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BACKGROUND: The interleukin (IL)-1 receptor accessory protein (IL1RAP) is an essential coreceptor required for signalling through the IL-1, IL-33 and IL-36 receptors. Here, we investigate the antifibrotic potential of the combined inhibition of these cytokines by an anti-IL1RAP antibody to provide a scientific background for clinical development in systemic sclerosis (SSc). METHODS: The expression of IL1RAP-associated signalling molecules was determined by data mining of publicly available RNA sequencing (RNAseq) data as well as by imaging mass cytometry. The efficacy of therapeutic dosing of anti-IL1RAP antibodies was determined in three complementary mouse models: sclerodermatous chronic graft-versus-host disease (cGvHD), bleomycin-induced dermal fibrosis model and topoisomerase-I (topo)-induced fibrosis. RESULTS: SSc skin showed upregulation of IL1RAP and IL1RAP-related signalling molecules on mRNA and protein level compared with normal skin. IL-1, IL-33 and IL-36 all regulate distinct gene sets related to different pathophysiological processes in SSc. The responses of human fibroblasts and endothelial cells to IL-1, IL-33 and IL-36 were completely blocked by treatment with an anti-IL1RAP antibody in vitro. Moreover, anti-IL1RAP antibody treatment reduced dermal and pulmonary fibrosis in cGvHD-induced, bleomycin-induced and topoisomerase-induced fibrosis. Importantly, RNAseq analyses revealed effects of IL1RAP inhibition on multiple processes related to inflammation and fibrosis that are also deregulated in human SSc skin. CONCLUSION: This study provides the first evidence for the therapeutic benefits of targeting IL1RAP in SSc. Our findings have high translational potential as the anti-IL1RAP antibody CAN10 has recently entered a phase one clinical trial.
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OBJECTIVES: Our previous studies have demonstrated that the Damage Associated Molecular Pattern (DAMP) protein, S100A4, is overexpressed in the involved skin and peripheral blood of patients with SSc. It is associated with skin and lung involvement, and disease activity. By contrast, lack of S100A4 prevented the development of experimental dermal fibrosis. Herein we aimed to evaluate the effect of murine anti-S100A4 mAb 6B12 in the treatment of preestablished experimental dermal fibrosis. METHODS: The effects of 6B12 were assessed at therapeutic dosages in a modified bleomycin-induced dermal fibrosis mouse model by evaluating fibrotic (dermal thickness, proliferation of myofibroblasts, hydroxyproline content, phosphorylated Smad3-positive cell count) and inflammatory (leukocytes infiltrating the lesional skin, systemic levels of selected cytokines and chemokines) outcomes, and transcriptional profiling (RNA sequencing). RESULTS: Treatment with 7.5 mg/kg 6B12 attenuated and might even reduce pre-existing dermal fibrosis induced by bleomycin as evidenced by reduction in dermal thickness, myofibroblast count and collagen content. These antifibrotic effects were mediated by the downregulation of TGF-ß/Smad signalling and partially by reducing the number of leukocytes infiltrating the lesional skin and decrease in the systemic levels of IL-1α, eotaxin, CCL2 and CCL5. Moreover, transcriptional profiling demonstrated that 7.5 mg/kg 6B12 also modulated several profibrotic and proinflammatory processes relevant to the pathogenesis of SSc. CONCLUSION: Targeting S100A4 by the 6B12 mAb demonstrated potent antifibrotic and anti-inflammatory effects on bleomycin-induced dermal fibrosis and provided further evidence for the vital role of S100A4 in the pathophysiology of SSc.
