A NOVEL ESTROGEN-ENHANCED TRANSCRIPT IDENTIFIED IN THE RAT UTERUS BY DIFFERENTIAL DISPLAY

Estrogen exerts its physiological effects in the uterus by inducing a cascade of transcriptional events; however, the number of genes known to be directly activated by estrogen in the uterus is small. In this s tudy, immature ovariectomized rats were treated with estrogen or vehic le, and 3 h later the uterine horns were flushed to extract epithelial RNA. This RNA was used in the differential display technique to searc h for estrogen-responsive genes. Products of reverse transcriptase-PCR , made with pairs of arbitrary and oligo-deoxythymidine primers, were separated on denaturing polyacrylamide gels; candidate bands were exci sed and reamplified to produce probes for use in Northern blot analysi s and screening of a lambda gt10 complementary DNA library made from r at uterus. A novel estrogen-enhanced transcript, designated EET-1, was identified from a differential display band, and the estrogen sensiti vity of its expression was verified in Northern analysis. Characteriza tion of EET-1 expression in the uterus showed that estrogen treatment resulted in a rapid and transient increase in EET-1 messenger RNA; ste ady state levels peaked between 2-3 h, returning to basal levels by 6 h. This increase was not abolished by pretreatment with cycloheximide, indicating that induction of EET-1 is a primary response to estrogen. Induction was specific to estrogen when extracts of whole uterus were examined; in the epithelium, there was also a slight response to prog esterone. Expression of the gene was found in all organs surveyed; how ever, hormonal regulation was observed only in tissues of the reproduc tive tract and in the kidney. Analysis of cloned EET-1 complementary D NA revealed a 2008-base sequence that showed 61% identity with a repor ted transcript that encodes a protein that plays a role in phorbol est er-induced regulation of the tumor necrosis factor-alpha gene. Potenti al casein kinase-2 and protein kinase C phosphorylation sites and a cy steine-rich region were identified in the amino acid sequence deduced from EET-1. Thus, it appears that EET-1 represents a primary estrogen response gene that may code for a phosphorylated protein involved in g ene regulation through a protein kinase C-activated pathway.