Nancy L. Weigel

Molecular interactions of steroid hormone receptor with its enhancer element: Evidence for receptor dimer formation

A steroid hormone responsive element (GRE/PRE), sufficient to confer glucocorticoid and progesterone inducibility when linked to a reporter gene, was used in band-shift assays to examine its molecular interactions with steroid hormone receptors. Both progesterone and glucocorticoid receptors bound directly and specifically to the GRE/PRE. The purine contact sites for both form A and form B chicken progesterone receptor, as well as those for rat glucocorticoid receptor, are identical. A peptide fragment produced in bacteria that primarily contain the DNA binding domain of the glucocorticoid receptor binds first to the TGTTCT half-site of the GRE/PRE, and a second molecule binds subsequently to the TGTACA (half-site) of the GRE/PRE in a cooperative manner. Utilizing the peptide fragment and the protein A-linked fragment, we demonstrated that the receptor interacts with its cognate enhancer as a dimer.

Activation of the Human Androgen Receptor through a Protein Kinase A Signaling Pathway

Aberrant activation of the androgen receptor through signaling pathways independent of androgen may be responsible for the progression of prostate tumors to the rapidly proliferating androgen-independent state. In this study, the effects of protein kinase A modulators on human androgen receptor activity were tested. Using an adenoviral DNA delivery system, we demonstrate that the androgen receptor can be activated by a protein kinase A activator, forskolin, in the absence of androgen when androgen receptor is co-transfected into monkey kidney CV1 cells or human prostate PC-3 cells with androgen-responsive reporters. Immunoblotting reveals that there is no significant change in androgen receptor protein level following forskolin treatment, suggesting that the enhanced activity is due to activation of the receptor. This activation can be blocked by a protein kinase A inhibitor peptide. Two potent anti-androgens, casodex and flutamide, can significantly reduce this activation, confirming that the ligand-independent pathway is an androgen receptor-mediated phenomenon. An intact DNA binding domain of the receptor is critical for this alternate signaling pathway since mutants with reduced DNA binding ability are inactive. The phosphorylation status of the androgen receptor or associated proteins may critically modulate receptor activity and should be considered when designing improved approaches to prostate cancer therapy.

Phosphorylation of the Nuclear Receptor SF-1 Modulates Cofactor Recruitment: Integration of Hormone Signaling in Reproduction and Stress

Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor that serves as an essential regulator of many hormone-induced genes in the vertebrate endocrine system. The apparent absence of a SF-1 ligand prompted speculation that this receptor is regulated by alternative mechanisms involving signal transduction pathways. Here we show that maximal SF-1-mediated transcription and interaction with general nuclear receptor cofactors depends on phosphorylation of a single serine residue (Ser-203) located in a major activation domain (AF-1) of the protein. Moreover, phosphorylation-dependent SF-1 activation is likely mediated by the mitogen-activated protein kinase (MAPK) signaling pathway. We propose that this single modification of SF-1 and the subsequent recruitment of nuclear receptor cofactors couple extracellular signals to steroid and peptide hormone synthesis, thereby maintaining dynamic homeostatic responses in stress and reproduction.

International Union of Pharmacology. LXV. The pharmacology and classification of the nuclear receptor superfamily: glucocorticoid, mineralocorticoid, progesterone, and androgen receptors.

The glucocorticoid receptor (GR[1][1]), mineralocorticoid receptor (MR), progesterone receptor (PR), and androgen receptor (AR) are classic members of the nuclear receptor superfamily, composing subfamily 3C. Members of this subfamily are among those receptors that were cloned the earliest, with the

The Nuclear Corepressors NCoR and SMRT Are Key Regulators of Both Ligand- and 8-Bromo-Cyclic AMP-Dependent Transcriptional Activity of the Human Progesterone Receptor

