RESUMEN
OBJECTIVE: To analyse pro-survival mechanisms elicited in RA synovial fibroblasts (RASFs) upon detachment from their extracellular matrix dependent on the disintegrin metalloproteinase ADAM15 and Yes-associated protein kinase 1 (YAP1). METHODS: Detachment-induced apoptosis was determined by caspase 3/7 assays. Immunofluorescent stainings, cell surface biotinylation and immunoblotting were applied to analyse phosphorylated kinases and subcellular localization of YAP1 and connective tissue growth factor (CTGF). Caspase and transwell transmigration assays served to study CTGF function. RESULTS: Silencing of ADAM15 or YAP1 in RASFs leads to significantly increased levels of detachment-induced caspase activity. In non-silenced RASFs detachment causes simultaneous ADAM15-enhanced phosphorylation of YAP1 at S127, known for promoting its cytoplasmic localization, and Src-dependent phosphorylation at tyrosine Y357. The majority of nuclear YAP1 leaves the nucleus shortly after cell detachment, but prolonged detachment causes a marked nuclear re-entry of YAP1, resulting in significantly increased synthesis of CTGF. The newly synthesized CTGF, however, is not detectable in the supernatant, but is bound to the outside of the plasma membrane. In vitro studies demonstrated autocrine binding of CTGF to the EGF receptor and ß1 integrin, with concomitant triggering of survival kinases, AKT1, ERK1/2, Src and focal adhesion kinase. Functional studies revealed anti-apoptotic effects of CTGF on detached RASFs and an enhancement of their potential for endothelial transmigration using HUVEC-coated transwells. CONCLUSION: The elucidation of a new molecular mechanism that protects RASFs in the highly pro-apoptotic environment of inflamed RA joints by promoting anoikis-resistance and transendothelial migration via ADAM15/YAP1-mediated CTGF upregulation uncovers potentially new targets for future therapeutic intervention.
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Artritis Reumatoide , Factor de Crecimiento del Tejido Conjuntivo , Humanos , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Anoicis , Transducción de Señal , Artritis Reumatoide/genética , Artritis Reumatoide/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Fibroblastos/metabolismo , Membrana Sinovial/metabolismo , Células Cultivadas , Proteínas de la Membrana/metabolismo , Proteínas ADAM/metabolismo , Proteínas ADAM/farmacologíaRESUMEN
BACKGROUND: Psoriatic Arthritis (PsA) is a chronic inflammatory disease of the joints. PsA is etiologically complex, and 11 susceptibility loci have been identified so far. Most of these overlap with loci associated with psoriasis vulgaris (PsV), the most common psoriatic skin manifestation which is also frequently seen in PsA patients. In addition, two copy number variants (CNVs) are associated with PsV, one of which, located within the LCE3 gene cluster, is also associated with PsA. Finally, an intergenic deletion has been reported as a PsA-specific CNV. METHODS: We performed a genome-wide association study (GWAS) of CNVs in PsA and assessed the contribution to disease risk by CNVs at known psoriasis susceptibility loci. RESULTS: After stringent quality assessment and validation of CNVs of the GWAS with an alternative quantitative method, two significantly associated CNVs remained, one near UXS1, the other one at the TRB locus. However, MLPA analysis did not confirm the CN state in ~1/3 of individuals, and an analysis of an independent case-control-study failed to confirm the initial associations. Furthermore, detailed PCR-based analysis of the sequence at TRB revealed the existence of a more complex genomic sequence most accurately represented by freeze hg18 which accordingly failed to confirm the hg19 sequence. Only rare CNVs were detected at psoriasis susceptibility loci. At three of 12 susceptibility loci with CNVs (CSMD1, IL12B, RYR2), CN variability was confirmed independently by MLPA. Overall, the rate of CNV confirmation by MLPA was strongly dependent upon CNV type, CNV size and the number of array markers involved in a CNV. CONCLUSION: Although we identified PsA associations at several loci and confirmed that the common CNVs at these sites were real, ~1/3 of the common CNV states could not be reproduced. Furthermore, replication analysis failed to confirm the original association. Furthermore, SNP array-based analyses of CNVs were found to be more reliable for deletions than duplications, independent of the respective CNV allele frequency. CNVs are thus good candidate disease variants, while the methods to detect them should be applied cautiously and reproduced by an independent method.
