Expression of tgf-β receptors in corneal myofibroblasts and fibroblasts

Purpose: We have reported that the differentiation of myofibroblasts from fibroblasts depends upon at least two factors: TGF-β and loss of cell-cell contact (PNAS, 1996; 93:4219-4223). In the current study, we examine whether the effect of TGF-β is the result of increased expression of TGF-β receptors and whether this is correlated with loss of cell-cell contact after plating fibroblasts at low density. Methods: Myofibroblast cultures, defined by smooth muscle a-actin (SM a-actin) protein expression, were prepared b/ plating corneal fibroblasts at low density (6 cells/mm2) with 10% FBS in DME-FI2 Fibroblast cultures were prepared by plating cells at high density (600 cells/mm2:. Total RNA was extracted from 70-80 % confluent cultures. Using RT-PCR we evaluated the abundance of mRNA for TGF-β receptor l, TGF-β receptor II and SM a-actin. These were compared to the abundance of mRNA of the house-keeping gene, GAPDH. Quantitative Northern blot analysis was performed. Surface expression of TGF-β receptors was determined by a radioligand assay. Results: SM a-actin mRNA was more abundant in myofibroblasts than in fibroblastï indicating transcriptional regulation of the phenotype. We also found greater expression of TGF-β receptor I and II mRNA in the myofibroblasts than in the fibroblasts. Conclusions: SM a-actin, previously characterized by Western blot analysis, and immunocytochemistry, is regulated at the transcriptional level in corneal myofibroblasts which differentiate from low density cultures of fibroblasts. The TGF-β I and II receptors are similarly up-regulated in myofibroblast cultures. We suggest tnat associated with decreased cell-cell contact in low density cultures, there is increased expression of TGF-β receptors which may mediate TGF-β induction of myofibroblast differentiation.