RESUMEN
AIMS: Glioblastoma multiforme (GBM) is the most aggressive type of human brain tumor, with a poor prognosis and a median overall survival of fewer than 15 months. Glioma stem cells (GSCs) have recently been identified as a key player in tumor initiation and therapeutic resistance in GBM. ADAMTS family of metalloproteinases is known to cleave a wide range of extracellular matrix substrates and has been linked to tissue remodeling events in tumor development. Here, we investigate that ADAMTS3 regulates GSC proliferation and self-renewal activities, and tumorigenesis in orthotopic xenograft models. METHODS: ADAMTS3 mRNA expression levels in normal human astrocyte (NHA), glioma, and GSCs cell lines were compared. After knockdown of ADAMTS3, alamarBlue assay, in vitro limiting dilution, and orthotopic xenograft assays were performed. To investigate the tumor-associated roles of ADAMTS3, several statistical assays were conducted using publicly available datasets. RESULTS: ADAMTS3 level was remarkably higher in GSCs than in NHA, glioma cell lines, and their matched differentiated tumor cells. Interestingly, knockdown of ADAMTS3 disrupted GSC's proliferation, self-renewal activity, and tumor formation in vivo. Furthermore, ADAMTS3 could be used as an independent predictor of malignancy progression in GBM. CONCLUSION: We identified ADAMTS3 as a potential therapeutic target for GBM.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Glioma , Humanos , Regulación hacia Abajo , Células Madre Neoplásicas/metabolismo , Glioma/metabolismo , Glioblastoma/patología , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Proteínas ADAMTS/genética , Proteínas ADAMTS/metabolismo , Procolágeno N-Endopeptidasa/genética , Procolágeno N-Endopeptidasa/metabolismo , Procolágeno N-Endopeptidasa/uso terapéuticoRESUMEN
ADAMTS-2 and ADAMTS-3, known as procollagen amino proteases (PNP), are primarily responsible for processing the amino ends of the fibrillar collagen precursors. ADAMTS-2 is a highly expressed gene in type I collagen-rich tissues, such as skin, bones, tendons, and aorta. ADAMTS-3 is mainly expressed in cartilage, where it colocalizes with type II procollagen and in the nervous system. Studies about ADAMTS-2 and ADAMTS-3 enzymes primarily focused on their collagen processing activity. Knowledge about the transcriptional regulations of these genes is rather limited. Here we analyzed the transcriptional regulations of ADAMTS-2 and ADAMTS-3 genes under chemically induced hypoxic conditions in endothelial cell model, HUVECs. We elucidated that hypoxia is the potent positive regulator of ADAMTS-2 and ADAMTS-3 genes. qRT-PCR and western blotting studies revealed that ADAMTS-2 and ADAMTS-3 expressions were increased at mRNA and protein levels under chemically induced hypoxic conditions in HUVECs. In addition, Transient transfection experiments of ADAMTS-2 and ADAMTS-3 promoter-reporter constructs indicated that low oxygen conditions increased ADAMTS-2 and ADAMTS-3 promoter activities. Furthermore, the DNA-protein interaction assay provided evidence of the functional binding of HIF-1α on bioinformatically determined HRE regions on the ADAMTS-2 and ADAMTS-3 promoters.
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Desintegrinas , Procolágeno , Humanos , Proteínas ADAM/genética , Proteína ADAMTS4 , Células Endoteliales/metabolismo , Hipoxia , Metaloproteinasas de la Matriz , Procolágeno N-Endopeptidasa/genética , Procolágeno N-Endopeptidasa/metabolismo , Trombospondinas , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismoRESUMEN
Osteoarthritis (OA) is the most common degenerative joint disease. In 1999, two members of the A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) family of metalloproteinases, ADAMTS4 and ADAMTS5, or aggrecanases, were identified as the enzymes responsible for aggrecan degradation in cartilage. The first aggrecanase inhibitors targeted the active site by chelation of the catalytic zinc ion. Due to the generally disappointing performance of zinc-chelating inhibitors in preclinical and clinical studies, inhibition strategies tried to move away from the active-site zinc in order to improve selectivity. Exosite inhibitors bind to proteoglycan-binding residues present on the aggrecanase ancillary domains (called exosites). While exosite inhibitors are generally more selective than zinc-chelating inhibitors, they are still far from fulfilling their potential, partly due to a lack of structural and functional data on aggrecanase exosites. Filling this gap will inform the design of novel potent, selective aggrecanase inhibitors.
