CAMP INFLUENCE ON TRANSCRIPTION OF THROMBOMODULIN IS DEPENDENT ON DE-NOVO SYNTHESIS OF A PROTEIN INTERMEDIATE - EVIDENCE FOR COHESIVE REGULATION OF MYOGENIC PROTEINS IN VASCULAR SMOOTH-MUSCLE

We have previously shown that cyclic adenosine monophosphate (cAMP) in creases thrombomodulin (TM) mRNA and protein in vascular smooth muscle cells (VSMCs). The mechanism of that enhancement is now further defin ed. A time course evaluation of this effect by Northern blot analysis showed that exposure to the cAMP analog dibutyryl-cAMP and theophyllin e (CT) amplified TM mRNA sixfold by 3 hours. This effect was sustained through 9 hours and began to decline by 24 hours of CT exposure. In v itro exposure of VSMCs either to CT and actinomycin D or to actinomyci n D alone showed equivalent hall-lives for TM mRNA, This indicates tha t the increase in TM mRNA with CT supplementation was not the result o f enhanced mRNA stability. Nuclear run-off analysis of VSMCs grown in the presence of control or CT-supplemented medium showed that the incr ease in TM mRNA in VSMCs with CT exposure was transcriptional. CT expo sure was associated with an eightfold increase in measured TM transcri ption at 90 minutes. As previously reported, cAMP induced a decrease i n tropomyosin and in alpha-actin mRNA species, a change that parallele d the enhancement of TM. Thus cAMP enhances transcription of this anti thrombotic species white simultaneously causing diminished expression of these myogenic mRNA species. Addition of cycloheximide prevented th e cAMP-mediated increase in TM mRNA and curtailed the down-regulation of myogenic mRNA species, alpha-actin, and tropomyosin. This suggests that the cAMP-mediated down-regulation of some smooth muscle-specific mRNA, including tropomyosin mRNA and alpha-actin mRNA, like the enhanc ement of TM transcription, is dependent on de novo protein synthesis. We postulate a protein or a family of proteins, acting as transcriptio nal regulators, that coordinately govern the phenotype in VSMCs and ar e influenced by cAMP.