPC5 but not to TRPC3, TRPC6, or TRPC7²⁶,²⁷ and that Orai1 interacts with TRPC6 as a regulatory subunit.²⁸ However, as TRPC1 silencing has no effect on SOCE amplitude and TRPC4 and TRPC5 are absent from Huh-7 cells, TRPC6, STIM1, and Orai1 may interact to form SOCE channels in these cells, as shown for TRPC1/STIM1/Orai1.²⁹ STIM1 indirectly regulates TRPC6 by mediating the heteromultimerization of TRPC4 with TRPC6,²⁷ but we found that TRPC4 was absent from Huh-7 cells. TRPC6 levels may also account for the role of this protein in SOCE in Huh-7 cells. It was recently suggested that increased TRPC3 levels lead to a switch from store-dependent to store-independent activation mechanisms.³⁰ However, the amplitude of OAG-induced $Ca^{2+}$ influx was unaffected by the over- or underexpression of TRPC6 in Huh-7 cells.

Cell proliferation depends on calcium influx and ER $Ca^{2+}$ content.³¹ Stable growth is characterized by optimal filling, due to balanced expression of the sarcoendoplasmic calcium ATPase (SERCA)2b $Ca^{2+}$ pump and ER leak channels. Increases in proliferation are characterized by overfilling, due to the overexpression of SERCA2b, and the inhibition of proliferation is characterized by underfilling because of the underexpression of SERCA2b.³² Our work shows that ER content is independent of TRPC6 expression, supporting a role for calcium influx in cell proliferation. We have recently shown that TRPC1/C4 is linked to apoptosis in prostate cancer epithelial cells, whereas TRPC6 activation induces cell proliferation.¹² Thus, the cell proliferation rate depends solely on the amount of calcium entering Huh-7 cells through TRPC6, providing further evidence that localized calcium entry plays a key role in determining cell fate.²¹

TRPC expression has not been studied in detail in the liver. Hepatocytes from adult liver are quiescent, blocked in the G₀ phase of the cell cycle.³³,³⁴ Low levels of cell proliferation occur in the adult liver to replace the small percentage of the hepatocyte population undergoing apoptosis. We found that adult hepatocytes expressed only STIM1, Orai1, and TRPC5. TRPC5 binds STIM1³⁵ and is associated with SOCE in several cell types. It may therefore be responsible for the calcium entry observed in
freshly dissociated hepatocytes.³⁶,³⁷ However, as in Huh-7 cells, the expression of EGF and HGF receptors in hepatocarcinomas probably induces TRPC6 expression, increasing the rate of cell proliferation. It would now be of interest to determine whether the reduction of SOCE activity through this specific channel slows tumor development in human livers.

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