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BACE1 is well-known for its role in the development of Alzheimer's disease. Recent publications, including our own, have demonstrated a role for this enzyme in other chronic diseases. The aim of this study was to investigate the role of BACE1 in the autoimmune disease systemic sclerosis (SSc). BACE1 protein levels were elevated in the skin of patients with SSc. Inhibition of BACE1 with small-molecule inhibitors or small interfering RNA blocked SSc and fibrotic stimuli-mediated fibroblast activation. Furthermore, we show that BACE1 regulation of dermal fibroblast activation is dependent on ß-catenin and Notch signaling. The neurotropic factor brain-derived neurotrophic factor negatively regulates BACE1 expression and activity in dermal fibroblasts. Finally, sera from patients with SSc show higher ß-amyloid and lower brain-derived neurotrophic factor levels than healthy controls. The ability of BACE1 to regulate SSc fibroblast activation reveals a therapeutic target in SSc. Several BACE1 inhibitors have been shown to be safe in clinical trials for Alzheimer's disease and could be repurposed to ameliorate fibrosis progression.
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BACKGROUND: Systemic sclerosis (SSc) is a heterogeneous disease, characterized by variable tissue and vascular fibrosis in the context of autoimmune activation. CCL24 (or Eotaxin2) has been shown to promote microangiopathic, proinflammatory, and profibrotic processes in preclinical models of SSc. Here, we study serum CCL24 levels in a real-life cohort of patients with SSc, to determine its distribution across disease features and its value in predicting disease progression and related mortality. METHODS: Serum CCL24 was assessed in an observational cohort of consecutively enrolled patients with SSc. A high CCL24 cutoff was defined based on its distribution in a matched cohort of healthy controls. Disease progression and mortality were analyzed from the date of serum assessment. RESULTS: Two-hundred thirteen consecutively enrolled patients with SSc were included in this analysis. Median disease duration was six years (interquartile range 3-14), 28.6% of patients presented with interstitial lung disease (ILD), 46.9% had digital ulcers, and 25.3% showed high CCL24 serum concentration. High-CCL24 patients were more frequently male and positive for anti-scl-70, with a diagnosis of ILD and synovitis (P < 0.05 for all). Notably, high-CCL24 patients had lower diffusion of carbon monoxide and higher prevalence of digital ulcers, telangiectasias, and calcinosis (P < 0.05 for all). In a longitudinal setting, high CCL24 was associated with greater lung function decline and with higher disease-related mortality. CONCLUSION: Serum CCL24 is a biomarker of disease severity across fibrotic and vascular disease manifestations. These data support the development of therapies targeting CCL24 as a novel comprehensive therapeutic target in SSc.
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BACKGROUND: Activation of melanocortin 1 receptor (MC1R) is known to exert broad anti-inflammatory and anti-fibrotic effects. The purpose of this study is to investigate the potential of dersimelagon, a novel oral MC1R agonist, as a therapeutic agent for systemic sclerosis (SSc). METHODS: The effects of dersimelagon phosphoric acid (MT-7117) on skin fibrosis and lung inflammation were evaluated in bleomycin (BLM)-induced SSc murine models that were optimized for prophylactic and therapeutic evaluation. Microarray-based gene expression analysis and serum protein profiling were performed in the BLM-induced SSc models. The effect of MT-7117 on transforming growth factor-ß (TGF-ß)-induced activation of human dermal fibroblasts was evaluated in vitro. Immunohistochemical analyses of MC1R expression in the skin of SSc patients were performed. RESULTS: Prophylactic treatment with MT-7117 (≥ 0.3 mg/kg/day p.o.) significantly inhibited skin fibrosis and lung inflammation, and therapeutic treatment with MT-7117 (≥ 3 mg/kg/day p.o.) significantly suppressed the development of skin fibrosis in the BLM-induced SSc models. Gene array analysis demonstrated that MT-7117 exerts an anti-inflammatory effect via suppression of the activation of inflammatory cells and inflammation-related signals; additionally, vascular dysfunction was extracted as the pathology targeted by MT-7117. Serum protein profiling revealed that multiple SSc-related biomarkers including P-selectin, osteoprotegerin, cystatin C, growth and differentiation factor-15, and S100A9 were suppressed by MT-7117. MT-7117 inhibited the activation of human dermal fibroblasts by suppressing TGF-ß-induced ACTA2 (encoding α-smooth muscle actin) mRNA elevation. MC1R was expressed by monocytes/macrophages, neutrophils, blood vessels (endothelial cells), fibroblasts, and epidermis (keratinocytes) in the skin of SSc patients, suggesting that these MC1R-positive cells could be targets for MT-7117. CONCLUSIONS: MT-7117 demonstrates disease-modifying effects in preclinical models of SSc. Investigations of its mechanism of action and target expression analyses indicate that MT-7117 exerts its positive effect by affecting inflammation, vascular dysfunction, and fibrosis, which are all key pathologies of SSc. The results of the present study suggest that MT-7117 is a potential therapeutic agent for SSc. A phase 2 clinical trial investigating the efficacy and tolerability of MT-7117 in patients with early, progressive diffuse cutaneous SSc is currently in progress.
