Browsing by Subject "Intercellular Signaling Peptides and Proteins"
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Item Cell Migration and Survival Pathways in Cardiac Development and Disease(2005-12-20) Saxena, Ankur; Srivastava, DeepakMammalian cardiac development is a complex process, rendering it susceptible to errors. As a result, congenital heart defects are the most common type of birth defect. Furthermore, heart disease in adults is the leading cause of mortality in the developed world. Given these statistics, gaining an understanding of cardiac development and disease is of paramount importance. Here, data are presented suggesting that the signaling molecules SDF-1, ephrin-B1, and ephrin-B2 play important roles in proper valve formation and maturation during cardiogenesis. Furthermore, another signaling molecule, thymosin ᴬ is implicated in promoting cell survival, potentially through an ILK-Akt pathway, as well as angiogenesis in the treatment of mice post-myocardial infarction. Finally, SDF-1 not only plays a role in cardiac development but, in a manner strikingly similar to the actions of thymosin ᴬ also appears to have therapeutic benefit post-myocardial infarction through the ILK-Akt pathway, reduced cell death, and increased angiogenesis. Together, these data and other information presented herein suggest new roles for signaling molecules in both cardiogenesis and cardiac therapy.Item From Bud to Organ: An In Depth Analysis of the Development of the Pancreas.(2009-09-04) Villasenor, Alethia; Cleaver, OndineIn this dissertation, a careful analysis of different aspects of pancreatic development was conducted in order to expand our understating of the biology of this organ. This thesis encompasses an in depth description of pancreas macro morphology throughout development as well as the analysis of the role of signaling molecules not previously studied in the pancreas. In brief, Chapter 2 presents a characterization of pancreatic branching and cellular polarization. It provides an anatomical model for branching of the pancreas and establishes the dynamics of cell polarity changes within the pancreatic epithelium throughout development. This chapter provides seminal work in an area that has received little attention in forwarding our understanding of how the epithelium reshapes itself to form a functional organ. Chapter 3 and Chapter 4 focus on the endocrine compartment of the pancreas. In Chapter 3, the expression of Neourogenin3, or Ngn3, a master gene regulator of endocrine fate was studied and a novel molecular correlation with the first and secondary transitions of pancreatic endocrine differentiation was demonstrated. In Chapter 4 it was shown that Rgs genes, specifically Rgs8 and Rgs16, are expressed in endocrine cells during pancreatic development and become quiescent during adulthood. Only under models of islet regeneration and pancreatic stress was a re-activation of Rgs8 and Rgs16 expression in endocrine cells observed. Our results suggest that Rgs16 and Rgs8 control aspects of islet progenitor cell activation, differentiation, and their actions might be required to compensate pancreatic metabolic stress. Finally, Chapter 5 analyzes the role Eph/ephrinB signaling in pancreatic development. Mice lacking signaling of EphB2 and EphB3 receptors showed fewer insulin-producing cells, abnormal islet distribution, anomalies in vasculogenesis and disrupted epithelial polarity and branching. In addition, they showed abnormal pancreatic function since the mutants are hypoglycemic after a glucose tolerance test. Studies in this chapter clearly reveal a role for Eph/ephrinB signaling during pancreatic morphogenesis, differentiation, and physiology. Moreover, since the ephrins (ligands) are expressed in the pancreatic mesoderm and blood vessels and the Eph (receptors) are expressed in the pancreatic endoderm; our results suggest that Eph/ephrinB-mediated tissue-cross-talk is required for proper pancreatic morphogenesis and islet formation. Overall, this thesis provides an in depth analysis of the biology of the developing pancreasItem HIF-2: Standing Guard at the Crossroads of Stress and Aging(2009-06-15) Dioum, El Hadji Mamadou; Garcia, Joseph A.The capacity of mammalian organisms to cope with hypoxic or ischemic stress is in part mediated by stress-induced transcription factors. Hypoxia-induced mediators include transcription factors, such as the α (alpha) subunit of Hypoxia inducible factors (HIF-1alpha and HIF-2 alpha). HIF-1 alpha and HIF-2 alpha have similar structural organization, and after forming an obligate heterodimer with the common partner ARNT/HIF-1 alpha, bind to the same recognition element located in target gene promoter or enhancer regions. However, despite these similarities, HIF-1 alpha and HIF-2 alpha regulate distinct target genes. In previous studies from the Garcia laboratory using mouse knockout studies, we demonstrated the importance of HIF-2 alpha in the in vivo regulation of genes involved in the cellular response to hypoxic and oxidative stress. These genes include Erythropoietin (epo), vascular endothelial cell growth factor (Vegf), superoxide dismutase 2 (Sod2) and other genes encoding major antioxidant enzymes (AOE). Novel roles for HIF-2 alpha have been found not only in hematopoiesis, but also in the control of reactive oxygen species and mitochondrial homeostasis. The molecular mechanism by which HIF-2 alpha selectively regulates its target genes remains an exciting area of research. In the first part of my thesis, I identified a novel molecular mechanism regulating activity of the enhancer region in the Epo gene. First, by using bioinformatics to perform an unbiased sequence comparison of several mammalian 3 prime Epo enhancer region, we identified a previously unrecognized evolutionary conserved region. Second, we determined the functional significance of these conserved sequences using transient transfection and mutation analyses in cell culture studies and determined that these sequences