Dual targeting of MEK and PI3K pathways attenuates established and progressive pulmonary fibrosis.
ABSTRACT: Pulmonary fibrosis is often triggered by an epithelial injury resulting in the formation of fibrotic lesions in the lung, which progress to impair gas exchange and ultimately cause death. Recent clinical trials using drugs that target either inflammation or a specific molecule have failed, suggesting that multiple pathways and cellular processes need to be attenuated for effective reversal of established and progressive fibrosis. Although activation of MAPK and PI3K pathways have been detected in human fibrotic lung samples, the therapeutic benefits of in vivo modulation of the MAPK and PI3K pathways in combination are unknown. Overexpression of TGF? in the lung epithelium of transgenic mice results in the formation of fibrotic lesions similar to those found in human pulmonary fibrosis, and previous work from our group shows that inhibitors of either the MAPK or PI3K pathway can alter the progression of fibrosis. In this study, we sought to determine whether simultaneous inhibition of the MAPK and PI3K signaling pathways is a more effective therapeutic strategy for established and progressive pulmonary fibrosis. Our results showed that inhibiting both pathways had additive effects compared to inhibiting either pathway alone in reducing fibrotic burden, including reducing lung weight, pleural thickness, and total collagen in the lungs of TGF? mice. This study demonstrates that inhibiting MEK and PI3K in combination abolishes proliferative changes associated with fibrosis and myfibroblast accumulation and thus may serve as a therapeutic option in the treatment of human fibrotic lung disease where these pathways play a role.
Project description:Pulmonary fibrosis is often triggered by an epithelial injury resulting in the formation of fibrotic lesions in the lung, which progress to impair gas exchange and ultimately cause death. Recent clinical trials using drugs that target either inflammation or a specific molecule have failed, suggesting that multiple pathways and cellular processes need to be attenuated for effective reversal of established and progressive fibrosis. Although activation of MAPK and PI3K pathways have been detected in human fibrotic lung samples, the therapeutic benefits of in vivo modulation of the MAPK and PI3K pathways in combination are unknown. Overexpression of TGFα in the lung epithelium of transgenic mice results in the formation of fibrotic lesions similar to those found in human pulmonary fibrosis, and previous work from our group shows that inhibitors of either the MAPK or PI3K pathway can alter the progression of fibrosis. In this study, we sought to determine whether simultaneous inhibition of the MAPK and PI3K signaling pathways is a more effective therapeutic strategy for established and progressive pulmonary fibrosis. Our results showed that inhibiting both pathways had additive effects compared to inhibiting either pathway alone in reducing fibrotic burden, including reducing lung weight, pleural thickness, and total collagen in the lungs of TGFα mice. This study demonstrates that inhibiting MEK and PI3K in combination abolishes proliferative gene changes associated with fibrosis and myfibroblast accumulation and thus may serve as a therapeutic option in the treatment of human fibrotic lung disease where these pathways play a role. mRNA profiles of CCSP/TGFalpha mice treated with vehicle, ARRY, PX-866, ARRY/PX-866
Project description:Myofibroblast apoptosis is critical for the normal resolution of wound repair responses, and impaired myofibroblast apoptosis is associated with tissue fibrosis. Lung expression of endothelin (ET)-1, a soluble peptide implicated in fibrogenesis, is increased in murine models of pulmonary fibrosis and in the lungs of humans with pulmonary fibrosis. Mechanistically, ET-1 has been shown to induce fibroblast proliferation, differentiation, contraction, and collagen synthesis. In this study, we examined the role ET-1 in the regulation of lung fibroblast survival and apoptosis. ET-1 rapidly activates the prosurvival phosphatidylinositol 3'-OH kinase (PI3K)/AKT signaling pathway in normal and fibrotic human lung fibroblasts. ET-1-induced activation of PI3K/AKT is dependent on p38 mitogen-activated protein kinase (MAPK), but not extracellular signal-regulated kinase (ERK) 1/2, JNK, or transforming growth factor (TGF)-beta1. Activation of the PI3K/AKT pathway by ET-1 inhibits fibroblast apoptosis, and this inhibition is reversed by blockade of p38 MAPK or PI3K. TGF-beta1 has been shown to attenuate myofibroblast apoptosis through the p38 MAPK-dependent secretion of a soluble factor, which activates PI3K/AKT. In this study, we show that, although TGF-beta1 induces fibroblast synthesis and secretion of ET-1, TGF-beta1 activation of PI3K/AKT is not dependent on ET-1. We conclude that ET-1 and TGF-beta1 independently promote fibroblast resistance to apoptosis through signaling pathways involving p38 MAPK and PI3K/AKT. These findings suggest the potential for novel therapies targeting the convergence of prosurvival signaling pathways activated by these two profibrotic mediators.
