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1.
Gene ; 659: 1-10, 2018 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-29518549

RESUMO

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 1380 bp upstream of the transcription start site. Transient transfection experiments showed that all truncated promoter constructs are active and 171 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.


Assuntos
Proteínas ADAMTS/genética , Proteínas ADAMTS/metabolismo , Neoplasias Ósseas/genética , Osteossarcoma/genética , Pró-Colágeno N-Endopeptidase/genética , Pró-Colágeno N-Endopeptidase/metabolismo , Fator de Transcrição Sp1/metabolismo , Proteínas ADAMTS/química , Sítios de Ligação , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Clonagem Molecular , Colágeno/genética , Simulação por Computador , Ilhas de CpG , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Modelos Biológicos , Osteossarcoma/metabolismo , Pró-Colágeno N-Endopeptidase/química , Regiões Promotoras Genéticas
2.
PLoS One ; 13(1): e0190999, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29364909

RESUMO

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.


Assuntos
Doença de Crohn/tratamento farmacológico , Fármacos Gastrointestinais/uso terapêutico , Infliximab/uso terapêutico , Enteropatias/complicações , Adulto , Doença de Crohn/complicações , Doença de Crohn/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Feminino , Fibrose , Humanos , Enteropatias/metabolismo , Enteropatias/patologia , Mucosa Intestinal/enzimologia , Masculino , Pessoa de Meia-Idade , Pró-Colágeno N-Endopeptidase/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto Jovem
3.
Hum Mol Genet ; 26(21): 4095-4104, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28985353

RESUMO

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.


Assuntos
Proteínas ADAMTS/deficiência , Proteínas ADAMTS/genética , Anormalidades Craniofaciais/genética , Linfangiectasia Intestinal/genética , Linfedema/genética , Pró-Colágeno N-Endopeptidase/deficiência , Pró-Colágeno N-Endopeptidase/genética , Proteínas ADAMTS/metabolismo , Adulto , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos , Criança , Sequência Conservada , Anormalidades Craniofaciais/metabolismo , Células Endoteliais/metabolismo , Feminino , Células HEK293 , Humanos , Linfangiectasia Intestinal/metabolismo , Linfedema/metabolismo , Masculino , Mutação de Sentido Incorreto , Linhagem , Pró-Colágeno N-Endopeptidase/metabolismo , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo
4.
J Neurosci ; 37(12): 3181-3191, 2017 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-28213441

RESUMO

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.


Assuntos
Proteínas ADAMTS/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Córtex Cerebral/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Pró-Colágeno N-Endopeptidase/metabolismo , Serina Endopeptidases/metabolismo , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Ativação Enzimática , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Ligação Proteica
5.
J Clin Invest ; 126(6): 2167-80, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27159393

RESUMO

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.


Assuntos
Linfangiogênese/fisiologia , Fator C de Crescimento do Endotélio Vascular/metabolismo , Fator D de Crescimento do Endotélio Vascular/metabolismo , Proteínas ADAMTS/deficiência , Proteínas ADAMTS/genética , Proteínas ADAMTS/metabolismo , Animais , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proliferação de Células , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células HEK293 , Humanos , Ligantes , Linfangiogênese/genética , Vasos Linfáticos/metabolismo , Camundongos , Camundongos Knockout , Modelos Biológicos , Peptídeo Hidrolases/metabolismo , Pró-Colágeno N-Endopeptidase/genética , Pró-Colágeno N-Endopeptidase/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Fator C de Crescimento do Endotélio Vascular/deficiência , Fator C de Crescimento do Endotélio Vascular/genética , Fator D de Crescimento do Endotélio Vascular/deficiência , Fator D de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo
6.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 32(3): 393-6, 2016 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-26927563

RESUMO

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.


Assuntos
Proteínas ADAM/imunologia , Anticorpos Monoclonais/imunologia , Soros Imunes/imunologia , Pró-Colágeno N-Endopeptidase/imunologia , Proteínas Recombinantes/imunologia , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteínas ADAMTS , Proteína ADAMTS4 , Animais , Especificidade de Anticorpos/imunologia , Western Blotting , Ensaio de Imunoadsorção Enzimática , Feminino , Masculino , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Pró-Colágeno N-Endopeptidase/genética , Pró-Colágeno N-Endopeptidase/metabolismo , Coelhos , Proteínas Recombinantes/metabolismo
7.
Stem Cell Res Ther ; 7: 34, 2016 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-26921206

RESUMO

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.


