Transcriptional Roles of Nuclear Factor B and Nuclear Factor-Interleukin-6 in the Tumor Necrosis Factor -Dependent Induction of Cyclooxygenase-2 in MC3T3-E1 Cells

doi:10.1074/jbc.270.52.31315

Transcriptional Roles of Nuclear Factor B and Nuclear Factor-Interleukin-6 in the Tumor Necrosis Factor -Dependent Induction of Cyclooxygenase-2 in MC3T3-E1 Cells (*)

From the Department of Biochemistry, The University of Tokushima, School of Medicine, Tokushima 770, Japan

** To whom correspondence should be addressed:
Dept. of Biochemistry, The University of Tokushima, School of Medicine, Kuramoto-cho, Tokushima 770, Japan
. Tel.: 81-886-31-3111 (ext. 2220); Fax: 81-886-33-6409.

↵^¶ Present address: Dept. of Biochemistry, Michigan State University, East Lansing, MI 48824.

Abstract

When a mouse osteoblastic cell line MC3T3-E1 was cultured in the presence of tumor necrosis factor α (TNFα), the release of prostaglandin E² and the cyclooxygenase activity increased in a dose- and time-dependent manner. The increase of the enzyme activity was attributed mostly to the induction of cyclooxygenase-2 rather than cyclooxygenase-1 as judged by the inhibitory effect of NS398, Western blotting, and Northern blotting. In this system we attempted to elucidate the transcriptional regulation of the cyclooxygenase-2 gene. As examined by the luciferase assay, two positive regulatory regions (−186 to −131 and −512 to −385 base pairs) were found in the 5′-flanking promoter region of the mouse cyclooxygenase-2 gene in the TNFα-stimulated cells. The former included putative NF-IL6 (C/EBPβ) and AP2 elements, and the latter contained the NFκB motif. A DNA probe including the NF-IL6 and AP2 sites gave positive bands upon electrophoretic mobility shift assay using the nuclear extracts of MC3T3-E1 cells. The bands were supershifted by the addition of anti-NF-IL6 antibody but not by anti-AP2 antibody. A probe including the NFκB site also gave positive bands, which were supershifted by anti-NFκB p50 and p65 antibodies. Furthermore, when the motif of NF-IL6 or NFκB or both was subjected to point mutation, the luciferase activity was markedly reduced. These data suggested a potential role of both NF-IL6 and NFκB in the induction of cyclooxygenase-2 by TNFα.

Footnotes

↵^§ On leave from the Dept. of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Minami Josanjima-cho, Tokushima, 770.
↵* This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan, the Japanese Foundation of Metabolism and Disease, the Japan Foundation for Applied Enzymology, Ono Pharmaceutical Company, Kissei Pharmaceutical Company, Sankyo Company, Takeda Pharmaceutical Industry, and the Japan Research Foundation for Clinical Pharmacology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵⁽¹⁾ The abbreviations used are:

PG

prostaglandin

CRE

cyclic AMP response element

NF-IL6

nuclear factor IL-6

C/EBP

CCAAT enhancer binding protein

NFκB

nuclear factor κB

α-MEM

α-modified Eagle's minimum essential medium

TNFα

tumor necrosis factor α

bp

base pairs

IL

interleukin.
- Received August 21, 1995.
- Revision received October 6, 1995.