Over 200,000 cases of breast cancer are diagnosed every year. Nearly 20% of these patients supplement their diets with some form of vitamin D. This high frequency of vitamin D supplement use may be due in part to research suggesting that cancer patients with higher serum vitamin D3 levels have better prognoses than patients with low serum vitamin D3. However, double-blind clinical trials on the efficacy of vitamin D3 supplementation in breast cancer have been inconclusive. A recent meta-analysis showed evidence of reduced cancer recurrence in patients taking vitamin D3 supplements who had ‘estrogen receptor positive’ (ERα66+) breast cancer, but not those who had estrogen receptor negative’ (ERα66-) breast cancer. Once ingested, vitamin D3 is metabolized in the liver into the circulating pre-hormone 25(OH)D3, which is then further metabolized into 1a,25(OH)2D3 and 24R,25(OH)2D3. 24R,25(OH)2D3 has been shown to activate a number of membrane signaling pathways, some of which overlap with 17b-estradiol (E2) signaling through ERα36, a membrane isoform of ERα66. The central hypothesis of this thesis was that 24R,25(OH)2D3 is tumorigenic in certain cancers and that this tumorigenicity is mediated in part by ERa isoforms. E2 signaling through ERa36 has been described in the ERa66-, ERa36+ breast cancer cell line HCC381. Specific aim 1 determined whether E2 signaling through ERa36 was tumorigenic other cancers with different ERa profiles. Specific aim 2 determined how 24R,25(OH)2D3 affected tumorigenicity in breast cancer using the common breast cancer cell line MCF7 (ERa66+, ERa36+) as a model. Specific aim 3 investigated the role of ERa isoforms in 24R,25(OH)2D3 signaling in breast cancer cell lines by comparing the tumorigenic effects of 24R,25(OH)2D3 in MCF7 cells (ERa66+, ERa36+) and HCC38 cells (ERa66-, ERa36+). To determine whether ERa66 regulates the effects of 24R,25(OH)2D3, ERa66 was expressed in two ERα66- cell lines. The effect of 24R,25(OH)2D3 on apoptosis was assessed in wild-type and ERa-expressing cell lines.

The steroid/thyroid hormone receptor superfamily is a large class of ligand-dependent transcription factors that plays a critical role in regulating the expression of genes involved in a broad range of physiological functions, including development, homeostasis, and reproduction. In the absence of cognate hormone, several receptors are able to repress transcription below the basal level via the recruitment of the nuclear receptor corepressors SMRT and NCoR. Upon hormone binding by the receptor, the corepressor complex is dissociated and a coactivator complex is subsequently recruited. This thesis details the mechanisms by which receptor-associated coactivator 3 (RAC3) interacts with nuclear receptors, particularly the vitamin D, estrogen, and retinoid receptors, and modulates their transcriptional activity. It was discovered that these receptors interact with different α-helical LXXLL motifs of RAC3 in vitro. Mutation of specific motifs differentially impairs the ability of RAC3 to enhance transcription by the receptors in vivo. In addition, the intrinsic transcriptional activation function of RAC3 was also characterized. Here, a single LXXLL motif, NR box v, was found to be essential to activation by serving as a binding surface for the general transcriptional integrator CBP/p300. Finally, the cofactor binding pocket of retinoid receptors was characterized. It was demonstrated that, to a large extent, the coactivator pocket of RARα overlaps with the corepressor pocket, with the exception of helix 12, which is required for coactivator, but not corepressor binding. Recruitment of RAC3 or SMRT also correlates directly with the ability of RARα to activate or repress transcription, respectively. Intriguingly, it was discovered that the AF-2 domain of RXRα inhibited cofactor binding to RXRα heterodimers, for deletion of this domain dramatically enhanced RAC3 and SMRT binding. In addition, it was demonstrated that the RXRα cofactor binding pocket contributed minimally to recruitment of cofactors. Conversely, the AF-2 domain of the partnering monomer and its cofactor pocket were required for these interactions. These findings suggest that the partner of RXRα is the primary docking point for cofactors at RXRα heterodimeric complexes. Taken together, this work contributes significantly to the field of nuclear receptor function in detailing the mechanisms by which the coactivator RAC3 is recruited to nuclear receptors and regulates their transcriptional activity.

