Transforming growth factor (TGF)-α and its receptor, the epidermal growth factor receptor, are induced after lung injury and are associated with remodeling in chronic pulmonary diseases, such as pulmonary fibrosis and asthma. Expression of TGF-α in the lungs of adult mice causes fibrosis, pleural thickening, and pulmonary hypertension, in addition to increased expression of a transcription factor, early growth response-1 (Egr-1). Egr-1 was increased in airway smooth muscle (ASM) and the vascular adventitia in the lungs of mice conditionally expressing TGF-α in airway epithelium (Clara cell secretory protein–rtTA+/−/[tetO]7–TGF-α+/−). The goal of this study was to determine the role of Egr-1 in TGF-α–induced lung disease. To accomplish this, TGF-α–transgenic mice were crossed to Egr-1 knockout (Egr-1ko/ko) mice. The lack of Egr-1 markedly increased the severity of TGF-α–induced pulmonary disease, dramatically enhancing airway muscularization, increasing pulmonary fibrosis, and causing greater airway hyperresponsiveness to methacholine. Smooth muscle hyperplasia, not hypertrophy, caused the ASM thickening in the absence of Egr-1. No detectable increases in pulmonary inflammation were found. In addition to the airway remodeling disease, vascular remodeling and pulmonary hypertension were also more severe in Egr-1ko/ko mice. Thus, Egr-1 acts to suppress epidermal growth factor receptor–mediated airway and vascular muscularization, fibrosis, and airway hyperresponsiveness in the absence of inflammation. This provides a unique model to study the processes causing pulmonary fibrosis and ASM thickening without the complicating effects of inflammation.
transforming growth factor-α; pulmonary fibrosis; asthma; pulmonary hypertension; vascular remodeling
By stimulating collagen synthesis and myofibroblasts differentiation, transforming growth factor-β (TGF- β) plays a pivotal role in tissue repair and fibrosis. The early growth response-1 (Egr-1) transcription factor mediates profibrotic TGF-β responses, and its expression is elevated in biopsies from patients with scleroderma. NGF1-A-binding protein 2 (Nab2) is a conserved transcriptional cofactor that directly binds to Egr-1 and positively or negatively modulates Egr-1 target gene transcription. Despite the recognized importance of Nab2 in governing the intensity of Egr-1-dependent responses, the regulation and function of Nab2 in the context of fibrotic TGF-β signaling is unknown. Here we show that TGF-β caused a time-dependent stimulation of Nab2 protein and mRNA in normal fibroblasts. Ectopic expression of Nab2 in these cells blocked Egr-1-dependent transcriptional responses, and abrogated TGF-β-induced stimulation of collagen synthesis and myofibroblasts differentiation. These inhibitory effects of Nab2 involved recruitment of the NuRD chromatin remodeling complex to the COL1A2 promoter and were accompanied by reduced histone H4 acetylation. Mice with targeted deletion of Nab2 displayed increased collagen accumulation in the dermis, and genetic or siRNA-mediated loss of Nab2 in fibroblasts was associated with constitutively elevated collagen synthesis and accentuation of Egr-1-dependent TGF-β responses in vitro. Expression of Nab2 was markedly up-regulated in skin biopsies from patients with scleroderma, and was localized primarily to epidermal keratinocytes. In contrast, little Nab2 could be detected in dermal fibroblasts. These results identify Nab2 as a novel endogenous negative regulator of Egr-1-dependent TGF-β signaling responsible for setting the intensity of fibrotic responses. Defective Nab2 expression or function in dermal fibroblasts might play a role in persistent fibrotic responses in scleroderma.
Collagen-containing leukocytes (CD45+Col-I+) accumulate in diseased and fibrotic tissues. However, the precise identity of these cells and whether injury is required for their recruitment remain unknown. Using a murine model of pulmonary fibrosis in which an inducible, bioactive form of the human transforming growth factor (TGF)-β1 gene is targeted to the lung, we characterized the cell surface phenotype of collagen-containing CD45+ cells in the lung and tested the hypothesis that apoptotic cell death responses are essential to the accumulation of CD45+Col-I+ cells.
Our studies demonstrate that CD45+Col-I+ cells appearing in the TGF-β1-exposed murine lung express markers of the monocyte lineage. Inhibition of apoptosis via pharmacological caspase blockade led to a significant reduction in CD45+Col-I+ cells, which appear to accumulate independently of alternatively activated macrophages. There are also increased levels of apoptosis and greater numbers of CD45+Col-I+ in the lung tissue of patients with two distinct forms of fibrotic lung disease, idiopathic pulmonary fibrosis and connective tissue disease-related interstitial lung disease, when compared to lung from healthy normal controls. These findings are accompanied by an increase in collagen production in cultured monocytes obtained from subjects with fibrotic lung disease. Treatment of these cultured cells with the caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD/fmk) reduces both apoptosis and collagen production in all subjects.