contribute to HIF-2 alpha selectivity. Finally, using a candidate factor strategy, we determined that members of the early growth response (Egr) transcription factor family bind to these elements and act synergistically with HIF-2 alpha to augment Epo gene expression. In the second part of my thesis, we demonstrate that the redox-sensing, NAD+ dependent deacetylase enzyme Sirtuin 1, also known as Sirt1 or silent mating type information regulator 2 (Sir2) homolog 1, selectively stimulates HIF-2 alpha signaling during hypoxia. In lower organisms and cell culture models, the FoxO family of transcription factor regulates the transcription of SOD2 and other major AOE. During oxidative stress, Sirt1 modulates FoxO transcriptional activity, promoting the protective cellular response to oxidative stress. We hypothesized that Sirt1 would be activated by redox changes induced by hypoxia and that activated Sirt1 would in turn modulate HIF-2 signaling. We determined that HIF-2 alpha signaling is indeed increased by Sirt1 in transfection assays. Sirt1/HIF-2 alpha signaling does not involve previously described oxygen-dependent HIF-2 alpha modifications. Sirt1 augmentation of HIF-2 alpha transcriptional activity involves direct binding to and deacetylation of HIF-2 alpha. In cultured cells and in mice models, interventions that decrease or increase Sirt1 activity affect expression of the HIF-2 alpha target gene epo accordingly. Thus, Sirt1 is a molecular switch that promotes HIF-2 signaling during hypoxia and likely other environmental stresses.Item Progranulin Biology: Small Molecule Enhancers of Progranulin Expression and Biochemical Analysis of Granulin Receptors(2012-12-05) Cenik, Basar 1981-; Terman, Jonathan R.; Herz, Joachim; Yu, Gang; Bezprozvanny, IlyaFrontotemporal dementia (FTD) is the second most common presenile dementia syndrome. Mutations in the GRN gene account for about 20% of patients with familial FTD. The protein encoded by GRN, progranulin, is a secreted glycoprotein with growth factor-like and immunomodulatory activities. Human progranulin contains seven granulin domains (denoted granulins A through F) that can be individually liberated following proteolytic cleavage. It is uncertain whether the holoprotein, the granulins or both mediate the biological effects of progranulin. All pathogenic GRN mutations result in haploinsufficiency and decreased extracellular progranulin. Therefore, increasing progranulin expression from the wild-type allele or (pro)granulin receptor agonists may be therapeutic in FTD. The overall goals of the work presented here were to identify small molecule enhancers of progranulin expression and (pro)granulin receptors that can be drug targets for the treatment and prevention of GRN deficient FTD. As described here, I discovered that suberoylanilide hydroxamic acid (SAHA), an FDA-approved histone deacetylase (HDAC) inhibitor, enhances GRN expression and nearly normalizes progranulin levels in haploinsufficient primary human cells from GRN mutation carriers. I also discovered that granulin A binds three proteins in solubilized extracts of rodent brain membranes: wolframin, excitatory amino acid transporter 1 (EAAT1), and the α3 subunit of the Na+/K+ ATPase. I argue that these proteins are candidates for a putative granulin receptor.Item Studies on Cellular Nutrient Responses and Protein Degradation(2015-06-01) Ghosh, Anwesha; Goodman, Joel M.; Cobb, Melanie H.; Albanesi, Joseph P.; Sternweis, Paul C.I have worked on two projects. The first project investigates mechanisms involved in cellular responses to amino acids. Amino-acid abundance promotes protein synthesis and cell growth via activation of the protein kinase mTOR, while amino-acid deprivation promotes protein degradation by autophagy. The heterodimeric G protein coupled receptor (GPCR) T1R1-T1R3 can act as an extracellular sensor for amino acids, promoting mTOR activity while repressing autophagy in cells. Quantitative PCR analysis revealed that T1R3 depletion increases mRNA expression of amino acid transporters as a compensatory mechanism induced by perceived starvation. The arrestin proteins can bind GPCRs to mediate their internalization or to facilitate downstream signaling. I tested the hypothesis that β-arrestin 2 might participate in regulation of mTOR activity and autophagy by amino acids. siRNA-mediated β-arrestin 2 depletion decreased T1R1-T1R3 protein expression, reduced mTOR activity and increased autophagy in different cell types. β-arrestin 2 loss increased phosphorylation of the MAP kinase ERK1/2, which may play a role in promoting autophagy. Taken together, these findings demonstrate a role for β-arrestin 2 in promoting mTOR activity and suppressing autophagy. The second project examined the role of different protein degradation pathways and an E3 ubiquitin ligase UBR5 in regulating the stability of the protein kinase WNK1, a key regulator of cellular ion homeostasis. Mutations that increase WNK1 protein expression cause familial hypertension, highlighting the importance of understanding the regulation of WNK1 protein expression. Cycloheximide chase experiments revealed that WNK1 degradation may be complex, as it does not follow simple exponential decay kinetics. Pharmacological inhibition of different protein degradation pathways showed that autophagy and the calpain system of non-lysosomal cysteine proteases, but not the proteasome, can promote WNK1 degradation. Inhibition of the protein chaperone Hsp90 increased WNK1 protein levels, possibly through stabilization of WNK1 by Hps70. Immunoprecipitation experiments demonstrated that UBR5 can associate with WNK1. siRNA-mediated silencing of UBR5 increased WNK1 stability, decreased the ubiquitination of an overexpressed N terminal fragment of WNK1, and reduced the levels of KLHL3, an adaptor protein that recruits WNK1 to the Cullin3-RBX1 E3 ligase complex for ubiquitination and degradation. Taken together, these findings identify degradation pathways and molecular players that regulate WNK1 stability.