Browsing by Author "Clay, Colin, committee member"
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Item Open Access Cholinergic synaptic homeostasis is regulated by Drosophila α7 nicotinic acetylcholine receptors and Kv4 potassium channels(Colorado State University. Libraries, 2021) Eadaim, Abdunaser Omar, author; Tsunoda, Susan, advisor; Tamkun, Michael, committee member; Amberg, Gergory, committee member; Bouma, Gerrit, committee member; Clay, Colin, committee member; DeLuca, Jennifer, committee memberHomeostatic synaptic plasticity (HSP) is an important mechanism that stabilizes neural activity during changes that occur during development and learning and memory formation, and some pathological conditions. HSP in cholinergic neurons has been implicated in pathological conditions, such as Alzheimer's disease and nicotine addiction. In a previous study in primary Drosophila neuron culture, cholinergic activity was blocked using pharmacological tools and this induced a homeostatic response that was mediated by an increase in the Drosophila α7 (Dα7) nAChR, which was subsequently tuned by an increase in the voltage-dependent potassium channel, Kv4/Shal. In this study, we inhibit cholinergic activity in live flies using temperature-sensitive mutant alleles of the choline acetyltransferase gene (Chats2 mutants). We show that this in vivo activity inhibition induces HSP similarly mediated by Dα7 nAChRs followed by an up-regulation of Kv4/Shal. We show that the up-regulation of Dα7 nAChRs alone is sufficient to induce an increase in Kv4/Shal protein, as well as mRNA. Finally, we test the involvement of transcription factors, dCREB2 and nuclear factor of activated T cells (NFAT) in the up-regulation of Kv4/Shal. In particular, we find that NFAT is required for the inactivity-induced up-regulation of Kv4/Shal channels. Our studies reveal a novel receptor-ion channel system transcriptionally coupled to prevent over-excitation.Item Open Access Contributions of specific TRPS to myometrial calcium entry(Colorado State University. Libraries, 2010) Ulloa, Aida Erendira, author; Sanborn, Barbara M., advisor; Clay, Colin, committee member; Curthoys, Norman P., committee member; Kinnamon, Sue C., committee member; Roess, Deborah A., committee memberUnderstanding the mechanisms that regulate contractions in the myometrium during pregnancy may help avoid scenarios such as premature births. During labor, increases in intracellular calcium ([Ca 2+],) have been closely correlated with human myometrium contractions. Extracellular calcium enters the cell through voltage-operated and signal-regulated Ca2+-entry (SRCE) mechanisms and is involved in actions such as stimulating the contractile apparatus and replenishing intracellular Ca^^ stores. In SRCE, activation of some receptors and/or depletion of agonist-sensitive [Ca^^]j-stores stimulates Ca -uptake from the extracellular solution. This and other SRCE mechanisms are all important in providing regulation of calcium homeostasis. Ion channels potentially responsible for SRCE are the canonical transient receptor potential (TRPC) channels. The seven members (TRPC 1-7) are postulated to form hetero- or homotetramers, thus allowing for the formation of a variety of channels possessing different physiological properties. Additional TRPC channel regulation is also provided by STIMl, an endoplasmic reticulum Ca2+ sensor. My studies show that human myometrium expresses higher concentrations of TRPC4 and TRPCl mRNAs relative to other TRPCs as well as expressing STIMl proteins. Furthermore, I have found that specific TRPC proteins are involved in the SRCE pathways when studied in immortalized myometrial PHMl cells. The main objective of this work was to understand the functional role of TRPC 1 and TRPC4 channels in relation to Ca^^ signaling in order to elucidate their relative significance in myometrium. The individual roles for TRPCl and TRPC4 as well as the potential additive effects that a TRPCl plus TRPC4 double knockdown exerted on SRCE were studied in PHMl and primary human uterine smooth muscle (UtSMC) cells. This was achieved through RNAi mechanisms by expression of shRNAs targeting TRPCl, TRPC4, or TRPCl plus TRPC4. The role of STIMl in myometrial Ca2+ dynamics was also investigated by use of STIM1AERM, a dominant negative form of STIM1. The data presented here suggest that both TRPCl and TRPC4 are activated by similar G protein coupled receptor-stimulated Ca^^ entry mechanisms; however, no additive effects were observed by their combined knockdown. Additionally, thapsigargin- and OAGstimulated Ca entry