Sp1/Sp3 transcriptional regulation of the elastin gene in aortic smooth muscle cells.

摘要

Elastin mRNA levels increase more than 10-fold between days 2 and 8 in culture. Nuclear run-on analyses show that the increase in mRNA is accompanied by a comparable increase in transcription. Elastin gene transcription begins to increase after day 4 with a maximum increase occurring between days 6 and 8. Transient transfection of SMC with a −195 to +2 by human elastin promoter construct within this period of transcriptional activation suggests that the proximal promoter region plays a role in the upregulation. Mutation of Sp1/Sp3 sites localized to this region diminishes the increase in promoter activity of the −195 pCAT construct observed at day 8. Gel shift and Southwestern analyses suggest that an initial decrease in Sp3 and/or Sp1 binding to the proximal −152 to −118 by promoter sequence initiates the upregulation of elastin gene transcription. This decrease is followed by an increase in Sp3 and/or Sp1 binding between days 6 and 8. Western blot analyses show that the ratio of the phosphorylated (105 kDa) to non-phosphorylated forms (95 kDa) of Sp1 increases by day 6. Expression of full-length (115 kDa) and truncated (70 and 68 kDa) forms of Sp3 decrease initially and increase after day 6. Chromatin immunoprecipitation analyses of the endogenous rat elastin promoter demonstrate an increase in Sp3 binding during the 6–8 day transition and an increase in acetylated histone H3 binding from days 4–8. We hypothesize that Sp3 (115 kDa) acts as a transcriptional activator of elastin gene transcription by binding to the proximal promoter. This mediates the initial burst of elastin gene expression displayed by confluent smooth muscle cells. Previous studies have implicated Sp3 as a repressor of elastin transcription in day 14 cultures. Therefore, Sp3 appears to be an important factor that differentially regulates elastin gene transcription depending upon the state of the smooth muscle cell.