We have characterized the function of connexin (Cx) 32 gene mutations found in X-linked dominant Charcot-Marie-Tooth disease with respect to their ability to form functional gap junctions among themselves and to inactivate wild-type Cx32 by a dominant negative mechanism. We prepared four types of Cx32 mutant cDNAs and transfected them into HeLa cells, which do not show detectable levels of gap junctional intercellular communication (GJIC), nor expression of any connexins examined. Cells transfected with the wild-type Cx32 gene, but not those transfected with three different base substitution mutations (i.e. Cys 60 to Phe, Val 139 to Met, and Arg 215 to Trp), restored GJIC. Unexpectedly, in cells transfected with a nonsense mutant at codon 220, there was also restored GJIC. When we double-transfected these mutant constructs into the HeLa cells that had already been transfected with the wild-type Cx32 gene and thus were GJIC proficient, three base substitution mutants inhibited GJIC, suggesting that these three mutants can eliminate the function of wild-type Cx32 in a dominant negative manner. The nonsense mutation at codon 220 did not show such a dominant negative effect. Since both mutant and wild-type Cx32 mRNAs were detected, but only poor Cx32 protein expression at cell-cell contact areas was observed in the double transfectants, it is suggested that certain mutants form nonfunctional chimeric connexons with wild-type connexins, which are not properly inserted into the cytoplasmic membrane.
Gap junctions are intercellular plasma membrane domains enriched in channels that allow direct exchange of ions and small molecules between adjacent cells. Gap junction channels are composed of a family of transmembrane proteins called connexin. Connexins play important roles in the regulation of cell growth and differentiation. Cancer cells usually have downregulated levels of gap junctions, and several lines of evidence suggest that loss of gap junctional intercellular communication is an important step in carcinogenesis. In support of this hypothesis are studies showing that reexpression of connexins in cancer cells causes normalization of cell growth control and reduced tumor growth. To gain a more detailed understanding of the role of connexins as tumor suppressors, a clearer picture of the mechanisms involved in loss of gap junctions in cancer cells is needed. Furthermore, defining the mechanisms involved in downregulation of connexins in carcinogenesis will be an important step toward utilizing the potential of connexins as targets in cancer prevention and therapy. Various mechanisms are involved in the loss of gap junctions in cancer cells, ranging from loss of connexin gene transcription to aberrant trafficking of connexin proteins. This review will discuss our current knowledge on the molecular mechanisms involved in the downregulation of gap junctions in cancer cells.
We investigated the mechanism of phenotypic plasticity of hepatocytes in a three-dimensional organoid culture system, in which hepatocytic spheroids were embedded within a collagen gel matrix. Hepatocytes expressed several bile duct markers including cytokeratin (CK) 19 soon after culture and underwent branching morphogenesis within the matrix in the presence of insulin and epidermal growth factor. Cultured hepatocytes did not express Delta-like, a specific marker for oval cells and hepatoblasts. Furthermore, hepatocytes isolated from c-kit mutant rats (Ws/Ws), which are defective in proliferation of oval cells, showed essentially the same phenotypic changes as those isolated from control rats. The bile duct-like differentiation of hepatocytes was associated with increased expression of Jagged1, Jagged2, Notch1, and several Notch target genes. CK19 expression and branching morphogenesis were inhibited by dexamethasone, a mitogen-activated protein kinase kinase 1 (MEK1) inhibitor (PD98059), and a phosphatidyl inositol 3-kinase inhibitor (LY294002). After being cultured for more than 3 weeks within the gels, hepatocytes transformed into ductular structures surrounded by basement membranes. Our results suggest that hepatocytes might have the potential to transdifferentiate into bile duct-like cells without acquiring a stem-like phenotype and that this is mediated through specific protein tyrosine phosphorylation pathways.
Connexin genes expressing gap junction proteins have tumor-suppressive effects on primary cancers with certain cell specificity, but the suppressive effects on metastatic cancers are still conflicting. In this study, we show that connexin32 (Cx32) has a strong tumor-suppressive effect on a human metastatic renal cell carcinoma cell line (Caki-1 cell). Cx32 expression in Caki-1 cells reduced in vitro malignant phenotypes of the cells such as anchorage independency and invasion capacity. Furthermore, the Cx32 expression drastically reduced the development of Caki-1 cells in nude mice. We also determined that Cx32 reduced the malignant phenotypes in Caki-1 cells mainly through the inactivation of Src signaling. Especially, Cx32-dependent inactivation of Src decreased the production of vascular epithelial growth factor (VEGF) via the suppression of signal transducers and activators of transcription 3 (Stat3) activation, and we confirmed this result using short interfering RNA. In nude mice, Cx32-transfected Caki-1 cells showed lower serum level of VEGF comparing mock transfectant, and the development of the cells in nude mice positively related to the VEGF level. These data suggest that Cx32 acts as a tumor suppressor gene in Caki-1 cells and that the tumor-suppressive effect partly depends on the inhibition of Src-Stat3-VEGF signal pathway.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.