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Alarminas , Piel , Animales , Humanos , Ratones , Anticuerpos Monoclonales/farmacología , Bleomicina/toxicidad , Modelos Animales de Enfermedad , Proteína de Unión al Calcio S100A4/genética , Piel/patología , FibrosisRESUMEN
Chronic graft-versus-host disease (cGVHD) is a major life-threatening complication of allogeneic hematopoietic stem cell transplantation. The molecular mechanisms underlying cGVHD remain poorly understood, and targeted therapies for clinical use are not well established. Here, we examined the role of the canonical WNT pathway in sclerodermatous cGVHD (sclGVHD). WNT signaling was activated in human sclGVHD with increased nuclear accumulation of the transcription factor ß-catenin and a WNT-biased gene expression signature in lesional skin. Treatment with the highly selective tankryase inhibitor G007-LK, the CK1α agonist pyrvinium, or the LRP6 inhibitor salinomycin abrogated the activation of WNT signaling and protected against experimental cGVHD, without a significant impact on graft-versus-leukemia effect (GVL). Treatment with G007-LK, pyrvinium, or salinomycin almost completely prevented the development of clinical and histological features in the B10.D2 (H-2d) â BALB/c (H-2d) and LP/J (H-2b) â C57BL/6 (H-2b) models of sclGVHD. Inhibition of canonical WNT signaling reduced the release of extracellular matrix from fibroblasts and reduced leukocyte influx, suggesting that WNT signaling stimulates fibrotic tissue remodeling by direct effects on fibroblasts and by indirect inflammation-dependent effects in sclGVHD. Our findings may have direct translational potential, because pyrvinium is in clinical use, and tankyrase inhibitors are in clinical trials for other indications.
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Enfermedad Injerto contra Huésped/prevención & control , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Piranos/farmacología , Compuestos de Pirvinio/farmacología , Esclerodermia Sistémica/prevención & control , Sulfonas/farmacología , Triazoles/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Animales , Antibacterianos/farmacología , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/metabolismo , Enfermedad Injerto contra Huésped/patología , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Persona de Mediana Edad , Esclerodermia Sistémica/etiología , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patologíaRESUMEN
OBJECTIVE: X-linked inhibitor of apoptosis protein (XIAP) is a multifunctional protein with important functions in apoptosis, cellular differentiation and cytoskeletal organisation and is emerging as potential target for the treatment of various cancers. The aim of the current study was to investigate the role of XIAP in the pathogenesis of systemic sclerosis (SSc). METHODS: The expression of XIAP in human skin samples of patients with SSc and chronic graft versus host disease (cGvHD) and healthy individuals was analysed by quantitative PCR, immunofluorescence (IF) and western blot. XIAP was inactivated by siRNA-mediated knockdown and pharmacological inhibition. The effects of XIAP inactivation were analysed in cultured fibroblasts and in the fibrosis models bleomycin-induced and topoisomerase-I-(topoI)-induced fibrosis and in Wnt10b-transgenic mice. RESULTS: The expression of XIAP, but not of other inhibitor of apoptosis protein family members, was increased in fibroblasts in SSc and sclerodermatous cGvHD. Transforming growth factor beta (TGF-ß) induced the expression of XIAP in a SMAD3-dependent manner. Inactivation of XIAP reduced WNT-induced fibroblast activation and collagen release. Inhibition of XIAP also ameliorated fibrosis induced by bleomycin, topoI and overexpression of Wnt10b in well-tolerated doses. The profibrotic effects of XIAP were mediated via WNT/ß-catenin signalling. Inactivation of XIAP reduces binding of ß-catenin to TCF to in a TLE-dependent manner to block WNT/ß-catenin-dependent transcription. CONCLUSIONS: Our data characterise XIAP as a novel link between two core pathways of fibrosis. XIAP is overexpressed in SSc and cGvHD in a TGF-ß/SMAD3-dependent manner and in turn amplifies the profibrotic effects of WNT/ß-catenin signalling on fibroblasts via transducin-like enhancer of split 3. Targeted inactivation of XIAP inhibits the aberrant activation of fibroblasts in murine models of SSc.
