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Regulation of c-Jun N-terminal Kinase and p38 Kinase Pathways in Endothelial Cells

Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University, Baltimore, Maryland; and Children's Hospital and Harvard Medical School, Boston, Massachusetts

Address correspondence to: Raj Wadgaonkar, Ph.D., Division of Pulmonary and Critical Care Medicine, Johns Hopkins University, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. E-mail: raj.wadgaonkar{at}downstate.edu

The rapid and transient induction of E-selectin gene expression by inflammatory tumor necrosis factor (TNF)– in endothelial cells is mediated by signaling pathways which involve c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) kinase pathways. To explore this regulation, we first observed that in the continuous presence of cytokine TNF, activation of JNK-1 in both nuclear and cytoplasmic compartments peaked at 15–30 min, with activity returning to uninduced levels by 60 min. Phosphorylation of both the p38 kinase and its molecular target, the nuclear transcription factor, activating transcription factor–2, were transient after TNF- or interleukin (IL)-1ß induction. However, cycloheximide treatment prolonged the TNF-–induced JNK-1 kinase activity beyond 60 min, suggesting that protein synthesis is required to limit this signaling cascade. We investigated the possible role of the dual-specificity phosphatases MAPK phosphatase (MKP)-1 and MKP-2 in limiting cytokine-induced MAPK signaling. Maximum induction of MKP-1 mRNA and nuclear protein levels by TNF- or IL-1ß were noted at 60 min and their expression correlated with the termination of JNK kinase activity, whereas nuclear levels of MKP-2 were not significantly affected by treatment with TNF- or IL-1ß. Transient overexpression of MKP-1 demonstrated significant specific inhibition of E-selectin promoter activity consistent with a regulatory role for dual-specificity phosphatases. Inhibition of MKP-1 expression through the use of small interfering RNAs prolonged the cytokine-induced p38 and JNK kinase phosphorylation. Our results suggest that endogenous inhibitors of the MAPK cascade, such as the dual-specificity phosphatases like MKP-1 may be important for the postinduction repression of MAPK activity and E-selectin transcription in endothelial cells. Thus, these inhibitors may play an important role in limiting the inflammatory effects of TNF- and IL-1ß.

Abbreviations: activating transcription factor, ATF • cAMP response element, CRE • extracellular signal-regulated kinase, ERK • glyceraldehyde-3-phosphate dehydrogenase, GAPDH • green fluorescence protein, GFP • human umbilical vein endothelial cells, HUVEC • human VH-1 homologue, hVH • c-Jun N-terminal kinase, JNK • mitogen-activated protein kinase, MAPK • MAPK phosphatase, MKP • nuclear factor–B, NF-B • scrambled RNA sequence, Scr-RNA • sodium dodecyl sulfate, SDS • serine, Ser • silencing or inhibitory RNA, SiRNA • threonine, Thr • thymidine kinase, TK • tumor necrosis factor, TNF • ultraviolet, UV • vaccinia virus phosphatase, VH-1

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