ABSTRACT: Idiopathic pulmonary fibrosis (IPF) is a complex disease of unknown etiology. Environmental factors can affect disease susceptibility via epigenetic effects. Few studies explore global DNA methylation in lung fibroblasts, but none have focused on transforming growth factor beta-1 (TGF-b1) as a potential modifier of the DNA methylome. Here we analyzed changes in methylation and gene transcription in normal and IPF fibroblasts following TGF-b1 treatment. Overall design: Primary human lung fibroblasts were obtained from normal and IPF lungs. Control, treated for 24 hours and 5 days fibroblasts of both cell lines were selected for DNA extraction, bisulphite conversion, and further hybridization to the Illumina Infinium Human Methylation 450k array.
Project description:Idiopathic pulmonary fibrosis (IPF) is a complex disease of unknown etiology. Environmental factors can affect disease susceptibility via epigenetic effects. Few studies explore global DNA methylation in lung fibroblasts, but none have focused on transforming growth factor beta-1 (TGF-b1) as a potential modifier of the DNA methylome. Here we analyzed changes in methylation and gene transcription in normal and IPF fibroblasts following TGF-b1 treatment. Overall design: Primary human lung fibroblasts were obtained from normal and IPF lungs. Control, treated for 24 hours and 5 days fibroblasts of both cell lines were selected for RNA extraction and hybridization on Affymetrix microarrays.
Project description:This SuperSeries is composed of the SubSeries listed below. Idiopathic pulmonary fibrosis (IPF) is a complex disease of unknown etiology. Environmental factors can affect disease susceptibility via epigenetic effects. Few studies explore global DNA methylation in lung fibroblasts, but none have focused on transforming growth factor beta-1 (TGF-b1) as a potential modifier of the DNA methylome. Here we analyzed changes in methylation and gene transcription in normal and IPF fibroblasts following TGF-b1 treatment. Overall design: Refer to individual Series. Primary human lung fibroblasts were obtained from normal and IPF lungs. Control, treated for 24 hours and 5 days fibroblasts of both cell lines were selected for analyzing the level of gene expression and DNA methylation.
Project description:Idiopathic pulmonary fibrosis is a complex disease of unknown etiology. Environmental factors can affect disease susceptibility via epigenetic effects. Few studies explore global DNA methylation in lung fibroblasts, but none have focused on transforming growth factor beta-1 (TGF-?1) as a potential modifier of the DNA methylome. Here we analyzed changes in methylation and gene transcription in normal and IPF fibroblasts following TGF-?1 treatment. We analyzed the effects of TGF-?1 on primary fibroblasts derived from normal or IPF lungs treated for 24 hours and 5 days using the Illumina 450k Human Methylation array and the Prime View Human Gene Expression Array. TGF-?1 induced an increased number of gene expression changes after short term treatment in normal fibroblasts, whereas greater methylation changes were observed following long term stimulation mainly in IPF fibroblasts. DNA methyltransferase 3 alpha (DMNT3a) and tet methylcytosine dioxygenase 3 (TET3) were upregulated after 5-days TGF-?1 treatment in both cell types, whereas DNMT3a was upregulated after 24h only in IPF fibroblasts. Our findings demonstrate that TGF-?1 induced the upregulation of DNMT3a and TET3 expression and profound changes in the DNA methylation pattern of fibroblasts, mainly in those derived from IPF lungs.
Project description:Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease. Although the pathogenesis is poorly understood, evidence suggests that genetic and epigenetic alterations, such as DNA methylation, may play a key role. Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-? (TGF-?) superfamily and are important regulators in IPF. Here we identified BMP endothelial cell precursor-derived regulator (BMPER) as a key regulator of fibroblast activation. BMPER is a secreted glycoprotein that binds directly to BMPs and may regulate TGF-?/BMP signaling, but its role in lung fibrosis is not clear. BMPER is highly expressed in human IPF lung fibroblasts compared to normal lung fibroblasts. Demethylation agent 5'-azacytidine decreased BMPER expression in fibroblasts, and attenuated the invasion and migration of IPF lung fibroblasts. Furthermore, siRNA-mediated reduction of BMPER in the human lung fibroblasts impaired cell migration and invasion. 5'-azacytidine treatment additionally regulated BMPER expression and reduced lung fibrosis in mice in vivo. These findings demonstrate that methylation of specific genes in fibroblasts may offer a new therapeutic strategy for IPF by modulating fibroblast activation.