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Esclerodermia Sistémica , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , beta Catenina , Animales , Bleomicina/farmacología , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Fibrosis , Humanos , Ratones , Esclerodermia Sistémica/patología , Piel/patología , Factor de Crecimiento Transformador beta/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidores , beta Catenina/metabolismoRESUMEN
OBJECTIVES: Interstitial lung disease (ILD) is a key driver of mortality in patients with systemic sclerosis (SSc). A lack of approved treatments encompasses a high unmet medical need. Nintedanib has recently been approved for treatment in SSc-associated ILD (SSc-ILD) following SENSCIS®, a Phase III clinical trial showing that nintedanib slows the loss of pulmonary function in patients with SSc-ILD relative to placebo, as measured by annual rate of decline in forced vital capacity over 52 weeks. The aim of this study was to compare the activity of nintedanib and mycophenolate mofetil (MMF) in a transgenic Fra2 mouse model of SSc-ILD. METHODS: Fra2 transgenic mice were treated with MMF or nintedanib. Haematoxylin and Eosin and Sirius Red staining were used to identify pulmonary fibrosis and vascular remodelling in whole lung sections. Fibrosis was quantified by Ashcroft scoring, fold change in fibrotic area, and hydroxyproline. Ki67, SM22a, CD31, and caspase-3 staining was used to quantify proliferating vascular smooth muscle cells and apoptotic endothelial cells. RESULTS: Nintedanib effectively ameliorated pulmonary vascular remodelling and fibrosis in Fra2 transgenic mice. Pulmonary fibrotic and vascular remodelling parameter scores and the apoptosis of dermal endothelial cells were significantly reduced compared with vehicle-treated Fra2 transgenic mice. Treatment with MMF had only mild antifibrotic effects and no effect on pulmonary vascular remodelling. CONCLUSIONS: In this model of SSc-ILD, nintedanib ameliorated pulmonary fibrosis, remodelling of pulmonary vasculature, and the apoptosis of endothelial cells. In contrast, MMF had minor effects on pulmonary fibrosis and no effects on vascular manifestations.
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Enfermedades Pulmonares Intersticiales , Esclerodermia Sistémica , Animales , Células Endoteliales , Humanos , Indoles , Pulmón , Enfermedades Pulmonares Intersticiales/tratamiento farmacológico , Enfermedades Pulmonares Intersticiales/etiología , Ratones , Ratones Transgénicos , Ácido Micofenólico , Esclerodermia Sistémica/complicaciones , Esclerodermia Sistémica/tratamiento farmacológico , Capacidad VitalRESUMEN
OBJECTIVES: Coactivators are a heterogeneous family of transcriptional regulators that are essential for modulation of transcriptional outcomes and fine-tune numerous cellular processes. The aim of the present study was to evaluate the role of the coactivator peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in the pathogenesis of systemic sclerosis (SSc). METHODS: Expression of PGC-1α was analysed by real-time PCR, western blot and immunofluorescence. Modulation of autophagy was analysed by reporter studies by expression of autophagy-related genes. The effects of PGC-1α knockdown on collagen production and myofibroblast differentiation were analysed in cultured human fibroblasts and in two mouse models with fibroblast-specific knockout of PGC-1α. RESULTS: The expression of PGC-1α was induced in dermal fibroblasts of patients with SSc and experimental murine fibrosis. Transforming growth factor beta (TGFß), hypoxia and epigenetic mechanisms regulate the expression of PGC-1α in fibroblasts. Knockdown of PGC-1α prevented the activation of autophagy by TGFß and this translated into reduced fibroblast-to-myofibroblast differentiation and collagen release. Knockout of PGC-1α in fibroblasts prevented skin fibrosis induced by bleomycin and by overexpression of a constitutively active TGFß receptor type I. Moreover, pharmacological inhibition of PGC-1α by SR18292 induced regression of pre-established, bleomycin-induced skin fibrosis. CONCLUSION: PGC-1α is upregulated in SSc and promotes autophagy to foster TGFß-induced fibroblast activation. Targeting of PGC-1α prevents aberrant autophagy, inhibits fibroblast activation and tissue fibrosis and may over therapeutic potential.
