Human Endometrial cells in primary tissue culture: Modulation of the progesterone receptor level by natural and synthetic estrogens in vitro

Richard L. Eckert, Benita S Katzenellenbogen

Research output: Contribution to journalArticle

Abstract

Primary cultures of human endometrial cells were prepared from surgical biopsy specimens (premenopausal) and used to examine the direct regulatory effects of estrogen on the progesterone receptor (PR) level in vitro. Cells were grown as monolayer cultures in Dulbecco's modified Eagle's medium with 10% steroid-depleted serum from castrate adrenalectomized calves. Maximal stimulation of the PR level was obtained after a 3- to 4-day incubation with 10 nM estradiol. A dose-dependent increase in PR was produced by estradiol, with half-maximal stimulation at 1 nM added hormone and optimal increases (up to 7-fold) at 10 nM. The PR had a K d of 2.5 ± 0.4 × 10 −10 M, as determined by Scatchard analysis using17, 21-dimethyl-19-norpregna- 4, 9-diene-3, 20-dione ([ 3 H]R5020), and sedimentedpredominantly as a 7S species on low salt sucrose density gradients. Estrogen stimulation increased the number of progestin-binding sites without altering the receptor affinity. A variety of estrogens, including the synthetic zearalanol estrogen P1496 [6-(6, 10-dihydroxyundecyl)β- resorcylic acid μ-lactone] and the antiestrogen CI628 [α-(4- pyrrolidinoethoxy) phenyl-4-methoxy-α -nitrostilbene] increased PR levels. Dose-response studies indicate the effectiveness in stimulating the level of progesterone receptor to be P1496 = 17β-estradiol ≥ 17α-estradiol = estriol = estrone » CI628. Stimulation of PR correlated with the accumulation of estrogen receptor sites in the nucleus. An understanding of the relative potencies of the different estrogens and CI628 in PRstimulation has been obtained by assessing their affinities for estrogen receptor, theirmetabolism by the cell cultures, and the extent of serum binding of each compound in theculture medium. Their relative affinities for the cytoplasmic estrogen receptor, determined by competitive binding analyses, were: P1496, 30% estrone, 28% 17α-estradiol, 18% estriol, 12% and CI628, 9%, where 17β-estradiolis set at 100%. 17β-Estradiol was extensively (<90% by24 h) metabolized to estrone by the cell cultures, with little conversion of estrone (=5%) to estradiol. However, despite marked metabolism to estrone, analyses of nuclear associated radioactivity at 24 h still showed substantial nuclear estradiol localization. Likewise, the low level of estradiol generated in the medium from cells incubated with estrone was substantially sequestered in the nucleus. No metabolic conversionof the other compounds was detected. Equilibrium dialysis measurements revealed marked binding of compounds to serum components in the culture medium (CI628, <90%bound; 17β-estradiol, 17α-estradiol, P1496, and estrone, 70–80% bound; estriol, 30% bound), such that the biologically effective (free) concentration of each was substantially less than the added concentration. These studies provide direct evidence that physiological concentrations of a variety of estrogens promote dose-dependent increases in PR levels in human endometrial cells and that these increases are correlated with increases in the nuclear accumulation of estrogen receptor sites. This system should provide a useful in vitro modelfor the study of human uterine function.

Original languageEnglish (US)
Pages (from-to)699-708
Number of pages10
JournalJournal of Clinical Endocrinology and Metabolism
Volume52
Issue number4
DOIs
StatePublished - Apr 1981

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Biochemistry
  • Endocrinology
  • Clinical Biochemistry
  • Biochemistry, medical

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