Claude-Luvier Bonnet

The Genetic Underpinnings of Glucocorticoid Receptor Polymorphisms in Obese Populations

Obesity is a product of multiple comorbidities ,such as elevated blood pressure, serum triglycerides, and blood sugar. All of these factors are affected by nuclear hormone receptors that regulate gene expression. The glucocorticoid receptor (GR), is one example of these nuclear hormone receptors. The GR binds glucocorticoids, most importantly, cortisol, which controls factors that contribute to obesity. Specific polymorphisms of the GR, the mineralocorticoid receptor (MR), heat shock protein 90 (HSP90) and 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) have been found in our labs and others to be present at high frequency in obese populations. One new gene of interest is the FK506 binding protein (FKBP), a co-chaperone that works in conjunction with HSP90 to negatively regulate GR activity. It is hypothesized that polymorphisms exist in the FKBP51 gene that would result in decreased activity of FKBP and subsequent overactivity of GR resulting in phenotypic hypercortisolemia. Analysis of the FKBP51 single nucleotide polymorphism RS1360780 showed a correlation with serum triglycerides, blood pressure, BMI, in bariatric surgery patients. This finding, in combination with previous results, will lead to a better understanding of the genetic underpinnings of obesity.

Ayanah Dowdye

Assessing Glucocorticoid Receptor Polymorphisms in Obese Populations

The glucocorticoid receptor (GR) is part of a family of nuclear receptors that control gene expression. In the presence of the steroid hormone cortisol, certain genes are expressed; the products of which control certain features of the body, including but not limited to, blood pressure, serum triglycerides, and blood sugar. There is evidence that these features are major contributors to obesity. Specific polymorphisms of the GR and other regulators of either GR or the closely related mineralocorticoid receptor such as heat shock protein 90 (HSP90) and 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) have been found in our labs and others to be present at a higher frequency in obese populations. Our current research is investigating polymorphisms in these genes as well as the gene encoding the FK506 binding protein (FKBP) which works in conjunction with HSP90 to negatively regulate GR activity. This finding in combination with previous results will lead to a better understanding of the genetic underpinnings of obesity and may reveal novel therapeutic approaches. These polymorphisms will be assessed in relation to post-operative features of bariatric surgery, analyzing whether or not there is a correlation with weight regain after bariatric surgery in the presence of certain mutations.

Claire Kelly

Impact of Single Nucleotide Polymorphisms on HPA Axis Functionality in Depression

The hypothalamic-pituitary-adrenal (HPA) axis plays a primary role in stress response through the regulated secretion of the glucocorticoid hormone cortisol. Diseases of cortisol dysregulation such as Cushing’s syndrome (hypercortisolemia) and Addison’s Disease (hypocortisolemia) are both associated with depression. Based on this we, and others, have hypothesized that mutations in the genes for the glucocorticoid receptor (GR), the closely related mineralocorticoid receptor (MR), and regulatory proteins associated with cortisol or GR function may contribute to depression in the absence of hyper- or hypo-cortisolemia. Our study investigated the genotypic frequency in the clinical population of several single nucleotide polymorphisms (SNPs) that affect GR and MR sensitivity to cortisol binding. Buccal swab DNA samples were acquired from patients clinically diagnosed with depression and from a random population. Extracted DNA was analyzed utilizing multiple allele-specific polymerase chain reactions to determine genotypic frequency of SNPs associated with hypersensitivity or resistance to cortisol. In addition, patients suffering from depression completed the multiple measures of depression and anxiety. Preliminary results have shown that a specific allele of the GR BclI polymorphism (rs41423247) is more frequently seen in depressed patients compared to a random population and is also associated with elevated measures of depression. Understanding the role of geneotypic variation in cortisol function could lead to more specific and targeted therapies for depression with the goal of improving patient outcomes.

Annie Leamon

FSH Dependent Activation of Proteins in Murine Osteoclast and Osteoclast Precursor Cells

Osteoporosis, an illness resulting in weak and brittle bones, will soon cause over $25 billion dollars of economic damage to the US economy each year. For decades, menopausal osteoporosis was thought to be effect of bone turnover as a result of the loss of estrogen due to depletion of ovarian follicles. Recent research, however, has shown that the hormone primarily responsible for estrogen synthesis, follicle stimulating hormone (FSH), can contribute to bone degeneration. What is unclear is the specific mechanism by which FSH has this effect. We hypothesized that FSH may contribute to monocyte differentiation to osteoclasts. To test this, we used the RAW 246.7 murine monocyte cell line which can differentiate into osteoclasts in the presence of RANKL. We were able to find evidence of FSH-dependent signaling in cells FSH treated cells with or without RANKL. In particular, time dependent activation of the cyclic AMP Response Element Binding Protein (CREB) was observed along with other proteins utilized by FSH signaling and osteoclast differentiation. Interestingly, RANKL treatment changed the pattern of phosphorylation of CREB and the related transcription factor ATF1. Since CREB partially regulates osteoclast differentiation and function this could be one part of the mechanism by which FSH stimulates osteoclastogenesis and bone demineralization. With this in mind, future therapies targeting FSH action could be developed to prevent the onset of osteoporosis.