were not affected by either the individual or combined knockdown of TRPCl and TRPC4. In contrast, STIMAERM appeared to induce an inhibitory effect on all three types of SRCE stimulation. These contributions, in addition to the important role in Ca2+ homeostasis played by voltage-operated channels and the influences of Ca^^ pumps, exchangers and potassium channels, provide the myometrium with the ability to respond in specific and precise ways to influences in its environment, both during pregnancy and at the time of parturition.Item Open Access Estradiol exposure alters gonadothropin-releasing hormone (GNRH) induced gonadotrope plasticity(Colorado State University. Libraries, 2010) Hartshorn, Cheryl, author; Tobet, Stuart, advisor; Clay, Colin, committee member; Hentges, Shane, committee member; Tjalkens, Ron, committee memberThe reproductive axis is dependent upon communication among the hypothalamus, pituitary and gonads. For successful ovulation, a large increase in circulating estradiol provides positive feedback at both the hypothalamic and pituitary levels to promote an luteinizing hormone (LH) surge. An LH surge is necessary for the final maturation of the pre-ovulatory follicle and ovulation. The cellular and molecular events underlying estradiol’s action(s) upon the anterior pituitary gland, specifically gonadotropes, remain elusive. Recent video microscopy experiments showed that pituitary cells in vitro in slice culture move in response to GnRH [Navratil, et al., 2007]; presumably these cells were gonadotropes. The current study utilized a novel transgenic animal model that has gonadotrope specific fluorescence provided by yellow fluorescent protein (YFP) [Wen et al., 2008]. I sought to determine if 17(3-estradiol (E2) working through either a genomic or non-genomic mechanism affected gonadotrope specific movements in response to GnRH. Consistent with earlier studies [Navratil et al., 2007], application of GnRH [100nM] altered the cytoarchitecture of gonadotropes with observable cell process extensions. Using live video- microscopy, exposure to 10nM E2 for fourteen hours significantly enhanced the ability of gonadotropes to extend processes in response to GnRH compared to short-term exposure of E2 (1.5 hours) or vehicle. There was no demonstrable effect of 1.5 hours of E2 exposure on GnRH-induced process extensions. I hypothesize that the differential effect of short-term versus long-term E2 exposure is due to a genomic mechanism that may underlie the ability of E2 to enhance GnRH induced cellular plasticity. Thus, E2 and GnRH may cooperate to maximize the secretory interface between gonadotropes and the adjacent vasculature during the pre-ovulatory LH surge.Item Embargo IQGAP1 is a novel effector of gonadotropin-releasing hormone receptor signaling(Colorado State University. Libraries, 2023) Alqahtani, Huda A., author; Amberg, Gregory, advisor; Clay, Colin, committee member; Tamkun, Michael, committee member; DeLuca, Jennifer, committee memberStimulation of gonadotropin-releasing hormone (GnRH) receptors on the surface of anterior pituitary gonadotrope cells is a key signaling event for the hypothalamic-pituitary-gonadal axis. One important downstream component of GnRH receptor signaling is activation of the mitogen-activated protein kinase ERK (extracellular signal-regulated kinase), which is essential for the production of the gonadotropin luteinizing hormone. Evidence suggests that GnRH receptors reside in low-density plasma membrane domains where they participate in multiprotein signaling complexes. Here we used quantitative proteomics to identify proteins associated with low-density plasma membrane domains and to measure changes in their relative abundance in these domains in response to GnRH. Using αT3-1 gonadotropes, we identified 537 proteins in detergent-free subcellular fractions containing low-density plasma membranes. SILAC (stable isotope labeling by amino acids in cell culture) in combination with mass spectrometry demonstrated that GnRH, within 10 min, altered the association of 87 proteins with this plasma membrane fraction. Ontology analysis revealed that GnRH promoted an enrichment of actin cytoskeletal and adherens junction-related proteins including the molecular scaffold IQGAP1 and the small GTPase Rac1. Subsequent investigation revealed that the association between Rac1 and IQGAP1 increased with GnRH receptor stimulation and that GnRH increased Rac1 activity. Demonstrating functional relevance, inhibiting Rac1 reduced GnRH-dependent ERK activation. Our data reveals an upstream activation of signaling and