Regulation of collagen synthesis and messenger RNA levels in normal and hypertrophic scar fibroblasts in vitro by interferon alfa‐2b

Abstract

Hypertrophic scars, which commonly occur after thermal and traumatic injury of the skin, are a fibroproliferative disorder of the dermal matrix wherein components of the inflammatory process, including the fibrotic growth factor, transforming growth factor-beta, appear to activate dormant fibroblasts leading to cellular proliferation and excessive matrix synthesis. To investigate the potential beneficial role and mechanism of interferon alfa-2b in controlling excessive collagen production in hypertrophic scar, we measured dose response, time of onset, and duration of action in hypertrophic scar fibroblasts in vitro and compared them with those of site-matched normal fibroblasts obtained from four patients after thermal injury. Interferon alfa-2b reduced collagen protein synthesis and type I messenger RNA levels in both hypertrophic scar and normal fibroblasts after treatment, but these changes were apparent only after approximately 72 hours. Significant reductions in collagen synthesis occurred in four pairs of normal and hypertrophic scar fibroblasts (p < 0.05), accompanied by significant reductions in type I (p < 0.05) but not type III procollagen messenger RNA. Hypertrophic scar fibroblasts recovered completely from the effects of interferon alfa-2b on procollagen type I messenger RNA within 48 hours of cessation of treatment in contrast to normal skin fibroblasts, in which the reduction in type I procollagen messenger RNA by interferon alfa-2b persisted beyond 72 hours after treatment. These data suggest that interferon alfa-2b reduces collagen synthesis in both normal and hypertrophic fibroblasts but the hypertrophic fibroblast may remain less sensitive to its effects.

Induction of Collagenase mRNA Expression in Dermal Fibroblasts by IFN-α2b and Determination of the IFN-α2b Responsive Element on 5′-Flanking Regions of Collagenase Promoter

“…(10)(11)(12)(13) The results of our previous studies also showed that total collagen synthesis and mRNA expression for fibronectin are reduced in response to IFN-a2b in dermal fibroblasts. (21,22) As our findings and those of others (10)(11)(12)(13) suggest that these cytokines can be used as therapeutic agents to treat FPD, the expression of collagenase, as the other side of the equation involved in collagen metabolism, in response to these cytokines needs to be investigated. Duncan and Berman (27) showed an increase in collagenase activity in conditioned medium after treatment with either IFN-a or IFN-g.…”

“…The response of postburn HSc fibroblasts to these cytokines appears similar to that of normal dermal fibroblasts obtained from the same patient. (20,21) We have also provided evidence that IFN-a2b significantly decreases the expression of fibronectin mRNA in human HSc and normal dermal fibroblasts. (21,22) These findings and those of others (10)(11)(12)(13) suggest that these cytokines can be used as therapeutic agents to normalize the accumulation of collagen in FPD.…”

“…(20,21) We have also provided evidence that IFN-a2b significantly decreases the expression of fibronectin mRNA in human HSc and normal dermal fibroblasts. (21,22) These findings and those of others (10)(11)(12)(13) suggest that these cytokines can be used as therapeutic agents to normalize the accumulation of collagen in FPD. As the other side of the equation involved in collagen metabolism, in this study the effects of various concentrations of IFN-a2b on the expression of collagenase mRNA and activity of eight different regions of collagenase promoter in dermal fibroblasts were examined.…”

“…(21,22) Cultures at passages 3-7 were used for IFN-a2b treatment, analysis of mRNA for collagenase and 18S ribosomal RNA, promoter gene transfection, and CAT assay.…”

“…(21) Extraction of total RNA from each individual cell lysate was performed using the GITC/CsCl procedure of Chirgwin et al (24) Total RNA was separated by electrophoresis and blotted onto nitrocellulose filters, (22) which were baked under vacuum for 2 h at 80°C and prehybridized in a prehybridization solution for 4-6 h at 42°C. Hybridization was performed in the same solution at 42°C for 16-20 h using cDNA probes for either human collagenase or 18S ribosomal RNA.…”