Steven Berberich (Committee Member), Nancy Bigley (Committee Member), David Cool (Committee Member), Saber Hussain (Committee Member), Courtney Sulentic (Advisor)
Doctor of Philosophy (PhD)
Reactive Oxygen Intermediates (ROI) can significantly affect B Lymphocyte function, but the effects on key signaling pathways and gene expression in activated and non-activated B lymphocytes are largely undefined. This study demonstrates a concentration dependent effect of exogenous hydrogen peroxide on the transcriptional regulation of a 3' regulatory region of the Immunoglobulin heavy chain gene (3'IghRR). Specifically, low μM hydrogen peroxide induces an enhancing effect while higher μM hydrogen peroxide (greater than or equal to 100 μM) is suppressive. The enhancing effects of hydrogen peroxide on 3'IghRR activity are dependent on the NFκB/Rel pathway, and appear to be most prominent in antigen activated B lymphocytes. Moreover, treatment of activated B lymphocytes with a chemical inhibitor of ROI producing enzymes inhibited 3'IghRR activity, Ig production, and NFκB/Rel activity, suggesting that endogenous ROI in part facilitate activation-induced Ig expression and NFκB/Rel activity. Furthermore, exposure of non-activated B lymphocytes with higher μM hydrogen peroxide concentrations suppressed NFκB/Rel activity and caused dramatic decreases in cellular viability. This effect of hydrogen peroxide on viability was suppressed by co-treatment with a pan caspase inhibitor, thus indicating an important role for caspases in the hydrogen peroxide-mediated cell death. Hydrogen peroxide-induced caspase activity was found to directly target critical components of the NFκB/Rel pathway, which is involved in maintaining cell survival signals in the B lymphocyte. We observed that hydrogen peroxide exposure induced caspase-dependent cleavage of a critical NFκB/Rel regulatory protein, IκBα that can result in the ΔN-IκBα form and most notably a novel 19 kDa form of IκBα. Hydrogen peroxide exposure also induced two caspase-dependent cleavage products of a prominent NFκB/Rel transcription factor, p65/RelA. We believe hydrogen peroxide mobilizes caspases activity as part of a mechanism to suppress the NFκB/Rel pathway during oxidative stress as co-treatment of hydrogen peroxide-exposed B lymphocytes with a pan caspase inhibitor markedly increased the expression of an NFκB/Rel transcriptional reporter. These observations demonstrate that ROI can modulate critical functions in the B lymphocyte such as Ig expression and cellular viability in a manner that is dependent upon the activation state of the cell, and at least in part, involves the modulation of the NFκB/Rel pathway. These findings may be highly relevant to further understanding the effects of ROI on NFκB/Rel-dependent B lymphocyte function in adaptive immunity and many disease states and treatments known to involve ROI.
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