structural molecules, including Ca2+, CDC42 and Rac1, E-cadherin, N-cadherin, and β-catenin. We also identified interactions between the scaffold protein IQGAP1 and these molecules, indicating that IQGAP1 is a fundamental regulator of GnRH-dependent signaling in gonadotropes. Furthermore, our data shows that IQGAP1 has a transcriptional regulatory role in gonadotropes treated with GnRH. In sum, these data indicate that IQGAP1 complexed with Rac1 modulates ERK activity and as such serves as an essential effector in modulating cell polarity and cell-cell contacts in gonadotropes. Altogether, our proteomics data show that acute stimulation of GnRH receptors (3 nM for 10 min) alters the PAM fraction abundance of proteins, such as IQGAP1, mechanistically linked to gonadotrope activation.Item Open Access L-type calcium channel-dependent signaling impacts GnRH receptor function and intercellular communication in cultured gonadotropes(Colorado State University. Libraries, 2020) Drennan, Meggan L., author; Amberg, Gregory, advisor; Clay, Colin, committee member; Garrity, Deborah, committee member; Kelp, Nicole, committee memberThe hypothalamic-pituitary-gonadal (HPG) axis is a negative feedback biological system critical in fertility, reproduction and development. Gonadotropin-releasing hormone (GnRH) is first released by the hypothalamus and binds to GnRH receptors (GnRH-R) on gonadotrope cells of the anterior pituitary gland where the receptors must mediate a variety of pulsatile signals. The gonadotropin hormones, luteinizing hormone (LH) and follicle stimulating hormone (FSH), are subsequently released by the pituitary and act upon the ovaries and testes, further producing gonadal steroids to be circulated throughout the body. GnRH pulse frequency and amplitude determine successful gonadotropin release, which is ultimately regulated by the GnRH-R. The GnRH-R is a heterotrimeric G-protein coupled 7-transmembrane domain receptor with Gα, β, and γ subunits. Ligand binding initiates an intracellular cascade that leads to a global increase of cytosolic calcium concentration by way of calcium influx through voltage-gated calcium (Cav) channels, and intracellular calcium release from endoplasmic reticulum (ER) stores. Gonadotropes depend on intracellular calcium concentration to carry out their specific physiological function, such as transcription of gonadotropin subunits, hormone biosynthesis and release. Calcium flux is a normal and important aspect of cellular function, including cell-cell communication. Calcium oscillations have been well documented in multiple cell types, with different patterns being induced with distinct treatments. Observations in this line of research include the following: different oscillatory patterns lead to different physiological outcomes, the rate at which internal calcium is secreted from the ER can greatly impact these patterns, and IP3 receptor clustering on the ER results in localized changes in calcium concentration rather than a marked global difference, implicating a spatial stochasticity. These oscillations have shown evidence of paracellular coupling at gap junctions, as well as synchrony following extracellular diffusion. Chapter two of this thesis details experiments investigating calcium oscillations using a membrane-targeted calcium indicator. Immortalized αT3-1 cells were transfected with a membrane-targeted GCaMP and TIRF microscopy was used to capture fluorescent calcium activity. Cells were treated with GnRH as well as various pharmaceutical treatments that would exploit L-type Cav channel function and manipulate normal intracellular calcium release. An array of observations was recorded. Qualitatively, there was an overall increase in calcium activity in the majority of cells after GnRH treatment. Drug-induced inhibition of calcium influx and intracellular calcium release diminished calcium activity entirely. Further, synchronized activity was captured among several cell groups, showing both pre-established synchrony and GnRH-induced synchronized peaking. Further research should be conducted to better understand the full mechanism underlying these behavioral responses, but these experiments provide a foundation for this work. Chapter three highlights experiments using a GFP-tagged GnRH-R in αT3-1 gonadotropes in order to investigate GnRH binding-induced receptor mobility and clustering. Treatment groups were identical to the previous chapter. SRRF and binary analysis were used to characterize receptor activity. Descriptively, clustering of receptors was seen, especially when calcium activity was