Regulation Of Myofibroblast Differentiation Research Articles

Collagen-Binding α11 Integrin Expression in Human Myometrium and Fibroids Utilizing a Novel RNA In Situ Probe

Integrin α11β1 is a collagen receptor specific to fibroblasts that regulates myofibroblast differentiation. We sought to determine whether α11β1 is expressed in myometrium and fibroids and whether tissue expression varies. Comparison of α11 in human myometrium and fibroids using Western blot and RNA in situ hybridization. Specimens were obtained from fibroid and myometrium. For Western blots, we used a polyclonal antibody to integrin α11. RNA in situ hybridization was performed using a custom RNA probe for α11 subunit. Myometrium and fibroids express α11 integrin, with expression 2-fold greater in fibroids. The RNA probe offers a more precise method compared to Western blot using polyclonal human antibody. The difference in expression in myometrium and fibroids suggests that α11 is involved in the formation of myofibroblasts and fibroid development.

Do laser and led phototherapies influence mast cells and myofibroblasts to produce collagen?

Laser and LED phototherapies accelerate tissue repair. Mast cells induce the proliferation of fibroblasts and the development of local fibrosis. Increased numbers of myofibroblasts and mast cells are frequently found together in a normal wound repair, suggesting that mediators produced by the mast cells could play a role in the regulation of myofibroblast differentiation and function. The aim of this study was to analyze the involvement of mast cells on the synthesis of collagen and their influence on myofibroblast differentiation in the late phase of tissue repair on wounds treated with LLLT (λ 660 nm, 10 J/cm(2), 40 mW, 252 s) or LED (λ 630 ± 10 nm, 10 J/cm(2), 115 mW, 87 s). A 1 × 1-cm surgical wound was created on the dorsum of 30 rats divided into three groups of ten animals each: control, laser, and LED. The animals of each group were irradiated and sacrificed 7 and 14 days after injury. The statistical analysis was performed using the Mann-Whitney and Spearman correlation tests. Laser light improved the collagen deposition rate along the time points (p = 0.22), but when compared to the control groups during the periods studied, the number of mast cells decreased significantly (p ≤ 0.05). With respect to myofibroblasts, the results showed a trend to their reduction. No statistical significances were observed for LED light according to the parameters used in this study. It is concluded that the mast cell and myofibroblast population might participate in the collagen formation of irradiated wounds particularly in relation to laser phototherapy.

Negative regulation of GADD34 on myofibroblasts during cutaneous wound healing.

The growth arrest and DNA damage-inducible protein, GADD34, has been proved to be involved in TGF-β signaling pathway and correlates with cell death, which are two important mechanisms in regulating myofibroblast differentiation and apoptosis during tissue repair. But roles of GADD34 in myofibroblasts differentiation and apoptosis remain unknown. To investigate the function of GADD34 in these processes, we subjected WT and GADD34−/− mice to dermal wound healing. Here we show that GADD34−/− mice exhibited accelerated wound closure compared with WT mice. In addition, GADD34−/− mice showed increased number of myofibroblasts, elevated collagen production, and decreased cell apoptosis during wound healing. Moreover, we found that GADD34−/− mice showed increased phosphorylation of Smad3 and lower level of cleaved caspase-3. Thus these results indicate that GADD34 appears to suppress myofibroblast differentiation through inhibiting Smad3-dependent TGFβ signal pathway and promote its apoptosis by activating caspase-3 pathway.

Redox signaling as a therapeutic target to inhibit myofibroblast activation in degenerative fibrotic disease.

