Thy-1 expression regulates the ability of rat lung fibroblasts to activate transforming growth factor-beta in response to fibrogenic stimuli

Distinct subpopulations of fibroblasts contribute to lung fibrosis, although the mechanisms underlying fibrogenesis in these subpopulations are not clear. Differential expression of the glycophosphatidylinositol-linked protein Thy-1 affects proliferation and myofibroblast differentiation. Lung fibroblast populations selected on the basis of Thy-1 expression by cell sorting were examined for responses to fibrogenic stimuli. Thy-1 (-) and Thy-1 (+) fibroblast populations were treated with platelet-derived growth factor-BB, interleukin-1beta, interleukin-4, or bleomycin and assessed for activation of transforming growth factor (TGF)-beta, Smad3 phosphorylation, and alpha-smooth muscle actin and fibronectin expression. Thy-1 (-) fibroblasts responded to these stimuli with increased TGF-beta activity, Smad3 phosphorylation, and expression of alpha-smooth muscle actin and fibronectin, whereas Thy-1 (+) fibroblasts resisted stimulation. The unresponsiveness of Thy-1 (+) cells is not because of defective TGF-beta signaling because both subsets respond to exogenous active TGF-beta. Rather, Thy-1 (-) fibroblasts activate latent TGF-beta in response to fibrogenic stimuli, whereas Thy-1 (+) cells fail to do so. Defective activation is common to multiple mechanisms of TGF-beta activation, including thrombospondin 1, matrix metalloproteinase, or plasmin. Thy-1 (-) lung fibroblasts transfected with Thy-1 also become resistant to fibrogenic stimulation, indicating that Thy-1 is a critical biological response modifier that protects against fibrotic progression by controlling TGF-beta activation. These studies provide a molecular basis for understanding the differential roles of fibroblast subpopulations in fibrotic lung disease through control of latent TGF-beta activation.     

Enhanced myofibroblastic differentiation and survival in Thy-1(-) lung fibroblasts

Thy-1 is a glycosylphosphatidyl-inositol-linked cell surface glycoprotein whose exact biological role remains unclear. Differential expression of Thy-1 affects fibroblast proliferation and fibrogenic signaling. In idiopathic pulmonary fibrosis, the proliferating myofibroblasts within the fibroblastic foci are Thy-1(-), whereas normal lung fibroblasts are predominantly Thy-1(+). In this study, we used rat lung fibroblasts sorted for Thy-1 expression to examine myofibroblastic differentiation in response to fibrogenic stimuli. We examined the effects of transforming growth factor-beta, endothelin-1, and connective tissue growth factor on the expression of myofibroblast proteins and myogenic regulatory factors by real-time RT-PCR and immunoblotting. Thy-1(-) cells have significantly higher myofibroblast and myogenic regulatory factor gene and protein expression compared with Thy-1(+) cells, confirmed by immunofluorescence. We also used floating collagen matrix contraction assays to assess the functional differentiation of the fibroblasts. At baseline and after stimulation with transforming growth factor-beta and endothelin-1, Thy-1(-) cells caused significantly greater collagen contraction than did Thy-1(+) cells, supporting the hypothesis that Thy-1(-) cells are more fully differentiated myofibroblasts. Because apoptosis has been implicated in the regression of myofibroblasts, we examined the percentage of apoptotic cells in the contracted collagen matrices at baseline and after stimulation with fibrogenic agents. A significantly greater proportion of Thy-1(+) cells underwent apoptosis in all conditions compared with Thy-1(-) fibroblasts. Transfection of Thy-1 into Thy-1(-) cells inhibits collagen matrix contraction and reduces cell survival. Our data indicate that Thy-1 regulates myogenic gene expression, myofibroblastic differentiation, and survival in lung fibroblasts.     