Project description:Pulmonary fibrosis is a progressive and fatal disorder. In our previous study, we found that the Yiqihuoxue formula (YQHX), a prescription of Traditional Chinese Medicine, had a curative effect on scleroderma, a typical fibrotic disease. The aim of this study was to determine the key ingredient mediating the therapeutic effects of YQHX and to examine its effect on pulmonary fibrosis, including its mechanism. Luciferase reporter assays showed that the most important anti-fibrotic component of the YQHX was Salviae miltiorrhiza (SM). Experiments performed using a bleomycin-instilled mouse model of pulmonary fibrosis showed that Salvianolic acid B (SAB), the major ingredient of SM, had strong anti-inflammatory and anti-fibrotic effects through its inhibition of inflammatory cell infiltration, alveolar structure disruption, and collagen deposition. Furthermore, SAB suppressed TGF-?-induced myofibroblastic differentiation of MRC-5 fibroblasts and TGF-?-mediated epithelial-to-mesenchymal transition of A549 cells by inhibiting both Smad-dependent signaling and the Smad-independent MAPK pathway. Taken together, our results suggest that SM is the key anti-fibrotic component of the YQHX and that SAB, the major ingredient of SM, alleviates experimental pulmonary fibrosis both in vivo and in vitro by inhibiting the TGF-? signaling pathway. Together, these results suggest that SAB potently inhibits pulmonary fibrosis.
Project description: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-? (TGF-?)-induced differentiation and proliferation of myofibroblasts is recognized as a primary event. The molecular pathways involved in TGF-? 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-?: fibroblast proliferation and differentiation into myofibroblasts. Here we show that the pan-inhibitor of PI3Ks LY294002 is able to abrogate the TGF-?-induced increase in cell proliferation, in ?- 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 p110? and p110? are functionally expressed in human lung fibroblasts, in addition to the ubiquitously expressed p110? and ?. Finally, results obtained with both selective inhibitors and gene knocking-down experiments demonstrate a major role of p110? and p110? in both TGF-?-induced fibroblast proliferation and differentiation. This finding suggests that specific PI3K isoforms can be pharmacological targets in IPF.
Project description:Pulmonary fibrosis is common in a variety of inflammatory lung diseases, there is currently no effective clinical drug treatment. It has been reported that the ethanol extract of Eclipta prostrata L. can improve the lung collagen deposition and fibrosis pathology induced by bleomycin (BLM) in mice. In the present study, we studied whether wedelolactone (WEL), a major coumarin ingredient of E. prostrata, provided protection against BLM-induced pulmonary fibrosis. ICR or C57/BL6 strain mice were treated with BLM to establish lung fibrosis model. WEL (2 or 10 mg/kg) was given daily via intragastric administration for 2 weeks starting at 7-day after intratracheal instillation. WEL at 10 mg/kg significantly reduced BLM-induced inflammatory cells infiltration, pro-inflammatory factors expression, and collagen deposition in lung tissues. Additionally, treatment with WEL also impaired BLM-induced increases in fibrotic marker expression (collagen I and ?-SMA) and decrease in an anti-fibrotic marker (E-cadherin). Treatment with WEL significantly prevented BLM-induced increase in TGF-?1 and Smad2/3 phosphorylation in the lungs. WEL administration (10 mg/kg) also significantly promoted AMPK activation compared to model group in BLM-treated mice. Further investigation indicated that activation of AMPK by WEL can suppressed the transdifferentiation of primary lung fibroblasts and the epithelial mesenchymal transition (EMT) of alveolar epithelial cells, the inhibitive effects of WEL was significantly blocked by an AMPK inhibitor (compound C) in vitro. Together, these results suggest that activation of AMPK by WEL followed by reduction in TGF?1/Raf-MAPK signaling pathways may have a therapeutic potential in pulmonary fibrosis.