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Neumonía , Esclerodermia Sistémica , Animales , Bleomicina/toxicidad , Proteínas Sanguíneas , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Fibroblastos/metabolismo , Fibrosis , Humanos , Inflamación/patología , Ratones , Neumonía/metabolismo , Receptor de Melanocortina Tipo 1/metabolismo , Esclerodermia Sistémica/inducido químicamente , Esclerodermia Sistémica/tratamiento farmacológico , Esclerodermia Sistémica/metabolismo , Transducción de Señal/fisiología , Piel/patología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Systemic sclerosis (SSc) is a terminal disease characterized by vasculopathy, tissue fibrosis, and autoimmunity. Although the exact etiology of SSc remains unknown, endothelial dysfunction, oxidative stress, and calcium handling dysregulation have been associated with a large number of SSc-related complications such as neointima formation, vasculogenesis, pulmonary arterial hypertension, impaired angiogenesis, and cardiac arrhythmias. Hemeoxygenase-1 (HO-1) is an antioxidant enzyme involved in multiple biological actions in the cardiovascular system including vascular tone, angiogenesis, cellular proliferation, apoptosis, and oxidative stress. The aim of this work was to investigate the physiological role of HO-1 and its relevance in the cardiovascular complications occurring in SSc. We found that, in early phases of SSc, the expression of HO-1 in dermal fibroblast is lower compared to those isolated from healthy control individuals. This is particularly relevant as reduction of the HO-1/CO signaling pathway is associated with endothelial dysfunction and vasculopathy. We show evidence of the role of HO-1/carbon monoxide (CO) signaling pathway in calcium handling. Using an in vitro model of pulmonary arterial hypertension (PAH) we investigated the role of HO-1 in Ca2+ mobilization from intracellular stores. Our results indicate that HO-1 regulates calcium release from intracellular stores of human pulmonary arterial endothelial cells. We interrogated the activity of HO-1 in angiogenesis using an organotypic co-culture of fibroblast-endothelial cell. Inhibition of HO-1 significantly reduced the ability of endothelial cells to form tubules. We further investigated if this could be associated with cell motility or migration of endothelial cells into the extracellular matrix synthesized by fibroblasts. By mean of holographic imaging, we studied the morphological and functional features of endothelial cells in the presence of an HO-1 activator and selective inhibitors. Our results demonstrate that inhibition of HO-1 significantly reduces cell proliferation and cell motility (migration) of cultured endothelial cells, whilst activation of HO-1 does not modify either morphology, proliferation or motility. In addition, we investigated the actions of CO on the Kv7.1 (KCQN1) channel current, an important component of the cardiac action potential repolarization. Using electrophysiology (whole-cell patch-clamp in a recombinant system overexpressing the KCQN1 channel), we assessed the regulation of KCQN1 by CO. CORM-2, a CO donor, significantly reduced the Kv7.1 current, suggesting that HO-1/CO signaling may play a role in the modulation of the cardiac action potential via regulation of this ion channel. In summary, our results indicate a clear link between: 1) downregulation of HO-1/CO signaling; and 2) pathophysiological processes occurring in early phases of SSc, such as calcium homeostasis dysregulation, impaired angiogenesis and cardiac arrhythmias. A better understanding of the canonical actions (mainly due to the biological actions of CO), and non-canonical actions of HO-1, as well as the interaction of HO-1/CO signaling with other gasotransmitters in SSc will contribute to the development of novel therapeutic approaches.
