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Oculodentodigital dysplasia (ODDD) is mainly an autosomal dominant human disease caused by mutations in the GJA1 gene, which encodes the gap junction protein connexin43 (Cx43). Surprisingly, there have been two autosomal recessive mutations reported that cause ODDD: a single amino acid substitution (R76H) and a premature truncation mutation (R33X). When expressed in either gap junctional intercellular communication (GJIC)-deficient HeLa cells or Cx43-expressing NRK cells, the R76H mutant trafficked to the plasma membrane to form gap junction-like plaques, whereas the R33X mutant remained diffusely localized throughout the cell, including the nucleus. As expected, the R33X mutant failed to form functional channels. In the case of the R76H mutant, dye transfer studies in HeLa cells and electrical conductance analysis in GJIC-deficient N2a cells revealed that this mutant could form functional gap junction channels, albeit with reduced macroscopic and single channel conductance. Alexa 350 dye transfer studies further revealed that the R76H mutant had no detectable negative effect on the function of co-expressed Cx26, Cx32, Cx37 or Cx40, whereas the R33X mutant exhibited significant dominant or trans-dominant effects on Cx43 and Cx40 as manifested by a reduction in wild-type connexin gap junction plaques. Taken together, our results suggest that the trans-dominant effect of R33X together with its complete inability to form a functional channel may explain why patients harboring this autosomal recessive R33X mutant exhibit greater disease burden than patients harboring the R76H mutant [ABSTRACT FROM AUTHOR]

Background: Connexins (Cx) are the basic units of gap junctions and contribute to cellular integrity by promoting intercellular communication. Disruption of the retinal pigment epithelial monolayer may be an early event in the pathogenesis of age-related macular degeneration, a condition in which vascular endothelial growth factor (VEGF) is known to be of importance. This study was designed to assess the effect of connexin43 (Cx43) expression and gap junctional intercellular communication (GJIC) on the expression and secretion of VEGF from the retinal pigment epithelium under normal cell culture and oxidative stress conditions. Methods: Stable cell lines of ARPE-19 were produced in which wild-type Cx43 was either over-expressed, down-regulated by targeted shRNA, or functionally inhibited by co-expression of a disease-linked dominant-negative mutant (G21R). Pharmacologic blockade of GJIC was accomplished with flufenamic acid. Oxidant challenge was performed with tert-butyl hydroperoxide (tBH). VEGF gene expression and secretion were assessed by real-time PCR and ELISA respectively. Results: Over-expression of Cx43 in ARPE-19 cells reduced both gene expression and secretion of VEGF. Down-regulation of Cx43 increased gene expression and secretion of VEGF. Increased secretion of VEGF was also observed in ARPE-19 cells expressing a dominant-negative mutant of Cx43, and when GJIC was blocked. Over-expression of Cx43 reduced tBH-induced secretion of VEGF from ARPE-19 cells. Conclusions: These studies show that Cx43 protects against oxidative stress-induced VEGF secretion in ARPE-19 cells, and thus has important implications in understanding the pathogenesis of age-related macular degeneration. [ABSTRACT FROM AUTHOR]