Inhibition of mechanosensitive signaling in myofibroblasts ameliorates experimental pulmonary fibrosis
01 Mar 2013-Journal of Clinical Investigation (American Society for Clinical Investigation)-Vol. 123, Iss: 3, pp 1096-1108
TL;DR: It is indicated that targeting mechanosensitive signaling in myofibroblasts to trigger the intrinsic apoptosis pathway may be an effective approach for treatment of fibrotic disorders.
Abstract: Matrix stiffening and myofibroblast resistance to apoptosis are cardinal features of chronic fibrotic diseases involving diverse organ systems. The interactions between altered tissue biomechanics and cellular signaling that sustain progressive fibrosis are not well defined. In this study, we used ex vivo and in vivo approaches to define a mechanotransduction pathway involving Rho/Rho kinase (Rho/ROCK), actin cytoskeletal remodeling, and a mechanosensitive transcription factor, megakaryoblastic leukemia 1 (MKL1), that coordinately regulate myofibroblast differentiation and survival. Both in an experimental mouse model of lung fibrosis and in human subjects with idiopathic pulmonary fibrosis (IPF), we observed activation of the Rho/ROCK pathway, enhanced actin cytoskeletal polymerization, and MKL1 cytoplasmic-nuclear shuttling. Pharmacologic disruption of this mechanotransduction pathway with the ROCK inhibitor fasudil induced myofibroblast apoptosis through a mechanism involving downregulation of BCL-2 and activation of the intrinsic mitochondrial apoptotic pathway. Treatment with fasudil during the postinflammatory fibrotic phase of lung injury or genetic ablation of Mkl1 protected mice from experimental lung fibrosis. These studies indicate that targeting mechanosensitive signaling in myofibroblasts to trigger the intrinsic apoptosis pathway may be an effective approach for treatment of fibrotic disorders.
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TL;DR: This disease has improved understanding of the mechanisms of lung fibrosis, and offers hope that similar approaches will transform the management of patients with other progressive fibrotic lung diseases.
986 citations
TL;DR: Together, these results identify YAP and TAZ as mechanoactivated coordinators of the matrix-driven feedback loop that amplifies and sustains fibrosis.
Abstract: Pathological fibrosis is driven by a feedback loop in which the fibrotic extracellular matrix is both a cause and consequence of fibroblast activation. However, the molecular mechanisms underlying this process remain poorly understood. Here we identify yes-associated protein (YAP) (homolog of drosophila Yki) and transcriptional coactivator with PDZ-binding motif (TAZ) (also known as Wwtr1), transcriptional effectors of the Hippo pathway, as key matrix stiffness-regulated coordinators of fibroblast activation and matrix synthesis. YAP and TAZ are prominently expressed in fibrotic but not healthy lung tissue, with particularly pronounced nuclear expression of TAZ in spindle-shaped fibroblastic cells. In culture, both YAP and TAZ accumulate in the nuclei of fibroblasts grown on pathologically stiff matrices but not physiologically compliant matrices. Knockdown of YAP and TAZ together in vitro attenuates key fibroblast functions, including matrix synthesis, contraction, and proliferation, and does so exclusively on pathologically stiff matrices. Profibrotic effects of YAP and TAZ operate, in part, through their transcriptional target plasminogen activator inhibitor-1, which is regulated by matrix stiffness independent of transforming growth factor-β signaling. Immortalized fibroblasts conditionally expressing active YAP or TAZ mutant proteins overcome soft matrix limitations on growth and promote fibrosis when adoptively transferred to the murine lung, demonstrating the ability of fibroblast YAP/TAZ activation to drive a profibrotic response in vivo. Together, these results identify YAP and TAZ as mechanoactivated coordinators of the matrix-driven feedback loop that amplifies and sustains fibrosis.
541 citations
TL;DR: The hypothesis that the mechanical state of the extracellular matrix and the pro-fibrotic transforming growth factor (TGF)-β1 cooperate to regulate the remodeling activities of stromal cells is developed.
430 citations
TL;DR: It is determined that genes encoding IPF-associated ECM proteins are targets for miR-29, which was downregulated in fibroblasts grown on IPf-derived ECM, and baseline expression of ECM targets could be restored by overexpression of miR -29.
Abstract: Pathological remodeling of the extracellular matrix (ECM) by fibroblasts leads to organ failure. Development of idiopathic pulmonary fibrosis (IPF) is characterized by a progressive fibrotic scarring in the lung that ultimately leads to asphyxiation; however, the cascade of events that promote IPF are not well defined. Here, we examined how the interplay between the ECM and fibroblasts affects both the transcriptome and translatome by culturing primary fibroblasts generated from IPF patient lung tissue or nonfibrotic lung tissue on decellularized lung ECM from either IPF or control patients. Surprisingly, the origin of the ECM had a greater impact on gene expression than did cell origin, and differences in translational control were more prominent than alterations in transcriptional regulation. Strikingly, genes that were translationally activated by IPF-derived ECM were enriched for those encoding ECM proteins detected in IPF tissue. We determined that genes encoding IPF-associated ECM proteins are targets for miR-29, which was downregulated in fibroblasts grown on IPF-derived ECM, and baseline expression of ECM targets could be restored by overexpression of miR-29. Our data support a model in which fibroblasts are activated to pathologically remodel the ECM in IPF via a positive feedback loop between fibroblasts and aberrant ECM. Interrupting this loop may be a strategy for IPF treatment.
