pannexin3inhibitsproliferationofosteoprogenitorcellsby ... · background: the mechanism of the...

Pannexin 3 Inhibits Proliferation of Osteoprogenitor Cells byRegulating Wnt and p21 Signaling*

Received for publication, October 1, 2013, and in revised form, December 2, 2013 Published, JBC Papers in Press, December 12, 2013, DOI 10.1074/jbc.M113.523241

Masaki Ishikawa‡1, Tsutomu Iwamoto§, Satoshi Fukumoto¶, and Yoshihiko Yamada‡2

From the ‡Laboratory of Cell and Developmental Biology, NIDCR, National Institutes of Health, Bethesda, Maryland 20892-4370,the §Department of Pediatric Dentistry, University of Tokushima Graduate School, Tokushima 770-8504, Japan, and the¶Department of Pediatric Dentistry, Tohoku University Graduate School of Dentistry, Sendai 980-8576, Japan

Background: The mechanism of the transition from osteoprogenitor cell proliferation to differentiation is unclear.Results: Panx3 inhibits osteoprogenitor proliferation by blocking canonical Wnt signaling and promoting p21 activation.Conclusion: A Panx3 hemichannel induces multiple Panx3 signaling pathways critical for the cell cycle exit.Significance: Our findings reveal that Panx3 is a new regulator to switch the stage from proliferation to differentiation inosteoprogenitor cells.

Canonical Wnt signaling and BMP promote the proliferationand differentiation of osteoprogenitors, respectively. However,the regulatory mechanism involved in the transition from pro-liferation to differentiation is unclear. Here, we show that Panx3(pannexin 3) plays a key role in this transition by inhibiting theproliferation and promoting the cell cycle exit. Using primarycalvarial cells and explants, C3H10T1/2 cells, and C2C12 cells,we found that Panx3 expression inhibited cell growth, whereasthe inhibition of endogenous Panx3 expression increased it. Wealso found that the Panx3 hemichannel inhibited cell growth bypromoting �-catenin degradation through GSK3� activation.Additionally, the Panx3 hemichannel inhibited cyclin D1 tran-scription and Rb phosphorylation through reduced cAMP/PKA/CREB signaling. Furthermore, the Panx3 endoplasmicreticulum Ca2� channel induced the transcription and phos-phorylation of p21, through the calmodulin/Smad pathway, andresulted in the cell cycle exit. Our results reveal that Panx3 is anew regulator that promotes the switch from proliferation todifferentiation of osteoprogenitors via multiple Panx3 signalingpathways.

Highly coordinated proliferation and differentiation pro-grams regulate bone development and homeostasis. CanonicalWnt signaling plays an important role in osteoprogenitor pro-liferation and bone mass (see Refs. 15–22). Wnt proteins aresecreted signaling molecules that regulate many biological pro-cesses, such as proliferation, differentiation, maintenance, andsurvival, through �-catenin-dependent (canonical) and -inde-pendent pathways (noncanonical) (1–3). Canonical Wnt sig-naling involves protein stabilization and nuclear translocationof the downstream �-catenin protein, from its multimeric pro-

tein complex consisting of glycogen synthase kinase-3�(GSK3�),3 APC, and Axin (4, 5). In the absence of Wnt,�-catenin is phosphorylated by GSK3� in the complex anddegraded through ubiquitination. Upon Wnt binding to a friz-zled receptor, and its co-receptors LRP5/6, Axin, and GSK3�are recruited to the plasma membrane with the scaffold proteindisheveled, which disrupts the protein complexes (6, 7). Thisdisruption of the protein complexes leads to the phosphoryla-tion of GSK3� and inhibition of �-catenin phosphorylation,resulting in the stabilization of �-catenin and its translocationinto the nucleus. �-Catenin in the nucleus binds to TCF/LEFtranscription factors to activate Wnt/�-catenin-responsivegenes, such as CDK1 and cyclin D1, which are required for cellcycle progression (1, 8, 9). In addition to the membrane associ-ation mechanism, Wnt signaling is regulated by PKA and PI3K/Akt, which phosphorylate and inactivate GSK3�, resulting inthe stabilization of �-catenin (10 –13).

Genetic studies in human patients with the osteoporosis-pseudoglioma syndrome (14) showed that loss and gain muta-tions in LRP5 or LRP6 result in low (14) and high bone mass (15,16), respectively. Studies in mice also support the crucial role ofcanonical Wnt signaling in bone development. LRP5 KO micedisplay inhibition of bone formation and osteoblast prolifera-tion (17). Mice with the mutation of Dkk1, which prevents Wntsignaling by binding LRP5/6, have high bone density and anincreased number of osteoblasts (18). Conditional �-cateninKO mice show low bone mass (19 –21). Although canonicalWnt signaling is required for osteoprogenitor cell proliferation,the mechanism of how Wnt signaling is regulated during osteo-genesis is still not fully understood. Osterix (Osx) negativelyregulates canonical Wnt signaling by promoting the expressionof Dkk1 and inhibits osteoblast proliferation (22). However,Osx is expressed in differentiating osteoblasts, not in the tran-sitional stage from osteoprogenitor proliferation to osteoblasts(23).

Pannexins (Panxs) were recently identified as a new gap junc-tion protein family (24). The Panx family consists of three

* This work was supported, in whole or in part, by National Institutes of HealthIntramural Research Program at the NIDCR. This work was also supportedby Grant-in-Aid 20679006 from the Ministry of Education, Science, andCulture of Japan (to S. F.) and NEXT program Grant LS010 (to S. F.).

1 Supported in part by the Research Fellowship of the Japan Society for thePromotion of Science for Young Scientists.

2 To whom correspondence should be addressed: 30 Convent Dr., MSC 4370,Bldg. 30, Rm. 407, NIDCR, NIH, Bethesda, MD 20892-4370. Tel.: 301-496-2111; Fax: 301-402-0897; E-mail: [email protected].

3 The abbreviations used are: GSK, glycogen synthase kinase; Panx3, pan-nexin 3; Osx, Osterix; ER, endoplasmic reticulum; �-MEM, �-minimumessential medium; CaM, calmodulin.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 5, pp. 2839 –2851, January 31, 2014Published in the U.S.A.

