Induction of Collagenase mRNA Expression in Dermal Fibroblasts by IFN- α 2b and Determination of the IFN- α 2b Responsive Element on 5′-Flanking Regions of Collagenase Promoter

Abstract

We have previously demonstrated that interferon-α2b (IFN-α2b) markedly depresses the expression of mRNA for type I procollagen in dermal fibroblasts. In the present study, the effect of various concentrations of IFN-α2b 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-α2b on collagenase mRNA stability and promoter activity were evaluated to determine the mechanism by which IFN-α2b increases the expression of collagenase mRNA. IFN-α2b-treated and untreated fibroblasts were treated with α-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-α2b-treated cells relative to untreated control cells. This difference was more pronounced in cells treated with α-amanitin at either 12 or 24 h. To determine the regions of the collagenase promoter that might function as IFN-α2b 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-α2b 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-α2b 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-α2b treatment. In conclusion, IFN-α2b 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-α2b responsive element on collagenase promoter.