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OBJECTIVES: To assess antibody and T cell responses to SARS-CoV-2 vaccination in patients with rheumatoid arthritis (RA) on disease-modifying antirheumatic drugs (DMARDs). METHODS: This prospective study recruited 100 patients with RA on a variety of DMARDs for antibody and T cell analysis, pre-vaccination and 4 weeks post-vaccination. Positive antibody response was defined as sera IgG binding to ≥1 antigen. Those that remained seronegative after first vaccination were retested 4 weeks after second vaccination; and if still seronegative after vaccination three. A T cell response was defined an ELISpot count of ≥7 interferon (IFN)γ-positive cells when exposed to spike antigens. Type I IFN activity was determined using the luminex multiplex assay IFN score. RESULTS: After vaccine one, in patients without prior SARS-CoV-2 exposure, 37/83 (45%) developed vaccine-specific antibody responses, 44/83 (53%) vaccine-specific T cell responses and 64/83 (77%) developed either antibody or T cell responses. Reduced seroconversion was seen with abatacept, rituximab (RTX) and those on concomitant methotrexate (MTX) compared to 100% for healthy controls (p<0.001). Better seroconversion occurred with anti-tumour necrosis factor (TNF) versus RTX (p=0.012) and with age ≤50 (p=0.012). Pre-vaccine SARS-CoV-2 exposure was associated with higher quantitative seroconversion (≥3 antibodies) (p<0.001). In the subgroup of non-seroconverters, a second vaccination produced seroconversion in 54% (19/35), and after a third in 20% (2/10). IFN score analysis showed no change post-vaccine. CONCLUSION: Patients with RA on DMARDs have reduced vaccine responses, particularly on certain DMARDs, with improvement on subsequent vaccinations but with approximately 10% still seronegative after three doses.
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Antirreumáticos , Artritis Reumatoide , COVID-19 , Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , COVID-19/prevención & control , Vacunas contra la COVID-19 , Humanos , Estudios Prospectivos , SARS-CoV-2 , Linfocitos T , VacunaciónRESUMEN
Secreted Frizzled Receptor Protein 4 (SFRP4) has been shown to be increased in Scleroderma (SSc). To determine its role in immune-driven fibrosis, we analysed SSc and sclerotic Chronic Graft Versus Host Disease (sclGVHD) biosamples; skin biopsies (n = 24) from chronic GVHD patients (8 with and 5 without sclGVHD), 8 from SSc and 3 healthy controls (HC) were analysed by immunofluorescence (IF) and SSc patient sera (n = 77) assessed by ELISA. Epithelial cell lines used for in vitro Epithelial-Mesenchymal-Transition (EMT) assays and analysed by Western Blot, RT-PCR and immunofluorescence. SclGVHD skin biopsies resembled pathologic features of SSc. IF of fibrotic skin biopsies indicated the major source of SFRP4 expression were dermal fibroblasts, melanocytes and vimentin positive/caveolin-1 negative cells in the basal layer of the epidermis. In vitro studies showed increased vimentin and SFRP4 expression accompanied with decreased caveolin-1 expression during TGFß-induced EMT. Additionally, SFRP4 serum concentration correlated with severity of lung and skin fibrosis in SSc. In conclusion, SFRP4 expression is increased during skin fibrosis in two different immune-driven conditions, and during an in vitro EMT model. Its serum levels correlate with skin and lung fibrosis in SSc and may function as biomarker of EMT. Further studies are warranted to elucidate the role of SFRP4 in EMT within the pathogenesis of tissue fibrosis.
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Skeletal muscle damage is a common clinical manifestation of systemic sclerosis (SSc). C-X-C chemokine ligand 10 (CXCL10) is involved in myopathy and cardiomyopathy development and is associated with a more severe SSc prognosis. Interestingly, the phosphodiesterase type 5 inhibitor (PDE5i) sildenafil reduces CXCL10 sera levels of patients with diabetic cardiomyopathy and in cardiomyocytes. Here, we analyzed the levels of CXCL10 in the sera of 116 SSc vs. 35 healthy subjects and explored differences in 17 SSc patients on stable treatment with sildenafil. CXCL10 sera levels were three-fold higher in SSc vs. healthy controls, independent of subset and antibody positivity. Sildenafil treatment was associated with lower CXCL10 sera levels. Serum CXCL10 strongly correlated with the clinical severity of muscle involvement and with creatine kinase (CK) serum concentration, suggesting a potential involvement in muscle damage in SSc. In vitro, sildenafil dose-dependently reduced CXCL10 release by activated myocytes and impaired cytokine-induced Signal transducer and activator of transcription 1 (STAT1), Nuclear factor-κB (NFκB) and c-Jun N-terminal kinase (JNK) phosphorylation. This was also seen in cardiomyocytes. Sildenafil-induced CXCL10 inhibition at the systemic and human muscle cell level supports the hypothesis that PDE5i could be a potential therapeutic therapy to prevent and treat muscle damage in SSc.
