CCN3 (NOV) Interacts with Connexin43 in C6 Glioma Cells
2004; Elsevier BV; Volume: 279; Issue: 35 Linguagem: Inglês
10.1074/jbc.m403952200
ISSN1083-351X
AutoresChristine Fu, John F. Bechberger, Mark A. Ozog, Bernard Perbal, Christian C. Naus,
Tópico(s)Biomarkers in Disease Mechanisms
ResumoMany tumor cells exhibit aberrant gap junctional intercellular communication, which can be restored by transfection with connexin genes. We have previously discovered that overexpression of connexin43 (Cx43) in C6 glioma cells not only reduces proliferation but also leads to production of soluble growth-inhibitory factors. We identified that several members of the CCN (Cyr61/connective tissue growth factor/nephroblastoma-overexpressed) family are up-regulated following Cx43 expression, including CCN3 (NOV). We now report evidence for an association between CCN3 and Cx43. Western blot analysis demonstrated that the 48-kDa full-length CCN3 protein was present in the lysate and conditioned medium of growth-suppressed C6-Cx43 cells, as well as primary astrocytes, but not in C6 parental and human glioma cells. Immunocytochemical examination of CCN3 revealed diffuse localization in parental C6 cells, whereas transfection of C6 cells with Cx43 (C6-Cx43) or with a modified Cx43 tagged to green fluorescent protein on its C terminus (Cx43-GFP) resulted in punctate staining, suggesting that CCN3 co-localizes with Cx43 in plaques at the plasma membrane. In cells expressing a C-terminal truncation of Cx43 (Cx43Δ244-382), this co-localization was lost. Glutathione S-transferase pull-down assay and co-immunoprecipitation demonstrated that CCN3 was able to physically interact with Cx43. In contrast, CCN3 was not found to associate with Cx43Δ244-382. Similar experiments revealed that CCN3 did not co-localize or associate with other connexins, including Cx40 or Cx32. Taken together, these data support an interaction of CCN3 with the C terminus of Cx43, which could play an important role in mediating growth control induced by specific gap junction proteins. Many tumor cells exhibit aberrant gap junctional intercellular communication, which can be restored by transfection with connexin genes. We have previously discovered that overexpression of connexin43 (Cx43) in C6 glioma cells not only reduces proliferation but also leads to production of soluble growth-inhibitory factors. We identified that several members of the CCN (Cyr61/connective tissue growth factor/nephroblastoma-overexpressed) family are up-regulated following Cx43 expression, including CCN3 (NOV). We now report evidence for an association between CCN3 and Cx43. Western blot analysis demonstrated that the 48-kDa full-length CCN3 protein was present in the lysate and conditioned medium of growth-suppressed C6-Cx43 cells, as well as primary astrocytes, but not in C6 parental and human glioma cells. Immunocytochemical examination of CCN3 revealed diffuse localization in parental C6 cells, whereas transfection of C6 cells with Cx43 (C6-Cx43) or with a modified Cx43 tagged to green fluorescent protein on its C terminus (Cx43-GFP) resulted in punctate staining, suggesting that CCN3 co-localizes with Cx43 in plaques at the plasma membrane. In cells expressing a C-terminal truncation of Cx43 (Cx43Δ244-382), this co-localization was lost. Glutathione S-transferase pull-down assay and co-immunoprecipitation demonstrated that CCN3 was able to physically interact with Cx43. In contrast, CCN3 was not found to associate with Cx43Δ244-382. Similar experiments revealed that CCN3 did not co-localize or associate with other connexins, including Cx40 or Cx32. Taken together, these data support an interaction of CCN3 with the C terminus of Cx43, which could play an important role in mediating growth control induced by specific gap junction proteins. Gap junctions have long been thought to play an integral part in cellular growth control, both in normal and tumor cells. Loewenstein and Kanno (1Loewenstein W.R. Kanno Y. Nature. 