01 RHOA-DEPENDENT REGULATION OF CYR61 GENE EXPRESSION INVOLVES AP-1- AND CREB-DEPENDENT PROMOTER ACTIVATION IN SMOOTH MUSCLE CELLS

B. Chaqour. Dept of Anatomy and Cell Biology, University of Pennsylvania, Philadelphia, PA, USA

We have previously demonstrated that mechanical strain strongly upregulates the expression of the Cyr61 gene in cultured smooth muscle cells and suggested a potential key role of the small G-protein RhoA in strain-induced Cyr61 gene expression. Since the activation of the RhoA signaling pathway is a critical mechanism for regulating smooth muscle cell differentiation, expression of the Cyr61 gene is probably involved in the progression of these cells to hypertrophy. In this study, we sought to investigate further the mechanisms whereby RhoA activation regulates Cyr61 gene expression and the signaling machinery involved in such a process.

First, we showed that constitutive expression of an active form of RhoA increased endogenous levels of Cyr61 mRNA in a dose-dependent manner. Similarly, incubation of the cells with exogenous inducers of Rho activation such as sphingosine 1-phosphate (S1P) or lysophosphatidic acid (LPA) upregulate the expression of the Cyr61 gene at both the mRNA and protein levels. Y-27632, a well known RhoA inhibitor, abolished their effects. When actinomycin D was added to the cells concomitantly with either S1P or LPA, the decay in Cyr61 transcripts was not altered, indicating the lack of independent effects on transcript stability. Nuclear run-on analysis further indicated that RhoA-dependent Cyr61 gene expression occurs at the level of transcription.

We further studied the transcriptional requirements for RhoA-induced Cyr61 gene expression by using a construct containing a 936-bp DNA fragment of the promoter region of the human Cyr61 gene cloned upstream of a CAT reporter. The relative CAT activities driven by this promoter region were measured in transiently transfected cells. We showed that coexpression of constitutively active RhoA but not Rac 1 or cdc 42, significantly increased (11- to 15-fold) the activity of the Cyr61 promoter. The latter …