Project description:BACKGROUND:Pirfenidone, an antifibrotic agent used for the treatment of idiopathic pulmonary fibrosis (IPF), functions by inhibiting myofibroblast differentiation, which is involved in transforming growth factor (TGF)-β1-induced IPF pathogenesis. However, unlike normal lung fibroblasts, the relationship between pirfenidone responses of TGF-β1-induced human fibrotic lung fibroblasts and lung fibrosis has not been elucidated. METHODS:The effects of pirfenidone were evaluated in lung fibroblasts isolated from fibrotic human lung tissues after TGF-β1 exposure. The ability of two new pharmacological targets of pirfenidone, collagen triple helix repeat containing protein 1(CTHRC1) and four-and-a-half LIM domain protein 2 (FHL2), to mediate contraction of collagen gels and migration toward fibronectin were assessed in vitro. RESULTS:Compared to control lung fibroblasts, pirfenidone significantly restored TGF-β1-stimulated fibroblast-mediated collagen gel contraction, migration, and CTHRC1 release in lung fibrotic fibroblasts. Furthermore, pirfenidone attenuated TGF-β1- and CTHRC1-induced fibroblast activity, upregulation of bone morphogenic protein-4(BMP-4)/Gremlin1, and downregulation of α-smooth muscle actin, fibronectin, and FHL2, similar to that observed post-CTHRC1 inhibition. In contrast, FHL2 inhibition suppressed migration and fibronectin expression, but did not downregulate CTHRC1. CONCLUSIONS:Overall, pirfenidone suppressed fibrotic fibroblast-mediated fibrotic processes via inverse regulation of CTHRC1-induced lung fibroblast activity. Thus, CTHRC1 can be used for predicting pirfenidone response and developing new therapeutic targets for lung fibrosis.
Project description:TGF?1 signaling is a critical driver of collagen accumulation in pulmonary fibrotic diseases and a well-characterized regulator of cancer associated fibroblasts (CAF) activation in lung cancer. Myofibroblasts induced by TGF?1 and other factors are key players in the pathogenesis of lung fibrosis and tumor. Tremendous attention has been gained to targeting myofibroblasts in order to inhibit the progression of fibrosis and myofibroblast-induced tumor progression and metastasis. Here we determined the therapeutic efficacy of simultaneously targeting PI3K and HDAC pathways in lung myofibroblasts and CAF with a single agent and to evaluate biomarkers of treatment response. CUDC-907 is a first-in-class compound, functioning as a dual inhibitor of HDACs and PI3K/AKT pathway. We investigated its effects in counteracting the activity of TGF?1-induced myofibroblasts/CAF in regard to cell proliferation, migration, invasion, apoptosis in vitro antifibrosis efficiency in vivo. We found that CUDC-907 inhibited myofibroblasts/CAF cell proliferation, migration and apoptosis in a dose-dependent manner and caused cell cycle arrest at G1-S phase. CUDC-907 not only inhibited myofibroblasts markers expression, but also significantly inhibited the phosphorylation level of AKT, mTOR, Smad2/3, and promoted acetylation of histones. Furthermore, the observed inhibitory effect was also confirmed in bleomycin-induced mice lung fibrosis and nude mouse transplanted tumor model. Overall, these data suggest that dual inhibition of HDAC and the tyrosine kinase signaling pathways with CUDC-907 is a promising treatment strategy for TGF?1-induced lung and tumor fibrosis.
