Apoptotic cell death plays an important role in the pathogenesis of tubular epithelial damage following oxidant injury such as, ischemia/reflow and radiation nephropathy. Elucidating the cell signaling pathways involved in apoptosis will define sites for cytotoxic chemotherapy and modulation of cell sensitivity to apoptosis. Redox- regulation of protein phosphatase activity is a cellular response pathway that provides an attractive link between apoptosis, cell proliferation, and oxidant stress. Protein phosphatases play regulatory roles in mitosis. The protein phosphatase 1 and 2A inhibitor and tumor promoter Okadaic acid, induces apoptosis in normal rat kidney epithelial cells in culture. Mitosis and apoptosis have similar morphologic, molecular, and biochemical characteristics, such as chromatin condensation and gene expression. Therefore, it is reasonable to expect that the cell's decision to divide or die may depend on pathways regulated by protein phosphatases. Protein phosphatase activity depends on maintenance of the enzyme's reduced cysteine or sulfhydryl groups. Therefore, changes in glutathione, the major non-protein thiol, may modulate phosphatase activity. Cellular glutathione decreases in response to oxidant stress. Since decreases in cellular redox status may affect protein phosphatase activity, and protein phosphatases play a role in apoptosis and mitosis, then protein phosphatases may be a mechanistic link between oxidant injury, apoptosis, and cell proliferation. The broad objectives of this proposal are to elucidate the role(s) of protein phosphatases in apoptosis, to examine the effect of cellular thiol redox state on protein phosphatases involved in apoptotic signaling, and to ultimately identify regulatory signals common to cell proliferation, oxidant stress and apoptosis.
The specific aims are (1) to determine the phosphorylation state and activity of cell proliferation modulators such as c-Jun, p34/CDC2 kinase/cyclin B, CDK2, and MAP kinases in okadaic- acid treated NRK-52E cells (2) to determine the occurrence of mitosis specific events such as mitosis-specific protein expression (MPM-2) in okadaic acid- treated NRK-52E cells. Since cellular glutathione decreases in response to oxidant stress and protein phosphatase activity is redox sensitive this study will also (3) examine the effects of decreased cellular glutathione on protein phosphatase 1 and 2A activity (4) to determine the effects of thiol elevating compounds on okadaic acid induced -morphology, protein phosphorylation, and cell death and (5) to determine the effects of oxidants on protein phosphatase 1 and 2A activity in NRK-52E cells. The information obtained from these studies will demonstrate the role of cellular thiols and protein phosphatases in apoptosis. Redox regulation of protein phosphatase activity in apoptosis will also define a physiologically relevant pathway by which cellular responses occur in vivo. Most importantly, redox-regulation of protein phosphatase activity provides an attractive cellular response mechanism linking apoptosis, cell proliferation, and oxidant stress.
| Davis, M A; Chang, S H; Trump, B F (1996) Differential sensitivity of normal and H-ras oncogene-transformed rat kidney epithelial cells to okadaic acid-induced apoptosis. Toxicol Appl Pharmacol 141:93-101 |
