The mammalian cell cycle and its points-of-entry are well characterized pathways. These points-of-entry are normally regulated via mitogens and include, amongst others, the ERK, JNK and p38 mitogen-activated protein kinase (MAPK) pathways. However, while the restriction point(R-point), the temporal switch-point at which a cell becomes irrevocably committed to division irrespective of mitogenic stimulus, is known among other cell types, its position within the murine myoblast line C2C12 is currently unknown. Similarly, while MAPK pathways, such as JNK and ERK, have been modeled computationally, no model yet exists of p38 MAPK as stimulated by mitogens. The aims of this dissertation, then, were to determine the R-point within the C2C12 cell cycle and construct a computational mitogen-stimulated p38 MAPK model. It was found that a synchronous C2C12 population, when stimulated to divide, took 7 to 9 hours to reach S-phase from G0, consistent with data from the literature. The R-point was determined to lie between 6 and 7 hours post G1-re-entry stimulation,which was consistent with studies in other cell types. Core modeling of the p38 MAPK pathway revealed that ultrasensitivitywas inherent within the pathway structure. Further, a branching/re-converging structure within the pathway imparted greater responsiveness to signal upon the pathway. A realistic p38 MAPK model demonstrated good responsiveness to signal, its output matched that of several other MAPK models, and it was capable of replicating previous in vitro data. This model can be used as a tool for further investigation of the mammalian cell cycle by linking it to other cell cycle models. The predictions by an expanded model may be better suited for understanding the effects of mitogen stimulus on the cell cycle in situ. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Fibroblast growth factor (FGF) signalling plays many fundamentally important roles during the development of the mammalian embryo. However, its effects on pluripotent stem cells derived from mouse and human embryos appear to be markedly different. FGF2 is routinely added to culture medium for propagating undifferentiated human (hES) cells, whereas in mouse (mES) cell cultures FGFs have been described as regulators of their differentiated progeny. To assess the effect of FGF signalling on undifferentiated mES cells, the effects of FGF2 and 4 were analysed in the presence of saturating and sub-saturating levels of the inhibitor of differentiation, leukaemia inhibitory factor (LIF). Mouse ES cell self-renewal was quantified by measuring the expression of the stem cell specific reporter Oct4-LacZ in biochemical and fluorometric assays. Treatment with FGF reduced the expression of the OCT4-LacZ reporter, even under saturating concentrations of LIF and this was mirrored by decreased levels of OCT4 protein. Furthermore, treatment with FGF leads to upregulation of the ectodermal differentiation marker Pax6. These results suggest that FGF signalling has a direct impact on undifferentiated mES cells, and actively promotes their differentiation. To asses the effect of FGF signalling on hES cells without the influence of undefined factors, a feeder and serum free system was developed. Cells growing in this conditions for >20 passages maintained expression of surface (SSEA3 and TRA1-60 and 81) and internal (OCT4) markers specific for undifferentiated hES cells. Expression of these markers was dependant on the continuous presence of FGF2. Indeed, withdrawal of FGF2 resulted in a rapid decrease of in hES cell growth and of the emergence of cell flattened morphology and of the surface marker SSEA1, changes typically associated with differentiation. Two important signals activated by FGF in hES cells are the ERK/MAPK and PI3K pathways. To assess their functional relevance, hES cell cultures were treated with the drugs UO126 and LY294002, inhibitors of the MAPK and PI3K pathways respectively. Drug mediated suppression of the phosphorylation of these pathways, correlated with a reduction in cell growth, flattening of the colonies and reduction in SSEA4 expression. Use of SB431542, specific inhibitor of TGFβ/activin type I receptor kinase (Alk5) also resulted in the flattening of the colonies and the appearance of dispersed cells. Therefore, inhibition of MAPK and PI3K appears to impair growth and self-renewal in hES cells and this may be happening in conjunction with TGFβ/Activin pathway. Taken together, these results suggest that FGF signalling has opposite effects in mouse and human ES cells: inducing differentiation in mES and sustaining self-renewal in hES.