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Osteoartritis , Procolágeno N-Endopeptidasa , Humanos , Procolágeno N-Endopeptidasa/metabolismo , Agrecanos/metabolismo , Proteína ADAMTS5 , Proteína ADAMTS4 , Zinc , Desintegrinas , Osteoartritis/metabolismo , Proteínas ADAM/metabolismo , TrombospondinasRESUMEN
Proteolytical processing of the growth factor VEGFC through the concerted activity of CCBE1 and ADAMTS3 is required for lymphatic development to occur. How these factors act together in time and space, and which cell types produce these factors is not understood. Here we assess the function of Adamts3 and the related protease Adamts14 during zebrafish lymphangiogenesis and show both proteins to be able to process Vegfc. Only the simultaneous loss of both protein functions results in lymphatic defects identical to vegfc loss-of-function situations. Cell transplantation experiments demonstrate neuronal structures and/or fibroblasts to constitute cellular sources not only for both proteases but also for Ccbe1 and Vegfc. We further show that this locally restricted Vegfc maturation is needed to trigger normal lymphatic sprouting and directional migration. Our data provide a single-cell resolution model for establishing secretion and processing hubs for Vegfc during developmental lymphangiogenesis.
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Fibroblastos/metabolismo , Linfangiogénesis/genética , Neuronas/metabolismo , Factor C de Crecimiento Endotelial Vascular/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Proteínas ADAMTS/genética , Proteínas ADAMTS/metabolismo , Animales , Animales Modificados Genéticamente , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Humanos , Vasos Linfáticos/embriología , Vasos Linfáticos/metabolismo , Microscopía Confocal , Procolágeno N-Endopeptidasa/genética , Procolágeno N-Endopeptidasa/metabolismo , Factor C de Crecimiento Endotelial Vascular/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
Reelin is a large secreted protein that is essential for the brain development and function. Reelin is negatively regulated by the specific cleavage by a disintegrin and metalloproteinase with thrombospondin type 1 motifs 3 (ADAMTS-3) which is also secreted from neurons. It is likely that there are other proteases that can cleave Reelin. This chapter describes the protocol for expression and handling of recombinant Reelin and ADAMTS-3 proteins to facilitate investigation of these proteins.
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Proteínas ADAMTS/genética , Moléculas de Adhesión Celular Neuronal/genética , Proteínas de la Matriz Extracelular/genética , Expresión Génica , Proteínas del Tejido Nervioso/genética , Procolágeno N-Endopeptidasa/genética , Serina Endopeptidasas/genética , Proteínas ADAMTS/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Células HEK293 , Humanos , Proteínas del Tejido Nervioso/metabolismo , Procolágeno N-Endopeptidasa/metabolismo , Ingeniería de Proteínas , Proteínas Recombinantes/metabolismo , Proteína Reelina , Serina Endopeptidasas/metabolismoRESUMEN
Proteolytic cleavage of the secreted signaling protein Reelin has been suggested to play causative roles in many neuropsychiatric and neurodegenerative disorders. Therefore, characterization of the proteolytic activity against Reelin is important not only for understanding how the brain works but also for the development of novel therapy for these disorders. Notably, ADAMTS family proteases are the primary suspects of Reelin-cleaving proteases under many, though not all, circumstances. Here we describe how to measure the Reelin-cleaving activity of ADAMTS (or of any other protease that may cleave Reelin), how to purify the Reelin-cleaving protease ADAMTS-3 from the culture supernatant of cortical neurons, and how to detect endogenous Reelin protein and its fragments in the brain.
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Proteínas ADAMTS/metabolismo , Encéfalo/metabolismo , Moléculas de Adhesión Celular Neuronal/química , Corteza Cerebral/citología , Proteínas de la Matriz Extracelular/química , Proteínas del Tejido Nervioso/química , Procolágeno N-Endopeptidasa/metabolismo , Serina Endopeptidasas/química , Animales , Células Cultivadas , Corteza Cerebral/metabolismo , Células HEK293 , Humanos , Ratones , Neuronas/citología , Neuronas/metabolismo , Proteolisis , Proteína ReelinaRESUMEN
ADAMTS3 is a member of procollagen N-proteinase subfamily of ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family. It has an important function in the procollagen maturation process. The removal of N-peptidases is required for the accurate processing of fibrillar collagens. Otherwise, several disorders can occur that is related with the collagenous tissues. ADAMTS3 mainly maturates type II collagen molecule which is the main component of the bone and cartilage. There are several expression studies about ADAMTS3 gene however its transcriptional regulation has not been lightened up, yet. Here we first time cloned and functionally analyzed the promoter region of ADAMTS3 gene, approximately 1380â¯bp upstream of the transcription start site. Transient transfection experiments showed that all truncated promoter constructs are active and 171â¯bp fragment is sufficient to activate gene expression in both Saos-2 and MG63 cells. In silico analysis showed that ADAMTS3 has a TATA-less promoter and contains several SP1/GC box binding motifs and a CpG island. Therefore we mainly investigated the SP1 dependent regulation of ADAMTS3 promoter. SP1 downregulated ADAMTS3 transcriptional activity. As consistent with the transcriptional activity, mRNA, and protein expression levels were also decreased by SP1. On the other hand, functional binding of the SP1 on multiple regions of ADAMTS3 promoter was confirmed by EMSA studies. As ADAMTS3 is responsible for the collagen maturation and biosynthesis, further we investigated the effect of SP1 on type I-II and III collagen gene expressions. We point out that SP1 increased type II and III collagen expression and in contrast decreased type I collagen expression levels in Saos-2 cells. mRNA expression level was decreased for all collagen types in MG63 model. Decrease in the type II collagen expression was also demonstrated at the protein level by SP1. Collectively these results provide first findings for the SP1-related transcriptional regulation of ADAMTS3 and collagen genes in osteosarcoma cell lines.