Interventions that prevent collagen production by monocytes via modulation of caspase activation and of apoptosis may be ameliorative in monocyte-associated, TGF-β1-driven processes such as pulmonary fibrosis.
Fibrotic reactions in the airways of the lung or the pulmonary interstitium are a common pathologic outcome after exposure to a wide variety of toxic agents, including metals, particles or fibers. The survival of mesenchymal cells (fibroblasts and myofibroblasts) is a key factor in determining whether a fibroproliferative response that occurs after toxic injury to the lung will ultimately resolve or progress to a pathologic state. Several polypeptide growth factors, including members of the platelet-derived growth factor (PDGF) family and the epidermal growth factor (EGF) family, are prosurvival factors that stimulate a replicative and migratory mesenchymal cell phenotype during the early stages of lung fibrogenesis. This replicative phenotype can progress to a matrix synthetic phenotype in the presence of transforming growth factor-β1 (TGF-β1). The resolution of a fibrotic response requires growth arrest and apoptosis of mesenchymal cells, whereas progressive chronic fibrosis has been associated with mesenchymal cell resistance to apoptosis. Mesenchymal cell survival or apoptosis is further influenced by cytokines secreted during Th1 inflammation (e.g., IFN-γ) or Th2 inflammation (e.g., IL-13) that modulate the expression of growth factor activity through the STAT family of transcription factors. Understanding the mechanisms that regulate the survival or death of mesenchymal cells is central to ultimately developing therapeutic strategies for lung fibrosis.
Increased production of EGF or TGF-alpha by the respiratory epithelial cells has been associated with the pathogenesis of various forms of lung injury. Growth factors and cytokines are thought to act locally, via paracrine and autocrine mechanisms, to stimulate cell proliferation and matrix deposition by interstitial lung cells resulting in pulmonary fibrosis. To test whether TGF-alpha mediates pulmonary fibrotic responses, we have generated transgenic mice expressing human TGF-alpha under control of regulatory regions of the human surfactant protein C (SP-C) gene. Human TGF-alpha mRNA was expressed in pulmonary epithelial cells in the lungs of the transgenic mice. Adult mice bearing the SP-C-TGF-alpha transgene developed severe pulmonary fibrosis. Fibrotic lesions were observed in peribronchial, peribronchiolar, and perivascular regions, as well as subjacent to pleural surfaces. Lesions consisted of fibrous tissue that included groups of epithelial cells expressing endogenous SP-C mRNA, consistent with their identification as distal respiratory epithelial cells. Peripheral fibrotic regions consisted of thickened pleura associated with extensive collagen deposition. Alveolar architecture was disrupted in the transgenic mice with loss of alveoli in the lung parenchyma. Pulmonary epithelial cell expression of TGF-alpha in transgenic mice disrupts alveolar morphogenesis and produces fibrotic lesions mediated by paracrine signaling between respiratory epithelial and interstitial cells of the lung.
Transforming growth factor-ß (TGF-ß) signaling is implicated in the pathogenesis of fibrosis in scleroderma or systemic sclerosis (SSc), but the precise mechanisms are poorly understood. The immediate-early gene Egr-1 is an inducible transcription factor with key roles in mediating fibrotic TGF-ß responses. To elucidate Egr-1 function in SSc-associated fibrosis, we examined change in gene expression induced by Egr-1 in human fibroblasts at the genome-wide level. Using microarray expression analysis, we derived a fibroblast “Egr-1-responsive gene signature” comprising over 600 genes involved in cell proliferation, TGF-ß signaling, wound healing, extracellular matrix synthesis and vascular development. The experimentally derived “Egr-1-responsive gene signature” was then evaluated in an expression microarray dataset comprising skin biopsies from 27 patients with localized and systemic forms of scleroderma and six healthy controls. We found that the “Egr-1 responsive gene signature” was substantially enriched in the “diffuse-proliferation” subset comprising exclusively of patients with diffuse cutaneous SSc (dcSSc) of skin biopsies. A number of Egr-1-regulated genes was also associated with the “inflammatory” intrinsic subset. Only a minority of Egr-1-regulated genes was concordantly regulated by TGF-ß. These results indicate that Egr-1 induces a distinct profibrotic/wound healing gene expression program in fibroblasts that is associated with skin biopsies from SSc patients with diffuse cutaneous disease. These observations suggest that targeting Egr-1 expression or activity might be a novel therapeutic strategy to control fibrosis in specific SSc subsets.
