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Differential-display polymerase chain reaction identifies nucleophosmin as an estrogen-regulated gene in human
1Department of Medicine, Molecular Cardiology Research Center, Tufts University Medical School, Boston, MA, USA.
Purpose:
Atheroprotective effects of estrogen (E2) are well documented and are due in part to direct effects of E2 on vascular cells. We recently demonstrated that human vascular smooth muscle cells (VSMCs) express a functional E2 receptor capable of activatine gene expression. This study was designed to identify E2-regulated genes in human VSMCs.
Methods:
VSMC mRNA was screened by differential-display polymerase chain reaction (ddPCR) to identify E2-regulated genes. Quiescent human VSMCs were stimulated with 10% fetal bovine serum in the absence or presence of 10(-8) mol/L E2, 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 were confirmed by dot blotting and positive bands were identified by sequence analysis. E2-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 E2- 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 E2 treatment (p < 0.05 vs control). Sequence analysis identified EAo8 as nucleophosmin, a nucleolar phosphprotein implicated in the regulation of cell growth and protein synthesis. E2-induced expression of nucleophosmin protein was demonstrated by immunoblotting in both saphenous vein and mammary artery VSMCs.
Conclusions:
E2 induces nucleophosmin expression in human VSMCs, and ddPCR is a useful approach for studying estrogen-regulated gene expression in VSMCs.