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The current study aimed to understand the anti-apoptotic effect of overexpressed gap junction forming protein connexin (Cx) 43 in C6 glioma cells. C6 cells exposed to hydrogen peroxide (H2O2) or staurosporine demonstrated morphological and biochemical changes consistent with apoptosis, whereas C6 cells expressing Cx43 demonstrated relative resistance to H2O2, but not to staurosporine. This selective protection against H2O2 was due to inhibition of caspase 3 activation in Cx43 expressing cells. siRNA knockdown experiments in rat primary astrocytes confirmed the presence of endogenous Cx43-mediated anti-apoptotic effect. Cx43 interacts with the upstream apoptosis signal-regulating kinase 1 known to mediate H2O2-induced apoptosis providing a possible mechanism for protection. These findings provided new evidence for regulation of the mitogen activated protein kinase pathway and apoptosis by Cx43 implicating this protein in intracellular signaling beyond its role as a gap junction forming protein on the plasma membrane.

T-cadherin is a 95kDa glycoprotein member of the cadherin family of adhesion molecules attached to the extracellular surface of the cell membrane through a glycosyl-phosphatidylinositol (GPI)-anchor. Whether a T-cadherin ectodomain apical targeting signal or the GPI-anchor itself targets this protein to the apical membrane is not known. Chimeras of the reporter EGFP and T-cadherin have demonstrated that a minimal construct consisting of the C-terminal 25 amino acids including the N690 (omega-site) of T-cadherin was sufficient to GPI-anchor the EGFP protein. However, efficient GPI-anchor with minimal secretion of the protein required an additional 5 residues (omega-1 to omega-5). The GPI-anchored chimeras fractionated to the Triton X-100 detergent insoluble fraction and were released to the cell culture supernatant by phosphoinositide-specific phospho-lipase C digestion. When expressed in MDCK cells, all GPI-anchored chimeras targeted to the basolateral membrane, while the T/N-chimera and the wild-type T-cadherin targeted to the apical membrane. Therefore, T-cadherin is an example of another rare GPI-anchored protein where the anchor itself is not sufficient for apical targeting in MDCK cells.

We attempted to attenuate neointimal formation following vascular injury using a retroviral suicide vector. Epicardial coronary arteries of adult miniature swine were injured by deployment of oversized tantalum stents. One week later, injured segments were exposed to packaged retroviral constructs with or without the herpes simplex virus thymidine kinase gene. Ganciclovir treatments were initiated 48 hours later in both control and experimental swine and continued for two weeks. Four weeks following vascular injury, both control and experimental arteries were harvested and histologically prepared for image analysis. Despite adequate marker gene expression, there was no significant difference in the neointimal area or neointimal/media ratio between control and experimental groups. While the HSVtk ganciclovir system attenuates cell proliferation in other systems, retroviral vector targeting of vascular smooth muscle cells for elimination may be too inefficient to prevent restenosis following angioplasty.