Assuntos
Matriz Extracelular/enzimologia , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Fenômenos Biomecânicos , Células Cultivadas , Indução Enzimática , Matriz Extracelular/genética , Feminino , Expressão Gênica , Humanos , Disco Intervertebral/enzimologia , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/enzimologia , Degeneração do Disco Intervertebral/patologia , Masculino , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Pessoa de Meia-Idade , Pró-Colágeno N-Endopeptidase/genética , Pró-Colágeno N-Endopeptidase/metabolismo
8.
J Ovarian Res ; 9: 9, 2016 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-26916548

RESUMO

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.


Assuntos
Proteínas ADAM/metabolismo , Pró-Colágeno N-Endopeptidase/metabolismo , Progesterona/fisiologia , Receptores de Progesterona/metabolismo , Proteínas ADAM/genética , Proteína ADAMTS1 , Proteína ADAMTS4 , Linhagem Celular Tumoral , Indução Enzimática , Feminino , Expressão Gênica , Humanos , Mifepristona/farmacologia , Neoplasias Ovarianas , Pró-Colágeno N-Endopeptidase/genética , Receptores Androgênicos/metabolismo , Receptores Estrogênicos/metabolismo
9.
Development ; 143(4): 648-57, 2016 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-26755702

RESUMO

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.


Assuntos
Cartilagem/patologia , Condrócitos/metabolismo , Efrina-B2/metabolismo , Osteoclastos/patologia , Osteogênese , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Animais , Animais Recém-Nascidos , Cartilagem/metabolismo , Adesão Celular , Diferenciação Celular , Condrócitos/patologia , Feminino , Regulação da Expressão Gênica , Imuno-Histoquímica , Integrases/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Tamanho do Órgão , Osteoblastos/patologia , Osteoclastos/metabolismo , Osteoclastos/ultraestrutura , Osteogênese/genética , Osteopetrose/genética , Osteopetrose/patologia , Fenótipo , Pró-Colágeno N-Endopeptidase/metabolismo , Tíbia/metabolismo , Tíbia/patologia
10.
Mol Neurodegener ; 11: 10, 2016 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-26809777

RESUMO

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.


Assuntos
Proteínas ADAM/metabolismo , Esclerose Amiotrófica Lateral/metabolismo , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Pró-Colágeno N-Endopeptidase/metabolismo , Medula Espinal/metabolismo , Proteína ADAMTS4 , Esclerose Amiotrófica Lateral/patologia , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Masculino , Camundongos Transgênicos , Superóxido Dismutase/metabolismo
11.
FASEB J ; 30(5): 1741-56, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26740262

RESUMO

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.


Assuntos
Proteínas ADAMTS/metabolismo , Matriz Extracelular/metabolismo , Pró-Colágeno N-Endopeptidase/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas ADAMTS/genética , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Ligação a TGF-beta Latente/genética , Proteínas de Ligação a TGF-beta Latente/metabolismo , Pró-Colágeno N-Endopeptidase/genética , Proteoglicanas/genética , Proteoglicanas/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/genética
12.
Endocrinology ; 157(2): 942-55, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26672804

RESUMO

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.


Assuntos
Efrina-A5/genética , Fertilidade/genética , Ovário/metabolismo , RNA Mensageiro/metabolismo , Superovulação/genética , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Animais , Betacelulina/genética , Betacelulina/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Corpo Lúteo/patologia , Células do Cúmulo/patologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Efrina-A5/metabolismo , Epirregulina/genética , Epirregulina/metabolismo , Feminino , Gonadotropinas , Células da Granulosa/patologia , Infertilidade/genética , Luteinização , Camundongos , Camundongos Knockout , Folículo Ovariano/patologia , Ovário/patologia , Ovulação/genética , Pró-Colágeno N-Endopeptidase/genética , Pró-Colágeno N-Endopeptidase/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
Mol Cells ; 39(2): 103-10, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26608362

RESUMO

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.