ABSTRACT Previously, we defined a novel class of ligands for the human progesterone receptor (PR) which function as mixed agonists. These compounds induce a conformational change upon binding the receptor that is different from those induced by agonists and antagonists. This establishes a correlation between the structure of a ligand-receptor complex and its transcriptional activity. In an attempt to define the cellular components which distinguish between different ligand-induced PR conformations, we have determined, by using a mammalian two-hybrid assay, that the nuclear receptor corepressor (NCoR) and the silencing mediator for retinoid and thyroid hormone receptor (SMRT) differentially associate with PR depending upon the class of ligand bound to the receptor. Specifically, we observed that the corepressors preferentially associate with antagonist-occupied PR and that overexpression of these corepressors suppresses the partial agonist activity of antagonist-occupied PR. Binding studies performed in vitro, however, reveal that recombinant SMRT can interact with PR in a manner which is not influenced by the nature of the bound ligand. Thus, the inability of SMRT or NCoR to interact with agonist-activated PR when assayed in vivo may relate more to the increased affinity of PR for coactivators, with a subsequent displacement of corepressors, than to an inherent low affinity for the corepressor proteins. Previous work from other groups has shown that 8-bromo-cyclic AMP (8-bromo-cAMP) can convert the PR antagonist RU486 into an agonist and, additionally, can potentiate the transcriptional activity of agonist-bound PR. In this study, we show that exogenous expression of NCoR or SMRT suppresses all 8-bromo-cAMP-mediated potentiation of PR transcriptional activity. Further analysis revealed that 8-bromo-cAMP addition decreases the association of NCoR and SMRT with PR. Thus, we propose that 8-bromo-cAMP-mediated potentiation of PR transcriptional activity is due, at least in part, to a disruption of the interaction between PR and the corepressors NCoR and SMRT. Cumulatively, these results suggest that NCoR and SMRT expression may play a pivotal role in PR pharmacology.

Phosphorylation of steroid receptor coactivator-1. Identification of the phosphorylation sites and phosphorylation through the mitogen-activated protein kinase pathway.

Steroid receptor coactivator-1 (SRC-1) is a member of a coactivator family that enhance the activation of the steroid/nuclear receptor superfamily of ligand-stimulated transcription factors. To study the regulation of SRC-1 by signaling pathways in the cell, the major phosphorylation sites of SRC-1 were identified in COS-1 cells using a combination of in vivo labeling with [32P]H3PO4, modified manual Edman degradation, phosphoamino acid analysis, endoproteinase digestion, and mutagenesis of the SRC-1 phosphorylation sites. Seven phosphorylation sites were identified in SRC-1: serine 372, serine 395, serine 517, serine 569, serine 1033, threonine 1179, and serine 1185. All the sites contained consensus sequences for the serine/threonine-proline-directed family of protein kinases, and two sites (serine 395 and threonine 1179) contained a perfect consensus sequence for the mitogen-activated protein kinase family (Erk-1 and Erk-2). Furthermore, Erk-2 phosphorylated threonine 1179 and serine 1185 (and to a lesser extent, serine 395) in vitro, suggesting the importance of this pathway for SRC-1 regulation. Treatment of cells expressing SRC-1 with epidermal growth factor enhanced the ligand-dependent, progesterone receptor-mediated activation of a target reporter gene. These results identify phosphorylation as a regulatory modification of SRC-1 and provide a basis upon which to identify signaling pathways that regulate SRC-1 function and, consequently, modify steroid/nuclear receptor action.