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:Interstitial lung fibrosis, a frequently idiopathic and fatal disease, has been linked to the increased expression of profibrotic transforming growth factor (TGF)-βs. P311 is an RNA-binding protein that stimulates TGF-β1, -β2, and -β3 translation in several cell types through its interaction with the eukaryotic translation initiation factor 3b. We report that P311 is switched on in the lungs of patients with idiopathic pulmonary fibrosis (IPF) and in the mouse model of bleomycin (BLM)-induced pulmonary fibrosis. To assess the in vivo role of P311 in lung fibrosis, BLM was instilled into the lungs of P311-knockout mice, in which fibrotic changes were significantly decreased in tandem with a reduction in TGF-β1, -β2, and -β3 concentration/activity compared with BLM-treated wild-type mice. Complementing these findings, forced P311 expression increased TGF-β concentration/activity in mouse and human lung fibroblasts, thereby leading to an activated phenotype with increased collagen production, as seen in IPF. Consistent with a specific effect of P311 on TGF-β translation, TGF-β1-, -β2-, and -β3-neutralizing antibodies downregulated P311-induced collagen production by lung fibroblasts. Furthermore, treatment of BLM-exposed P311 knockouts with recombinant TGF-β1, -β2, and -β3 induced pulmonary fibrosis to a degree similar to that found in BLM-treated wild-type mice. These studies demonstrate the essential function of P311 in TGF-β-mediated lung fibrosis. Targeting P311 could prove efficacious in ameliorating the severity of IPF while circumventing the development of autoimmune complications and toxicities associated with the use of global TGF-β inhibitors.
Project description:Vascular endothelial growth factor (VEGF)-D, a member of the VEGF family, induces both angiogenesis and lymphangiogenesis by activating VEGF receptor-2 (VEGFR-2) and VEGFR-3 on the surface of endothelial cells. Transforming growth factor (TGF)-?1 has been shown to stimulate VEGF-A expression in human lung fibroblast via the Smad3 signaling pathway and to induce VEGF-C in human proximal tubular epithelial cells. However, the effects of TGF-?1 on VEGF-D regulation are unknown. To investigate the regulation of VEGF-D, human lung fibroblasts were studied under pro-fibrotic conditions in vitro and in idiopathic pulmonary fibrosis (IPF) lung tissue. We demonstrate that TGF-?1 downregulates VEGF-D expression in a dose- and time-dependent manner in human lung fibroblasts. This TGF-?1 effect can be abolished by inhibitors of TGF-? type I receptor kinase and Jun NH2-terminal kinase (JNK), but not by Smad3 knockdown. In addition, VEGF-D knockdown in human lung fibroblasts induces G1/S transition and promotes cell proliferation. Importantly, VEGF-D protein expression is decreased in lung homogenates from IPF patients compared with control lung. In IPF lung sections, fibroblastic foci show very weak VEGF-D immunoreactivity, whereas VEGF-D is abundantly expressed within alveolar interstitial cells in control lung. Taken together, our data identify a novel mechanism for downstream signal transduction induced by TGF-?1 in lung fibroblasts, through which they may mediate tissue remodeling in IPF.
Project description:Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterized by the pathological remodeling of air sacs as a result of excessive accumulation of extracellular matrix (ECM) proteins, but the mechanism governing the robust protein expression is poorly understood. Our recent findings demonstrate that alternative polyadenylation (APA) caused by NUDT21 reduction is important for the increased expression of fibrotic mediators and ECM proteins in lung fibroblasts by shortening the 3'-untranslated regions (3'-UTRs) of mRNAs and stabilizing their transcripts, therefore activating pathological signaling pathways. Despite the importance of NUDT21 reduction in the regulation of fibrosis, the underlying mechanisms for the depletion are unknown. We demonstrate here that NUDT21 is depleted by TGF?1. We found that miR203, which is increased in IPF, was induced by TGF?1 to target the NUDT21 3'-UTR, thus depleting NUDT21 in human and mouse lung fibroblasts. TGF?1-mediated NUDT21 reduction was attenuated by the miR203 inhibitor antagomiR203 in fibroblasts. TGF?1 transgenic mice revealed that TGF?1 down-regulates NUDT21 in fibroblasts in vivo Furthermore, TGF?1 promoted differential APA of fibrotic genes, including FGF14, RICTOR, TMOD2, and UCP5, in association with increased protein expression. This unique differential APA signature was also observed in IPF fibroblasts. Altogether, our results identified TGF?1 as an APA regulator through NUDT21 depletion amplifying pulmonary fibrosis.