L'étirement est un stimulus hypertrophique qui active de nombreuses voies de signalisation similaires à celles mises en évidence lors de l'étude de l'hypertrophie cellulaire. L'objectif principal de mon travail de thèse était de caractériser les évènements moléculaires impliqués dans l'activation des MAPKinases (MAPK), ERK et JNK lors de l'étirement. Nous avons étudié ces protéines par 2 approches différentes. D'une part, nous nous sommes intéressés aux rôles de protéines potentiellement nécessaires à l'activation des MAPK. D'autre part, nous avons cherché à mettre en évidence des interconnexions moléculaires entre les différentes voies de signalisation activées par l'étirement cellulaire, en montrant notamment la formation de complexes protéiques nécessaires à l'activation des différents partenaires. Nous montrons ainsi que deux protéines à activité tyrosine kinase, l'Epidermal Growth Factor Receptor (EGFR) et la Proline-rich tyrosine kinase 2 (Pyk2), sont respectivement nécessaires à l'activation de ERK et de JNK lors de l'étirement. Ces cascades de transduction peuvent être dépendantes de la petite protéine G Ras. Bien que les voies des MAPK et de PI3K/Akt soient considérées comme indépendantes, nous montrons également que Akt participe à l'activation de ERK par l'étirement. Enfin, nous avons montré la formation d'un complexe Protein Kinase C (PKC)/Calcineurine nécessaire à l'activation et à la translocation de la PKC lors de l'étirement. Cette étude de différentes voies de signalisation et des interactions protéiques apporte une meilleure connaissance des mécanismes activés par l'étirement cellulaire et permet donc de mieux comprendre la signalisation impliquée dans l'hypertrophie ventriculaire / Cardiomyocyte stretch is a major determinant of ventricular hypertrophy. It stimulates numerous signalling pathways leading to the Mitogen Activated Protein kinases (MAPK) activation. The objective of this thesis was to evaluate the molecular events involved in MAPK ERK and JNK activations during stretch. We studied these pathways by 2 different approaches. We analysed the role of several pivotal proteins involved in ERK and JNK activations and next we evaluated the molecular interactions between different signalling pathways by protein complexes formation induced by stretch and necessary for protein activations. We show that 2 tyrosine Kinases, the Epidermal Growth Factor Receptor (EGFR) and the Proline-rich tyrosine kinase 2 (Pyk2) are necessary for ERK and JNK activations respectively during stretch with a possible involvement of the small G protein Ras. MAPK and PI3/Akt pathways are generally considered independent but we show that ERK activation is PI3K/Akt dependent during stretch. Thus, we demonstrate that 2 other pathways are associated since PKC and calcineurin form a complex necessary for PKC activation and translocation. This study of signalling pathways and protein interactions sheds a new light on intracellular pathways leading to MAPK activation and may have implications for the development of new drugs in the management of cardiac hypertrophy and failure

by Ho Wai Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 207-243). / Abstracts in English and Chinese. / Title --- p.i / Abstract --- p.ii / 摘要 --- p.v / Acknowledgements --- p.viii / Table of Contents --- p.x / List of Abbreviations --- p.xviii / List of Figures --- p.xxiv / List of Tables --- p.xxix / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Traumatic brain injury --- p.1 / Chapter 1.2 --- Ceils of the nervous system: glia --- p.1 / Chapter 1.2.1 --- Astroglia - / Chapter 1.2.1.1 --- Molecular markers of astroglia --- p.3 / Chapter 1.2.1.2 --- Functions of astroglia --- p.3 / Chapter 1.2.2 --- Oligodendrocyte --- p.5 / Chapter 1.2.2.1 --- Molecular markers of oligodendrocyte --- p.6 / Chapter 1.2.2.2 --- Functions of oligodendrocyte --- p.6 / Chapter 1.2.3 --- Microglia --- p.7 / Chapter 1.2.3.1 --- Molecular markers of