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Autofagia/genética , Fibroblastos/fisiología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/fisiología , Esclerodermia Sistémica/genética , Esclerodermia Sistémica/patología , Animales , Bleomicina/farmacología , Western Blotting , Colágeno/biosíntesis , Modelos Animales de Enfermedad , Fibrosis , Técnica del Anticuerpo Fluorescente , Humanos , Ratones , Reacción en Cadena de la Polimerasa , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia ArribaRESUMEN
OBJECTIVES: Fibrosis is a complex pathophysiological process involving interplay between multiple cell types. Experimental modelling of fibrosis is essential for the understanding of its pathogenesis and for testing of putative antifibrotic drugs. However, most current models employ either phylogenetically distant species or rely on human cells cultured in an artificial environment. Here we evaluated the potential of vascularised in vitro human skin equivalents as a novel model of skin fibrosis and a platform for the evaluation of antifibrotic drugs. METHODS: Skin equivalents were assembled on a three-dimensional extracellular matrix by sequential seeding of endothelial cells, fibroblasts and keratinocytes. Fibrotic transformation on exposure to transforming growth factor-ß (TGFß) and response to treatment with nintedanib as an established antifibrotic agent were evaluated by quantitative polymerase chain reaction (qPCR), capillary Western immunoassay, immunostaining and histology. RESULTS: Skin equivalents perfused at a physiological pressure formed a mature, polarised epidermis, a stratified dermis and a functional vessel system. Exposure of these models to TGFß recapitulated key features of SSc skin with activation of TGFß pathways, fibroblast to myofibroblast transition, increased release of collagen and excessive deposition of extracellular matrix. Treatment with the antifibrotic agent nintedanib ameliorated this fibrotic transformation. CONCLUSION: Our data provide evidence that vascularised skin equivalents can replicate key features of fibrotic skin and may serve as a platform for evaluation of antifibrotic drugs in a pathophysiologically relevant human setting.
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Indoles/uso terapéutico , Enfermedades de la Piel/tratamiento farmacológico , Piel/patología , Células Cultivadas , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Fibrosis/tratamiento farmacológico , Fibrosis/patología , Humanos , Inhibidores de Proteínas Quinasas/uso terapéutico , Piel/irrigación sanguínea , Piel/efectos de los fármacos , Enfermedades de la Piel/patologíaRESUMEN
OBJECTIVES: Wnt signalling has been implicated in activating a fibrogenic programme in fibroblasts in systemic sclerosis (SSc). Porcupine is an O-acyltransferase required for secretion of Wnt proteins in mammals. Here, we aimed to evaluate the antifibrotic effects of pharmacological inhibition of porcupine in preclinical models of SSc. METHODS: The porcupine inhibitor GNF6231 was evaluated in the mouse models of bleomycin-induced skin fibrosis, in tight-skin-1 mice, in murine sclerodermatous chronic-graft-versus-host disease (cGvHD) and in fibrosis induced by a constitutively active transforming growth factor-ß-receptor I. RESULTS: Treatment with pharmacologically relevant and well-tolerated doses of GNF6231 inhibited the activation of Wnt signalling in fibrotic murine skin. GNF6231 ameliorated skin fibrosis in all four models. Treatment with GNF6231 also reduced pulmonary fibrosis associated with murine cGvHD. Most importantly, GNF6231 prevented progression of fibrosis and showed evidence of reversal of established fibrosis. CONCLUSIONS: These data suggest that targeting the Wnt pathway through inhibition of porcupine provides a potential therapeutic approach to fibrosis in SSc. This is of particular interest, as a close analogue of GNF6231 has already demonstrated robust pathway inhibition in humans and could be available for clinical trials.
