Role of a mitogen-activated protein kinase pathway in the induction of phase II detoxifying enzymes by chemicals.

Mitogen-activated protein kinase (MAPK) cascades are activated by diverse extracellular signals and participate in the regulation of an array of cellular programs. In this study, we investigated the roles of MAPKs in the induction of phase II detoxifying enzymes by chemicals. Treatment of human hepatoma (HepG2) and murine hepatoma (Hepa1c1c7) cells with tert-butylhydroquinone (tBHQ) or sulforaphane (SUL), two potent phase II enzyme inducers, stimulated the activity of extracellular signal-regulated protein kinase 2 (ERK2) but not c-Jun N-terminal kinase 1. tBHQ and SUL also activated MAPK kinase. Inhibition of MAPK kinase with its inhibitor, PD98059, abolished ERK2 activation and impaired the induction of quinone reductase, a phase II detoxifying enzyme, and antioxidant response element (ARE)-linked reporter gene by tBHQ and SUL. Overexpression of a dominant-negative mutant of ERK2 also attenuated tBHQ and SUL induction of ARE reporter gene activity. Interestingly, although expression of Ras and its mutant forms showed distinct effects on basal ARE reporter gene activity, they did not affect the activation of reporter gene by the inducers. Furthermore, a dominant-negative mutant of Ras had little effect on ERK2 activation by tBHQ and SUL, implicating a Ras-independent mechanism. Indeed, both tBHQ and SUL were able to stimulate Raf-1 kinase activity in vivo as well as in vitro. Thus, our results indicate that the induction of ARE-dependent phase II detoxifying enzymes is mediated by a MAPK pathway, which may involve direct activation of Raf-1 by the inducers.     

Phosphatidylinositol 3-kinase, not extracellular signal-regulated kinase, regulates activation of the antioxidant-responsive element in IMR-32 human neuroblastoma cells

The antioxidant-responsive element (ARE) plays an important role in the induction of phase II detoxifying enzymes including NADPH:quinone oxidoreductase (NQO1). We report herein that activation of the human NQO1-ARE (hNQO1-ARE) by tert-butylhydroquinone (tBHQ) is mediated by phosphatidylinositol 3-kinase (PI3-kinase), not extracellular signal-regulated kinase (Erk1/2), in IMR-32 human neuroblastoma cells. Treatment with tBHQ significantly increased NQO1 protein without activation of Erk1/2. In addition, PD 98059 (a selective mitogen-activated kinase/Erk kinase inhibitor) did not inhibit hNQO1-ARE-luciferase expression or NQO1 protein induction by tBHQ. Pretreatment with LY 294002 (a selective PI3-kinase inhibitor), however, inhibited both hNQO1-ARE-luciferase expression and endogenous NQO1 protein induction. In support of a role for PI3-kinase in ARE activation we show that: 1) transfection of IMR-32 cells with constitutively active PI3-kinase selectively activated the ARE in a dose-dependent manner that was completely inhibited by treatment with LY 294002; 2) pretreatment of cells with the PI3-kinase inhibitors, LY 294002 and wortmannin, significantly decreased NF-E2-related factor 2 (Nrf2) nuclear translocation induced by tBHQ; and 3) ARE activation by constitutively active PI3-kinase was blocked completely by dominant negative Nrf2. Taken together, these data clearly show that ARE activation by tBHQ depends on PI3-kinase, which lies upstream of Nrf2.     

Role of protein kinase C delta in curcumin-induced antioxidant response element-mediated gene expression in human monocytes

The Nrf2/antioxidant response element (ARE) signaling pathway plays a key role in activating cellular antioxidants, including heme oxygenase-1 (HO-1), NADPH quinone oxidoreductase-1 (NQO1), and glutathione. Protein kinase C (PKC) may also regulate these antioxidants, as PKC phosphorylates Nrf2 in vitro. This study examined the role of PKC in ARE-mediated gene regulation in human monocytes by curcumin, a potent inducer of the Nrf2/ARE pathway. Curcumin increased HO-1 and glutamyl cysteine ligase modulator (GCLM) expression and stimulated Nrf2 binding to the ARE. Curcumin also rapidly stimulated PKC phosphorylation and Ro-31-8220, a pan-PKC inhibitor, decreased curcumin-induced GCLM and HO-1 mRNA expression and ARE binding. Rottlerin (a PKC delta inhibitor) and PKC delta antisense oligonucleotides significantly inhibited curcumin-induced GCLM and HO-1 mRNA expression and ARE binding. Furthermore, a p38 MAP kinase inhibitor reduced GCLM and HO-1 expression and rottlerin inhibited curcumin-induced p38 phosphorylation. In summary, curcumin activates ARE-mediated gene expression in human monocytes via PKC delta, upstream of p38 and Nrf2.     

The p38/RK mitogen-activated protein kinase pathway regulates interleukin-6 synthesis response to tumor necrosis factor.

