Estrogen action in vitro. Induction of the synthesis of a specific uterine protein.
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Animals↗
Biomedical subjects
B S Katzenellenbogen
Publications and source records attributed to B S Katzenellenbogen.
Estrogen regulation of human osteoblast function is determined by the stage of differentiation and the estrogen receptor isoform.
Although osteoblasts have been shown to respond to estrogens and express both isoforms of the estrogen receptor (ER alpha and ER beta), the role each isoform plays in osteoblast cell function and differentiation is unknown. The two ER isoforms are known to differentially regulate estrogen-inducible promoter-reporter gene constructs, but their individual effects on endogenous gene expression in osteoblasts have not been reported. We compared the effects of 17 beta-estradiol (E) and tamoxifen (TAM) on gene expression and matrix formation during the differentiation of human osteoblast cell lines stably expressing either ER alpha (hFOB/ER alpha 9) or ER beta (hFOB/ER beta 6). Expression of the appropriate ER isoform in these cells was confirmed by northern and western blotting and the responses to E in the hFOB/ER beta 6 line were abolished by an ER beta-specific inhibitor. The data demonstrate that (1) in both the hFOB/ER cell lines, certain responses to E or TAM (including alkaline phosphatase, IL-6 and IL-11 production) are more pronounced at the late mineralization stage of differentiation compared to earlier stages, (2) E exerted a greater regulation of bone nodule formation and matrix protein/cytokine production in the ER alpha cells than in ER beta cells, and (3) the regulated expression of select genes differed between the ER alpha and ER beta cells. TAM had no effect on nodule formation in either cell line and was a less potent regulator of gene/protein expression than E. Thus, both the ER isoform and the stage of differentiation appear to influence the response of osteoblast cells to E and TAM.
Alkaline Phosphatase↗
[125I]iododesethyl tamoxifen aziridine: synthesis and covalent labeling of the estrogen receptor with an iodine-labeled affinity label.
Iododesethyl tamoxifen aziridine (I-Tam-Az), an analog of the estrogen receptor-affinity label tamoxifen aziridine (Tam-Az) in which the ethyl group has been replaced by an iodine, has been prepared by two routes: (a) metallation of a bromotriarylethylene system, followed by reaction with iodine, and aziridinylation, and (b) direct iodination of a trimethylstannyl triarylethylene system that is the immediate precursor of I-Tam-Az. The latter method can be used to prepare [125I]I-Tam-Az rapidly and in good yield, both at carrier-added and no-carrier-added levels; specific activities greater than 200 Ci/mmol have been obtained. In competitive radiometric binding assays with the estrogen receptor, I-Tam-Az has an apparent affinity of ca. 20%, equivalent to that of Tam-Az. It also undergoes rapid and selective time-dependent, irreversible binding to the estrogen receptor. [125I]I-Tam-Az reacts covalently with estrogen receptor in uterine cytosol preparations; its attachment is rapid and efficient, but somewhat less selective than that of Tam-Az. Estrogen receptor in intact MCF-7 human breast cancer cells can also be labeled with [125I]I-Tam-Az, and autoradiographic analysis of salt extracts of labeled nuclear estrogen receptor on SDS-polyacrylamide slab gels shows highly selective labeling of a 65K protein. [125I]I-Tam-Az is an efficient, selective affinity label for the estrogen receptor, available at high specific activity, and should be useful in studies on estrogen receptor structure, dynamics, and chromatin interactions.
Affinity Labels↗
Stilbestrols and stilbestrol derivatives: estrogenic potency and temporal relationships between estrogen receptor binding and uterine growth.
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Animals↗
Determinants for the repression of estrogen receptor transcriptional activity by ligand-occupied progestin receptors.
