Tumor Necrosis Factor α-Induced E-selectin Expression Is Activated by the Nuclear Factor-κB and c-JUN N-terminal Kinase/p38 Mitogen-activated Protein Kinase Pathways

doi:10.1074/jbc.272.5.2753

Tumor Necrosis Factor α-Induced E-selectin Expression Is Activated by the Nuclear Factor-κB and c-JUN N-terminal Kinase/p38 Mitogen-activated Protein Kinase Pathways*

From the^‡ Vascular Research Division, Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115 and
the** Howard Hughes Medical Institute, and Program in Molecular Medicine, Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605

^§§ Established Investigator of the American Heart Association. To whom correspondence should be addressed:
Dept. of Pathology, Brigham and Women's Hospital, 221 Longwood Ave., Boston, MA 02115.
Tel. 617-732-5990; Fax: 617-278-6990; E-mail: tcollins{at}bustoff.bwh.harvard.edu

↵^§ Current address: ProScript, 38 Sidney St., Cambridge, MA 02138.
↵^∥ Current address: Genetics Institute, 87 Cambridge Park Dr., Cambridge, MA 02140.

Abstract

E-selectin expression by endothelium is crucial for leukocyte recruitment during inflammatory responses. Transcriptional regulation of the E-selectin promoter by tumor necrosis factor α (TNFα) requires multiple nuclear factor-κB (NF-κB) binding sites and a cAMP-responsive element/activating transcription factor-like binding site designated positive domain II (PDII). Here we characterize the role of the stress-activated family of mitogen-activated protein (MAP) kinases in induced expression of this adhesion molecule. By UV cross-linking and immunoprecipitation, we demonstrated that a heterodimer of transcription factors ATF-2 and c-JUN is constitutively bound to the PDII site. TNFα stimulation of endothelial cells induces transient phosphorylation of both ATF-2 and c-JUN and induces marked activation of the c-JUN N-terminal kinase (JNK1) and p38 but not extracellular signal-regulated kinase (ERK1). JNK and p38 are constitutively present in the nucleus, and DNA-bound c-JUN and ATF-2 are stably contacted by JNK and p38, respectively. MAP/ERK kinase kinase 1 (MEKK1), an upstream activator of MAP kinases, increases E-selectin promoter transcription and requires an intact PDII site for maximal induction. MEKK1 can also activate NF-κB -dependent gene expression. The effects of dominant interfering forms of the JNK/p38 signaling pathway demonstrate that activation of these kinases is critical for cytokine-induced E-selectin gene expression. Thus, TNFα activates two signaling pathways, NF-κB and JNK/p38, which are both required for maximal expression of E-selectin.

Footnotes

↵^¶ These authors contributed equally to this work.
↵^‡‡ Investigator of the Howard Hughes Medical Institute.
↵* This work was supported by Research Grants HL 35716, HL 45462, and PO1 HL 36028 from the National Institutes of Health (to T. C.) and the National Cancer Institute Grants CA58396 and CA65861 (to R. J. D.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

TNFα

tumor necrosis factor-α

BAEC

bovine aortic endothelial cells

HUVEC

human umbilical vein endothelial cell

JNK

c-JUN N-terminal kinase, IκBα, inhibitor κB-α

MAP

mitogen activated protein kinase

MEKK

MAP kinase-kinase-kinase, MKK, MAP kinase-kinase

MKP

MAP kinase phosphatase

NF-κB

nuclear factor-κB

PD

positive regulatory domain

PMSF

phenylmethylsulfonyl fluoride

DTT

dithiothreitol

CRE/ATF

cAMP response element/activating transcription factor element

PP2A

protein phosphatase 2A.
↵² M. Z. Whitley and T. Collins, unpublished observations.
↵³ J. W. Pierce, J. Best, and T. Collins, unpublished data.
↵⁴ S. Gupta and R. Davis, unpublished observations.
- Received August 29, 1996.
- Revision received October 10, 1996.