Latent Transforming Growth Factor-β-Binding Protein-4 Regulates Transforming Growth Factor-β1 Bioavailability for Activation by Fibrogenic Lung Fibroblasts in Response to Bleomycin

Recent evidence suggests that subsets of lung fibroblasts differentially contribute to fibrogenic progression. We have previously shown that a subset of rat lung fibroblasts with fibrogenic characteristics [Thy-1 (−) fibroblasts] responds to stimuli (bleomycin, interleukin-4, etc) with increased latent transforming growth factor (TGF)-β activation, whereas non-fibrogenic Thy-1-expressing [Thy-1 (+)] fibroblasts do not. Activation of latent TGF-β1 by interstitial lung fibroblasts is critical for fibrogenic responses. To better understand the susceptibility of fibrogenic fibroblasts to the stimulation of TGF-β activation, we examined the role of latent TGF-β-binding proteins (LTBPs), key regulators of TGF-β bioavailability and activation, in TGF-β1 activation by these fibroblasts. Treatment of fibroblasts with bleomycin up-regulated LTBP-4 mRNA, protein, and soluble LTBP-4-bound large latent TGF-β1 complexes in Thy-1 (−) fibroblasts to significantly higher levels than in Thy-1 (+) fibroblasts. Bleomycin-induced TGF-β1 activation required LTBP-4, since lung fibroblasts deficient in LTBP-4 did not activate TGF-β1. Expression of LTBP-4 restored TGF-β1 activation in response to bleomycin, but expression either of LTBP-4 lacking the TGF-β-binding site or only the TGF-β-binding domain did not. Bleomycin treatment of mice increased LTBP-4 expression in the lung. Thy-1 knockout mice had increased levels of both LTBP-4 expression and TGF-β activation, as well as enhanced Smad3 phosphorylation compared with wild-type mice. Together, these data identify a critical role for LTBP-4 in the regulation of latent TGF-β1 activation in bleomycin-induced lung fibrosis.     

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.     

Sphingosine kinase 1 regulates differentiation of human and mouse lung fibroblasts mediated by TGF-beta1

Transforming growth factor beta (TGF-beta) contributes to the progression of pulmonary fibrosis through up-regulation of alpha-smooth muscle actin (alpha-SMA) as lung myofibroblast differentiation. Bioactive sphingosine 1-phosphate (S1P) has been shown to mimic TGF-beta signals; however, the function of S1P in lung fibrotic process has not been well documented. We found, in a mouse model of bleomycin lung fibrosis, that SPHK1 and alpha-SMA were colocalized within lung fibrotic foci and that these expressions were significantly increased in primary cultured fibroblasts. Using human lung fibroblasts WI-38, we explored the rationale of sphingosine kinase (SPHK) with TGF-beta1 stimulation. SPHK inhibitors and small interference RNA (siRNA) targeted SPHK1 decreased alpha-SMA and fibronectin expression up-regulated by TGF-beta1. In the meantime, SPHK1 inhibition did not affect smad2 phosphorylation in response to TGF-beta1. Then we examined whether S1P receptors transactivation may affect TGF-beta signals. siRNA against S1P(2) and S1P(3), but not S1P(1), reduced alpha-SMA expression as well as Y-27632, Rho kinase inhibitor. We also detected activation of Rho GTPase upon stimulation of TGF-beta1 on the cell membrane where S1P(2) or S1P(3) was overexpressed. These data suggested that SPHK1 activation by TGF-beta1 leads to Rho-associated myofibroblasts differentiation mediated by transactivated S1P receptors in the lung fibrogenic process.     