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Artritis Psoriásica/genética , Variaciones en el Número de Copia de ADN , Estudio de Asociación del Genoma Completo , Población Blanca/genética , Artritis Psoriásica/diagnóstico , Estudios de Casos y Controles , Eliminación de Gen , Frecuencia de los Genes , Sitios Genéticos , Predisposición Genética a la Enfermedad , Genoma Humano , Técnicas de Genotipaje , Alemania , Humanos , Polimorfismo de Nucleótido Simple , Reproducibilidad de los ResultadosRESUMEN
OBJECTIVE: To study the contribution of ADAM15, a disintegrin metalloproteinase that is up-regulated in the rheumatoid arthritis (RA) synovial membrane, to the characteristic resistance of RA synovial fibroblasts (RASFs) to apoptosis induction by genotoxic stress or stimulation with proapoptotic FasL, which is present at high concentrations in RA synovial fluid. METHODS: Caspase 3/7 activity and the total apoptosis rate in RASFs upon exposure to the DNA-damaging agent camptothecin or FasL were determined using enzyme assays and annexin V staining. Phosphorylated signaling proteins were analyzed by immunoblotting. RNA interference was used to silence ADAM15 expression. NF-κB activity was determined by enzyme-linked immunosorbent assay. RESULTS: RASFs displayed significantly higher caspase 3/7 activity upon camptothecin and FasL exposure when ADAM15 had been down-regulated by specific small interfering RNAs. Upon FasL stimulation, RASFs phosphorylated focal adhesion kinase (FAK) and c-Src (Src), and activated phosphatidylinositol 3-kinase as well as the transcription factor NF-κB. This ADAM15-dependent, FasL-induced activation of antiapoptotic kinases and NF-κB was demonstrated by a marked reduction of apoptosis upon knockdown of ADAM15 protein expression. Inhibitors specifically interfering with FAK and Src signaling, such as FAK inhibitor 14 and dasatinib, potently induce apoptosis in RASFs, with significant enhancement by the silencing of ADAM15. CONCLUSION: ADAM15 contributes to apoptosis resistance in RASFs by activating the Src/FAK pathway upon FasL exposure, rendering the FAK/Src signaling pathway an interesting target for potential therapeutic intervention in RA.
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Proteínas ADAM/metabolismo , Apoptosis/fisiología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Quinasa 1 de Adhesión Focal/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal/fisiología , Proteínas ADAM/genética , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Biopsia , Camptotecina/farmacología , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Proteína Ligando Fas/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Proteínas de la Membrana/genética , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Regulación hacia Arriba/fisiologíaRESUMEN
OBJECTIVE: Psoriatic arthritis (PsA) is a common inflammatory joint disease distinct from other chronic arthritides and frequently accompanied by psoriasis vulgaris. In a first genome-wide association study (GWAS), we were able to identify several genetic risk factors. However, even combined with previously identified factors, the genetic contribution to disease was not fully explained. Therefore, we undertook this study to investigate further 17 loci from our GWAS that did not reach genome-wide significance levels of association in the initial analysis. METHODS: Twenty-one of 22 single-nucleotide polymorphisms were successfully genotyped in independent cohorts of 1,398 PsA patients and 6,389 controls and in a group of 964 German patients with psoriasis vulgaris. RESULTS: Association with a RUNX3 variant, rs4649038, was replicated in independent patients and controls and resulted in a combined P value of 1.40 × 10(-8) by Cochran-Mantel-Haenszel test and an odds ratio (OR) of 1.24 (95% confidence interval [95% CI] 1.15-1.33). Further analyses based on linkage disequilibrium (LD) at RUNX3 refined the most significant association to an LD block located in the first intron of one isoform. Weaker evidence for association was detected in German patients with psoriasis vulgaris (P = 5.89 × 10(-2) ; OR 1.13 [95% CI 1.00-1.28]), indicating a role in the skin manifestations of psoriasis. CONCLUSION: Our analyses identified variants in RUNX3 as susceptibility factors for PsA. RUNX3 has already been implicated in susceptibility to ankylosing spondylitis, another spondyloarthritis, although its risk allele is independent from the one for PsA. RUNX-3 is involved in CD8+ T lymphocyte differentiation and is therefore a good candidate for involvement in PsA and psoriasis vulgaris as T cell-mediated diseases.