Transforming growth factor-β (TGF-β) is a cytokine implicated in wound healing and in the pathogenesis of pulmonary fibrosis. TGF-β stimulates myofibroblast differentiation characterized by expression of contractile smooth muscle (SM)-specific proteins such as SM–α-actin. In the present study, we examined the role of serum response factor (SRF) in the mechanism of TGF-β–induced pulmonary myofibroblast differentiation of human lung fibroblasts (HLF). TGF-β stimulated SM–α-actin expression in HLF, which paralleled with a profound induction of SRF expression and activity. Inhibition of SRF by the pharmacologic SRF inhibitor (CCG-1423), or via adenovirus-mediated transduction of SRF short hairpin RNA (shSRF), blocked the expression of both SRF and SM–α-actin in response to TGF-β without affecting Smad-mediated signaling of TGF-β. However, forced expression of SRF on its own did not promote SM–α-actin expression, whereas expression of the constitutively transactivated SRF fusion protein (SRF-VP16) was sufficient to induce SM–α-actin expression, suggesting that both expression and transactivation of SRF are important. Activation of protein kinase A (PKA) by forskolin or iloprost resulted in a significant inhibition of SM–α-actin expression induced by TGF-β, and this was associated with inhibition of both SRF expression and activity, but not of Smad-mediated gene transcription. In summary, this is the first direct demonstration that TGF-β–induced pulmonary myofibroblast differentiation is mediated by SRF, and that inhibition of myofibroblast differentiation by PKA occurs through down-regulation of SRF expression levels and SRF activity, independent of Smad signaling.
transforming growth factor-β; serum response factor; myofibroblast; protein kinase A; Smad
Transforming growth factor (TGF)-α is a ligand for the epidermal growth factor receptor (EGFR). EGFR activation is associated with fibroproliferative processes in human lung disease and animal models of pulmonary fibrosis. Overexpression of TGF-α in transgenic mice causes progressive and severe pulmonary fibrosis; however, the intracellular signaling pathways downstream of EGFR mediating this response are unknown. Using a doxycycline-regulatable transgenic mouse model of lung-specific TGF-α expression, we observed increased PCNA protein and phosphorylation of Akt and p70S6K in whole lung homogenates in association with induction of TGF-α. Induction in the lung of TGF-α caused progressive pulmonary fibrosis over a 7-week period. Daily administration of rapamycin prevented accumulation of total lung collagen, weight loss, and changes in pulmonary mechanics. Treatment of mice with rapamycin 4 weeks after the induction of TGF-α prevented additional weight loss, increases in total collagen, and changes in pulmonary mechanics. Rapamycin prevented further increases in established pulmonary fibrosis induced by EGFR activation. This study demonstrates that mammalian target of rapamycin (mTOR) is a major effector of EGFR-induced pulmonary fibrosis, providing support for further studies to determine the role of mTOR in the pathogenesis and treatment of pulmonary fibrosis.
epidermal growth factor receptor; PI3K; Akt; mTOR
Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic dysregulated response to alveolar epithelial injury with differentiation of epithelial cells and fibroblasts into matrix-secreting myofibroblasts resulting in lung scaring. The prognosis is poor and there are no effective therapies or reliable biomarkers. Galectin-3 is a β-galactoside binding lectin that is highly expressed in fibrotic tissue of diverse etiologies.
Objectives: To examine the role of galectin-3 in pulmonary fibrosis.
Methods: We used genetic deletion and pharmacologic inhibition in well-characterized murine models of lung fibrosis. Further mechanistic studies were performed in vitro and on samples from patients with IPF.
Measurements and Main Results: Transforming growth factor (TGF)-β and bleomycin-induced lung fibrosis was dramatically reduced in mice deficient in galectin-3, manifest by reduced TGF-β1–induced EMT and myofibroblast activation and collagen production. Galectin-3 reduced phosphorylation and nuclear translocation of β-catenin but had no effect on Smad2/3 phosphorylation. A novel inhibitor of galectin-3, TD139, blocked TGF-β–induced β-catenin activation in vitro and in vivo and attenuated the late-stage progression of lung fibrosis after bleomycin. There was increased expression of galectin-3 in the bronchoalveolar lavage fluid and serum from patients with stable IPF compared with nonspecific interstitial pneumonitis and controls, which rose sharply during an acute exacerbation suggesting that galectin-3 may be a marker of active fibrosis in IPF and that strategies that block galectin-3 may be effective in treating acute fibrotic exacerbations of IPF.
