The role of 4-hydroxynonenal in cellular signaling mechanisms
Brad Allen Patrick
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One of the critical steps involved in cellular oxidative stress is the peroxidation of membrane lipids. The downstream effects of the autocatalytic lipid peroxidation (LPO) cycle are widespread and involve several processes. Using lens epithelial cells (HLE B-3) and retinal pigment epithelial cells (RPE28 SV4) as models, in proposed studies we addressed the hypothesis that a physiological level of LPO product 4-hydroxynonenal (HNE) is necessary for maintaining key proliferation, adhesion, and survival signaling by regulating expression of genes involved in these pathways, and that alteration of that level either up or down will result in induction of genes pro-apoptotic or pro-carcinogenic signaling pathways, respectively.\r\n After stable transfection of hGSTA4 in HLE B-3, cells underwent a morphological transformation becoming smaller and rounded, and lost anchorage dependence. Additionally they were observed to divide much more rapidly and acquired resistance to apoptosis via oxidative stress. In an attempt to provide a mechanistic explanation for this phenotypic set of changes, cells were assayed for changes in expression for a wide set of genes via gene microarray studies. Results showed that over 6900 genes were strongly modulated in hGSTA4-transformed cells compared to controls. This prompted further verification in a subset of well-known and important genes involved in cell cycle control, survival, and cell adhesion via quantitative RT-PCR and Western blot studies. In these cells, considerably lower levels of transcription and translation of key genes regulating those affected processes were observed. \r\n Further exploration of HLE B-3 showed that expression of Fas correlated with HNE concentration, an observation found both before and after HNE treatment. This is strengthened by observations that transiently-transfected HLE B-3 overexpress hGSTA4 along with a depletion of intracellular HNE and depletion of Fas expression and further strengthened in a mouse mGSTA4¬ knockout model with high steady-state HNE tissue levels, where Fas expression is found to be elevated in several tissues above WT. Together these studies implicate HNE as an important mediator of expression of several key genes responsible for processes underlying cell cycle regulation, survival, and adhesion, and lay a foundation upon which further investigation can be performed.