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Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple/genética , Espondilitis Anquilosante/genética , Estudios de Casos y Controles , Estudios de Cohortes , Frecuencia de los Genes , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Desequilibrio de LigamientoRESUMEN
ADAM15, a disintegrin and metalloproteinase, is capable of counteracting genotoxic stress-induced apoptosis by the suppression of caspase-3 activation. A cell line expressing the membrane-bound ADAM15 without its cytoplasmic tail, however, lost this anti-apoptotic property, suggesting a crucial role of the intracellular domain as a scaffold for recruitment of survival signal-transducing kinases. Accordingly, an enhanced phosphorylation of FAK at Tyr-397, Tyr-576, and Tyr-861 was detected upon genotoxic stress by camptothecin in ADAM15-transfected T/C28a4 cells, but not in transfectants expressing an ADAM15 mutant without the cytoplasmic tail. Accordingly, a specific binding of the cytoplasmic ADAM15 domain to the C terminus of FAK could be shown by mammalian two-hybrid, pulldown, and far Western studies. In cells expressing full-length ADAM15, a concomitant activation of Src at Tyr-416 was detected upon camptothecin exposure. Cells transfected with a chimeric construct consisting of the extracellular IL-2 receptor α-chain and the cytoplasmic ADAM15 domain were IL-2-stimulated to prove that the ADAM15 tail can transduce a percepted extracellular signal to enhance FAK and Src phosphorylation. Our studies further demonstrate Src binding to FAK but not a direct Src interaction with ADAM15, suggesting FAK as a critical intracellular adaptor for ADAM15-dependent enhancement of FAK/Src activation. Moreover, the apoptosis induction elicited by specific inhibitors (PP2, FAK 14 inhibitor) of FAK/Src signaling was significantly reduced by ADAM15 expression. The newly uncovered counter-regulatory response to genotoxic stress in a chondrocytic survival pathway is potentially also relevant to apoptosis resistance in neoplastic growth.
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Proteínas ADAM/metabolismo , Condrocitos/enzimología , Daño del ADN , Quinasa 1 de Adhesión Focal/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal , Proteínas ADAM/genética , Caspasa 3/genética , Caspasa 3/metabolismo , Línea Celular , Activación Enzimática/genética , Inhibidores Enzimáticos/farmacología , Quinasa 1 de Adhesión Focal/genética , Humanos , Interleucina-2/genética , Interleucina-2/metabolismo , Subunidad alfa del Receptor de Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Proteínas de la Membrana/genética , Fosforilación , Estructura Terciaria de Proteína , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
OBJECTIVE: To investigate the capacity of ADAM15, a disintegrin metalloproteinase that is up-regulated in osteoarthritic (OA) cartilage, to protect chondrocytes against apoptosis induced by growth factor deprivation and genotoxic stress. METHODS: Caspase 3/7 activity was determined in primary OA and ADAM15-transfected T/C28a4 chondrocytes upon exposure to the DNA-damaging agent camptothecin or serum withdrawal. Camptothecin-induced cytotoxicity was determined by measuring cellular ATP content. (Anti-)apoptotic proteins were analyzed by immunoblotting, and levels of messenger RNA (mRNA) for X-linked inhibitor of apoptosis (XIAP) were determined using real-time polymerase chain reaction. RNA interference was applied for down-regulation of ADAM15 and XIAP expression. Immunohistochemistry analysis of normal and OA cartilage samples was performed using XIAP- and ADAM15-specific antibodies. RESULTS: ADAM15-transfected chondrocytes cultured on a collagen matrix displayed significantly reduced caspase 3/7 activity upon serum or intermittent matrix withdrawal, compared with vector-transfected control cells. Apoptosis induction by camptothecin exposure also led to significantly elevated caspase 3/7 activity and reduced cell viability of the vector-transfected compared with ADAM15-transfected chondrocytes. Increased levels of activated caspase 3 and cleaved poly(ADP-ribose) polymerase were detected in the vector controls. XIAP, an inhibitor of activated caspase 3, was significantly up-regulated ( approximately 3-fold) at the protein and mRNA levels in ADAM15-transfected chondrocytes upon camptothecin treatment. Specific down-regulation of either ADAM15 or XIAP in OA chondrocytes led to significant sensitization to camptothecin-induced caspase 3/7 activity. Immunohistochemical analysis revealed low to moderate XIAP expression in normal specimens and markedly increased XIAP staining, colocalizing with ADAM15, in OA cartilage. CONCLUSION: ADAM15 conveys antiapoptotic properties to OA chondrocytes that might sustain their potential to better resist the influence of death-inducing stimuli under pathophysiologic conditions.