1966; 209: 1248-1249Crossref PubMed Scopus (324) Google Scholar) were first to observe a decrease in gap junctional intercellular communication (GJIC) 1The abbreviations used are: GJIC, gap junctional intercellular communication; GST, glutathione S-transferase; PBS, phosphate-buffered saline; CIP, calf intestinal phosphatase.1The abbreviations used are: GJIC, gap junctional intercellular communication; GST, glutathione S-transferase; PBS, phosphate-buffered saline; CIP, calf intestinal phosphatase. in tumor cells when compared with their nontumorigenic counterpart cells. Since then, many tissues and cell lines have been analyzed in which the hypothesis of GJIC down-regulation in tumor tissues is upheld (2Yamasaki H. Naus C.C. 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In contrast, other connexins fail to exhibit this growth-suppressive phenotype (2Yamasaki H. Naus C.C. Carcinogenesis. 1996; 17: 1199-1213Crossref PubMed Scopus (451) Google Scholar).Functional gap junctions may not be completely necessary for growth-regulating effects, but simply the expression of connexin genes may be sufficient. Supporting evidence shows that medium conditioned by C6 cells transfected with Cx43 is capable of suppressing the growth rate of the control tumor cells (18Zhu D.G. Kidder G.M. Caveney S. Naus C.C.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10218-10221Crossref PubMed Scopus (127) Google Scholar). Goldberg et al. (17Goldberg G.S. Bechberger J.F. Tajima Y. Merritt M. Omori Y. Gawinowicz M.A. Narayanan R. Tan Y. Sanai Y. Yamasaki H. Naus C.C. Tsuda H. Nicholson B.J. Cancer Res. 2000; 60: 6018-6026PubMed Google Scholar) suggested that cells transfected with connexin genes may secrete soluble growth-inhibitory factors into the surrounding environment in order to potentiate any effects on growth of the neighboring cells. Recently, several reports have described that Cx43 plays a role in suppressing tumor growth independent of gap junction formation (19Moorby C. Patel M. Exp. Cell Res. 2001; 271: 238-248Crossref PubMed Scopus (198) Google Scholar, 20Zhang Y.W. Kaneda M. Morita I. J. Biol. Chem. 2003; 278: 44852-44856Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 21Zhang Y.W. Nakayama K. Nakayama K. Morita I. Cancer Res. 2003; 63: 1623-1630PubMed Google Scholar, 22Huang R.P. Fan Y. Hossain M.Z. Peng A. Zeng Z.L. Boynton A.L. Cancer Res. 1998; 58: 5089-5096PubMed Google Scholar). The C-terminal domain of Cx43 seems to be important in this tumor-inhibiting effect (20Zhang Y.W. Kaneda M. Morita I. J. Biol. Chem. 2003; 278: 44852-44856Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Cx43 may also regulate the cell cycle by acting on the S-phase kinase-associated protein (21Zhang Y.W. Nakayama K. Nakayama K. Morita I. Cancer Res. 2003; 63: 1623-1630PubMed Google Scholar).To date, the mechanism by which connexins may suppress growth of tumor cells is unknown. Connexins may elicit growth-altering effects by influencing the expression of other downstream genes. In the pursuit of genes possibly regulated by connexins, we identified members of the CCN (Cyr61/connective tissue growth factor/nephroblastoma-overexpressed) family (23Naus C.C. Bond S.L. Bechberger J.F. Rushlow W. Brain Res. Brain Res. Rev. 2000; 32: 259-266Crossref PubMed Scopus (53) Google Scholar). These genes are involved in numerous cellular processes including cell proliferation, differentiation, and development (reviewed in Ref. 24Perbal B. Lancet. 2004; 363: 62-64Abstract Full Text Full Text PDF PubMed Scopus (600) Google Scholar). They have also been shown to play a role in tumorigenesis and growth control. One of these, CCN3 (NOV), showed substantial increase in expression in C6 glioma cells following transfection with Cx43, and immunocytochemistry indicated co-localization at gap junction plaques (25McLeod T.L. Bechberger J.F. Naus C.C. Cell Commun. Adhes. 