limited, but more appropriate methods of quantitative analysis should be explored in order to go beyond these observations in processed images. This thesis concludes overall that GnRH-induced calcium oscillation patterning and receptor clustering are far more complex and difficult to study than initially thought. Much more research is needed to determine any conclusive findings, however, these experiments may serve as a stepping stone toward obtaining the answers sought.Item Open Access Physical and molecular characteristics of day 75 nuclear transfer cloned bovine conceptuses(Colorado State University. Libraries, 2012) De Lille, Alexandra, author; Seidel, George, advisor; Anthony, Russell, committee member; Clay, Colin, committee member; Garry, Franklyn, committee memberThis study was designed to measure fetal and placental characteristics in bovine day 75 nuclear transfer and control pregnancies. Responses included mRNA concentration of the insulin-like growth factor (IGF) system [IGF-1, IGF-2, IGF1R, IGF2R, IGFBBP-1, -2, -3] and the vascular endothelial growth factor (VEGF) system [VEGF, PlGF, VEGF1R, and VEGF2R]. Fetal attrition of the cloned pregnancies up to day 75 was high (89%, 63 out of 71 frozen embryos transferred; 8 of 16 cloned conceptuses present on day 30 survived to day 75, as did 5 of 5 controls). No significant differences in mean weights of large and medium placentomes were observed between 8 clones and 5 controls. However, the variance of mean weight of large placentomes was greater in clones than in controls; one gestation had placentomes six standard deviations larger than controls. Interestingly, the mean umbilical cord weight/length ratio was significantly greater for clones (P < 0.05). Mean fetal length, fetal weight, fetal weight/length index and mean weights for heart, brain, liver, kidneys and the mean brain/liver index did not differ between cloned and control day 75 conceptuses, but numbers per group were limited. Northern blot analysis, revealed the presence of three transcripts of 3.7kb, 2.2kb and 1.7kb for VEGF and one 1.7 kb transcript for PlGF mRNA in the cotyledons and allantochorion of day 45 cloned and control gestations. All three VEGF bands were present in both cloned and control day 75 cotyledons and caruncles, but the PlGF transcript was barely detectable, except for the cotyledons of one clone. mRNA for all of genes studied could be detected with real time PCR in day 75 cotyledons and caruncles, and fetal livers contained mRNA for all IGF's and IGFBP's evaluated. In all placentomal tissues, PlGF mRNA concentration was 100-fold less than VEGF mRNA, which seems to be the driving force for placentomal vascularization at day 75. There was a trend for a reduction by half of the PlGF mRNA concentration in caruncle of clones vs. controls (P = 0.06). VEGF2R (KDR) mRNA was abundant, but VEGF1R (Flt-1), was only present in very low concentrations; our primer set did not distinguish between soluble versus membrane bound receptor mRNA for VEGF1R. Four of the cloned conceptuses contained substantially less cotyledonary IGF1R mRNA than the other clones and controls. IGFBP-3 mRNA concentrations were very high in placentomes; IGFBP-1 and -2 mRNA concentration on the other hand was very low for clones and controls. mRNA for IGFBP-1, -2, -3, however, was abundant in day 75 fetal livers, while IGF-1 mRNA was scarce in this tissue. Fetal livers from cloned pregnancies contained 4-fold more IGF-2 mRNA than controls (P<0.01). We observed that liver IGF-2 mRNA concentration and liver weight increased with weight of the largest placentome; in clones these increases were associated with a decrease in cotyledonary IGF-2 mRNA, while the opposite occurred with controls. Interestingly, there was a trend to lower IGF2R mRNA concentrations (P = 0.09), and IGF-1 mRNA was twofold higher in cotyledons of clones (P= 0.03) compared to controls. For many measurements, means were not significantly different (P > 0.1) between clones and controls. However, unequal variances were common, and data points with statistical outlier behavior were observed for clones, which highlights the heterogeneity of the cloned population. This variation in gene expression may exacerbate abnormal placentation later in gestation, and might explain some of the increased morbidity and mortality seen in calves resulting from cloning by nuclear transfer.Item Open Access Proopiomelanocortin neuron manipulation in mouse models of energy balance disorders(Colorado State University. Libraries, 2021) Daimon, Caitlin Mieko, author; Hentges, Shane, advisor; Clay, Colin, committee