Degenerative fibrotic diseases encompass numerous systemic and organ-specific disorders. Despite their associated significant morbidity and mortality, there is currently no effective antifibrotic treatment. Fibrosis is characterized by the development and persistence of myofibroblasts, whose unregulated deposition of extracellular matrix components disrupts signaling cascades and normal tissue architecture leading to organ failure and death. The profibrotic cytokine transforming growth factor beta (TGFβ) is considered the foremost inducer of fibrosis, driving myofibroblast differentiation in diverse tissues. This review summarizes recent in vitro and in vivo data demonstrating that TGFβ-induced myofibroblast differentiation is driven by a prooxidant shift in redox homeostasis. Elevated NADPH oxidase 4 (NOX4)-derived hydrogen peroxide (H2O2) supported by concomitant decreases in nitric oxide (NO) signaling and reactive oxygen species scavengers are central factors in the molecular pathogenesis of fibrosis in numerous tissues and organs. Moreover, complex interplay between NOX4-derived H2O2 and NO signaling regulates myofibroblast differentiation. Restoring redox homeostasis via antioxidants or NOX4 inactivation as well as by enhancing NO signaling via activation of soluble guanylyl cyclases or inhibition of phosphodiesterases can inhibit and reverse myofibroblast differentiation. Thus, dysregulated redox signaling represents a potential therapeutic target for the treatment of wide variety of different degenerative fibrotic disorders.

Akt1 Mediates α-Smooth Muscle Actin Expression and Myofibroblast Differentiation via Myocardin and Serum Response Factor

Myofibroblast (MF) differentiation, marked by the de novo expression of smooth muscle α-actin (αSMA) stress fibers, plays a central role in wound healing and its persistence is a hallmark of fibrotic diseases. We have previously shown that Akt1 is necessary for wound healing through matrix regulation. However, the role of Akt1 in regulating MF differentiation with implications in fibrosis remains poorly defined. Here, we show that sustained activation of Akt1 was associated with a 6-fold increase in αSMA expression and assembly; an effect that is blunted in cells expressing inactive Akt1 despite TGFβ stimulation. Mechanistically, Akt1 mediated TGFβ-induced αSMA synthesis through the contractile gene transcription factors myocardin and serum response factor (SRF), independent of mammalian target of rapamycin in mouse embryonic fibroblasts and fibroblasts overexpressing active Akt1. Akt1 deficiency was associated with decreased myocardin, SRF, and αSMA expressions in vivo. Furthermore, sustained Akt1-induced αSMA synthesis markedly decreased upon RNA silencing of SRF and myocardin. In addition to its integral role in αSMA synthesis, we also show that Akt1 mediates fibronectin splice variant expression, which is required for MF differentiation, as well as total fibronectin, which generates the contractile force that promotes MF differentiation. In summary, our results constitute evidence that sustained Akt1 activation is crucial for TGFβ-induced MF formation and persistent differentiation. These findings highlight Akt1 as a novel potential therapeutic target for fibrotic diseases.

Collagen-binding α11 integrin expression in human myometrium and fibroids utilizing a novel RNA in situ probe

Integrins are α and β heterodimer cell membrane receptors that bind to ECM involved in signaling across the cell membrane. When compared to myometrium, fibroids contain excessive collagen and demonstrate a disordered structure and orientation consistent with fibrosis. Fibroids have increased β1 and α6 subunits, and similar expression of other α subunits compared to myometrium. Integrin α11β1 is a collagen receptor specific to fibroblasts and regulates myofibroblast differentiation in some tissues.

Abstract 353: Hdac Class I Inhibition Diminished Cd90+ Cardiac Fibroblast Activation Via Gsk3b/β-catenin Pathway.