Conclusions: This study identifies galectin-3 as an important regulator of lung fibrosis and provides a proof of principle for galectin-3 inhibition as a potential novel therapeutic strategy for IPF.
fibrosis; epithelial cells; fibroblasts
Idiopathic pulmonary fibrosis (IPF) is a progressive fibroproliferative disease characterized by an accumulation of fibroblasts and myofibroblasts in the alveolar wall. Even though the pathogenesis of this fatal disorder remains unclear, transforming growth factor-β (TGF-β)-induced differentiation and proliferation of myofibroblasts is recognized as a primary event. The molecular pathways involved in TGF-β signalling are generally Smad-dependent yet Smad-independent pathways, including phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt), have been recently proposed. In this research we established ex-vivo cultures of human lung fibroblasts and we investigated the role of the PI3K/Akt pathway in two critical stages of the fibrotic process induced by TGF-β: fibroblast proliferation and differentiation into myofibroblasts. Here we show that the pan-inhibitor of PI3Ks LY294002 is able to abrogate the TGF-β-induced increase in cell proliferation, in α- smooth muscle actin expression and in collagen production besides inhibiting Akt phosphorylation, thus demonstrating the centrality of the PI3K/Akt pathway in lung fibroblast proliferation and differentiation. Moreover, for the first time we show that PI3K p110δ and p110γ are functionally expressed in human lung fibroblasts, in addition to the ubiquitously expressed p110α and β. Finally, results obtained with both selective inhibitors and gene knocking-down experiments demonstrate a major role of p110γ and p110α in both TGF-β-induced fibroblast proliferation and differentiation. This finding suggests that specific PI3K isoforms can be pharmacological targets in IPF.
Transforming growth factor (TGF)-β1 is an essential regulatory cytokine that has been implicated in the pathogenesis of diverse facets of the injury and repair responses in the lung. The types of responses that it elicits can be appreciated in studies from our laboratory that demonstrated that the transgenic (Tg) overexpression of TGF-β1 in the murine lung causes epithelial apoptosis followed by fibrosis, inflammation, and parenchymal destruction. Because a cyclin-dependent kinase inhibitor, p21, is a key regulator of apoptosis, we hypothesized that p21 plays an important role in the pathogenesis of TGF-β1–induced tissue responses. To test this hypothesis we evaluated the effect of TGF-β1 on the expression of p21 in the murine lung. We also characterized the effects of transgenic TGF-β1 in mice with wild-type and null mutant p21 loci. These studies demonstrate that TGF-β1 is a potent stimulator of p21 expression in the epithelial cells and macrophages in the murine lung. They also demonstrate that TGF-β1–induced lung inflammation, fibrosis, myofibroblast accumulation, and alveolar destruction are augmented in the absence of p21, and that these alterations are associated with exaggerated levels of apoptosis and caspase-3 activation. Finally, our studies further demonstrated that TGF-β1 induces p21 via a TNF-α–signaling pathway and that p21 is a negative modulator of TGF-β1–induced TNF-α expression. Collectively, our studies demonstrate that p21 regulates TGF-β1–induced apoptosis, inflammation, fibrosis, and alveolar remodeling by interacting with TNF-α–signaling pathways.
TGF-β; p21; apoptosis; fibrosis; emphysema
Myofibroblasts are pathogenic in pulmonary fibrotic disease due to their exuberant production of matrix rich in collagen that interferes with gas exchange and the ability of these cells to contract and distort the alveolar space. Transforming growth factor-β1 (TGF-β1) is a well-known inducer of myofibroblast differentiation. TGF-β1-induced transformation of fibroblasts to apoptosis-resistant myofibroblasts is adhesion-dependent and focal adhesion kinase (FAK)-mediated. Prostaglandin E2 (PGE2) inhibits this differentiation via E prostanoid receptor 2 (EP2) signaling and cAMP elevation, but whether PGE2 does so by interfering with TGF-β1 signaling is unknown. Thus we examined the effects of PGE2 in the presence and absence of TGF-β1 stimulation on candidate signaling pathways in human lung fibroblasts. We now demonstrate that PGE2 does not interfere with TGF-β1-induced Smad phosphorylation or its translocation to the nucleus. Rather, PGE2 has dramatic effects on cell shape and cytoskeletal architecture and disrupts the formation of appropriate focal adhesions. PGE2 treatment diminishes TGF-β1-induced phosphorylation of paxillin, STAT-3, and FAK and, in turn, limits activation of the protein kinase B (PKB/Akt) pathway. These alterations do not, however, result in increased apoptosis within the first 24 h of treatment. Interestingly, the effects of PGE2 stimulation alone do not always mirror the effects of PGE2 in the presence of TGF-β1, indicating that the context for EP2 signaling is different in the presence of TGF-β1. Taken together, our results demonstrate that PGE2 has the potential to limit TGF-β1-induced myofibroblast differentiation via adhesion-dependent, but Smad-independent, pathways.