member; Myers, Brent, committee member; Vandewoude, Susan, committee memberProopiomelanocortin (POMC) neurons in the arcuate nucleus (ARC) of the hypothalamus are critical regulators of energy balance. Highly conserved amongst mammalian species, POMC neurons release peptide transmitters to help an organism maintain appropriate levels of food intake and bodyweight by inhibiting feeding and facilitating metabolism of consumed nutrients. Disruptions in POMC signaling are thought to underlie aspects of energy balance disorders. There are two kinds of energy balance disorders: those of positive energy balance, which includes diseases like obesity, and those of negative energy balance, which includes eating disorders like anorexia nervosa (AN). Given that POMC neurons are believed to be dysregulated in energy balance disorders, treatment strategies for these disorders have focused on POMC neurons or their targets. The goal of the studies discussed herein was to determine whether manipulation of POMC neurons could improve pathophysiological alterations in bodyweight and food intake in mouse models of energy balance disorders. Mouse models of AN and obesity were used in the current studies. AN was mimicked in the mouse via the well-validated activity-based anorexia (ABA) behavioral paradigm. The results shown in chapters 2 and 3 indicate that POMC neurons are selectively involved in generating food anticipatory activity (FAA) in mice undergoing ABA as disruption of either the POMC peptide product β-endorphin or inhibition of the entire POMC neuron resulted in decreased FAA. As FAA is the primary output of the food entrainable oscillator (FEO), the circadian clock that allows an organism to anticipate the daily arrival of meals, these results suggest that POMC neurons via the peptide product β-endorphin are possibly involved in the expression of the FEO. As the identity of the FEO has yet to be determined, future studies should further characterize the contribution of β-endorphin and POMC neurons to the FEO. To determine whether manipulation of POMC neurons is beneficial in a mouse model of obesity, mice fed an obesogenic diet were subjected to chronic POMC neuron stimulation for one month. The unexpected finding that sustained stimulation leads to weight gain as opposed to weight loss indicates that chronic stimulation of POMC neurons may not be a viable option for weight loss, at least under the dosing scheme used in the current study. How POMC neurons adapt to chronic stimulation remains unknown and should be the focus of future work.Item Open Access The role of proline rich 15 in trophoblast cell development(Colorado State University. Libraries, 2012) Gates, Katherine C., author; Anthony, Russell V., advisor; Clay, Colin, committee member; Duval, Dawn L., committee member; Hansen, Thomas R., committee memberMaintenance of pregnancy in eutherian mammals requires a sophisticated and tightly regulated program of gene expression in order to develop a fully functional placenta. This transient organ mediates nutrient and gas exchange between the mother and fetus while protecting the fetus from the maternal immune system. Deviations from the normal regulation of gene expression during early pregnancy can lead to early embryonic loss as well as dysfunctional placentation, which can cause significant maternal and fetal morbidity and mortality. Proline rich 15 (PRR15) is a low molecular weight nuclear protein expressed by the trophoblast during early gestation in several mammalian species, including humans, mice, cattle, sheep, and horses. Immunohistochemistry revealed localization of PRR15 to the trophectoderm and extraembryonic endoderm of day 15 sheep conceptuses. In humans, PRR15 is localized in the nuclei of both first and second trimester trophoblast cells. Additional research has shown increased PRR15 transcription in colorectal cancers with mutations in the adenomatous polyposis coli (Apc) protein, suggesting a link to the Wnt signaling pathway. PRR15 mRNA concentrations increase when trophoblast cells, both sheep (oTR) and human (ACH-3P), are cultured on Matrigel, a basement membrane matrix. The expression profile in the sheep conceptus during pregnancy revealed a rise in PRR15 mRNA concentrations during the period of conceptus elongation with a peak in expression at day 16 of gestation, followed by a decline to day 30 of gestation. This peak coincides with a halt in elongation of the conceptus, and the initial period of apposition to the uterine luminal epithelium. Lentiviral-mediated knockdown of PRR15 in ovine trophectoderm at