Myofibroblast activation is one of the major mechanisms of scar formation and interstitial myocardial fibrosis after myocardial infarction (MI) and subsequent heart failure. Elucidating the mechanisms of myofibroblast activation will provide a strategy to treat heart failure. Histone Deacetylases (HDAC) were reported to regulate myofibroblast differentiation and extracellular matrix deposition in lung, kidney and liver fibrosis. Furthermore, HDAC inhibitors were shown to retard myocardial remodeling, interstitial fibrosis and improve cardiac function after MI. However, the mechanisms are unclear. Here we examined whether HDAC1/2 are up-regulated after MI, identify which myocardial cell type is predominately expressing HDAC, and examine the effects of HDAC inhibition on myocardial fibrosis. MI was created in rats by ligation of the left coronary artery. We found that, in sham rats, HDACs were uniformly expressed in both right and left ventriculars. On the other hand, we observed up-regulation of both HDAC1 and 2 in the infarcted LV following MI. In addition, HDAC1 and 2 were co-localized with cardiac fibroblast markers CD90, Vimentin, and αSMA in the infarcted area. Treatment of CD90+ cells isolated from both CHF and sham atrial culture with pan HDAC inhibitor (100 nM, or 300 nM) TSA diminished cardiac fibroblasts activation as indicated by the decrease in αSMA and the increase in E-cadherin gene expressions. Similarly, treatment of CD90 cells with a specific HDAC class I inhibitor (2 μM, Mocetinostat) upregulated E-cadherin and diminished the markers of fibrosis (αSMA, MMP-2, and Collagen III).The decrease in fibroblast activation by HDAC inhibition was associated with increase in p-Gsk3β/Gsk3β ratio and β-catenin protein content. In addition, Mocetinostat treatment of CD90+ cells up-regulated cleaved-Caspase3, an indicator of apoptosis. Taken together, diminishing cardiac fibroblast activation using HDAC class I inhibitor appears to be dependent on Gsk3β/β-catenin pathway.

Mechanoregulation of the Myofibroblast in Wound Contraction, Scarring, and Fibrosis: Opportunities for New Therapeutic Intervention

Myofibroblasts are responsible for wound closure that occurs in healed acute wounds. However, their actions can result in disfiguring scar contractures, compromised organ function, and a tumor promoting stroma. Understanding the mechanisms regulating their contractile machinery, gene expression, and lifespan is essential to develop new therapies to control their function. Mechanical stress and transforming growth factor beta-1 (TGF-β1) regulate myofibroblast differentiation from mesenchymal progenitors. As these precursor cells differentiate, they assemble a contractile apparatus to generate the force used to contract wounds. The mechanisms by which mechanical stress promote expression of contractile genes through the TGF-β1 and serum response factor pathways and offer therapeutic targets to limit myofibroblast function are being elucidated. Emerging evidence suggests that the integration of mechanical cues with intracellular signaling pathways is critical to myofibroblast function via its effects on gene expression, cellular contraction, and paracrine signaling with neighboring cells. In addition, while apoptosis is clearly one pathway that can limit myofibroblast lifespan, recent data suggest that pathogenic myofibroblasts can become senescent and adopt a more beneficial phenotype, or may revert to a quiescent state, thereby limiting their function. Given the important role that myofibroblasts play in pathologies as disparate as cutaneous scarring, organ fibrosis, and tumor progression, knowledge gained in the areas of intracellular signaling networks, mechanical signal transduction, extracellular matrix biology, and cell fate will support efforts to develop new therapies with a wide impact.

Regulation of myofibroblast differentiation and bleomycin-induced pulmonary fibrosis by adrenomedullin

Myofibroblast differentiation induced by transforming growth factor-β (TGF-β) is characterized by the expression of smooth muscle α-actin (SMA) and extracellular matrix proteins. We and others have previously shown that these changes are regulated by protein kinase A (PKA). Adrenomedullin (ADM) is a vasodilator peptide that activates cAMP/PKA signaling through the calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying proteins (RAMP). In this study, we found that recombinant ADM had little effect on cAMP/PKA in quiescent human pulmonary fibroblasts, whereas it induced a profound activation of cAMP/PKA signaling in differentiated (by TGF-β) myofibroblasts. In contrast, the prostacyclin agonist iloprost was equally effective at activating PKA in both quiescent fibroblasts and differentiated myofibroblasts. TGF-β stimulated a profound expression of CRLR with a time course that mirrored the increased PKA responses to ADM. The TGF-β receptor kinase inhibitor SB431542 abolished expression of CRLR and attenuated the PKA responses of cells to ADM but not to iloprost. CRLR expression was also dramatically increased in lungs from bleomycin-treated mice. Functionally, ADM did not affect initial differentiation of quiescent fibroblasts in response to TGF-β but significantly attenuated the expression of SMA, collagen-1, and fibronectin in pre-differentiated myofibroblasts, which was accompanied by decreased contractility of myofibroblasts. Finally, sensitization of ADM signaling by transgenic overexpression of RAMP2 in myofibroblasts resulted in enhanced survival and reduced pulmonary fibrosis in the bleomycin model of the disease. In conclusion, differentiated pulmonary myofibroblasts gain responsiveness to ADM via increased CRLR expression, suggesting the possibility of using ADM for targeting pathological myofibroblasts without affecting normal fibroblasts.