Project description:BACKGROUND:A critical feature for fibroblasts differentiation into myofibroblasts is the expression of alpha-smooth muscle actin (?-SMA) during the tissue injury and repair process. The epigenetic mechanism, DNA methylation, is involved in regulating ?-SMA expression. It is not clear how methyl-CpG-binding protein 2 (MeCP2) interacts with CpG-rich region in ?-SMA, and if the CpG methylation status would affect MeCP2 binding and regulation of ?-SMA expression. METHODS:The association of MeCP2 with ?-SMA CpG rich region were examined by chromatin immunoprecipitation (ChIP) assays in primary fibroblasts from idiopathic pulmonary fibrosis (IPF) and non-IPF control individuals, and in the lung fibroblasts treated with profibrotic cytokine transforming growth factor ?1 (TGF-?1). The regulation of ?-SMA by MeCP2 was examined by knocking down MeCP2 with small interfering RNA (siRNA). To explore the effects of the DNA methylation status of the CpG rich region on ?-SMA expression, the cells were treated with DNA methyltransferase inhibitor, 5'-azacytidine (5'-aza). The expression of ?-SMA was examined by Western blot and quantitative polymerase chain reaction, the association with MeCP2 was assessed by ChIP assays, and the methylation status was checked by bisulfate sequencing. RESULTS:The human lung fibroblasts with increased ?-SMA showed an enriched association of MeCP2, while knockdown MeCP2 by siRNA reduced ?-SMA upregulation by TGF-?1. The 5'-Aza-treated cells have decreased ?-SMA expression with reduced MeCP2 association. However, bisulfite sequencing revealed that most CpG sites are unmethylated despite the different expression levels of ?-SMA after being treated by TGF-?1 or 5'-aza. CONCLUSION:Our data indicate that the methyl-binding protein MeCP2 is critical for ?-SMA expression in human lung myofibroblast, and the DNA methylation status at the CpG rich region of ?-SMA is not a determinative factor for its inducible expression.
Project description:<h4>Background</h4>Idiopathic pulmonary fibrosis (IPF) is a poorly understood progressive disease characterized by the recurrent damage of alveolar epithelial cells as well as inappropriate expansion and activation of fibroblasts resulting in pronounced extracellular matrix (ECM) deposition. Although recent studies have indicated the involvement of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein regulating ECM deposition, in the pathogenesis of fibrosis, factors regulating SPARC expression or roles of SPARC in fibrosis have not been fully elucidated.<h4>Results</h4>Among the profibrotic factors examined in cultured fibroblasts, we showed that SPARC expression was upregulated mainly by transforming growth factor (TGF)-?. We also showed that expression of SPARC in the lung was upregulated in the murine bleomycin-induced pulmonary fibrosis model, which was inhibited by TGF-? receptor I inhibitor. Knockdown of SPARC in fibroblasts using siRNA or treatment with the antioxidant N-acetylcysteine attenuated epithelial cell injury induced by TGF-?-activated fibroblasts in a coculture system. We also demonstrated that SPARC was required for hydrogen peroxide (H2O2) production in fibroblasts treated with TGF-?. Furthermore, TGF-? activated integrin-linked kinase (ILK), which was inhibited by SPARC siRNA. Knockdown of ILK attenuated extracellular H2O2 generation in TGF-?-stimulated fibroblasts. Our results indicated that SPARC is upregulated by TGF-? and is required for TGF-?-induced H2O2 production via activation of ILK, and this H2O2 production from fibroblasts is capable of causing epithelial cell injury.<h4>Conclusions</h4>The results presented in this study suggest that SPARC plays a role in epithelial damage in the IPF lung via enhanced H2O2 production from fibroblasts activated by TGF-?. Therefore, SPARC inhibition may prevent epithelial injury in IPF lung and represent a potential therapeutic approach for IPF.