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members, Panx1, 2, and 3. Panx1 is ubiquitously expressed withparticularly strong expression in the central nervous system.Panx2 is expressed in the central nervous system and is shownto modulate Panx1 channel activities (24 –27). Panx3 is themember that was most recently identified by genome bioinfor-matic analysis (24). Although Panx3 is expressed in certain softtissues, such as skin and coronary arteries (28, 29), we foundhigh levels of Panx3 expression in developing hard tissues,including cartilage and bone (23, 30).

Previously, we demonstrated that Panx3 is induced in theprehypertrophic zone and in the perichondrium of the growthplate. Panx3 inhibits PTH-mediated chondrocyte proliferationby its hemichannel activity and promotes the differentiation ofchondrocytes (30). We also showed that Panx3 promotes osteo-blast differentiation through its multiple pathways (23). In thisstudy, we demonstrate that Panx3 inhibits osteoprogenitorproliferation by inhibiting Wnt/�-catenin and PKA/CREB sig-naling and promotes the cell cycle exit by increasing p21 activ-ity. Our results demonstrate that Panx3 is a new regulator thatpromotes the switch from proliferation to differentiation inosteoblasts.

EXPERIMENTAL PROCEDURES

Reagents—The rabbit anti-Panx3 antibody, Panx3 expres-sion vector (pEF1/Panx3), control vector (pEF1), shRNA vectorfor Panx3 (shPanx3), and control vector (sh control) weredescribed previously (23, 30). The control adenovirus (AdCont)and Panx3 expression adenovirus (AdPanx3) were prepared andpurified by Welgen, Inc. The Akt-CA and Akt-DN vectors andTCF-luciferase reporter plasmids (Topflash and Fopflash)were obtained from Addgene. The antibodies for P-�-catenin,P-GSK3�, GSK3�, PKA, P-PKA, CREB, P-CREB, cyclin D1, P-Rb,Smad1, and P-Smad1/5 were obtained from Cell Signaling Tech-nology, Inc. P-p21 and Rb were obtained from Santa Cruz, p21 wasfrom BD Biosciences, �-catenin and �-tubulin were from Sigma,and Ki67 was from Dako. Dkk1 was obtained from Invitrogen, andWnt3a was from R&D Systems. The PPDA was obtained fromSigma, BMP2 was from Humanzyme, and iQ SYBR GreenSupermix was from Bio-Rad. The HRP-conjugated goat anti-mouse and goat anti-rabbit IgG were obtained from UnitedStates Biological (Swampscott, MA). The inhibitory Panx3 pep-tide and its control scrambled peptide were described previ-ously (23, 30).

Cell Culture—C2C12 cells were grown in DMEM (Invitro-gen) containing 10% FBS (HyClone) at 37 °C under 5% CO2. Forthe proliferation assay, the cells (2.5 � 103/ml) stably trans-fected with either pEF1/Panx3 or control pEF1 were cultured inDMEM in 96-well plates for up to 5 days. C2C12 cells stablytransfected with the Panx3 shRNA vector were incubated withBMP2 (300 ng/ml) in 96-well plates and cultured for up to 5days. The C3H10T1/2 cells were grown in DMEM/F-12 (Invit-rogen) containing 10% FBS. For the proliferation assay,C3H10T1/2 cells transiently transfected with either pEF1/Panx3 or pEF1 were cultured in normal media in 96-well platesfor up to 4 days. The cells transiently transfected with the Panx3shRNA vector were incubated with an osteogenic medium(DMEM/F-12, 10% FBS, 100 �g/ml ascorbic acid, 10 mM

�-glycerol phosphate, and 100 ng/ml BMP2). The cell prolifer-

ation activity was evaluated using a cell counting kit (Dojindo).The absorbance was measured using a microplate reader. Theprimary calvarial cells were prepared from the calvaria of new-born mice and cultured in �-minimum essential medium(�-MEM; Invitrogen) with 10% FBS, 100 units/ml of penicillin,and 100 �g/ml of streptomycin as previously described (31).For the proliferation assay, primary calvarial cells transientlytransfected with pEF1/Panx3 or pEF1 were cultured in �-MEMin 96-well plates for 2 days. For the TOPflash/FOPflashreporter assays, luciferase reporter plasmids were co-trans-fected with the pRLSV40 plasmid (32) as an internal control fortransfection efficiency. Luciferase activities were assayed usingthe Dual-Luciferase ReporterTM assay system (Promega). Rela-tive luciferase activities were expressed as ratios of luciferaseactivities of the experimental vectors to the internal controlvector.

Ex Vivo Calvarial Organ Culture—Calvarial bones were iso-lated from either newborn C57BL/6 mice or heterozygousAxin2LacZ mice obtained from Jackson Labs (33) and were cul-tured in DMEM containing 5% FBS, 50 �g/ml ascorbic acid(Sigma), and 1 mM �-glycerol phosphate (Sigma) at 37 °C in ahumidified atmosphere of 5% CO2 as previously described (34,35). One day after starting the culture, the calvarial bones wereinfected with either recombinant adenovirus AdCont orAdPanx3 (1 � 109 pfu/ml) for 2 days. For the peptide inhibitionassay, the Panx3 inhibitory peptide or scramble peptide (100�g/ml) was added to the calvarial culture, and the culture wasincubated for 2 days. All experimental procedures wereapproved by the Animal Care and Use Committee of theNational Institute of Dental and Craniofacial Research.

Immunostaining and X-Gal Staining—For X-gal staining, thecalvarial samples were fixed with 4% paraformaldehyde over-night, then equilibrated in sucrose, and embedded in an O.C.Tcompound (Tissue-Tek) for cryosection. X-gal staining wasperformed as described previously. For immunostaining, afterdeparaffinization and rehydration, the sections were treatedwith heat-induced epitope retrieval in a pH 6.0 citrate buffer(Dako). The quantification of LacZ- and Ki67-positive cells wasanalyzed using ImageJ 1.40g. For the staining of cultured cells,the cells were blocked with Power block (Biocare Medical) andreacted for 2 h at room temperature with primary antibodies.Primary antibodies were detected by Alexa 488 (Invitrogen) orby Cy-3 (Jackson ImmunoResearch Laboratories)-conjugatedsecondary antibodies. Nuclear staining was performed usingHoechst dye (Sigma-Aldrich). The analysis was performed onan LSM 710 inverted confocal microscope (Carl Zeiss Micro-Imaging, Inc.), and co-localization was analyzed by MetaMorph(Molecular Devices).