Calcitriol-induced apoptosis in LNCaP cells is blocked by overexpression of Bcl-2

While the role of vitamin D in bone and mineral metabolism has been investigated extensively, the role of the vitamin D receptor in other tissues is less well understood. 1,25-dihydroxyvitamin D3 (calcitriol) can act as a differentiating agent in normal tissues and can inhibit the growth of many cancer cell lines including LNCaP prostate cancer cells. We have shown previously that calcitriol causes LNCaP cell accumulation in the G0/G1 phase of the cell cycle. In this study, we demonstrate that calcitriol also induces apoptosis of LNCaP cells. The calcitriol-induced apoptosis is accompanied by a down-regulation of Bcl-2 and Bcl-XL proteins, both of which protect cells from undergoing apoptosis. Other proteins important in apoptotic control, Bax, Mcl-1, and Bcl-Xs, are unaffected by calcitriol treatment. We find that overexpression of Bcl-2 blocks calcitriol-induced apoptosis and reduces, but does not eliminate, calcitriol-induced growth inhibition. We conclude that both regulation of cell cycle and the apo...

Role of SRC-1 in the Promotion of Prostate Cancer Cell Growth and Tumor Progression

Prostate cancer is initially androgen dependent and there is evidence that androgen receptor continues to play a role in androgen-independent prostate cancer. Androgen receptor activity depends both on the level of androgens and on the level of coactivators that interact with androgen receptor. Our goal was to evaluate the role of the androgen receptor coactivator SRC-1 in prostate cancer progression. Using tissue arrays to measure SRC-1 protein levels, we found that increased SRC-1 expression in clinically localized, androgen-dependent cancer is associated with clinical and pathologic variables of increased tumor aggressiveness. Interestingly, there was variable expression of SRC-1 in normal prostate tissue which correlated with the staining intensity of the corresponding cancer tissue. To test the contribution of SRC-1, we examined its role in androgen-dependent LNCaP and androgen-independent C4-2 prostate cancer cell lines. Using small interfering RNA to reduce expression of androgen receptor, we found that androgen receptor was required both for cell growth and for basal expression of prostate-specific antigen in the androgen-independent C4-2 cell line. Thus, although the cells can grow in an androgen-depleted medium, they remained androgen receptor dependent. Reduction of SRC-1 expression significantly reduced growth and altered androgen receptor target gene regulation in both LNCaP and C4-2 cell lines whereas it had no effect on the growth of the androgen receptor-negative PC-3 and DU145 prostate cancer cell lines. Although the requirement for androgens and androgen receptor in the development of prostate cancer is well established, our study implicates enhanced androgen receptor activity through elevated expression of SRC-1 in the development of more aggressive disease in men with prostate cancer.

The progesterone receptor stimulates cell-free transcription by enhancing the formation of a stable preinitiation complex

Highly purified chicken progesterone receptor (cPR) is shown to stimulate RNA synthesis directly in an in vitro transcription assay. Stimulation of transcription by cPR requires the presence of progesterone response elements (PREs) in the template and can be specifically inhibited by addition of competitor oligonucleotides containing PREs. Binding of receptor to two PREs is cooperative and leads to synergistic (27-fold) stimulation of transcription. A purified fusion protein containing the DNA binding domain of cPR linked to yeast ubiquitin was produced in E. coli and also functions in the transcription assay. Using this in vitro transcription system, we demonstrate that hormone-free cPR activated by salt treatment induces transcription of a test gene in a hormone-independent manner. Finally, we present evidence that the progesterone receptor acts by facilitating the formation of a stable preinitiation complex at the target gene promoter and thus augments the initiation of transcription by RNA polymerase II.

Androgen receptor action in hormone-dependent and recurrent prostate cancer.

The importance of androgens and androgen receptors (AR) in primary prostate cancer is well established. Metastatic disease is usually treated with some form of androgen ablation, which is effective for a limited amount of time. The role of AR in prostate cancers that recur despite androgen ablation therapy is less certain. Most of these tumors express prostate specific antigen (PSA), an androgen‐regulated gene; moreover, AR is generally highly expressed in recurrent prostate cancer. We propose that AR continues to play a role in many of these tumors and that it is not only the levels of AR, ligands, and co‐regulators, but also the changes in cell signaling that induce AR action in recurrent prostate cancer. These pathways are, therefore, potential therapeutic targets. J. Cell. Biochem. 99: 362–372, 2006.