Induction of collagenase mRNA expression in dermal fibroblasts by IFN-alpha 2b and determination of the IFN-alpha 2b responsive element on 5'-flanking regions of collagenase promoter.
We have previously demonstrated that interferon-alpha2b (IFN-alpha2b) markedly depresses the expression of mRNA for type I procollagen in dermal fibroblasts. In the present study, the effect of various concentrations of IFN-alpha2b on the expression of collagenase mRNA and activity of 5'-flanking regions of collagenase promoter in dermal fibroblasts are presented. The results showed at least a 2-fold increase in the expression of collagenase mRNA in fibroblasts grown at either 70% confluency (40.9 +/- 4.6 vs. 18.5 +/- 1.6, n=4, p<0.05) or 95% confluency (24.7 +/- 6.7 vs. 4.5 +/- 1.6, n=4, p<0.05). The effects of IFN-alpha2b on collagenase mRNA stability and promoter activity were evaluated to determine the mechanism by which IFN-alpha2b increases the expression of collagenase mRNA. IFN-alpha2b-treated and untreated fibroblasts were treated with alpha-amanitin to arrest collagenase mRNA transcription, and total RNA was then harvested at 0, 3, 6, 12, and 24 h. The decay curves of collagenase mRNA as a function of time showed a greater rate of degradation for collagenase mRNA in IFN-alpha2b-treated cells relative to untreated control cells. This difference was more pronounced in cells treated with alpha-amanitin at either 12 or 24 h. To determine the regions of the collagenase promoter that might function as IFN-alpha2b responsive elements, eight different fragments of the collagenase promoter, -518, -300, -171, -161, -127, -91, -74, and -66 to +63 nucleotide (nt), were constructed in a chloramphenicol acetyltransferase (CAT) expression vector. The results of CAT activity of cells transfected with these construct identified three constructs, 171/+63, -161/+63, and -127/+63, as being responsive to IFN-alpha2b treatment in dermal fibroblasts. The CAT activity was increased 279%, 163%, and 261% in -171/+63, -161/+63, and -127/+63-transfected fibroblasts, respectively, in response to IFN-alpha2b treatment relative to untreated control. No significant increase in CAT activity was found in cells transfected with the other constructs of the collagenase promoter. A time response experiment showed a marked increase in CAT activity of cells transfected with either 127/+63 or -171/63 constructs within 6-12 hr of IFN-alpha2b treatment. In conclusion, IFN-alpha2b significantly increases the expression of collagenase mRNA in dermal fibroblasts probably through stimulation of the -127/-91 region of the collagenase promoter. Thus, this region may function as an IFN-alpha2b responsive element on collagenase promoter.
Ghahary
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Wound Healing Research Group, Department of Surgery, Division of Critical Care, University of Alberta, Edmonton, Alberta, Canada, T6G 2B7. agahary@ualberta.ca
Tredget
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Shen
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D020121:5' Untranslated Regions; D002478:Cells, Cultured; D017364:Collagenases / Q000096:biosynthesis / Q000235:genetics*; D020405:Dermis / Q000166:cytology / Q000201:enzymology*; D004790:Enzyme Induction / Q000235:genetics / Q000276:immunology; D004795:Enzyme Stability / Q000235:genetics / Q000276:immunology; D005347:Fibroblasts / Q000201:enzymology* / Q000276:immunology; D015971:Gene Expression Regulation, Enzymologic / Q000276:immunology; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000235:genetics* / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000276:immunology; D020871:RNA Stability / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis*; D011994:Recombinant Proteins; D020218:Response Elements / Q000276:immunology
D020121:5' Untranslated Regions; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D012333:RNA, Messenger; D011994:Recombinant Proteins; D017364:Collagenases