The synapse is the primary locus of cell-cell communication in the nervous system. The elaboration of a functional synapse requires both a specialized structure and an efficient communication system. For my thesis work, I studied proteins implicated in each of these functions: the structural molecules dystroglycan and dystrophin, and the signaling elements Insulin Receptor Substrate p58/53 and insulin receptor. The α/β-dystroglycan complex, believed to be the heart of cellmatrix adhesion in muscle and other tissues, provides a link between dystrophin, a cytoskeletal protein at the base of the muscle cell's Dystrophin Associated Protein Complex, and the extracellular matrix. In addition, dystrophin is found at central synapses, tightly associated with the postsynaptic density. The absence of dystrophin and the secondary loss of its associated proteins causes the genetic disease Duchenne Muscular Dystrophy. DMD affects both muscle and brain, causing a severe muscular dystrophy and lower IQs than control groups. In the first portion of my thesis work, I sought to determine the role of dystroglycan, dystrophin's peripheral partner, at central synapses. I probed Northern blots of brain regions to delineate the distribution of brain β-dystroglycan mRNA and to uncover any β-dystroglycan-related transcripts in brain. Then, using subcellular brain fractions, and cultured hippocampal neurons, I determined that whereas α-dystroglycan is associated with central synapses, β-dystroglycan is not. This discovery is surprising, and differs from the finding that dystrophin and α- and β-dystroglycan colocalize at the presynaptic membrane of retinal photoreceptors. In the course of the above mentioned work, using the anti-β-dystroglycan antiserum Ab98, I discovered a pair of proteins that were tightly associated with the postsynaptic density. These polypeptides of 58 kDa and 53 kDa (p58/53) were highly enriched in postsynaptic density (PSD) fractions from rat cerebral cortex, hippocampus, and cerebellum. In pursuit of a potential synapse-specific dystroglycan relative, I purified p58 and p53 by a combination of hydrophobic interaction chromatography and two-dimensional gel electrophoresis. Mass spectroscopy and peptide microsequencing revealed that p58/53 is identical to the insulin receptor tyrosine kinase substrate p58/53 (IRSp53). Whereas IRSp58/53 has no significant homology to β-dystroglycan other than the one span of peptides that confers its antibody cross-reactivity, its localization to the PSD newly implicates insulin signaling at synapses. Analysis of IRSp58/53 mass profiles, peptides, and mRNA indicated that IRSp58 and IRSp53 are the product of the same coding sequence. Immunolocalization showed that IRSp58/53 is expressed in the synapserich molecular layer of the cerebellum. Immunostaining of cultured hippocampal neurons showed that both IRSp58/53 and insulin receptor are highly concentrated at synapses. Like IRSp58/53, insulin receptors are a component of the PSD fraction. Together, these data suggest that the synapse is a specialized site for insulin signaling in the brain.

One important objective of prostate cancer (PCa) research is to understand the molecular basis underlying the progression of these cancers from an androgen dependent to an androgen independent state. Hypermethylation of the promoter CpG islands is associated with the transcriptional silencing of specific gene sets in each tumor type and subtype. Transcriptional silencing of antitumor genes via CpG island hypermethylation could be a mechanism mediating PCa progression from an androgen-dependent to an androgen-independent state. Hypermethylation associated gene silencing has been reported for a great number of genes in PCa with the exception of the genes that undergo methylation associated silencing specifically during cancer development to androgen independence. The first aim of this thesis is to identify novel glenes which undergo DNA hypermethylation associated gene silencing during the cancer progression. The androgen-dependent (AD, as defined as the inability of celill to proliferate in the absence of androgen) PCa cell line LNCaP gives rise to the androgen-independent (AI) subline LNCaPcs generated by maintaining LNCaP in medium with charcoal-stripped (CS) serum for over 30 passages. This LNCaP cell model was used to identify differentially methylated sequences between the two genomes using the Methylation-Sensitive Restriction Fingerprinting (MSRF) technique. One sequence identified is located in a 5' CpG island, which encompasses part of the promoter, exon 1, and part of intron 1, of the Peroxisomal Membrane Protein 24 KD (PMP24) gene. PMP24 is silenced in concert with the hypermethylation of its CpG island in AI LNCaPcsand PC-3 cell lines. The silencing is reactivated by the treatment with a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5AZAdC). PMP24 is specifically silenced in PCa cancer cell lines and shows potential antitumor properties. These results demonstrate the utility of MSRF in the identification of novel, differentially methylated DNA sequences in the genome and suggest that hypermethylation-mediated silencing of PMP24 is an epigenetic event involved in PCa progression to androgen independence. The next study investigated the molecular mechanism for DNA methylation associated gene silencing of PMP24 in AI LNCaPcs and PC-3 cell lines. We demonstrated that PMP24 transcription is repressed by the disruption of transcription factor binding to a critical cis-element by hypermethylation of its promoter CpG island. We found a CpG containing activator protein 2 (AP-2) cis-element in the intron 1 of PMP24 whose first CpG dinucleotidle is essential for the sequence-specific protein binding and the promoter activity of the gene. We presented first in cellulo evidence that the methylation of AP-2 cis-element alone but not the whole CpG island, using a newly developed methylated oligonucleotides treatment, is sufficient for the downregulation of PMP24. Our study is the first to report that the silencing mechanism for PMP24 in AI LNCaPcs and PC-3 is mediated by the complete methylation of a single GpG site of AP-2 cis-element in the intron 1 part of the CpG island, which interferes with transcription factor binding. Most interestingly, the promoter CpG island of PMP24 is hypermethylated in AD LNCaP cells with the incomplete methylation specifically at the AP-2 cis-element. The silencing of PMP24 in AD LNCaP cells was reactivated not by the 5AZAdC treatment but by the treatment with Trichostatin A (TSA), a histone deacetylase inhibitor. An alternative silencing mechanism for PMP24 other than the interference with transcription factor binding by methylation is therefore likely involved at this androgen-dependent stage. During the androgen ablation process, this mechanism is either evolved by the spread of methylation in the promoter CpG island or selected against, leading to the methylation-dominant silencing mechanism in the AI cells as seen in LNCaPcsand PC-3 cells. Taken together, this thesis emphasized the important role of DNA methylation in the progression of PCa into androgen independence. Particular respect should be paid to the specific CpG dinucleotides in cis-elements critical for the promoter activity, whose complete methylation could dominate the silencing mechanism which is independent of androgen. This thesis also pointed to the importance of monitoring the effects of cell culture on the methylation status of genes. Most importantly, this thesis raised the possibility that the silencing mechanisms for PMP24 could be different in AD LNCaP cells as compared to AI LNCaPcs and PC-3 cells. Either the evolution of such mechanism or the selectivity against it during the androgen ablation process would result in a methylation-dominant silencing mechanism of the genes such as PMP24 in AI cells and may contribute to the overall androgen independence of the cells.