RT-PCR—The total RNA was extracted using QG-810 andthe QuickGene RNA cultured cell HC kit S (Fujifilm). The totalRNA (1 �g) was used for reverse transcription to generatecDNA, which was used as a template for the PCRs with gene-specific primers (Table 1), as previously described (30). Realtime PCR amplification was performed with iQ SYBR GreenSupermix (Bio-Rad) and the Eco real time PCR system (Illu-mina). Real time PCR was performed for 40 cycles of 95 °C for15 s and 60 °C for 1 min. Gene expression was normalized to thehousekeeping gene Hprt.

Pannexin 3 Promotes Osteoprogenitor Cell Cycle Exit

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Flow Cytometry Analysis—Stably transfected C2C12 cells(1.0 � 104 cells) were seeded in a 60-mm dish and cultured withor without BMP2 (300 ng/ml) for 2 days. For the Panx3 hemi-channel blocking experiment, the cells were cultured with thePanx3 antibody (1.5 �g/ml) for 2 days without BMP2. For theprimary calvarial cells, 8.0 � 104 transiently transfected cellswere cultured for 2 days with or without the Panx3 antibody(1.5 �g/ml). The cells were then collected by centrifugation at120 � g for 5 min. DNA content was analyzed by propidiumiodide staining (EMD Biosciences) with CellQuest software onFACSCalibur Station (Becton Dickinson).

Measurement of Intracellular cAMP—The cells were seededat 1.0 � 104 cells/well in a 96-well plate and cultured for 1day with either DMEM for the C2C12 cells or �-MEM forprimary calvarial cells. The cells were then incubated withmedia containing 0.1% albumin medium for 12 h, followedby incubation in media containing 10% serum for 1 h. Thelevel of cAMP was determined with a Bridge-It cAMPdesigner fluorescence assay kit (Mediomics) and measuredas previously described (30).

Western Blot Analysis—The cell lysates were prepared as pre-viously described (30). Ten �g of each protein was electro-phoresed in 4 –12% SDS-polyacrylamide gel (Invitrogen) andtransferred onto a polyvinylidene difluoride membrane usingiBlot (Invitrogen). The membranes were immunoblotted withantibodies.

Data Analysis—Each experiment was repeated several times,and the data were analyzed using Prism 5 software. Statisticaldifferences between two groups of data were analyzed with theStudent’s t test. One-way analysis of variance was used for cellproliferation assays with Wnt3a and Dkk1 (see Fig. 3A). p �0.05 was considered to be statistically significant.

RESULTS

Panx3 Inhibits Osteoprogenitor Cell Proliferation—Panx3 isinduced during the transition from proliferation to differentia-tion in osteoprogenitor cells and promotes osteoblast differen-tiation (23, 30); therefore, we hypothesized that Panx3 regulatesosteoprogenitor cell proliferation. We first examined theeffects of Panx3 overexpression on the proliferation of primarycalvarial cells from newborn mice. The neonatal calvarium con-tains progenitor cells for osteoblasts and adipocytes, of whichthe majority are osteoprogenitors (31, 36, 37). Transfection of aPanx3 expression vector (pEF1/Panx3) into primary calvarialcells cultured in proliferation media reduced proliferation com-pared with that of a control vector (pEF1) (Fig. 1A, panel a). Wealso tested the effect of Panx3 overexpression in multipotentC3H10T1/2 cells, which differentiate into osteoblasts withBMP2. Panx3 overexpression reduced the proliferation of

C3H10T1/2 cells cultured in proliferation media (Fig. 1A, panelb). C2C12 cells are osteogenic and myogenic, depending on theculture conditions. C2C12 cells differentiate into osteoblastswith BMP2 in FBS-containing media, whereas they differenti-ate into myoblasts in horse serum containing media. As withprimary calvarial cells and C3H10T1/2 cells, Panx3 overexpres-sion reduced proliferation of the C2C12 cells cultured in FBS-containing media (Fig. 1A, panel c).

We next analyzed the inhibitory activity of Panx3 for prolif-eration in neonatal mouse calvarial organ culture using arecombinant adenovirus system (Fig. 1B). The control adeno-virus infection (AdCont) showed staining of endogenousPanx3-expressing cells and Ki67-positive proliferating cells.The merged images revealed that these positive staining cellsdid not overlap (Fig. 1B, panels a– d). With a Panx3 adenovirus(AdPanx3) infection, the number of Panx3-expressing cellsincreased, whereas the number of Ki67-positive proliferatingcells decreased (Fig. 1B, panels e– h). The Panx3-positive cellsin either AdCont-infected (Fig. 1B, panels a– d) or AdPanx3-infected (Fig. 1B, panels e– h) calvaria did not overlap Ki67-positive cells, suggesting that the proliferation of Panx3-ex-pressing cells is inhibited. The quantification also showedreduced numbers of Ki67-positive cells with the AdPanx3infection (Fig. 1B, panel i). We next examined the effect of thesuppression of endogenous Panx3 expression by Panx3 shRNA(shPanx3) on the proliferation of C3H10T1/2 and C2C12 cellscultured in the presence of BMP2, which induces endogenousPanx3 expression. The transfection of C3H10T1/2 and C2C12cells with shPanx3 increased the proliferation of these cellscompared with that in the control shRNA transfection cells(Fig. 1C, panels a and b). These results indicate that Panx3inhibits osteoprogenitor proliferation.

Panx3 Promotes Cell Cycle Arrest at G0/G1 Phase—Weexamined the inhibitory activity of Panx3 in proliferation usingthe FACS analysis (Fig. 2). Calvarial cells overexpressing Panx3accumulated in the G0/G1 phase and were reduced in both theS and G2/M phases (Fig. 2A). We also found a similar cell cyclearrest at the G0/G1 phase in C2C12 cells overexpressing Panx3(Fig. 2B, panel a). Furthermore, shPanx3 transfected cells werereduced in the G0/G1 phases and increased in the G2/M phases(Fig. 2B, panel b). These results suggest that Panx3 expressionin both primary calvarial cells and C2C12 cells inhibits prolif-eration by arresting the cell cycle at the G0/G1 phase.