Thy-1 expression in human fibroblast subsets defines myofibroblastic or lipofibroblastic phenotypes

Fibroblasts represent a dynamic population of cells, exhibiting functional heterogeneity within and among tissues. Fibroblast heterogeneity also results from phenotypic differences and may arise from activation or differentiation processes taking place in the cells. We previously reported that human fibroblasts were heterogeneous with respect to surface Thy-1 expression and that separation into Thy-1(+) and Thy-1(-) subsets resulted in functionally distinct subpopulations, leading to the concept of fibroblast subset specialization. In this report we investigated whether Thy-1(+) and/or Thy-1(-) fibroblasts were capable of differentiating into myofibroblasts or lipofibroblasts. Fibroblast subsets were used from human myometrium and orbit to test this hypothesis. Only Thy-1(+) human myometrial and orbital fibroblasts were capable of myofibroblast differentiation after treatment with TGFbeta or platelet concentrate supernatant, assessed by alpha smooth muscle actin expression. Interestingly, only Thy-1(-), but not Thy-1(+) subsets differentiated to lipofibroblasts, as determined by the accumulation of cytoplasmic lipid droplets after treatment with 15-deoxy-Delta(12, 14)-PGJ(2) or ciglitazone. We propose that fibroblast Thy-1 display pre-determines lineage to a contractile or lipid-like phenotype in the human myometrium and orbit. This additional distinction between Thy-1(+) and Thy-1(-) human fibroblast subtypes has important consequences in normal tissue homeostasis and in pathogenesis of orbital and myometrial diseases characterized by persistent myofibroblasts or fat accumulation, such as occurs in Graves' ophthalmopathy, tissue fibrosis, abnormal wound healing, and scarring.     

Thy-1 promoter hypermethylation: a novel epigenetic pathogenic mechanism in pulmonary fibrosis

Mechanisms regulating myofibroblastic differentiation of fibroblasts within fibroblastic foci in idiopathic pulmonary fibrosis (IPF) remain unclear. Epigenetic processes, including DNA methylation, produce heritable but potentially reversible changes in DNA or its associated proteins and are prominent in development and oncogenesis. We have shown that Thy-1 suppresses myofibroblastic differentiation of lung fibroblasts and that fibroblasts in fibroblastic foci are Thy-1(-). Epigenetic down-regulation of Thy-1 has been demonstrated in cellular transformation and clinical cancer. We hypothesized that epigenetic regulation of Thy-1 affects the lung fibroblast fibrogenic phenotype. RT-PCR, methylation-specific PCR (MSP), and bisulfite genomic sequencing were used to determine the methylation status of the Thy-1 promoter in Thy-1(+) and Thy-1(-) lung fibroblasts, and MSP-in situ hybridization (MSPISH) was performed on fibrotic tissue. Thy-1 gene expression is absent in Thy-1(-) human and rat fibroblasts despite intact Thy-1 genomic DNA. Cytosine-guanine islands in the Thy-1 gene promoter are hypermethylated in Thy-1(-), but not Thy-1(+), fibroblasts. RT-PCR and MSP demonstrate that, in IPF samples in which Thy-1 expression is absent, the Thy-1 promoter region is methylated, whereas in lung samples retaining Thy-1 expression, the promoter region is unmethylated. MSPISH confirms methylation of the Thy-1 promoter in fibroblastic foci in IPF. Treatment with DNA methyltransferase inhibitors restores Thy-1 expression in Thy-1(-) fibroblasts. Epigenetic regulation of Thy-1 is a novel and potentially reversible mechanism in fibrosis that may offer the possibility of new therapeutic options.     

Thy-1 signals through PPARγ to promote lipofibroblast differentiation in the developing lung

