Thesis (Ph. D.)--Virginia Commonwealth University, 2010. / Prepared for: Dept. of Pathology. Title from resource description page. Includes bibliographical references . Unavailable until 5/13/2105.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Not embargoed. Vita. Bibliography: 105-114.

Thesis (Ph. D.)--Case Western Reserve University, 2006. / [School of Medicine] Department of Physiology and Biophysics. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Abstract Cardiac hypertrophy provides an adaptive mechanism to maintain cardiac output in response to increased workload, and although initially beneficial, hypertrophy eventually leads to heart failure, a major cause of morbidity and mortality in Western countries. The hypertrophic response in cardiac myocytes is accompanied by e.g. activation of signal transduction pathways, such as mitogen-activated protein kinases (MAPKs), and complex changes in gene programming. The purpose of this study was to characterize gene expression patterns in experimental models of cardiac load by using high-throughput DNA microarray technologies. In the present study, changes in gene expression were evaluated in response to acute pressure overload and prolonged hypertension as well as during the development of left ventricular hypertrophy (LVH) and the transition to diastolic heart failure in an animal model of genetic hypertension, the spontaneously hypertensive rat (SHR). Increased expression of several immediate early genes was seen in response to acute hemodynamic overload in vivo. The transition from LVH to diastolic hypertensive heart failure was almost exclusively associated with changes in genes encoding extracellular matrix proteins and their regulatory processes showing the importance of progressive extracellular matrix remodeling. The effect of p38 MAPK activation on gene expression patterns in vivo was elucidated. Cardiac-specific overexpression of p38 MAPK resulted in upregulation of genes controlling cell division and inflammation as well as cell signaling and adhesion. Accordingly, the functional role of p38 MAPK was related to myocardial cell proliferation, inflammation and fibrosis. Finally, temporal analysis of mechanical stretch induced gene expression changes in neonatal rat cardiomyocyte cultures in vitro indicated that mechanical stretch induced complex gene expression profiles, demonstrating that both positive and negative regulators are involved in the hypertrophic process. Many novel stretch responsive genes were identified, and a subset of them may be putative downstream targets of p38 MAPK. In conclusion, in the present study a number of well-established gene expression changes of cardiac hypertrophy were observed and novel modulators associated with increased cardiac load, such as thrombospondin-4, were identified. The study provides a better understanding of molecular mechanisms associated with increased cardiac load, and may indicate potential targets for novel therapeutic interventions.

Alpha B Crystallin (αBC) is a putative effector protein of ischemic preconditioning (IPC). that is phosphorylated on Ser 45 by ERK1/2 and Set 59 by the p38 MAPK substrate, MAPKAPK-2. Translocation and phosphorylation of αBC was determined in cytosolic and cytoskeletal fractions by 1D SDS-PAGE and IEF, or using Ser 45 and Set 59 phospho-specific antibodies in: (1) control rabbit cardiomyocytes; (2) cells preconditioned by 10 min in vitro ischemia; or after pre-treatment with specific inhibitors of (3) Ser/Thr protein phosphatase 1/2A (calyculin A); (4) p38 MAPK (SB203580); or (5) ERK 1/2 (PD98059); all prior to 180 min ischemia. Ischemia induced a cytosolic to cytoskeletal translocation of αBC, which was similar in all the groups. Highly phosphorylated isoforms (D1/2) of αBC were present in cytosolic but not cytoskeletal fractions at 0 min ischemia. By 60-90 min ischemia. D1/2 isoforms had translocated to the cytoskeletal fraction. Calyculin A maintained D1/2 levels throughout prolonged ischemia. SB203580 decreased αBC phosphorylation. Neither PD98059 nor IPC altered αBC phosphorylation during prolonged ischemia. It is concluded that αBC phosphorylation during ischemia is regulated by p38 MAPK but not by ERK 1/2. The inability to detect a correlation between IPC protection and either αBC translocation or phosphorylation suggests that the proteins in the highly phosphorylated isoform bands of αBC quantitated in this study are not protective end effectors of classical IPC.