The insulin/IGF-1 signaling pathway plays a pivotal role in life span regulation in diverse organisms. In Caenorhabditis elegans, a PI 3-kinase signaling cascade downstream of DAF-2, an ortholog of the mammalian insulin and insulin-like growth factor-1 (IGF-1) receptor, negatively regulates DAF-16/forkhead transcription factor. DAF-16 then regulates a wide variety of genes involved in longevity, stress response, metabolism and development. DAF-16 also receives signals from other pathways regulating life span and development. However, the precise mechanism by which DAF-16 directs multiple functions is poorly understood. First, in Chapter II, we demonstrate that JNK is a novel positive regulator of DAF-16 in both life span regulation and stress resistance. Our genetic analysis suggests that the JNK pathway acts in parallel with the insulin-like signaling pathway to regulate life span and both pathways converge onto DAF-16. We also show that JNK-1 directly interacts with and phosphorylates DAF-16. Moreover, in response to heat stress, JNK-1 promotes the translocation of DAF-16 into thc nucleus. Our findings define a novel interaction between the stress response pathway (JNK) and the master regulator of life span (DAF-16), and provide a mechanism by which JNK regulates longevity and stress resistance. Next, in Chapter III, we focus on the downstream targets of DAF-16. Here, we used a modified chromatin immunoprecipitation (ChIP) method to identify direct target promoters of DAF-16. We cloned 103 target sequences containing consensus DAF-16 binding sites and randomly selected 33 targets for further analysis. The expression of majority of these genes is regulated in a DAF-16-dependent manner. Moreover, inactivation of more than 50% of these genes significantly altered DAF-16-dependent functions such as longevity, fat storage and dauer diapause. Our results show that the ChIP-based cloning strategy leads to greater enrichment of DAF-16 target genes, compared to previous studies using DNA micro array or bioinformatics. We also demonstrate that DAF-16 is recruited to multiple promoters to coordinate regulation of its downstream target genes. In summary, we identified the JNK signaling pathway as a novel input into DAF-16 to adapt animals to the environmental stresses. We also revealed a large number of novel outputs of DAF-16. Taken together, these studies provide insight into the complex regulation by DAF-16 to control diverse biological functions and eventually broaden our understanding of aging.