There is considerable evidence for cross-talk between the estrogen and progestin signaling pathways, including examples of repression or attenuation of estrogen-stimulated endpoints by progestin receptor (PR) agonists and antagonists. We have previously described an experimental system for examining aspects of this cross-talk, namely the repression of estrogen receptor (ER) transcriptional activity by liganded PR (Kraus, W. L., Weis, K. E., Katzenellenbogen, B. S., Mol. Cell. Biol. 15 (1995) 1847-1857). Under promoter and cell type conditions where liganded PR was not a good activator of transcription, PR isoforms were shown to act as potent ligand-dependent repressors of ER transcriptional activity. In the current study, we have identified multiple determinants of this repression by systematically manipulating potentially important variables in this system (e.g. PR A:PR B ratio, sequence of the response elements, receptor structure, and ligand type). Alterations in several of these parameters had profound effects on the ability of PR to repress the activity of ER. Decreases in the PR A:PR B ratio and changes in the sequence of the progestin response element in the reporter gene construct abolished the repressive action of agonist-occupied PR A on ER transcriptional activity. In addition, point or deletion mutations in the amino-terminal A/B region of ER, including a triple point mutation which eliminates phosphorylation sites previously shown to be important in the activity of the receptor, made the ER more sensitive to the repressive actions of liganded PR. The PR ligands that promoted the most potent repression of ER activity were those with 11beta phenyl substitutions, suggesting that the phenyl moiety in the 11beta position is the important structural feature leading to strong repression. Interestingly, changes in the structure of the ER ligand and the sequence of the estrogen response element did not influence the magnitude of repression by PR. The fact that alterations in these check points along the estrogen signaling pathway had little or no effect on the magnitude of repression suggests that liganded PR interferes with the ability of ER to interact productively with the transcriptional machinery; in other words, PR-mediated repression occurs downstream of the events leading to the ligand-dependent conversion of ER to a transcriptionally active form. Our results indicate that a number of parameters which are naturally varied in vivo, such as the sequence of PR DNA binding sites and the PR A:PR B ratio, can dramatically alter the repression of ER activity by liganded PR, and may explain the differential affects of progestin-occupied PR on the expression of different estrogen regulated genes.
3T3 Cells↗
Mechanisms of action and cross-talk between estrogen receptor and progesterone receptor pathways.
The intriguing biology of estrogens and progestins in their diverse target cells is determined by the structure of the hormonal ligand, the receptor subtype or isoform involved, the nature of the hormone-responsive gene promoter, and the character and balance of coactivators and corepressors that modulate the cellular response to the receptor-ligand complex. Estrogens regulate the growth, differentiation, and functioning of diverse target tissues, both within and outside of the reproductive system. Most of the actions of estrogens appear to be exerted through the estrogen receptor (ER) of target cells, an intracellular receptor that is a member of a large superfamily of proteins, which function as ligand-activated transcription factors, regulating the synthesis of specific RNAs and proteins. To understand how the ER discriminates between estrogen ligands, which activate the ER, and antiestrogen ligands, which fail to effectively activate the ER, we have generated and analyzed human ERs with mutations or other alterations in portions of the receptor. These studies provide evidence for the promoter-specific and cell-specific actions of the estrogen-occupied and antiestrogen-occupied ER, highlight a regional dissociation of the hormone-binding and transcription activation functions in domain E of the receptor, and indicate that some of the contact sites of estrogens and antiestrogens in the ER are likely different. In addition, multiple interactions among different cellular signaling pathways are involved in the regulation of gene expression and cell proliferation by the ER. In several cell types, protein kinase activators and some growth factors enhance the transcriptional activity of the ER. Cyclic AMP also alters the agonist/antagonist balance of some antiestrogens. Estrogens, and antiestrogens to a lesser extent, as well as protein kinase activators and growth factors, increase phosphorylation of the ER and possibly other proteins involved in the ER-specific response pathway, suggesting that changes in cellular phosphorylation state will be important in determining the biologic activity of the ER and the effectiveness of antiestrogens as estrogen antagonists. The ER also has important interrelationships with the progesterone receptor (PR) system in modulation of biologic responses. Liganded PR-A and PR-B can each suppress estradiol-stimulated ER activity, with the magnitude of repression dependent on the PR isoform, progestin ligand, promoter, and cell type. These findings underscore the mounting evidence for the importance of interactions between members of the steroid hormone receptor family.
Animals↗