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Proteínas ADAM/metabolismo , Apoptosis/fisiología , Condrocitos/citología , Proteínas de la Membrana/metabolismo , Osteoartritis/metabolismo , Osteoartritis/patología , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Proteínas ADAM/genética , Apoptosis/efectos de los fármacos , Proteínas Sanguíneas/farmacología , Camptotecina/farmacología , Proteínas Portadoras/farmacología , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 7/genética , Caspasa 7/metabolismo , Células Cultivadas , Condrocitos/efectos de los fármacos , Condrocitos/fisiología , Regulación hacia Abajo/fisiología , Inhibidores Enzimáticos/farmacología , Humanos , Péptidos y Proteínas de Señalización Intercelular , Proteínas de la Membrana/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Estrés Fisiológico/fisiología , Transfección , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , Proteína Inhibidora de la Apoptosis Ligada a X/genéticaRESUMEN
Mechanotransduction is elicited in cells upon the perception of physical forces transmitted via the extracellular matrix in their surroundings and results in signaling events that impact cellular functions. This physiological process is a prerequisite for maintaining the integrity of diarthrodial joints, while excessive loading is a factor promoting the inflammatory mechanisms of joint destruction. Here, we describe a mechanotransduction pathway in synovial fibroblasts (SF) derived from the synovial membrane of inflamed joints. The functionality of this pathway is completely lost in the absence of the disintegrin metalloproteinase ADAM15 strongly upregulated in SF. The mechanosignaling events involve the Ca2+-dependent activation of c-Jun-N-terminal kinases, the subsequent downregulation of long noncoding RNA HOTAIR, and upregulation of the metabolic energy sensor sirtuin-1. This afferent loop of the pathway is facilitated by ADAM15 via promoting the cell membrane density of the constitutively cycling mechanosensitive transient receptor potential vanilloid 4 calcium channels. In addition, ADAM15 reinforces the Src-mediated activation of pannexin-1 channels required for the enhanced release of ATP, a mediator of purinergic inflammation, which is increasingly produced upon sirtuin-1 induction.
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Proteínas ADAM/metabolismo , Inflamación/genética , Mecanotransducción Celular/genética , Proteínas de la Membrana/metabolismo , Membrana Sinovial/patología , Proteínas ADAM/genética , Acetilación , Adenosina Trifosfato/metabolismo , Anciano , Señalización del Calcio , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Conexinas/metabolismo , Regulación hacia Abajo/genética , Humanos , Inflamación/patología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteínas de la Membrana/genética , Persona de Mediana Edad , NAD/metabolismo , Proteínas del Tejido Nervioso/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Sirtuina 1/metabolismo , Estrés Mecánico , Canales Catiónicos TRPV/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
In a mouse melanoma metastasis model it has been recently shown that ADAM15 overexpression in melanoma cells significantly reduced the number of metastatic nodules on the lung. Unfortunately, the expression of ADAM15 in human melanoma tissue has not been determined so far. In our study, we characterized the expression of ADAM15 in tissue micro-arrays of patients with primary melanoma with melanoma metastasis. ADAM15 was expressed in melanocytes and endothelial cells of benign nevi and melanoma tissue. Importantly, ADAM15 was significantly downregulated in melanoma metastasis compared to primary melanoma. We further demonstrate that IFN-γ and TGF-ß downregulate ADAM15 protein levels in melanoma cells. To investigate the role of ADAM15 in melanoma progression, we overexpressed ADAM15 in melanoma cells. Importantly, overexpression of ADAM15 in melanoma cells reduced the migration, invasion and the anchorage dependent and independent cell growth of melanoma cells. In summary, the downregulation of ADAM15 plays an important role in melanoma progression and ADAM15 act as a tumorsuppressor in melanoma.