MiR‐145 regulates myofibroblast differentiation and lung fibrosis

The expression of smooth muscle actin-α (SMA-α) by fibroblasts defines phenotypic transition to myofibroblasts and is a primary contributor to contractile force generation by these differentiated cells. Although the regulation of SMA-α expression has been the focus of many studies, there is presently only limited information concerning miRNA regulation of lung myofibroblast differentiation and the involvement of these miRNAs in pulmonary fibrosis. To determine the role of miR-145 in regulating lung myofibroblast differentiation and pulmonary fibrosis. Wild-type and miR-145(-/-) mice were studied. Lung fibrosis models and cell culture systems were employed. miR-145 mimics or inhibitors were transfected into pulmonary fibroblasts. Fibrogenic and contractile activities of lung fibroblasts were determined. We found that miR-145 expression is upregulated in TGF-β1-treated lung fibroblasts. miR-145 expression is also increased in the lungs of patients with idiopathic pulmonary fibrosis as compared to in normal human lungs. Overexpression of miR-145 in lung fibroblasts increased SMA-α expression, enhanced contractility, and promoted formation of focal and fibrillar adhesions. In contrast, miR-145 deficiency diminished TGF-β1 induced SMA-α expression. miR-145 did not affect the activity of TGF-β1, but promoted the activation of latent TGF-β1. miR-145 targets KLF4, a known negative regulator of SMA-α expression. Finally, we found that miR-145(-/-) mice are protected from bleomycin-induced pulmonary fibrosis. miR-145 plays an important role in the differentiation of lung myofibroblasts. miR-145 deficiency is protective against bleomycin-induced lung fibrosis, suggesting that miR-145 may be a potential target in the development of novel therapies to treat pathological fibrotic disorders.

Inhibition of mechanosensitive signaling in myofibroblasts ameliorates experimental pulmonary fibrosis

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.

Epigenetic Regulation of Myofibroblast Differentiation and Extracellular Matrix Production in Nasal Polyp-Derived Fibroblasts

Epigenetic Regulation of Myofibroblast Differentiation and Extracellular Matrix Production in Nasal Polyp-Derived Fibroblasts

Biomechanical regulation of mesenchymal cell function

Cells of mesenchymal origin are strongly influenced by their biomechanical environment. They also help to shape tissue architecture and reciprocally influence tissue mechanical environments through their capacity to deposit, remodel, and resorb extracellular matrix and to promote tissue vascularization. Although mechanical regulation of cell function and tissue remodeling has long been appreciated in other contexts, the purpose of this review is to highlight the increasing appreciation of its importance in fibrosis and hypertrophic scarring. Experiments in both animal and cellular model systems have demonstrated pivotal roles for the biomechanical environment in regulating myofibroblast differentiation and contraction, endothelial barrier function and angiogenesis, and mesenchymal stem cell fate decisions. Through these studies, a better understanding of the molecular mechanisms transducing the biomechanical environment is emerging, with prominent and interacting roles recently identified for key network components including transforming growth factor-β/SMAD, focal adhesion kinase, MRTFs, Wnt/β-catenin and YAP/TAZ signaling pathways. Progress in understanding biomechanical regulation of mesenchymal cell function is leading to novel approaches for improving clinical outcomes in fibrotic diseases and wound healing. These approaches include interventions aimed at modifying the tissue biomechanical environment, and efforts to target mesenchymal cell activation by, and reciprocal interactions with, the mechanical environment.

