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.

Osteoblasts are highly active cells that are responsible for secreting bone forming components such as collagen type I and matricellular proteins that mediate collagen deposition and mineralisation. SPARC and osteopontin are matricellular proteins that are involved in bone regulation and cell-matrix interactions and are also upregulated in metastatic disease. Secretion of these proteins results in changes to the stromal environment that includes cell migration, angiogenesis, matrix degradation, matrix deposition, bone mineralisation and bone resorption. Signalling pathways not only lead to the expression of target proteins, but also have immediate early effects, for example, on cell adhesion. We asked if the ERK 1 and 2 module of the MAPK pathway was involved in the intracellular trafficking of SPARC and Osteopontin. Membrane trafficking is an essential process that ensures newly synthesised proteins pass from their site of synthesis to the extracellular environment. Using an inhibitor of ERK 1 and 2 activation (U0126), as well as siRNA directed against ERK 1 or 2 individually, a change in intracellular localisation of SPARC and osteopontin was observed in cells treated with U0126 and siRNA against ERK 2 alone, likely in or around the Golgi apparatus. Consistent with the observation above, analysis of protein secretion showed that there was a reduction of total protein secreted (30% reduction) when ERK 1 and 2 activation was prevented together or knock down of ERK 2 alone. A mechanism is proposed where ERK 2 is likely activating a substrate that is allowing SPARC and osteopontin to continue along the secretory pathway. This directly implicates ERK 2 as an important regulator of matricellular protein secretion in osteoblasts. In cancer, Ras mutations can lead to permanent activation of the MAPK pathway leading to cancer cell proliferation and survival, however, we propose another mechanism important in metastasis whereby ERK 2 activation is manipulated to facilitate secretion of matricellular proteins which can then mediate changes to the stromal environment that allow the tumour to metastasise successfully.

Abstract Cardiac myocytes react to increased workload and hypertrophic neurohumoral stimuli by increasing protein synthesis, reinitiating expression of fetal forms of structural genes, α-skeletal actin (α-SkA) and β-myosin heavy chain (β-MHC), and by increasing expression and secretion of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP). Initially, the response is beneficial, but when prolonged, it leads to pathological cardiomyocyte hypertrophy. In this study, cardiomyocyte hypertrophy was initiated by hypertrophic agonists, endothelin-1 (ET-1) and phenylephrine (PE), and by increased stretching of atrial wall. Transcription factor GATA4 was studied to identify the mechanism leading to increased gene expression of BNP. In BNP promoter, GATA4 binds to cis elements mediating hypertrophic response. Eliminating GATA4 binding by using the decoy approach, basal BNP gene expression was reduced. To identify mechanisms regulating GATA4, the roles of mitogen-activated protein kinases (MAPKs) were studied. Activation of p38 MAPK increased GATA4 binding to BNP gene and led to increased GATA4 dependent BNP gene expression. p38 MAPK was required for ET-1 induced GATA4 binding, whereas extracellular signal-regulated kinase (ERK) was required for maintaining basal GATA4 binding activity. PE and ET-1 activated protein kinase C (PKC) signaling in cardiac myocytes. Antisense oligonucleotide inhibition of PKCα markedly reduced PE induced ANP secretion and ET-1 induced BNP secretion, whereas gene expression of natriuretic peptides was not affected. Antisense PKCα treatment inhibited PE induced expression of α-SkA, while increased protein synthesis or β-MHC gene expression were not affected. Sretching of the perfused rat atria increased BNP, c-fos and BNP gene expression via mechanism involving p38 MAP kinase activation of transcription factor Elk-1. In cultured neonatal rat atrial myocytes stretch induced BNP gene expression was dependent upon transcription factor Elk-1 binding sites within the BNP gene promoter. In conclusion, hypertrophic signaling in cardiac myocytes involves multiple signaling cascades. Activation of p38 MAPK is required for the development of ET-1 induced hypertrophic phenotype and GATA4 mediated BNP gene expression in cultured ventricular myocytes, and for stretch induced Elk-1 dependent BNP gene expression in atrial myocytes. PKCα is involved in PE induced hypertrophic response and PE induced switch in gene programming inducing expression of α-SkA, the fetal form of cardiac α-actin.