fibrosis; eicosanoid; lung; signal transduction
Transforming growth factor (TGF)-beta1 has been implicated in the pathogenesis of fibrosis based upon its matrix-inducing effects on stromal cells in vitro, and studies demonstrating increased expression of total TGF-beta1 in fibrotic tissues from a variety of organs. The precise role in vivo of this cytokine in both its latent and active forms, however, remains unclear. Using replication-deficient adenovirus vectors to transfer the cDNA of porcine TGF-beta1 to rat lung, we have been able to study the effect of TGF-beta1 protein in the respiratory tract directly. We have demonstrated that transient overexpression of active, but not latent, TGF-beta1 resulted in prolonged and severe interstitial and pleural fibrosis characterized by extensive deposition of the extracellular matrix (ECM) proteins collagen, fibronectin, and elastin, and by emergence of cells with the myofibroblast phenotype. These results illustrate the role of TGF-beta1 and the importance of its activation in the pulmonary fibrotic process, and suggest that targeting active TGF-beta1 and steps involved in TGF-beta1 activation are likely to be valuable antifibrogenic therapeutic strategies. This new and versatile model of pulmonary fibrosis can be used to study such therapies.
Transforming growth factor‐β1 (TGF‐β1) has the potential to induce acute inflammation and apoptosis in lung epithelial cells and plays a central role in subsequent fibrosis.
To examine a new anti‐TGF‐β1 therapy against lung injury and fibrosis, which comprises the transfection of soluble TGF type II receptor (sTGFRII) gene into skeletal muscles by in vivo electroporation.
Soluble TGFRII was detectable between 1 and 14 days in the serum and significantly increased between 3 and 10 days after gene transfer into muscles. Based on these findings, the sTGFRII gene was injected at 3 days before or 4 days after the bleomycin instillation in order to examine the significance of TGF‐β1 on the early inflammatory phase (day 0 to day 7) or the fibrotic phase (day 7 to day 14) in this model.
Transfection of sTGFRII gene at 3 days before or 4 days after bleomycin instillation significantly attenuated apoptosis, injury, and fibrosis at 7 or 14 days, respectively. This method does not require the use of viral vector or neutralising antibody, and it is therefore possible to avoid problems regarding the pathogenicity of the viral vector or immunocomplex.
This novel anti‐TGF‐β1 strategy may have clinical application in the treatment of lung injury and fibrosis.
in vivo electroporation; pulmonary fibrosis; transforming growth factor‐β1; apoptosis; gene therapy
Recent studies suggest the importance of oxidant stress in the progression of pulmonary fibrosis. The aim of this study was to investigate extracellular superoxide dismutase (ECSOD), the major antioxidant enzyme of the extracellular matrix of human lung, in biopsy-proven idiopathic pulmonary fibrosis (IPF) related to usual interstitial pneumonia (UIP).
Methods and results
Fibrotic areas and fibroblastic foci in UIP lungs were notable for absence of ECSOD by immunohistochemistry. Western blotting showed significantly lowered immunoreactivity of ECSOD in fibrotic compared with non-fibrotic areas of the diseased lung. The only cell type that showed intense ECSOD positivity in UIP was the interstitial mast cell. In order to investigate the mechanism for ECSOD depletion in fibrotic areas, alveolar epithelial cells were exposed to tumour necrosis factor-α and transforming growth factor (TGF)-β1; TGF-β suggested a trend towards decreased synthesis. Patients with UIP were also assessed to determine whether this disease is associated with a naturally occurring mutation in ECSOD (Arg213Gly) which leads to a loss of tissue binding of ECSOD. No significant differences could be found in the allele or genotype frequencies of this polymorphism between 63 UIP patients and 61 control subjects.
Overall, consistent with several other antioxidant enzymes, ECSOD is very low in fibrotic areas of UIP, which may further increase the oxidant burden in this disease.
antioxidant; extracellular superoxide dismutase; fibrosis; oxidant; usual interstitial pneumonia
Semaphorin (SEMA) 7A regulates neuronal and immune function. In these studies, we tested the hypothesis that SEMA 7A is also a critical regulator of tissue remodeling. These studies demonstrate that SEMA 7A and its receptors, plexin C1 and β1 integrins, are stimulated by transforming growth factor (TGF)-β1 in the murine lung. They also demonstrate that SEMA 7A plays a critical role in TGF-β1–induced fibrosis, myofibroblast hyperplasia, alveolar remodeling, and apoptosis. TGF-β1 stimulated SEMA 7A via a largely Smad 3–independent mechanism and stimulated SEMA 7A receptors, matrix proteins, CCN proteins, fibroblast growth factor 2, interleukin 13 receptor components, proteases, antiprotease, and apoptosis regulators via Smad 2/3–independent and SEMA 7A–dependent mechanisms. SEMA 7A also played an important role in the pathogenesis of bleomycin-induced pulmonary fibrosis. TGF-β1 and bleomycin also activated phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB)/AKT via SEMA 7A–dependent mechanisms, and PKB/AKT inhibition diminished TGF-β1–induced fibrosis. These observations demonstrate that SEMA 7A and its receptors are induced by TGF-β1 and that SEMA 7A plays a central role in a PI3K/PKB/AKT-dependent pathway that contributes to TGF-β1–induced fibrosis and remodeling. They also demonstrate that the effects of SEMA 7A are not specific for transgenic TGF-β1, highlighting the importance of these findings for other fibrotic stimuli.
