Selective inhibition of activin receptor-like kinase 5 signaling blocks profibrotic transforming growth factor beta responses in skin fibroblasts.
OBJECTIVE
Members of the transforming growth factor beta (TGFbeta) cytokine superfamily play critical roles in both homeostasis and disease. In light of their profibrotic effects, these molecules are implicated in the pathogenesis of fibrosis. In fibroblasts, TGFbeta signals through the activin receptor-like kinase 5 (ALK-5) type I TGFbeta and triggers Smad and MAP kinase signaling pathways. Because targeting of TGFbeta signaling represents a potential approach to the treatment of systemic sclerosis (SSc) and other fibrotic disorders, we investigated the modulation of intracellular TGFbeta signal transduction by SB431542, the first small-molecule inhibitor of ALK-5 to be described.


METHODS
Ligand-induced activation of the Smad signaling pathway in human dermal fibroblasts was examined by Western blot analysis and confocal immunocytochemistry. Modulation of profibrotic gene expression was investigated using Northern blot analysis, transient transfection assays, and confocal microscopy. Induction of TGFbeta production was evaluated by enzyme-linked immunosorbent assay.


RESULTS
SB431542 abrogated TGFbeta-induced phosphorylation and nuclear importation of endogenous Smad2/3 and Smad4, and inhibited Smad3- and Smad2-dependent gene transcription. Treatment with SB431542 prevented TGFbeta-induced stimulation of collagen, fibronectin, plasminogen activator inhibitor 1, and connective tissue growth factor gene expression, TGFbeta autoinduction, and myofibroblast transdifferentiation, and it could reverse stimulation even when added to the cultures after TGFbeta. In contrast, STAT-6-mediated stimulation of collagen gene expression induced by interleukin-13 was not prevented by SB431542, indicating the specificity of blockade for ALK-5-dependent signaling. Furthermore, in contrast to its effects on receptor-activated Smad activation, SB431542 failed to prevent TGFbeta-induced activation of MAP kinases.


CONCLUSIONS
The results indicate that SB431542 is a potent inhibitor of intracellular TGFbeta signaling in normal fibroblasts through selective interference with ALK-5-mediated Smad activation and Smad-dependent transcriptional responses. Therefore, SB431542 is useful as a novel experimental tool for gaining a detailed understanding of normal and aberrant TGFbeta signaling in SSc. Furthermore, as an anti-TGFbeta agent, SB431542 may represent a potential new approach to the treatment of fibrosis.
Mori
Yasuji
Y
University of Illinois College of Medicine, Chicago.
Ishida
Wataru
W
Bhattacharyya
Swati
S
Li
Yongzhong
Y
Platanias
Leonidas C
LC
Varga
John
J
D030201:Activin Receptors, Type I / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000328:Adult; D001549:Benzamides / Q000494:pharmacology*; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004149:Dioxoles / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology*; D005355:Fibrosis / Q000188:drug therapy; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D006801:Humans; D017403:In Situ Hybridization; D007231:Infant, Newborn; D008297:Male; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger / Q000378:metabolism; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015398:Signal Transduction; D012867:Skin / Q000187:drug effects / Q000201:enzymology*; D051785:Smad Proteins; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors* / Q000378:metabolism
C459179:4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D001549:Benzamides; D004268:DNA-Binding Proteins; D004149:Dioxoles; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D018125:Receptors, Transforming Growth Factor beta; D051785:Smad Proteins; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I; C000625841:TGFBR1 protein, human