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

Jiro Usuki, Kuniko Matsuda, Arata Azuma, Shoji Kudoh and Akihiko Gemma

Division of Pulmonary Medicine, Infection Diseases and Oncology, Graduate School of Medicine, Nippon Medical School

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-β (TGF-β)/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 α-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-β1 on myofibroblast differentiation and on type I collagen expression in a murine lung fibroblast cell line (MLg2908). TGF-β1 induced rapid expression of p-Smad2/3 in nuclei, after which ASMA organization in the cytoplasm of fibroblasts was observed. However, TGF-β1 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-β1 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-β/Smad pathway.

J Nippon Med Sch 2012; 79: 46-59

Keywords
myofibroblast differentiation, transforming growth factor-β/Smad pathway, pulmonary fibrosis

Correspondence to
Jiro Usuki, MD, Divisions of Pulmonary Medicine, Infectious Diseases and Oncology, Department of Internal Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan
usukij@nms.ac.jp

Received, May 20, 2011
Accepted, August 15, 2011