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ALK-5 Mediates Endogenous and TGF-1–Induced Expression of Connective Tissue Growth Factor in Embryonic Lung

Department of Pediatrics, Division of Neonatology, University of Miami School of Medicine, Miami, Florida; and Lovelace Respiratory Research Institute, Albuquerque, New Mexico

Correspondence and requests for reprints should be addressed to Shu Wu, Department of Pediatrics, Division of Neonatology, University of Miami School of Medicine, P.O. Box 016960, Miami, FL 33101. E-mail: swu2{at}med.miami.edu

Transforming growth factor-1 (TGF-1) has been implicated as a major negative regulator of lung branching morphogenesis. Since connective tissue growth factor (CTGF) is a downstream mediator of TGF-1 effects on mesenchymal cells, we hypothesized that TGF-1 induces CTGF expression in mouse embryonic lung explants and that CTGF mediates TGF-1 inhibition of branching morphogenesis. We show that addition of TGF-1 to the serum-free medium of embryonic day (E)12.5 lung explant cultures inhibited branching morphogenesis and induced CTGF mRNA expression in time- and dose-dependent manners. In contrast to basal endogenous CTGF protein, which was exclusively localized in the distal airway epithelium, TGF-1–induced CTGF protein was localized in both the epithelium and the mesenchyme. Addition of exogenous CTGF to culture medium directly inhibited branching morphogenesis. To identify the signal transduction pathway through which TGF-1 induces CTGF, we used SB431542, a specific inhibitor for TGF- type I receptor (TRI)/ALK-5 to block TGF-1–induced Smad2/3 phosphorylation. Consequently, SB431542 stimulated normal branching morphogenesis and blocked TGF-1 inhibition of branching. Furthermore, SB-431542 blocked both endogenous and TGF-1–induced expression of CTGF mRNA and protein. These results demonstrate for the first time that TGF-1 induces CTGF expression in mouse embryonic lung explants, that CTGF inhibits branching morphogenesis, and that both endogenous and TGF-1–induced CTGF expression are mediated by the TRI/ALK-5–dependent Smad2 signaling pathway.

Key Words: connective tissue growth factor • TGF- • 1 • ALK-5 • Smad2 • lung

CLINICAL RELEVANCE This research will enhance our knowledge of the regulation of lung branching morphogenesis. It may also provide better understanding of lung diseases that are caused by abnormal development and repair processes.