A novel estrogen-enhanced transcript identified in the rat uterus by differential display

Lynn M. Everett, Aixin Li, Gayatri Devaraju, Andrea Caperell-Grant, Robert Bigsby

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

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 Agt10 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-α gene. Potential casein kinase-2 and protein kinase C phosphorylation sites and a cysteine-rich region were identified in the amine 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.

Original languageEnglish
Pages (from-to)3836-3841
Number of pages6
JournalEndocrinology
Volume138
Issue number9
DOIs
StatePublished - 1997

Fingerprint

Uterus
Estrogens
Genes
Protein Kinase C
Complementary DNA
RNA
Casein Kinase II
Phorbol Esters
Cycloheximide
Reverse Transcriptase Polymerase Chain Reaction
Gene Library
Northern Blotting
Thymidine
Amines
Progesterone
Cysteine
Proteins
Epithelium
Tumor Necrosis Factor-alpha
Phosphorylation

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

A novel estrogen-enhanced transcript identified in the rat uterus by differential display. / Everett, Lynn M.; Li, Aixin; Devaraju, Gayatri; Caperell-Grant, Andrea; Bigsby, Robert.

In: Endocrinology, Vol. 138, No. 9, 1997, p. 3836-3841.

Research output: Contribution to journalArticle

Everett, Lynn M. ; Li, Aixin ; Devaraju, Gayatri ; Caperell-Grant, Andrea ; Bigsby, Robert. / A novel estrogen-enhanced transcript identified in the rat uterus by differential display. In: Endocrinology. 1997 ; Vol. 138, No. 9. pp. 3836-3841.
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