Thy-1 is a glycosylphosphytidylinositol-linked cell-surface glycoprotein present on a subset of lung fibroblasts, which plays an important role in postnatal alveolarization. In the present study, we define the role of Thy-1 in pulmonary lipofibroblast differentiation and in the regulation of lipid homeostasis via peroxisome proliferator-activated receptor-γ (PPARγ). Thy-1 was associated with interstitial cells containing lipid droplets in vivo. The transfection of Thy-1 into Thy-1 (-) fibroblasts increased triglyceride content, fatty-acid uptake, and the expression of the lipofibroblast marker adipocyte differentiation-related protein. Thy-1 (+) fibroblasts exhibited 2.4-fold higher PPARγ activity, and the inhibition or activation of PPARγ reduced and increased triglyceride content, respectively. Thy-1 (-) fibroblasts were not responsive to either of the PPARγ agonists ciglitazone or prostaglandin J(2), supporting the importance of Thy-1 in signaling via PPARγ. Thy-1 (+) fibroblasts expressed significantly higher concentrations of fatty-acid transporter protein-3 mRNA, and demonstrated higher rates of fatty-acid uptake and increased triglyceride content. The inhibition of fatty-acid transporter protein function reduced Thy-1 (+) fibroblast lipid content. The expression of Thy-1 in C57BL/6 lung fibroblasts increased during the neonatal period, coinciding with the onset of alveolarization. Thy-1 promoted lipofibroblast differentiation via the expression of PPARγ, stimulated lipid accumulation via fatty-acid esterification, and enhanced the fatty-acid uptake mediated by fatty-acid transporter proteins. Thy-1 is important in the regulation of lipofibroblast differentiation in the developing lung.     

Absence of Thy-1 results in TGF-β induced MMP-9 expression and confers a profibrotic phenotype to human lung fibroblasts

Fibroblasts differ in a variety of phenotypic features, including the expression of Thy-1 a glycophosphatidylinositol-linked glycoprotein. Fibroblasts in idiopathic pulmonary fibrosis (IPF) are Thy-1 negative, whereas most fibroblasts from normal lungs are Thy-1 positive. However, the functional consequences of the absence of Thy-1 are not fully understood. We analyzed the expression of Thy-1 in several primary fibroblasts lines derived from IPF, hypersensitivity pneumonitis (HP), and normal human lungs. We found that a high proportion, independently of their origin, expressed Thy-1 in vitro. We identified a primary culture of HP fibroblasts, which did not express Thy-1, and compared several functional activities between Thy-1 (-) and Thy-1 (+) fibroblasts. Thy-1 (-) fibroblasts were smaller (length: 41.3±20.8?μ versus 83.1±40 μ), showed increased proliferative capacity and enhanced PDGF-induced transmigration through collagen I (59.9% versus 42.2% over control under basal conditions, P<0.01). Likewise, Thy-1 (-) fibroblasts either spontaneously or after TGF-β stimulation demonstrated stronger contraction of collagen matrices (eg, 0.17±0.03 versus 0.6±0.05 cm2 after TGF-β stimulation at 24 h; P<0.01). Thy-1 (-) lung fibroblasts stimulated with TGF-β1 expressed MMP-9, an enzyme that is usually not produced by lung fibroblasts. TGFβ-induced MMP-9 expression was reversible upon re-expression of Thy-1 after transfection with full-length Thy-1. β-glycan, a TGF-β receptor antagonist abolished MMP-9 expression. TGF-β1-induced MMP-9 in Thy-1 (-) fibroblasts depended on the activation of ERK1/2 signaling pathway. Finally, we demonstrated that fibroblasts from IPF fibroblastic foci, which do not express Thy-1 exhibit strong staining for immunoreactive MMP-9 protein in vivo. These findings indicate that loss of Thy-1 in human lung fibroblasts induces a fibrogenic phenotype.     

Telomerase regulation of myofibroblast differentiation

Telomerase activity, which has wide expression in cancerous cells, is induced in lung proliferating fibroblasts. It is preferentially expressed in fibroblasts versus myofibroblasts. It is unknown whether regulation of telomerase expression is related to the process of fibroblast differentiation into myofibroblasts. The objective of this study was to clarify such a potential link between telomerase expression and myofibroblast differentiation. Telomerase inhibitor, 3'-azido-2',3'-dideoxythymidine, or antisense oligonucleotide to the telomerase RNA component was used to inhibit the induced fibroblast telomerase activity. The results showed that inhibition of induced telomerase increased alpha-smooth muscle actin expression, an indicator of myofibroblast differentiation. In contrast, induction of telomerase by basic fibroblast growth factor inhibited alpha-smooth muscle actin expression. These findings suggest that the loss of telomerase activity is closely associated with myofibroblast differentiation and possibly functions as a trigger for myofibroblast differentiation. Conversely, expression of telomerase suppresses myofibroblast differentiation.     