2001; 8: 441-445Crossref PubMed Scopus (14) Google Scholar). Here we show that Cx43 interacts with CCN3, as demonstrated by glutathione S-transferase (GST) pull-down and co-immunoprecipitation. Furthermore, this association is lost when the C terminus of Cx43 is deleted. It is also connexin-specific, since both Cx40 and Cx32 were not found to associate with CCN3. In addition, CCN3 is secreted from C6 cells transfected with Cx43. These findings have implications for understanding the tumor-suppressive role of connexins.EXPERIMENTAL PROCEDURESCell Culture and Bacterial Strains—C6 (ATCC, Manassas, VA) and C6 cells transfected with Cx43 (C6-Cx43) (13Zhu D. Caveney S. Kidder G.M. Naus C.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1883-1887Crossref PubMed Scopus (304) Google Scholar) were maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal calf serum (Sigma), 10 units/ml penicillin, and 10 μg/ml streptomycin (Invitrogen). Primary astrocyte cultures were prepared from wild type and Cx43 null mice, and human glioma cell lines (CRL-1718, SF188, SF539, U251, U87, and XF498) (26Naus C.C.G. Bechberger J.F. Bond S.L. Spray D.C. Dermietzel R. Gap Junctions in the Nervous System. R. G. Landes Co., Georgetown, TX1996Google Scholar) were cultured as previously described. Cells were incubated at 37 °C with 100% humidity and 5% CO2. Cloning and amplification of various plasmids were performed in Escherichia coli JM109. The protease-deficient E. coli strain BL21 was used in the expression of GST and GST fusion protein.Antibodies—An antibody was raised in rabbit against the C-terminal 21 amino acid sequence of human CCN3 (NH2-CPKNNEAFLQELELKTTRGKM-COOH) (Genemed, San Franciso, CA). This rabbit antiserum was purified with the ImmunoPure Protein A kit (Pierce). To confirm specificity, purified CCN3 IgG was preabsorbed with a 50-fold excess of peptide in phosphate-buffered saline (PBS) overnight at 4 °C prior to Western blotting. Several additional commercially available antibodies were used according to recommended dilutions, including Cx43 (rabbit (Sigma) and mouse (Chemicon, Temecula, CA)), Cx32 (rabbit (Zymed Laboratories Inc.) and mouse (Chemicon)), and GFP (mouse (Chemicon)). Horseradish peroxidase-labeled secondary antibodies were used at a 1:10,000 dilution (Cedarlane, Hornby, Canada), whereas fluorescence-labeled secondary antibodies were diluted 1:500 (Molecular Probes, Inc., Eugene, OR).Retroviral Infection of C6 Cells—The retroviral vector AP2-Cx40GFP was generated and used as previously described for AP2-Cx43GFP (27Mao A.J. Bechberger J. Lidington D. Galipeau J. Laird D.W. Naus C.C. J. Biol. Chem. 2000; 275: 34407-34414Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The AP2-Cx43Δ244-382-GFP vector was generously provided by Dr. Dale Laird (University of Western Ontario, London, Canada). Delivery of genes was performed by exposing 60% confluent cultures of C6 cells to viral medium for 24 h.GST Pull-down Assay—A fusion protein of GST and full-length human CCN3 was produced in E. coli BL21 and solubilized with 1% SDS using a protocol modified from Ref. 28Huang Y. Vanscheeuwijck P. Regan J.W. BioTechniques. 1993; 15: 989-992PubMed Google Scholar. The fusion protein was purified with glutathione-Sepharose beads (Amersham Biosciences) and incubated with cell lysates for 2.5 h at 4 °C. The beads were subsequently washed in PBS, and isolated protein was subjected to SDS-PAGE followed by immunoblot analysis.Immunocytochemistry—Cells grown on coverslips were fixed in 80% methanol at -20 °C for 10 min followed by rehydration in PBS (pH 7.4). Coverslips were blocked in 10% normal goat serum and 1% bovine serum albumin in PBS for 30 min and incubated with primary antibody for 1 h. After three washes in PBS, secondary antibody was applied for 1 h, followed by further washes. 