The Gap Junction-independent Tumor-suppressing Effect of Connexin 43

Abstract

The gap junction gene connexin 43 (Cx43) showed tumor-suppressing effects on various tumor cell lines. We have previously demonstrated that Cx43 inhibited expression of S phase kinase-associated protein 2 (Skp2), the human F-box protein that regulates the ubiquitination of p27. Cx43 did not alter the mRNA level of SKP2, but it promoted the degradation of the Skp2 proteins (Zhang, Y. W., Nakayama, K., Nakayama K. I., and Morita, I. (2003) Cancer Res. 63, 1623-1630). In this study, we showed that the specific gap junction inhibitor 18 β-glycyrrhetinic acid did not influence the inhibitory effect of Cx43 on Skp2 expression. Further, the deletion mutation analyses demonstrated that the C-terminal domain of Cx43 that did not form gap junctions was sufficient to inhibit expression of Skp2, whereas the N-terminal domain that formed the gap junctions did not show such an effect. Like the full-length Cx43, the C-terminal domain also increased the protein instability of Skp2, whereas the N terminus did not. Moreover, the C-terminal domain was as effective as the full-length Cx43 in inhibiting cell proliferation; however, the N-terminal domain did not show any inhibitory effect on cell proliferation. Therefore, these data revealed a gap junction-independent pathway for Cx43 to inhibit tumor growth by suppressing the Skp2 expression. The gap junction gene connexin 43 (Cx43) showed tumor-suppressing effects on various tumor cell lines. We have previously demonstrated that Cx43 inhibited expression of S phase kinase-associated protein 2 (Skp2), the human F-box protein that regulates the ubiquitination of p27. Cx43 did not alter the mRNA level of SKP2, but it promoted the degradation of the Skp2 proteins (Zhang, Y. W., Nakayama, K., Nakayama K. I., and Morita, I. (2003) Cancer Res. 63, 1623-1630). In this study, we showed that the specific gap junction inhibitor 18 β-glycyrrhetinic acid did not influence the inhibitory effect of Cx43 on Skp2 expression. Further, the deletion mutation analyses demonstrated that the C-terminal domain of Cx43 that did not form gap junctions was sufficient to inhibit expression of Skp2, whereas the N-terminal domain that formed the gap junctions did not show such an effect. Like the full-length Cx43, the C-terminal domain also increased the protein instability of Skp2, whereas the N terminus did not. Moreover, the C-terminal domain was as effective as the full-length Cx43 in inhibiting cell proliferation; however, the N-terminal domain did not show any inhibitory effect on cell proliferation. Therefore, these data revealed a gap junction-independent pathway for Cx43 to inhibit tumor growth by suppressing the Skp2 expression. As one of the cell-cell interaction structures, the gap junctions (GJs) 1The abbreviations used are: GJ, gap junction; GJIC, gap junctional intercellular communication; BFA, brefeldin A; Cx, connexin; GA, 18 β-glycyrrhetinic acid; Skp2, S phase kinase-associated protein; PBS, phosphate-buffered saline; FL, full-length. provide direct transfer of small hydrophilic molecules (e.g. ions and metabolites) up to ∼1 kDa in size from the cytoplasm of one cell to that of adjacent cells. This process is called gap junctional intercellular communication (GJIC). GJs are composed of connexin (Cx) proteins. Currently, at least 14 Cxs have been cloned and identified (1Goodenough D.A. Goliger J.A. Paul D.L. Ann. Rev. Biochem. 1996; 65: 475-502Crossref PubMed Scopus (1088) Google Scholar, 2Kumar N.M. Gilula N.B. Cell. 1996; 84: 381-388Abstract Full Text Full Text PDF PubMed Scopus (1652) Google Scholar). A large number of studies have indicated that certain Cxs (e.g. Cx26, Cx32, and Cx43) have tumor-suppressing effects (3Yamasaki H. Naus C.C. Carcinogenesis. 1996; 17: 1199-1213Crossref PubMed Scopus (452) Google Scholar, 4Yamasaki H. Krutovskikh V. Mesnil M. Tanaka T. Zaidan-Dagli M.L. Omori Y. C. R. Acad. Sci. III. 1999; 322: 151-159Crossref PubMed Scopus (160) Google Scholar, 5Zhang Y.W. Morita I. Ikeda M. Ma K.W. Murota S. Oncogene. 