Control of Steroid Hormone Receptor Action
By Bekir Cinar, DVM, and Robert A. Sikes, PhD
Steroid hormones regulate many physiological processes that include: male and female reproduction, cellular proliferation, cellular differentiation, cell death, and homeostasis. The action of these lipophilic steroid hormones is mediated by steroid hormone receptors (SHRs) that, when bound to their cognate steroidal ligand, are transmuted into activated transcription factors. Steroids have long been thought to play a role in the etiology of cancers arising in hormonally responsive tissues like breast and prostate.
In breast cancer, one of the longest standing prognostic markers is the estrogen receptor (ER).1 The presence of the ER in a breast cancer biopsy is associated with a better prognosis than in the absence or loss of the ER. In cervical-vaginal cancer, one hypothesis is that a neonatal exposure to estrogen disrupts ER signaling so that transcription that would normally be ER-regulated is now ER-independent and constitutively activated.2
As prostate cancer (PCa) progresses the loss of the androgen receptor (AR) is rare; however, the sensitivity to androgen can change dramatically. In metastatic PCa, the AR has been found to undergo mutations (Thr>Ala codon 877) in the AR’s ligand binding domain (LBD) that change the affinity of the AR for steroid hormones, making it responsive to other steroid hormones in addition to androgen and anti-androgens.3,4 This promiscuity for other steroid ligands has been proposed as one mechanism by which PCa can escape dependence upon androgen. Additionally, most metastatic PCa will amplify the AR, which could result in a greater sensitivity to androgens. Finally, in androgen independent PCa, the AR appears to be involved in a general up regulation of the basal transcription from androgen responsive promoter elements. This seems to require little or no ligand. Since the control of gene transcription by steroids is not solely dependent on the action of the SHRs as transcription factors, recent efforts have focused on the interaction of SHRs with transcriptional cofactors. The rationale being that altered cofactors, either coactivators or repressors, could be the source for the transcriptional changes seen in hormonally independent, SHR positive disease, like PCa. Herein we summarize recent reports on SHR/coactivator-corepressor interactions and their effect on transcriptional control by SHRs.
Background
Steroid hormone receptors (androgen, estrogen, progesterone, glucocorticoid, and mineralocorticoid receptors) are members of the nuclear hormone receptor superfamily. All members of the SHR family have a similar structural organization with a highly variable N-terminal trans-activation domain (NT-TAD), followed by a highly conserved DNA-binding domain (DBD) consisting of two zinc fingers, a hinge region, and a moderately conserved C-terminal ligand-binding domain (CT-LBD). The transcriptional specificity of SHRs is mediated by a constitutively active N-terminal localized activation function (AF-1) domain and a ligand-dependent AF-2 domain arising in the CT-LBD.5 (See Figure.)
Mechanism of SHR Action
Steroid receptors activate target gene transcription upon ligand activation by binding as homodimers to their cognate DNA sequence, or hormone response elements (HRE), followed by the initiation, assembly, and stabilization of the preinitiation complex (PIC). How are steroid receptors able to initiate or repress target gene transcription? Current data suggest that steroid receptors modulate gene expression using the following mechanisms: a) steroid receptors induce nucleosome rearrangement, thereby allowing other essential transcription factors to bind and activate transcription; b) direct interaction between the steroid receptor and components of the PIC, that include the transcription factors TFllB, TFllF, and the TATA-box binding protein (TBP) and TBP-associated factors (TAFs); and c) linking the receptors to the PIC by co-factors, termed coactivators and co-repressors.6-8
Steroid Receptors and the Target Gene
Discovery of coactivators and corepressors proteins has enhanced our understanding of how steroid receptors activate or inhibit transcription of the target gene.8,9 Several proteins that interact with steroid receptors in a ligand-dependent manner have been identified. Among these are RIP140/160 for estrogen receptors, GRIP1 for glucocorticoid receptors, and ARA70 for the androgen receptor.10-12 In addition to steroid receptor coactivator-1 (SRC-1) and c-AMP response element binding protein (CREP or CBP), two other proteins, p300 and P/CIP, have been shown to interact with steroid receptors upon ligand administration and found to enhance the transcriptional activity of the interacting SHR.8,13 Coactivators themselves are not DNA-binding proteins; they modulate transcriptional activity of the SHR of preference through protein-protein interactions with other transcriptional apparatuses like the aforementioned PIC. More interestingly, these transcriptional coactivators display histone acetyl transferase (HAT) activity and are able to acetylate histones. The acetylation of a conserved lysine residue in the N-terminal domain of histones loosens the nucleosome structure, making the DNA more accessible to transcription factors.6,7,14 Therefore, in response to hormone signaling, steroid receptors and coactivators form a multimeric complex that is able to activate specific gene transcription through chromatin remodeling. This explains, in part, the nucleosome displacement observed with SHR activation.