Myofibroblasts

Interest in the myofibroblast as a key player in propagation of chronic progressive fibrosis continues to elicit many publications, with focus on its cellular origins and the mechanisms underpinning their differentiation and/or transition. The objective of the review is to highlight this recent progress. The epithelial origin of the myofibroblast in fibrosis has been challenged by recent studies, with the pericyte suggested as a possible precursor instead. Additional signaling pathways, including Notch, Wnt, and hedgehog, are implicated in myofibroblast differentiation. The importance of NADPH oxidase 4 was highlighted recently to suggest a potential link between cellular/oxidative stress and the genesis of the myofibroblast. Recent observations on the importance of lysophosphatidic acid in fibrosis suggest that this may be due, in part, to its ability to regulate myofibroblast differentiation. Finally, there is increasing evidence for the role of epigenetic mechanisms in regulating myofibroblast differentiation, including DNA methylation and miRNA regulation of gene expression. These recent discoveries open up a whole new array of potential targets for novel antifibrotic therapies. This is of special importance given the current bleak outlook for chronic progressive fibrotic diseases, such as scleroderma, due to lack of effective therapies.

Regulation of Myofibroblast Differentiation by Poly(ADP-Ribose) Polymerase 1

Poly(ADP-ribosyl)ation (PARylation) is a post-translational protein modification effected by enzymes belonging to the poly(ADP-ribose) polymerase (PARP) superfamily, mainly by PARP-1. The key acceptors of poly(ADP-ribose) include PARP-1 itself, histones, DNA repair proteins, and transcription factors. Because many of these factors are involved in the regulation of myofibroblast differentiation, we examined the role of PARylation on myofibroblast differentiation. Overexpression of PARP-1 with an expression plasmid activated expression of the α-SMA gene (Acta2), a marker of myofibroblast differentiation in lung fibroblasts. Suppression of PARP-1 activity or gene expression with PARP-1 inhibitors or siRNA, respectively, had the opposite effect on these cells. PARP-1-deficient cells also had reduced α-SMA gene expression. DNA pyrosequencing identified hypermethylated regions of the α-SMA gene in PARP-1-deficient cells, relative to wild-type cells. Interestingly, and of potential relevance to human idiopathic pulmonary fibrosis, PARP activity in lung fibroblasts isolated from idiopathic pulmonary fibrosis patients was significantly higher than that in cells isolated from control subjects. Furthermore, PARP-1-deficient mice exhibited reduced pulmonary fibrosis in response to bleomycin-induced lung injury, relative to wild-type controls. These results suggest that PARylation is important for myofibroblast differentiation and the pathogenesis of pulmonary fibrosis.

Abstract 259: Histone Deacetylase1 and 2 Are Increased in Myofibroblasts/Fibroblasts After MI

Myofibroblast activation after myocardial infarction (MI) is one of the major mechanisms of myocardial fibrosis and subsequent heart failure. Elucidating the mechanisms of myofibroblast activation will provide a strategy to treat heart failure. Histone Deacetylases (HDAC) were reported to regulate myofibroblast differentiation and extracellular matrix deposition in idiopathic pulmonary fibrosis. Furthermore, HDAC inhibitors were shown to retards myocardial remodeling and improve cardiac function after MI. Therefore, here we examined whether HDAC1/2 are up-regulated after MI, and identify which myocardial cell type is predominately expressing HDAC. MI was created in rats by ligation of the left coronary artery. First, we assessed expression of HDAC 1 and 2 following MI in heart sections. Both HDACs uniformly expressed in myocardium in right ventricular (LV) and left ventricular (LV) in sham hearts. On the other hand, we observed up-regulation of both HDAC1 and 2 in the infarcted LV following MI. In addition, HDAC1 and 2 were co-localized with fibroblast marker Vimentin in the infarcted area. Next, we performed western blot analysis to investigate protein levels of HDAC1 and HDAC2 in right ventricular (RV), septum (S) and LV (infarcted and remote area). We found up-regulation of HDAC1 and HDAC2 only in the infarcted area compared to sham rats. Altogether these results suggest HDAC1 and 2 play a role in fibrosis following MI.

