Several Dual Specificity Phosphatases Coordinate to Control the Magnitude and Duration of JNK Activation in Signaling Response to Oxidative Stress
ABSTRACT Mitogen-activated protein kinases (MAPKs) are important mediators that integrate signaling from upstream pathways in response to various environmental cues. In order to control appropriate gene expression through phosphorylation of transcription factors, the activity of MAPKs must be tightly regulated by the actions coordinated between protein kinases and phosphatases. In this study, we explore the underlying mechanism through which the oxidative stress-activated c-Jun N-terminal kinases (JNKs), members of MAPKs, are regulated by dual specificity phosphatases (DUSPs). DUSPs are a group of enzymes that belong to the superfamily of protein-tyrosine phosphatases. They are able to recognize phospho-Ser/Thr and phospho-Tyr residues in substrates. Using quantitative real time PCR, we found that stimulation of human embryonic kidney 293T cells with H(2)O(2) or xanthine/xanthine oxidase led to inducible expression of multiple DUSPs. We used RNA interference to characterize the functional role of these DUSPs and found rapid and transient induction of DUSP1 and DUSP10 to be essential for determining the appropriate magnitude of JNK activation in response to oxidative stress. The transcription factor ATF2, which is phosphorylated and activated by JNK, is a critical mediator for inducible expression of DUSP1 and DUSP10 in this signaling pathway. We further demonstrated that DUSP4 and DUSP16, both showing significant late phase induction, dephosphorylate JNK effectively, causing the down-regulation of the signaling cascade. Thus, this study provides new insights into the role of several DUSPs that coordinate with each other to control the magnitude and duration of JNK activity in response to oxidative stress.
- SourceAvailable from: Shigeo Muro[Show abstract] [Hide abstract]
ABSTRACT: Chronic obstructive pulmonary disease (COPD) is characterized by airflow obstruction and persistent inflammation in the airways and lung parenchyma. Oxidative stress contributes to the pathogenesis of COPD. Interleukin (IL)-32 expression has been reported to increase in the lung tissue of patients with COPD. Here, we show that IFNγ upregulated IL-32 expression and that oxidative stress augmented IFNγ-induced-IL-32 expression in airway epithelial cells. We further investigated transcriptional regulation responsible for IFNγ induced IL-32 expression in human airway epithelial cells. Human bronchial epithelial (HBE) cells were stimulated with H2O2 and IFNγ, and IL-32 expression was evaluated. The cell viability was confirmed by MTT assay. The intracellular signaling pathways regulating IL-32 expression were investigated by examining the regulatory effects of MAPK inhibitors and JAK inhibitor after treatment with H2O2 and IFNγ, and by using a ChIP assay to identify transcription factors (i.e. c-Jun, CREB) binding to the IL-32 promoter. Promoter activity assays were conducted after mutations were introduced into binding sites of c-Jun and CREB in the IL-32 promoter. IL-32 expression was also examined in HBE cells in which the expression of either c-Jun or CREB was knocked out by siRNA of indicated transcription factors. There were no significant differences of cell viability among groups. After stimulation with H2O2 or IFNγ for 48 hours, IL-32 expression in HBE cells was increased by IFNγ and synergistically upregulated by the addition of H2O2. The H2O2 augmented IFNγ induced IL-32 mRNA expression was suppressed by a JNK inhibitor, but not by MEK inhibitor, p38 inhibitor, and JAK inhibitor I. Significant binding of c-Jun and CREB to the IL-32 promoter was observed in the IFNγ + H2O2 stimulated HBE cells. Introducing mutations into the c-Jun/CREB binding sites in the IL-32 promoter prominently suppressed its transcriptional activity. Further, knocking down CREB expression by siRNA resulted in significant suppression of IL-32 induction by IFNγ and H2O2 in HBE cells. IL-32 expression in airway epithelium may be augmented by inflammation and oxidative stress, which may occur in COPD acute exacerbation. c-Jun and CREB are key transcriptional factors in IFNγ and H2O2 induced IL-32 expression.Respiratory research 03/2012; 13(1):19. DOI:10.1186/1465-9921-13-19 · 3.38 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: In the diabetic kidney, stimulation of mitogen-activated protein kinases (MAPKs) leads to extracellular matrix protein synthesis. In the proximal tubule, angiotensin-(1-7) [Ang-(1-7)] blocks activation of MAPKs by angiotensin II. We studied the effect of Ang-(1-7) on signalling responses in LLC-PK(1) cells in normal (5 mM) or high (25 mM) glucose. The p38 MAPK was assayed by immunoblot, Src homology 2-containing protein-tyrosine phosphatase-1 (SHP-1) activity was measured after immunoprecipitation, cell protein synthesis was determined by [(3)H]-leucine incorporation and transforming growth factor-beta1 (TGF-beta1), fibronectin and collagen IV were assayed by immunoblots and/or ELISA. High glucose stimulated p38 MAPK. This response was inhibited by Ang-(1-7) in a concentration-dependent fashion, an effect reversed by the receptor Mas antagonist A-779. Ang-(1-7) increased SHP-1 activity, via the receptor Mas. An inhibitor of tyrosine phosphatase, phenylarsine oxide, reversed the inhibitory effect of Ang-(1-7) on high glucose-stimulated p38 MAPK. Ang-(1-7) inhibited high glucose-stimulated protein synthesis, and blocked the stimulatory effect of glucose on TGF-beta1. Conversely, Ang-(1-7) had no effect on glucose-stimulated synthesis of fibronectin or collagen IV. These data indicate that in proximal tubular cells, binding of Ang-(1-7) to the receptor Mas stimulates SHP-1, associated with the inhibition of glucose-stimulated p38 MAPK. Ang-(1-7) selectively inhibits glucose-stimulated protein synthesis and TGF-beta1. In diabetic nephropathy, Ang-(1-7) may partly counteract the profibrotic effects of high glucose.Nephrology Dialysis Transplantation 02/2009; 24(6):1766-73. DOI:10.1093/ndt/gfn736 · 3.49 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Critical tumor suppression pathways in brain tumors have yet to be fully defined. Along with mutational analyses, genome-wide epigenetic investigations may reveal novel suppressor elements. Using differential methylation hybridization, we identified a CpG-rich region of the promoter of the dual-specificity mitogen-activated protein kinase phosphatase-2 gene (DUSP4/MKP-2) that is hypermethylated in gliomas. In 83 astrocytic gliomas and 5 glioma cell lines examined, hypermethylation of the MKP-2 promoter was found to occur relatively more frequently in diffuse or anaplastic astrocytomas and secondary glioblastomas relative to primary glioblastomas. MKP-2 hypermethylation was associated with mutations in TP53 and IDH1, exclusive of EGFR amplification, and with prolonged survival of patients with primary glioblastoma. Expression analysis established that promoter hypermethylation correlated with reduced expression of MKP-2 mRNA and protein. Consistent with a regulatory role, reversing promoter hypermethylation by treating cells with 5-aza-2'-deoxycytidine increased MKP-2 mRNA levels. Furthermore, we found that glioblastoma cell growth was inhibited by overexpression of exogenous MKP-2. Our findings reveal MKP-2 as a common epigenetically silenced gene in glioma, the inactivation of which may play a significant role in glioma development.Cancer Research 02/2010; 70(4):1689-99. DOI:10.1158/0008-5472.CAN-09-3218 · 9.28 Impact Factor