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Proteínas ADAMTS/genética , Proteínas ADAMTS/metabolismo , Neoplasias Óseas/genética , Osteosarcoma/genética , Procolágeno N-Endopeptidasa/genética , Procolágeno N-Endopeptidasa/metabolismo , Factor de Transcripción Sp1/metabolismo , Proteínas ADAMTS/química , Sitios de Unión , Neoplasias Óseas/metabolismo , Línea Celular Tumoral , Clonación Molecular , Colágeno/genética , Simulación por Computador , Islas de CpG , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Humanos , Modelos Biológicos , Osteosarcoma/metabolismo , Procolágeno N-Endopeptidasa/química , Regiones Promotoras GenéticasRESUMEN
INTRODUCTION: Overt fibrostenotic disease is a relative contraindication for anti-TNF therapy in Crohn's disease. We hypothesized that subclinical fibrosis may also contribute to an incomplete response to anti-TNF therapy before the onset of symptomatic stenosis. METHODS: In a previous trial, patients with ileocecal Crohn's disease were randomized to either immediate ileocecal resection or medical treatment with Infliximab. In case of insufficient response to Infliximab, the latter underwent secondary ileocecal resection. We compared specimens from those patients undergoing immediate resection (Infliximab naïve, n = 20) to those who failed Infliximab therapy (n = 20). RESULTS: Infliximab naïve and Infliximab failure patients had similar severity of inflammation when assessed by CRP levels (median 14 vs 9 mg/L) and histology (Geboes-D'Haens-score, median 10 vs 11 points). On immunohistochemistry, collagen-III and fibronectin depositions were increased in patients previously exposed to Infliximab compared to patients naïve to Infliximab. On mRNA level, procollagen peptidase showed significantly more mucosal mRNA expression in Crohn's disease patients who failed Infliximab. Infliximab responders showed no increase of this marker after 4 weeks of successful Infliximab treatment. DISCUSSION: Failure to Infliximab therapy is associated with subclinical fibrosis in Crohn's disease.
Asunto(s)
Enfermedad de Crohn/tratamiento farmacológico , Fármacos Gastrointestinales/uso terapéutico , Infliximab/uso terapéutico , Enfermedades Intestinales/complicaciones , Adulto , Enfermedad de Crohn/complicaciones , Enfermedad de Crohn/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Fibrosis , Humanos , Enfermedades Intestinales/metabolismo , Enfermedades Intestinales/patología , Mucosa Intestinal/enzimología , Masculino , Persona de Mediana Edad , Procolágeno N-Endopeptidasa/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto JovenRESUMEN
Primary lymphedema is due to developmental and/or functional defects in the lymphatic system. It may affect any part of the body, with predominance for the lower extremities. Twenty-seven genes have already been linked to primary lymphedema, either isolated, or as part of a syndrome. The proteins that they encode are involved in VEGFR3 receptor signaling. They account for about one third of all primary lymphedema cases, underscoring the existence of additional genetic factors. We used whole-exome sequencing to investigate the underlying cause in a non-consanguineous family with two children affected by lymphedema, lymphangiectasia and distinct facial features. We discovered bi-allelic missense mutations in ADAMTS3. Both were predicted to be highly damaging. These amino acid substitutions affect well-conserved residues in the prodomain and in the peptidase domain of ADAMTS3. In vitro, the mutant proteins were abnormally processed and sequestered within cells, which abolished proteolytic activation of pro-VEGFC. VEGFC processing is also affected by CCBE1 mutations that cause the Hennekam lymphangiectasia-lymphedema syndrome syndrome type1. Our data identifies ADAMTS3 as a novel gene that can be mutated in individuals affected by the Hennekam syndrome. These patients have distinctive facial features similar to those with mutations in CCBE1. Our results corroborate the recent in vitro and murine data that suggest a close functional interaction between ADAMTS3 and CCBE1 in triggering VEGFR3 signaling, a cornerstone for the differentiation and function of lymphatic endothelial cells.
