Contrary effects of sphingosine-1-phosphate on expression of α-smooth muscle actin in transforming growth factor β1-stimulated lung fibroblasts

Transforming growth factor-β1 (TGFβ1) plays a pivotal role in fibrosis in various organs including the lung. Following pulmonary injury, TGFβ1 stimulates conversion of fibroblasts to myofibroblasts that are mainly characterized by up-regulation of α-smooth muscle actin (αSMA) expression, and the resulting excess production of extracellular matrix proteins causes fibrosis with loss of alveolar function. The present study was undertaken to define the role of the sphingosine-1-phosphate (S1P) pathway in TGFβ1-induced expression of αSMA in human fetal lung fibroblasts, HFL1 cells. Analysis of mRNA revealed the existence of S1P1, S1P2, and S1P3 receptor mRNAs. Treatment with TGFβ1 increased sphingosine kinase (SphK) activity and S1P3 receptor mRNA at 24 h after stimulation, and pharmacological data showed the involvement of sphingomyelinase, SphK, and S1P3 receptor in the TGFβ1-induced up-regulation of αSMA with and without serum. Treatment with pertussis toxin and S1P1 receptor antagonist W146 enhanced αSMA expression by TGFβ1/serum, and S1P decreased and increased αSMA levels with and without serum, respectively. TGFβ1 increased cyclooxygenase-2 expression in a manner dependent on serum and the sphingomyelinase/SphK pathway, and the response was decreased by pertussis toxin. Prostaglandin E2, formed by TGFβ1/serum stimulation, decreased the TGFβ1-induced expression of αSMA via EP prostanoid receptor. These data suggest that S1P formed by TGFβ1 stimulation has diverse effects on the expression of αSMA, inhibition via the S1P1 receptor-mediated and serum-dependent expression of cyclooxygenase-2 and the resulting formation of prostaglandin E2, and stimulation via the S1P3 receptor in a serum-independent manner.