microglia --- p.7 / Chapter 1.2.3.2 --- Functions of microglia --- p.8 / Chapter 1.3 --- Cytokine and brain injury --- p.8 / Chapter 1.4 --- Tumor necrosis factor alpha (TNF-α) --- p.9 / Chapter 1.5 --- TNF-α receptor --- p.10 / Chapter 1.6 --- Biological activities of TNF-α --- p.11 / Chapter 1.7 --- Signaling mechanism --- p.13 / Chapter 1.7.1 --- Protein kinase C --- p.13 / Chapter 1.7.2 --- Protein kinase A --- p.14 / Chapter 1.7.3 --- p38 mitogen-activated protein kinase (p38 MAPK) --- p.15 / Chapter 1.7.3.1 --- Biological activities of p38 MAPK --- p.18 / Chapter 1.7.4 --- Inducible nitric oxide synthase (iNOS) --- p.20 / Chapter 1.7.5 --- cAMP responsive element binding protein (CREB) --- p.21 / Chapter 1.7.6 --- Transcription factor c-fos --- p.23 / Chapter 1.7.7 --- Nuclear factor kappa-B (NF-kB) --- p.24 / Chapter 1.8 --- "Brain injury, astrogliosis and scar formation" --- p.26 / Chapter 1.9 --- β-adrenergic receptor (β-AR) --- p.28 / Chapter 1.9.1 --- Functions of β-AR in astrocytes --- p.29 / Chapter 1.10 --- Why do we use C6 glioma cell? --- p.31 / Chapter 1.11 --- Fluorescent differential display (FDD) --- p.34 / Chapter 1.12 --- Aims and Scopes of this project --- p.36 / Chapter Chapter 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Material --- p.40 / Chapter 2.1.1 --- Cell line --- p.40 / Chapter 2.1.2 --- Cell culture reagents --- p.40 / Chapter 2.1.2.1 --- Complete Dulbecco's modified Eagle medium (CDMEM) --- p.40 / Chapter 2.1.2.2 --- Rosewell Park Memorial Institute (RPMI) medium --- p.41 / Chapter 2.1.2.3 --- Phosphate buffered saline (PBS) --- p.41 / Chapter 2.1.3 --- Recombinant cytokines --- p.41 / Chapter 2.1.4 --- Chemicals for signal transduction study --- p.42 / Chapter 2.1.4.1 --- Modulators of p38 mitogen-activated protein kinase (p38 MAPK) --- p.42 / Chapter 2.1.4.2 --- Modulators of protein kinase C (PKC) --- p.42 / Chapter 2.1.4.3 --- Modulators of protein kinase A (PKA) --- p.42 / Chapter 2.1.4.4 --- β-Adrenergic agonist and antagonist --- p.43 / Chapter 2.1.5 --- Antibodies --- p.44 / Chapter 2.1.5.1 --- Anti-p38 mitogen-activated protein kinase (p38 MAPK) antibody --- p.44 / Chapter 2.1.5.2 --- Anti-phosporylation p38 mitogen-activated protein kinase (p-p38 MAPK) antibody --- p.44 / Chapter 2.1.5.3 --- Antibody conjugates --- p.44 / Chapter 2.1.6 --- Reagents for RNA isolation --- p.45 / Chapter 2.1.7 --- Reagents for DNase I treatment --- p.45 / Chapter 2.1.8 --- Reagents for reverse transcription of mRNA and fluorescent PCR amplification --- p.45 / Chapter 2.1.9 --- Reagents for fluorescent differential display --- p.46 / Chapter 2.1.10 --- Materials for excision of differentially expressed cDNA fragments --- p.46 / Chapter 2.1.11 --- Reagents for reamplification of differentially expressed cDNA fragments --- p.46 / Chapter 2.1.12 --- Reagents for subcloning of reamplified cDNA fragments --- p.47 / Chapter 2.1.13 --- Reagents for purification of plasmid DNA from recombinant clones --- p.47 / Chapter 2.1.14 --- Reagents for DNA sequencing of differentially expressed cDNA fragments --- p.47 / Chapter 2.1.15 --- Reagents for reverse transcription-polymerase chain reaction (RT-PCR) --- p.48 / Chapter 2.1.16 --- Reagents for electrophoresis --- p.50 / Chapter 2.1.17 --- Reagents and buffers for Western blot --- p.50 / Chapter 2.1.18 --- Other chemicals and reagents --- p.50 / Chapter 2.2 --- Maintenance of rat C6 glioma cell line --- p.51 / Chapter 2.3 --- RNA isolation --- p.52 / Chapter 2.3.1 --- Measurement of RNA yield --- p.53 / Chapter 2.4 --- DNase I treatment --- p.53 / Chapter 2.5 --- Reverse transcription of mRNA and fluorescent PCR amplification --- p.54 / Chapter 2.6 --- Fluorescent differentia display --- p.55 / Chapter 2.7 --- Excision of differentially expressed cDNA fragments --- p.59 / Chapter 2.8 --- Reamplification of differentially expressed cDNA fragments --- p.59 / Chapter 2.9 --- Subcloning of reamplified cDNA fragments --- p.60 / Chapter 