Asunto(s)
Proteínas ADAMTS/deficiencia , Proteínas ADAMTS/genética , Anomalías Craneofaciales/genética , Linfangiectasia Intestinal/genética , Linfedema/genética , Procolágeno N-Endopeptidasa/deficiencia , Procolágeno N-Endopeptidasa/genética , Proteínas ADAMTS/metabolismo , Adulto , Alelos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Niño , Secuencia Conservada , Anomalías Craneofaciales/metabolismo , Células Endoteliales/metabolismo , Femenino , Células HEK293 , Humanos , Linfangiectasia Intestinal/metabolismo , Linfedema/metabolismo , Masculino , Mutación Missense , Linaje , Procolágeno N-Endopeptidasa/metabolismo , Factor C de Crecimiento Endotelial Vascular/genética , Factor C de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismoAsunto(s)
Proteínas ADAMTS/genética , Enfermedad Coronaria/genética , ADN/genética , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Procolágeno N-Endopeptidasa/genética , Fumar/efectos adversos , Proteínas ADAMTS/metabolismo , Enfermedad Coronaria/metabolismo , Vasos Coronarios/metabolismo , Humanos , Procolágeno N-Endopeptidasa/metabolismo , Factores de RiesgoRESUMEN
The secreted glycoprotein Reelin regulates embryonic brain development and adult brain functions. It has been suggested that reduced Reelin activity contributes to the pathogenesis of several neuropsychiatric and neurodegenerative disorders, such as schizophrenia and Alzheimer's disease; however, noninvasive methods that can upregulate Reelin activity in vivo have yet to be developed. We previously found that the proteolytic cleavage of Reelin within Reelin repeat 3 (N-t site) abolishes Reelin activity in vitro, but it remains controversial as to whether this effect occurs in vivo Here we partially purified the enzyme that mediates the N-t cleavage of Reelin from the culture supernatant of cerebral cortical neurons. This enzyme was identified as a disintegrin and metalloproteinase with thrombospondin motifs-3 (ADAMTS-3). Recombinant ADAMTS-3 cleaved Reelin at the N-t site. ADAMTS-3 was expressed in excitatory neurons in the cerebral cortex and hippocampus. N-t cleavage of Reelin was markedly decreased in the embryonic cerebral cortex of ADAMTS-3 knock-out (KO) mice. Importantly, the amount of Dab1 and the phosphorylation level of Tau, which inversely correlate with Reelin activity, were significantly decreased in the cerebral cortex of ADAMTS-3 KO mice. Conditional KO mice, in which ADAMTS-3 was deficient only in the excitatory neurons of the forebrain, showed increased dendritic branching and elongation in the postnatal cerebral cortex. Our study shows that ADAMTS-3 is the major enzyme that cleaves and inactivates Reelin in the cerebral cortex and hippocampus. Therefore, inhibition of ADAMTS-3 may be an effective treatment for neuropsychiatric and neurodegenerative disorders.SIGNIFICANCE STATEMENT ADAMTS-3 was identified as the protease that cleaves and inactivates Reelin in the cerebral cortex and hippocampus. ADAMTS-3 was expressed in the excitatory neurons of the embryonic and postnatal cerebral cortex and hippocampus. Cleavage by ADAMTS-3 is the major contributor of Reelin inactivation in vivo Tau phosphorylation was decreased and dendritic branching and elongation was increased in ADAMTS-3-deficient mice. Therefore, inhibition of ADAMTS-3 upregulates Reelin activity and may be a potential therapeutic strategy for the prevention or treatment of neuropsychiatric and neurodegenerative disorders, such as schizophrenia and Alzheimer's disease.
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Proteínas ADAMTS/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Corteza Cerebral/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Hipocampo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Procolágeno N-Endopeptidasa/metabolismo , Serina Endopeptidasas/metabolismo , Transducción de Señal/fisiología , Animales , Células Cultivadas , Activación Enzimática , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Unión Proteica , Proteína ReelinaRESUMEN
Lymphangiogenesis is supported by 2 homologous VEGFR3 ligands, VEGFC and VEGFD. VEGFC is required for lymphatic development, while VEGFD is not. VEGFC and VEGFD are proteolytically cleaved after cell secretion in vitro, and recent studies have implicated the protease a disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAMTS3) and the secreted factor collagen and calcium binding EGF domains 1 (CCBE1) in this process. It is not well understood how ligand proteolysis is controlled at the molecular level or how this process regulates lymphangiogenesis, because these complex molecular interactions have been difficult to follow ex vivo and test in vivo. Here, we have developed and used biochemical and cellular tools to demonstrate that an ADAMTS3-CCBE1 complex can form independently of VEGFR3 and is required to convert VEGFC, but not VEGFD, into an active ligand. Consistent with these ex vivo findings, mouse genetic studies revealed that ADAMTS3 is required for lymphatic development in a manner that is identical to the requirement of VEGFC and CCBE1 for lymphatic development. Moreover, CCBE1 was required for in vivo lymphangiogenesis stimulated by VEGFC but not VEGFD. Together, these studies reveal that lymphangiogenesis is regulated by two distinct proteolytic mechanisms of ligand activation: one in which VEGFC activation by ADAMTS3 and CCBE1 spatially and temporally patterns developing lymphatics, and one in which VEGFD activation by a distinct proteolytic mechanism may be stimulated during inflammatory lymphatic growth.
