.. PHB Tg/Nrf2?/? mice show increased ERK1/2 activation and AP-1 expression during colitis. In addition to Nrf2, the transcription factor AP-1 binds to some ARE sites and activates transcription of antioxidant/phase II detoxifying enzymes, including HO-1 and NQO-1 (18, 33). Furthermore, ERK Lapatinib Ditosylate signaling has been identified as an upstream mediator involved in AP-1 activation (17). AP-1 is a dimeric transcription factor composed of c-Jun, c-Fos, or activating transcription factor subunits (28). Since increased expression of HO-1 and NQO-1 was sustained in PHB Tg mice, even with Nrf2 deletion, we next assessed activation of ERK, as well as protein levels of c-Fos and c-Jun. PHB Tg and PHB Tg/Nrf2?/? mice exhibited increased colonic phosphorylated ERK protein levels during colitis induced by DSS (Fig.

7A) and TNBS (Fig. 7C) compared with WT and Nrf2?/? mice. Colonic c-Jun protein levels were increased in PHB Tg and PHB Tg/Nrf2?/? mice treated with DSS (Fig. 7B) or TNBS (Fig. 7D), while c-Fos protein levels were increased predominantly in PHB Tg/Nrf2?/? mice during colitis. Fig. 7. PHB Tg/Nrf2?/? mice show increased ERK1/2 activation and activator protein 1 (AP-1) expression during colitis. A and C: colonic phosphorylated ERK1/2 (pERK1/2) expression quantified by Western blot in DSS- and TNBS-treated mice. B and … ERK signaling contributes to PHB-induced ARE activation during TNF�� treatment. To assess the role of ERK in PHB-induced ARE activation during proinflammatory signaling and Nrf2 knockdown, ERK signaling was inhibited using PD-98059 in Caco-2-BBE cells. As shown in Fig.

8, addition of the ERK inhibitor PD-98059 in cells overexpressing PHB decreased TNF��-induced ARE4 activation by 41% (16.80 �� 3.3 and 9.83 �� 0.3 with TNF�� and TNF�� + PD-98059, respectively, P < 0.01) compared with only 21% in vector-transfected cells (8.46 �� 1.4 and 6.60 �� 0.2 with TNF�� and TNF�� + PD-98059, respectively, P < 0.05). Vector-transfected cells with Nrf2 knockdown showed a 70% reduction in TNF��-induced ARE4-luciferase expression (8.46 �� 1.4 and 2.50 �� 0.6 with TNF�� and TNF�� + Nrf2 siRNA, respectively, P < 0.01; Fig. 8), whereas PHB-overexpressing cells showed only a reduction of 41% (16.80 �� 3.3 and 9.98 �� 1.7 with TNF�� and TNF�� + Nrf2 siRNA, respectively, P < 0.01), which are similar to results shown in Fig. 2A.

Addition of PD-98059 during Nrf2 knockdown decreased TNF��-induced ARE4 activation by an additional 38% compared with Nrf2 knockdown alone in cells overexpressing PHB (9.98 �� 1.7 and 3.47 �� 1.9 with TNF�� + Nrf2 siRNA and TNF�� + Nrf2 siRNA + PD-98059, respectively, P < 0.01) vs. an AV-951 additional 15% in cells overexpressing control vector (2.50 �� 0.6 and 1.3 �� 0.5 with TNF�� + Nrf2 siRNA and TNF�� + Nrf2 siRNA + PD-98059, respectively, P < 0.05; Fig. 8).