Dynamic Regulation of Platelet-Derived Growth Factor Receptor α Expression in Alveolar Fibroblasts during Realveolarization

Although the importance of platelet-derived growth factor receptor (PDGFR)-α signaling during normal alveogenesis is known, it is unclear whether this signaling pathway can regulate realveolarization in the adult lung. During alveolar development, PDGFR-α-expressing cells induce α smooth muscle actin (α-SMA) and differentiate to interstitial myofibroblasts. Fibroblast growth factor (FGF) signaling regulates myofibroblast differentiation during alveolarization, whereas peroxisome proliferator-activated receptor (PPAR)-γ activation antagonizes myofibroblast differentiation in lung fibrosis. Using left lung pneumonectomy, the roles of FGF and PPAR-γ signaling in differentiation of myofibroblasts from PDGFR-α-positive precursors during compensatory lung growth were assessed. FGF receptor (FGFR) signaling was inhibited by conditionally activating a soluble dominant-negative FGFR2 transgene. PPAR-γ signaling was activated by administration of rosiglitazone. Changes in α-SMA and PDGFR-α protein expression were assessed in PDGFR-α-green fluorescent protein (GFP) reporter mice using immunohistochemistry, flow cytometry, and real-time PCR. Immunohistochemistry and flow cytometry demonstrated that the cell ratio and expression levels of PDGFR-α-GFP changed dynamically during alveolar regeneration and that α-SMA expression was induced in a subset of PDGFR-α-GFP cells. Expression of a dominant-negative FGFR2 and administration of rosiglitazone inhibited induction of α-SMA in PDGFR-α-positive fibroblasts and formation of new septae. Changes in gene expression of epithelial and mesenchymal signaling molecules were assessed after left lobe pneumonectomy, and results demonstrated that inhibition of FGFR2 signaling and increase in PPAR-γ signaling altered the expression of Shh, FGF, Wnt, and Bmp4, genes that are also important for epithelial-mesenchymal crosstalk during early lung development. Our data demonstrate for the first time that a comparable epithelial-mesenchymal crosstalk regulates fibroblast phenotypes during alveolar septation.

Abstract 4265: Human prostate mesenchymal stem cells in prostate cancer: Mechanisms of TGF-β1 promoted myofibroblast/CAF differentiation

Abstract Reactive stroma in prostate cancer is typified by the co-evolution of myofibroblasts and carcinoma-associated fibroblasts (CAFs), yet the origin and biology of myofibroblast stem/progenitor cells are not known. TGF-β1 is overexpressed in prostate cancer cells and affects myofibroblast differentiation, a key step in formation of a tumor-promoting reactive stroma. However, specific mechanisms of how TGF-β1 regulates recruitment, activation and differentiation of myofibroblast stem/progenitor cells are poorly understood. Human prostate mesenchymal stem cell (hpMSC) lines were initiated from organ cultures of normal human prostate gland and characterized by a novel 3D co-culture model. Differentially engineered LNCaP cells to overexpress active TGF-β1 were cultured as organoids on CM-inserts and hpMSCs were co-cultured with engineered LNCaP cells either in direct contact or on laminin-coated coverslips in the bottom chamber. 3D organoids grown in serum-free media for 72 hours were analyzed by IHC, ELISA and qRT-PCR. In addition, RNAi gene knockdown experiments were performed in hpMSC to identify key transcription factors involved in myofibroblast/CAF differentiation. hpMSCs were CD44+/CD90+ and showed characteristics and gene expression similar to human mesenchymal stem cells. In the 3D model, LNCaP cells and hpMSCs self-organized into a free-floating organoid. The organoids exhibited a periphery of LNCaP cells and a core of reactive stroma-appearing cells. Significantly, LNCaP cells induced myofibroblast differentiation of hpMSC and this was promoted by LNCaP expression of active TGF-β1. Gene expression analysis identified the RUNX1 and ID1 transcription factors as upregulated in hpMSC by TGF-β1. Knockdown of RUNX1 and ID1 are being used to assess the role and mechanisms of how these transcription factors regulate myofibroblast differentiation. Together, these data suggest that the genesis and co-evolution of reactive stroma in prostate cancer results from activation and differentiation of CD44+/CD90+ endogenous hpMSC via upregulation of specific transcription factors by TGF-β1. Defining key mechanisms that recruit, activate and differentiate the origin of myofibroblasts/CAFs could provide clues to develop new strategies to target the microenvironment niche. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4265. doi:1538-7445.AM2012-4265

