ALK-5 mediates endogenous and TGF-beta1-induced expression of connective tissue growth factor in embryonic lung.
Transforming growth factor-beta1 (TGF-beta1) has been implicated as a major negative regulator of lung branching morphogenesis. Since connective tissue growth factor (CTGF) is a downstream mediator of TGF-beta1 effects on mesenchymal cells, we hypothesized that TGF-beta1 induces CTGF expression in mouse embryonic lung explants and that CTGF mediates TGF-beta1 inhibition of branching morphogenesis. We show that addition of TGF-beta1 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-beta1-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-beta1 induces CTGF, we used SB431542, a specific inhibitor for TGF-beta type I receptor (TbetaRI)/ALK-5 to block TGF-beta1-induced Smad2/3 phosphorylation. Consequently, SB431542 stimulated normal branching morphogenesis and blocked TGF-beta1 inhibition of branching. Furthermore, SB-431542 blocked both endogenous and TGF-beta1-induced expression of CTGF mRNA and protein. These results demonstrate for the first time that TGF-beta1 induces CTGF expression in mouse embryonic lung explants, that CTGF inhibits branching morphogenesis, and that both endogenous and TGF-beta1-induced CTGF expression are mediated by the TbetaRI/ALK-5-dependent Smad2 signaling pathway.
Wu
Shu
S
Department of Pediatrics, Division of Neonatology, University of Miami School of Medicine, P.O. Box 016960, Miami, FL 33101, USA. swu2@med.miami.edu
Peng
Jinghong
J
Duncan
Matthew R
MR
Kasisomayajula
Kalyani
K
Grotendorst
Gary
G
Bancalari
Eduardo
E
D030201:Activin Receptors, Type I / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D055513:Connective Tissue Growth Factor; D003513:Cycloheximide / Q000494:pharmacology; D004149:Dioxoles / Q000494:pharmacology; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects*; D017874:Immediate-Early Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D008168:Lung / Q000166:cytology / Q000187:drug effects / Q000196:embryology* / Q000378:metabolism*; D008297:Male; D051379:Mice; D009024:Morphogenesis / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D014176:Protein Biosynthesis / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051899:Smad2 Protein / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
C459179:4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D001549:Benzamides; C526318:CCN2 protein, mouse; D004149:Dioxoles; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D018125:Receptors, Transforming Growth Factor beta; D051899:Smad2 Protein; D053773:Transforming Growth Factor beta1; D055513:Connective Tissue Growth Factor; D003513:Cycloheximide; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I; C000625842:Tgfbr1 protein, mouse