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Quimiocina CXCL10/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/genética , Cardiomiopatías Diabéticas/tratamiento farmacológico , Esclerodermia Sistémica/tratamiento farmacológico , Citrato de Sildenafil/farmacología , Cardiomiopatías Diabéticas/sangre , Cardiomiopatías Diabéticas/patología , Femenino , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Masculino , Persona de Mediana Edad , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Miocitos Cardíacos/efectos de los fármacos , FN-kappa B , Inhibidores de Fosfodiesterasa 5/farmacología , Factor de Transcripción STAT1/genética , Esclerodermia Sistémica/sangre , Esclerodermia Sistémica/patologíaRESUMEN
OBJECTIVES: Plasmacytoid dendritic cells (pDC) have been implicated in the pathogenesis of autoimmune diseases, such as scleroderma (SSc). However, this has been derived from indirect evidence using ex vivo human samples or mouse pDC in vivo. We have developed human-specific pDC models to directly identify their role in inflammation and fibrosis, as well as attenuation of pDC function with BDCA2-targeting to determine its therapeutic application. METHODS: RNAseq of human pDC with TLR9 agonist ODN2216 and humanised monoclonal BDCA2 antibody, CBS004. Organotypic skin rafts consisting of fibroblasts and keratinocytes were stimulated with supernatant from TLR9-stimulated pDC and with CBS004. Human pDC were xenotransplanted into Nonobese diabetic/severe combined immunodeficiency (NOD SCID) mice treated with Aldara (inflammatory model), or bleomycin (fibrotic model) with CBS004 or human IgG control. Skin punch biopsies were used to assess gene and protein expression. RESULTS: RNAseq shows TLR9-induced activation of human pDC goes beyond type I interferon (IFN) secretion, which is functionally inactivated by BDCA2-targeting. Consistent with these findings, we show that BDCA2-targeting of pDC can completely suppress in vitro skin IFN-induced response. Most importantly, xenotransplantation of human pDC significantly increased in vivo skin IFN-induced response to TLR agonist and strongly enhanced fibrotic and immune response to bleomycin compared with controls. In these contexts, BDCA2-targeting suppressed human pDC-specific pathological responses. CONCLUSIONS: Our data indicate that human pDC play a key role in inflammation and immune-driven skin fibrosis, which can be effectively blocked by BDCA2-targeting, providing direct evidence supporting the development of attenuation of pDC function as a therapeutic application for SSc.
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Células Dendríticas/inmunología , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Inmunológicos/metabolismo , Esclerodermia Localizada/inmunología , Esclerodermia Localizada/patología , Animales , Células Dendríticas/patología , Modelos Animales de Enfermedad , Fibrosis , Xenoinjertos , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Esclerodermia Localizada/metabolismo , Piel/inmunología , Piel/metabolismo , Piel/patologíaRESUMEN
OBJECTIVES: Tissue fibrosis in SSc is driven by active fibroblasts (myofibroblasts). Previous studies have shown the intracellular chloride channel 4 (CLIC4) mediates the activation of cancer-associated fibroblasts. In this study we investigated the role of CLIC4 in SSc fibroblast activation. METHODS: Fibroblasts were obtained from full thickness skin biopsies from SSc patients (early-diffuse). RNA and protein were collected from the fibroblasts and CLIC4 transcript and protein levels were assessed by qPCR and western blot. SSc patient fibroblasts were treated with the chloride channel inhibitors nitro-2-(3-phenylpropylamino)benzoic acid and indyanyloxyacetic acid 94. RESULTS: CLIC4 was expressed at significantly higher levels in SSc patients' fibroblasts compared with healthy controls, at both the transcript (3.7-fold) and protein (1.7-fold) levels. Inhibition of the TGF-ß receptor and its downstream transcription factor SMAD3 led to a reduction in CLIC4 expression, confirming this pathway as the main driver of CLIC4 expression. Importantly, treatment of SSc fibroblasts with known pharmacological inhibitors of CLIC4 led to reduced expression of the myofibroblast markers collagen type 1 and α-smooth muscle actin, inferring a direct role for CLIC4 in disease pathogenesis. CONCLUSIONS: We have identified a novel role for CLIC4 in SSc myofibroblast activation, which strengthens the similarities of SSc fibroblasts with cancer-associated fibroblasts and highlights this channel as a novel target for therapeutic intervention.