4′,6-Diamidino-2-phenylindole was used to stain nuclei where indicated. Coverslips were mounted onto glass slides using Vectashield fluorescent mounting medium (Vector Laboratories, Burlingame, CA) and viewed under a fluorescent microscope (Axioskop 2; Zeiss).Protein Isolation and Immunoprecipitation—Cells on a confluent 100-mm plate were lysed in 800 μl of radioimmune precipitation lysis buffer (0.1% SDS, 1% IGEPAL, 0.5% Sarkosyl, 50 mm Tris-HCl (pH 8.0), 150 mm NaCl) supplemented with MiniComplete protease inhibitors (Roche Applied Science) and phosphatase inhibitors (Sigma). Protein concentration was determined using the BCA protein quantification kit (Pierce). Following preclearing with 10 μl of preimmune serum and 10 μl of protein A-Sepharose (Sigma) at 4 °C for 30 min, cell lysates were incubated with 10 μl of antiserum for 1 h and 20 μl of protein A beads for 2 h at 4 °C with gentle agitation. Beads were washed three times in radioimmune precipitation buffer and boiled in SDS sample buffer to release bound proteins. For alkaline phosphatase treatment, half of the lysate collected from a 100-mm plate was adjusted to pH 7.5 and incubated with 4 μl of calf intestinal phosphatase (CIP; Sigma) at 30 °C for 15 min. CIP-treated lysate was compared with the untreated half by Western analysis.Precipitation of Protein from Conditioned Culture Medium—Conditioned media were collected following culturing of C6 and C6-Cx43 cells in serum-free media for 24 h. Methanol precipitation (29Wessel D. Flugge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3127) Google Scholar) was performed on 200 μl of each sample. Briefly, 3 volumes of methanol, 1 volume of chloroform, and 4 volumes of H2O were added, mixed well, and centrifuged at maximum speed for 1 min. Upper aqueous phase was removed without disturbing the protein at the interphase. Subsequently, 8 volumes of methanol were added and vortexed to mix. Following precipitation at -20 °C for 30 min, samples were centrifuged at maximum speed for 15 min to pellet the protein. Proteins were then air-dried, resuspended in radioimmune precipitation buffer, and quantitated using the BCA kit (Pierce).Western Blotting—Protein samples were boiled for 5 min in SDS sample buffer and separated on a 10% polyacrylamide gel. Subsequently, proteins were transferred to polyvinylidene difluoride membrane (Amersham Biosciences) at 100 V for 1 h. Following incubation with primary antibody at 4 °C overnight and horseradish peroxidase-linked secondary antibody for 1 h at room temperature, detection was achieved with SuperSignal chemiluminescent substrate (Pierce) on x-ray film.RESULTSA 48-kDa Isoform of CCN3 Appears in C6 Cells Transfected with Cx43—Lysates of C6 and transfected cells were analyzed for CCN3 expression with Western blotting. The appearance of a 48-kDa isoform of CCN3 was readily apparent in C6-Cx43 cells, whereas it was absent in parental C6 cells (Fig. 1A, solid arrow). In C6 cells virally infected with Cx43 carrying a C-terminal GFP tag, this 48-kDa CCN3 was also observed, although at a lower level compared with C6-Cx43 cells. On the contrary, C6 cells infected with a retroviral construct of Cx43 deleted on the C terminus at amino acid 244 and fused C-terminally with GFP (Cx43Δ244-382-GFP) was also examined and found to express no 48-kDa CCN3 isoform (Fig. 1A).In addition, a 27-kDa band was abundant in all cell lines examined (Fig. 1A, open arrow). The level of this truncated CCN3 isoform rose in C6-Cx43 cells, whereas no noticeable up-regulation could be observed in other tranfectant cells. Both the 48- and 27-kDa bands disappeared following preabsorption of CCN3 IgG with specific immunogen peptide used to raise the antibody (Fig. 1B, arrows). Other bands that were present in all samples represented nonspecific cross-reactivity of the polyclonal antibody, since these were still observed following preabsorption. The size of CCN3 isoforms detected here corresponds with those identified in previous studies (30Su B.Y. Cai W.Q. Zhang C.G. Martinez V. Lombet A. Perbal B. Mol. Pathol. 