05 was considered statistically significant in all analyses. RESULTS Caco-2-BBE Wortmannin mw cells overexpressing PHB exhibit increased TNF��-induced Nrf2 nuclear localization, ARE activation, and HO-1 and NQO-1 expression. Our previous study suggested that PHB overexpression in intestinal epithelial cells protected against inflammation and oxidative stress in association with increased Nrf2 activation (52). It is widely accepted that TNF�� is a central mediator of the proinflammatory response during intestinal inflammation (3). TNF��-induced ROS generation occurs primarily in the mitochondria, and signal transduction pathways that delineate with TNF��, including NF-��B activation and apoptosis, are reliant on mitochondrial ROS production (12, 13, 15).

Furthermore, mitochondrial function and intracellular ROS levels are regulated by relative concentrations of PHB (20). To determine whether PHB overexpression induces Nrf2 during TNF�� signaling, Caco-2-BBE cells were transfected with GFP-tagged PHB or GFP control vector and treated with TNF��. Protein expression of endogenous PHB, predominantly localized in the cytosolic fraction, is decreased by TNF�� treatment (Fig. 1A). Caco-2-BBE cells overexpressing PHB show increased Nrf2 nuclear translocation 60 min after TNF�� treatment compared with control vector-expressing cells. Blots were probed with an anti-GFP antibody to confirm expression of exogenous GFP-tagged PHB, which localized in the cytosol (Fig. 1A). PHB-overexpressing cells cotransfected with an ARE4-luciferase reporter construct exhibited more relative luciferase activity induced by TNF�� than control vector-transfected cells (Fig.

1B). Although the mRNA (Fig. 1C) and protein (Fig. 1D) expression patterns of NQO-1 and HO-1 in vector-transfected cells are similar to those in PHB-overexpressing cells following TNF�� treatment, the induction is significantly more robust and occurs more rapidly in cells overexpressing PHB. TNF�� treatment decreases endogenous PHB protein expression to the same magnitude in control vector- and PHB-transfected cells (Fig. 1D). Blots were probed with an anti-GFP antibody to confirm expression of exogenous GFP-tagged PHB (Fig. 1D). Fig. 1. Caco-2-BBE cells overexpressing prohibitin (PHB) exhibit increased TNF��-induced nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear localization, antioxidant response element (ARE) activation, and heme oxygenase-1 (HO-1) and NAD(P)H quinone .

.. PHB-induced ARE activation, NQO-1 and HO-1 protein expression, and decreased intracellular ROS levels during TNF�� treatment are not exclusively mediated via Nrf2. To assess the role of Nrf2 in PHB-induced antioxidant responses during proinflammatory signaling, Nrf2 expression in Caco-2-BBE cells was knocked down using siRNA. Drug_discovery Basal ARE4-luciferase expression was unaffected by Nrf2 knockdown in vector- and PHB-overexpressing cells (Fig. 2A).