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Canales de Cloruro/metabolismo , Fibroblastos/metabolismo , Miofibroblastos/metabolismo , Esclerodermia Sistémica/metabolismo , Línea Celular , Canales de Cloruro/genética , Humanos , Esclerodermia Sistémica/genética , Transducción de Señal/genéticaRESUMEN
OBJECTIVES: Systemic sclerosis (SSc) is characterised by tissue fibrosis of the major organs of the body including the skin, lungs and heart. We have previously reported that the lncRNA HOTAIR plays a central role in the activation of SSc myofibroblasts, the key cellular elements of fibrosis. HOTAIR induces fibroblast activation through H3K27me3-mediated activation of the Notch signalling pathway. Here we aimed to identify the signalling events downstream of Notch that drive SSc myofibroblast activation. METHODS: Patient fibroblasts were obtained from full-thickness forearm skin biopsies of 3 adult patients with SSc of recent onset. The lncRNA HOTAIR was expressed in healthy dermal fibroblasts by lentiviral transduction. Hedgehog signalling pathway was inhibited with GANT61 and GLI2 siRNA. Gamma secretase inhibitors RO4929097 and DAPT were used to block Notch signalling. GSK126 was used to inhibit Enhancer of Zeste 2 (EZH2). RESULTS: Overexpression of HOTAIR in dermal fibroblasts induced the expression of the Hedgehog pathway transcription factor GLI2. This is mediated by activation of Notch signalling following epigenetic downregulation of miRNA-34a expression. Inhibition of H3K27 methylation and Notch signalling reduced expression of GLI2 in HOTAIR-expressing fibroblasts as well as in SSc dermal fibroblasts. Importantly, the inhibition of GLI2 function using GANT61 or siRNA mitigates the pro-fibrotic phenotype induced by HOTAIR. CONCLUSIONS: Our data indicates that GLI2 expression is stably upregulated in SSc myofibroblasts through HOTAIR and that GLI2 mediates the expression of pro-fibrotic markers downstream of Notch.
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ARN Largo no Codificante , Receptores Notch , Esclerodermia Sistémica , Transducción de Señal , Proteína Gli2 con Dedos de Zinc , Adulto , Fibroblastos/patología , Fibrosis , Proteínas Hedgehog , Humanos , Proteínas Nucleares , ARN Largo no Codificante/genética , Esclerodermia Sistémica/genética , Esclerodermia Sistémica/patología , Piel/patología , Proteína Gli2 con Dedos de Zinc/genéticaRESUMEN
BACKGROUND: Systemic sclerosis (SSc) is characterised by autoimmune activation, tissue and vascular fibrosis in the skin and internal organs. Tissue fibrosis is driven by myofibroblasts, that are known to maintain their phenotype in vitro, which is associated with epigenetically driven trimethylation of lysine 27 of histone 3 (H3K27me3). METHODS: Full-thickness skin biopsies were surgically obtained from the forearms of 12 adult patients with SSc of recent onset. Fibroblasts were isolated and cultured in monolayers and protein and RNA extracted. HOX transcript antisense RNA (HOTAIR) was expressed in healthy dermal fibroblasts by lentiviral induction employing a vector containing the specific sequence. Gamma secretase inhibitors were employed to block Notch signalling. Enhancer of zeste 2 (EZH2) was blocked with GSK126 inhibitor. RESULTS: SSc myofibroblasts in vitro and SSc skin biopsies in vivo display high levels of HOTAIR, a scaffold long non-coding RNA known to direct the histone methyltransferase EZH2 to induce H3K27me3 in specific target genes. Overexpression of HOTAIR in dermal fibroblasts induced EZH2-dependent increase in collagen and α-SMA expression in vitro, as well as repression of miRNA-34A expression and consequent NOTCH pathway activation. Consistent with these findings, we show that SSc dermal fibroblast display decreased levels of miRNA-34a in vitro. Further, EZH2 inhibition rescued miRNA-34a levels and mitigated the profibrotic phenotype of both SSc and HOTAIR overexpressing fibroblasts in vitro. CONCLUSIONS: Our data indicate that the EZH2-dependent epigenetic phenotype of myofibroblasts is driven by HOTAIR and is linked to miRNA-34a repression-dependent activation of NOTCH signalling.