To review the scientific literature supporting the participation of caveolin-1 in the pathogenesis of tissue fibrosis and that modulation of the caveolin-1 pathway may represent a novel treatment for systemic sclerosis (SSc) and other fibrotic diseases.
Caveolin-1 plays an important role in the regulation of transforming growth factor β (TGF-β) signaling owing to its participation in TGF-β receptor (TβR) internalization. TβR internalized through caveolin-1 lipid rafts undergoes rapid degradation, effectively decreasing TGF-β signaling. Studies have shown that caveolin-1 knockdown in vitro markedly increased collagen gene expression in normal human lung fibroblasts. Caveolin-1 was reduced in affected SSc lungs and skin and in idiopathic pulmonary fibrosis (IPF) lung tissues and fibroblasts. Increasing caveolin-1 expression markedly improved bleomycin-induced pulmonary fibrosis. Restoration of caveolin bioavailability employing penetratin, a cell-permeable peptide carrier for a bioactive caveolin-1 fragment abrogated TGF-β activation of cultured human dermal fibroblasts. Systemic administration of penetratin-caveolin-1 peptide to mice with bleomycin-induced lung fibrosis reduced fibrosis.
Caveolin-1 plays an important role in the regulation of TGF-β signaling and participates in the pathogenesis of SSc and IPF. Restoration of caveolin function employing active caveolin-1 fragments coupled to cell-permeable carrier peptides may represent a novel approach for their treatment.
Caveolin-1; TGF-β; fibrosis; collagen; systemic sclerosis; idiopathic pulmonary fibrosis
Rationale: Asthma is characterized by increases in airway resistance, pulmonary remodeling, and lung inflammation. The cytokine transforming growth factor (TGF)-β has been shown to have a central role in asthma pathogenesis and in mouse models of allergic airway disease.
Objectives: To determine the contribution of TGF-β to airway hyperresponsiveness (AHR), we examined the time course, source, and isoform specificity of TGF-β production in an in vivo mouse asthma model. To then elucidate the function of TGF-β in AHR, inflammation, and pulmonary fibrosis, we examined the effects of blocking TGF-β signaling with neutralizing antibody.
Methods: Mice were sensitized and challenged with ovalbumin (OVA) to establish allergic airway disease. TGF-β activity was neutralized by intranasal administration of monoclonal antibody.
Measurements and Main Results: TGF-β1 protein levels were increased in OVA-challenged lungs versus naive controls, and airway epithelial cells were shown to be a likely source of TGF-β1. In addition, TGF-β1 levels were elevated in OVA-exposed IL-5–null mice, which fail to recruit eosinophils into the airways. Neutralization of TGF-β1 with specific antibody had no significant effect on airway inflammation and eosinophilia, although anti–TGF-β1 antibody enhanced OVA-induced AHR and suppressed pulmonary fibrosis.
Conclusions: These data show that TGF-β1 is the main TGF-β isoform produced after OVA challenge, with a likely cellular source being the airway epithelium. The effects of blocking TGF-β1 signaling had differential effects on AHR, fibrosis, and inflammation. While TGF-β neutralization may be beneficial to abrogating airway remodeling, it may be detrimental to lung function by increasing AHR.
lung; mice; hypersensitivity; cytokines
Fibrosis is a deregulated and ultimately defective form of tissue repair that underlies a large number of chronic human diseases, as well as obesity and aging. The pathogenesis of fibrosis involves multiple cell types and extracellular signals, of which transforming growth factor- β (TGF-β) is pre-eminent. The prevalence of fibrosis is rising worldwide, and to date no agents has shown clinical efficacy in the attenuating or reversing the process. Recent studies implicate the immediate-early response transcription factor Egr-1 in the pathogenesis of fibrosis. Egr-1 couples acute changes in the cellular environment to sustained alterations in gene expression, and mediates a broad spectrum of biological responses to injury and stress. In contrast to other ligand-activated transcription factors such as NF-κB, c-jun and Smad2/3 that undergo post-translational modification such as phosphorylation and nuclear translocation, Egr-1 activity is regulated via its biosynthesis. Aberrant Egr-1 expression or activity is implicated in cancer, inflammation, atherosclerosis, and ischemic injury and recent studies now indicate an important role for Egr-1 in TGF-β-dependent profibrotic responses. Fibrosis in various animal models and human diseases such as scleroderma (SSc) and idiopathic pulmonary fibrosis (IPF) is accompanied by aberrant Egr-1 expression. Moreover Egr-1 appears to be required for physiologic and pathological connective tissue remodeling, and Egr-1-null mice are protected from fibrosis. As a novel profibrotic mediator, Egr-1 thus appears to be a promising potential target for the development of anti-fibrotic therapies.