the blastocyst stage led to demise of the embryo by day 15 of gestation. This provides compelling evidence that PRR15 is a critical factor during this precarious window of development when initial attachment and implantation begin. The first aim of this research was to determine the effect of PRR15 deficiency on trophoblast gene expression, as well as trophoblast proliferation and survival. The human first trimester trophoblast cell line, ACH-3P, was infected with control lentivirus (LL3.7) and lentivirus expressing a short hairpin (sh)RNA to target PRR15 mRNA for degradation, resulting in a 68% decrease in PRR15 mRNA (p<0.01). Microarray analysis of these cell lines revealed differential expression of genes related to cancer, focal adhesion, and p53 signaling. We selected 21 genes for validation of mRNA levels by quantitative real-time RT-PCR, 18 (86%) of which gave results consistent with the microarray analysis, with similar direction and magnitude fold changes. This included significant up-regulation of GDF15, a cytokine increased in pregnancies with preeclampsia. GDF15 mRNA concentrations were examined more extensively during early ovine gestation, which revealed that GDF15 was low during peak PRR15 expression, then increased significantly at day 30 when PRR15 was nearly undetectable. Proliferation, as measured by cell metabolic activity and bromodeoxyuridine (BrdU) uptake, decreased in the PRR15-deficient cells, which was consistent with a decrease observed in cell cycle-related genes CCND1 and CDK6, and an increase in CCNG2 and CDKN1A in the PRR15-deficient cells. TNFSF10, a tumor necrosis factor superfamily member known to induce apoptosis, and its receptor, TNFRSF10b, increased significantly in the PRR15-deficient cells, suggesting trophoblast cells may be more susceptible to apoptosis when depleted of PRR15. Assays for caspase activity and annexin V staining revealed an increased population of apoptotic cells when treated with shRNA to target PRR15. These results suggest that PRR15 is required for driving trophoblast proliferation and survival during early development of the placenta, functions that are critical to early embryonic survival and successful placentation. The second experimental aim was to examine regions of the PRR15 promoter that are necessary for regulating its expression in trophoblast cells and to identify the role of Wnt signaling in PRR15 transcription. The 5'-flanking sequences from -824, -640, -424, -326, and -284 bp to +7 bp relative to the annotated transcription start site were amplified by PCR and ligated into the pGL3-Basic plasmid. These vectors were co-transfected into the first trimester human trophoblast cell line, ACH-3P, HT29 (human colorectal carcinoma), oTR, and BHK-21 (hamster kidney fibroblast) cells with a RSV-β-galactosidase vector control. In ACH-3P cells, transactivation of the luciferase reporter was maximal following transfections with the -326 construct (15.4 ± 4.8-fold). Significant promoter activity was absent in the -284, -424, and -640 constructs, but regained with the -824 construct (14.8 ± 5.8-fold). These results suggest that cis-acting elements within the proximal promoter of the PRR15 gene are essential for expression in trophoblast cells, requiring the regions from -284 to -326 and -640 to -824. DNase I footprinting and electrophoretic mobility shift assays were performed to identify transcription factor binding sites within these regions. Due to the potential link to the Wnt signaling pathway, cells were treated with an inhibitor to GSK3β, the kinase responsible for phosphorylation and proteasomal degradation of β-catenin. Inhibition of GSK3β decreased PRR15 mRNA concentrations and decreased transactivation of the luciferase reporter in all proximal promoter reporter constructs; this effect was mediated through β-catenin activity in the proximal 284 bases of the PRR15 5'-flanking region. Furthermore, trophoblast cell proliferation decreased after treatment with the GSK3β inhibitor. Electrophoretic mobility shift assays on the region from -98 to -68 revealed differential binding of nuclear proteins derived from ACH-3P cells grown in the presence or absence of the GSK3β inhibitor. These results reveal that canonical Wnt signaling inhibits the transcription of PRR15, mediated in part through the -98 to -68 region of the 5'-flanking region, and decreases proliferation in trophoblast cells. This indicates that suppression of Wnt signaling may be crucial during early trophectoderm outgrowth in order to allow significant transcriptional activation of PRR15 and conceptus survival.