2001; 54: 184-191Crossref PubMed Scopus (52) Google Scholar, 31Ellis P.D. Chen Q. Barker P.J. Metcalfe J.C. Kemp P.R. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1912-1919Crossref PubMed Scopus (55) Google Scholar, 32Martinerie C. Gicquel C. Louvel A. Laurent M. Schofield P.N. Le Bouc Y. J. Clin. Endocrinol. Metab. 2001; 86: 3929-3940Crossref PubMed Scopus (38) Google Scholar).CCN3 Is Present in Medium Conditioned by C6-Cx43 but Not C6 Cells—Conditioned medium collected from C6 and C6-Cx43 cells was concentrated by methanol precipitation and subjected to Western blot analysis. Three bands were detected in medium conditioned by C6-Cx43 that were absent in the C6 sample (Fig. 2A, solid arrow). Better separation on SDS-PAGE resolved their molecular masses to be 56, 51, and 46 kDa, respectively (Fig. 2B). The 51-kDa protein probably corresponds to the 52-kDa isoform of CCN3 observed in serum (33Thibout H. Martinerie C. Creminon C. Godeau F. Boudou P. Le Bouc Y. Laurent M. J. Clin. Endocrinol. Metab. 2003; 88: 327-336Crossref PubMed Scopus (31) Google Scholar). The 46-kDa isoform has been found in various samples including rat brain extract and vascular endothelial cells (30Su B.Y. Cai W.Q. Zhang C.G. Martinez V. Lombet A. Perbal B. Mol. Pathol. 2001; 54: 184-191Crossref PubMed Scopus (52) Google Scholar, 31Ellis P.D. Chen Q. Barker P.J. Metcalfe J.C. Kemp P.R. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1912-1919Crossref PubMed Scopus (55) Google Scholar). The increase in molecular mass of CCN3 in conditioned medium is not unexpected. Chevalier et al. (34Chevalier G. Yeger H. Martinerie C. Laurent M. Alami J. Schofield P.N. Perbal B. Am. J. Pathol. 1998; 152: 1563-1575PubMed Google Scholar) previously noticed an increase of ∼4 kDa in apparent molecular mass as CCN3 was secreted from the cell, suggesting progressive N-glycosylation. In addition, a 27-kDa isoform was present in both conditioned medium samples at a similar level (Fig. 2A, open arrow). Following peptide neutralization, all four CCN3 bands were removed, confirming their specificity (Fig. 2A).Fig. 2Analysis of CCN3 protein secreted into the conditioned medium of C6 and C6-Cx43 cells. A, equal amounts of protein (13 μg) precipitated from conditioned media were analyzed by Western blotting. Four bands of 56, 51, 46, and 27 kDa were present in medium conditioned by C6-Cx43. No slowly migrating CCN3 proteins were detected in the conditioned medium of C6 cells except for the 27-kDa isoform (open arrow). To permit better resolution of the three high molecular weight bands that are similar in size (solid arrow), proteins were separated on an 8% polyacrylamide gel for a longer period of time in B. Only the upper portion of the Western blot is shown here.View Large Image Figure ViewerDownload (PPT)CCN3 Co-localizes with Cx43—Immunocytochemistry was used to determine the subcellular localization of CCN3 in C6 and C6-Cx43 cells. Previous reports have demonstrated that CCN3 is in general diffusely localized within various cell types (34Chevalier G. Yeger H. Martinerie C. Laurent M. Alami J. Schofield P.N. Perbal B. Am. J. Pathol. 1998; 152: 1563-1575PubMed Google Scholar, 35Perbal B. J. Clin. Pathol. Mol. Pathol. 1999; 52: 84-91Crossref Scopus (57) Google Scholar, 36Maillard M. Cadot B. Ball R.Y. Sethia K. Edwards D.R. Perbal B. Tatoud R. Mol. Pathol. 2001; 54: 275-280Crossref PubMed Scopus (65) Google Scholar, 37Kocialkowski S. Yeger H. Kingdom J. Perbal B. Schofield P.N. Anat. Embryol. 