2.10 --- Purification of plasmid DNA from recombinant clones --- p.63 / Chapter 2.11 --- DNA sequencing of differentially expressed cDNA fragments --- p.64 / Chapter 2.12 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.66 / Chapter 2.13 --- Western bolt analysis --- p.67 / Chapter Chapter 3 --- RESULTS / Chapter 3.1 --- DNase I treatment --- p.71 / Chapter 3.2 --- FDD RT-PCR and band excision --- p.71 / Chapter 3.3 --- Reamplification of excised cDNA fragments --- p.74 / Chapter 3.4 --- Subcloning of reamplified cDNA fragments --- p.77 / Chapter 3.5 --- DNA sequencing of subcloned cDNA fragments --- p.77 / Chapter 3.6 --- Confirmation of the differentially expressed cDNA fragments by RT-PCR and Western blotting --- p.84 / Chapter 3.6.1 --- Effects of TNF-α on p38a mitogen protein kinase (p38 α MAPK) --- p.84 / Chapter 3.6.2 --- Effects of TNF-α on p38 a MAPK and p-p38 α MAPK protein level --- p.86 / Chapter 3.7 --- Effects of TNF-α on p38 MAPK --- p.88 / Chapter 3.7.1 --- "Effects of TNF-α on p38 α, β,γ andδ MAPK" --- p.88 / Chapter 3.7.2 --- Role of TNF-receptor (TNF-R) subtype in the TNF-α-induced p3 8 MAPK expression in C6 cells --- p.89 / Chapter 3.7.3 --- The signaling system mediating TNF-α-induced p38 a MAPK expression in C6 cells --- p.92 / Chapter 3.7.3.1 --- The involvement of PKC in TNF-α-induced p38 MAPK expression in C6 cells --- p.92 / Chapter 3.7.3.2 --- The involvement of PKC in TNF-α-induced p38 MAPK expression in C6 cells --- p.98 / Chapter 3.7.4 --- The relationship between p38 MAPK and β-adrenergic mechanisms in C6 cells --- p.99 / Chapter 3.7.4.1 --- Effects of isoproterenol and propanol on p38 MAPK mRNA levels in C6 cells --- p.103 / Chapter 3.7.4.2 --- Effects of β1-agonist and -antagonist on p38 MAPK mRNA levels in C6 cells --- p.106 / Chapter 3.7.4.3 --- Effects of β2-agonist and -antagonist on p38 MAPK mRNA levels in C6 cells --- p.107 / Chapter 3.8 --- The relationship between p3 8 MAPK and inducible nitric oxide synthase (iNOS) expression --- p.113 / Chapter 3.8.1 --- Effects of TNF-α on the iNOS expression in C6 cells --- p.113 / Chapter 3.8.2 --- Role of TNF-receptors (TNF-R) subtypes in the TNF-α- induced iNOS expression in C6 cells --- p.115 / Chapter 3.8.3 --- The signaling system mediating TNF-α-induced iNOS expression in C6 cells --- p.115 / Chapter 3.8.3.1 --- The involvement of p38 MAPK in the TNF-α-induced iNOS expression in C6 cells --- p.117 / Chapter 3.8.3.2 --- The involvement of PKA in the TNF-α-induced iNOS expression in C6 cells --- p.119 / Chapter 3.9 --- The relationship between p38 MAPK and cAMP-responsive element binding protein (CREB) expression --- p.120 / Chapter 3.9.1 --- Effects of TNF-α on the CREB expression in C6 cells --- p.120 / Chapter 3.9.2 --- Role of TNF-receptors (TNF-R) subtypes in the TNF-α- induced CREB expression in C6 cells --- p.124 / Chapter 3.9.3 --- The signaling system mediating TNF-α-induced CREB expression in C6 cells --- p.126 / Chapter 3.9.3.1 --- The involvement of p38 MAPK in the TNF-α-induced CREB expression in C6 cells --- p.126 / Chapter 3.9.3.2 --- The involvement of PKC in the TNF-α-induced CREB expression in C6 cells --- p.128 / Chapter 3.9.3.3 --- The involvement of PKA in TNF-α-induced CREB expression in C6 cells --- p.129 / Chapter 3.9.4 --- The relationship between CREB and β-adrenergic mechanisms in C6 cells --- p.136 / Chapter 3.9.4.1 --- Effects of isoproterenol and propanol on CREB mRNA levels in C6 cells --- p.136 / Chapter 3.9.4.2 --- Effects of β1-agonist and -antagonist on CREB mRNA levels in C6 cells --- p.139 / Chapter 3.9.4.3 --- Effects of (32-agonist and -antagonist on CREB mRNA levels in C6 cells --- p.142 / Chapter 3.10 --- The relationship between p38 MAPK and transcription factor c-fos expression --- p.146 / Chapter 3.10.1 --- Effects of TNF-α on the c-fos expression in C6 cells --- p.146 / Chapter 3.10.2 --- Role of TNF-receptors (TNF-R) subtypes in the TNF-α- induced c-fos expression in C6 