Corticosteroid ligands activate the glucocorticoid receptor (GR). GR plays a role in glucose homeostasis, adipogenesis, inflammation, and mood and cognitive functions. Understanding the interplay of diverse forms of receptor regulation (including post-translational modification, cofactor interactions, ligand binding, and receptor localization) and their effects is important for understanding and developing more effective treatment for a variety of conditions. Prior to ligand binding, the naïve GR is primarily cytoplasmic, residing in a chaperone complex containing heat-shock proteins and immunophilins. Upon ligand-binding, alterations to the complex allow the receptor to dimerize and import into the nucleus. Nuclear GR interacts with transcriptional regulatory sequences and recruits cofactors to regulate specific gene expression. Upon hormone withdrawal, the original chaperone complex is reassembled and the receptor is exported to the cytoplasm. Interestingly, while the import of GR into the nucleus occurs very rapidly (t ½ = 5 min), the re-export is significantly slower (t ½ = 12-24h). Previous work by our lab and others has indicated the existence of a nuclear retention signal (NRS) within the GR. The NRS sequence of the GR, its interaction partners, and the role it might play in the activity of the receptor have not yet been fully defined. Work in the Hache lab indicates that mutation of the GR nuclear localization signal 1 (NL1) increases the export rate of nuclear GR to the cytoplasm, as well as compromising receptor import, suggesting that the NL1 overlapped an NRS sequence. In this work, I made a series of GR mutants, based on sequence from the SV40 large T antigen NLS, which lacks nuclear retention activity. Using these mutants, I found that GR nuclear retention is influenced by both specific residues within the hinge region and the location of the sequence within the receptor, as reintroduction of the NLS sequence at the N-terminus of the receptor retention mutant failed to reconstitute the retention activity. Agonist liganded and hormone-withdrawn receptor mutants showed a similar decrease in retention. By contrast, antagonist-withdrawn GR mutants were retained in the nucleus, possibly due to altered receptor configuration and interactions. Assays of GR-responsive promoter activation by receptor retention mutants showed that while no difference in the ability of retention mutants to activate transcription was seen at a simple promoter, activation of a complex promoter was compromised. This impaired transactivation for the SV506-523 mutant correlated with decreased histone H4 acetylation and PolII recruitment, while GR DNA-binding at the target promoter appeared to be unaffected. These results suggested that promoter-specific cofactor interactions might be implicated in GR nuclear retention. Loss of GR hinge interaction with Oct cofactors produced an incomplete loss of retention, suggesting overlapping signals, but not supporting Oct as a primary factor in GR retention. The overlap between important residues in GR nuclear retention and localization signals and the lack of retention shown by the SV40 NLS suggested that retention might be intrinsic to the sequence of particular NLS. Preliminary results suggest that the KT511-512 residues of GR may be of general importance in protein nuclear retention, while the role of proline is likely more variable. My research has focused on increasing our understanding of glucocorticoid receptor nuclear retention and its possible implications. I have determined that the KT511-512 residues of GR play an important role in its retention, and possibly also figure in nuclear retention of other proteins. These residues are involved in interactions which affect promoter-specific histone acetylation and transcriptional activation in GR, suggesting a reason for the existence of nuclear retention.

