2 PROXIMAL CARG ELEMENTS REGULATE SM ALPHA-ACTIN PROMOTER, A GENETIC-MARKER OF ACTIVATED PHENOTYPE OF MESANGIAL CELLS

Mesangial cells express smooth muscle alpha-actin (SM alpha-actin) in response to glomerular injury in vivo, and SM alpha-actin gene express ion serves as a genetic marker characterizing the activated phenotype of mesangial cells. We used a molecular genetic approach to analyze th e SM alpha-actin promoter and evaluate transcriptional mechanisms that might direct the genetic switch of mesangial cells to the activated p henotype. The sequence spanning -894 to +1 of the SM alpha-actin promo ter directed high levels of transcription that were attenuated in seru m-restricted cells and upregulated upon treatment with serum or endoth elin-1. Deletional analysis revealed a core promoter fragment, from po sitions -122 to +1, that was necessary and sufficient for transcriptio n. This core activity was modulated by upstream sequences between -670 and -122. The 122-bp core promoter contains two highly conserved CArG box motifs (designated CB1 and CB2), and introduction of deletion mut ations of either CB1 or CB2 reduced transcription in mesangial cells t o near basal levels. Further analysis revealed that CB1 and CB2 acted synergistically when subcloned upstream of a heterologous, minimal thy midine kinase promoter. CB2 alone was sufficient to confer serum induc ibility to a heterologous promoter, but both CB2 and CB1 were required for maximal levels of serum-induced transcription. Collectively, thes e results demonstrate that CB1 and CB2 cooperate to mediate serum-indu ced activation of the SM alpha-actin promoter in mesangial cells.