Hepatocyte growth factor inhibits epithelial to myofibroblast transition in lung cells via Smad7

Idiopathic pulmonary fibrosis is a lethal parenchymal lung disease characterized by denudation of the lung epithelium, fibroblast proliferation, and collagen deposition. Cellular changes underlying disease progression involve injury to alveolar epithelial cells, epithelial to mesenchymal transition, proliferation of alpha-smooth muscle actin (alpha-SMA)-expressing myofibroblasts and of fibroblasts resulting in enhanced deposition of extracellular matrix proteins. Hepatocyte growth factor (HGF) inhibits progression of bleomycin-induced pulmonary fibrosis in mice. The mechanism underlying the inhibitory effect of HGF was investigated in an in vitro model. We show that HGF markedly antagonizes basal and transforming growth factor (TGF)-beta-induced expression of myofibroblast markers such as alpha-SMA, collagen type 1, and fibronectin in rat alveolar epithelial cells. HGF also inhibited TGF-beta-induced alpha-SMA expression in primary murine alveolar epithelial cells. Since TGF-beta is known to regulate alpha-SMA expression, the effect of HGF on components of TGF-beta signaling was investigated. HGF induced expression of Smad7, an inhibitor of TGF-beta signaling, in a mitogen-activated protein kinase-dependent manner. HGF also induced the nuclear export of Smad7 and Smad ubiquitin regulatory factor 1 (Smurf1) to the cytoplasm. HGF-dependent decrease in alpha-SMA was abolished with specific siRNAs targeted to Smad7. Thus, induction of Smad7 by HGF serves to limit acquisition of the myofibroblast phenotype in alveolar epithelial cells.     

Plasminogen activation induced pericellular fibronectin proteolysis promotes fibroblast apoptosis

Apoptosis of fibroblasts/myofibroblasts is a critical event in the resolution of tissue repair responses; however, mechanisms for the regulation of (myo)fibroblast apoptosis/survival remain unclear. In this study, we demonstrate counter-regulatory interactions between the plasminogen activation system and transforming growth factor-beta1 (TGF-beta1) in the control of fibroblast apoptosis. Plasmin treatment induced fibroblast apoptosis in a time- and dose-dependent manner in association with proteolytic degradation of extracellular matrix proteins, as detected by the release of soluble fibronectin peptides. Plasminogen, which was activated to plasmin by fibroblasts, also induced fibronectin proteolysis and fibroblast apoptosis, both of which were blocked by alpha2-antiplasmin but not by inhibition of matrix metalloproteinase activity. TGF-beta1 protected fibroblasts from apoptosis induced by plasminogen but not from apoptosis induced by exogenous plasmin. The protection from plasminogen-induced apoptosis conferred by TGF-beta1 is associated with the up-regulation of plasminogen activator-1 (PAI-1) expression and inhibition of plasminogen activation. Moreover, lung fibroblasts from mice genetically deficient in PAI-1 lose the protective effect of TGF-beta1 against plasminogen-induced apoptosis. These findings support a novel role for the plasminogen activation system in the regulation of fibroblast apoptosis and a potential role of TGF-beta1/PAI-1 in promoting (myo)fibroblast survival in chronic fibrotic disorders.     

