Endothelial Cell Inflammatory Responses to Tumor Necrosis Factor α

doi:10.1074/jbc.271.22.13094

Endothelial Cell Inflammatory Responses to Tumor Necrosis Factor α

CERAMIDE-DEPENDENT AND -INDEPENDENT MITOGEN-ACTIVATED PROTEIN KINASE CASCADES*

From the Departments of ^¶ Medicine and
^‡ Pathology, the
Nora Eccles Harrison Cardiovascular Research and Training Institute, and the
^∥ Program in Human Molecular Biology and Genetics, University of Utah, Salt Lake City, Utah 84112

^″ To whom correspondence should be addressed:
Bldg. 500, CVRTI, University of Utah, Salt Lake City, UT 84112.
Tel.: 801-581-8183; Fax: 801-581-3128; E-mail: mcintyre{at}cvrti.utah.edu

Abstract

Ceramide generation by stimulated sphingomyelinase activity has been implicated in tumor necrosis factor α (TNF) signaling of apoptosis and differentiation. We examined the role of ceramide in a major action of TNF: the initiation of inflammatory events. Sphingomyelinase C at high levels induced inflammatory protein expression in endothelial cells resulting in leukocyte adhesion, but the pattern of induction of adhesion molecules (E-selectin, ICAM-1, VCAM-1) and cytokines (interleukins 6 and 8) differed from that induced by TNF. TNF induced only a small increase in ceramide: using lower doses of sphingomyelinase to mimic this we found that small amounts of ceramide did not induce protein expression, but still rapidly activated Raf-1, mitogen-activated protein/extracellular regulated kinase (ERK) kinase (MEK) and ERKs. TNF additionally caused rapid p38 and JNK-1 mitogen-activated protein kinase activation and efficient NF-κB translocation, which could not be achieved even by high levels of ceramide. Thus activation of the ERK cascade alone is an incomplete endothelial cell stimulus, and the TNF receptor generates at least two signals: Raf-1 activation, which could be ceramide-dependent; and ceramide-independent efficient NF-κB translocation and activation of p38 and JNK-1 mitogen-activated kinases.

Footnotes

↵* This work was supported by the Nora Eccles Treadwell Foundation and Grant HL 50513 (SCOR) from the National Institutes of Health. The Flow Cytometry Facility was supported by National Institutes of Health NCI Grant CCSG 3P30 CA 42014-09. 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:

TNF

tumor necrosis factor α

MAP

mitogen-activated protein

IL

interleukin

ELISA

enzyme-linked immunosorbent assay

PMSF

phenylmethylsulfonyl fluoride

DTT

dithiothreitol

PAGE

polyacrylamide gel electrophoresis

PMA

phorbol 12-myristate 13-acetate

ICAM-1

intercellular adhesion molecule 1

VCAM-1

vascular cell adhesion molecule-1

MEK

MAP/ERK kinase

ERK

extracellular regulated kinase.
↵² M. Feldhaus, G. A. Zimmerman, S. M. Prescott, and T. M. McIntyre, unpublished results.
- Received November 17, 1995.
- Revision received March 1, 1996.