Estrogen Regulates the Synthesis of Epidermal Growth Factor in Mouse Uterine Epithelial Cells

Abstract

Immunocytochemical analyses, using several mouse epidermal growth factor (EGF) polyclonal antibodies, detected immunoreactivity only in uterine luminal and glandular epithelia on late proestrus, estrus, and early on day 1 of pregnancy, but not late on day 1. This immunoreactivity was not detected in the ovariectomized uterus, but after estrogen stimulation it was detected first in the luminal epithelium between 12-24 h and then also in the glandular epithelium by 48 h. After 72 h of estrogen withdrawal, EGF immunoreactivity was no longer detected. This response was specific for estrogen and did not occur after progesterone injection (2 mg/day for 4 days). Using antipeptide antibodies specific for prepro-EGF, no immunoreactivity was detected in the ovariectomized uterus, weak reactivity was detected in the estrogenized uterus and submandibular gland, and strong reactivity was detected in the kidney. Northern blot analysis of uterine RNA failed to detect the expected 4.8-kilobase prepro-EGF mRNA, but, instead, a rare transcript of 2.4 kilobases was detected, which suggests that EGF mRNA is alternately processed in the uterus. The presence of an EGF-coding uterine transcript was further documented by hybridization of an EGF-coding region-specific oligodeoxyribonucleotide (oligo) to polymerase chain reaction-amplified uterine cDNA. In situ hybridization, using a prepro-EGF cRNA probe as well as an EGF-coding region-specific oligo, showed hybridization that colocalized with the EGF immunostaining (epithelia) and was absent from non-EGF-immunoreactive cells. Pulse labeling experiments coupled with immunoaffinity chromatography showed that estrogen induced an increase in the relative rate of synthesis of an acid-soluble immunoreactive protein which was the same size as authentic EGF. Furthermore, analysis of acid-soluble uterine proteins fractionated by DEAE-cellulose chromatography demonstrated a single coincident peak of antigenic activity and receptor-binding activity which coeluted from the column with authentic EGF. Electron microscopy localized EGF immunoreactivity to the Golgi of luminal epithelial cells. Taken together these results suggest that estrogen regulates expression of the EGF gene specifically in uterine epithelial cells. Increased expression of this gene results in an increase in the relative rate of synthesis of this protein and the accumulation of mature EGF.