cells --- p.146 / Chapter 3.10.3 --- The signaling system mediating TNF-α-induced c-fos expression in C6 cells --- p.149 / Chapter 3.10.3.1 --- The involvement of p38 MAPK in the TNF-α-induced c-fos expression in C6 cells --- p.149 / Chapter 3.10.3.2 --- The involvement of PKC in the TNF-α-induced c-fos expression in C6 cells --- p.151 / Chapter 3.10.3.3 --- The involvement of PKA in TNF-α-induced c-fos expression in C6 cells --- p.154 / Chapter 3.10.4 --- The relationship between c-fos and β-adrenergic mechanisms in C6 cells --- p.157 / Chapter 3.10.4.1 --- Effects of isoproterenol and propanolol on c-fos mRNA levels in C6 cells --- p.157 / Chapter 3.10.4.2 --- Effects of β1-agonist and -antagonist on c-fos mRNA levels in C6 cells --- p.160 / Chapter 3.10.4.3 --- Effects of β2-agonist and -antagonist on c-fos mRNA levels in C6 cells --- p.164 / Chapter 3.11 --- The relationship between p38 MAPK and transcription factor NF-kB expression --- p.168 / Chapter 3.11.1 --- Effects of TNF-α on the NF-kB expression in C6 cells --- p.168 / Chapter 3.11.2 --- Role of TNF-receptors (TNF-R) subtypes in the TNF-α- induced NF-kB expression in C6 cells --- p.168 / Chapter 3.11.3 --- The signaling system mediating TNF-α-induced NF-kB expression in C6 cells --- p.171 / Chapter 3.11.3.1 --- The involvement of p38 MAPK in the TNF-α-induced NF-kB expression in C6 cells --- p.171 / Chapter 3.11.3.2 --- The involvement of PKC in the TNF-α-induced NF-kB expression in C6 cells --- p.173 / Chapter Chapter 4 --- DISCUSSION AND CONCLUSION / Chapter 4.1 --- Effects of tumor-necrosis factor-alpha (TNF-α) on C6 cell proliferations --- p.176 / Chapter 4.2 --- The Signaling System Involved in TNF-α-Induced p38 MAPK Expression in C6 cells --- p.178 / Chapter 4.3 --- The Signaling System Involved in TNF-α-Induced iNOS Expression in C6 cells --- p.184 / Chapter 4.4 --- The Signaling System Involved in TNF-α-Induced CREB Expression in C6 cells --- p.186 / Chapter 4.5 --- The Signaling System Involved in TNF-α-Induced c-fos Expressionin in C6 cells --- p.190 / Chapter 4.6 --- The Signaling System Involved in TNF-α-Induced NF-kB Expression in C6 cells --- p.193 / Chapter 4.7 --- Conclusions --- p.195 / Chapter 4.8 --- Possible application / References

Cells process information from their environment, such as the stimuli to grow, divide, or die, via cell signaling. Deregulated processing of extracellular stimuli can lead to aberrant cell responses and cause cancer. Given that the in vivo cell environment constantly changes, it is important to understand how cells incorporate the context of their environment into their decision making processes. The idea of responding to relative, not absolute, changes in stimuli was first proposed in studies of human perception and became known as Weber's Law. Although, evidence of Weber's Law at the molecular level has been previously presented in studies of several organisms, to the best of our knowledge, it has never been explored in the case of relative sensing of extracellular stimuli in mammalian signaling cascades. The Mitogen-Activated Protein Kinase (MAPK) signaling pathway has been implicated in multiple human diseases, including cancers, and therefore cell signaling through this pathway is an important subject of research. Here we present a theoretical framework and an experimental validation of the mechanism of Weber's Law in the ability of cells to sense relative changes in the levels of extracellular stimuli in the MAPK signaling pathway. In particular, in this work we consider relative sensing in levels of Epidermal Growth Factor (EGF) in the MAPK pathway. We derive an analytical model of steady state behavior of the MAPK signaling pathway stimulated with constant doses of EGF. We demonstrate a mechanism that produces phosphorylation responses proportional to relative changes in ligand concentrations. The mechanism of Weber's Law presented here entails the retention of memory of the dose of