Thrombin Differentiates Normal Lung Fibroblasts to a Myofibroblast Phenotype via the Proteolytically Activated Receptor-1 and a Protein Kinase C-dependent Pathway

Abstract

Myofibroblasts are ultrastructurally and metabolically distinctive fibroblasts that express smooth muscle (SM)-α actin and are associated with various fibrotic lesions. The present study was undertaken to investigate the myofibroblast phenotype that appears after activation of normal lung fibroblasts by thrombin. We demonstrate that thrombin induces smooth muscle-α actin expression and rapid collagen gel contraction by normal lung fibroblasts via the proteolytically activated receptor-1 and independent of transforming growth factor-β pathway. Using antisense oligonucleotides we demonstrate that a decreased level of PKCε abolishes SM-α actin expression and collagen gel contraction induced by thrombin in normal lung fibroblasts. Inhibition of PKCε translocation also abolishes thrombin-induced collagen gel contraction, SM-α actin increase, and its organization by normal lung fibroblasts, suggesting that activation of PKCε is required for these effects. In normal lung fibroblasts PKCε binds to SM-α actin after thrombin treatment, but in activated fibroblasts derived from scleroderma lung they associate even in untreated cells. This suggests that SM-α actin may serve as a substrate for PKCε in lung fibroblasts when activated by thrombin. We propose that thrombin differentiates normal lung fibroblasts to a myofibroblast phenotype via a PKC-dependent pathway. Thrombin-induced differentiation of normal lung fibroblasts to a myofibroblast phenotype resembles the phenotype observed in scleroderma lung fibroblasts. Therefore, we conclude that chronic exposure to thrombin after microvascular injury leads to activation of normal lung fibroblasts and to the appearance of a myofibroblast phenotype in vivo. Our study provides novel, compelling evidence that thrombin is an important mediator of the interstitial lung fibrosis associated with scleroderma.