Project description:Idiopathic pulmonary fibrosis (IPF) is a fatal disease in which the intricate alveolar network of the lung is progressively replaced by fibrotic scars. Myofibroblasts are the effector cells that excessively deposit extracellular matrix proteins thus compromising lung structure and function. Emerging literature suggests a correlation between fibrosis and metabolic alterations in IPF. In this study, we show that the first-line antidiabetic drug metformin exerts potent antifibrotic effects in the lung by modulating metabolic pathways, inhibiting TGF?1 action, suppressing collagen formation, activating PPAR? signaling and inducing lipogenic differentiation in lung fibroblasts derived from IPF patients. Using genetic lineage tracing in a murine model of lung fibrosis, we show that metformin alters the fate of myofibroblasts and accelerates fibrosis resolution by inducing myofibroblast-to-lipofibroblast transdifferentiation. Detailed pathway analysis revealed a two-arm mechanism by which metformin accelerates fibrosis resolution. Our data report an antifibrotic role for metformin in the lung, thus warranting further therapeutic evaluation.
Project description:Pulmonary fibrosis remains a significant public health burden with no proven therapies. The mitogen-activated protein kinase (MAPK)/MAPK kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling cascade is a major pathway controlling cellular processes associated with fibrogenesis, including growth, proliferation, and survival. Activation of the MAPK/ERK pathway is detected in the lungs of human fibrosis samples; however, the effect of modulating the pathway in vivo is unknown. Overexpression of transforming growth factor (TGF)-? in the lung epithelium of transgenic mice causes a progressive pulmonary fibrosis associated with increased MEK/ERK activation localized primarily in mesenchymal cells. To determine the role of the MEK pathway in the induction of TGF-?-induced lung fibrosis, TGF-? was overexpressed for 4 weeks while mice were simultaneously treated with the specific MEK inhibitor, ARRY-142886 (ARRY). Treatment with ARRY prevented increases in lung cell proliferation and total lung collagen, attenuated production of extracellular matrix genes, and protected mice from changes in lung function. ARRY administered as a rescue treatment after fibrosis was already established inhibited fibrosis progression, as assessed by lung histology, changes in body weights, extracellular matrix gene expression, and lung mechanics. These findings demonstrate that MEK inhibition prevents progression of established fibrosis in the TGF-? model, and provides proof of concept of targeting the MEK pathway in fibrotic lung disease.
Project description:Silicosis is a very serious occupational disease and it features pathological manifestations of inflammatory infiltration, excessive proliferation of fibroblasts and massive depositions of the extracellular matrix in the lungs. Recent studies described the roles of a variety of microRNAs (miRNAs) in fibrotic diseases. Here, we aimed to explore the potential mechanism of miR-542-5p in the activation of lung fibroblasts. To induce a pulmonary fibrosis mouse model, silica suspension and the miR-542-5p agomir were administered to mice by intratracheal instillation and tail vein injection. We found that miR-542-5p was significantly decreased in mouse fibrotic lung tissues and up-regulation of miR-542-5p visually attenuated a series of fibrotic lesions, including alveolar structural damage, alveolar interstitial thickening and silica-induced nodule formation. The down-regulation of miR-542-5p was also observed in mouse fibroblast (NIH-3T3) treated with transforming growth factor ?1 (TGF-?1). The proliferation and migration ability of NIH-3T3 cells were also inhibited by the transfection of miR-542-5p mimic. Integrin ?6 (Itga6), reported as a cell surface protein associated with fibroblast proliferation, was confirmed to be a direct target of miR-542-5p. The knockdown of Itga6 significantly inhibited the phosphorylation of FAK/PI3K/AKT. In conclusion, miR-542-5p has a potential function for reducing the proliferation of fibroblasts and inhibiting silica-induced pulmonary fibrosis, which might be partially realized by directly binding to Itga6. Our data suggested that miR-542-5p might be a new therapeutic target for silicosis or other pulmonary fibrosis.