Asunto(s)
Linfangiogénesis/fisiología , Factor C de Crecimiento Endotelial Vascular/metabolismo , Factor D de Crecimiento Endotelial Vascular/metabolismo , Proteínas ADAMTS/deficiencia , Proteínas ADAMTS/genética , Proteínas ADAMTS/metabolismo , Animales , Proteínas de Unión al Calcio/deficiencia , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proliferación Celular , Células Endoteliales/citología , Células Endoteliales/metabolismo , Células HEK293 , Humanos , Ligandos , Linfangiogénesis/genética , Vasos Linfáticos/metabolismo , Ratones , Ratones Noqueados , Modelos Biológicos , Péptido Hidrolasas/metabolismo , Procolágeno N-Endopeptidasa/genética , Procolágeno N-Endopeptidasa/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Factor C de Crecimiento Endotelial Vascular/deficiencia , Factor C de Crecimiento Endotelial Vascular/genética , Factor D de Crecimiento Endotelial Vascular/deficiencia , Factor D de Crecimiento Endotelial Vascular/genética , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVE: To obtain recombinant mouse collagenase ADAMTS2 (ADAM metallopeptidase with thrombospondin type 1 motif, 2) C terminal (1109-1213), and prepare the corresponding rabbit anti-ADAMTS2 polyclonal antibodies. METHODS: The recombinant expression plasmid pGEX-6p-1-ADAMTS2 (1109-1213) was transformed into E.coli. The target protein was induced by IPTG and identified by mass spectrometry following affinity purification. The expressed and purified ADAMTS2 (1109-1213) protein was used to immunize New Zealand rabbits to prepare anti-ADAMTS2 polyclonal antibodies. The antibody titers were detected by ELISA and the antibody specificity by Western blotting. RESULTS: The protein ADAMTS2 (1109-1213) was expressed in E.coli after IPTG induction, and with the purified protein, we prepared antiserum in the immunized rabbits. The titer of the antiserum reached over 1:160 000. The antiserum showed a good specificity. CONCLUSION: The high titer and specific rabbit anti-ADAMTS2 antibody has been prepared successfully.
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Proteínas ADAM/inmunología , Anticuerpos Monoclonales/inmunología , Sueros Inmunes/inmunología , Procolágeno N-Endopeptidasa/inmunología , Proteínas Recombinantes/inmunología , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteínas ADAMTS , Proteína ADAMTS4 , Animales , Especificidad de Anticuerpos/inmunología , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Femenino , Masculino , Ratones Endogámicos C57BL , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Procolágeno N-Endopeptidasa/genética , Procolágeno N-Endopeptidasa/metabolismo , Conejos , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: Mechanical loading plays an important role in the regulation of extracellular matrix (ECM) homeostasis as well as pathogenesis of intervertebral disc (IVD) degeneration. The human annulus fibrosus (hAF) in the IVD is subjected to contact shear stress during body motion. However, the effects of shear stress on hAF cells remain unclear. This aim of the study was to investigate the expression of the ECM (COLI, COLIII and aggrecan) and matrix metalloproteinase (MMP-1, MMP-3 and ADAMTS-4) genes in hAF cells following fluid-induced shear stress in a custom-fabricated bio-microfluidic device. METHODS: hAF cells were harvested from degenerated disc tissues in routine spine surgery, staged by magnetic resonance imaging, expanded in monolayers and then seeded onto the bio-microfluidic device. The experimental groups were subjected to 1 and 10 dyne/cm(2) shear stress for 4 h, and no shear stress was applied to the control group. We used real time polymerase chain reaction for gene expression. RESULTS: Shear stress of 1 dyne/cm(2) exerted an anabolic effect on COLI and COLIII genes and catabolic effects on the aggrecan gene, while 10 dyne/cm(2) had an anabolic effect on the COLI gene and a catabolic effect on COLIII and aggrecan genes. The COLI gene was upregulated in a stress-dependent manner. Expression of MMP-1 was significantly higher in the 10 dyne/cm(2) group compared to the control group (P < 0.05), but was similar in the control and 1 dyne/cm(2) groups. Expression of MMP-3 and ADAMTS-4 were similar in all three groups. CONCLUSION: Taken together, hAF cells responded to shear stress. The findings help us understand and clarify the effects of shear stress on IVD degeneration as well as the development of a new therapeutic strategy for IVD degeneration.