2001; 20: 4138-4149Crossref PubMed Scopus (110) Google Scholar). Either the Cx-modulated GJs or the Cx protein itself contributed to the effects of tumor growth inhibition (4Yamasaki H. Krutovskikh V. Mesnil M. Tanaka T. Zaidan-Dagli M.L. Omori Y. C. R. Acad. Sci. III. 1999; 322: 151-159Crossref PubMed Scopus (160) Google Scholar). It seems that the Cx species and the cell types used determined whether the GJs or the Cx proteins regulate the tumor growth (4Yamasaki H. Krutovskikh V. Mesnil M. Tanaka T. Zaidan-Dagli M.L. Omori Y. C. R. Acad. Sci. III. 1999; 322: 151-159Crossref PubMed Scopus (160) Google Scholar, 6Mesnil M. Krutovskikh V. Piccoli C. elfgang C. Traub O. Willecke K. Yamasaki H. Cancer Res. 1995; 55: 629-639PubMed Google Scholar). Recently, we reported that Cx43 suppressed expression of S phase kinase-associated protein 2 (Skp2), the human F-box protein that regulates p27 ubiquitination (7Zhang Y.W. Nakayama K. Nakayama K.I. Morita I. Cancer Res. 2003; 63: 1623-1630PubMed Google Scholar); through it, Cx43 increased the level of p27 and inhibited cell proliferation of the osteosarcoma U2OS cells (5Zhang Y.W. Morita I. Ikeda M. Ma K.W. Murota S. Oncogene. 2001; 20: 4138-4149Crossref PubMed Scopus (110) Google Scholar, 7Zhang Y.W. Nakayama K. Nakayama K.I. Morita I. Cancer Res. 2003; 63: 1623-1630PubMed Google Scholar). Here we report that Cx43 inhibited Skp2 expression via a GJ-independent pathway. Construction of the Cx43 Mutants—The rat heart cDNA library (TaKaRa, Tokyo, Japan) was used to generate the Cx43 mutants by PCR. The primers will be provided upon request. TaRaKa Ex Taq polymerase (TaKaRa) was used to attach an additional A at the 3′ end of the PCR products. Then the PCR products were ligated into the pcDNA3.1/V5-HisTOPO (Invitrogen) vector using the TA cloning techniques. Sense insertion and the DNA sequence were confirmed by both restriction enzyme reaction and sequencing. Cell Culture and Transfection—U2OS and COS-7 cells were cultivated in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum. Cells were transfected using the FuGENE 6 reagent (Roche Applied Science), as recommended by the manufacturer. Cell Proliferation—Cell proliferation was estimated by counting the cell number as previously described (5Zhang Y.W. Morita I. Ikeda M. Ma K.W. Murota S. Oncogene. 2001; 20: 4138-4149Crossref PubMed Scopus (110) Google Scholar). GJIC Analysis—A cell-coupling assay was performed as previously described using the gap fluorescence recovery after photobleaching (gap-FRAP) technique (8Zhang Y.W. Morita I. Nishida M. Murota S. J. Cell. Physiol. 1999; 180: 305-313Crossref PubMed Scopus (30) Google Scholar, 9Zhang Y.W. Morita I. Zhang L. Gang S. Yao X.S. Murota S. Planta Med. 2000; 66: 119-123Crossref PubMed Scopus (36) Google Scholar). In brief, the cells were rinsed twice with PBS containing 1.25 mm CaCl2 and 0.5 mm MgCl2 (PBS (+)) and stained with 5-10 μm 5,6-carboxyfluorescence diacetate (CFDA) in PBS (+) for 10-15 min. The cells were then rinsed five times with PBS (+) to remove the extra dye and covered with PBS (+) for FRAP analysis. The fluorescence of several randomly selected cells that had been stained with CFDA was bleached with 488-nm 600 mW visible laser beams, and then the recovery of fluorescence was monitored over the subsequent 15 min. An unbleached cell served as the 100% fluorescence control and was used to correct for the loss of fluorescence due to background leakage and photobleaching. This technique involves analyzing the increase of fluorescence over time in a photobleached living cell adjacent to unbleached dye-labeled living cells, so it is able to yield quantitative data. The fluorescence recovery of the photobleached cell reflects the level of GJIC that existed between the photobleached cell and neighbor cells. Immunofluorescence Staining—Immunofluorescence staining was performed according to a modified procedure (5Zhang Y.W. Morita I. Ikeda M. Ma K.W. Murota S. Oncogene. 