CYCLIC-AMP MEDIATED GENE-EXPRESSION IN BOVINE CORNEAL ENDOTHELIAL-CELLS

Purpose. Agents that increase intracellular levels of cAMP mediate gen e expression associated with cellular morphology, growth, and/or diffe rentiation via the cAMP response element (CRE). The cAMP element bindi ng protein (CREB) is a transcriptional activator that binds and stimul ates gene expression from the CRE in the promoters of cAMP responsive genes. This study was designed to characterize the cyclic AMP (cAMP) t ranscription apparatus in bovine corneal endothelial cells (BCE). Meth ods. CRE transcriptional activity was determined by transient transfec tion assays using the CRE-chloramphenicol acetyl transferase gene (CRE -CAT) fusion reporter construct. Western blot analyses were performed to determine whether CREB was present in BCE. Mobility shift DNA-bindi ng assay using gel electrophoresis and DNase I protection assays were performed to exclude the possibility of other CRE-binding factors. Res ults. The authors identified the transcription factor, CREB, in nuclea r extracts from BCE by Western blot analysis and showed that its DNA-b inding characteristics are identical to the previously characterized C REB protein by DNase I protection and mobility shift DNA-binding studi es. Transient transfection studies using the CRE-CAT reporter construc ts revealed that the beta-adrenergic receptor agonist, isoproterenol, stimulates gene expression to levels similar to those induced by forsk olin, a direct activator of adenylate cyclase (6.0- and 7.2-fold, resp ectively). Conclusions. The results suggest that agents that modulate receptors coupled to adenylate cyclase may effect the corneal endothel ium by altering gene expression through the second messenger, cAMP.