Egr-1; TGF-β; fibrosis; Scleroderma (systemic sclerosis); fibroblast
Pulmonary fibrosis is a progressive scarring disease with no effective treatment. Transforming growth factor (TGF)-β is up-regulated in fibrotic diseases, where it stimulates differentiation of fibroblasts to myofibroblasts and production of excess extracellular matrix. Peroxisome proliferator–activated receptor (PPAR) γ is a transcription factor that regulates adipogenesis, insulin sensitization, and inflammation. We report here that a novel PPARγ ligand, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), is a potent inhibitor of TGF-β–stimulated differentiation of human lung fibroblasts to myofibroblasts, and suppresses up-regulation of α–smooth muscle actin, fibronectin, collagen, and the novel myofibroblast marker, calponin. The inhibitory concentration causing a 50% decrease in aSMA for CDDO was 20-fold lower than the endogenous PPARγ ligand, 15-deoxy-Δ12,14-prostaglandin J2 (15 d-PGJ2), and 400-fold lower than the synthetic ligand, rosiglitazone. Pharmacologic and genetic approaches were used to demonstrate that CDDO mediates its activity via a PPARγ-independent pathway. CDDO and 15 d-PGJ2 contain an α/β unsaturated ketone, which acts as an electrophilic center that can form covalent bonds with cellular proteins. Prostaglandin A1 and diphenyl diselenide, both strong electrophiles, also inhibit myofibroblast differentiation, but a structural analog of 15 d-PGJ2 lacking the electrophilic center is much less potent. CDDO does not alter TGF-β–induced Smad or AP-1 signaling, but does inhibit acetylation of CREB binding protein/p300, a critical coactivator in the transcriptional regulation of TGF-β–responsive genes. Overall, these data indicate that certain PPARγ ligands, and other small molecules with electrophilic centers, are potent inhibitors of critical TGF-β–mediated profibrogenic activities through pathways independent of PPARγ. As the inhibitory concentration causing a 50% decrease in aSMA for CDDO is 400-fold lower than that in rosiglitazone, the translational potential of CDDO for treatment of fibrotic diseases is high.
myofibroblast; peroxisome proliferator–activated receptor γ; 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid; 15-deoxy-Δ12,14-prostaglandin J2; α-smooth muscle actin
Connective tissue growth factor (CTGF) is widely thought to promote the development of fibrosis in collaboration with transforming growth factor (TGF)-β; however, most of the evidence for its involvement comes from correlative and culture-based studies. In this study, the importance of CTGF in tissue fibrosis was directly examined in three murine models of fibrotic disease: a novel model of multiorgan fibrosis induced by repeated intraperitoneal injections of CTGF and TGF-β2; the unilateral ureteral obstruction (UUO) renal fibrosis model; and an intratracheal bleomycin instillation model of pulmonary fibrosis.
Intraperitoneal coadministration of CTGF and TGF-β2 elicited a profound fibrotic response that was inhibited by the human anti-CTGF antibody FG-3019, as indicated by the ability of FG-3019 to ameliorate the histologic signs of fibrosis and reduce the otherwise increased hydroxyproline:proline (Hyp:Pro) ratios by 25% in kidney (P < 0.05), 30% in liver (P < 0.01) and 63% in lung (P < 0.05). Moreover, administration of either cytokine alone failed to elicit a fibrotic response, thus demonstrating that CTGF is both necessary and sufficient to initiate fibrosis in the presence of TGF-β and vice versa. In keeping with this requirement for CTGF function in fibrosis, FG-3019 also reduced the renal Hyp:Pro response up to 20% after UUO (P < 0.05). In bleomycin-injured animals, a similar trend towards a FG-3019 treatment effect was observed (38% reduction in total lung Hyp, P = 0.056). Thus, FG-3019 antibody treatment consistently reduced excessive collagen deposition and the pathologic severity of fibrosis in all models.
Cooperative interactions between CTGF and TGF-β signaling are required to elicit overt tissue fibrosis. This interdependence and the observed anti-fibrotic effects of FG-3019 indicate that anti-CTGF therapy may provide therapeutic benefit in different forms of fibroproliferative disease.