2001; 20: 4138-4149Crossref PubMed Scopus (110) Google Scholar, 8Zhang Y.W. Morita I. Nishida M. Murota S. J. Cell. Physiol. 1999; 180: 305-313Crossref PubMed Scopus (30) Google Scholar). The cells were seeded into 35-mm glass-cover dishes 1 day prior to transfection with the Cx43 mutants. After 48 h of transfection, the cells were rinsed twice with PBS and fixed in cold methanol/acetone (1:1) for 15 min at room temperature. After thorough washing, the cells were blocked in PBS containing 10% fetal bovine serum and 0.2% saponin for 10 min, washed twice with PBS, and incubated with mouse anti-His6 or anti-Cx43 antibodies (1:150) for 1 h at room temperature. Then the cells were washed five times with PBS, exposed to goat anti-mouse IgG conjugated with Rhodamine (1:200) for 45 min, washed extensively with PBS, and viewed with the fluorescent microscope. 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nuclei. Antibodies and Immunoblotting—Anti-Skp2 (H-435) was from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-Cx43 was purchased from Transduction Laboratories (Lexington, KY) and Chemicon International (Temecula, CA). Anti-His6 monoclonal antibody was purchased from Genzyme Techne (Minneapolis, MN) and Qiagen (Stanford, CA). Immunoblotting was performed as previously described (8Zhang Y.W. Morita I. Nishida M. Murota S. J. Cell. Physiol. 1999; 180: 305-313Crossref PubMed Scopus (30) Google Scholar). Northern Blotting—The Northern blot analysis was performed as previously described (7Zhang Y.W. Nakayama K. Nakayama K.I. Morita I. Cancer Res. 2003; 63: 1623-1630PubMed Google Scholar). Statistical Analyses—The Student's t test was performed for statistical evaluation of a difference; p < 0.001 was considered to be statistically significant. The Specific GJ Inhibitors Did Not Alter the Effects of Cx43 on Skp2 Expression—In our previous study (5Zhang Y.W. Morita I. Ikeda M. Ma K.W. Murota S. Oncogene. 2001; 20: 4138-4149Crossref PubMed Scopus (110) Google Scholar), we reported that stable-transfection of Cx43 elevated the level of the cyclin-dependent kinase inhibitor p27 in the human osteosarcoma U2OS cells. We recently demonstrated that Cx43 suppressed the expression of Skp2 (7Zhang Y.W. Nakayama K. Nakayama K.I. Morita I. Cancer Res. 2003; 63: 1623-1630PubMed Google Scholar), the human F-box protein that regulates the ubiquitin-dependent p27 degradation. Because both the GJ-dependent and the GJ-independent pathways were involved in the Cx43-induced p27 up-regulation (5Zhang Y.W. Morita I. Ikeda M. Ma K.W. Murota S. Oncogene. 2001; 20: 4138-4149Crossref PubMed Scopus (110) Google Scholar), we then asked whether Cx43 inhibited expression of Skp2 via the GJ-dependent pathway or not. We first examined effects of two GJ inhibitors on the down-regulated Skp2 expression by Cx43. Brefeldin A (BFA) blocks the transfer of the Cx43 proteins from the Golgi apparatus to the cellular membranes, thus preventing formation of both the GJs and the hemi-channels by Cx43 (5Zhang Y.W. Morita I. Ikeda M. Ma K.W. Murota S. Oncogene. 2001; 20: 4138-4149Crossref PubMed Scopus (110) Google Scholar). However, 18 β-glycyrrhetinic acid (GA) specifically inhibits the GJ function whereas it does not affect the GJ formation (5Zhang Y.W. Morita I. Ikeda M. Ma K.W. Murota S. Oncogene. 2001; 20: 4138-4149Crossref PubMed Scopus (110) Google Scholar). Thus, both BFA and GA significantly inhibited the GJIC level of the Cx43-transfected cells (Fig. 1A) (5Zhang Y.W. Morita I. Ikeda M. Ma K.W. Murota S. Oncogene. 