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 study, immature ovariectomized rats were treated with estrogen or vehicle, and 3 h later the uterine horns were flushed to extract epithelial RNA. This RNA was used in the differential display technique to search 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 excised and reamplified to produce probes for use in Northern blot analysis and screening of a lambda gt10 complementary DNA library made from rat uterus. A novel estrogen-enhanced transcript, designated EET-1, was identified from a differential display band, and the estrogen sensitivity of its expression was verified in Northern analysis. Characterization of EET-1 expression in the uterus showed that estrogen treatment resulted in a rapid and transient increase in EET-1 messenger RNA; steady 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 progesterone. Expression of the gene was found in all organs surveyed; however, hormonal regulation was observed only in tissues of the reproductive tract and in the kidney. Analysis of cloned EET-1 complementary DNA revealed a 2008-base sequence that showed 61% identity with a reported transcript that encodes a protein that plays a role in phorbol ester-induced regulation of the tumor necrosis factor-alpha gene. Potential casein kinase-2 and protein kinase C phosphorylation sites and a cysteine-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 gene regulation through a protein kinase C-activated pathway.
Everett
L M
LM
Department of Obstetrics and Gynecology, Indiana University School of Medicine, Indianapolis 46202-5196, USA.
Li
A
A
Devaraju
G
G
Caperell-Grant
A
A
Bigsby
R M
RM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D018076:DNA, Complementary / Q000737:chemistry; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D010052:Ovariectomy; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics*; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017385:Sequence Homology; D014599:Uterus / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism
D018076:DNA, Complementary; C107874:Litaf protein, rat; D010750:Phosphoproteins; D012333:RNA, Messenger; D004958:Estradiol