Panx3 Inhibits Wnt/�-Catenin Signaling—Because canoni-cal Wnt signaling promotes the proliferation of osteoprogeni-tor cells (17, 22, 38 – 41), Panx3 may block the Wnt/�-cateninpathway. To explore this possibility, we examined the effect ofWnt signaling on the proliferation of Panx3-overexpressingC2C12 cells (Fig. 3A). C2C12 cells produce canonical and non-canonical Wnts (data not shown) (42, 43). Without exogenousWnt3a, the proliferation of Panx3-overexpressing cells wasreduced when compared with that of the control cells similar tothat shown in Fig. 1A. The addition of Wnt3a increased prolif-eration of the control cells but did not promote proliferation ofthe Panx3-overexpressing cells, which suggests that Panx3inhibited Wnt signaling. Dkk-1, an antagonist of Wnt3a/LRP5receptor interactions, inhibited proliferation even in theabsence of exogenous Wnt3a. This occurred because the cells

TABLE 1Primer sequences for quantitative RT-PCR

Gene name Sequence

�-Catenin forward 5�-TTTTCACTCTGGTGGATACGGC-3��-Catenin reverse 5�-CCCATCAACTGGATAGTCAGCAC-3�E2F1 forward 5�-TTTGACTGTGACTTTGGGGACC-3�E2F1 reverse 5�-AATGAGGCAGGAAGGATGGC-3�p21 forward 5�-CCCTCTATTTTGGAGGGTTAATCT-3�p21 reverse 5�-GTACCCTGCATATACATTCCCTTC-3�




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produce endogenous Wnts (43). Dkk-1 blocked the effect ofWnts in a dose-dependent manner. However, even at the high-est dose (100 ng/ml) of Dkk-1, the inhibition level of the controlcell proliferation by Dkk-1 was lower than that of the Panx3-overexpressing cells with or without Dkk-1 (last and first pairsof bars in Fig. 3A). This suggests that Panx3 inhibits cell prolif-eration through not only Wnt signaling, but also otherpathways.

�-Catenin is involved in downstream canonical Wnt signal-ing (1, 44, 45). We therefore examined the expression and local-ization of the �-catenin protein in Panx3-overexpressingC2C12 cells and in control cells (Fig. 3B). In the control cells,�-catenin was localized in the plasma membrane and in thenucleus, whereas in Panx3-overexpressing cells, the proteinlevel and nuclear localization of �-catenin were reduced (Fig.3B, panels a–f). The addition of Wnt3a to the control cells

increased the �-catenin localization in the nucleus. In contrast,in Panx3-overexpressing cells, the nuclear localization of�-catenin was reduced (Fig. 3B, panels g–l). Quantitative anal-ysis confirmed the reduced nuclear localization level of�-catenin in Panx3-overexpressing cells, compared with that inthe control cells (Fig. 3B, panel m). Western blot analysisrevealed that Panx3 reduced the protein level of �-catenin,especially in both the cytoplasm and nucleus (Fig. 3C). Theseresults suggested that Panx3 inhibited canonical Wnt signalingby reducing �-catenin activity. Moreover, we analyzed �-cateninactivity using a TOPflash reporter vector (Fig. 3D) and found thatTOPflash reporter activity was reduced in the Panx3-overexpress-ing cells compared with that in the control cells (Fig. 3D, panel a).The reporter activity was strongly activated in the control cells bythe addition of Wnt3a, whereas it was inhibited in the Panx3-over-expressing cells (Fig. 3D, panel b).

FIGURE 1. Panx3 inhibits cell proliferation. A, panel a, proliferation of Panx3-overexpressing primary calvarial cells. pEF1 or pEF1/Panx3 transiently trans-fected calvarial cells were cultured in �-MEM media for 2 days. Panels b and c, proliferation of Panx3-overexpressing or shPanx3 transfected C3H10T1/2 andC2C12 cells. Panel b, pEF1 or pEF1/Panx3 transiently transfected C3H10T1/2 cells were cultured in DMEM/F-12 media for the indicated days. Panel c, C2C12 cellswere stably transfected with control vector (pEF1) or Panx3 expression vector (pEF1/Panx3). The transfected cells were cultured in undifferentiated media forthe indicated days. B, newborn mouse calvarial bones were cultured and infected with AdPanx3 or AdCont for 2 days and were then immunostained. Panels aand e, images under light microscopy. Panels b and f, Panx3 antibody (green). Panels c and g, Ki67 antibody (red) and Hoechst nuclear staining (blue). Panels dand h, merged image. Panel i, quantification of Ki67-positive cells. C, proliferation of shPanx3 transfected osteogenic cell line. Panel a, sh control or shPanx3transiently transfected C3H10T1/2 cells were cultured in osteogenic media. Panel b, sh control or shPanx3 stably transfected C2C12 cells were cultured withBMP2 (300 ng/ml). *, p � 0.05; **, p � 0.01. Error bars represent the means � S.D., n � 7.




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We also examined �-catenin activity in an ex vivo culture ofcalvarial bone from heterozygous Axin2LacZ mice containing anAxin2-lacZ knock-in allele, which is a target gene of �-catenin(33). Infection with AdPanx3 reduced the number of LacZ-pos-itive cells compared with that of infection with AdCont (Fig. 3E,panel a and b). The quantification showed a lower percentageof LacZ-positive cells in the AdPanx3-infected cells (Fig. 3E,panel c). These results indicate that Panx3 inhibits Wnt signal-ing by reducing �-catenin activity.

Panx3 Promotes �-Catenin Degradation by GSK3� Activation—To identify the mechanism of inhibition of �-catenin activity byPanx3, we first analyzed the mRNA and protein levels of�-catenin as well as its activation level in Panx3-overexpressingC2C12 cells. We found that �-catenin mRNA levels did notchange significantly between the control cells and Panx3-over-expressing cells (Fig. 4A). We also examined the effect of theinhibition of endogenous Panx3 expression on �-cateninmRNA expression by shPanx3 in C2C12 cells that had beentreated with BMP2 for a short time to induce endogenousPanx3 expression. The shPanx3 did not change the �-cateninmRNA expression level when compared with that of the con-trol shRNA (Fig. 4A).