2001; 203: 417-427Crossref PubMed Scopus (50) Google Scholar). In the current study, we used a new rabbit polyclonal antibody that showed a diffuse staining pattern of CCN3 in the cytoplasm (Fig. 3B). On the other hand, C6-Cx43 cells exhibited punctate staining (Fig. 3, E and H). Co-labeling with Cx43 and CCN3 antibodies revealed that CCN3 co-localized with Cx43 in gap junction plaques (Fig. 3, F and I).Fig. 3CCN3 co-localizes with Cx43 in C6-Cx43 cells. A-C, immunocytochemistry for C6 cells. C6-Cx43 immunocytochemistry is shown in D-F. G-I are higher magnification images of C6-Cx43 cells. A, D, and G were reacted with Cx43 antibody. B, E, and H were reacted with CCN3 antibody. C, F, and I are overlays of the previous two images. F and I were counterstained with 4′,6-diamidino-2-phenylindole to show the location of nuclei. The arrows indicate co-localization as revealed by yellow staining. Bar, 10 μm.View Large Image Figure ViewerDownload (PPT)CCN3 Physically Interacts with Cx43—To examine whether physical interaction exists between co-localized CCN3 and Cx43, a fusion construct of GST and full-length human CCN3 (GST-CCN3) was employed (38Li C.L. Martinez V. He B. Lombet A. Perbal B. Mol. Pathol. 2002; 55: 250-261Crossref PubMed Scopus (53) Google Scholar). The GST-CCN3 fusion protein immobilized on glutathione beads sequestered Cx43 from the C6-Cx43 cell lysate (Fig. 4A). No Cx43 was detected in the lysate of C6 parental cells subjected to the GST pull-down assay, probably because the level of Cx43 in C6 cells was too low to be detected. Negative control experiments were conducted with GST protein only and showed no pull-down of Cx43 (data not shown).Fig. 4CCN3 physically interacts with Cx43. A, GST pull-down assay. In C6-Cx43 cell lysates, Cx43 was sequestered by the GST-CCN3 fusion protein, whereas no Cx43 was pulled down from the C6 parental cell lysate (right two lanes). Crude lysates from C6 and C6-Cx43 cells were loaded as controls (left two lanes). B, co-immunoprecipitation (IP) was performed with CCN3 antibody on C6, C6-Cx43, and C6-Cx43GFP lysates. Western blot analysis with Cx43 antibody showed that Cx43 (lane 1) and Cx43GFP (duplicate samples in lanes 2 and 3) were co-immunoprecipitated with CCN3 from transfected C6 cells. No Cx43 was co-immunoprecipitated from parental C6 cells (lanes 4 and 5). Corresponding crude lysates were loaded as controls (lanes 6-8). The arrow indicates cross-reactivity with the heavy chain of CCN3 antibody used in co-immunoprecipitation.View Large Image Figure ViewerDownload (PPT)The physical association of CCN3 with Cx43 was verified through co-immunoprecipitation experiments (Fig. 4B). Cx43 was found to co-immunoprecipitate with CCN3 antibody in lysates of C6-Cx43 as well as C6-Cx43GFP cells, but not in C6 parental cells. Negative control experiments were performed using C6 cells retrovirally infected with the GFP reporter alone, which failed to produce any identifiable bands by co-immunoprecipitation (data not shown).Interaction with CCN3 Occurs via the C Terminus of Cx43 but Is Phosphorylation-independent—To delineate the domain of Cx43 that interacts with CCN3, the aforementioned retroviral construct of Cx43 deprived of its C-terminal tail (Cx43Δ244-382-GFP) was utilized. C6-Cx43Δ244-382-GFP cells were examined for CCN3 localization in parallel with those expressing full-length Cx43GFP (C6-Cx43GFP). Immunocytochemical analysis revealed punctate staining of CCN3 co-localizing with Cx43GFP epifluorescence (Fig. 5, A-C). In contrast, although the level of gap junction plaque formation in C6-Cx43Δ244-382-GFP cells was comparable with that of C6-Cx43GFP, CCN3 was present only as diffuse cytoplasmic staining rather than localizing to areas where C6-Cx43Δ244-382-GFP accumulated (Fig. 5, D-F).Fig. 5CCN3 co-localizes with Cx43GFP