Assuntos
Condrócitos/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Interleucina-1beta/farmacologia , MicroRNAs/genética , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Agrecanas/genética , Agrecanas/metabolismo , Animais , Animais Lactentes , Proliferação de Células/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Colágeno Tipo X/genética , Colágeno Tipo X/metabolismo , Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Isoenzimas/genética , Isoenzimas/metabolismo , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Camundongos , MicroRNAs/metabolismo , Modelos Biológicos , Osteoartrite/genética , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteoartrite/terapia , Cultura Primária de Células , Pró-Colágeno N-Endopeptidase/genética , Pró-Colágeno N-Endopeptidase/metabolismo , Transdução de Sinais , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo
14.
Int Immunopharmacol ; 30: 36-42, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26637956

RESUMO

Sanmiao formula (SM) is a compound prescription, which has been used in traditional Chinese medicine since the Ming Dynasty for gouty and rheumatoid arthritis treatments. However, no evidence has been unfolded to show the relationship between SM and gouty arthritis (GA), particularly inhibiting cartilage matrix degradation. In the present study, we undertook a characterization of anti-GA activity of SM using an in vivo rat model induced by potassium oxonate and cold bath together with in vitro studies with chondrocytes for further molecular characterization. Potassium oxonate and cold bath rats were treated with SM at doses of 7.2g/kg per day for 5days. SM treatments significantly suppressed the swelling rate and the severe pathologic changes in the joints of the animals in gout model. Inflammatory factors count by ELISA analysis, SM exhibited inhibition on IL-1ß and TNF-α. Moreover, histological analysis of the joints and SM-serum substantially interfered with the MSU-induced expression of glycosaminoglycans (GAG), up-regulated the content of proteoglycan. Importantly, SM interfered with GA-augmented expression of matrix metalloproteinases (MMPs) -3 and aggrecanases (ADAMTS)-4, which are considered to be key enzymes in cartilage matrix degradation, and simultaneously augmented GA-reduced tissue inhibitors of metalloproteinases (TIMPs) -1 and -3 expression in the joints and chondrocytes. Therefore, SM is looking forward to be a potential novel agent that could prevent cartilage matrix degradation effectively in gouty arthritis, and this provides a new target for development of new medicines.


Assuntos
Artrite Gotosa/tratamento farmacológico , Condrócitos/efeitos dos fármacos , Misturas Complexas/administração & dosagem , Medicamentos de Ervas Chinesas/administração & dosagem , Articulações/efeitos dos fármacos , Proteínas Matrilinas/metabolismo , Medicina Tradicional Chinesa , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Animais , Artrite Gotosa/induzido quimicamente , Células Cultivadas , Condrócitos/fisiologia , Regulação para Baixo , Humanos , Hidrólise/efeitos dos fármacos , Hidrólise/efeitos da radiação , Interleucina-1beta/metabolismo , Articulações/patologia , Masculino , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Modelos Animais , Ácido Oxônico/administração & dosagem , Pró-Colágeno N-Endopeptidase/genética , Pró-Colágeno N-Endopeptidase/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/metabolismo
15.
BMC Genomics ; 16: 869, 2015 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-26503507

RESUMO

BACKGROUND: Idiopathic interstitial pneumonias (IIPs) are a group of heterogeneous, somewhat unpredictable diseases characterized by progressive scarring of the interstitium. Since lung function is a key determinant of survival, we reasoned that the transcriptional profile in IIP lung tissue would be associated with measures of lung function, and could enhance prognostic approaches to IIPs. RESULTS: Using gene expression profiling of 167 lung tissue specimens with IIP diagnosis and 50 control lungs, we identified genes whose expression is associated with changes in lung function (% predicted FVC and % predicted DLCO) modeled as categorical (severe vs mild disease) or continuous variables while adjusting for smoking status and IIP subtype; false discovery rate (FDR) approach was used to correct for multiple comparisons. This analysis identified 58 transcripts that are associated with mild vs severe disease (categorical analysis), including those with established role in fibrosis (ADAMTS4, ADAMTS9, AGER, HIF-1α, SERPINA3, SERPINE2, and SELE) as well as novel IIP candidate genes such as rhotekin 2 (RTKN2) and peptidase inhibitor 15 (PI15). Protein-protein interactome analysis of 553 genes whose expression is significantly associated with lung function when modeled as continuous variables demonstrates that more severe presentation of IIPs is characterized by an increase in cell cycle progression and apoptosis, increased hypoxia, and dampened innate immune response. Our findings were validated in an independent cohort of 131 IIPs and 40 controls at the mRNA level and for one gene (RTKN2) at the protein level by immunohistochemistry in a subset of samples. CONCLUSIONS: We identified commonalities and differences in gene expression among different subtypes of IIPs. Disease progression, as characterized by lower measures of FVC and DLCO, results in marked changes in expression of novel and established genes and pathways involved in IIPs. These genes and pathways represent strong candidates for biomarker studies and potential therapeutic targets for IIP severity.