Steroid hormones are reported to increase oxytocin (OXT) expression in skeletal muscle. It is possible that OXT may play essential roles in satellite cell (SC) proliferation and differentiation, which further contribute to skeletal muscle development and growth. In this dissertation, we tested this hypothesis with in-vivo and in-vitro studies in intrauterine growth restriction (IUGR) sheep, caloric restricted (CR) calves, tamoxifen (TAM) treated heifers, and bovine satellite cells (BSCs), respectively. In the in-vivo studies, we collected (1) longissimus dorsi muscle (LM) from IUGR sheep; (2) infraspinatus muscle (INF), LM, and semitendinosus muscle (ST) from CR calves; (3) LM muscle from TAM heifers. In all samples, mRNA abundance of targeted genes, such as OXT, were measured. Muscle fiber size and BSC number were also determined in skeletal muscle from TAM treated heifers. For the in-vitro studies, different treatments including 17β-estradiol (E2), trenbolone (TBA), TAM, OXT, and atosiban were applied to wild-type BSC and OXT knockout BSC (CRISPR-OXT) separately to investigate OXT's functions on BSC activity. For in-vivo studies, OXT expression significantly decreased (P<0.05) in IUGR LM muscle. Caloric restriction decreased OXT expression (P<0.05) in INF, LD, and ST muscle. Expression of OXT in LM from TAM animals decreased 50% (P<0.05). Moreover, TAM caused a small statistical reduction (P<0.05) in cross-sectional area (CSA). In in-vitro studies, OXT alone increased (P<0.05) fusion index but not proliferation in the wild-type BSC, whereas both proliferation and differentiation were stimulated (P<0.05) by OXT treatment in the CRISPR-OXT cell. By contrast, E2 and TBA increased (P<0.05) both proliferation rate and fusion index in wild-type BSC. However, E2 and TBA only stimulated proliferation rate (P<0.05) but not fusion index for CRISPR-OXT cells. Atosiban treatment resulted in lower proliferation and differentiation (P<0.05) in both wild-type BSC and CRISPR-OXT cell compared with OXT and E2 treatment groups. Together, our in-vivo studies indicate that OXT may play important roles in skeletal muscle development and growth. Our in-vitro studies demonstrate that OXT plays important roles in BSC proliferation and differentiation, and it is involved in steroid hormone stimulated BSC activity. / PHD / Oxytocin (OXT) is a mammalian neurohypophysial hormone. It has been demonstrated that OXT is highly expressed in skeletal muscle and satellite cell (SC) by steroid hormone administration. However, the role of OXT in SC proliferation and differentiation is not elucidated. In this dissertation, the in-vivo and in-vitro studies are combined and used to investigate if OXT is involved in bovine SC (BSC) activity. In the in-vivo studies, we collected muscle samples from intrauterine growth restriction (IUGR) sheep, caloric restricted (CR) calves, and tamoxifen (TAM) treated heifers. In all samples, mRNA abundance of OXT was measured. For the in-vitro studies, wild-type BSC and OXT knockout BSC (CRISPR-OXT) were treated with different factors including 17β-estradiol (E2), trenbolone (TBA), TAM, OXT, and atosiban separately to investigate OXT's functions on BSC activity. For in-vivo studies, OXT expression significantly decreased (P<0.05) in IUGR, CR, and TAM muscle. In in-vitro studies, OXT alone increased (P<0.05) fusion index but not proliferation in the wild-type BSC, whereas both proliferation and differentiation were stimulated (P<0.05) by OXT treatment in the CRISPR-OXT cell. By contrast, E2 and TBA, which can stimulate OXT expression in cultured BSC, increased (P<0.05) both proliferation rate and fusion index in wild-type BSC. However, E2 and TBA only stimulated proliferation rate (P<0.05) but not fusion index for CRISPR-OXT cells. Atosiban treatment resulted in lower proliferation and differentiation (P<0.05) in both wild-type BSC and CRISPR-OXT cell compared with OXT and E2 treatment groups. Together, our studies indicate that OXT plays important roles in BSC proliferation and differentiation, and it is involved in steroid hormone stimulated BSC activity. Studies to investigate specific biological mechanisms of steroid hormone stimulated OXT expression in SC are needed in the future.

The growth of many breast cancers is stimulated by the action of the hormone estrogen. Hormonal therapy used to treat these estrogen-dependent breast cancers acts by interfering with the action of estrogen. Current treatments, such as tamoxifen, are not consistently useful due to development of resistance to these drugs. Tamoxifen treatment can also lead to the development of other gynecological cancers, therefore the discovery of novel treatment options for breast cancer is critical. Vitamin B6 is well documented for its role as a modulator of steroid hormones. Pyridoxal phosphate (PLP), the active form of Vitamin B6, may interfere with the action of the estrogen receptor (ER) by blocking the hormone-binding and/or DNA-binding site of the ER. The objective of this study was to examine the effects of Vitamin B6 supplementation on cell proliferation and estrogen-dependent gene expression in breast cancer cells. To accomplish this, estrogen-dependent (MCF-7 and T-47D) and estrogen-independent (BT-20) breast cancer cells were grown in medium supplemented with 0,100, or 300 µM pyridoxal (PL) in the absence or presence of 0.01µM estradiol. Cell counts and [3H]-thymidine incorporation into DNA were assessed in all cell lines. The expression of pS2, an estrogen-sensitive gene, was performed using RNA extracted from MCF-7 cells. PL supplementation was found to significantly decrease total cell numbers and DNA synthesis in both the estrogen-dependent (ER+) and -independent (ER-) breast cancer cells, but did not alter the expression of pS2. These results indicate that PL significantly impairs growth of breast cancer cells and may be exerting its effects via a steroid-independent mechanism. / Master of Science