422 citations
TL;DR: Therapeutics targeting TGF-β-induced and ROS-dependent cellular signaling represents a novel approach in the treatment of fibrotic disorders.
Abstract: Transforming growth factor beta (TGF-β) is the most potent pro-fibrogenic cytokine and its expression is increased in almost all of fibrotic diseases. Although signaling through Smad pathway is believed to play a central role in TGF-β's fibrogenesis, emerging evidence indicates that reactive oxygen species (ROS) modulate TGF-β's signaling through different pathways including Smad pathway. TGF-β1 increases ROS production and suppresses antioxidant enzymes, leading to a redox imbalance. ROS, in turn, induce/activate TGF-β1 and mediate many of TGF-β's fibrogenic effects, forming a vicious cycle (see graphic flow chart on the right). Here, we review the current knowledge on the feed-forward mechanisms between TGF-β1 and ROS in the development of fibrosis. Therapeutics targeting TGF-β-induced and ROS-dependent cellular signaling represents a novel approach in the treatment of fibrotic disorders.
421 citations
References
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TL;DR: Cells undergoing apoptosis in vivo showed increased release of cy tochrome c to their cytosol, suggesting that mitochondria may function in apoptosis by releasing cytochrome c.
5,128 citations
"Inhibition of mechanosensitive sign..." refers background in this paper
...The release of cytochrome c from mitochondria is an essential step for activation of the intrinsic apoptosis pathway (31)....
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TL;DR: One possible role of Bcl-2 in prevention of apoptosis is to block cytochrome c release from mitochondria, which is normally located in the mitochondrial intermembrane space.
Abstract: Bcl-2 is an integral membrane protein located mainly on the outer membrane of mitochondria. Overexpression of Bcl-2 prevents cells from undergoing apoptosis in response to a variety of stimuli. Cytosolic cytochrome c is necessary for the initiation of the apoptotic program, suggesting a possible connection between Bcl-2 and cytochrome c, which is normally located in the mitochondrial intermembrane space. Cells undergoing apoptosis were found to have an elevation of cytochrome c in the cytosol and a corresponding decrease in the mitochondria. Overexpression of Bcl-2 prevented the efflux of cytochrome c from the mitochondria and the initiation of apoptosis. Thus, one possible role of Bcl-2 in prevention of apoptosis is to block cytochrome c release from mitochondria.
4,961 citations
TL;DR: It is clear that the understanding of the myofibroblast — its origins, functions and molecular regulation — will have a profound influence on the future effectiveness not only of tissue engineering but also of regenerative medicine generally.
Abstract: During the past 20 years, it has become generally accepted that the modulation of fibroblastic cells towards the myofibroblastic phenotype, with acquisition of specialized contractile features, is essential for connective-tissue remodelling during normal and pathological wound healing. Yet the myofibroblast still remains one of the most enigmatic of cells, not least owing to its transient appearance in association with connective-tissue injury and to the difficulties in establishing its role in the production of tissue contracture. It is clear that our understanding of the myofibroblast its origins, functions and molecular regulation will have a profound influence on the future effectiveness not only of tissue engineering but also of regenerative medicine generally.
3,836 citations
"Inhibition of mechanosensitive sign..." refers background in this paper
...Fibroblast-to-myofibroblast differentiation is characterized by α-SMA expression and enhanced contractile activity (4)....
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...(Myo)fibroblast contractility is associated with the phosphorylation of myosin light chain (MLC20) (4)....
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...MLC20 and myosin phosphatase target subunit 1 (MYPT-1) are ROCK-specific substrates (42–44)....
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...We detected the highest levels of MLC20 phosphorylation in fibroblasts grown on stiff matrix treated with TGF-β1, followed by fibroblasts grown on stiff matrix in the absence of treatment, then fibroblasts grown on soft matrix treated with TGF-β1; fasudil treatment inhibited these responses to both soluble and matrix signals (Figure 4D)....
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...Myofibroblasts also participate in normal wound healing by facilitating wound closure and synthesis of ECM proteins (4)....
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TL;DR: A core set of proteins including actin, Arp2/3 complex, profilin, capping protein, and ADF/cofilin can reconstitute the process in vitro, and mathematical models of the constituent reactions predict the rate of motion.
3,793 citations
TL;DR: Rho appears to inhibit myosin phosphatase through the action of Rho-kinase, which is activated by GTP·RhoA, phosphorylation of MBS and MLC in NIH 3T3 cells.
Abstract: The small guanosine triphosphatase Rho is implicated in myosin light chain (MLC) phosphorylation, which results in contraction of smooth muscle and interaction of actin and myosin in nonmuscle cells. The guanosine triphosphate (GTP)-bound, active form of RhoA (GTP.RhoA) specifically interacted with the myosin-binding subunit (MBS) of myosin phosphatase, which regulates the extent of phosphorylation of MLC. Rho-associated kinase (Rho-kinase), which is activated by GTP.RhoA, phosphorylated MBS and consequently inactivated myosin phosphatase. Overexpression of RhoA or activated RhoA in NIH 3T3 cells increased phosphorylation of MBS and MLC. Thus, Rho appears to inhibit myosin phosphatase through the action of Rho-kinase.
2,899 citations