Assuntos
Regulação da Expressão Gênica , Pneumonias Intersticiais Idiopáticas/genética , Pneumonias Intersticiais Idiopáticas/fisiopatologia , Pulmão/fisiopatologia , Proteínas/genética , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Proteína ADAMTS9 , Adulto , Idoso , Selectina E/genética , Selectina E/metabolismo , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Pessoa de Meia-Idade , Pró-Colágeno N-Endopeptidase/genética , Pró-Colágeno N-Endopeptidase/metabolismo , Receptor para Produtos Finais de Glicação Avançada/genética , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Serpina E2/genética , Serpina E2/metabolismo , Serpinas/genética , Serpinas/metabolismo
16.
Gene ; 573(2): 321-7, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26232334

RESUMO

Up-regulation of ADAMTS genes with proinflammatory cytokines is important for some pathological conditions such as osteoarthritis (OA) that is a disease based on ECM degradation in cartilage. IL-1α is a proinflammatory cytokine and important both to normal and pathophysiologic conditions in cartilage and bone. Effects of some proinflammatory cytokines such as TNF-α and IL-1ß on the some members of ADAMTS family have been investigated in some chondrocyte tissues or cell lines. However the effect of the IL-1α on the expression of ADAMTS-2 and ADAMTS-3 gene expression in osteoblast like cell lines, remains unclear. Therefore, the aim of this study is to investigate the effect of IL-1α on ADAMTS-2 and ADAMTS-3 gene expression in osteoblast like cells, Saos-2 and MG-63. The present study, for the first time, demonstrated that IL-1α increases ADAMTS-2 and ADAMTS-3 gene expressions in both Saos-2 and MG-63 cells. Having correlation to mRNA induction, the upregulation of ADAMTS-2,-3 protein levels by IL-1α stimulation is also observed. The inhibition studies showed that this upregulation occurred at the level of transcription, and there was no effect of IL-1α on ADAMTS-2 mRNA half-life in Saos-2 cells. Transactivation potential of IL-1α on ADAMTS-2 promoter was investigated by transient transfection assay. Specifically, IL-1α strongly increased -658/+112 and -530/+112 ADAMTS-2 promoter constructs. Further, we analyzed signaling pathways involved in ADAMTS-2 induction. Pathway inhibition studies revealed that this upregulation depends on the activation of MEK, JNK and PI3K pathways. These findings suggested that IL-1α is a strong positive regulator of ADAMTS-2 and ADAMTS-3 expression. These findings would provide novel insight into the pathophysiology of OA.


Assuntos
Proteínas ADAM/genética , Interleucina-1alfa/fisiologia , Sistema de Sinalização das MAP Quinases , Osteoblastos/enzimologia , Pró-Colágeno N-Endopeptidase/genética , Proteínas ADAM/metabolismo , Proteínas ADAMTS , Proteína ADAMTS4 , Linhagem Celular Tumoral , Indução Enzimática , Humanos , MAP Quinase Quinase Quinases/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Pró-Colágeno N-Endopeptidase/metabolismo , Regiões Promotoras Genéticas , Ativação Transcricional
17.
Osteoarthritis Cartilage ; 23(12): 2279-2287, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26165503