Assessing the genetics of wild animal populations aims to understand selective pressures, and factors whether it be inbreeding or adaptation, that affect the genome. Although numerous techniques are available for assessing population structure, a major obstacle in studying wild populations is obtaining samples from the animals without having to capture them, which can lead to undue distress and injury. Therefore, biologists often use non-invasive sampling methods (i.e., collection of feces, hair) to extract host DNA. In this study, new DNA extraction protocols were developed that improved the quality and quantity of DNA obtained from fecal matter. Fecal samples aged up to Day 6 as well as field samples with unknown days since defecation were successful in individualization of the contributors using microsatellites and were further used to demonstrate kinship. Neutral markers such as short tandem repeat, and mitochondrial D-loop sequences are used for assessing relatedness and evolutionary relationships and can mutate without detrimental effects on the organism. Loci, such as the major histocompatibility complex (MHC), adapt more rapidly under selective pressure such as parasite load, or resistance to diseases and support natural selection processes. Analysis of the neutral microsatellites in Big Summit feral horse population demonstrated a population lacking diversity and trending towards being an inbred population. However, examination of the MHC genes showed maintenance of greater variation that may be the result of selection pressures. The MHC similarity and lower genetic demarcation between geographically separated horse populations further indicated effect of selection pressures in preserving diversity at the MHC genes. Although such molecular markers are used in profiling populations, the current study was also successful in demonstrating the use of individual odor profiles as an additional profiling tool. Volatile organic compounds (VOC) obtained from hair of domestic horses were able to individualize horses as well as differentiate between horse breeds and display kinship. The relation of genetics to odor phenotype is of interest as the inherent polymorphic nature of MHC genes has the potential to generate unique combinations of genotypes that presumably produce distinct odor phenotypes. Subsequently, this study was able to show a significant correlation between MHC genotypes and VOC odor profiles in horses. Understanding the relationship between MHC and odor using domestic horses with known relatedness provides evidence that these same correlations may be applicable to wild equids and dictates their harem hierarchal social structure.

Chlamydia is the most reported sexually transmitted infection in the US and is caused by the obligate intracellular bacterium Chlamydia trachomatis. Typically, this presents as a lower genital tract infection (cervicitis or urethritis), but can ascend to the upper genital tract, causing pelvic inflammatory disease, tubal infertility, epididymitis, or ectopic pregnancy. While chlamydia infections can be cured with a single-dose oral antibiotic, repeat infections are common and having multiple chlamydial infections increases a woman’s risk of developing serious chronic conditions. Previous research has shown that estrogen has a positive effect on C. trachomatis infections—an important finding, connecting fluctuating estrogen levels in females to variance in pathogenesis.The mechanism behind this hormonal influence remains unknown; however, previous work in our laboratory indicates that estrogen-stimulated stromal cell effectors play a role in enhancing C. trachomatis infections in a polarized endometrial epithelial Ishikawa (IK)/stromal (SHT-290) cell co-culture model. Specifically, our data indicate that estrogen exposure stimulates osteopontin and vimentin release from stromal cells in co-culture with endometrial epithelial cells. Furthermore, we noted that Chlamydia-infected, polarized Ishikawa cells exposed to a combination of recombinant osteopontin and estrogen released significantly more infectious chlamydia than cultures exposed to estrogen alone. Most tissue culture models being used today employee non-polarized cells. Given the fact that epithelial cell polarization is known to impact C. trachomatis serovar E development, in the current study we sought to determine if the estrogen-induced stromal cell effectors, osteopontin and vimentin, affect C. trachomatis viability and infectivity in non-polarized Ishikawa cells. Non-polarized Ishikawa cells were exposed to osteopontin or vimentin in the presence or absence of estrogen, infected with C. trachomatis serovar E, and collected for examination of chlamydial infectivity and progeny production. Our initial data show that osteopontin and vimentin impact chlamydial progeny production in a concentration dependent fashion, with higher concentrations of recombinant effectors +/- estrogen significantly decreasing progeny production. These data suggest that polarization of host cells influences the way hormone-stimulated effectors interact with the cell to impact on chlamydial infection. Future research goals are to explore other stromal effectors such as fibronectin with estrogen and to study the cell signaling mechanism osteopontin and vimentin use to affect chlamydial infections in polarized epithelial cell cultures.

