Browsing by Subject "ERK"
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Item ERK/MAPK Pathway Regulation of GABA-A Receptor Function.(2009-12-01) Dohi, Akiko; Singh, MeharvanGABA-A receptor is a ligand-gated ion channel that conducts negatively charged chloride ions. Influx of this ion leads to hyperpolarization of neurons; thus, suppression of the neuronal excitability. Alterations in GABAergic neurotransmission may contribute to depression and anxiety. While neurosteroids can regulate the responsiveness of the GABAA receptor in allosteric manner, certain intracellular signaling pathways can also regulate the function of the GABA-A receptors through phosphorylation of its subunits. One pathway that is regulated by both neurotrophic factors and steroid hormones is the ERK/MAPK pathway. This pathway is involved in cell proliferation, maturation, and even, cell death. The role of this pathway in the regulation of the GABAA receptor, however, is not well studied and is the subject of my dissertation. Initial studies conducted by the Singh and Dillon laboratories showed that pharmacological inhibition of the ERK/MAPK pathway potentiated the α1β2γ2 configuration of the GABAA receptor, expressed in HEK-t cells. This suggested that the ERK/MAPK pathway was involved in the negative regulation of the GABAA receptor function. In silico analysis revealed that the Thr 375 residue within the α1 subunit was a plausible target of the ERK/MAPK pathway. As a result, I hypothesized that the activation of the ERK/MAPK pathway inhibited GABAA receptor function through the direct phosphorylation of the Thr 375 residue, resulting in receptor internalization. Supporting this hypothesis was data showing that mutation of the Thr 375 residue to Alanine prevented the enhancement of GABA-gated currents elicited by inhibiting the ERK/MAPK pathway. However, using the HEK-t cell line transfected with the α1β2γ2 configuration of the GABAA receptor, I determined that the activation of the ERK/MAPK pathway by HGF did not influence the peak amplitude of the GABA-gated currents. Further, the potentiation of the GABA-gated currents was apparently not due to internalization of the receptor. Collectively, while we believe that the Thr 375 within the α1 subunit is relevant to the effect of ERK/MAPK pathway inhibition, it was not a direct target of the ERK/MAPK pathway.Item Estrogen Signaling Protects Mitochondrial Membrane Potential Integrity from Oxidative Stress in Lens Epithelial Cells(2008-05-01) Flynn, James Martin; Cammarata, Patrick R.; Wordinger, Robert J.; Dimitrijevich, S. DanFlynn, James Martin, Estrogen Signaling Protects Mitochondrial Membrane Potential Integrity from Oxidative Stress in Lens Epithelial Cells. Doctor of Philosophy, (Cell Biology and Genetics) May, 2008, 265 pages, 36 figures, bibliography, 190 titles. Loss of mitochondrial membrane potential has been determined to be one of the initiating factors in activation of apoptosis after cellular damage. Estrogen and estrogen analogues have been shown to enhance cell survival in numerous tissues through rapid pro-survival cell signaling. This study was focused on elucidating mechanisms through which estrogen protects the cells by preventing the activation of mitochondrial permeability transition pores and the subsequent loss of mitochondrial membrane potential. It is hypothesized that the anti-apoptotic mitochondrial protein BAD, once phosphorylated by estrogen activated upstream kinases, can prevent the formation of the permeability transition pre via direct interaction. To address this, lens epithelial cells were used as a model system for the examination of mitochondrial depolarization during periods of either oxidative or hyperglycemic stress. Estrogen attenuated the loss of impermeability of the mitochondrial membrane, thus maintaining the cells during acute periods of stress. It was discovered that a number of the estrogen receptor isoforms are expressed in lens epithelium, and that the wild-type estrogen receptor-β1 isoform is localized to the mitochondria in lens epithelial cultures derived from both human males and females. siRNA treatment against estrogen receptor-β determined that is a required component to elicit estrogen’s protective abilities against oxidative stress induced mitochondrial depolarization. Furthermore, administration of exogenous estrogen rapidly activated signaling pathways, particularly ERK, which were shown to have influence over the loss of mitochondrial membrane potential. Studies using both pharmacological inhibitors of MAPK signaling, as well as siRNA of ERK2 kinase demonstrate a correlation between the activation of ERK and the severity of response to oxidative stress. Investigation of downstream substrates of ERK revealed that the mitochondrial protein BAD is phosphorylated after the administration of estrogen, yet it is not required for the prevention of mitochondrial depolarization as originally hypothesized. In conclusion, these studies have confirmed a mitochondrial targeted mechanism activated by estrogen which is rapid, gender independent, estrogen receptor-β mediated signal transduction pathway. The targeting of mitochondrial function to reduce oxidative or hyperglycemic stress, thereby preventing activation of the permeability transition pore, defines a novel concept which will contribute to innovative regimens for prevention or treatment of mitochondrial pathology.