When Y-1 mouse adrenal tumor cells were treated with sodium orthovanadate, an intermediate filament (IF) inhibitor in BHK21-F cells, there was no change in the amount of 20α-dihydroprogesterone produced. A neurofilament inhibitor, β, β'-iminodipropionitrile (IDPN), enhanced the ability of Y-1 cells to produce steroid in response to ACTH by acting on the plasma membrane. Electron microscopy of Y-1 cells extracted with Triton X-100 revealed that both vanadate and IDPN caused the aggregation of cytoskeletal and granular structures in the perinuclear area. The steroidogenic effects of IDPN suggest that the perinuclear aggrergation of cytoskeletal structures may result from the detachment of IF from the plasma membrane, while the reason for the cytoskeletal changes by vanadate is unknown.

WW-domain binding protein-2 (WBP-2) was cloned as an E6-associated protein (E6-AP) interacting protein and its role in steroid hormone receptor (SHR) function was investigated. We show that WBP-2 differs from other SHR coactivators, as it specifically enhanced the transactivation functions of progesterone receptor (PR) and estrogen receptor (ER alpha), whereas it had no significant effect on the androgen receptor, glucocorticoid receptor or the activation functions of p53 or VP-16. We also demonstrated that, like other well characterized coactivators, WBP-2 contains an intrinsic activation domain. Depletion of endogenous WBP-2 with small interfering RNAs indicated that normal physiological protein level of WBP-2 was required for the proper functioning of ER alpha and PR. Moreover, chromatin immunoprecipitation (ChIP) assays demonstrate the hormone-dependent recruitment of WBP-2 onto an estrogen-responsive promoter. As we initially identified WBP-2 as an E6-AP interacting protein, we investigated whether WBP-2 and E6-AP function in concert. Our data shows that WBP-2 and E6-AP each enhance PR function and when co-expressed they additively enhance the transactivation functions of PR. However, WBP-2 was also able to enhance the transactivation functions of ER alpha and PR in mouse embryonic fibroblast cells generated from E6-AP knockout mice lines, suggesting that the coactivation functions of WBP-2 was not dependent on E6-AP. The further elucidate the molecular mechanism of action of WBP-2; we dissected the functional importance of the polyproline (PY) motifs contained within the WBP-2 protein. Mutational analysis suggests that one of three PY motifs, PY3 of WBP-2 was essential for its coactivation and intrinsic activation functions. In this study, we also demonstrate that the WBP-2 binding protein, Yes-kinase associated protein 1 (YAP1) acts as a secondary coactivator of ER alpha and PR. However, the coactivation function of YAP1 is revealed only in the presence of wild-type WBP-2 and not with the PY motif 3 mutant WBP-2. This is consistent with our observations that, unlike the wild-type WBP-2, the PY motif 3 mutant WBP-2 does not interact with YAP1. Our quantitative reChIP assays demonstrates an estrogen-dependent recruitment and association of ER alpha with both WBP-2 and YAP1. The hormone-dependent recruitment of YAP1 to ER alpha responsive promoter is dependent on the physiological expression levels of WBP-2. This is consistent with, our observation that the coactivation functions of YAP1 is dependent on WBP-2, and is also in agreement with other known secondary coactivators that get recruited to SHR responsive promoter via their interaction with primary coactivators. Surprisingly, the association of WBP-2 with ER alpha and its recruitment to the ER alpha target promoter was abrogated by YAP1 knock-down, suggesting that WBP-2 and YAP1 may stabilize each other at the promoter, and consequently, are functionally interdependent. Taken together our data establish the role of WBP-2 and YAP1 as selective coactivators for ER alpha and PR transactivation pathways.