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Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Fibroblastos/metabolismo , MicroARNs/metabolismo , Miofibroblastos/metabolismo , ARN Largo no Codificante/metabolismo , Receptores Notch/metabolismo , Esclerodermia Sistémica/metabolismo , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Epigénesis Genética , Fibrosis , Código de Histonas , Humanos , Indoles/farmacología , Fenotipo , Piridonas/farmacología , Receptores Notch/antagonistas & inhibidores , Esclerodermia Sistémica/genética , Esclerodermia Sistémica/patología , Transducción de Señal , Piel/citología , Piel/metabolismo , Piel/patologíaRESUMEN
Serine hydroxymethyltransferase 2 (SHMT2) regulates one-carbon transfer reactions that are essential for amino acid and nucleotide metabolism, and uses pyridoxal-5'-phosphate (PLP) as a cofactor. Apo SHMT2 exists as a dimer with unknown functions, whereas PLP binding stabilizes the active tetrameric state. SHMT2 also promotes inflammatory cytokine signalling by interacting with the deubiquitylating BRCC36 isopeptidase complex (BRISC), although it is unclear whether this function relates to metabolism. Here we present the cryo-electron microscopy structure of the human BRISC-SHMT2 complex at a resolution of 3.8 Å. BRISC is a U-shaped dimer of four subunits, and SHMT2 sterically blocks the BRCC36 active site and inhibits deubiquitylase activity. Only the inactive SHMT2 dimer-and not the active PLP-bound tetramer-binds and inhibits BRISC. Mutations in BRISC that disrupt SHMT2 binding impair type I interferon signalling in response to inflammatory stimuli. Intracellular levels of PLP regulate the interaction between BRISC and SHMT2, as well as inflammatory cytokine responses. These data reveal a mechanism in which metabolites regulate deubiquitylase activity and inflammatory signalling.
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Enzimas Desubicuitinizantes/metabolismo , Glicina Hidroximetiltransferasa/metabolismo , Interferón Tipo I/inmunología , Complejos Multienzimáticos/inmunología , Complejos Multienzimáticos/metabolismo , Transducción de Señal/inmunología , Microscopía por Crioelectrón , Enzimas Desubicuitinizantes/antagonistas & inhibidores , Enzimas Desubicuitinizantes/química , Enzimas Desubicuitinizantes/ultraestructura , Glicina Hidroximetiltransferasa/ultraestructura , Células HEK293 , Humanos , Inflamación/inmunología , Modelos Moleculares , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Mutación , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Fosfato de Piridoxal/metabolismoRESUMEN
The p85α protein regulates flux through the PI3K/PTEN signaling pathway, and also controls receptor trafficking via regulation of Rab-family GTPases. In this report, we determined the impact of several cancer patient-derived p85α mutations located within the N-terminal domains of p85α previously shown to bind PTEN and Rab5, and regulate their respective functions. One p85α mutation, L30F, significantly reduced the steady state binding to PTEN, yet enhanced the stimulation of PTEN lipid phosphatase activity. Three other p85α mutations (E137K, K288Q, E297K) also altered the regulation of PTEN catalytic activity. In contrast, many p85α mutations reduced the binding to Rab5 (L30F, I69L, I82F, I177N, E217K), and several impacted the GAP activity of p85α towards Rab5 (E137K, I177N, E217K, E297K). We determined the crystal structure of several of these p85α BH domain mutants (E137K, E217K, R262T E297K) for bovine p85α BH and found that the mutations did not alter the overall domain structure. Thus, several p85α mutations found in human cancers may deregulate PTEN and/or Rab5 regulated pathways to contribute to oncogenesis. We also engineered several experimental mutations within the p85α BH domain and identified L191 and V263 as important for both binding and regulation of Rab5 activity.
