G A Gonzalez1, S T Feldman. 1. Department of Ophthalmology, University of California/San Diego, La Jolla 92093.
Abstract
PURPOSE: Agents that increase intracellular levels of cAMP mediate gene expression associated with cellular morphology, growth, and/or differentiation via the cAMP response element (CRE). The cAMP element binding protein (CREB) is a transcriptional activator that binds and stimulates gene expression from the CRE in the promoters of cAMP responsive genes. This study was designed to characterize the cyclic AMP (cAMP) transcription apparatus in bovine corneal endothelial cells (BCE). METHODS: CRE transcriptional activity was determined by transient transfection 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-binding assay using gel electrophoresis and DNase I protection assays were performed to exclude the possibility of other CRE-binding factors. RESULTS: The authors identified the transcription factor, CREB, in nuclear extracts from BCE by Western blot analysis and showed that its DNA-binding characteristics are identical to the previously characterized CREB protein by DNase I protection and mobility shift DNA-binding studies. Transient transfection studies using the CRE-CAT reporter constructs revealed that the beta-adrenergic receptor agonist, isoproterenol, stimulates gene expression to levels similar to those induced by forskolin, a direct activator of adenylate cyclase (6.0- and 7.2-fold, respectively). CONCLUSIONS: The results suggest that agents that modulate receptors coupled to adenylate cyclase may effect the corneal endothelium by altering gene expression through the second messenger, cAMP.
PURPOSE: Agents that increase intracellular levels of cAMP mediate gene expression associated with cellular morphology, growth, and/or differentiation via the cAMP response element (CRE). The cAMP element binding protein (CREB) is a transcriptional activator that binds and stimulates gene expression from the CRE in the promoters of cAMP responsive genes. This study was designed to characterize the cyclic AMP (cAMP) transcription apparatus in bovine corneal endothelial cells (BCE). METHODS: CRE transcriptional activity was determined by transient transfection 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-binding assay using gel electrophoresis and DNase I protection assays were performed to exclude the possibility of other CRE-binding factors. RESULTS: The authors identified the transcription factor, CREB, in nuclear extracts from BCE by Western blot analysis and showed that its DNA-binding characteristics are identical to the previously characterized CREB protein by DNase I protection and mobility shift DNA-binding studies. Transient transfection studies using the CRE-CAT reporter constructs revealed that the beta-adrenergic receptor agonist, isoproterenol, stimulates gene expression to levels similar to those induced by forskolin, a direct activator of adenylate cyclase (6.0- and 7.2-fold, respectively). CONCLUSIONS: The results suggest that agents that modulate receptors coupled to adenylate cyclase may effect the corneal endothelium by altering gene expression through the second messenger, cAMP.