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Matriz Extracelular/enzimología , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Fenómenos Biomecánicos , Células Cultivadas , Inducción Enzimática , Matriz Extracelular/genética , Femenino , Expresión Génica , Humanos , Disco Intervertebral/enzimología , Disco Intervertebral/patología , Degeneración del Disco Intervertebral/enzimología , Degeneración del Disco Intervertebral/patología , Masculino , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/metabolismo , Persona de Mediana Edad , Procolágeno N-Endopeptidasa/genética , Procolágeno N-Endopeptidasa/metabolismoRESUMEN
BACKGROUND: Ovarian carcinomas, usually associated with sex hormones dysregulation, are the leading cause of gynecological neoplastic death. In normal ovaries, hormones play a central role in regulating cell proliferation, differentiation, and apoptosis. On the other hand, hormonal alterations also play a variety of roles in cancer. Stimulation by sex hormones potentially affects gene expression, invasiveness, cell growth and angiogenesis. Proteases of the "a disintegrin and metalloproteinase with thrombospondin motifs" (ADAMTS) family are secreted by different cell types and become involved in collagen processing, cleavage of the proteoglycan matrix, and angiogenesis. We evaluated whether sex hormones affect ADAMTS 1 and 4 expression in ovarian cancer cells. METHODS: We analysed mRNA and protein levels in human ovarian tumor cells with different degrees of malignancy, NIH-OVCAR-3 and ES-2, that were treated or not with estrogen, testosterone and progesterone. RESULTS: Our results suggest that progesterone increases ADAMTS protein and mRNA levels in the lysates from ES-2 cells, and it increases ADAMTS protein in the lysates and conditioned media from NIH-OVCAR-3. Progesterone effects were reversed by RU486 treatment. CONCLUSION: We conclude that progesterone acts via the progesterone receptor to modulate ADAMTS 1 and 4 levels in ovarian cancer cell lines.
Asunto(s)
Proteínas ADAM/metabolismo , Procolágeno N-Endopeptidasa/metabolismo , Progesterona/fisiología , Receptores de Progesterona/metabolismo , Proteínas ADAM/genética , Proteína ADAMTS1 , Proteína ADAMTS4 , Línea Celular Tumoral , Inducción Enzimática , Femenino , Expresión Génica , Humanos , Mifepristona/farmacología , Neoplasias Ováricas , Procolágeno N-Endopeptidasa/genética , Receptores Androgénicos/metabolismo , Receptores de Estrógenos/metabolismoRESUMEN
A disintegrin and metalloproteinase with thrombospondin type I motif (ADAMTS)2, 3, and 14 are collectively named procollagen N-proteinases (pNPs) because of their specific ability to cleave the aminopropeptide of fibrillar procollagens. Several reports also indicate that they could be involved in other biological processes, such as blood coagulation, development, and male fertility, but the potential substrates associated with these activities remain unknown. Using the recently described N-terminal amine isotopic labeling of substrate approach, we analyzed the secretomes of human fibroblasts and identified 8, 17, and 22 candidate substrates for ADAMTS2, 3, and 14, respectively. Among these newly identified substrates, many are components of the extracellular matrix and/or proteins related to cell signaling such as latent TGF-ß binding protein 1, TGF-ß RIII, and dickkopf-related protein 3. Candidate substrates for the 3 ADAMTS have been biochemically validated in different contexts, and the implication of ADAMTS2 in the control of TGF-ß activity has been further demonstrated in human fibroblasts. Finally, the cleavage site specificity was assessed showing a clear and unique preference for nonpolar or slightly hydrophobic amino acids. This work shows that the activities of the pNPs extend far beyond the classically reported processing of the aminopropeptide of fibrillar collagens and that they should now be considered as multilevel regulators of matrix deposition and remodeling.-Bekhouche, M., Leduc, C., Dupont, L., Janssen, L., Delolme, F., Vadon-Le Goff, S., Smargiasso, N., Baiwir, D., Mazzucchelli, G., Zanella-Cleon, I., Dubail, J., De Pauw, E., Nusgens, B., Hulmes, D. J. S., Moali, C., Colige, A. Determination of the substrate repertoire of ADAMTS2, 3, and 14 significantly broadens their functions and identifies extracellular matrix organization and TGF-ß signaling as primary targets.