Cyclooxygenase-2 deficiency results in a loss of the anti-proliferative response to transforming growth factor-beta in human fibrotic lung fibroblasts and promotes bleomycin-induced pulmonary fibrosis in mice

Prostaglandin E(2) (PGE(2)) inhibits fibroblast proliferation and collagen production. Its synthesis by fibroblasts is induced by profibrotic mediators including transforming growth factor (TGF)-beta(1). However, in patients with pulmonary fibrosis, PGE(2) levels are decreased. In this study we examined the effect of TGF-beta(1) on PGE(2) synthesis, proliferation, collagen production, and cyclooxygenase (COX) mRNA levels in fibroblasts derived from fibrotic and nonfibrotic human lung. In addition, we examined the effect of bleomycin-induced pulmonary fibrosis in COX-2-deficient mice. We demonstrate that basal and TGF-beta(1)-induced PGE(2) synthesis is limited in fibroblasts from fibrotic lung. Functionally, this correlates with a loss of the anti-proliferative response to TGF-beta(1). This failure to induce PGE(2) synthesis is because of an inability to up-regulate COX-2 mRNA levels in these fibroblasts. Furthermore, mice deficient in COX-2 exhibit an enhanced response to bleomycin. We conclude that a decreased capacity to up-regulate COX-2 expression and COX-2-derived PGE(2) synthesis in the presence of increasing levels of profibrotic mediators such as TGF-beta(1) may lead to unopposed fibroblast proliferation and collagen synthesis and contribute to the pathogenesis of pulmonary fibrosis.     

Inhibition of myofibroblast apoptosis by transforming growth factor beta(1)

Fibroblast differentiation to the myofibroblast phenotype is associated with alpha-smooth-muscle actin (alpha-SMA) expression and regulated by cytokines. Among these, transforming growth factor (TGF)-beta(1) and interleukin (IL)-1beta can stimulate and inhibit myofibroblast differentiation, respectively. IL-1beta inhibits alpha-SMA expression by inducing apoptosis selectively in myofibroblasts via induction of nitric oxide synthase (inducible nitric oxide synthase [iNOS]). Because TGF-beta is known to inhibit iNOS expression, this study was undertaken to see if this cytokine can protect against IL-1beta-induced myofibroblast apoptosis. Rat lung fibroblasts were treated with IL-1beta and/or TGF-beta(1) and examined for expression of alpha-SMA, iNOS, and the apoptotic regulatory proteins bax and bcl-2. The results show that TGF-beta(1) caused a virtually complete suppression of IL-1beta-induced iNOS expression while preventing the decline in alpha-SMA expression or the myofibroblast subpopulation. TGF-beta(1) treatment also completely suppressed the IL-1beta-induced apoptosis in myofibroblasts. IL-1beta-induced apoptosis was associated with a significant decline in expression of the antiapoptotic protein bcl-2, which was prevented by concomitant TGF-beta(1) treatment. The level of the proapoptotic protein bax, however, was not significantly altered by either cytokine. These data suggest that TGF-beta(1) inhibits IL-1beta-induced apoptosis in myofibroblasts by at least two mechanisms, namely, the suppression of iNOS expression and the prevention of a decline in bcl-2 expression. Thus, TGF-beta(1) may be additionally important in fibrosis by virtue of this novel ability to promote myofibroblast survival by preventing the myofibroblast from undergoing apoptosis.     

Lung fibroblast alpha-smooth muscle actin expression and contractile phenotype in bleomycin-induced pulmonary fibrosis.

The emergence of the myofibroblast phenotype (characterized by alpha-smooth muscle actin expression) in wound healing and in tissues undergoing fibrosis is thought to be responsible for the increased contractility of the affected tissues. In bleomycin-induced pulmonary fibrosis, the myofibroblast is also responsible for the observed increase in collagen gene expression. To evaluate further these phenotypic changes in lung fibroblasts, contractile and other phenotypic properties of fibroblasts isolated from lungs of rats with bleomycin-induced fibrosis were compared with those of normal rats using in vitro models. Pulmonary fibrosis was induced in rats by endotracheal injection on day 0, and 7 and 14 days later the animals were sacrificed and lung fibroblasts isolated. Using immunofluorescence, < 10% of fibroblasts from control animals express alpha-smooth muscle actin when cultured as a monolayer. In contrast, 19% and 21% of cells from day 7 and day 14 bleomycin-treated animals, respectively, expressed this actin and with greater intensity than in control lung cells. This increase in actin expression was associated with enhanced contractility when evaluated using a three-dimensional cell culture model consisting of fibroblast-populated collagen gels. This enhanced contractility was abolished by treatment with antibody to transforming growth factor-beta (TGF-beta), whereas exogenous TGF-beta 1 and serum-stimulated contraction of control lung fibroblasts. TGF-beta 1 gene expression was greater in cells from bleomycin-treated animals than those from control lungs. These results show that cells with the myofibroblast phenotype are more abundant in fibrotic lung, and that these cells possess greater contractile capacity in vitro at least partly by virtue of their enhanced endogenous TGF-beta 1 gene expression.     