Tumour necrosis factor (TNF) is a pleiotropic cytokine, the activities of which include effects on gene expression, cell growth and cell death. The biological signalling mechanisms which are responsible for these TNF effects remain largely unknown. Here we demonstrate that the stress-responsive p38 mitogen-activated protein (MAP) kinase is involved in TNF-induced cytokine expression. TNF Treatment of cell activated the p38 MAP kinase pathway, as revealed by increased phosphorylation of p38 MAP kinase itself, activation of the substrate protein MAPKAP kinase-2, and culminating in the phosphorylation of the heat shock protein 27 (hsp27). Pretreatment of cells with the highly specific p38 MAP kinase inhibitor SB203580 completely blocked this TNF-induced activation of MAPKAP kinase-2 and hsp27 phosphorylation. Under the same conditions, SB203580 also completely inhibited TNF-induced synthesis of interleukin (IL)-6 and expression of a reporter gene that was driven by a minimal promoter containing two NF-Kappa B elements. However, neither TNF-induced DNA binding of TNF-Kappa B nor TNF-induced phosphorylation of its subunits was modulated by SB203580, suggesting that NF-Kappa B is not a direct target for the p38 MAP kinase pathway. Interestingly, TNF-induced cytotoxicity was not affected by SB203580, indicating that p38 MAP kinase might be an interesting target to interfere selectively with TNF-induced gene activation.     

Growth regulation via p38 mitogen-activated protein kinase in developing liver

During normal development in the rat, hepatocytes undergo marked changes in the rate of proliferation. We have previously observed transient G(1) growth arrest at term, re-activation of proliferation immediately after birth, and a gradual transition to the quiescent adult hepatocyte phenotype after postnatal day 4. We hypothesized that these changes in proliferation are due in part to growth inhibitory effects mediated by the p38 mitogen-activated protein kinase pathway. p38 kinase activity measurements showed an inverse relationship with hepatocyte proliferation during the perinatal and postnatal transitions, whereas p38 content remained constant. Anisomycin activated the p38 pathway in fetal hepatocyte cultures while inducing growth inhibition that was sensitive to the p38 inhibitor, SB203580. Activation of p38 in these cultures, via transient transfection with a constitutively active form of its upstream kinase MKK6, also inhibited DNA synthesis as well as reducing cyclin D1 content. Transfection with inactive MKK6 did neither. Furthermore, MKK6-induced growth arrest was sensitive to SB203580. Finally, administration of SB203580 to near-term fetal rats in utero abrogated the transient hepatocyte growth arrest that occurs at term. These findings indicate a role for the p38 mitogen-activated protein kinase pathway in the physiological regulation of hepatocyte proliferation during normal development in the rat.     

Smad3 mediates transforming growth factor-beta-induced collagenase-3 (matrix metalloproteinase-13) expression in human gingival fibroblasts. Evidence for cross-talk between Smad3 and p38 signaling pathways

Transforming growth factor-beta (TGF-beta) is a potent inducer of collagenase-3 (MMP-13) gene expression in human gingival fibroblasts, and this requires activation of the p38 mitogen-activated protein kinase pathway. Here, we have constructed recombinant adenoviruses harboring genes for hemagglutinin-tagged Smad2, Smad3, and Smad4 and used these in dissecting the role of Smads, the signaling mediators of TGF-beta, in regulation of endogenous MMP-13 gene expression in human gingival fibroblasts. Adenoviral expression of Smad3, but not Smad2, augmented the TGF-beta-elicited induction of MMP-13 expression. In addition, adenoviral gene delivery of dominant negative Smad3 blocked the TGF-beta-induced MMP-13 expression in gingival fibroblasts. Co-expression of Smad3 with constitutively active MKK3b and MKK6b, the upstream activators of p38, resulted in nuclear translocation of Smad3 in the absence of TGF-beta and in induction of MMP-13 expression. The induction of MMP-13 expression by Smad3 and constitutively active mutants of MKK3b or MKK6b was blocked by specific p38 inhibitor SB203580 and by the dominant negative form of p38alpha. These results show that TGF-beta-induced expression of human MMP-13 gene in gingival fibroblasts is dependent on the activation of two distinct signaling pathways (i.e. Smad3 and p38alpha). In addition, these findings provide evidence for a novel type of cross-talk between Smad and p38 mitogen-activated protein kinase signaling cascades, which involves activation of Smad3 by p38alpha.     

Differential regulation of ARE-mediated TNFalpha and IL-1beta mRNA stability by lipopolysaccharide in RAW264.7 cells

Messenger RNA degradation is a mechanism by which eukaryotic cells regulate gene expression and influence cell growth and differentiation. Many protooncogene, cytokine, and growth factor RNAs contain AU-rich element (AREs) in the 3'untranslated regions which enable them to be targeted for rapid degradation. To investigate the mechanism of ARE-mediated RNA stability, we demonstrate the expression and regulation of TNFalpha and IL-1beta mRNAs in LPS-stimulated macrophages. TNFalpha mRNA was rapidly induced by LPS and showed short half-life at 2-h induction, whereas IL-1beta mRNA was induced slowly and had longer half-life. Electrophoretic mobility shift assays showed that the LPS-induced destabilization factor tristetraprolin (TTP) could bind to TNFalpha ARE with higher affinity than to IL-1beta ARE. HuR was identified to interact with TNFalpha ARE to exert RNA stabilization activity. The expression and phosphorylation of TTP could be activated by p38 MAPK pathway during LPS stimulation. Moreover, ectopic expression with TTP and kinases in p38 pathway followed by biochemical assays showed that the activation of p38 pathway resulted in the phosphorylation of TTP and a decrease in its RNA-binding activity. The ARE-containing reporter assay presented that the p38 signal could reverse the inhibitory activity of TTP on IL-1beta ARE but not on TNFalpha ARE. The present results indicate that the heterogeneity of AREs from TNFalpha and IL-1beta could reflect distinct ARE-binding proteins to modulate their RNA expression.