The observations that substance P (SP) is localized in the anterior pituitary gland (AP) and is regulated by the hormonal status of the animal, as well as the demonstration of SP binding sites in the AP, have led to the idea that SP may participate in the regulation of AP function. Numerous and sometimes contradictory reports of SP effects on AP hormone secretion, particularly on luteinizing hormone (LH), left the question of whether SP acts directly at the level of the AP to regulate LH secretion still unanswered. To investigate a possible physiological function of SP in the AP, the effects of exogenous SP on LH secretion from AP cells from adult and prepubertal male and female rats in short term culture were studied. It was found that SP (100nM-1μM) significantly stimulates LH release in cultured AP cells and that this effect varies as a function of age and sex. SP has no significant effect on LH release from AP cells of male and female prepubertal rats. After day 30 a sharp increase in the response to SP occurs in both sexes. This level of responsiveness continues through adulthood in AP cells from the female rat. In contrast, AP cells from male rats failed to respond during adulthood (over 50 days of age) but were highly responsive during the peripubertal period (30-35 days). The possibility that the responsiveness to SP is influenced by the endocrine status of the animal was investigated by exposing AP cells from responding animals to androgens in vivo and in vitro. It was found that AP cells from female rats treated with androgen were less responsive to 100nM SP but did respond at higher doses of SP. SP effects on AP function were further analyzed in experiments using radioligand binding assays to assess possible changes in SP receptor number or affinity as related to age and sex. In AP membranes from female rats, maximum binding is 8-fold higher (Bmax=4.2 pmo1/mg membrane protein) than in AP membranes from male rats (Bmax=560fmo1/ mg membrane protein). These studies suggest a role for SP as a secondary regulator of LH secretion with possible physiological significance for reproductive function.

The 21st century brought about a dramatic increase in knowledge about genetic and molecular profiles of cancer. This information has validated the complexity of tumor cells and increased awareness of “nodal proteins”, but has yet to advance the development of rational targeted cancer therapeutics. Nodal proteins are critical cellular proteins that collect biological inputs and distribute the information across diverse biological processes. Survivin acts as a nodal protein by interfacing the multiple signals involved in mitosis and apoptosis and functionally integrate proliferation, cell death, and cellular homeostasis. By characterizing survivin as a target of both Type 1 Insulin-like Growth Factor (IGF-1) and Notch developmental signaling, we contribute to the paradigm of survivin as a nodal protein. The two signaling systems, Notch and IGF-1, regulate survivin by two independent mechanisms. Notch activation induces survivin transcription preferentially in basal breast cancer, a breast cancer subtype with poor prognosis and lack of molecular therapies. Activated Notch binds the transcription factor RBP-Jк and drives transcription from the survivin promoter. Notch mediated survivin expression increases cell cycle kinetics promoting tumor proliferation. Inhibition of Notch in a breast xenograft model reduced tumor growth and systemic metastasis. On the other hand, IGF-1 signaling drives survivin protein translation in prostate cancer cells. Binding of IGF-1 to its receptor activates downstream kinases, mammalian target of rapamycin (mTOR) and p70 S6 protein kinase (p70S6K), which modulates survivin mRNA translation to increase the apoptotic threshold. The multiple roles of survivin in tumorigenesis implicate survivin as a rational target for the “next generation” of cancer therapeutics.

Background Obesity is an important risk factor for resistance to insulin-mediated glucose disposal, and is a precursor of type 2 diabetes and other disorders. Objectives To identify molecular pathways in adipose tissue and inflammatory cells that may result in obesity-associated insulin resistance, we exploited the fact that not all obese individuals are prone to insulin resistance. Thus the degree of obesity as a variable was removed by studying obese subjects of similar body mass index (BMI) who are insulin-sensitive (IS) versus insulin-resistant (IR). Methods Combining gene expression profiling with computational approaches, we determined the global gene expression signatures of omental and subcutaneous adipose tissue samples obtained from 10 obese-IR and 10 obese-IS patients undergoing gastric bypass surgery. In a secondary study, we isolated monocytes from 4 obese-IR, 3 obese-IS, and 4 nonobese-IS adolescent and young adult subjects for purposes of assessing differences in expression of inflammatory genes in monocytes using RT-PCR. Results Gene sets related to chemokine activity and chemokine receptor-binding were identified as most highly enriched in the omental tissue from obese-IR compared to obese-IS subjects, independent of BMI. Strikingly, insulin resistance, but not BMI, was associated with increased macrophage infiltration in the omental adipose tissue, as was adipocyte size. In the adolescent and young adult cohort, expression of two cytokine signaling molecules (IL8, SOCS3) and two downstream products of the JNK pathway (JunB, c-Fos) showed increased expression in the obese-IR subjects compared to the obese-IS and nonobese-IS subjects, suggesting the presence of a proinflammatory phenotype in monocytes in obesity, which is exacerbated in the insulin resistant state. Conclusions Our findings demonstrate that inflammation of omental adipose tissue and activation of proinflammatory monocytes is strongly associated with insulin resistance in human obesity. Manipulation of these pathways may result in the prevention of or delay in the onset of obesity-related co-morbidities.