Regulation of the rat interstitial collagenase promoter by IL-1β, c-Jun, and ras-dependent signaling in growth plate chondrocytes

• Published in: Journal of cellular biochemistry 1997-10, Vol.67 (1), p.92-102
• Main Authors: Grumbles, R.M., Shao, L., Jeffrey, J.J., Howell, D.S.
• Format: Article
• Language: English
• Subjects: Animals; chondrocyte; Chondrocytes - enzymology; Chondrocytes - metabolism; collagenase promoter; Collagenases - genetics; DNA - metabolism; DNA binding; Gene Expression Regulation, Enzymologic - drug effects; Gene Expression Regulation, Enzymologic - physiology; Growth Plate - cytology; Interleukin-1 - pharmacology; interleukin-1β; Male; Matrix Metalloproteinase 1; Oligonucleotides, Antisense - pharmacology; Promoter Regions, Genetic - genetics; Proto-Oncogene Proteins c-jun - genetics; Proto-Oncogene Proteins c-jun - physiology; ras Proteins - physiology; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins; RNA, Messenger - analysis; Signal Transduction - genetics; Transfection; TRE
• ISSN: 0730-2312; 1097-4644
• DOI: 10.1002/(SICI)1097-4644(19971001)67:1<92::AID-JCB10>3.0.CO;2-M

In an attempt to better define molecular influences on rat interstitial collagenase gene expression in cartilage, the promoter function was characterized using transient transfection assay, electrophoresis mobility shift assay, and genetic analysis in isolated growth plate chondrocytes. Data from 5′‐flanking deletion and selected mutations suggest that multiple cis elements in both the proximal and distal regions of the promoter were important in the regulation of promoter activity. A proximal tumor response element (TRE) was shown to be necessary for basal and interleukin (IL)‐1β–inducible reporter gene activity. Cells stimulated by IL‐1β (1 ng/ml; 18 h) had elevated TRE binding activity, and one of the factors involved was identified as the nuclear protein, c‐Jun. Indeed, c‐Jun directed antisense oligonucleotides reduced rat interstitial collagenase mRNA. A sense oligonucleotide was ineffective. Regulation of promoter activity was susceptible to Ras‐dependent signaling as expression of dominant negative mutant of Ras kinase (pZIP‐RasN17) reduced reporter gene activity. In a comparison of proximal promoter reporter plasmid activity between proliferative and hypertrophic cells, inhibition of Ras‐dependent signaling was less effective in the later cell type. This study suggests that the activation of nuclear binding proteins that bind TRE may be a common event with IL‐1β regulation. Moreover, these data suggest that the regulation of rat interstitial collagenase gene expression is a combinatorial process and multiple cis‐acting regulatory sites may interact to exert different effects dependent on the stage of chondrocyte differentiation. J. Cell. Biochem. 67:92–102, 1997. Published 1997 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.