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

Abstract

We have previously shown that cyclic adenosine monophosphate (cAMP) increases thrombomodulin (TM) mRNA and protein in vascular smooth muscle cells (VSMCs). The mechanism of that enhancement is now further defined. A time course evaluation of this effect by Northern blot analysis showed that exposure to the cAMP analog dibutyryl-cAMP and theophylline (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 vitro exposure of VSMCs either to CT and actinomycin D or to actinomycin D alone showed equivalent half-lives for TM mRNA. This indicates that the increase in TM mRNA with CT supplementation was not the result of enhanced mRNA stability. Nuclear run-off analysis of VSMCs grown in the presence of control or CT-supplemented medium showed that the increase in TM mRNA in VSMCs with CT exposure was transcriptional. CT exposure was associated with an eightfold increase in measured TM transcription at 90 minutes. As previously reported, cAMP induced a decrease in tropomyosin and in alpha-actin mRNA species, a change that paralleled the enhancement of TM. Thus cAMP enhances transcription of this antithrombotic species while simultaneously causing diminished expression of these myogenic mRNA species. Addition of cycloheximide prevented the 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 enhancement of TM transcription, is dependent on de novo protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)