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Aminopiridinas/uso terapéutico , Proteínas de la Membrana/antagonistas & inhibidores , Piperazinas/uso terapéutico , Esclerodermia Localizada/prevención & control , Esclerodermia Sistémica/prevención & control , Piel/patología , Vía de Señalización Wnt/efectos de los fármacos , Aciltransferasas , Aminopiridinas/farmacología , Animales , Bleomicina , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Fibrosis , Enfermedad Injerto contra Huésped/complicaciones , Ratones Endogámicos BALB C , Piperazinas/farmacología , Proteínas Serina-Treonina Quinasas/genética , Fibrosis Pulmonar/etiología , Fibrosis Pulmonar/prevención & control , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Esclerodermia Localizada/etiología , Esclerodermia Localizada/metabolismo , Esclerodermia Sistémica/inducido químicamente , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Piel/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
OBJECTIVE: S100A4 is a DAMP protein. S100A4 is overexpressed in patients with systemic sclerosis (SSc), and levels correlate with organ involvement and disease activity. S100A4-/- mice are protected from fibrosis. The aim of this study was to assess the antifibrotic effects of anti-S100A4 monoclonal antibody (mAb) in murine models of SSc and in precision cut skin slices of patients with SSc. METHODS: The effects of anti-S100A4 mAbs were evaluated in a bleomycin-induced skin fibrosis model and in Tsk-1 mice with a therapeutic dosing regimen. In addition, the effects of anti-S100A4 mAbs on precision cut SSc skin slices were analyzed by RNA sequencing. RESULTS: Inhibition of S100A4 was effective in the treatment of pre-established bleomycin-induced skin fibrosis and in regression of pre-established fibrosis with reduced dermal thickening, myofibroblast counts, and collagen accumulation. Transcriptional profiling demonstrated targeting of multiple profibrotic and proinflammatory processes relevant to the pathogenesis of SSc on targeted S100A4 inhibition in a bleomycin-induced skin fibrosis model. Moreover, targeted S100A4 inhibition also modulated inflammation- and fibrosis-relevant gene sets in precision cut SSc skin slices in an ex vivo trial approach. Selected downstream targets of S100A4, such as AMP-activated protein kinase, calsequestrin-1, and phosphorylated STAT3, were validated on the protein level, and STAT3 inhibition was shown to prevent the profibrotic effects of S100A4 on fibroblasts in human skin. CONCLUSION: Inhibition of S100A4 confers dual targeting of inflammatory and fibrotic pathways in complementary mouse models of fibrosis and in SSc skin. These effects support the further development of anti-S100A4 mAbs as disease-modifying targeted therapies for SSc.
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Anticuerpos Monoclonales , Bleomicina , Modelos Animales de Enfermedad , Fibrosis , Proteína de Unión al Calcio S100A4 , Esclerodermia Sistémica , Piel , Esclerodermia Sistémica/tratamiento farmacológico , Esclerodermia Sistémica/genética , Animales , Proteína de Unión al Calcio S100A4/genética , Proteína de Unión al Calcio S100A4/metabolismo , Humanos , Ratones , Piel/patología , Piel/efectos de los fármacos , Piel/metabolismo , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Factor de Transcripción STAT3/metabolismo , FemeninoRESUMEN
Fibrotic diseases impose a major socioeconomic challenge on modern societies and have limited treatment options. Adropin, a peptide hormone encoded by the energy homeostasis-associated (ENHO) gene, is implicated in metabolism and vascular homeostasis, but its role in the pathogenesis of fibrosis remains enigmatic. Here, we used machine learning approaches in combination with functional in vitro and in vivo experiments to characterize adropin as a potential regulator involved in fibroblast activation and tissue fibrosis in systemic sclerosis (SSc). We demonstrated consistent down-regulation of adropin/ENHO in skin across multiple cohorts of patients with SSc. The prototypical profibrotic cytokine TGFß reduced adropin/ENHO expression in a JNK-dependent manner. Restoration of adropin signaling by therapeutic application of bioactive adropin34-76 peptides in turn inhibited TGFß-induced fibroblast activation and fibrotic tissue remodeling in primary human dermal fibroblasts, three-dimensional full-thickness skin equivalents, mouse models of bleomycin-induced pulmonary fibrosis and sclerodermatous chronic graft-versus-host-disease (sclGvHD), and precision-cut human skin slices. Knockdown of GPR19, an adropin receptor, abrogated the antifibrotic effects of adropin in fibroblasts. RNA-seq demonstrated that the antifibrotic effects of adropin34-76 were functionally linked to deactivation of GLI1-dependent profibrotic transcriptional networks, which was experimentally confirmed in vitro, in vivo, and ex vivo using cultured human dermal fibroblasts, a sclGvHD mouse model, and precision-cut human skin slices. ChIP-seq confirmed adropin34-76-induced changes in TGFß/GLI1 signaling. Our study characterizes the TGFß-induced down-regulation of adropin/ENHO expression as a potential pathomechanism of SSc as a prototypical systemic fibrotic disease that unleashes uncontrolled activation of profibrotic GLI1 signaling.
