title: Delayed stress fiber formation mediates pulmonary myofibroblast differentiation in response to TGF-β.
abstract: Myofibroblast differentiation induced by transforming growth factor-β (TGF-β) and characterized by de novo expression of smooth muscle (SM)-specific proteins is a key process in wound healing and in the pathogenesis of fibrosis. We have previously shown that TGF-β-induced expression and activation of serum response factor (SRF) is required for this process. In this study, we examined the signaling mechanism for SRF activation by TGF-β as it relates to pulmonary myofibroblast differentiation. TGF-β stimulated a profound, but delayed (18-24 h), activation of Rho kinase and formation of actin stress fibers, which paralleled SM α-actin expression. The translational inhibitor cycloheximide blocked these processes without affecting Smad-dependent gene transcription. Inhibition of Rho kinase by Y-27632 or depolymerization of actin by latrunculin B resulted in inhibition TGF-β-induced SRF activation and SM α-actin expression, having no effect on Smad signaling. Conversely, stabilization of actin stress fibers by jasplakinolide was sufficient to drive these processes in the absence of TGF-β. TGF-β promoted a delayed nuclear accumulation of the SRF coactivator megakaryoblastic leukemia-1 (MKL1)/myocardin-related transcription factor-A, which was inhibited by latrunculin B. Furthermore, TGF-β also induced MKL1 expression, which was inhibited by latrunculin B, by SRF inhibitor CCG-1423, or by SRF knockdown. Together, these data suggest a triphasic model for myofibroblast differentiation in response to TGF-β that involves 1) initial Smad-dependent expression of intermediate signaling molecules driving Rho activation and stress fiber formation, 2) nuclear accumulation of MKL1 and activation of SRF as a result of actin polymerization, and 3) SRF-dependent expression of MKL1, driving further myofibroblast differentiation.
authors.affiliation: Univ. of Wisconsin School of Medicine and Public Health, 5229 MFCB 1685 Highland Ave, Madison, WI 53705, USA. nsandbo@medicine.wisc.edu
authors.forename: Nathan
authors.initials: N
authors.lastname: Sandbo
authors.forename: Andrew
authors.initials: A
authors.lastname: Lau
authors.forename: Jacob
authors.initials: J
authors.lastname: Kach
authors.forename: Caitlyn
authors.initials: C
authors.lastname: Ngam
authors.forename: Douglas
authors.initials: D
authors.lastname: Yau
authors.forename: Nickolai O
authors.initials: NO
authors.lastname: Dulin
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000256:Adenoviridae; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000032:analysis; D008168:Lung / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D058628:Myofibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D015514:Oncogene Proteins, Fusion / Q000235:genetics / Q000378:metabolism; D061251:Primary Cell Culture; D011658:Pulmonary Fibrosis / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D060888:Real-Time Polymerase Chain Reaction; D026362:Serum Response Factor / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D051785:Smad Proteins / Q000235:genetics / Q000378:metabolism; D022502:Stress Fibers / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D015534:Trans-Activators; D014161:Transduction, Genetic; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D054460:rho-Associated Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: D000199:Actins; D004268:DNA-Binding Proteins; C431575:MRTFA protein, human; D015514:Oncogene Proteins, Fusion; D026362:Serum Response Factor; D051785:Smad Proteins; D015534:Trans-Activators; D053773:Transforming Growth Factor beta1; D008156:Luciferases; D054460:rho-Associated Kinases