Differential-display polymerase chain reaction identifies nucleophosmin as an estrogen-regulated gene in human vascular smooth muscle cells

Purpose: Atheroprotective effects of estrogen (E 2) are well documented and are due in part to direct effects of E 2 on vascular cells. We recently demonstrated that human vascular smooth muscle cells (VSMCs) express a functional E 2 receptor capable of activating gene expression. This study was designed to identify E 2-regulated genes in human VSMCs. Methods: VSMC mRNA was screened by differential-display polymerase chain reaction (ddPCR) to identify E 2-regulated genes. Quiescent human VSMCs were stimulated with 10% fetal bovine serum in the absence or presence of 10 −8 mol/L E 2, and total cellular RNA was harvested. ddPCR was performed in triplicate on each RNA sample and differentially expressed candidate genes were isolated. Differential expression of candidate genes was confirmed by dot blotting, and positive bands were identified by sequence analysis. E 2-mediated regulation at the protein level was investigated by immunoblotting. Results: ddPCR of RNA harvested from aortic VSMCs identified a 462 bp gene product, EAo8, as a candidate E 2-regulated gene. Dot blotting with probes derived from four independent VSMC cultures demonstrated a 5.8±3.9-fold increase in EAo8 expression in response to E 2 treatment ( p