2001; 20: 4138-4149Crossref PubMed Scopus (110) Google Scholar). Then we examined the expression of Skp2 in the presence of these two GJ inhibitors. As reported (7Zhang Y.W. Nakayama K. Nakayama K.I. Morita I. Cancer Res. 2003; 63: 1623-1630PubMed Google Scholar), the level of Skp2 was clearly reduced in the Cx43-transfected cells (Fig. 1A, lanes 1-3). Nevertheless, treatment with neither BFA nor GA changed the reduction in the level of Skp2 in the Cx43-transfected cells (Fig. 1A, lanes 3-5). Because Cx43 reduced the level of Skp2 via increasing the protein degradation (7Zhang Y.W. Nakayama K. Nakayama K.I. Morita I. Cancer Res. 2003; 63: 1623-1630PubMed Google Scholar), we also examined effects of BFA and GA on the Skp2 protein stability. Fig. 1B showed that the Skp2 degradation was clearly enhanced when the Cx43-transfected cells were treated with the protein synthesis inhibitor cycloheximide. However, BFA or GA did not alter the enhanced effect of Cx43 on the degradation of Skp2. Thus, these data suggest that Cx43 inhibited the Skp2 expression likely through a GJ (also including the hemi-channel-)-independent pathway, which may be ascribed from the biological role of the Cx43 protein itself. The Cytoplasmic Carboxyl Terminal Domain Was the Active Part of Cx43 in Suppressing Skp2 Expression—Cx43, like other connexin proteins, goes through the cellular membranes four times, leading to a short N-terminal and a relatively long C-terminal domain within the cytoplasm (3Yamasaki H. Naus C.C. Carcinogenesis. 1996; 17: 1199-1213Crossref PubMed Scopus (452) Google Scholar) (Fig. 2A). The above mentioned data indicate that the inhibitory effect of Cx43 on Skp2 expression is likely GJ-independent (Fig. 1). To confirm this hypothesis, we generated four deletion mutants that contained part of either N-terminal or C-terminal domain of Cx43. All these mutants were tagged at the C terminus with an additional His6 tail. To eliminate the possibility that the His6 tail might influence the function of these mutants, we generated the full-length (FL) construct of Cx43, which was also tagged with the His6 tail at the C terminus (Fig. 2A). We first analyzed the cellular distribution of these mutants. In comparison with the parental cells that did not show any GJ staining in the membranes (Fig. 2B, a), the un-tagged Cx43 proteins, the FL and the N3 constructs (all contain the four trans-membrane domains of Cx43) went into the membranes and formed GJs (Fig. 2B, b-d). However, the N1, N2, and C mutants (all contain only a part of the four trans-membrane domains of Cx43) did not form GJs (Fig. 2B, e-g). Next we examined the GJIC levels in cells transiently transfected with these mutants. Like Cx43, the FL and the N3 constructs showed very high level of GJIC, whereas the N1, N2, and C mutants did not (Fig. 3). Together with Fig. 2, these data indicate that the four trans-membrane domains are necessary for the formation of functional GJs. Immunoblotting with the monoclonal anti-His6 antibodies demonstrated that cells transfected with these constructs expressed the similar levels of proteins (Fig. 4A). In addition, FL and C, but not N1, N2, and N3, were also recognized by the anti-Cx43 antibody (Fig. 4A), because only FL and C contained the amino acid sequence a of Cx43, from which the mouse anti-Cx43 antibody was generated (Fig. 2A). Then we examined effects of these constructs on the Skp2 expression. Cx43 suppressed expression of Skp2 (Fig. 4A, lanes 1 and 2). Likewise, this inhibitory effect was observed in the FL transiently transfected cells (Fig. 4A, lanes 1-4), indicating that the His6 tag at the C terminus does not obviously disturb the effect of Cx43 on Skp2 expression (also see below, Fig. 4C and Fig. 5). The cells transfected with the mutants N1, N2, and N3 did not show any reduction in the level of Skp2 (Fig. 4A, lanes 1 and 5-10). However, expression of Skp2 was significantly inhibited in the mutant C transfected cells (Fig. 4A, lanes 1, 11, and 12); moreover, the inhibitory effect was quite close to that of the full-length Cx43 (Fig. 4A, lanes 2, 3, 11, and 12), indicating that the C-terminal domain (amino acids 242-382) was not only required but also sufficient for Cx43 to inhibit Skp2 expression. Together with Figs. 1,2,3, these data suggest that Cx43 did not require the GJ forming domain (amino acids 1-242) to inhibit Skp2 expression. In other words, the inhibitory