the past chronic stimulation with EGF. The molecular mechanisms responsible for Weber's Law in MAPK signaling are likely to contribute to many other receptors signaling systems. Therefore, the mechanism of relative sensing of extracellular ligand concentrations derived here can be generalized beyond the EGF-activated MAPK signaling pathway to many other cell signaling systems. This thesis also presents a probabilistic framework to explore the parameter space of a detailed mechanistic ODE model of EGFR signaling cascades. The application of the model simulation allows us to generate probabilistic predictions of EGFR system behavior and to explore structure-to-function relationships between the model's parameter space and EGFR system responses. Overall, this work suggests an alternative view on the role of cellular endocytosis in the MAPK signaling in vivo. Specifically, traditionally viewed as a mechanism to downregulate and terminate cell signaling, endocytosis may enable cells to dynamically adjust their sensitivity to extracellular stimuli, and hence allow cells to integrate information about the past stimulations into the cell responses to the consequent stimulations and thus, cell fate decisions.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

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.

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.

Una de las vías que se postula que tienen una mayor importancia en las enfermedades neurodegenerativas es la de los inositoles fosfato. Para el estudio de esta vía se ha utilizado un inhibidor farmacológico de la fosfoinositol 3 cinasa (PI3K), el LY294002, en un modelo in vitro de células granulares de cerebelo de rata (CGC). Al tratar las CGC con una dosis de 30μM de LY294002 se produce una muerte celular por apoptosis que es independiente de calpaínas y dependiente de caspasas, además no se observa la fragmentación de p35 ni de α espectrina que se da por activación de las calpaínas. Los ensayos de actividad caspasa nos muestran un incremento significativo de la actividad de las caspasas 6 y 9 pero no de la 3 como sucede en otros modelos de apoptosis como la deprivación de S/K+. Nuestros estudios muestran que aunque existen algunas similitudes entre los modelos de inhibición de la PI3K y la deprivación de S/K+ también existen importantes diferencias. En ambos se produce una desfosforilación de AKT en Ser476 y consecuentemente una desfosforilación de GSK3β en Ser9, lo que indica la activación de GSK3β. Respecto a la proteína Rb en ambos modelos se observa un incremento de su fosforilación, si bien su papel es distinto ya que en la deprivación de S/K+ conduce a la liberación del E2F y a la transcripción de proteínas relacionadas con el ciclo celular. Además, se observó un incremento de la síntesis de DNA. Por el contrario el tratamiento con LY294002, pese a provocar un incremento en la fosforilación del Rb, no lleva a la expresión de ciclinas, CDKs ni un aumento de la síntesis de DNA.. Sin embargo el uso de inhibidores de CDK como flavopiridol y roscovitina muestran una protección significativa frente a la apoptosis inducida por LY294002, nuestros estudios muestran por vez primera que, no solo flavopiridol sino también otros inhibidores de CDK como la roscovitina tienen capacidad para inhibir la actividad GSK3β. Rb puede ser fosforilado por p38, un miembro de la vía de las MAPK las cuales son inhibidas por AKT. Nuestros resultados indican que LY294002 produce un incremento de la actividad de p38, pero no de JNK. Además, los cultivos Knockout de JNK3 no muestran una protección frente al tratamiento con LY294002, lo que refuerza la idea de que JNK no juega un papel central en este modelo. El incremento de actividad de p38 fue revertido con SB203580, un inhibidor de p38, así como por SP600125, inhibidor de JNK. Ambos fármacos mostraron una protección significativa frente a la apoptosis inducida por LY294002 y una reducción de la fosforilación del