Western blot analysis showed that, in contrast to the mRNAlevels, �-catenin protein levels were reduced in Panx3-overex-

pressing cells and were higher in shPanx3 transfected cells (Fig.4B, panel a). Furthermore, the phosphorylation of �-cateninwas promoted in Panx3-overexpressing cells, whereas its phos-phorylation levels were reduced by shPanx3 (Fig. 4B, panel a).We found that the phosphorylation of GSK3� was inhibited inPanx3-overexpressing cells, whereas shPanx3 produced theopposite effect (Fig. 4B, panel a). Additionally, we observedsimilar results in primary calvarial cells (Fig. 4B, panel b). Theseresults suggest that that Panx3 induces �-catenin degradationvia increasing GSK3� activity.

Panx3 Hemichannels Inhibit Osteoprogenitor Proliferation—Hemichannels link the cytoplasm with the extracellular spaceand regulate cellular signaling through the release of small mol-ecules such as ATP, and Panx3 functions as a hemichannel (23,30). Therefore, the Panx3 hemichannel may be involved in theinhibition of osteoprogenitor proliferation. To address thispossibility, we first examined whether I-peptide, which is aninhibitor specific to Panx3 hemichannel function (23, 30), abro-gates the Panx3-mediated inhibition of cell proliferation in anex vivo calvarial culture. The addition of I-peptide increased thenumber of Ki67-positive proliferating cells (Fig. 5A, panelse– h). We found that many Ki67-positive cells were also positivefor Panx3. This is because the I-peptide inhibited the Panx3hemichannel function but not the Panx3 expression andincreased the proliferation of Panx3-positive cells. A controlscramble peptide (S-peptide) containing the same amino acidcontents of the I-peptide did not affect the proliferation state(Fig. 5A, panels a– d). Under this condition, the Panx3- andKi67-positive cells did not overlap. The quantification con-firmed the increased number of Ki67-positive cells as a result ofthe I-peptide addition (Fig. 5A, panel i). These results suggestthat the Panx3 hemichannel plays an important role in the inhi-bition of osteoprogenitor proliferation.

We confirmed this Panx3 hemichannel function using thePanx3 antibody, which reacts with the extracellular domain ofPanx3 and inhibits the Panx3 hemichannel (23, 30). We showedthat the addition of the Panx3 antibody to the culture abrogatedthe inhibition of Panx3-overexpressing C2C12 cell prolifera-tion (Fig. 5B). Because intracellular ATP plays an importantrole in cell proliferation by regulating the intracellular cAMPlevels (46, 47) (see Fig. 8), we examined the intracellular cAMPlevels in C2C12 cells. Panx3 overexpression reduced the intra-cellular cAMP levels caused by the release of intracellular ATPvia the Panx3 hemichannel (Fig. 5C, panel a), which is consis-tent with our previous findings (23, 30). We showed that theanti-Panx3 antibody abrogated the reduced cAMP levels byinhibiting the Panx3 hemichannel function (Fig. 5, C, panel a,and D, panel a), whereas shPanx3 increased the cAMP level inC2C12 cells and in primary calvarial cells (Fig. 5, C, panel b, andD, panel b). These cAMP levels correlate with the proliferationstates of pEF1/Panx3 and Panx3 shRNA cells (Fig. 1), and ourresults suggest that Panx3 reduces cell proliferation in partthrough the ATP/cAMP pathway by the Panx3 hemichannel.

Panx3 Hemichannels Reduce PKA/CREB Signaling and�-Catenin Activity—Intracellular cAMP activates downstreamPKA/CREB signaling, which induces the expression of genesinvolved in the progression of cell proliferation (48). To furtherdelineate the Panx3 hemichannel pathway, which inhibits cell

FIGURE 2. Panx3 arrests cell cycle at G0/G1 phase. FACS analysis of cell cyclewas used. A, transiently transfected calvarial cells with pEF1 or pEF1/Panx3were cultured in �-MEM for 2 days. B, 1 day after seeding, stably transfectedC2C12 cells with pEF1, pEF1/Panx3, sh control, or shPanx3 vectors were incu-bated in serum-free 0.1% albumin containing DMEM for 12 h. pEF1- andPanx3-overexpressing cells were cultured in undifferentiated media for 2days (panel a), whereas sh control and shPanx3 transfected cells were cul-tured with BMP2 (300 ng/ml) for 2 days (panel b). The cells were stained withpropidium iodide, and cell cycle stages were measured by FACS analysis. Thepanels represent distribution of cells (%) in the G0/G1, S, and G2/M phases.*, p � 0.05; **, p � 0.01. Error bars represent means � S.D. of three independ-ent experiments.




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proliferation, we analyzed the downstream molecules of cAMPsignaling in either pEF1/Panx3 or shPanx3 transfected C2C12cells (Fig. 6A) and in primary calvarial cells (Fig. 6B). Panx3overexpression reduced the phosphorylation of PKA and CREB(Fig. 6, A, panel a, and B, panel a, left panels; A, panel b, and B,panel b, left panels), whereas the addition of the Panx3 antibodyto the Panx3-overexpressing C2C12 cells blocked the reductionin the PKA/CREB phosphorylation levels (Fig. 6A, panel a, mid-dle panel, and panel b, left panels). The inhibition of endoge-nous Panx3 expression by Panx3 shRNA in C2C12 and primarycalvarial cells increased the levels of both PKA and CREB phos-phorylation, compared with that of the control shRNA trans-fected cells (Fig. 6, A, panel a, and B, panel a, right panels; A,

panel b, and B, panel b, right panels). These results suggest thatPanx3 inhibits proliferation of both C2C12 and primary calvar-ial cells through the PKA/CREB signaling pathway.