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Proteínas ADAM/biosíntesis , Melanoma/patología , Proteínas de la Membrana/biosíntesis , Neoplasias Cutáneas/patología , Proteínas Supresoras de Tumor/biosíntesis , Proteínas ADAM/antagonistas & inhibidores , Proteínas ADAM/genética , Animales , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Humanos , Melanoma/enzimología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Ratones , Metástasis de la Neoplasia , Neoplasias Cutáneas/enzimología , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genéticaRESUMEN
INTRODUCTION: Psoriasis susceptibility locus 4 (PSORS4) is a susceptibility locus for psoriasis vulgaris (PsV), a common inflammatory, hyperproliferative skin disorder. Recently, a deletion of 2 late cornified envelope (LCE) genes within epidermal differentiation complex on chromosome 1 was shown to be enriched in 1426 patients with PsV, suggesting compromised barrier function in deletion carriers. This genetic association was subsequently confirmed in a German cohort. METHODS: In order to investigate whether this variant also predisposes to psoriatic arthritis (PsA), this deletion and 3 single nucleotide polymorphisms (SNPs) in strong linkage disequilibrium with it were genotyped in a case-control cohort of 650 patients and 937 control individuals of German origin. RESULTS: LCE deletion frequency did not significantly differ between patients with PsA and controls (65.0% vs 65.5%). Similarly, no evidence for association to the three SNPs was observed. DISCUSSION: This is the first non-human leucocyte antigen (HLA) risk factor predisposing only to skin type of psoriasis, supporting the concept of partially overlapping but different aetiological factors underlying skin and joint manifestations.
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Artritis Psoriásica/genética , Proteínas Ricas en Prolina del Estrato Córneo/genética , Eliminación de Gen , Adolescente , Adulto , Estudios de Casos y Controles , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Alemania , Humanos , Desequilibrio de Ligamiento , Masculino , Adulto JovenRESUMEN
ADAM15 belongs to a family of transmembrane multi-domain proteins implicated in proteolysis, cell-cell and cell-matrix interactions in various disease conditions. In osteoarthritis (OA), ADAM15 is up-regulated in the chondrocytes already at early stages of cartilage degeneration where it seems to exert homeostatic effects likely associated with its ability to enhance integrin-mediated chondrocyte adhesion to the surrounding collagen matrix. The aim of our present study was, therefore, to characterize functional domains of ADAM15 involved in collagen II (CII) interaction and to analyse associated outside-in signalling events. Accordingly, ADAM15 and respective deletion mutants were stably transfected into the chondrocyte cell line T/C28a4. Transfected cells were adhered to CII and phosphoproteins analysed by Western blotting. Co-immunoprecipitation served to identify protein binding to ADAM15. Our results elucidate the prodomain as critical for the capacity of ADAM15 to enhance CII adhesion, thereby identifying for the first time a cell-adhesive role of a metalloproteinase prodomain. Moreover, the cytoplasmic tail of ADAM15 confers a modulatory effect on the autophosphorylation site Y397 of the focal adhesion kinase (FAK) during chondrocyte-collagen interaction. In conclusion, the newly uncovered impact of ADAM15 on signalling events that arise from chondrocyte interactions with its collagen matrix might contribute to the elucidation of the mechanism underlying its proposed chondroprotective role in degenerative cartilage disease.