Androgens and the androgen receptor (AR) play important roles in virilization, spermatogenesis, and sexual behavior in vertebrates. An understanding of the distribution and levels of expression of the ARs on the cellular and tissue level demonstrates the pattern of responsiveness to the androgenic hormones in a given organism. In this study, a fragment of the AR gene was cloned and sequenced from the bonnethead shark, Sphyrna tiburo, an elasmobranch species with a well-defined annual reproductive cycle. Acquiring this gene sequence facilitated the construction of species-specific AR polymerase chain reaction (PCR) primers and species-specific AR mRNA probes that were used to screen reproductive tissues for evidence of AR gene expression using reverse transcription (RT)-PCR and in situ hybridization (ISH), respectively. The RT-PCR screens demonstrated AR gene expression in the testes, epididymides, seminal vesicles, and claspers of male sharks. The use of relative PCR revealed that these organs have variable levels of AR gene expression that significantly differ with the stage of the shark's seasonal reproductive cycle. ISH results localized the AR RNA in the interstitial cells, Sertoli cells, and developing sperm of the testes, and mature spermatozoa within the seminal vesicles and the epididymides. Immunocytochemical methods used to detect the AR protein using a rabbit polyclonal antibody, PG-21, produced comparable results in the shark testes but did not yield positive results in the seminal vesicles or the epididymides. However, the Leydig gland, whose secretions contribute to the seminal fluid, demonstrated consistent AR immunoreactivity. Results of ICC in male and female embryos of S. tiburo revealed AR protein in the developing kidney but not in the embryonic reproductive structures. By characterizing AR distribution in the reproductive tract of male S. tiburo, this study provides the basis for future research on the direct and indirect effects of androgenic hormones in this species.

Vaginal atrophy symptoms such as dryness, irritation, and itching, are common after menopause. Vaginal estrogen therapy is the most effective treatment but not appropriate for all women. Women with estrogen-responsive breast cancer treated with aromatase inhibitor (AI) treatment, suppressing estrogen levels, often suffer from more pronounced vaginal atrophy symptoms. However, vaginal estrogen treatment is not recommended, leaving them without effective treatment options. The aim of this thesis was to study the effect of long-term anti-estrogen therapy on circulating estrogen levels and biochemical factors in vaginal mucosa in relation to morphological changes and clinical signs of vaginal atrophy. Circulating estrogen levels were analyzed by use of mass spectrometry and radioimmunoassay. Immunohistochemistry was used to study vaginal proliferation and steroid hormone receptors in vaginal mucosa. Vaginal gene expression was studied by use of microarray technology and bioinformatic tools, and validated by use of quantitative real-time PCR and immunohistochemistry. An estrogenic regulation of aquaporins and a possible role in vaginal dryness was investigated in vaginal mucosa and in Vk2E6E7 cells. Aromatase inhibitor-treated women had higher than expected estradiol and estrone levels but still significantly lower than other postmenopausal women. Aromatase was detected in vaginal tissue, the slightly stronger staining in vaginal mucosa from AI-treated women, suggest a local inhibition of vaginal aromatase in addition to the systemic suppression. Vaginal mucosa from AI-treated women had weak progesterone receptor, and strong androgen receptor staining intensity. Low estrogen levels lead to low expression of genes involved in cell adhesion, proliferation, and differentiation as well as weak aquaporin 3 protein immunostaining. The higher than expected estrogen levels in AI-treated women suggest that estrogen levels might previously have been underestimated. Systemic estrogen suppression by treatment with AIs, and possibly also by local inhibition of vaginal aromatase, results in reduced cell adhesion, proliferation, differentiation, and weak aquaporin 3 protein staining. Low proliferation and poor differentiation leads to fewer and less differentiated superficial cells affecting epithelial function and possibly also causing vaginal symptoms. Aquaporin 3 with a possible role in vaginal dryness, cell proliferation, and differentiation should be further explored for the development of non-hormonal treatment options for vaginal symptoms.

For my dissertation research I explored the diversity and functional evolution of steroid hormone receptors (SRs) in animals and the physiological implications of the evolution of antifreeze proteins in Antarctic notothenioid fishes. For the former, I discovered multiple new SRs from the vast and under-sampled swath of animal diversity known as invertebrates. I used the sequences of these and other newly discovered related receptors in combination with genomic data and molecular phylogenetic techniques to revise the understanding of the evolutionary history of this important gene family. While previous studies have suggested that vertebrate SR diversity arose from a gene duplication in an ancestor of all bilaterian animals, my work presents strong evidence that this duplication occurred much later, at the base of the chordates. Furthermore, to determine the implications of added diversity and a revised phylogeny on inferences of the functional evolution of SRs, I functionally characterized heretofore-unknown SRs from hemichordates, an acoelomate flatworm, and a chaetognath and statistically reconstructed and functionally characterized ancestral SRs. My results expand the known sequence and functional repertoire of SRs in animals while reinforcing the previous inference that all SRs evolved from an estrogen-sensitive ancestral receptor. I also explored the consequences of the evolution of antifreeze proteins in Antarctic notothenioid fishes, a crucial adaptation to their icy, polar environment. These special proteins adsorb to ice crystals that enter a fish's body and prevent further growth, thereby averting death. I discovered that, in addition to their lifesaving growth-inhibiting ability, AFPs also prevent the melting of internal ice crystals at temperatures above the expected equilibrium melting point. Together with a decade-long temperature record of one of the coldest fish habitats on earth, my experimental results show that the evolution and expression of antifreeze proteins is accompanied by a potentially detrimental consequence: the lifelong accumulation of ice inside these fishes' bodies. This dissertation includes previously published co-authored material as well as unpublished co-authored material. / 2017-01-14