Because GSK3� kinase activity is inhibited through the phos-phorylation of GSK3� by cAMP-dependent PKA (11, 12),Panx3-mediated �-catenin degradation may be regulatedthrough decreased cAMP/PKA signaling by the Panx3 hemi-channel, which promotes GSK3� kinase activity resulting in anincrease in �-catenin phosphorylation for the degradation. Weexamined �-catenin activity in an ex vivo calvarial culture usingAxinLacZ mice with I-peptide to inhibit the Panx3 hemichannel.We found that an addition of I-peptide increased the number ofthe LacZ-positive cells, indicating increased �-catenin activity

FIGURE 3. Panx3 inhibits Wnt/�-catenin signaling. A, proliferation of Panx3-overexpressing C2C12 cells with Wnt3a or Dkk1. pEF1 and pEF1/Panx3 trans-fected cells were cultured in DMEM with either Wnt3a (100 ng/ml) or several doses of Dkk1 for 2 days. B, panels a–l, cellular localization of �-catenin inPanx3-overexpressing cells with (panels g–l) or without (panels a–f) Wnt3a. Fluorescent confocal images showed Panx3 (red), �-catenin (green), and Hoechstnuclear staining (blue). Panel m, measurements show the percentage of �-catenin nuclear localization. C, �-catenin expression in cell membrane, cytoplasmic,and nuclear fractions. D, cells stably transfected with pEF1 and pEF1/Panx3 were co-transfected with either the TOPflash reporter construct or the FOPflashreporter construct as a negative control. The RLSV40 construct was co-transfected as an internal control. After transfection, cells were cultured in media with(panel b) or without (panel a) Wnt3a (100 ng/ml) for 1 day. The next day, the cell lysate was subjected to the dual luciferase reporter assay system. E, panels a andb, X-gal staining of ex vivo calvarial culture of Axin2LacZ mice infected with AdCont (panel a) or AdPanx3 (panel b). Panel c, quantification of LacZ-positive cells.Arrowheads show LacZ-positive cells. *, p � 0.05; **, p � 0.01. Error bars represent the means � S.D., n � 3. IB, immunoblot.




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in the ex vivo calvarial culture (Fig. 6C, panels a and b). Thequantification confirmed the increased number of LacZ-posi-tive cells by the addition of I-peptide (Fig. 6C, panel c). Thisresult indicates that the Panx3 hemichannel inhibits �-cateninsignaling through the cAMP/PKA pathway.

Panx3 Inhibits the Activity of Cell Cycle Regulators—We fur-ther analyzed the activity of cell cycle regulators downstream ofthe PKA/CREB pathway (Fig. 7). PKA/CREB signaling inducesthe expression of E2F1, cyclin D1, and CDK4, which promotecell cycle progression (49). The cyclin D1-CDK4 complexinduces the phosphorylation of Rb, which dissociates Rb fromthe Rb-E2F1 complex. Free E2F1 induces gene expression forcell cycle progression (50). Wnt/�-catenin signaling activatesgenes for cell cycle molecules, such as c-Myc and cyclin D1, andpromotes cell growth (8, 22). In both the Panx3-overexpressingprimary calvarial cells and C2C12 cells, cyclin D1 protein levelsand Rb phosphorylation levels were reduced, whereas Panx3shRNA cells showed the opposite effects (Fig. 7A, panels a andb). Panx3 overexpression also reduced E2F1 mRNA levels (Fig.7A, panel c). These results suggest that Panx3 promotes cellcycle arrest at the G0/G1 phase by inhibiting the activity of thecell cycle molecules involved in cell cycle progression from theG1 to the S phase.

Panx3 ER Ca2� Channel Promotes the Smad1/5 and p21Pathway for Cell Cycle Exit—The Panx3 ER Ca2� channel isactivated by PI3K/Akt signaling and increases intracellularCa2� levels, ([Ca2�]i), following the activation of calmodulin(CaM) signaling (23). In these processes, the external ATPreleased from the cells through the Panx3 hemichannel binds topurinergic P2 receptors, which activate PI3K/Akt signaling. It isknown that the CaM pathway activates Smad1/5 signaling,which regulates p21, a cell cycle inhibitor (51, 52). Therefore,we examined whether Panx3 promotes p21 activation via Smadsignaling in primary calvarial cells and in C2C12 cells. ThePanx3 overexpression resulted in the activation of Smad1/5 andp21 (Fig. 7B, panels a– c). The activation of these proteins wasblocked by PPADS, an inhibitor of the P2 receptors, and by thePanx3 antibody, which inhibited the Panx3 hemichannel (Fig.7B, panels b and c). Panx3 overexpression also promoted theexpression of p21 mRNA (Fig. 7B, panel d) and protein levels(Fig. 7B, panels a– c). Because the Panx3 ER Ca2� channel pro-motes [Ca2�]i by Akt signaling, we examined the effects of Aktactivation on the phosphorylation levels of the Smad1/5 andp21 pathway by transfecting the Akt constitutive active (Akt-CA) and dominant negative (Akt-DN) vectors to Panx3-over-expressing C2C12 cells. The Akt-CA promoted increased phos-phorylation of the Smad1/5 and p21 over that of the mockvector transfection. In contrast, Akt-DN inhibited the activa-tion of the Smad1/5 and p21 by Panx3 overexpression (Fig. 7C).These results suggest that Panx3 promotes the cell cycle exit bythe activation of Smad1/5 and p21 through the Panx3 functionas an ER Ca2� channel.

DISCUSSION

In this study, we demonstrate that Panx3 plays an importantrole in the transition from proliferation to differentiation inosteoprogenitor cells. We found that Panx3 inhibits osteopro-genitor proliferation and promotes the cell cycle exit. Fig. 8presents a schematic diagram of the mechanism of Panx3actions that negatively regulate the proliferation state of osteo-progenitor cells. At the proliferation stage, canonical Wnt sig-naling stabilizes and translocates �-catenin to the nucleus,where it activates genes for cell cycle progression (Fig. 8A).When Panx3 is induced by BMP at the early transition stage,Panx3 functions as a hemichannel to release intracellular ATPinto the extracellular space. This Panx3 hemichannel activityinduces all subsequent Panx3 signaling pathways involved inthe inhibition of proliferation. It reduces cAMP/PKA signaling,resulting in �-catenin degradation via GSK3� activation, whichleads to a reduction in the �-catenin/TCF/Lef-dependent tran-scription of the genes necessary for proliferation. The Panx3hemichannel also reduces activity of CREB, a DNA-bindingprotein factor downstream of cAMP/PKA, resulting in thereduced transcription of the CREB target genes required for cellcycle progression. In addition, the binding of ATP releasedfrom the Panx3 hemichannel to ATP receptors activates thePanx3 ER Ca2�channel/CaM/CaM kinase/Smad1/5 pathwayvia PI3K/Akt signaling, following an increase in the transcrip-tion and activation of p21. All of these signaling pathways areabrogated by the inhibition of the Panx3 hemichannel. Thus,