Matrix Stiffness–Induced Myofibroblast Differentiation Is Mediated by Intrinsic Mechanotransduction

The mechanical properties of the extracellular matrix have recently been shown to promote myofibroblast differentiation and lung fibrosis. Mechanisms by which matrix stiffness regulates myofibroblast differentiation are not fully understood. The goal of this study was to determine the intrinsic mechanisms of mechanotransduction in the regulation of matrix stiffness-induced myofibroblast differentiation. A well established polyacrylamide gel system with tunable substrate stiffness was used in this study. Megakaryoblastic leukemia factor-1 (MKL1) nuclear translocation was imaged by confocal immunofluorescent microscopy. The binding of MKL1 to the α-smooth muscle actin (α-SMA) gene promoter was quantified by quantitative chromatin immunoprecipitation assay. Normal human lung fibroblasts responded to matrix stiffening with changes in actin dynamics that favor filamentous actin polymerization. Actin polymerization resulted in nuclear translocation of MKL1, a serum response factor coactivator that plays a central role in regulating the expression of fibrotic genes, including α-SMA, a marker for myofibroblast differentiation. Mouse lung fibroblasts deficient in Mkl1 did not respond to matrix stiffening with increased α-SMA expression, whereas ectopic expression of human MKL1 cDNA restored the ability of Mkl1 null lung fibroblasts to express α-SMA. Furthermore, matrix stiffening promoted production and activation of the small GTPase RhoA, increased Rho kinase (ROCK) activity, and enhanced fibroblast contractility. Inhibition of RhoA/ROCK abrogated stiff matrix-induced actin cytoskeletal reorganization, MKL1 nuclear translocation, and myofibroblast differentiation. This study indicates that actin cytoskeletal remodeling-mediated activation of MKL1 transduces mechanical stimuli from the extracellular matrix to a fibrogenic program that promotes myofibroblast differentiation, suggesting an intrinsic mechanotransduction mechanism.

Relaxin Regulates Myofibroblast Contractility and Protects against Lung Fibrosis

Myofibroblasts are specialized contractile cells that participate in tissue fibrosis and remodeling, including idiopathic pulmonary fibrosis (IPF). Mechanotransduction, a process by which mechanical stimuli are converted into biochemical signals, regulates myofibroblast differentiation. Relaxin is a peptide hormone that mediates antifibrotic effects through regulation of collagen synthesis and turnover. In this study, we demonstrate enhanced myofibroblast contraction in bleomycin-induced lung fibrosis in mice and in fibroblastic foci of human subjects with IPF, using phosphorylation of the regulatory myosin light chain (MLC(20)) as a biomarker of in vivo cellular contractility. Compared with wild-type mice, relaxin knockout mice express higher lung levels of phospho-MLC(20) and develop more severe bleomycin-induced lung fibrosis. Exogenous relaxin inhibits MLC(20) phosphorylation and bleomycin-induced lung fibrosis in both relaxin knockout and wild-type mice. Ex vivo studies of IPF lung myofibroblasts demonstrate decreases in MLC(20) phosphorylation and reduced contractility in response to relaxin. Characterization of the signaling pathway reveals that relaxin regulates MLC(20) dephosphorylation and lung myofibroblast contraction by inactivating RhoA/Rho-associated protein kinase through a nitric oxide/cGMP/protein kinase G-dependent mechanism. These studies identify a novel antifibrotic role of relaxin involving the inhibition of the contractile phenotype of lung myofibroblasts and suggest that targeting myofibroblast contractility with relaxin-like peptides may be of therapeutic benefit in the treatment of fibrotic lung disease.