Idiopathic pulmonary fibrosis is a progressive and fatal fibrotic disease of the lungs with unclear etiology. Prior efforts to treat idiopathic pulmonary fibrosis that focused on anti-inflammatory therapy have not proven to be effective. Recent insight suggests that the pathogenesis is mediated through foci of dysregulated fibroblasts driven by profibrotic cytokine signaling. TGF-β and PDGF are 2 of the most potent of these cytokines. In the current study, we investigated the role of TGF-β–induced fibrosis mediated by activation of the Abelson (Abl) tyrosine kinase. Our data indicate that fibroblasts respond to TGF-β by stimulating c-Abl kinase activity independently of Smad2/3 phosphorylation or PDGFR activation. Moreover, inhibition of c-Abl by imatinib prevented TGF-β–induced ECM gene expression, morphologic transformation, and cell proliferation independently of any effect on Smad signaling. Further, using a mouse model of bleomycin-induced pulmonary fibrosis, we found a significant inhibition of lung fibrosis by imatinib. Thus, Abl family members represent common targets for the modulation of profibrotic cytokine signaling.
Fibrosis involves an orchestrated cascade of events including activation of fibroblasts, increased production and deposition of extracellular matrix components, and differentiation of fibroblasts into myofibroblasts. Epithelial-mesenchymal cross-talk plays an important role in this process, and current hypotheses of organ fibrosis liken it to an aberrant wound healing response in which epithelial-mesenchymal transition (EMT) and cellular senescence may also contribute to disease pathogenesis. The fibrotic response is associated with altered expression of growth factors and cytokines, including increased levels of transforming growth factor-β1 (TGF-β1) and the more recent observation that increased levels of several insulin-like growth factor binding proteins (IGFBPs) are associated with a number of fibrotic conditions. IGFBPs have been implicated in virtually every cell type and process associated with the fibrotic response, making the IGFBPs attractive targets for the development of novel anti-fibrotic therapies. In this review, the current state of knowledge regarding the classical IGFBP family in organ fibrosis will be summarized and the clinical implications considered.
Extracellular matrix; fibrosis; IGFBP; senescence; systemic sclerosis; idiopathic pulmonary fibrosis.
The number of mesenchymal cells, as well as their ability to synthesize extracellular matrix (ECM) components, greatly increase in the interstitium of fibrotic lungs. We have previously shown that the transcription of type I procollagen and fibronectin genes in the lungs is preferentially elevated during the early stages of bleomycin-induced pulmonary fibrosis (Raghow, R., S. Lurie, J. M. Seyer, and A. H. Kang. 1985, J. Clin. Invest. 76:1734-1739. Since a cytokine-like transforming growth factor beta (TGF beta) that is capable of enhancing mesenchymal cell proliferation and ECM synthesis could be potentially involved in this process, we investigated the temporal relationship between the regulation of TGF beta gene transcription and cellular proliferation in the bleomycin-treated hamster lungs. We observed a transient 5-7-fold increase in the accumulation of TGF beta transcripts, a concomitant 3-4-fold elevation in the cellular proliferation, and 8-10-fold stimulation of DNA synthesis in these lungs; all three parameters peaked around day 10 after bleomycin administration. Based on these results, we conclude that regulation of TGF beta gene expression may contribute significantly to the early events that lead to bleomycin-induced pulmonary fibrosis.
Pulmonary fibrosis, in particular idiopathic pulmonary fibrosis (IPF), results from aberrant wound healing and scarification. One population of fibroblasts involved in the fibrotic process is thought to originate from lung epithelial cells via epithelial-mesenchymal transition (EMT). Indeed, alveolar epithelial cells (AECs) undergo EMT in vivo during experimental fibrosis and ex vivo in response to TGF-β1. As the ECM critically regulates AEC responses to TGF-β1, we explored the role of the prominent epithelial integrin α3β1 in experimental fibrosis by generating mice with lung epithelial cell–specific loss of α3 integrin expression. These mice had a normal acute response to bleomycin injury, but they exhibited markedly decreased accumulation of lung myofibroblasts and type I collagen and did not progress to fibrosis. Signaling through β-catenin has been implicated in EMT; we found that in primary AECs, α3 integrin was required for β-catenin phosphorylation at tyrosine residue 654 (Y654), formation of the pY654–β-catenin/pSmad2 complex, and initiation of EMT, both in vitro and in vivo during the fibrotic phase following bleomycin injury. Finally, analysis of lung tissue from IPF patients revealed the presence of pY654–β-catenin/pSmad2 complexes and showed accumulation of pY654–β-catenin in myofibroblasts. These findings demonstrate epithelial integrin–dependent profibrotic crosstalk between β-catenin and Smad signaling and support the hypothesis that EMT is an important contributor to pathologic fibrosis.