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Esclerodermia Sistémica , Ratones , Animales , Humanos , Proteína con Dedos de Zinc GLI1/metabolismo , Proteína con Dedos de Zinc GLI1/farmacología , Fibrosis , Esclerodermia Sistémica/metabolismo , Fibroblastos/patología , Factor de Crecimiento Transformador beta/metabolismo , Piel/patología , Células Cultivadas , Modelos Animales de Enfermedad , Bleomicina/metabolismo , Bleomicina/farmacología , Proteínas del Tejido Nervioso/metabolismo , Receptores de Neurotransmisores/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Transforming growth factor ß (TGF-ß) signaling is a core pathway of fibrosis, but the molecular regulation of the activation of latent TGF-ß remains incompletely understood. Here, we demonstrate a crucial role of WNT5A/JNK/ROCK signaling that rapidly coordinates the activation of latent TGF-ß in fibrotic diseases. WNT5A was identified as a predominant noncanonical WNT ligand in fibrotic diseases such as systemic sclerosis, sclerodermatous chronic graft-versus-host disease, and idiopathic pulmonary fibrosis, stimulating fibroblast-to-myofibroblast transition and tissue fibrosis by activation of latent TGF-ß. The activation of latent TGF-ß requires rapid JNK- and ROCK-dependent cytoskeletal rearrangements and integrin αV (ITGAV). Conditional ablation of WNT5A or its downstream targets prevented activation of latent TGF-ß, rebalanced TGF-ß signaling, and ameliorated experimental fibrosis. We thus uncovered what we believe to be a novel mechanism for the aberrant activation of latent TGF-ß in fibrotic diseases and provided evidence for targeting WNT5A/JNK/ROCK signaling in fibrotic diseases as a new therapeutic approach.
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Fibroblastos , Fibrosis , Factor de Crecimiento Transformador beta , Proteína Wnt-5a , Quinasas Asociadas a rho , Proteína Wnt-5a/metabolismo , Proteína Wnt-5a/genética , Animales , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/genética , Ratones , Humanos , Fibroblastos/metabolismo , Fibroblastos/patología , Quinasas Asociadas a rho/metabolismo , Quinasas Asociadas a rho/genética , Esclerodermia Sistémica/patología , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/genética , Ratones Noqueados , Proteínas Wnt/metabolismo , Proteínas Wnt/genética , Sistema de Señalización de MAP Quinasas , Miofibroblastos/metabolismo , Miofibroblastos/patología , Transducción de Señal , Fibrosis Pulmonar Idiopática/patología , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/genéticaRESUMEN
OBJECTIVE: Fibrotic tissues are characterized by excessive crosslinking between extracellular matrix (ECM) proteins, rendering them more resistant to degradation. Although increased crosslinking of ECM is thought to play an important role for progression of tissue fibrosis, enhanced ECM crosslinking has not yet been targeted therapeutically in systemic sclerosis (SSc). Here, we investigated the role of transglutaminase 2 (TG2), a central crosslinking enzyme, in the activation of SSc fibroblasts. METHODS: We assessed TG2 expression and activity using TG2 staining, Western blotting, and TG2 activity assays. We inhibited TG2 in fibroblasts cultured under standard 2-dimensional conditions and in a 3-dimensional full-thickness equivalent skin model using monoclonal inhibitory anti-TG2 antibodies. RESULTS: TG2 expression was increased in the skin of patients with SSc compared with healthy controls, with levels particularly high in patients with SSc-associated interstitial lung disease. TG2 expression and TG2 activity were also increased in SSc dermal fibroblasts. Moreover, the levels of circulating TG2 in the plasma samples from SSc patients were increased versus samples from healthy controls. Anti-TG2 antibodies did not show consistent antifibrotic effects across different fibroblast cell lines under 2-dimensional culture conditions; however, anti-TG2 antibodies effectively reduced transforming growth factor ß-induced dermal thickening, myofibroblast differentiation, and collagen accumulation in the 3-dimensional full-thickness model of human skin. CONCLUSION: We provide the first evidence, to our knowledge, that inhibition of TG2 might be a potential antifibrotic approach in SSc. Our findings have translational potential as anti-TG2 antibodies are currently evaluated in a phase II clinical trial in chronic allograft injury and would thus be available for clinical studies in SSc (ClinicalTrials.gov identifier: NCT04335578).