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Proteínas ADAM/fisiología , Condrocitos/metabolismo , Matriz Extracelular/metabolismo , Proteínas de la Membrana/fisiología , Transducción de Señal/fisiología , Proteínas ADAM/genética , Secuencia de Bases , Western Blotting , Línea Celular , Condrocitos/citología , Cartilla de ADN , Humanos , Inmunoprecipitación , Proteínas de la Membrana/genéticaRESUMEN
OBJECTIVE: To investigate mechanisms underlying the capability of ADAM15 to transform FasL-mediated death-inducing signals into prosurvival activation of Src and focal adhesion kinase (FAK) in rheumatoid arthritis synovial fibroblasts (RASFs). METHODS: Caspase 3/7 activity and apoptosis rate were determined in RASFs and ADAM15-transfected T/C28a4 cells upon Fas/CD95 triggering using enzyme assays and annexin V staining. Phosphorylated Src and FAK were analyzed by immunoblotting. Interactions of ADAM15 and CD95 with calmodulin (CaM), Src, or FAK were analyzed by pull-downs using CaM-Sepharose and coimmunoprecipitations with specific antibodies. Protein binding assays were performed using recombinant CaM and ADAM15. Immunofluorescence was performed to investigate subcellular colocalization of ADAM15, Fas/CD95, and CaM. RESULTS: The antiapoptotic effect of ADAM15 in FasL-stimulated cells was demonstrated either by increased apoptosis of cells transfected with an ADAM15 construct lacking the cytoplasmic domain compared to cells transfected with full-length ADAM15 or by reduced apoptosis resistance of RASFs upon RNA interference silencing of ADAM15. Fas ligation triggered a Ca2+ release-activated Ca2+ /calcium release-activated calcium channel protein 1 (CRAC/Orai1) channel-dependent CaM recruitment to Fas/CD95 and ADAM15 in the cell membrane. Simultaneously, Src associated with CaM was shown to become engaged in the ADAM15 complex also containing cytoplasmic-bound FAK. Accordingly, Fas ligation in RASFs led to ADAM15-dependent phosphorylation of Src and FAK, which was associated with increased survival. Pharmacologic interference with either the CaM inhibitor trifluoperazine or the CRAC/Orai inhibitor BTP-2 simultaneously applied with FasL synergistically enhanced Fas-mediated apoptosis in RASFs. CONCLUSION: ADAM15 provides a scaffold for formation of CaM-dependent prosurvival signaling complexes upon CRAC/Orai coactivation by FasL-induced death signals and a potential therapeutic target to break apoptosis resistance in RASFs.
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Proteínas ADAM/genética , Apoptosis/genética , Artritis Reumatoide/metabolismo , Calmodulina/metabolismo , Condrocitos/metabolismo , Proteína Ligando Fas/metabolismo , Fibroblastos/metabolismo , Proteínas de la Membrana/genética , Proteínas ADAM/metabolismo , Anilidas/farmacología , Apoptosis/efectos de los fármacos , Calmodulina/antagonistas & inhibidores , Línea Celular , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Proteína ORAI1/antagonistas & inhibidores , Proteína ORAI1/metabolismo , Fosforilación , Interferencia de ARN , Membrana Sinovial/citología , Tiadiazoles/farmacología , Trifluoperazina/farmacología , Receptor fas/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
The regulation of temporo-spatial compartmentalization of protein synthesis is of crucial importance for a variety of physiologic cellular functions. Here, we demonstrate that the cell membrane-anchored disintegrin metalloproteinase ADAM15, upregulated in a variety of aggressively growing tumor cells, in the hyperproliferative synovial membrane of inflamed joints as well as in osteoarthritic chondrocytes, transiently binds to poly(A) binding protein 1 (PABP) in cells undergoing adhesion. The cytoplasmic domain of ADAM15 was shown to selectively interact with the proline-rich linker of PABP. Immunostainings of adhesion-triggered cells demonstrate an ADAM15-dependent recruitment of PABP to cell membrane foci coinciding with ongoing mRNA translation as visualized by the detection of puromycin-terminated polypeptides. Moreover, the increase in cell membrane-associated neosynthesis of puromycylated proteins upon induction of cell adhesion was proven linked to ADAM15 expression in HeLa and ADAM15-transfected chondrocytic cells. Thus, down regulation of ADAM15 by siRNA and/or the use of a cell line transfected with a mutant ADAM15-construct lacking the cytoplasmic tail resulted in a considerable reduction in the amount of cell membrane-associated puromycylated proteins formed during induced cell adhesion. These results provide first direct evidence for a regulatory role of ADAM15 on mRNA translation at the cell membrane that transiently emerges in response to triggering cell adhesion and might have potential implications under pathologic conditions of matrix remodeling associated with ADAM15 upregulation.
