Insights into ectopic estrogen receptor expression, nucleocytoplasmic distribution and interaction with chromatin obtained with new antibodies to estrogen receptors α and ß.

Recent reports have indicated that in cells ectopically expressing only ERα or the full-length hormone-binding isoform of ERß (ERß1), the receptors interact with chromatin with different efficacies and that antibodies capable of probing such interactions by chromatin immunoprecipitation (ChIP) are scarce. We therefore produced nine subtype and isoform-specific antibodies to ERα or ERß and validated their performance in receptor probing in cell lines and tissue biopsies by various immunochemical methods, including ChIP. We also produced clones of HEK-293 cells stably transfected with an estrogen response element (ERE)-dependent luciferase reporter and ERα or ERß1, in order to comparatively study their interaction with reporter ERE. We show that ERα was located in the nucleus and ERß1 in the cytoplasm as well as the nucleus of the stably transfected cells, while both receptors were found predominantly in the nucleus in transiently transfected cells and in all estrogen target tissues examined using the same antibodies. The cells displayed wild-type transcriptional activity and canonical regulation of ERE-dependent luciferase expression by estrogen agonists and antagonists. However, unlike ERα, ERß1 recruitment to the reporter ERE could be probed only by sequential ChIP with antibodies to receptor N- and C-terminus. These data suggest that in HEK-293 cells stably expressing ERα or ERß1, ER subtype-specific constraints apply to ERß1 nuclear entry; and that in cells displaying cytoplasmic as well as nuclear localization of ERß1, sequential ChIP with different antibodies to the receptor is the method of choice for probing its interaction with chromatin.

Estrogenic activity of isoflavonoids from Onobrychis ebenoides.

Fractionation of the neutral extract of Onobrychis ebenoides (Leguminosae) yielded a new isoflavone, named ebenosin (1), in addition to the known ones, afrormosin (2), formononetin (3) and daidzein (4). Although the relative binding affinities of 1 - 4 for estrogen receptor alpha (ERalpha) were nearly comparable and matched those of 1-3 for ERbeta, that of 4 for the latter receptor was significantly higher than any of the other. Compounds 1 - 4 induced cell proliferation and gene expression in breast and endometrial cancer cells in an ER-dependent manner. Nonetheless, the rank order of induction potencies ( 4 > 3 >or= 2 >or= 1) matched better that of affinities for ERbeta ( 4 > 3 >or= 2 >or= 1) rather than ERalpha ( 4 >or= 3 >or= 2 >or= 1). While the antiestrogen ICI 182,780 could inhibit the induction of proliferation of ER-positive breast cancer cells by 1-4, it could not prevent 1 from exhibiting significant ER-independent cytotoxicity at 10 microM. By contrast, 1 was much less cytotoxic and only weakly estrogenic for ER-positive endometrial adenocarcinoma cells. In conclusion, our data suggest that the C-8 isoprenyl substituent of 1 renders it cytotoxic and/or estrogenic in a cell-dependent manner.

Pronounced enhancement of glucocorticoid-induced gene expression following severe heat shock of heat-conditioned cells hints to intricate cell survival tactics.

We have previously reported that severe heat shock of HeLa cells stably transfected with a chloramphenicol acetyltransferase (CAT) gene, transcription of which is controlled by two glucocorticoid-responsive elements and a minimal promoter, pronouncedly enhanced glucocorticoid-induced CAT expression compared to that of non-heated cells, in spite of the glucocorticoid-receptor-mediated transcription of the gene being temporarily compromised by the shock. We now report that prolonged severe heat shock of properly heat-conditioned cells resulted in far more pronounced enhancement of glucocorticoid-induced CAT mRNA and protein expressions, in spite of a similar heat-induced loss of receptor-mediated CAT gene transcription. During recovery from the shock the hormonal activation of transcription exceeded that of non-heated cells. While CAT mRNA translation was restored appreciably later than CAT gene transcription, mRNA and protein expressions were thermally enhanced to a comparable extent, consistent with the integrity of CAT mRNA being preserved during recovery. CAT mRNA turnover was fully impaired during early recovery, suggesting that stabilisation of CAT mRNA as well as stimulation of the hormonal activation of CAT gene transcription account for the thermal enhancement of glucocorticoid-induced CAT expression. This data hint to cell survival tactics designed to safeguard high expression of genes of stress-enduring function.

Maintenance of glucocorticoid receptor function following severe heat-shock of heat-conditioned cells.

The competence of the glucocorticoid receptor to regulate gene expression is thought to depend on Hsp70-driven continuous reactivation following spontaneous inactivation of its hormone-binding state. We show here that the glucocorticoid-binding capacity of HeLa cells fell with increasing temperature in the range 43-45 degrees C in a manner that closely paralleled the loss of soluble receptor protein. Receptor activity was maintained during moderate (43 degrees C) but not severe (45 degrees C) heat shock. Hsp70 was rapidly rendered insoluble and was replenished by soluble chaperone at 43 but not 45 degrees C. In heat-conditioned cells expressing different levels of Hsp70, we observed a positive correlation between the concentration of active receptor and the amount of Hsp70 rendered insoluble by heat shock. Much higher amounts of Hsp70 were rendered insoluble and receptor competence to regulate gene expression was preserved after severe heat shock of appropriately heat-conditioned cells. An excess of Hsp90 was found associated with resolubilized heat-inactivated receptor from severely heat-shocked cells. The data indicate that GR activity is maintained, provided that denaturation and/or aggregation of the receptor is prevented by Hsp70; and that the concentration of the chaperone is the limiting determinant of receptor activity in heat-shocked HeLa cells.