but not Cx43Δ244-382-GFP. A-C demonstrate immunocytochemistry of C6-Cx43GFP cells. D-F, immunocytochemistry of C6-Cx43Δ244-382-GFP cells. A and D were visualized with GFP epifluorescence. B and E were reacted with CCN3 antibody. C and F show merged images. Co-localization of CCN3 with Cx43 in C6-Cx43GFP cells was evidenced by yellow labeling (arrows). CCN3 was diffusely distributed in C6-Cx43Δ244-382-GFP cells instead of co-localizing with gap junction plaques at the plasma membrane. Bar, 10 μm.View Large Image Figure ViewerDownload (PPT)Whereas Cx43GFP co-immunoprecipitated with CCN3 antibody, Cx43Δ244-382-GFP failed to co-immunoprecipitate, indicating that C-terminal truncation of Cx43 abolished its interaction with CCN3 (Fig. 6A). Since the C terminus of connexins contains numerous sites of phosphorylation (39Saez J.C. Berthoud V.M. Branes M.C. Martinez A.D. Beyer E.C. Physiol. Rev. 2003; 83: 1359-1400Crossref PubMed Scopus (969) Google Scholar), alkaline phosphatase CIP treatment was conducted to study the effect of dephosphorylation on the CCN3-Cx43 interaction. Both the phosphorylated (Cx43-P1) and dephosphorylated Cx43 (Cx43-NP) in C6-Cx43 lysates were pulled down by GSTCCN3 (Fig. 6B). Co-immunoprecipitation of CIP-treated Cx43 (NP) with CCN3 (Fig. 6C) concurred with the GST pull-down study, suggesting that the interaction of Cx43 with CCN3 was not critically dependent on the phosphorylation state of Cx43. Although the most highly phosphorylated species of Cx43 (Cx43-P2) did not appear to be pulled down by GST-CCN3 (Fig. 6B), a co-immunoprecipitation experiment that is considered more physiologically relevant demonstrated the ability of Cx43-P2 to associate with CCN3 in vivo (Fig. 6C). The failure of Cx43-P2 to interact with GST-CCN3 might reflect the lack of post-translational modification (such as glycosylation) of CCN3 in the bacterial expression system, or steric hindrance might have resulted from the GST tag.Fig. 6Western analysis to assess the role of Cx43 C terminus in its interaction with CCN3. A, Cx43Δ244-382-GFP failed to co-immunoprecipitate (IP) with CCN3. Lysates of C6-Cx43GFP and C6-Cx43Δ244-382-GFP were subjected to co-immunoprecipitation with CCN3 antibody. GFP antibody was used to visualize the bands. Whereas Cx43GFP and Cx43Δ244-382-GFP were expressed at similar levels (compare lanes 1 and 3), only Cx43GFP was co-immunoprecipitated with CCN3 (compare lanes 2 and 4). B, CCN3 interacts with both the phosphorylated and unphosphorylated Cx43. GST pull-down assay was performed with C6-Cx43 lysate treated with CIP. Cx43 was pulled down by GST-CCN3 both in the presence (lane 3) and absence (lane 4) of phosphorylation. C, all three Cx43 species (NP, P1, and P2) were co-immunoprecipitated with CCN3 (lane 3). Dephosphorylation by CIP prior to immunoprecipitation did not abolish the ability of Cx43 to interact with CCN3. In B and C, crude lysates were presented as control to illustrate the phosphorylation profile of untreated and treated Cx43 (lanes 1 and 2, respectively).View Large Image Figure ViewerDownload (PPT)CCN3 Does Not Interact with Cx40 or Cx32—In order to investigate whether interaction with CCN3 is specific to Cx43 or shared among other members of the connexin family, we studied Cx40 and Cx32, members of the α and β subfamilies, respectively. Immunocytochemistry revealed that CCN3 was not found co-localized with Cx40GFP (Fig. 7, A-C). In accordance with this observation, Cx40GFP failed to co-immunoprecipitate with CCN3 (Fig. 8A).Fig. 7CCN3 does not co-localize with Cx40 or Cx32 at the plasma membrane. A, GFP epifluorescence showing the localization of Cx40 in C6-Cx40GFP cells. B, CCN3 in C6-Cx40GFP cells; C, merged image of A and B; D, Cx32 in C6-Cx32 cells; E, CCN3 staining in C6-Cx32; F, merged image of D and E. C
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