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Proteínas ADAM/metabolismo , Proteínas ADAM/fisiología , Adhesión Celular/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Comunicación Celular , Línea Celular , Membrana Celular/metabolismo , Condrocitos/metabolismo , Humanos , Osteoartritis/genética , Osteoartritis/fisiopatología , Proteínas de Unión a Poli(A)/metabolismo , Biosíntesis de Proteínas/fisiología , ARN Mensajero/metabolismo , Transducción de Señal/fisiología , Membrana Sinovial/metabolismo , TransfecciónRESUMEN
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic, Th17-derived cytokine thought to critically contribute to the pathogenesis of diverse autoimmune diseases, including rheumatoid arthritis and psoriasis. Treatment with monoclonal antibodies that block GM-CSF activity is associated with favorable therapeutic effects in patients with rheumatoid arthritis. We evaluated the role of GM-CSF as a potential target for therapeutic interference in psoriasis using a combined pharmacologic and genetic approach and the mouse model of imiquimod-induced psoriasiform dermatitis (IMQPD). Neutralization of murine GM-CSF by an anti-GM-CSF antibody ameliorated IMQPD. In contrast, genetic deficiency in GM-CSF did not alter the course of IMQPD, suggesting the existence of mechanisms compensating for chronic, but not acute, absence of GM-CSF. Further investigation uncovered an alternative pathogenic pathway for IMQPD in the absence of GM-CSF characterized by an expanded plasmacytoid dendritic cell population and release of IFNα and IL-22. This pathway was not activated in wild-type mice during short-term anti-GM-CSF treatment. Our investigations support the potential value of GM-CSF as a therapeutic target in psoriatic disease. The discovery of an alternative pathogenic pathway for psoriasiform dermatitis in the permanent absence of GM-CSF, however, suggests the need for monitoring during therapeutic use of long-term GM-CSF blockade.
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Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/farmacología , Modelos Animales de Enfermedad , Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Psoriasis/tratamiento farmacológico , Aminoquinolinas/toxicidad , Animales , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Imiquimod , Inductores de Interferón/toxicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Psoriasis/inducido químicamente , Psoriasis/inmunología , Psoriasis/patologíaAsunto(s)
Artritis Psoriásica/genética , Predisposición Genética a la Enfermedad , Peroxidasa/genética , Psoriasis/genética , Artritis Psoriásica/diagnóstico , Artritis Psoriásica/epidemiología , Pruebas Genéticas , Alemania/epidemiología , Humanos , Polimorfismo de Nucleótido Simple , Psoriasis/diagnóstico , Psoriasis/epidemiología , Índice de Severidad de la EnfermedadRESUMEN
The gene for solute carrier family 12 member A8 has recently been proposed as a candidate gene for psoriasis susceptibility (PSORS5) on chromosome 3q based on association of five single nucleotide polymorphisms (SNP) in Swedish patients. To investigate whether this locus is relevant for German psoriasis vulgaris (PsV) patients, we analyzed a group of 210 trios and a case-control group including 375 patients. Based on our investigation of the linkage disequilibrium (LD) structure of SLC12A8, we assayed 35 haplotype tag SNP and grouped them into nine LD-blocks. In the case-control study, we detected an association for six SNP and three LD-based haplotypes. Association was strongest for ss35527511 (chi2 = 11.224, p = 0.0008) and haplotype E-2 (chi2 = 11.788, p = 0.00059) and independent of the presence of an HLA-associated PSORS1 risk allele. Through extended haplotype analysis, we could show that two independent association signals exist in SLC12A8, suggesting allelic heterogeneity. None of the SNP showed association in trios, apart from a weak association of rs2228674 (transmission disequilibrium test statistics p = 0.048), probably due to insufficient power. We conclude that SLC12A8 is a susceptibility locus for PsV. In order to establish the exact nature of this association, efforts to identify the disease-causing variants are ongoing.