FIGURE 4. Panx3 promotes �-catenin degradation and GSK3� activity. A,quantitative RT-PCR for �-catenin mRNA expression in C2C12 cells stablytransfected with either pEF1, pEF1/Panx3, sh control, or shPanx3. pEF1 andpEF1/Panx3 transfected cells were cultured in DMEM for 1 day, whereas shcontrol and shPanx3 transfected cells were cultured with BMP2 (300 ng/ml) inDMEM for 1 day. B, phosphorylation of �-catenin and GSK3�. Control andPanx3 overexpressed cells were cultured for 1 day in DMEM containing 10%FBS, whereas sh control and shPanx3 transfected cells were cultured withBMP2 for 1 day. Lysates were analyzed by Western blotting with antibodies tophospho-�-catenin (P-�-catenin), �-catenin, phospho-GSK3� (P-GSK3�),GSK3�, and �-tubulin (panel a). Calvarial cells transfected with pEF1 or pEF1/Panx3 were cultured in �-MEM for 2 days (panel b). Error bars represent themeans � S.D., n � 3.




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the Panx3 hemichannel is the most critical first step to switchproliferation to the cell cycle exit.

Wnt signaling plays an important role in many aspects oftissue development, including osteogenesis (45). In bone for-mation, Wnt signaling promotes osteoprogenitor cell prolifer-ation and mineralization while inhibiting osteoclastogenesis(40, 53). Wnt3a enhances the cell proliferation of the osteoblas-tic cell lines C2C12, MC3T3-E1, and C3H10T1/2 (39, 54, 55).Wnt1, 2, and 3a also induce alkaline phosphatase activity inosteogenic cells (55). Thus, Wnt signaling regulates osteopro-genitor cell proliferation and differentiation. However, themechanisms of Wnt signaling regulation in the cell prolifera-tion stage and in the late differentiation stage remain unclear.BMPs and Wnt signaling interactions regulate cell proliferation

and differentiation during development (56). BMPs antagonizethe Wnt-mediated proliferation of osteoprogenitor cells andpromote osteoblast differentiation by inducing Runx2 (57, 58).Subsequently, Runx2 induces Osx that inhibits Wnt signalingby inducing Dkk1 (22). In this paper, our data show a differentpathway for Wnt signaling inhibition by Panx3. We previouslydemonstrated that BMP2 induces Panx3 during osteoblast dif-ferentiation (23). The suppression and overexpression of Panx3inhibits and promotes the expression of Osx and other osteo-blast markers, respectively (23). These results indicate thatPanx3 is an upstream molecule of Osx. Thus, Panx3 inhibitsWnt signaling first, followed by Osx-mediated inhibition dur-ing osteoblast differentiation to ensure the differentiationprocess.

FIGURE 5. Panx3 reduces intracellular cAMP levels and the PKA/CREB signaling pathway. A, calvarial cultures were incubated with either I-peptide orS-peptide for 2 days followed by immunostaining. Panels a and e, image under light microscopy. Panels b and f, Panx3 antibody (green). Panels e and g, Ki67antibody (red) and Hoechst nuclear staining (blue). Panels d and h, merged image. Panel i, quantification of Ki67-positive cells. B, cell proliferation in the presenceof Panx3 antibody. pEF1 and pEF1/Panx3 transfected C2C12 cells were cultured with anti-Panx3 antibody for 2 days. C, the intracellular cAMP level. Panel a,pEF1 and pEF1/Panx3 transfected cells were incubated in serum-free DMEM with 0.1% albumin for 8 h and then incubated in 10% FBS-containing DMEM withor without Panx3 antibody for 1 day. Panel b, sh control and shPanx3 transfected cells were cultured with BMP2 (300 ng/ml) for 2 days. D, intracellular cAMPlevels in Panx3-overexpressing or shPanx3 transfected calvarial cells. Panel a, At 1 day after transfection, the cells were cultured in 10% FBS-containing �-MEMwith or without Panx3 antibody (1.5 �g/ml) for 1 day. Panel b, at 1 day after transfection, the cells were induced in 5% FBS-containing �-MEM with BMP2 (300ng/ml) for 1 day. *, p � 0.05; **, p � 0.01. NS, nonsignificant. Error bars represent the means � S.D., n � 3.




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[Ca2�]i plays a critical role in cell proliferation and differentia-tion (59). The ER serves as the major Ca2� storage space in the cell.Panx3 functions as an ER Ca2� channel, which promotes theosteoprogenitor cell cycle exit (Fig. 7). CaM is the major trans-ducer of Ca2� signaling in many cell types. In osteoblasts, uponbinding to Ca2�, CaM interacts with and activates protein factorssuch as calmodulin kinase II and calcineurin and induces differen-tiation (60–62). We found that Panx3 stimulated the phosphoryl-ation of Smad1/5 and increased both the protein levels and phos-phorylation levels of p21 (Fig. 7B). Our results indicate that the

Panx3 ER Ca2� channel regulates the CaM/Smads/p21 signalingpathway (Fig. 8). The anti-Panx3 antibody and PPADS inhibitedthe Panx3-promoted Smad1/5 activation and p21 expression, sug-gesting that the Panx3 hemichannels and P2 receptors areinvolved in these processes. The increase in p21 contributes to thePanx3-promoted cell cycle exit (Fig. 2). These results suggest thatPanx3-promoted Ca2� signaling activates Smad/p21 signaling,which promotes the cell cycle exit.