factor de transcripción c‐Jun, implicado en la apoptosis. La activación de c‐Jun conduce a la expresión de genes proapoptóticos como dp5 relacionados con la vía intrínseca, la inhibición de p38 previno del aumento de expresión de dp5. Por el contrario otras proteínas implicadas en la vía como Bim no están reguladas por c‐Jun ya que la inhibición de esta vía no reduce su activación. En nuestro estudio podemos concluir que, LY294002 produce una apoptosis dependiente de caspasas 6 y 9, sin implicación ni de calpaínas ni de proteínas del ciclo celular. La inhibición de AKT lleva a la activación de GSK3β y de p38. Además, p38 es capaz de fosforilar c‐Jun que regula la expresión de genes relacionados con la apoptosis por la vía intrínseca. / The inositol pathway has been reported that plays a key role in neurodegenerative diseases We study the mechansims involved in the apoptosis induced by inhibiting the phosphoinositol 3 kinase (PI3K) using a pharmacological inhibitor named LY294002 in an in vitro model of rat cerebellar granule cells (CGC). LY294002 induced apoptotic cell death through calpain independent and caspase dependent. Furthermore, we could not observed neither fragmentation of of p35 or α espectrin which is caused by calpains. The caspase activity assays showed a significant increase in caspase 6 and 9 but not in caspasa 3, in contrast with other apoptotic models such as de S/K+ deprivation. Our studies show that although exist several common points between inhibition of PI3K and S/K+ deprivation, also exist important differences between them. In both cases it has been observed AKT dephosphorylation at Ser476 and consequently GSK3β dephosphorylation at Ser9, which indicates GSK3β activation. On the other side, it was observed an increase of Rb phosphorylation in both models. However, it seems that the role played by this protein is different since in the de S/K+ deprivation leads to E2F released which participates in the transcription of proteins related to cell cycle. Moreover, the BrdU assay showed an increase in DNA synthesis. On the contrary, the LY294002 treatment, in spite of the fact that induced an increase of Rb phosphorylation, it did not induce any change of the levels neither cell cycle proteins or However, CDK inhibitors such as flavopiridol and roscovitine protected from the apoptosis induced by LY294002, our studies showed for the first time, that not only flavopiridol, but also other CDK inhibitors such as roscovitine could inhibit the GSK3β activity. Furthermore Rb can be phosphorylated by p38, which is a protein of MAPK pathway that is down‐regulated by AKT. Our results showed that LY294002 produced an increase of p38 activity, but not of JNK. Moreover, JNK3 Knockout cultures were not significantly protected from LY294002 treatment, this reinforces the idea that JNK is not the main target involved in this model. The increase of p38 activity was prevented with SB203580, a specific p38 inhibitor, and either with SP600125, a JNK inhibitor. Both drugs shown a significant protection from the apoptosis induced by LY294002 and prevented from c‐Jun phosphorylation, a transcription factor implied in apoptosis. The activation of c‐Jun triggered the expression of proapoptotic genes such as dp5 which is related to the intrinsic pathway, p38 inhibition prevented from the increase in dp5 expression. On the contrary, other proapoptotic proteins related to this pathway such as Bim was not regulated by c‐Jun since the inhibition of p38 pathway did not reduce its expression. In our study we can conclude that LY294002 induced apoptosis mediated by caspasas 6 and 9. Neither calpains nor cell cycle proteins were involved in this apoptotic model. The inhibition of AKT leaded to GSK3β and p38 activation. Moreover, p38 was able to phosphorylate c‐Jun that triggers the expression of proapoptotic genes implied in the apoptotic intrinsic pathway.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 78-93).