Dynamics of estrogen receptor turnover in uterine cells in vitro and in uteri in vivo

Ann M. Nardulli, Benita S Katzenellenbogen

Research output: Contribution to journalArticle

Abstract

The rate at which the estrogen receptor (ER) is synthesized and degraded (turns over) in target cells is a major factor regulating the levels of ER and cell sensitivity to estrogen. We have used the ability of tamoxifen aziridine [(TAZ)(Z)-l-[4-(2-[N-aziridinyl]ethoxy)phenyl]1,2-diphenyl-l-butene] to affinity label the ER in intact cells to study the dynamics of ER turnover in uterine cells in vitro and in intact rat uteri in vivo. In primary cultures of rat uterine cells (from 21-day-old rats), ER is degraded with a half-life of 3–4 h, as determined by pulse-chase experiments in which ER in cells are covalently labeled with [3H]TAZ (20 nm) and then exposed to a chase of 10−6 m estradiol. Density shift experiments, in which uterine cells are exposed for different time periods to medium containing dense (15N,13C,2H) amino acids and the shift of receptor from a normal density to a more dense species is analyzed on sucrose gradients, also confirm this rapid turnover for the uterine ER. A similar half-life is obtained for ER in rat uteri in vivo, after intralumenal installation of [3H]TAZ to label effectively ER covalently. Analysis of nuclear ER on sodium dodecyl sulfate-polyacrylamide gels during the chase period reveals the loss of the 65,000 mol wt [3H]TAZ-labeled receptor species; no lower mol wt [3H]TAZ-labeled fragments are observed. Likewise, immunoblot analyses of receptor with the ER monoclonal antibody H222Sp7 reveal only the 65,000 mol wt receptor species, with no evidence of any smaller receptor forms in cells. Interestingly, incubation of uterine cells with 10−5 m cycloheximide (which inhibits 98% of protein synthesis) completely arrests ER turnover, suggesting that ER turnover, which is rapid, is probably dependent upon the synthesis of other rapidly turning over proteins. Hence, we conclude that ER turnover is rapid in uterine cells, that turnover of uterine ER occurs at a rate similar to that previously reported for ER turnover in MCF-7 human breast cancer cells, and that ER turnover is similar in uterine cells under in vitro and in vivo conditions. These findings indicate that the receptor is a dynamic and rapidly turning over protein whose level could be adjusted quickly by alterations in the rate of synthesis and/or degradation.

Original languageEnglish (US)
Pages (from-to)2038-2046
Number of pages9
JournalEndocrinology
Volume119
Issue number5
DOIs
StatePublished - Nov 1986

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Estrogen Receptors
Uterus
In Vitro Techniques
Half-Life
Affinity Labels
Amino Acid Receptors
Proteins
Cycloheximide
Sodium Dodecyl Sulfate
Sucrose
Estradiol
Estrogens

ASJC Scopus subject areas

  • Endocrinology

Cite this

Dynamics of estrogen receptor turnover in uterine cells in vitro and in uteri in vivo. / Nardulli, Ann M.; Katzenellenbogen, Benita S.

In: Endocrinology, Vol. 119, No. 5, 11.1986, p. 2038-2046.

Research output: Contribution to journalArticle

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abstract = "The rate at which the estrogen receptor (ER) is synthesized and degraded (turns over) in target cells is a major factor regulating the levels of ER and cell sensitivity to estrogen. We have used the ability of tamoxifen aziridine [(TAZ)(Z)-l-[4-(2-[N-aziridinyl]ethoxy)phenyl]1,2-diphenyl-l-butene] to affinity label the ER in intact cells to study the dynamics of ER turnover in uterine cells in vitro and in intact rat uteri in vivo. In primary cultures of rat uterine cells (from 21-day-old rats), ER is degraded with a half-life of 3–4 h, as determined by pulse-chase experiments in which ER in cells are covalently labeled with [3H]TAZ (20 nm) and then exposed to a chase of 10−6 m estradiol. Density shift experiments, in which uterine cells are exposed for different time periods to medium containing dense (15N,13C,2H) amino acids and the shift of receptor from a normal density to a more dense species is analyzed on sucrose gradients, also confirm this rapid turnover for the uterine ER. A similar half-life is obtained for ER in rat uteri in vivo, after intralumenal installation of [3H]TAZ to label effectively ER covalently. Analysis of nuclear ER on sodium dodecyl sulfate-polyacrylamide gels during the chase period reveals the loss of the 65,000 mol wt [3H]TAZ-labeled receptor species; no lower mol wt [3H]TAZ-labeled fragments are observed. Likewise, immunoblot analyses of receptor with the ER monoclonal antibody H222Sp7 reveal only the 65,000 mol wt receptor species, with no evidence of any smaller receptor forms in cells. Interestingly, incubation of uterine cells with 10−5 m cycloheximide (which inhibits 98{\%} of protein synthesis) completely arrests ER turnover, suggesting that ER turnover, which is rapid, is probably dependent upon the synthesis of other rapidly turning over proteins. Hence, we conclude that ER turnover is rapid in uterine cells, that turnover of uterine ER occurs at a rate similar to that previously reported for ER turnover in MCF-7 human breast cancer cells, and that ER turnover is similar in uterine cells under in vitro and in vivo conditions. These findings indicate that the receptor is a dynamic and rapidly turning over protein whose level could be adjusted quickly by alterations in the rate of synthesis and/or degradation.",
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