Hannah Madden

Human Follicle Stimulating Hormone Receptor Lipid Raft Residency is Hormone and Caveolin Dependent
Human follicle stimulating hormone (hFSH) is a gonadotropin responsible for the maturation of ovarian follicles in women and production of sperm in men. The receptor (hFSHR) is a G protein-coupled receptor (GPCR); a class of receptors found embedded in the cell membrane to receive the hormone resulting in initiation of a complex downstream signaling cascade. Through previous research in the Cohen lab, hFSHR has been shown to be located within compartments of the cell membrane known as lipid rafts. Lipid rafts are microdomains of the membrane containing a higher concentration of cholesterol, glycosphingolipids, and a protein known as caveolin. Current research is focused on evaluating the mechanisms and effects of hFSHR lipid raft residency. Using discontinuous sucrose gradients we have shown that hFSH stimulation of an HEK293 cell line stably transfected with hFSHR cDNA results in alteration of hFSHR lipid raft residency in a time dependent manner.
These results suggest that hFSHR residency in lipid rafts is dependent on hormone stimulation potentially through interaction with caveolin. Through studying hFSHR lipid raft residency and signaling we hope to identify ways to control the receptor for improved methods for contraception and treatment of infertility.

Angelina Stewart

Modulation of Human Follicle Stimulating Hormone Receptor Action Using Small Molecules

Human follicle stimulating hormone (hFSH) is a gonadotropin involved in the stimulation of ovarian follicles in women and the proper maturation of sperm in men. The receptor, hFSHR, belongs to the G protein-coupled receptor class of hormone receptors and is found embedded in cell membranes. Binding of hFSH to its receptor initiates a complex downstream signaling pathway, the precise intricacies of which are still being determined. Previous research in has suggested that hFSHR is located in lipid rafts, microdomains of the membrane that are made up of a higher concentration of cholesterol, glycosphingolipids, and the protein caveolin.
To better understand the mechanism and effect of hFSHR lipid raft residency, cells from the HEK293 line were treated with a peptide mimetic of transmembrane domain 4 of hFSHR. This peptide includes a sequence consistent with a motif shown in other proteins to interact with caveolin. The putative caveolin interaction motif (phiXphiXXXXphiXXphi) was synthesized along with the HIV-TAT leader sequence to allow transmembrane passage. A second peptide where the aromatic amino acids were replaced with leucine was also used. Cells treated with the wild type peptide showed elevated basal signaling in the absence of FSH, an effect not observed with the mutant peptide.
The same cell line was also treated with two negative allosteric receptor modulators (NAMs) that have been shown to induce biased signaling of the receptor in other experiments. These modulators had an effect on hFSH action in our HEK293 cell line as well. These results suggest that receptor action can be modified using small molecules. Through further studying this signaling we hope to identify ways to control the receptor for improved methods for contraception and treatment of infertility.

Megan Wells

Follicle Stimulating Hormone Receptor Signaling is Regulated by Lipid Raft Residency

Infertility affects 7.4 million women and their partners in the United States. One cause for infertility in women is human follicle stimulating hormone (hFSH) dysfunction. hFSH is involved in follicle maturation and estrogen production in females and spermatogenesis in males. Lack of normal hFSH function in either the ligand or receptor leads to infertility in women, however does not fully inhibit fertility in males. hFSH is secreted from the pituitary gland and binds to its g-protein coupled receptor (hFSHR) located in the ovaries or the testis. Previous research in our lab has shown that hFSHR resides in cholesterol, sphingolipid, and caveolin enriched microdomains called lipid rafts. It has been previously shown that hFSHR interacts with caveolin although the specific mechanism of this interaction is unknown. One possibility is that the interaction is through a specific amino acid sequence in hFSHR consistent with caveolin interaction motifs identified in other proteins. The current study investigated how lipid rafts regulate hFSHR signaling. It was hypothesized that disruption of lipid rafts or interference with the caveolin interaction motif will alter hFSHR signaling. Lipid rafts in human granulosa cells (the hGrC1 cell line) were disrupted through cholesterol depletion with methyl-β-cyclodextrin (MβCD) and FSH signaling differences were observed by western blot. It was observed that MβCD treatment resulted in an increase of cAMP signaling compared to control cells. In parallel, mutations in the hFSHR caveolin interaction motif were made and expression plasmids were transiently transfected into HEK 293 cells. Western blot analysis revealed that some of the mutants showed increased basal signaling relative to the wild type receptor. Taken together, these results suggest that lipid rafts may be acting act as an inhibitory mechanism for hFSHR and that caveolin may play a role in receptor recruitment to the lipid raft. Researching hFSH signaling after lipid raft manipulation can provide a better understanding of the cellular mechanisms of hFSHR, which could lead to innovative ideas in infertility treatment for women.