These findings are in agreement with emerging data that suggest a role of PHB in alleviating oxidative stress in multiple cell types (14, 29, 35, 45, 53). Gene silencing of PHB in cultured intestinal epithelial cells induces mitochondrial membrane depolarization, increases intracellular ROS and mitophagy, and reduces cell viability (20), compound library suggesting that PHB is involved in epithelial cell homeostasis. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a basic leucine zipper, redox-sensitive transcription factor that plays a role in cellular defense against oxidative and electrophilic stress through the induction of antioxidant and phase II detoxification enzymes. These cytoprotective genes are regulated through an antioxidant response element (ARE) in their promoter region to which Nrf2 binds, thereby activating transcription (19).

ARE-regulated genes include NAD(P)H quinone oxidoreductase-1 (NQO-1), heme oxygenase-1 (HO-1), peroxiredoxin 1, ��-glutamylcysteine ligase, glutathione peroxidase, and glutathione disulfide reductase (26). Nrf2 also attenuates early-phase tissue damage during inflammation in multiple organs through regulation of the innate immune response and repression of proinflammatory mediators (26). Nrf2 knockout mice show increased susceptibility to colitis and colitis-associated tumorigenesis (24, 25, 40). Our previous study showed that PHB-mediated protection from dextran sodium sulfate (DSS)-induced colitis in mice was associated with increased colonic Nrf2 expression and nuclear localization (52).

This study investigates the role of Nrf2 in mediating PHB-induced protection against colitis and expression of the ARE-responsive antioxidant enzymes HO-1 and NQO-1. METHODS AND MATERIALS Cell culture. The Caco-2-BBE human intestinal epithelial cell line (American Type Culture Collection, Manassas, VA) was used as an in vitro model of polarized intestinal epithelium. All cells were grown as a confluent monolayer in Dulbecco’s modified Eagle’s medium supplemented with 40 mg/l penicillin, 90 mg/l streptomycin, and 10% fetal calf serum. All experiments were performed on Caco-2-BBE cells between passages 30 and 40. Caco-2-BBE cells were plated onto permeable supports (0.4-��m pore size; Transwell-Clear polyester membranes, Costar Life Sciences, Acton, MA). Cells were transiently cotransfected with pEGFPN1 expression vector or pEGFPN1-PHB (20) and 20 ��M Nrf2 small interfering RNA (siRNA) (Santa Cruz Biotechnology, Santa Cruz, CA) or 20 ��M Stealth RNAi negative control medium GC (Invitrogen, Carlsbad, CA) by nucleofection (Amaxa Carfilzomib transfection, cell line kit T; Lonza, Basel, Switzerland).