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Fosfohidrolasa PTEN/química , Fosfatidilinositol 3-Quinasas/química , Conformación Proteica , Proteínas de Unión al GTP rab5/química , Animales , Bovinos , Dicroismo Circular , Fosfatidilinositol 3-Quinasa Clase Ia , Cristalografía por Rayos X , Humanos , Mutación , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/genética , Unión Proteica/genética , Transporte de Proteínas/genética , Transducción de Señal/genética , Proteínas de Unión al GTP rab5/genéticaRESUMEN
OBJECTIVE: Aberrant activation of Wnt signaling has been observed in tissues from patients with systemic sclerosis (SSc). This study aimed to determine the role of transforming growth factor ß (TGFß) in driving the increased Wnt signaling, through modulation of axis inhibition protein 2 (Axin-2), a critical regulator of the Wnt canonical pathway. METHODS: Canonical Wnt signaling activation was analyzed by TOPflash T cell factor/lymphoid enhancer factor promoter assays. Axin-2 was evaluated in vitro by analysis of Axin-2 primary/mature transcript expression and decay, TGFß receptor type I (TGFßRI) blockade, small interfering RNA-mediated depletion of tristetraprolin 1, and XAV-939-mediated Axin-2 stabilization. In vivo, Axin-2 messenger RNA (mRNA) and protein expression was determined in skin and lung biopsy samples from mice that express a kinase-deficient TGFßRII specifically on fibroblasts (TßRIIΔk-fib-transgenic mice) and from littermate controls. RESULTS: SSc fibroblasts displayed an increased response to canonical Wnt ligands despite basal levels of Wnt signaling that were comparable to those in healthy control fibroblasts in vitro. Notably, we showed that SSc fibroblasts had reduced basal expression of Axin-2, which was caused by an endogenous TGFß-dependent increase in Axin-2 mRNA decay. Accordingly, we observed that TGFß decreased Axin-2 expression both in vitro in healthy control fibroblasts and in vivo in TßRIIΔk-fib-transgenic mice. Additionally, using Axin-2 gain- and loss-of-function experiments, we demonstrated that the TGFß-induced increased response to Wnt activation characteristic of SSc fibroblasts depended on reduced bioavailability of Axin-2. CONCLUSION: This study highlights the importance of reduced bioavailability of Axin-2 in mediating the increased canonical Wnt response observed in SSc fibroblasts. This novel mechanism extends our understanding of the processes involved in Wnt/ß-catenin-driven pathology and supports the rationale for targeting the TGFß pathway to regulate the aberrant Wnt signaling observed during fibrosis.
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Proteína Axina/fisiología , Fibroblastos/metabolismo , Esclerodermia Sistémica/metabolismo , Factor de Crecimiento Transformador beta/fisiología , Vía de Señalización Wnt/genética , Animales , Regulación hacia Abajo , Humanos , Pulmón/citología , Ratones , Ratones Transgénicos , Piel/citologíaRESUMEN
Deregulation of proliferation and differentiation-dependent signalling pathways is a hallmark of human papillomavirus (HPV) infection. Although the manipulation of these pathways by E6 and E7 has been extensively studied, controversies surround the role of the E5 oncoprotein during a productive virus life cycle. By integrating primary keratinocytes harbouring wild type or E5 knockout HPV18 genomes with pharmacological and gain/loss of function models, this study aimed to provide molecular information about the role of E5 in epithelial proliferation and differentiation. We show that E5 contributes to cell cycle progression and unscheduled host DNA synthesis in differentiating keratinocytes. E5 function correlates with increased EGFR activation in differentiating cells and blockade of this pathway impairs differentiation-dependent cell cycle progression of HPV18 containing cells. Our findings provide a functional requirement of enhanced EGFR signalling for suprabasal cellular DNA synthesis during the virus life cycle. They also reveal an unrecognised contribution of E5 towards the impaired keratinocyte differentiation observed during a productive HPV infection. E5 suppresses a signalling axis consisting of the keratinocyte growth factor receptor (KGFR) pathway. Inhibition of this pathway compensates for the loss of E5 in knockout cells and re-instates the delay in differentiation. The negative regulation of KGFR involves suppression by the EGFR pathway. Thus our data reveal an unappreciated role for E5-mediated EGFR signalling in orchestrating the balance between proliferation and differentiation in suprabasal cells.
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Exercise intolerance, as evidenced by a worsening of pain, fatigue, and stiffness after novel exertion, is a key feature of fibromyalgia (FM). In this pilot study, we investigate whether; insufficient muscle repair processes and impaired anti-inflammatory mechanisms result in an exaggerated pro-inflammatory cytokine response to exhaustive exercise, and consequently a worsening of muscle pain, stiffness and fatigue in the days post-exercise. We measured changes in muscle pain and tenderness, fatigue, stiffness, and serum levels of neuroendocrine and inflammatory cytokine markers in 20 women with FM and 16 healthy controls (HCs) before and after exhaustive treadmill exercise. Compared to HCs, FM participants failed to mount the expected anti-inflammatory response to exercise and experienced a worsening of symptoms post-exercise. However, changes in post-exertional symptoms were not mediated by post-exertional changes in pro-inflammatory cytokine levels. Implications of these findings are discussed.