Immunomodulator FTY720 induces myofibroblast differentiation via the lysophospholipid receptor S1P3 and Smad3 signaling

The novel immunomodulator FTY720 is an effective immunosuppressive agent in experimental models of transplantation and autoimmunity and is currently undergoing phase III clinical trials for multiple sclerosis. Phosphorylated FTY720 is a structural analogue of sphingosine 1-phosphate (S1P) and therefore acts as a high-affinity agonist at four of the five G protein-coupled S1P receptors. It has been well established that there exists a crosstalk between S1P and transforming growth factor (TGF)-beta signaling. Because TGF-beta is the most prominent inductor of fibrosis and myofibroblasts are primarily responsible for excessive matrix protein formation, we examined whether FTY720, in analogy to TGF-beta, induces differentiation of fibroblasts into myofibroblasts. Indeed, FTY720 provoked myofibroblast differentiation comparable with that of TGF-beta. For biological efficacy, FTY720 required endogenous phosphorylation because inhibition of sphingosine kinase completely prevented FTY720 from inducing the differentiation process. Moreover, we identified the lysophospholipid receptor S1P3 as the crucial receptor subtype for FTY720-induced myofibroblast differentiation because the effect was abolished in fibroblasts isolated from S1P3 knockout mice. Finally, we determined that downstream of S1P3 signaling Smad3 activation is essential for myofibroblast differentiation in response to FTY720. Thus, FTY720 may have adverse fibrotic effects related to its activity on S1P3 signaling.     

Lack of MK2 inhibits myofibroblast formation and exacerbates pulmonary fibrosis

Fibroblasts play a major role in tissue repair and remodeling. Their differentiation into myofibroblasts, marked by increased expression of smooth muscle-specific alpha-actin (alpha-SMA), is believed to be important in wound healing and fibrosis. We have recently described a role for MK2 in this phenotypic differentiation in culture. In this article, we demonstrate that MK2 also regulates myofibroblasts in vivo. Disruption of MK2 in mice prevented myofibroblast formation in a model of pulmonary fibrosis. However, MK2 disruption and consequent lack of myofibroblast formation exacerbated fibrosis rather than ameliorated it as previously postulated. When mice lacking MK2 (MK2-/-) were exposed to bleomycin, more collagen accumulated and more fibroblasts populated fibrotic regions in their lungs than in similarly treated wild-type mice. While there were many vimentin-positive cells in the bleomycin-treated MK2-/- mouse lungs, few alpha-SMA-positive cells were observed in these lungs compared with wild-type mouse lungs. siRNA against MK2 reduced alpha-SMA expression in wild-type mouse embryonic fibroblasts (MEF), consistent with its suppression in MK2-/- MEF. On the other hand expressing constitutively active MK2 in MK2-/- MEF significantly increased alpha-SMA expression. MK2-/-MEF proliferated at a faster rate and produced more collagen; however, they migrated at a slower rate than wild-type MEF. Overexpressing phosphomimicking HSP27, a target of MK2, did not reverse the effect of MK2 disruption on fibroblast migration. MK2 disruption did not affect Smad2 activation by transforming growth factor-beta. Thus, MK2 appears to mediate myofibroblast differentiation, and inhibiting that differentiation might contribute to fibrosis rather than protect against it.