Indiana University-Purdue University Indianapolis (IUPUI) / MicroRNAs are small RNA molecules that regulate expression of genes involved in development, cell differentiation, proliferation and death. It has been estimated that in eukaryotes, approximately 0.5 to 1% of predicted genes encode a microRNA, which in humans, regulate at least 30% of genes at an average of 200 genes per miRNA. Some microRNAs are tissue-specific, while others are ubiquitously expressed. In liver, a few microRNAs have been identified that regulate specialized functions. The best known is miR-122, the most abundant liver-specific miRNA, which regulates cholesterol biosynthesis and other genes of fatty acid metabolism; it also regulates the cell cycle through inhibition of cyclin G1. To discover other miRNAs with relevant function in liver, we characterized miRNA profiles in normal tissue and identified miR-709. Our data indicates this is a highly abundant hepatic miRNA and is dysregulated in an animal model of type 2 diabetes. To understand its biological role, miR-709 gene targets were identified by analyzing the transcriptome of primary hepatocytes transfected with a miR-709 mimic. The genes identified fell within four main categories: cytoskeleton binding, extracellular matrix attachment, endosomal recycling and fatty acid metabolism. Thus, similar to miR-122, miR-709 downregulates genes from multiple pathways. This would be predicted, given the abundance of the miRNA and the fact that the estimated number of genes targeted by a miRNA is in the hundreds. In the case of miR-709, these suggested a coordinated response during cell proliferation, when cytoskeleton remodeling requires substantial changes in gene expression. Consistently, miR-709 was found significantly upregulated in an animal model of hepatocellular carcinoma. Likewise, in a mouse model of liver regeneration, mature miR-709 was increased. To study the consequences of depleting miR-709 in quiescent and proliferating cells, primary hepatocytes and hepatoma cells were cultured with antagomiRs (anti-miRs). The presence of anti-miR-709 caused cell death in proliferating cells. Quiescent primary hepatocytes responded by upregulating miR-709 and its host gene, Rfx1. These studies show that miR-709 targets genes relevant to cystokeleton structural genes. Thus, miR-709 and Rfx1 may be needed to facilitate cytoskeleton reorganization, a process that occurs after liver injury and repopulation, or during tumorigenesis.

Indiana University-Purdue University Indianapolis (IUPUI) / More than 300 million people world-wide suffer from breathlessness, wheezing, chest tightness, and coughing characteristic of chronic bronchial asthma, the global incidence of which is on the rise. Allergen-sensitization and challenge elicits pulmonary expression of chemoattractants that promote a chronic eosinophil-rich infiltrate. Eosinophils are increasingly recognized as important myeloid effectors in chronic inflammation characteristic of asthma, although few eosinophil molecular signaling pathways have successfully been targeted in asthma therapy. p21 activated kinases (PAKs), members of the Ste-20 family of serine/threonine kinases, act as molecular switches in cytoskeletal-dependent processes involved in cellular motility. We hypothesized that PAK1 modulated eosinophil infiltration in an allergic airway disease (AAD) murine model. In this model, Pak1 deficient mice developed reduced inflammatory AAD responses in vivo with notable decreases in eosinophil infiltration in the lungs and broncho-alveolar lavage fluids (BALF). To test the importance of PAK1 in hematopoietic cells in AAD we used complementary bone marrow transplant experiments that demonstrated decreased eosinophil inflammation in hosts transplanted with Pak1 deficient bone marrow. In in vitro studies, we show that eotaxin-signaling through PAK1 facilitated eotaxin-mediated eosinophil migration. Ablating PAK1 expression by genetic deletion in hematopoietic progenitors or siRNA treatment in derived human eosinophils impaired eotaxin-mediated eosinophil migration, while ectopic PAK1 expression promoted this migration. Together these data suggest a key role for PAK1 in the development of atopic eosinophil inflammation and eotaxin-mediated eosinophil migration.

Indiana University-Purdue University Indianapolis (IUPUI) / Several common diseases such as atherosclerosis, post-angioplasty restenosis, and graft vasculopathies, are associated with the changes in the structure and function of smooth muscle cells. During the pathogenesis of these diseases, smooth muscle cells have a marked alteration in the expression of many smooth muscle-specific genes and smooth muscle cells undergo a phenotypic switch from the contractile/differentiated status to the proliferative/dedifferentiated one. Serum response factor (SRF) is the major transcription factor that plays an essential role in coordinating a variety of transcriptional events during this phenotypic change. The first goal of my thesis studies is to determine how SRF regulates the expression of smooth muscle myosin light chain kinase (smMLCK) to mediate changes in contractility. Using a combination of transgenic reporter mouse and knockout mouse models I demonstrated that a CArG element in intron 15 of the mylk1 gene is necessary for maximal transcription of smMLCK. SRF binding to this CArG element modulates the expression of smMLCK to control smooth muscle contractility. A second goal of my thesis work is to determine how SRF coordinates the activity of chromatin remodeling enzymes to control expression of microRNAs that regulate the phenotypes of smooth muscle cells. Using both mouse knockout models and in vitro studies in cultured smooth muscle cells I showed how SRF acts together with Brg1-containing chromatin remodeling complexes to regulate expression of microRNAs-143, 145, 133a and 133b. Moreover, I found that SRF transcription cofactor myocardin acts together with SRF to regulate expression of microRNAs-143 and 145 but not microRNAs-133a and 133b. SRF can, thus, further modulate gene expression through post-transcriptional mechanisms via changes in microRNA levels. Overall my research demonstrates that through direct interaction with a CArG box in the mylk1 gene, SRF is important for regulating expression of smMLCK to control smooth muscle contractility. Additionally, SRF is able to harness epigenetic mechanisms to modulate expression of smooth muscle contractile protein genes directly and indirectly via changes in microRNA expression. Together these mechanisms permit SRF to coordinate the complex phenotypic changes that occur in smooth muscle cells.