Asunto(s)
Proteínas ADAMTS/metabolismo , Matriz Extracelular/metabolismo , Procolágeno N-Endopeptidasa/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteínas ADAMTS/genética , Proteínas Adaptadoras Transductoras de Señales , Quimiocinas , Regulación de la Expresión Génica/fisiología , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de Unión a TGF-beta Latente/genética , Proteínas de Unión a TGF-beta Latente/metabolismo , Procolágeno N-Endopeptidasa/genética , Proteoglicanos/genética , Proteoglicanos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/genéticaRESUMEN
The majority of the skeleton arises by endochondral ossification, whereby cartilaginous templates expand and are resorbed by osteoclasts then replaced by osteoblastic bone formation. Ephrin B2 is a receptor tyrosine kinase expressed by osteoblasts and growth plate chondrocytes that promotes osteoblast differentiation and inhibits osteoclast formation. We investigated the role of ephrin B2 in endochondral ossification using Osx1Cre-targeted gene deletion. Neonatal Osx1Cre.Efnb2(Δ/Δ) mice exhibited a transient osteopetrosis demonstrated by increased trabecular bone volume with a high content of growth plate cartilage remnants and increased cortical thickness, but normal osteoclast numbers within the primary spongiosa. Osteoclasts at the growth plate had an abnormal morphology and expressed low levels of tartrate-resistant acid phosphatase; this was not observed in more mature bone. Electron microscopy revealed a lack of sealing zones and poor attachment of Osx1Cre.Efnb2(Δ/Δ) osteoclasts to growth plate cartilage. Osteoblasts at the growth plate were also poorly attached and impaired in their ability to deposit osteoid. By 6 months of age, trabecular bone mass, osteoclast morphology and osteoid deposition by Osx1Cre.Efnb2(Δ/Δ) osteoblasts were normal. Cultured chondrocytes from Osx1Cre.Efnb2(Δ/Δ) neonates showed impaired support of osteoclastogenesis but no significant change in Rankl (Tnfsf11) levels, whereas Adamts4 levels were significantly reduced. A population of ADAMTS4(+) early hypertrophic chondrocytes seen in controls was absent from Osx1Cre.Efnb2(Δ/Δ) neonates. This suggests that Osx1Cre-expressing cells, including hypertrophic chondrocytes, are dependent on ephrin B2 for their production of cartilage-degrading enzymes, including ADAMTS4, and this might be required for attachment of osteoclasts and osteoblasts to the cartilage surface during endochondral ossification.
Asunto(s)
Cartílago/patología , Condrocitos/metabolismo , Efrina-B2/metabolismo , Osteoclastos/patología , Osteogénesis , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Animales , Animales Recién Nacidos , Cartílago/metabolismo , Adhesión Celular , Diferenciación Celular , Condrocitos/patología , Femenino , Regulación de la Expresión Génica , Inmunohistoquímica , Integrasas/metabolismo , Ratones Endogámicos C57BL , Modelos Biológicos , Tamaño de los Órganos , Osteoblastos/patología , Osteoclastos/metabolismo , Osteoclastos/ultraestructura , Osteogénesis/genética , Osteopetrosis/genética , Osteopetrosis/patología , Fenotipo , Procolágeno N-Endopeptidasa/metabolismo , Tibia/metabolismo , Tibia/patologíaRESUMEN
BACKGROUND: A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) proteoglycanases are specialized in the degradation of chondroitin sulfate proteoglycans and participate in mechanisms mediating neuroplasticity. Despite the beneficial effect of ADAMTS-4 on neurorepair after spinal cord injury, the functions of ADAMTS proteoglycanases in other CNS disease states have not been studied. Therefore, we investigated the expression, effects and associated mechanisms of ADAMTS-4 during amyotrophic lateral sclerosis (ALS) in the SOD1(G93A) mouse model. RESULTS: ADAMTS-4 expression and activity were reduced in the spinal cord of SOD1(G93A) mice at disease end-stage when compared to WT littermates. To counteract the loss of ADAMTS-4, SOD1(G93A) and WT mice were treated with saline or a recombinant ADAMTS-4 before symptom onset. Administration of ADAMTS-4 worsened the prognosis of SOD1(G93A) mice by accelerating clinical signs of neuromuscular dysfunctions. The worsened prognosis of ADAMTS-4-treated SOD1(G93A) mice was accompanied by increased degradation of perineuronal nets enwrapping motoneurons and increased motoneuron degeneration in the lumbar spinal cord. Motoneurons of ADAMTS-4-treated SOD1(G93A) mice were more vulnerable to degeneration most likely due to the loss of their extracellular matrix envelopes. The decrease of neurotrophic factor production induced by ADAMTS-4 in vitro and in vivo may also contribute to a hostile environment for motoneuron especially when devoid of a net. CONCLUSIONS: This study suggests that the reduction of ADAMTS-4 activity during the progression of ALS pathology may be an adaptive change to mitigate its neurodegenerative impact in CNS tissues. Therapies compensating the compromized ADAMTS-4 activity are likely not promising approaches for treating ALS.
