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 document ed 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 express ion. This study was designed to identify E(2)-regulated genes in human VSMCs. Methods: VSMC mRNA was screened by differential-display polyme rase chain reaction (ddPCR) to identify E(2)-regulated genes. Quiescen t human VSMCs were stimulated with 10% fetal bovine serum in the absen ce or presence of 10(-8) mol/L E(2), and total cellular RNA was harves ted. ddPCR was performed in triplicate on each RNA sample and differen tially expressed candidate genes were isolated. Differential expressio n 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 R NA 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 in crease in EAo8 expression in response to E(2) treatment (p < 0.05 vs c ontrol). Sequence analysis identified EAo8 as nucleophosmin, a nucleol ar phosphoprotein implicated in the regulation of cell growth and prot ein synthesis. E(2)-induced expression of nucleophosmin protein was de monstrated by immunoblotting in both saphenous vein and mammary artery VSMCs. Conclusions: E(2) induces nucleophosmin expression in human VS MCs, and ddPCR is a useful approach for studying estrogen-regulated ge ne expression in VSMCs.