effect of Cx43 on Skp2 was GJ-independent. We have previously reported that Cx43 did not change the mRNA level of SKP2, but increased the protein instability (7Zhang Y.W. Nakayama K. Nakayama K.I. Morita I. Cancer Res. 2003; 63: 1623-1630PubMed Google Scholar). Likewise, all these constructs did not show obvious effects on the mRNA level of SKP2 (Fig. 4B). The effects of these constructs on the protein stability of Skp2 were also examined. The results showed that Cx43, Fl, and C increased the protein instability of Skp2 in the presence of cycloheximide, whereas N1, N2, and N3 did not show any effect (Fig. 4C). Thus, these data confirmed the negative role of the C terminus of Cx43 on Skp2 expression. They argue again that Cx43 inhibited Skp2 expression via mechanisms of the GJs. The Cytoplasmic C of Cx43 Cell we examined effects of these mutants on cell proliferation by counting the cell As reported previously (5Zhang Y.W. Morita I. Ikeda M. Ma K.W. Murota S. Oncogene. 2001; 20: 4138-4149Crossref PubMed Scopus (110) Google Scholar, 7Zhang Y.W. Nakayama K. Nakayama K.I. Morita I. Cancer Res. 2003; 63: 1623-1630PubMed Google Scholar), cell proliferation of the Cx43 transfected U2OS cells was significantly inhibited with the parental cells (Fig. 5). The FL and C constructs also showed inhibitory effects on cell proliferation of U2OS cells, the effects were that of transfected Cx43. However, the N1, N2, and N3 mutants did not inhibit cell proliferation at all (Fig. 5). data suggest that the C terminus also had the to inhibit proliferation of U2OS cells, that the GJ-independent role of Cx43 contributed to the Cx43-induced cell proliferation to the possibility that the inhibition of Cx43 on Skp2 expression were only observed in U2OS cells, we examined the effects of these mutants on other cell lines. As in Fig. the FL and C mutants, but not the N1, N2, and N3 mutants, clearly reduced the level of Skp2 in COS-7 cells. Moreover, the FL and C mutants also significantly the cell proliferation of COS-7 cells, whereas the N1, N2, and N3 mutants did not show any effect on the cell proliferation (Fig. We that the N3 mutant did not inhibit cell proliferation in both cell it formed GJs and showed high level of However, our previous data showed that the GJ function of Cx43 did a role in cell proliferation may be that the GJs formed by the N3 mutant are not the as formed by the full-length Cx43. these data suggest a gap junction-independent pathway for Cx43 to inhibit cell proliferation in certain cell by of Skp2. In the study, we provided that Cx43 inhibited expression of the human F-box protein Skp2 by mechanisms of the GJs. the specific GJ inhibitors did not affect the inhibitory effect of Cx43 on Skp2 expression. the C-terminal domain of Cx43 (amino acids to that did not form the GJ the effects as that of the full-length Cx43 on Skp2 whereas the N-terminal part of Cx43 (amino acids 1 to that the four trans-membrane domains of Cx43 and formed the GJ did not. Thus, these data revealed of Cx43 in inhibiting tumor proliferation by negative of Skp2 via GJ-independent The role of the Cx proteins been to be the formation of the GJ that the Therefore, the mechanisms by which Cxs inhibit tumor growth were to be through the of growth inhibitory via the Cx-modulated GJs Cell PubMed Scopus Google Scholar). However, increasing that the GJ-independent are also involved in the growth inhibition (5Zhang Y.W. Morita I. Ikeda M. Ma K.W. Murota S. Oncogene. 2001; 20: 4138-4149Crossref PubMed Scopus (110) Google Scholar, 6Mesnil M. Krutovskikh V. Piccoli C. elfgang C. Traub O. Willecke K. Yamasaki H. Cancer Res. 1995; 55: 629-639PubMed Google Scholar, Y. Cancer Res. Google Scholar, Y. Yamasaki H. J. PubMed Scopus Google Scholar, C. M. Cell Res. 2001; PubMed Scopus Google Scholar, H. H. J. T. J. Full Text Full Text PDF PubMed Scopus Google Scholar). Further, it was reported that the C-terminal domain showed the growth inhibition effects as the full-length Cx43 in certain cell C. M. Cell Res. 2001; PubMed Scopus Google Scholar, Cell Biochem. 