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Esclerodermia Sistémica , Humanos , Fibrosis , Esclerodermia Sistémica/patología , Colágeno/metabolismo , Proteínas de la Matriz Extracelular , Fibroblastos/metabolismo , Piel/patología , Células CultivadasRESUMEN
OBJECTIVE: Mitochondrial transcription factor A (TFAM) controls the transcription of core proteins required for mitochondrial homeostasis. This study was undertaken to investigate changes in TFAM expression in systemic sclerosis (SSc), to analyze mitochondrial function, and to evaluate the consequences for fibroblast activation. METHODS: TFAM expression was analyzed by immunofluorescence and Western blotting. The effects of TFAM knockout were investigated in cultured fibroblasts and in murine models of bleomycin-induced skin fibrosis, bleomycin-induced lung fibrosis, and skin fibrosis induced by overexpression of constitutively active transforming growth factor ß type I receptor (TGFßRΙ). RESULTS: TFAM expression was down-regulated in fibroblasts in SSc skin and in cultured SSc fibroblasts. The down-regulation of TFAM was associated with decreased mitochondrial number and accumulation of damaged mitochondria with release of mitochondrial DNA (mtDNA), accumulation of deletions in mtDNA, metabolic alterations with impaired oxidative phosphorylation, and release of the mitokine GDF15. Normal fibroblasts subjected to long-term, but not acute, exposure to TGFß mimicked SSc fibroblasts, with down-regulation of TFAM and accumulation of mitochondrial damage. Down-regulation of TFAM promoted fibroblast activation with up-regulation of fibrosis-relevant Gene Ontology terms in RNA-Seq, partially in a reactive oxygen species-dependent manner. Mice with fibroblast-specific knockout of Tfam were prone to fibrotic tissue remodeling, with fibrotic responses even to NaCl instillation and enhanced sensitivity to bleomycin injection and overexpression of constitutively active TGFßRI. TFAM knockout fostered Smad3 signaling to promote fibroblast activation. CONCLUSION: Alterations in the key mitochondrial transcription factor TFAM in response to prolonged activation of TGFß and associated mitochondrial damage induce transcriptional programs that promote fibroblast-to-myofibroblast transition and drive tissue fibrosis.
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Esclerodermia Sistémica , Enfermedades de la Piel , Animales , Bleomicina/toxicidad , Células Cultivadas , ADN Mitocondrial , Proteínas de Unión al ADN , Fibroblastos/metabolismo , Fibrosis , Ratones , Proteínas Mitocondriales , Esclerodermia Sistémica/patología , Piel/patología , Enfermedades de la Piel/patología , Factores de Transcripción , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Bone mass is maintained by the balance between osteoclast-induced bone resorption and osteoblast-triggered bone formation. In inflammatory arthritis such as rheumatoid arthritis (RA), however, increased osteoclast differentiation and activity skew this balance resulting in progressive bone loss. O-GlcNAcylation is a posttranslational modification with attachment of a single O-linked ß-D-N-acetylglucosamine (O-GlcNAc) residue to serine or threonine residues of target proteins. Although O-GlcNAcylation is one of the most common protein modifications, its role in bone homeostasis has not been systematically investigated. We demonstrate that dynamic changes in O-GlcNAcylation are required for osteoclastogenesis. Increased O-GlcNAcylation promotes osteoclast differentiation during the early stages, whereas its downregulation is required for osteoclast maturation. At the molecular level, O-GlcNAcylation affects several pathways including oxidative phosphorylation and cell-cell fusion. TNFα fosters the dynamic regulation of O-GlcNAcylation to promote osteoclastogenesis in inflammatory arthritis. Targeted pharmaceutical or genetic inhibition of O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) arrests osteoclast differentiation during early stages of differentiation and during later maturation, respectively, and ameliorates bone loss in experimental arthritis. Knockdown of NUP153, an O-GlcNAcylation target, has similar effects as OGT inhibition and inhibits osteoclastogenesis. These findings highlight an important role of O-GlcNAcylation in osteoclastogenesis and may offer the potential to therapeutically interfere with pathologic bone resorption.