IP3 receptors (IP3Rs), which consist of three members(IP3R1, 2, and 3), are ubiquitous ER Ca2� channels, and they

FIGURE 6. Panx3 hemichannels reduce PKA/CREB signaling and �-catenin activity. A, pEF1 and pEF1/Panx3 transfected C2C12 cells were cultured for 1 dayin DMEM containing 10% FBS with or without Panx3 antibody, whereas stable sh control and shPanx3 transfected cells were cultured with BMP2 for 1 day. Panela, Western blotting was performed with antibodies to Panx3, phospho-PKA (P-PKA), PKA, phospho-CREB (P-CREB), CREB, and �-tubulin. Panel b, quantificationof the ratios of P-PKA/PKA (upper panel) and P-CREB/CREB (lower panel). B, panel a, pEF1 and pEF1/Panx3 transfected calvarial cells were cultured in �-MEM for2 days, whereas sh control and shPanx3 transfected cells were cultured with BMP2 (300 ng/ml) for 2 days. Panel b, quantification of the ratios of P-PKA/PKA (leftpanel) and P-CREB/CREB (right panel). C, panels a and b, Axin2LacZ ex vivo calvarial cultures with either S-peptide (panel a) or I-peptide (panel b) were stained forX-gal. Panel c, quantification of LacZ-positive cells. **, p � 0.01. NS, nonsignificant. Error bars represent the means � S.D., n � 3.




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release Ca2� from the ER upon its binding to IP3 (63). Threetypes of IP3Rs are expressed in osteogenic C2C12 cells (data notshown). The expression levels of IP3Rs did not change duringthe osteoblast differentiation (data not shown). Ryanodine

receptors, which are another ER Ca2� channel, are notexpressed in C2C12 cells and are not induced during osteoblastdifferentiation (23). We previously showed that PPADSstrongly blocks the Panx3 ER Ca2� channel, but not IP3Rs, in

FIGURE 7. Panx3 reduces cell cycle signaling pathways and increases p21 activation. A, Western blotting with antibodies to Panx3, cyclin D1, phospho-Rb(P-Rb), Rb, and �-tubulin. Panels a and b, pEF1 and pEF1/Panx3 transfected cells were cultured for 2 days in �-MEM for primary calvarial cells (panel a) or for 1day in DMEM for C2C12 cells (panel b). sh control and shPanx3 transfected cells were cultured with BMP2 (300 ng/ml) for 2 days for calvarial cells (panel a) or 1day for C2C12 cells (panel b). pEF1 and pEF1/Panx3 transfected C2C12 cells were cultured in DMEM for 2 days. sh control and shPanx3 transfected cells werecultured with BMP2 (300 ng/ml) for 2 days. Panel c, E2F1 mRNA levels were then observed by quantitative PCR. B, Panels a– c, phosphorylation of Smad1/5 andp21 cultured in �-MEM for pEF1 and pEF1/Panx3 transfected calvarial cells (panel a) and DMEM with PPADS or anti-Panx3 antibody in pEF1 and pEF1/Panx3transfected C2C12 cells (panels b and c). Panel c, quantification of protein levels for P-Smad1/5 (upper panel) and P-p21 (lower panel). Panel d, quantitative PCRfor p21 mRNA levels on the same experimental condition as E2F1. C, Panx3-promoted Smad1/5 and p21 activation via Akt signaling pathways. Panx3overexpressed C2C12 cells transfected with Akt CA or Akt DN or mock vector were cultured in DMEM for 1 day. **, p � 0.01. Error bars represent the means �S.D., n � 3.




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C2C12 cells. The reduction of Smad1/5 and p21 activity byPPADS indicates that the Panx3 ER Ca2� channel primarilyregulates p21 activation to inhibit osteoprogenitor cell prolifer-ation (Fig. 7B). Mice lacking either IP3R2 or IP3R3 are viableand show no obvious abnormalities in skeletal development.Mice lacking both IP3R2 and IP3R3 are born with a normalappearance but begin losing body weight after weaning becauseof defects in exocrine secretion (64). Thus, the Panx3 ER Ca2�

channel likely plays a major role in osteogenic proliferation aswell as in osteogenic differentiation.

In summary, we have shown that Panx3 inhibits osteopro-genitor proliferation through the multiple signaling pathwaysinduced by the Panx3 hemichannel. Our results reveal thatPanx3 promotes the switch from osteoprogenitor proliferationto osteoblast differentiation.

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FIGURE 8. Panx3 signaling pathways inhibit osteoprogenitor cell proliferation and promote cell cycle exit. A, Wnt signaling in the proliferation stage ofosteoprogenitor cells. Wnt binding to a frizzled receptor and its co-receptors LRP5/6 stabilizes the �-catenin protein. The inactivation of GSK-3� by phosphor-ylation through PKA is involved in this �-catenin stabilization process. The stable �-catenin is translocated to the nucleus and activates gene expressionnecessary for cell proliferation. PKA also activates CREB by phosphorylation, which induces expression of cell cycle genes for proliferation. B, Panx3 signalingin the transition from proliferation to differentiation of osteoprogenitor cells. Panx3 is induced by BMP and functions as a hemichannel. The Panx3 hemichannelreleases intracellular ATP, resulting in a decrease in cAMP/PKA signaling, which in turn reduces proliferation. The reduced PKA activity promotes GSK3�activation, which phosphorylates �-catenin for its degradation, leading to the inhibition of cell growth. An increase in the inactive CREB by the reduced PKAactivity results in reduced proliferation. In addition, ATP released from the Panx3 hemichannel binds to ATP receptors (P2Rs) in its own cell and/or inneighboring cells and activates PI3K and Akt signaling. Akt activation promotes the Panx3 ER Ca2� channel to increase [Ca2�]i levels, which leads to activationof the CaM/calmodulin kinase (CaMK) pathway. The Panx3-mediated CaM kinase activation promotes Smad/p21 signaling for cell cycle exit. Dotted arrows inthe ATP/cAMP/PKA pathways indicate reduced signaling by the Panx3 hemichannel. Red crossed bars indicate that the reduced PKA activity increases the activeform of GSK3� and inactive form of CREB.




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Masaki Ishikawa, Tsutomu Iwamoto, Satoshi Fukumoto and Yoshihiko Yamadap21 Signaling

Pannexin 3 Inhibits Proliferation of Osteoprogenitor Cells by Regulating Wnt and

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