2003; PubMed Scopus Google Scholar) the We have previously reported that stable-transfection of Cx43 increased the level of the cyclin-dependent kinase inhibitor p27 (5Zhang Y.W. Morita I. Ikeda M. Ma K.W. Murota S. Oncogene. 2001; 20: 4138-4149Crossref PubMed Scopus (110) Google Scholar, 7Zhang Y.W. Nakayama K. Nakayama K.I. Morita I. Cancer Res. 2003; 63: 1623-1630PubMed Google Scholar). This increase was the of the increased synthesis and the reduced degradation of the p27 proteins. the GJ-dependent and the GJ-independent mechanisms contributed to the increase of p27 (5Zhang Y.W. Morita I. Ikeda M. Ma K.W. Murota S. Oncogene. 2001; 20: 4138-4149Crossref PubMed Scopus (110) Google Scholar). We have that was involved in the GJ-dependent pathway (5Zhang Y.W. Morita I. Ikeda M. Ma K.W. Murota S. Oncogene. 2001; 20: 4138-4149Crossref PubMed Scopus (110) Google Scholar), whereas our study, with the suggest that a GJ-independent inhibition of Skp2 expression was for the reduced p27 degradation (7Zhang Y.W. Nakayama K. Nakayama K.I. Morita I. Cancer Res. 2003; 63: 1623-1630PubMed Google Scholar). Therefore, we a pathway for Cx43 to inhibit tumor cell at least in certain cell (Fig. Because we have observed that Skp2 the in the Cx43-induced p27 increase and cell growth inhibition (7Zhang Y.W. Nakayama K. Nakayama K.I. Morita I. Cancer Res. 2003; 63: 1623-1630PubMed Google Scholar), we suggest that the GJ-independent pathway contributed the GJ-dependent in the Cx43-induced cell proliferation In comparison with cells that have a high level of human a level of and the reduced p27 protein level with high and of in J. Cell Physiol. 2000; PubMed Scopus Google Scholar, J. M. J. Physiol. 2000; PubMed Scopus Google Scholar, S. and C. J. Cell Scholar). The reduced level of p27 was reported to at least in the of increased expression of Skp2 R. M. Acad. Sci. S. 2001; PubMed Scopus Google Scholar, M. R. M. C. J. J. Acad. Sci. S. 2001; PubMed Scopus Google Scholar, O. M. 2001; PubMed Scopus Google Scholar, Y. S. S. M. I. T. Cancer Res. 2001; Google Scholar). the of Skp2 is in may be is that the the level of Skp2 is in It been reported that the human F-box protein Skp2 C. H. M. M. M. J. 2000; PubMed Scopus Google Scholar) and the F-box proteins Cell. Full Text Full Text PDF PubMed Scopus Google Scholar) and M. Acad. Sci. S. 1999; PubMed Scopus Google Scholar) were to degradation by the Thus, it is that the mechanisms the level of Skp2 are in which results in the elevated level of Skp2. this to be in human The other is that Skp2 may be the of tumor-suppressing of the or of the tumor-suppressing the level of Skp2 via which may of the of Skp2. data the A increase in the level of Skp2 was observed in of R. M. Acad. Sci. S. 2001; PubMed Scopus Google Scholar). of form of the elevated the level of Skp2 by reduced expression of p27 L. C. J. 2003; Full Text Full Text PDF PubMed Scopus (30) Google Scholar). In the tumor R. H. Zhang H. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) and the acid R. J. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, M. T. J. L. Cell 2003; PubMed Scopus Google Scholar), R. 2003; PubMed Scopus Google Scholar), and H. M. M. S. H. S. K. R. T. M. 2003; PubMed Scopus Google Scholar) in Skp2 levels with of p27 proteins in tumor cells. these studies and our we that one of the for Cx43 to function as a tumor-suppressing gene may be through the SKP2, and increasing the level of the cell p27. The mechanisms by which Cx43 promoted the Skp2 degradation Immunoblotting showed that the Cx43 proteins expressed both in the cytoplasm and in the not it is that a protein to form the cell-cell interaction in the membranes was involved in in the cytoplasm nuclei. Cx43, or the C may the through the sequence C. M. Cell Res. 2001; PubMed Scopus Google Scholar) to the ubiquitination of Skp2, or increase the degradation of the Skp2 proteins in the study is to show Cx43 increased the Skp2 degradation.