Studies on calcium(2+) signaling in corneal endothelial cells and molecular characterization of a novel apoptosis-inducing factor like gene.

摘要

The corneal endothelium maintains corneal transparency. Calcium signaling is important for corneal function. I explored calcium signaling in bovine corneal endothelial cells (BCEC) and cloned a novel apoptosis-inducing factor like gene (AIFL). A combinatorial approach was taken to investigate the mechanism of CCE. Induction of P-450s by beta-naphthoflavone (BN) enhanced CCE after store depletion. The BN-enhanced CCE was inhibited by a CCE blocker, indicating a role for P-450s in CCE. Treatment with calyculin A, which causes condensation of cortical cytoskeleton, inhibited CCE, suggesting a secretion-like coupling mechanism. Since transient receptor potential channel 4 (TRPC4) was a putative candidate for CCE or receptor operated calcium entry (ROC), I examined the expression of TRPC4 and evaluated the potential involvement of TRPC4 in CCE or ROC. An antibody was raised by using a purified GST-TRPC4C antigen. The TRPC4 antibody detected the expression of TRPC4 in BCEC. RNAi knocked down the expression of TRPC4 in cultured BCEC. Ca2+ entry induced by the purinergic receptor agonist ATP, was increased in TRPC4-siRNA transfected cells compared with the scrambled siRNA control, while TRPC4-siRNA did not affect CCE. Taken together, (1) an integrative model is suggested that both calcium influx factor (CIF) from cytochrome P-450 metabolism and secretion-like coupling mechanisms play roles in CCE in BCEC; (2) TRPC4 protein is expressed in BCEC and may negatively regulate ROC. I also cloned and characterized a novel gene: AIFL. RT-PCR analysis and immunohistochemistry showed the expression of AIFL in all tissues examined. AIFL is localized to the mitochondria. Overexpression of AIFL induced apoptosis, as shown by increased cytoplasmic nucleosomes and subdiploid cell populations in AIFL transfected cells. In conclusion, AIFL is a novel apoptogenic protein.