Abstract 473: Estradiol inhibits Arterial Smooth Muscle Cell Migration by Regulating Osteopontin Expression
Background: Several studies indicate that estrogens exert various beneficial effects on the vascular wall, namely the inhibition of arterial smooth muscle cell (SMC) migration involved in intimal lesion formation. We previously demonstrated that the extracellular nucleotide UTP induces SMC migration via osteopontin (OPN) production. The aim of this study is to identify the effects of 17 beta-estradiol (E2) both on the UTP-induced SMC migration and on the regulation of the OPN gene transcription.
Methods and results: Addition of E2 (10−8 or 10−10 M) to the culture medium inhibits UTP-induced SMC migration assayed in the Transwell system (58%±2 and 45%±1 respectively). Moreover, E2 also inhibits UTP-induced OPN expression (mRNA and protein). To understand the molecular mechanisms of this regulation, gene reporter assays using different OPN promoter constructions are performed. We first demonstrated that the three estrogen response elements (SFRE-like site) previously described on OPN promoter are not involved in E2 inhibition. In addition to classical effects of estrogens on the Estrogen Responsive Element, it has been recently reported that they can also act via the formation of a complex between estrogen receptor and other transcription factors. In the study we show that E2 loses its entire inhibitory effect when either the -76 AP-1 or the -1768 Ebox are mutated. This lost is also obtained when AP-1 or Ebox sites are inhibited by A-Fos or A-USF dominant negative forms respectively. Since same effects are obtained when each site is mutated individually, we hypothesize that AP-1 and USF could interact. This hypothesis is verified by chromatin immunoprecipitation assay demonstrating that a c-Fos antibody immunoprecipitates the -1768 Ebox sequence in UTP-stimulated cells and that this interaction is abolished by E2.
Conclusion: This study shows for the first time that E2 concomitantly inhibits UTP-induced SMC migration and OPN expression. Moreover, it strongly suggests that the interaction between a bHLHZip factor (USF) and a bZip factor (c-Fos) is necessary for OPN expression and is the target for E2 effect.