RESUMO

OBJECTIVE: Glucocorticoids (GCs) have been widely used in the management of osteoarthritis (OA) and rheumatoid arthritis (RA). Nevertheless, there has been some concern about their ability of increasing reactive oxygen species (ROS) in the cartilage. Forkhead-box class O (FOXO) transcription factors have been proved to have a protective role in chondrocytes through regulation of autophagy and defending oxidative stress. The objective of this study was to investigate the role of FOXO3 in Dex-induce up-regulation of ROS. DESIGN: Healthy cartilages debris from six patients were used for chondrocytes culture. After the treatment of dexamethasone (Dex), the ROS levels, autophagic flux, the expression of FOXO3 in chondrocytes were measured. RNA interference technique was also used to determine the role of FOXO3 in Dex-induced autophagy. The metabolism of the extra-cellular matrix was also investigated. THE RESULTS: Dex increased intracellular ROS level, the expression of Akt, FOXO3 as well as autophagy flux in human chondrocytes. The expression of aggrecanases also increased after the treatment of Dex. Catalase, the ROS scavenger, suppressed Dex-induced up-regulation of autophagy flux and expression of aggrecanases and Akt. MK-2206 and LY294002, the PI3K/Akt inhibitors, repressed Dex-induced up-regulation of FOXO3. Silencing FOXO3 resulted in down-regulation of Dex-induced autophagy. Moreover, knockdown of FOXO3 increased Dex-induced apoptosis as well as ROS levels in chondrocytes. In addition, up-regulation of autophagy by Rapamycin resulted in decreasing ROS level in chondrocytes. CONCLUSION: Dex could advance the degenerative process in cartilage. Autophagy was induced in response to Dex-induced up-regulation of ROS via ROS/Akt/FOXO3 signal pathway.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Impacto Femoroacetabular/metabolismo , Fatores de Transcrição Forkhead/efeitos dos fármacos , Glucocorticoides/farmacologia , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Proteína ADAMTS5 , Adolescente , Adulto , Cartilagem Articular/citologia , Estudos de Casos e Controles , Condrócitos/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Dexametasona/farmacologia , Regulação para Baixo , Feminino , Impacto Femoroacetabular/genética , Impacto Femoroacetabular/cirurgia , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Articulação do Quadril , Humanos , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Pró-Colágeno N-Endopeptidase/genética , Pró-Colágeno N-Endopeptidase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima , Adulto Jovem
18.
FASEB J ; 29(10): 4107-21, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26092928

RESUMO

We investigated the role of periostin, an extracellular matrix protein, in the pathophysiology of osteoarthritis (OA). In OA, dysregulated gene expression and phenotypic changes in articular chondrocytes culminate in progressive loss of cartilage from the joint surface. The molecular mechanisms underlying this process are poorly understood. We examined periostin expression by immunohistochemical analysis of lesional and nonlesional cartilage from human and rodent OA knee cartilage. In addition, we used small interfering (si)RNA and adenovirus transduction of chondrocytes to knock down and up-regulate periostin levels, respectively, and analyzed its effect on matrix metalloproteinase (MMP)-13, a disintegrin and MMP with thrombospondin motifs (ADAMTS)-4, and type II collagen expression. We found high periostin levels in human and rodent OA cartilage. Periostin increased MMP-13 expression dose [1-10 µg/ml (EC50 0.5-1 µg/ml)] and time (24-72 h) dependently, significantly enhanced expression of ADAMTS4 mRNA, and promoted cartilage degeneration through collagen and proteoglycan degradation. Periostin induction of MMP-13 expression was inhibited by CCT031374 hydrobromide, an inhibitor of the canonical Wnt/ß-catenin signaling pathway. In addition, siRNA-mediated knockdown of endogenous periostin blocked constitutive MMP-13 expression. These findings implicate periostin as a catabolic protein that promotes cartilage degeneration in OA by up-regulating MMP-13 through canonical Wnt signaling.


Assuntos
Cartilagem Articular/metabolismo , Moléculas de Adesão Celular/metabolismo , Matriz Extracelular/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Osteoartrite/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Idoso , Idoso de 80 Anos ou mais , Animais , Western Blotting , Bovinos , Moléculas de Adesão Celular/genética , Células Cultivadas , Condrócitos/metabolismo , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Metaloproteinase 13 da Matriz/genética , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Osteoartrite/genética , Pró-Colágeno N-Endopeptidase/genética , Pró-Colágeno N-Endopeptidase/metabolismo , Interferência de RNA , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa
19.
PLoS One ; 10(4): e0122700, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25909781