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Aberrant activation of fibroblasts with progressive deposition of extracellular matrix is a key feature of systemic sclerosis (SSc), a prototypical idiopathic fibrotic disease. Here, we demonstrate that the profibrotic cytokine transforming growth factor ß selectively up-regulates fibroblast growth factor receptor 3 (FGFR3) and its ligand FGF9 to promote fibroblast activation and tissue fibrosis, leading to a prominent FGFR3 signature in the SSc skin. Transcriptome profiling, in silico analysis and functional experiments revealed that FGFR3 induces multiple profibrotic pathways including endothelin, interleukin-4, and connective tissue growth factor signaling mediated by transcription factor CREB (cAMP response element-binding protein). Inhibition of FGFR3 signaling by fibroblast-specific knockout of FGFR3 or FGF9 or pharmacological inhibition of FGFR3 blocked fibroblast activation and attenuated experimental skin fibrosis in mice. These findings characterize FGFR3 as an upstream regulator of a network of profibrotic mediators in SSc and as a potential target for the treatment of fibrosis.
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Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/metabolismo , Esclerodermia Sistémica , Animales , Células Cultivadas , Fibroblastos/patología , Fibrosis , Ratones , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , Esclerodermia Sistémica/patología , Transducción de Señal , Piel/patología , Factor de Crecimiento Transformador betaRESUMEN
Uncontrolled activation of TGFß signaling is a common denominator of fibrotic tissue remodeling. Here we characterize the tyrosine phosphatase SHP2 as a molecular checkpoint for TGFß-induced JAK2/STAT3 signaling and as a potential target for the treatment of fibrosis. TGFß stimulates the phosphatase activity of SHP2, although this effect is in part counterbalanced by inhibitory effects on SHP2 expression. Stimulation with TGFß promotes recruitment of SHP2 to JAK2 in fibroblasts with subsequent dephosphorylation of JAK2 at Y570 and activation of STAT3. The effects of SHP2 on STAT3 activation translate into major regulatory effects of SHP2 on fibroblast activation and tissue fibrosis. Genetic or pharmacologic inactivation of SHP2 promotes accumulation of JAK2 phosphorylated at Y570, reduces JAK2/STAT3 signaling, inhibits TGFß-induced fibroblast activation and ameliorates dermal and pulmonary fibrosis. Given the availability of potent SHP2 inhibitors, SHP2 might thus be a potential target for the treatment of fibrosis.
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Fibroblastos/metabolismo , Fibroblastos/patología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Adulto , Anciano , Animales , Línea Celular , Regulación hacia Abajo/efectos de los fármacos , Femenino , Fibroblastos/efectos de los fármacos , Fibrosis , Humanos , Janus Quinasa 2/metabolismo , Masculino , Ratones Noqueados , Persona de Mediana Edad , Especificidad de Órganos , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Quinolinas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Transducción de Señal/efectos de los fármacos , Adulto JovenRESUMEN
BACKGROUND: Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is a glycoprotein that has been approved by the FDA for the treatment of neutropenia and leukemia in combination with chemotherapies. Recombinant hGM-CSF is produced industrially using the baker's yeast, Saccharomyces cerevisiae, by large-scale fermentation. The methylotrophic yeast, Pichia pastoris, has emerged as an alternative host cell system due to its shorter and less immunogenic glycosylation pattern together with higher cell density growth and higher secreted protein yield than S. cerevisiae. In this study, we compared the pipeline from gene to recombinant protein in these two yeasts. RESULTS: Codon optimization in silico for both yeast species showed no difference in frequent codon usage. However, rhGM-CSF expressed from S. cerevisiae BY4742 showed a significant discrepancy in molecular weight from those of P. pastoris X33. Analysis showed purified rhGM-CSF species with molecular weights ranging from 30 to more than 60 kDa. Fed-batch fermentation over 72 h showed that rhGM-CSF was more highly secreted from P. pastoris than S. cerevisiae (285 and 64 mg total secreted protein/L, respectively). Ion exchange chromatography gave higher purity and recovery than hydrophobic interaction chromatography. Purified rhGM-CSF from P. pastoris was 327 times more potent than rhGM-CSF from S. cerevisiae in terms of proliferative stimulating capacity on the hGM-CSF-dependent cell line, TF-1. CONCLUSION: Our data support a view that the methylotrophic yeast P. pastoris is an effective recombinant host for heterologous rhGM-CSF production.