Nucleic Acids Research, 1994, Vol. 22, No. 22 4818-4827
© 1994
MOLECULAR BIOLOGY |
Regulation of collagenase gene expression by IL–1ß requires transcriptional and post-transcriptional mechanisms
1Department of Medicine Hanover, NH 03755, USA 2Department of Biochemistry, Dartmouth Medical School Hanover, NH 03755, USA
*To whom correpsondence should be addressed
Received April 22, 1994. Revised September 23, 1994. Accepted September 23, 1994.
lnterleukin-1ß is believed to contribute to the pathophysiology of rheumatoid arthritis by activating collagenase gene expression. We have used a cell culture model of rabbit synovial f ibroblasts to examine the molecular mechanisms of IL-1ß-mediated collagenase gene expression. Stimulation of rabbit synovial fibroblasts with 10 ng/ml recombinant human IL-1ß resulted in a 20-fold increase in collagenase mRNA by 12 h. Transient transfection studies using collagenase promoter – CAT constructs demonstrated that proximal sequences responded poorly to IL-1ß, possibly due to insufficient activation of AP-1 by this cytokine. More distal sequences were required for IL-1ß responsiveness, with a 4700 bp construct showing {small tilde}5-fold induction above control. To examine posttranscriptional mechanisms, transcript from a human collagenase cDNA was constitutively produced by the simian virus 40 early promoter. IL-1ß stabilized the constitutively expressed human transcript. Furthermore, mutation of the ATTTA motifs in the 3' untranslated region of the human gene also stabilized the transcript. Finally, the rabbit collagenase 3' untranslated region destabilized a constitutively transcribed chloramphenicol acetyltransferase transcript. These data indicate that in addition to activating transcription, IL-1ß increases collagenase transcript stability by reversing the destabilizing effects of sequences in the 3' untranslated region.
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