Asunto(s)
Proteínas ADAM/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Procolágeno N-Endopeptidasa/metabolismo , Médula Espinal/metabolismo , Proteína ADAMTS4 , Esclerosis Amiotrófica Lateral/patología , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Masculino , Ratones Transgénicos , Superóxido Dismutasa/metabolismoRESUMEN
Follicle growth and ovulation involve the coordinated expression of many genes, driven by FSH and LH. Reports indicate that Eph receptors and ephrins are expressed in the ovary, suggesting roles in follicle growth and/or ovulation. We previously reported FSH-induced expression of ephrin-A5 (EFNA5) and 4 of its cognate Eph receptors in mouse granulosa cells. We now report that female mice lacking EFNA5 are subfertile, exhibit a compromised response to LH, and display abnormal ovarian histology after superovulation. Efna5(-/-) females litters were 40% smaller than controls, although no difference in litter frequency was detected. The ovarian response to superovulation was also compromised in Efna5(-/-) females, with 37% fewer oocytes ovulated than controls. These results corresponded with a reduction in ovarian mRNA levels of several LH-responsive genes, including Pgr, Ptgs2, Tnfaip6, Ereg, Btc, and Adamts4, suggesting that Efna5(-/-) ovaries exhibit a partially attenuated response to LH. Histopathological analysis indicated that superovulated Efna5(-/-) females exhibited numerous ovarian defects, including intraovarian release of cumulus oocyte complexes, increased incidence of oocytes trapped within luteinized follicles, granulosa cell and follicular fluid emboli, fibrin thrombi, and interstitial hemorrhage. In addition, adult Efna5(-/-) ovaries exhibited a 4-fold increase in multioocyte follicles compared with controls, although no difference was detected in 3-week-old mice, suggesting the possibility of follicle merging. Our observations indicate that loss of EFNA5 in female mice results in subfertility and imply that Eph-ephrin signaling may also play a previously unidentified role in the regulation of fertility in women.
Asunto(s)
Efrina-A5/genética , Fertilidad/genética , Ovario/metabolismo , ARN Mensajero/metabolismo , Superovulación/genética , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Animales , Betacelulina/genética , Betacelulina/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Cuerpo Lúteo/patología , Células del Cúmulo/patología , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Efrina-A5/metabolismo , Epirregulina/genética , Epirregulina/metabolismo , Femenino , Gonadotropinas , Células de la Granulosa/patología , Infertilidad/genética , Luteinización , Ratones , Ratones Noqueados , Folículo Ovárico/patología , Ovario/patología , Ovulación/genética , Procolágeno N-Endopeptidasa/genética , Procolágeno N-Endopeptidasa/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
As a degenerative joint disease, osteoarthritis (OA) constitutes a major cause of disability that seriously affects the quality of life of a large population of people worldwide. However, effective treatment that can successfully reverse OA progression is lacking until now. The present study aimed to determine whether two small non-coding RNAs miR-29a and miR-140, which are significantly down-regulated in OA, can be applied together as potential therapeutic targets for OA treatment. MiRNA synergy score was used to screen the miRNA pairs that potentially synergistically regulate OA. An in vitro model of OA was established by treating murine chondrocytes with IL-1ß. Transfection of miR-29a and miR-140 via plasmids was investigated on chondrocyte proliferation and expression of nine genes such as ADAMTS4, ADAMTS5, ACAN, COL2A1, COL10A1, MMP1, MMP3, MMP13 and TIMP metal-lopeptidase inhibitor 1 (TIMP1). Western blotting was used to determine the protein expression level of MMP13 and TIMP1, and ELISA was used to detect the content of type II collagen. Combined use of miR-29a and miR-140 successfully reversed the destructive effect of IL-1ß on chondrocyte proliferation, and notably affected the MMP13 and TIMP1 gene expression that regulates extracellular matrix. Although co-transfection of miR-29a and miR-140 did not show a synergistic effect on MMP13 protein expression and type II collagen release, but both of them can significantly suppress the protein abundance of MMP13 and restore the type II collagen release in IL-1ß treated chondrocytes. Compared with single miRNA transfection, cotransfection of both miRNAs exceedingly abrogated the suppressed the protein production of TIMP1 caused by IL-1ß, thereby suggesting potent synergistic action. These results provided novel insights into the important function of miRNAs' collaboration in OA pathological development. The reduced MMP13, and enhanced TIMP1 protein production and type II collagen release also implies that miR-29a and miR-140 combination treatment may be a possible treatment for OA.