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Predisposición Genética a la Enfermedad , Desequilibrio de Ligamiento , Polimorfismo de Nucleótido Simple , Psoriasis/genética , Simportadores de Cloruro de Sodio-Potasio/genética , Alelos , Estudios de Casos y Controles , Cromosomas Humanos Par 3/genética , Genotipo , Alemania , Haplotipos , Humanos , Proteínas/genética , SueciaRESUMEN
A DNA variant, rs734232, altering a RUNX1 binding site was recently reported as susceptibility allele at PSORS2 (17q25) in cohorts of psoriasis patients from the US. A testing of this variant in psoriasis patients from Germany did not confirm this association in 300 trios nor in two case-control studies with 281 patients with psoriasis vulgaris and 375 patients with psoriatic arthritis, respectively. These results fail to support rs734232 as a psoriasis susceptibility factor in German psoriasis patients.
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Artritis Psoriásica/genética , Cromosomas Humanos Par 17 , Proteínas de Unión al ADN/genética , Proteínas Proto-Oncogénicas/genética , Psoriasis/genética , Factores de Transcripción/genética , Adolescente , Adulto , Artritis Psoriásica/epidemiología , Sitios de Unión/genética , Estudios de Casos y Controles , Cromosomas Humanos Par 6 , Estudios de Cohortes , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Frecuencia de los Genes , Alemania/epidemiología , Haplotipos , Humanos , Persona de Mediana Edad , Psoriasis/epidemiología , Factores de RiesgoRESUMEN
INTRODUCTION: In recent genome-wide association studies for psoriatic arthritis (PsA) and psoriasis vulgaris, common coding variants in the TRAF3IP2 gene were identified to contribute to susceptibility to both disease entities. The risk allele of p.Asp10Asn (rs33980500) proved to be most significantly associated and to encode a mutant protein with an almost completely disrupted binding property to TRAF6, supporting its impact as a main disease-causing variant and modulator of IL-17 signaling. METHODS: To identify further variants, exons 2-4 encoding both known TNF-receptor-associated factor (TRAF) binding domains were sequenced in 871 PsA patients. Seven missense variants and one three-base-pair insertion were identified in 0.06% to 1.02% of alleles. Five of these variants were also present in 931 control individuals at comparable frequency. Constructs containing full-length wild-type or mutant TRAF3IP2 were generated and used to analyze functionally all variants for TRAF6-binding in a mammalian two-hybrid assay. RESULTS: None of the newly found alleles, though, encoded proteins with different binding properties to TRAF6, or to the cytoplasmic tail of the IL-17-receptor α-chain, suggesting that they do not contribute to susceptibility. CONCLUSIONS: Thus, the TRAF3IP2-variant p.Asp10Asn is the only susceptibility allele with functional impact on TRAF6 binding, at least in the German population.
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Artritis Psoriásica/genética , Frecuencia de los Genes/genética , Código Genético/genética , Variación Genética/genética , Factor 6 Asociado a Receptor de TNF/genética , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/genética , Proteínas Adaptadoras Transductoras de Señales , Adolescente , Adulto , Artritis Psoriásica/metabolismo , Artritis Psoriásica/fisiopatología , Estudios de Casos y Controles , Estudios de Cohortes , Femenino , Humanos , Masculino , Factor 6 Asociado a Receptor de TNF/metabolismo , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismo , Adulto JovenRESUMEN
Psoriatic arthritis (PsA) is an inflammatory joint disease that is distinct from other chronic arthritides and which is frequently accompanied by psoriasis vulgaris (PsV) and seronegativity for rheumatoid factor. We conducted a genome-wide association study in 609 German individuals with PsA (cases) and 990 controls with replication in 6 European cohorts including a total of 5,488 individuals. We replicated PsA associations at HLA-C and IL12B and identified a new association at TRAF3IP2 (rs13190932, P = 8.56 × 10⻹7). TRAF3IP2 was also associated with PsV in a German cohort including 2,040 individuals (rs13190932, P = 1.95 × 10⻳). Sequencing of the exons of TRAF3IP2 identified a coding variant (p.Asp10Asn, rs33980500) as the most significantly associated SNP (P = 1.13 × 10⻲°, odds ratio = 1.95). Functional assays showed reduced binding of this TRAF3IP2 variant to TRAF6, suggesting altered modulation of immunoregulatory signals through altered TRAF interactions as a new and shared pathway for PsA and PsV.