Vitamin D-interacting protein 205 (DRIP205) coactivation of estrogen receptor alpha (ERalpha) involves multiple domains of both proteins

Vitamin D-interacting protein 205 (DRIP205) is a mediator complex protein that anchors the complex to the estrogen receptor (ER) and other nuclear receptors (NRs). In ZR-75 breast cancer cells treated with 17beta-estradiol (E2) and transfected with a construct containing three tandem estrogen responsive elements (pERE(3)), DRIP205 coactivates ERalpha-mediated transactivation. DRIP205Delta587-636 is a DRIP205 mutant in which both NR boxes within amino acids 587-636 have been deleted and, in parallel transfection studies, DRIP205Delta587-636 also coactivates ERalpha. Moreover, both wild-type and variant DRIP205 also colocalize with ERalpha in the nuclei of transfected cells. Extensive deletion analysis of DRIP205 shows that multiple domains of this protein play a role in coactivation of ERalpha and in interactions with ERalpha. Coactivation of ERalpha by DRIP205 does not require NR boxes, and variants with deletion of N-terminal (amino acids 1-639) and C-terminal (amino acids 576-1566) significantly coactivate ERalpha. DRIP205 resembles p160 coactivators that also interact with multiple regions of ERalpha; however, unlike p160 coactivators, DRIP205 coactivation of ERalpha does not require NR boxes.     

Calmodulin is a selective modulator of estrogen receptors

In the search for differences between ERalpha and ERbeta, we analyzed the interaction of both receptors with calmodulin (CaM) and demonstrated that ERalpha but not ERbeta directly interacts with CaM. Using transiently transfected HeLa cells, we examined the effect of the CaM antagonist N-(6-aminohexyl)-5-chloro-naphthalene sulfonilamide hydrochloride (W7) on the transactivation properties of ERalpha and ERbeta in promoters containing either estrogen response elements or activator protein 1 elements. Transactivation by ERalpha was dose-dependently inhibited by W7, whereas that of ERbeta was not inhibited or even activated at low W7 concentrations. In agreement with these results, transactivation of an estrogen response element containing promoter in MCF-7 cells (which express a high ERalpha/ERbeta ratio) was also inhibited by W7. In contrast, transactivation in T47D cells (which express a low ERalpha/ERbeta ratio) was not affected by this CaM antagonist. The sensitivity of MCF-7 cells to W7 was abolished when cells were transfected with increasing amounts of ERbeta, indicating that the sensitivity to CaM antagonists of estrogen-responsive tissues correlates with a high ERalpha/ERbeta ratio. Finally, substitution of lysine residues 302 and 303 of ERalpha for glycine rendered a mutant ERalpha unable to interact with CaM whose transactivation activity became insensitive to W7. Our results indicate that CaM antagonists are selective modulators of ER able to inhibit ERalpha-mediated activity, whereas ERbeta actions were not affected or even potentiated by W7.     

ERalpha gene expression in human primary osteoblasts: evidence for the expression of two receptor proteins.

The beneficial influence of E2 in the maintenance of healthy bone is well recognized. However, the way in which the actions of this hormone are mediated is less clearly understood. Western blot analysis of ERalpha in osteoblasts clearly demonstrated that the well characterized 66-kDa ERalpha was only one of the ERalpha isoforms present. Here we describe a 46-kDa isoform of ERalpha, expressed at a level similar to the 66-kDa isoform, that is also present in human primary osteoblasts. This shorter isoform is generated by alternative splicing of an ERalpha gene product, which results in exon 1 being skipped with a start codon in exon 2 used to initiate translation of the protein. Consequently, the transactivation domain AF-1 of this ERalpha isoform is absent. Functional analysis revealed that human (h)ERalpha46 is able to heterodimerize with the full-length ERalpha and also with ERbeta. Further, a DNA-binding complex that corresponds to hERalpha46 is detectable in human osteoblasts. We have shown that hERalpha46 is a strong inhibitor of hERalpha66 when they are coexpressed in the human osteosarcoma cell line SaOs. As a functional consequence, proliferation of the transfected cells is inhibited when increasing amounts of hERalpha46 are cotransfected with hERalpha66. In addition to human bone, the expression of the alternatively spliced ERalpha mRNA variant is also detectable in bone of ERalpha knockout mice. These data suggest that, in osteoblasts, E2 can act in part through an ERalpha isoform that is markedly different from the 66-kDa receptor. The expression of two ERalpha protein isoforms may account, in part, for the differential action that estrogens and estrogen analogs have in different tissues. In particular, the current models of the action of estrogens should be reevaluated to take account of the presence of at least two ERalpha protein isoforms in bone and perhaps in other tissues.     

Phytoestrogen-mediated inhibition of proliferation of the human T47D breast cancer cells depends on the ERalpha/ERbeta ratio

This study investigates the importance of the intracellular ratio of the two estrogen receptors ERalpha and ERbeta for the ultimate potential of the phytoestrogens genistein and quercetin to stimulate or inhibit cancer cell proliferation. This is of importance because (i) ERbeta has been postulated to play a role in modulating ERalpha-mediated cell proliferation, (ii) genistein and quercetin may be agonists for both receptor types and (iii) the ratio of ERalpha to ERbeta is known to vary between tissues. Using human osteosarcoma (U2OS) ERalpha or ERbeta reporter cells it was shown that compared to estradiol (E2), genistein and quercetin have not only a relatively greater preference for ERbeta but also a higher maximal potential for activating ERbeta-mediated gene expression. Using the human T47D breast cancer cell line with tetracycline-dependent ERbeta expression (T47D-ERbeta), the effect of a varying intracellular ERalpha/ERbeta ratio on E2- or pythoestrogen-induced cell proliferation was characterised. E2-induced proliferation of cells in which ERbeta expression was inhibited was similar to that of the T47D wild type cells, whereas this E2-induced cell proliferation was no longer observed when ERbeta expression was increased. With increased expression of ERbeta the phytoestrogen-induced cell proliferation was also reduced. These results point at the importance of the cellular ERalpha/ERbeta ratio for the ultimate effect of (phyto)estrogens on cell proliferation.     

S-palmitoylation modulates human estrogen receptor-alpha functions

17beta-Estradiol (E2)-induced rapid functions (from seconds to minutes) can be attributed to a fraction of nuclear estrogen receptor-alpha (ERalpha) localized at the plasma membrane. As a potential mechanism, we postulated that S-palmitoylation of the Cys447 residue may explain the ability of ERalpha to associate to plasma membrane making possible E2-dependent rapid functions [e.g., extracellular regulated kinase (ERK) activation]. Here, we report direct evidence that the mutation of the Cys447 residue to Ala impairs human ERalpha palmitoylation and E2-induced rapid ERK phosphorylation when transfected in ER-devoid HeLa cells. Moreover, the Cys447Ala mutation significantly decreases the E2-induced transactivation of an estrogen responsive element construct probe. Similar effects were obtained treating HeLa cells transfected with wild type ERalpha with the palmitoyl-acyltransferase inhibitor 2-bromo-hexadecanoic acid. Moreover, the deletion of the A-D domains (containing the DNA binding region) of ERalpha had no consequences on [(3)H]palmitate incorporation, whereas no palmitoylation occurred in the ERalpha mutant devoid of the E domain (i.e., ligand binding domain). These results point to the pivotal role of the Cys447 residue in ERalpha palmitoylation and in the modulation of E2-induced non-genomic functions.