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.

Metabolic Reprogramming Is Required for Myofibroblast Contractility and Differentiation

“…This contractile activity can be transduced to nuclear signaling events by a process known as mechanotransduction (58). Indeed, our group has previously shown that the transcription factor MKL1 translocates to the nucleus under conditions of stress fiber formation and active contraction; a Rho kinase inhibitor that depolymerizes filamentous actin (F-actin) to globular actin (G-actin) inhibits the cytoplasmic-nuclear translocation of MKL1 and suppresses myofibroblast differentiation (9,10). Whether the integration of cellular bioenergetics to signaling of myofibroblast differentiation in our system involves MKL1 requires further study.…”

“…Myofibroblast Contraction and Differentiation Are Dependent on Mitochondrial Bioenergetics and Glycolysis-Contraction of myofibroblasts has been shown to promote myofibroblast differentiation (9,10,56). We determined whether the increase in mitochondrial biogenesis/bioenergetics is necessary to mediate contractility of differentiated myofibroblasts.…”

Antifibrotic Effects of Noscapine through Activation of Prostaglandin E2 Receptors and Protein Kinase A

“…We and others have established previously that this process is initiated by Smad-dependent expression of intermediate signaling molecules driving the activation of SRF that is required for the expression of SM-␣-actin, collagen 1, and other myofibroblast differentiation markers (13,14,31). We also showed that activation of SRF by TGF-␤ in HLF is mediated by increased expression and nuclear accumulation of SRF co-activator MKL1 and that this process is blocked by microtubule stabilizer, Taxol (14,18). As shown in Fig.…”

“…In Vitro Isolation of Stress Fibers-Stress fibers were isolated as described previously (14,18). All of the procedures were performed on ice using buffers containing protease inhibitors (Sigma).…”

“…We have demonstrated previously that TGF-␤ utilizes stress fiberdependent signaling driving nuclear accumulation of megakaryoblastic leukemia-1 (MKL1) 2 and activation of serum response factor (SRF) to fully induce the myofibroblast phenotype, and that activators of protein kinase A (PKA) attenuate myofibroblast differentiation through inhibition of SRF, but not Smad signaling (13,14). These data suggest that targeting the MKL1/SRF pathway could be an attractive approach for preventing myofibroblast differentiation and pulmonary fibrosis without affecting the initial Smad signaling of TGF-␤.…”