Aberrant androgen receptor (AR) activity plays a critical role in the development and progression of both early-staged organ-confined and late-staged metastatic human prostate cancer. Recent large-scale genomic sequencing studies showed that ~50% of organ-confined prostate cancer patients have genetic rearrangements that placed the ETS transcription factors (e.g. ERG, ETV1) under the control of androgen-regulated gene promoters such as TMPRSS2. This results in the upregulation of the ETS transcription factors’ expressions in the presence of androgens. The aberrant overexpression of the ETS transcription factors are shown to induce the expression of genes that promote the cellular motility and invasive potential of prostate-tumor cells. Moreover, the improved therapeutic outcome of the second-generation anti-androgen therapies (e.g. abiraterone and enzalutamide) are encouraging, and prove that aberrant AR activity still drives the progression of metastatic prostate cancer. Although these treatments are initially effective, these cancer cells eventually develop resistance to these AR-targeted therapies termed castration-resistant prostate cancer (CRPC). Since the molecular steps involved in AR activation is still not clearly defined, it is critical to define the interactions required for AR activation prostate cancer cells, which will provide a framework for establishing more effective treatments to inhibit aberrant AR activity in human prostate cancer cells. Here, I developed a cellular system to isolate ligand-dependent interactions of AR in prostate-tumor cells. A siRNA luciferase screen was also developed and identified novel modulators of AR-mediated transcription selected from the proteomic dataset. Further biochemical studies showed that AR is associated with the Golgi membrane in a ligand-sensitive manner. And that the nuclear localization of ARA160, an AR coactivator, is regulated by the COPI retrograde trafficking machinery. Collectively, these results support the use of this cellular system to decipher the known AR-interacting proteins and novel components involved in AR signaling in prostate-tumor cells. I next investigated the androgen-sensitive AR transcriptional complexes and androgen-sensitive microsomes isolated from LNCaP prostate-tumor cells. Both studies yielded results that would further strengthen the diverse AR actions mediated within the cell. These results further support the notion that there is significant crosstalk amongst different cell surface receptor signaling pathways with AR. An extension of the androgen-sensitive microsome findings also led us to study the androgen-sensitive G-protein coupled receptor, CXCR7. I showed that androgens regulate the expressions of CXCR7 and CXCR4 and in turn modulated CXCL12-mediated motility in prostate tumor cells. Lastly, biochemical strategies were developed to detect differences in glycoprotein expression of frozen prostate cancer tissues isolated from human patients. I showed that the workflow successfully solubilized and isolated N- and O-linked glycoproteins from the frozen tissue samples and can be analyzed by quantitative mass spectrometry. This workflow would thus facilitate future biomarker studies. In summary, these data demonstrate the utility of developing methods for the comprehensive mapping of AR-mediated signaling in prostate cancer cells, and thus provide novel target candidates for the therapeutic treatment of metastatic or CRPC.

Liquid chromatography, coupled with tandem mass spectrometry, presents a powerful tool for the quantification of the sex steroid hormones 17-β estradiol, progesterone and testosterone from biological matrices. The importance of accurate quantification with these hormones, even at endogenous levels, has evolved with our understanding of the role these regulators play in human development, fertility and disease risk and manifestation. Routine monitoring of these analytes can be accomplished by immunoassay techniques, which face limitations on specificity and sensitivity, or using gas chromatography–mass spectrometry. LC–MS/MS is growing in capability and acceptance for clinically relevant quantification of sex steroid hormones in biological matrices and is able to overcome many of the limitations of immunoassays. Analyte specificity has improved through the use of novel derivatizing agents, and sensitivity has been refined through the use of high-resolution chromatography and mass spectrometric technology. This review highlights these innovations, among others, in LC–MS/MS steroid hormone analysis captured in the literature over the last decade.