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.

The incidence of chronic kidney disease (CKD) is increasing. CKD is characterized by a gradual decrease in renal function leading to end stage renal disease (ESRD). Damage to the glomerular podocytes, is one of the first hallmarks of CKD. We hypothesized that podocyte prostaglandin E2 (PGE2) receptors contribute to the progression of glomerular injury in models of CKD. To test this hypothesis, transgenic mice were generated with either podocyte-specific overexpression or deletion of the PGE2 EP4 receptor (EP4pod+and EP4pod-/- respectively). Mice were next tested in the 5/6 nephrectomy (5/6 Nx) or angiotensin II (Ang II) models of CKD. These studies revealed increased proteinuria and decreased survival for EP4pod+ mice while EP4pod-/- mice were protected against the development of glomerular injury. Furthermore, our findings were supported by in vitro studies using cultured mouse podocytes where an adhesion defect was uncovered for cells overexpressing the EP4 receptor. Additionally, our investigations have demonstrated a novel synergy between angiotensin II AT1 receptors and prostaglandin E2 EP4 receptors. This was revealed by in vitro studies using isolated mouse glomeruli. There we were able to show that Ang II stimulation leads to increased expression of cyclooxygenase 2 (COX-2), the enzyme responsible for synthesis of PGE2, in a p38 mitogen activated protein kinase (MAPK) dependent fashion. Moreover increased PGE2 synthesis was measured in response to Ang II stimulation. We confirmed the presence of this synergy in our cultured mouse podocytes and showed an adhesion defect in response to Ang II stimulation which was COX-2 and EP4 dependent. These findings suggest that Ang II AT1 receptors and PGE2 EP4 receptors act in concert to exacerbate glomerulopathies. Studies using mice with either podocyte-specific overexpression of a dominant negative p38 MAPK or mice with global deletion of the EP1 receptor did not provide conclusive results as to their respective signaling involvement in podocyte injury. Altogether our findings provide novel insight for podocyte PGE2 EP4 and Ang II AT1 receptor signaling in models of CKD. These studies provide novel avenues for pursuing therapeutic interventions for individuals with progressive kidney disease.

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.

The potassium channels IK and IK1, responsible for the action potential repolarization and resting potential respectively, are altered during cardiac hypertrophy. The activation of insulin-like growth factor-I (IGF-I) during hypertrophy may affect channel activity. The aim was to examine the modulatory effects of IGF-I on IK and IK1 through mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways during hypertrophy. With the use of specific inhibitors for ERK1/2 (PD98059), p38 MAPK (SB203580) and PI3K/Akt (LY294002), Western blot and whole cell patch-clamp were conducted on sham and aorto-caval shunt-induced hypertrophy adult rat myocytes. Basal activation levels of MAPKs and Akt were increased during hypertrophy. Acute IGF-I (10-8 M) enhanced basal activation levels of these kinases in normal hearts but only those of Akt in hypertrophied ones. IK and IK1 activities were lowered by IGF-I. Inhibition of ERK1/2, p38 MAPK, or Akt reduced basal IK activity by 70, 32, or 50%, respectively, in normal cardiomyocytes vs. 53, 34, or 52% in hypertrophied ones. However, basal activity of IK1 was reduced by 45, 48, or 45% in the former vs. 63, 43, or 24% in the latter. The inhibition of either MAPKs or Akt alleviated IGF-I effects on IK and IK1. We conclude that basal IK and IK1 are positively maintained by steady-state Akt and ERK activities. K+ channels seem to be regulated in a dichotomic manner by acutely stimulated MAPKs and Akt. Eccentric cardiac hypertrophy may be associated with a change in the regulation of the steady-state basal activities of K+ channels towards MAPKs, while that of the acute IGF-I-stimulated ones toward Akt. .

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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