Regulation of Smad3 expression in bleomycin-induced pulmonary fibrosis: a negative feedback loop of TGF-beta signaling.

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine involved in controlling critical cellular activities including proliferation, differentiation, extracellular matrix production, and apoptosis. TGF-beta signals are mediated by a family of Smad proteins, of which Smad2 and Smad3 are downstream intracellular targets of serine/threonine kinase receptors of TGF-beta. Although Smad2 and Smad3 are crucial for TGF-beta signaling, little is known about the regulation of their expression. In this study, we investigated the expression of Smad2 and Smad3 in an in vivo animal model of lung fibrosis induced by bleomycin. We found that the expression of Smad3 was regulated in lungs during bleomycin-induced pulmonary fibrosis. The decline of Smad3 mRNA was evident at day three of post-bleomycin instillation and the expression of Smad3 continually decreased during the reparative phase of lung injury (days 8 and 12), whereas the expression of Smad2 showed little change after bleomycin administration. We further investigated whether the expression of Smad3 was regulated by TGF-beta in an in vitro lung fibroblast culture system. Our results show an immediate translocation of Smad3 protein from the cytoplasm to the nucleus and a delayed down-regulation of Smad3 mRNA by TGF-beta in lung fibroblasts. These studies provide direct evidence for a differential regulation of Smad3 expression that is distinct from that of Smad2 during bleomycin-induced pulmonary fibrosis and suggest a ligand-induced negative feedback loop that modulates cellular TGF-beta signaling.