Sequential Analysis of Myofibroblast Differentiation and Transforming Growth Factor-β1/Smad Pathway Activation in Murine Pulmonary Fibrosis

Myofibroblasts play a critical role in tissue fibrosis. However, the intracellular signaling pathways in myofibroblast differentiation are poorly understood. Here, we studied the relationship between transforming growth factor-beta (TGF-beta)/Smad pathway activation and myofibroblast differentiation in both in vivo and in vitro experiments. In murine bleomycin-induced pulmonary fibrosis, nuclear localization of phosphorylated Smad2/3 (p-Smad2/3) was observed in pulmonary fibrotic lesions 7 days after bleomycin injection, whereas alpha-smooth muscle actin (ASMA)-positive myofibroblasts appeared in the lesions at 14 days, when the cytoplasmic localization of p-Smad2/3 was observed. We also compared the effects of TGF-beta 1 on myofibroblast differentiation and on type I collagen expression in a murine lung fibroblast cell line (MLg2908). TGF-beta 1 induced rapid expression of p-Smad2/3 in nuclei, after which ASMA organization in the cytoplasm of fibroblasts was observed. However, TGF-beta 1 produced no effect on the quantity of ASMA, either in mRNA levels or protein levels, even after the phosphorylation of Smad2/3. In contrast, TGF-beta 1 upregulated the expression of type I collagen mRNA. These findings suggest that in pulmonary fibrosis the molecular mechanism of myofibroblast differentiation is complex and that the difference between ASMA expression and type I collagen expression is mediated by the TGF-beta/Smad pathway. (J Nippon Med Sch 2012; 79: 46-59)