Transcriptional corepressors inhibit target gene transcription when the SHR is unliganded. To date, SHR-specific corepressors have not been reported. In the absence of ligand, SHRs are complexed with heat shock proteins (HSPs) in the cytosol and do not bind to their cognate steroid hormone response element for the initiation of transcription. Therefore, cytosolic, unliganded SHRs apparently do not to bind corepressors, and corepressors probably have no important function in this unliganded state. However, yeast 2-hybrid screening has identified a few nuclear localized SHR corepressors, such as "nuclear receptor corepressor" (N-CoR) that interacts with the thyroid hormone receptor and "silencing mediator for retinoid and thyroid hormone receptors" (SMRT).9 These two proteins share a high degree of homology, which includes a transcriptional repression domain that interacts directly with the hinge/ligand-binding domain of nuclear SHRs. Interestingly, the repressor activity is mediated through the recruitment of Sin3 and other histone deacetylases (HDAC),15 thereby having the effect of tightening the nucleosomal structure. Moreover, chicken ovalbumin upstream promoter transcription factor-1 (COUP-TFl) has been reported to repress the transcriptional activity of several target genes by making complexes with the N-CoR and SMRT corepressor proteins.9 The deacetylation of histones and subsequent compaction of nucleosomes would result in inaccessible promoter elements and would be a potent mechanism for shutting-down gene expression.
Although steroid receptors do not seem to be associated with corepressors in the absence of ligand, some steroid receptors recruit corepressors when they are occupied by an antagonist. The most extensively studied example is the antagonistic effect of mifepristone (RU486) on the progesterone receptor (PR). RU486 binds to the PR, which results in the release of HSPs and the binding of the PR to its response elements, with subsequent activation of progesterone responsive genes in some cell types.16 Interestingly, SMRT and N-CoR have been shown to interact with RU486-bound PRs, which keep the receptor from becoming transcriptionally active.15 The absence or low expression of these corepressors may explain why RU486 acts as an agonist in certain cell types. This same mechanism seems to be responsible for the partial agonistic effects of tamoxifen on the ER. The down regulation of SMRT and N-CoR expression could then explain the development of tamoxifen resistance and partial tamoxifen-dependent growth in breast cancer.15-17 As if to complicate things further, a protein identified as a switch protein for receptor antagonists (L7/SPA) has been discovered that functions as a transcriptional enhancer for antagonist-occupied ER and PR.16 Finally, some steroidal antagonists have inherent agonist activity. Tamoxifen can promote classic ER/ERE-mediated gene transcription despite effective blockade of estrogen binding to the receptor.18 For the androgen receptor (AR) things are even less clear; however, a point mutation acquired in the LBD of the AR in the LNCaP cell line and some androgen-independent PCa does allow some antagonists to behave as partial agonists with respect to gene transcription.3-4
Conclusion
The steroid hormone receptors are ligand (steroid hormone) activated transcription factors characterized by ligand specificity and DNA response element specificity for the initiation of specific target gene transcription in response to ligand activation. It has become clear in recent years that the transcriptional activation of steroid hormone receptors is influenced by the presence of ligand available interacting transcriptional cofactors. The discovery of steroid hormone receptor coactivators and corepressors has expanded our understanding of the control of steroid-regulated gene expression and has provided insight into the potential mechanisms for steroid insensitivity or antagonist failure in cancer therapy. Certainly, this area will continue to expand since there are still many orphan steroid hormone receptor family members to be studied and the number of steroid hormone receptor interacting factors continually expands. Hopefully, a clear picture of steroid receptor function and interaction will provide new targets for therapeutic intervention in steroid responsive tissue-derived tumors. (Dr. Cinar is a Doctor of Veterinary Medicine from Ankara, Turkey, and is currently a Doctoral Candidate at the University of Virginia Health System in the Departments of Biochemistry and Molecular Genetics and Urology; Robert A. Sikes, PhD, is an Assistant Professor, University of Virginia Health System, Molecular Urology and Therapeutics Program, Charlottesville, VA.)
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