Indiana University-Purdue University Indianapolis (IUPUI) / An excess of bone resorption over bone formation leads to osteoporosis, resulting in a reduction of bone mass and an increase in the risk of bone fracture. Anabolic and anti-resorptive drugs are currently available for treatment, however, none of these drugs are able to both promote osteoblastogenesis and reduce osteoclastogenesis. This thesis focused on the role of eukaryotic translation initiation factor 2 alpha (eIF2alpha), which regulates efficiency of translational initiation. The elevation of phosphorylated eIF2alpha was reported to stimulate osteoblastogenesis, but its effects on osteoclastogenesis have not been well understood. Using synthetic chemical agents such as salubrinal and guanabenz that are known to inhibit the de-phosphorylation of eIF2alpha, the role of phosphorylation of eIF2alpha in osteoclastogenesis was investigated in this thesis. The questions addressed herein were: Does the elevation of phosphorylated eIF2alpha (p-eIF2alpha) by salubrinal and guanabenz alter osteoclastogenesis? If so, what regulatory mechanism mediates the process? It was hypothesized that p-eIF2alpha could attenuate the development of osteoclast by regulating the transcription factor(s) amd microRNA(s) involved in osteoclastogenesis. To test this hypothesis, we conducted in vitro and in silico analysis of the responses of RAW 264.7 pre-osteoclast cells to salubrinal and guanabenz. First, the in vitro results revealed that the elevated level of phosphorylated eIF2alpha inhibited the proliferation, differentiation, and maturation of RAW264.7 cells and downregulated the expression of NFATc1, a master transcription factor of osteoclastogenesis. Silencing eIF2alpha by RNA interference suppressed the downregulation of NFATc1, suggesting the involvement of eIF2alpha in regulation of NFATc1. Second, the in silico results using genome-wide expression data and custom-made Matlab programs predicted a set of stimulatory and inhibitory regulator genes as well as microRNAs, which were potentially involved in the regulation of NFATc1. RNA interference experiments indicated that the genes such as Zfyve21 and Ddit4 were primary candidates as an inhibitor of NFATc1. In summary, the results showed that the elevation of p-eIF2alpha by salubrinal and guanabenz leads to attenuation of osteoclastogenesis through the downregulation of NFATc1. The regulatory mechanism is mediated by eIF2alpha signaling, but other signaling pathways are likely to be involved. Together with the previous data showing the stimulatory role of p-eIF2alpha in osteoblastogenesis, the results herein suggest that eIF2alpha-mediated signaling could provide a novel therapeutic target for treatment of osteoporosis by promoting bone formation and reducing bone resorption.

Indiana University-Purdue University Indianapolis (IUPUI) / The islet beta cell is unique in its ability to synthesize and secrete insulin for use in the body. A number of factors including proinflammatory cytokines, free fatty acids, and islet amyloid are known to cause beta cell stress. These factors lead to lipotoxic, inflammatory, and ER stress in the beta cell, contributing to beta cell dysfunction and death, and diabetes. While transcriptional responses to beta cell stress are well appreciated, relatively little is known regarding translational responses in the stressed beta cell. To study translation, I established conditions in vitro with MIN6 cells and mouse islets that mimicked UPR conditions seen in diabetes. Cell extracts were then subjected to polyribosome profiling to monitor changes to mRNA occupancy by ribosomes. Chronic exposure of beta cells to proinflammatory cytokines (IL-1 beta, TNF-alpha, IFN-gamma), or to the saturated free fatty acid palmitate, led to changes in global beta cell translation consistent with attenuation of translation initiation, which is a hallmark of ER stress. In addition to changes in global translation, I observed transcript specific regulation of ribosomal occupancy in beta cells. Similar to other privileged mRNAs (Atf4, Chop), Pdx1 mRNA remained partitioned in actively translating polyribosomes during the UPR, whereas the mRNA encoding a proinsulin processing enzyme (Cpe) partitioned into inactively translating monoribosomes. Bicistronic luciferase reporter analyses revealed that the distal portion of the 5’ untranslated region of mouse Pdx1 (between bp –105 to –280) contained elements that promoted translation under both normal and UPR conditions. In contrast to regulation of translation initiation, deoxyhypusine synthase (DHS) and eukaryotic translation initiation factor 5A (eIF5A) are required for efficient translation elongation of specific stress relevant messages in the beta cell including Nos2. Further, p38 signaling appears to promote translational elongation via DHS in the islet beta cell. Together, these data represent new insights into stress induced translational regulation in the beta cell. Mechanisms of differential mRNA translation in response to beta cell stress may play a key role in maintenance of islet beta cell function in the setting of diabetes.