Expression of fibronectin messenger RNA in hypertrophic and normal dermal tissues and in vitro regulation by interferon alfa‐2b

Abstract

After severe thermal injury, hypertrophic scarring which is associated with accumulation of extracellular matrix proteins including fibronectin, frequently develops. We have recently demonstrated that interferon alfa-2b significantly reduces the level of type 1 procollagen messenger RNA expressed by both hypertrophic and normal dermal fibroblasts. In this report, we provide evidence that this cytokine also significantly decreases the expression of fibronectin messenger RNA in human hypertrophic scar and normal dermal fibroblasts. Four dermal fibroblast cell strains were established in cell culture from four human postburn hypertrophic scar tissues with the use of normal dermal fibroblasts from the same patients as controls. These cells were then treated with 2000 U/ml interferon alfa-2b in culture medium at various times. The results of Northern analysis of interferon-treated dermal fibroblasts indicate that this cytokine reduced the expression of fibronectin messenger RNA as early as 12 hours after treatment and reached its lowest level (24% relative to untreated fibroblasts) after 96 hours. When the expression of fibronectin messenger RNA was quantified by densitometry for each individual paired cell strain, a differential response to interferon treatment was found among cell strains. The level of fibronectin messenger RNA expression decreased from 17.2% to 69% in hypertrophic scar fibroblasts and 47% to 83.7% in normal fibroblasts relative to that of untreated control values. Although this decrease was less pronounced in normal fibroblasts than in hypertrophic scar fibroblasts, this reduction was significant in both interferon alfa-2b treated hypertrophic scar fibroblasts (6.39 +/- 0.71 versus 2.88 +/- 0.9, n = 4, p < 0.05) and normal cells compared with untreated controls (5.47 +/- 0.89 versus 3.64 +/- 0.99, n = 4, p < 0.05) as assessed with Student's paired t test. Rehybridization of the RNA blot prepared from interferon alfa-2b treated and untreated hypertrophic scar fibroblasts with a complementary DNA for the tissue inhibitor of metalloproteinase type 2 gelatinase inhibitor showed no significant changes in abundance of this transcript. This result suggests that this cytokine selectively suppresses the expression of fibronectin messenger RNA and that this reduction is not due to RNA loading. A dot blot analysis of total RNA extracted from these tissues was carried out to compare the expression of fibronectin messenger RNA between human hypertrophic scar tissues and normal dermis obtained from the same patients. The blot was initially hybridized with fibronectin complementary DNA and subsequently with a complementary DNA for the tissue inhibitor of metalloproteinase type 2 to correct for RNA loading. When the ratio of fibronectin to tissue inhibitor or metalloproteinase type 2 messenger RNA expression for each hypertrophic scar tissue was compared with its normal control, this ratio was fourfold higher in human hypertrophic scar tissues relative to normal controls. In contrast, the expression of this message in cultured hypertrophic scar fibroblasts was not significantly different from that in normal fibroblasts. The results of this study suggest that hypertrophic scarring developing after thermal injury is associated with an overexpression of fibronectin messenger RNA, and interferon alfa-2b may be of therapeutic value to down-regulate the expression of this transcript.