NF-кB/Rel family proteins regulate genes that are critical for many cellular processes including apoptosis, inflammation, immune response, as well as development. NF-кB/Rel proteins function as homodimers or heterodimers, which recognize specific DNA sequences within target promoters. I examined the activity of different Drosophila Rel-related proteins in modulating Drosophila immunity genes by expressing the Rel proteins in stably transfected cell lines. I also compared how different combinations of these transcriptional regulators control the activity of various immunity genes. The results show that Rel proteins are directly involved in regulating the Drosophila antimicrobial response. Furthermore, expression of drosomycin and defensin is best induced by the Relish/Dif and the Relish/Dorsal heterodimers, respectively; whereas attacin activity can be efficiently up-regulated by the Relish homodimer and heterodimers. These results illustrate how the formation of Rel protein dimers differentially regulates target gene expression. Another area of my research is to investigate the function of p38 MAP kinase (mitogen-activated protein kinase) in Drosophila immune response. In vertebrates, one of the responses evoked by the pro-inflammatory cytokines and lipopolysaccharide (LPS) is the initiation of a kinase cascade that leads to the phosphorylation of p38 MAP kinase on Thr and Tyr within the motif Thr-Gly-Tyr, which is located within subdomain VIII. Two genes that are highly homologous to the mammalian p38 MAP kinases were molecularly cloned and characterized. Furthermore, genes that encode two novel Drosophila MAP kinase kinases, D-MKK3 and D-MKK4, were identified. D-MKK3 is an efficient activator of both Drosophila p38 MAP kinases, while D-MKK4 is an activator of D-JNK but not D-p38. These data establish that Drosophila indeed possesses a conserved p38 MAP kinase signaling pathway. We have examined the role of the D-p38 MAP kinases in the regulation of insect immunity. The results revealed that one of the functions of D-p38 is to attenuate antimicrobial peptide gene expression induced by LPS.

Polycystic kidney disease is an inherited degenerative disease in which the uriniferous tubules are replaced by expanding fluid-filled cysts that ultimately destroy organ function. Autosomal dominant polycystic kidney disease (ADPKD) is the most common form, afflicting approximately 1 in 1,000 people. It primarily is caused by mutations in the transmembrane proteins Polycystin-1 (PKD1) and Polycystin-2 (PKD2). The most proximal effects of polycystin mutations leading to cyst formation are not known, but pro-proliferative signaling must be involved for the tubule epithelial cells to increase in number over time. The stress-activated mitogen-activated protein kinase (MAPK) pathway c-Jun N-terminal kinase (JNK) promotes proliferation in specific contexts and is activated in acute and chronic kidney disease. Previous work found evidence of JNK activation in cystic tissues (Le et al., 2005) and others showed that JNK signaling is activated by aberrant expression of PKD1 and PKD2 in cell culture (Arnould et al., 1998; Arnould et al., 1999; Parnell et al., 2002; Yu et al., 2010) but the contribution of JNK signaling to cystic disease in vivo has not been investigated. This body of work describes the use of conditional and germline deletion of Pkd2, Jnk1 and Jnk2 to model ADPKD and JNK signaling inhibition in juvenile and adult mice. Immunoblots and histological staining were used to measure JNK activation and evaluate the effect of JNK deletion on cystic disease. Results show that Pkd2 deletion activated JNK signaling in juvenile and adult mice. Reduction of JNK activity significantly reduced cystic burden in kidneys of juvenile Pkd2 mutant mice. This correlated with reduced tubule cell proliferation and reduced kidney fibrosis. The improvement in cystic phenotype was driven primarily by Jnk1 deletion rather than Jnk2. JNK signaling inhibition in adult Pkd2 mutants significantly reduced liver cysts when mice were aged six months. JNK inhibition reduces the severity of cystic disease caused by the loss of Pkd2 suggesting that the JNK pathway should be explored as a potential therapeutic target for ADPKD.

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The c-Jun NH2-terminal kinase (JNK) group and the p38 group of mitogen-activated protein kinases (MAPK) are stress-activated protein kinases that regulate cell proliferation, differentiation, development, and apoptosis. These protein kinases are involved in a signal transduction cascade that includes a MAP kinase (MAPK), a MAP kinase kinase (MAP2K), and a MAP kinase kinase kinase (MAP3K). MAPK are phosphorylated and activated by the MAP2K, which are phosphorylated and activated by various MAP3K. The work presented in this dissertation focuses on understanding the regulation and function of the JNK and p38 MAPK pathways. Two different strategies were utilized. First, I used molecular and biochemical techniques to examine how MAP2K and MAP3K mediate signaling specificity and to define their role in the MAPK pathway. Second, I used gene targeted disruption studies to determine the in vivo role ofMAP2K and MAP3K in MAPK activation. I specifically used these approaches to examine: (1) docking interactions between p38 MAPK and MAP2K [MKK3 and MKK6 (Chapter II)]; (2) the differential activation of p38 MAPK by MAP2K [MKK3, MKK4, and MKK6 (Chapter III)]; and (3) the selective involvement of the mixed lineage kinase (MLK) group of MAP3K in JNK and p38 MAPK activation (Chapter IV and Appendix). In addition, I analyzed the role of the MKK3 and MKK6 MAP2K in cell proliferation and the role of the MLK MAP3K in adipocyte differentiation (Chapter III and Chapter IV). Together, these data provide insight into the regulation and function of the stress-activated MAPK signal transduction pathways.