The Proteinase-activated Receptor 2 Is Induced by Inflammatory Mediators in Human Endothelial Cells

doi:10.1074/jbc.271.25.14910

The Proteinase-activated Receptor 2 Is Induced by Inflammatory Mediators in Human Endothelial Cells

COMPARISON WITH THE THROMBIN RECEPTOR*

From the ^‡ Division of Molecular Neurobiology, The Wallenberg Laboratory, Lund University, Sweden and
^¶ COR Therapeutics Inc., San Francisco, California 94080

^§ To whom correspondence should be addressed:
Division of Molecular Neurobiology, The Wallenberg Neurocenter, Lund University, Sōlvegatan 17, S-22362 Lund, Sweden.
Tel.: 46 46 2220586; Fax: 46 46 2220568; E-mail sverker.nystedt{at}mphy.lu.se

Abstract

The proteinase-activated receptor 2 (PAR-2) belongs to the family of seven transmembrane region receptors, and, like the related thrombin receptor, it is activated by specific proteolytic cleavage of its extracellular amino terminus. It is not known which proteinase is the physiological activator of the PAR-2, but candidates can be found among the enzymes involved in the inflammatory cascade systems. Here, we have studied the effects of various mediators on the expression of the PAR-2 and the thrombin receptor in cultured human umbilical vein endothelial cells. Stimulation with the cytokines tumor necrosis factor α or interleukin-1 α as well as bacterial lipopolysaccharide elevated the expression of PAR-2 in a dose-dependent manner. The time course of induction after cytokine stimulation was similar to those published for the adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. After 20 h of stimulation, PAR-2 mRNA and protein levels were increased to 5–10-fold basal values, and, in the continued presence of tumor necrosis factor α, PAR-2 mRNA expression was found to remain elevated for up to 4 days. In contrast, the thrombin receptor gene was not induced by any of these inflammatory mediators. The responses to phorbol ester treatment also differed between the two genes. Thrombin receptor mRNA levels decreased steadily up to 20 h, whereas PAR-2 mRNA levels first rose to about 3-fold basal values at 4 h before decreasing again. Cell surface protein levels of both receptors were decreased after 20 h of phorbol ester stimulation. Elevating intracellular cAMP levels by treatment with forskolin resulted in decreased expression of both receptors, and inhibition of cAMP degradation appeared to blunt the cytokine-induced increase in PAR-2 expression. The induction of the PAR-2 by cytokine treatment supports the concept of PAR-2 involvement in the acute inflammatory response.

Footnotes

↵* This work was supported by the Swedish Medical Research Council (B96-13X-09467), the Alfred Österlund Trust, and the Medical Faculty, Lund University. 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

FACS

fluorescence-activated cell sorting

HUVE

human umbilical vein endothelial

IBMX

isobutylmethylxanthine

ICAM-1

intercellular adhesion molecule-1

IL-1

interleukin-1

PAR-2

proteinase-activated receptor 2

PMA

phorbol 12-myristate 13-acetate

VCAM-1

vascular cell adhesion molecule-1

ANOVA

analysis of variance

GAPDH

glyceraldehyde phosphodehydrogenase

LPS

lipopolysaccharide.
↵² S. Nystedt, V. Ramakrishnan, and J. Sundelin, unpublished observations.
↵³ Anna-Karin Larsson et al., manuscript in preparation.
↵⁴ C. Nilsson, S. Nystedt, and J. Sundelin, unpublished results.
- Received January 22, 1996.
- Revision received April 1, 1996.