p38 Mitogen-activated Protein Kinase Negatively Regulates the Induction of Phase II Drug-metabolizing Enzymes That Detoxify Carcinogens

Abstract

Phase II drug-metabolizing enzymes, such as glutathione S-transferase and quinone reductase, play an important role in the detoxification of chemical carcinogens. The induction of these detoxifying enzymes by a variety of agents occurs at the transcriptional level and is regulated by a cis-acting element, called the antioxidant response element (ARE) or electrophile-response element. In this study, we identified a signaling kinase pathway that negatively regulates ARE-mediated gene expression. Treatment of human hepatoma HepG2 and murine hepatoma Hepa1c1c7 cells with tert-butylhydroquinone (tBHQ) stimulated the activity of p38, a member of mitogen-activated protein kinase family. Inhibition of p38 activation by its inhibitor, SB203580, enhanced the induction of quinone reductase activity and the activation of ARE reporter gene by tBHQ. In contrast, SB202474, a negative analog of SB203580, had little effect. Consistent with this result, interfering with the p38 kinase pathway by overexpression of a dominant-negative mutant of p38 or MKK3, an immediate upstream regulator of p38, potentiated the activation of the ARE reporter gene by tBHQ, whereas the wild types of p38 and MKK3 diminished such activation. In addition, inhibition of p38 activity augmented the induction of ARE reporter gene activity by tert-butylhydroxyanisole, sulforaphane, and ␤-naphthoflavone. Thus, p38 kinase pathway functions as a negative regulator in the AREmediated induction of phase II detoxifying enzymes.

Regulation of the antioxidant response element by protein kinase C-mediated phosphorylation of NF-E2-related factor 2

“…Previous studies have demonstrated the involvement of MAPK pathways in ARE-mediated transcription (26,27). While it is well established that PKC can activate the MAPK pathways (47), our finding that specific inhibitors of ERK and p38 did not affect Nrf2 phosphorylation appears to rule out the possibility that the role of PKC in Nrf2 phosphorylation is through its indirect action on these downstream kinases.…”

“…Previous studies implicating the involvement of various kinase pathways in ARE-mediated gene expression have failed to identify any cellular kinase targets (26,27,31). To ascertain whether Nrf2 can be phosphorylated in vivo, we performed metabolic labeling of HepG2 cells with [ 32 P]orthophosphate, followed by immunoprecipitation with an anti-Nrf2 antibody of the cell lysates.…”

“…4A revealed that this PKC inhibitor effectively blocked the phosphorylation of Nrf2 in untreated, or tBHQ-, ␤NF-, and PMAtreated cells. Because the MAPK pathways have been shown to influence ARE-mediated transcription (26,27), we also tested whether selective inhibitors of the extracellular signal-regulated kinase 2 and p38 pathways might affect Nrf2 phosphorylation. In contrast to staurosporine, however, U0126 (10 M), which targets the MEK kinases (34), and SB203580 (10 M), a selective inhibitor of the p38 kinase (35), were both ineffective in preventing the tBHQ-induced stimulation of Nrf2 phosphorylation (Fig.…”

“…One likely mechanism involves regulation by phosphorylation (25). A role for mitogen-activated protein kinase (MAPK) pathways has been suggested by results from transfection and specific kinase inhibitor studies, which revealed that extracellular signal-regulated kinase 2 positively (26), whereas p38 negatively (27), regulated ARE-mediated transcription. In earlier studies, we and others had also shown that phorbol esters, potent tumor promoters that activate protein kinase C (PKC) (28), induced ARE-driven expression of the rat GST A1 (29), rat QR (30), and mouse GST A2 (31) genes.…”

p38 Mitogen-Activated Protein Kinase Contributes to Adenosine A1Receptor-Mediated Synaptic Depression in Area CA1 of the Rat Hippocampus

“…SB202474 is an inactive pyridinyl imidazole compound that has been used as a negative control in p38 MAPK studies (Lee et al, 1994;Yu et al, 2000). Slices were either preincubated with 25 M SB202474 or with normal aCSF (control).…”