RESUMO

OBJECTIVE: The specific degradation of type II collagen and aggrecan by matrix metalloproteinase (MMP)-9, -13 and ADAMTS-4 and -5 (aggrecanase-1 and -2) in the cartilage matrix is a critical step in pathology of osteoarthritis (OA). The aims of this study were: i) To investigate the relative contribution of ADAMTS-4 and ADAMTS-5 to cartilage degradation upon catabolic stimulation; ii) To investigate the effect of regulating the activities of key enzymes by mean of broad-spectrum inhibitors. METHODS: Bovine full-depth cartilage explants stimulated with tumor necrosis factor alpha (TNF-α) and Oncostatin M (OSM) were cultured for 21 days with or without a number of inhibitors targeting different types of proteases. Monoclonal antibodies were raised against the active sites of ADAMTS-4, -5, MMP-9 and -13, and 4 ELISAs were developed and technically validated. In addition, the established AGNxI (ADAMTS-degraded aggrecan), AGNxII (MMP-degraded aggrecan), and CTX-II (MMP-derived type II collagen) were quantified in the explants-conditioned media. RESULTS: We found that: i) Active ADAMTS-4, MMP-9, -13 were released in the late stage of TNF-α/ OSM stimulation, whereas no significant active ADAMTS-5 was detected in either extracts or supernatants; ii) Active ADAMTS-4 was primarily responsible for E373-374A bond cleavage in aggrecan in this setting; and iii) The compensatory mechanism could be triggered following the blockage of the enzyme caused by inhibitors. CONCLUSIONS: ADAMTS-4 appeared to be the major protease for the generation of 374ARGS aggrecan fragment in the TNF-α/OSM stimulated bovine cartilage explants. This study addresses the need to determine the roles of ADAMTS-4 and ADAMTS-5 in human articular degradation in OA and hence identify the attractive target for slowing down human cartilage breakdown.


Assuntos
Cartilagem Articular/metabolismo , Osteoartrite/metabolismo , Inibidores de Proteases/farmacologia , Proteínas ADAM/imunologia , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Agrecanas/metabolismo , Animais , Área Sob a Curva , Autoanticorpos/imunologia , Biomarcadores , Cartilagem Articular/patologia , Bovinos , Colágeno/metabolismo , Modelos Animais de Doenças , Metaloproteinases da Matriz/imunologia , Metaloproteinases da Matriz/metabolismo , Oncostatina M/metabolismo , Osteoartrite/imunologia , Osteoartrite/terapia , Pró-Colágeno N-Endopeptidase/imunologia , Pró-Colágeno N-Endopeptidase/metabolismo , Proteólise , Fator de Necrose Tumoral alfa/metabolismo
20.
Matrix Biol ; 44-46: 46-53, 2015 May-Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25863161

RESUMO

Collagen fibers are the main components of most of the extracellular matrices where they provide a structural support to cells, tissues and organs. Fibril-forming procollagens are synthetized as individual chains that associate to form homo- or hetero-trimers. They are characterized by the presence of a central triple helical domain flanked by amino and carboxy propeptides. Although there are some exceptions, these two propeptides have to be proteolytically removed to allow the almost spontaneous assembly of the trimers into collagen fibrils and fibers. While the carboxy-propeptide is mainly cleaved by proteinases from the tolloid family, the amino-propeptide is usually processed by procollagen N-proteinases: ADAMTS2, 3 and 14. This review summarizes the current knowledge concerning this subfamily of ADAMTS enzymes and discusses their potential involvement in physiopathological processes that are not directly linked to fibrillar procollagen processing.


Assuntos
Proteínas ADAM/metabolismo , Colágenos Fibrilares/metabolismo , Pró-Colágeno N-Endopeptidase/metabolismo , Proteínas ADAM/deficiência , Proteínas ADAM/genética , Proteínas ADAMTS , Proteína ADAMTS4 , Inibidores da Angiogênese/metabolismo , Animais , Vasos Sanguíneos/fisiologia , Doença/genética , Síndrome de Ehlers-Danlos/genética , Colágenos Fibrilares/química , Humanos , Linfangiogênese , Pró-Colágeno N-Endopeptidase/deficiência , Pró-Colágeno N-Endopeptidase/genética , Especificidade por Substrato
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