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Terapia por Ejercicio/métodos , Fibromialgia/complicaciones , Inflamación/etiología , Inflamación/rehabilitación , Adulto , Análisis de Varianza , Antropometría , Citocinas/sangre , Ayuno , Femenino , Fibromialgia/sangre , Fibromialgia/rehabilitación , Hormonas/sangre , Humanos , Inflamación/sangre , Masculino , Persona de Mediana Edad , Consumo de Oxígeno , Escalas de Valoración Psiquiátrica , Encuestas y CuestionariosRESUMEN
Bladder cancers commonly show genetic aberrations in the phosphatidylinositol 3-kinase signaling pathway. Here we have screened for mutations in PIK3R1, which encodes p85α, one of the regulatory subunits of PI3K. Two hundred and sixty-four bladder tumours and 41 bladder tumour cell lines were screened and 18 mutations were detected. Thirteen mutations were in C-terminal domains and are predicted to interfere with the interaction between p85α and p110α. Five mutations were in the BH domain of PIK3R1. This region has been implicated in p110α-independent roles of p85α, such as binding to and altering the activities of PTEN, Rab4 and Rab5. Expression of these mutant BH-p85α forms in mouse embryonic fibroblasts with p85α knockout indicated that all forms, except the truncation mutants, could bind and stabilize p110α but did not increase AKT phosphorylation, suggesting that BH mutations function independently of p110α. In a panel of 44 bladder tumour cell lines, 80% had reduced PIK3R1 mRNA expression relative to normal urothelial cells. This, along with mutation of PIK3R1, may alter BH domain functioning. Our findings suggest that mutant forms of p85α may play an oncogenic role in bladder cancer, not only via loss of ability to regulate p110α but also via altered function of the BH domain.
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Mutación , Fosfatidilinositol 3-Quinasas/genética , Neoplasias de la Vejiga Urinaria/genética , Urotelio/enzimología , Animales , Bovinos , Línea Celular Tumoral , Proliferación Celular , Fosfatidilinositol 3-Quinasa Clase Ia , GTP Fosfohidrolasas/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Modelos Moleculares , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Estabilidad Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Neoplasias de la Vejiga Urinaria/patología , Urotelio/patología , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
This article presents a brief review of the physiologic abnormalities seen in fibromyalgia, current theories of widespread pain, and treatment options, including emerging therapeutics, with a focus on the use of duloxetine to manage fibromyalgia symptoms. Major clinical trials that examine the efficacy and effectiveness of duloxetine to date are reviewed, and safety issues are discussed.
Asunto(s)
Fibromialgia/tratamiento farmacológico , Dolor , Tiofenos/uso terapéutico , Ensayos Clínicos como Asunto , Clorhidrato de Duloxetina , Fibromialgia/fisiopatología , Humanos , Dimensión del DolorRESUMEN
BACKGROUND: It has been postulated that atypical and melancholic depression subtypes exist in depressed fibromyalgia (FM) patients, yet no study has empirically tested this hypothesis. The purpose of this study is to determine whether major depressive disorder (MDD) with atypical features and MDD with melancholic features occurs in a FM sample and to describe their demographic, clinical and diagnostic characteristics. METHODS: An observational cohort study using a descriptive cross-sectional design recruited a convenience sample of 76 outpatients with FM from an academic rheumatology clinic and a community mental health practice. Diagnoses of FM were confirmed using the 1990 ACR classification guidelines. Diagnoses of MDD and diagnostic subtypes were determined using the DSM-IV-TR criteria. Clinical characteristics were measured using the Fibromyalgia Impact Questionnaire, Structured Interview Guide for the Hamilton Depression Rating Scale with Atypical Depression Supplement and other standardized instruments. Odds ratios were computed on subtype-specific diagnostic criteria. Correlations assessed associations between subtype diagnoses and diagnostic criteria. RESULTS: Of the 76 subjects with FM, 11.8% (n = 9) were euthymic, 52.6% (n = 40) met diagnostic criteria for MDD with atypical features and 35.6% (n = 27) for MDD with melancholic features. Groups did not differ on demographic characteristics except for gender (p = 0.01). The non-depressed and atypical groups trended toward having a longer duration of FM symptoms (18.05 yrs. +/- 12.83; 20.36 yrs. +/- 15.07) compared to the melancholic group (14.11 yrs. +/- 8.82; p = 0.09). The two depressed groups experienced greater severity on all clinical features compared to the non-depressed group. The atypical group did not differ clinically from the melancholic group except the latter experienced greater depression severity (p = 0.001). The atypical group demonstrated the highest prevalence and correlations with atypical-specific diagnostic criteria: (e.g., weight gain/ increased appetite: OR = 3.5, p = 0.02), as did the melancholic group for melancholic-specific criteria: (e.g., anhedonia: OR = 20, p < 0.001). CONCLUSION: Depressed fibromyalgia patients commonly experience both atypical and melancholic depressive features; however, in this study, atypical depression was 1.5 times more common than melancholic depression. This finding may have significant research and clinical implications.