Oculodentodigital dysplasia (ODDD) is an autosomal dominant disorder characterized by pleiotropic developmental anomalies of the limbs, teeth, face and eyes that was shown recently to be caused by mutations in the gap junction protein alpha 1 gene (GJA1), encoding connexin 43 (Cx43). In the course of performing an Nethyl-N-nitrosourea mutagenesis screen, we identified a dominant mouse mutation that exhibits many classic symptoms of ODDD, including syndactyly, enamel hypoplasia, craniofacial anomalies and cardiac dysfunction. Positional cloning revealed that these mice carry a point mutation in Gja1 leading to the substitution of a highly conserved amino acid (G60S) in Cx43. In vivo and in vitro studies revealed that the mutant Cx43 protein acts in a dominant-negative fashion to disrupt gap junction assembly and function. In addition to the classic features of ODDD, these mutant mice also showed decreased bone mass and mechanical strength, as well as altered hematopoietic stem cell and progenitor populations. Thus, these mice represent an experimental model with which to explore the clinical manifestations of ODDD and to evaluate potential intervention strategies.

Oculodentodigital dysplasia, a rare condition displaying congenital craniofacial deformities and limb abnormalities, has been associated with over 20 known human connexin43 (Cx43) mutations. The localization of two of these mutants, G21R and G138R, was examined in Cx43-positive normal rat kidney cells (NRK) and Cx43-negative gap junctional intercellular communication-deficient HeLa cells. Green fluorescent protein-tagged and untagged Cx43 G21R and G138R mutants were transported to the plasma membrane and formed punctate structures reminiscent of gap junction plaques in both NRK and HeLa cells. Further localization studies revealed no significant trafficking defects as subpopulations of Cx43 mutants were found in both the Golgi apparatus and lysosomes, not unlike wild-type Cx43. Dual patch clamp functional analysis of the mutants expressed in gap junctional intercellular communication-deficient N2A cells revealed that neither G21R nor G138R formed functional gap junction channels, although they successfully reached cell-cell interfaces between cell pairs. Importantly, when either mutant was expressed in NRK cells, dye coupling experiments revealed that both mutants inhibited endogenous Cx43 function. These studies suggest that, although patients suffering from oculodentodigital dysplasia possess one wild-type Cx43 allele, it is likely that Cx43-mediated gap junctional intercellular communication is reduced below 50% because of a dominant-negative effect of mutant Cx43 on wild-type Cx43.

Connexins are gap junction proteins that assemble into channels that mediate direct intercellular communication.Connexins are well-documented tumor suppressors and are thought to regulate both cell growth and differentiation. As previously reported, most human breast tumors and cell lines down-regulate gap junctions or have defective gap junctional intercellular communication. Furthermore, overexpression of connexins in breast cancer cells inhibits tumor growth in vivo. In this study, we hypothesize that controlled Cx43 down-regulation would induce breast tumor cells to acquire a more aggressive phenotype. Here we report that Cx43 was down-regulated in both normal rat kidney (NRK) cells and human breast cancer cell lines (MDA-MB-231 and Hs578T) by transfection with chemically synthesized small interfering RNA (siRNA) or short hairpin RNA generated from a retroviral infection. Furthermore, we show that retroviral delivery and expression of siRNA directed to different coding regions of Cx43 resulted in differential levels of Cx43 silencing and impaired gap junctional intercellular communication. Cx43-silenced Hs578T cells grew faster and were more migratory. Finally, Western blot analysis revealed that down-regulation of Cx43 resulted in decreased expression of thrombospondin-1, an antiangiogenesis molecule, and increased expression of vascular endothelial growth factor. Taken together, these results suggest that Cx43 is required for maintaining cell differentiation and the regulation of molecules important in angiogenesis. (Cancer Res 2005; 65(7): 2705-11)

Studies of mice with targeted disruptions of specific connexin genes have revealed that at least two connexins, connexin37 (Cx37) and connexin43 (Cx43), play essential roles in ovarian follicle development. To explore the respective roles of these two connexins in gap-junctional communication between the developing murine oocyte and its surrounding cumulus granulosa cells, we used confocal immunofluorescence microscopy and oocyte preloading functional assays. Immunofluorescence microscopy located Cx37 within gap-junction plaques between granulosa cells and the oocyte, and Cx43 between surrounding granulosa cells. Preloading assays combining denuded oocytes and cultured granulosa cells expressing or lacking Cx37 or Cx43 revealed that Cx37 must be present in both cell types for the establishment of heterocellular gap-junctional coupling. Furthermore, immunofluorescence microscopy of cultured granulosa cells after incubation with denuded oocytes showed that the oocyte induces the formation of gap junctions containing Cx37 at the surface of granulosa cells. Continuous Cx37 expression in granulosa cells was confirmed using RT-PCR. Together, these results indicate that the growing murine oocyte is functionally coupled with granulosa cells by homotypic gap junctions composed of Cx37, and that the formation and/or stabilization of Cx37 junctions is selectively induced at the oocyte-granulosa interface by cell contact.