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1.
FEBS J ; 289(1): 121-139, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34270864

RESUMO

Matrix metalloproteinase-13 (MMP-13) is a uniquely important collagenase that promotes the irreversible destruction of cartilage collagen in osteoarthritis (OA). Collagenase activation is a key control point for cartilage breakdown to occur, yet our understanding of the proteinases involved in this process is limited. Neutrophil elastase (NE) is a well-described proteoglycan-degrading enzyme which is historically associated with inflammatory arthritis, but more recent evidence suggests a potential role in OA. In this study, we investigated the effect of neutrophil elastase on OA cartilage collagen destruction and collagenase activation. Neutrophil elastase induced significant collagen destruction from human OA cartilage ex vivo, in an MMP-dependent manner. In vitro, neutrophil elastase directly and robustly activated pro-MMP-13, and N-terminal sequencing identified cleavage close to the cysteine switch at 72 MKKPR, ultimately resulting in the fully active form with the neo-N terminus of 85 YNVFP. Mole-per-mole, activation was more potent than by MMP-3, a classical collagenase activator. Elastase was detectable in human OA synovial fluid and OA synovia which displayed histologically graded evidence of synovitis. Bioinformatic analyses demonstrated that, compared with other tissues, control cartilage exhibited remarkably high transcript levels of the major elastase inhibitor, (AAT) alpha-1 antitrypsin (gene name SERPINA1), but these were reduced in OA. AAT was located predominantly in superficial cartilage zones, and staining enhanced in regions of cartilage damage. Finally, active MMP-13 specifically inactivated AAT by removal of the serine proteinase cleavage/inhibition site. Taken together, this study identifies elastase as a novel activator of pro-MMP-13 that has relevance for cartilage collagen destruction in OA patients with synovitis.


Assuntos
Inflamação/genética , Elastase de Leucócito/genética , Metaloproteinase 13 da Matriz/genética , Osteoartrite/genética , alfa 1-Antitripsina/genética , Cisteína/genética , Humanos , Inflamação/metabolismo , Inflamação/patologia , Metaloproteinase 3 da Matriz/genética , Neutrófilos/enzimologia , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Sinovite/genética , Sinovite/metabolismo , Sinovite/patologia , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/patologia
2.
J Biol Chem ; 294(26): 10266-10277, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31110047

RESUMO

The collagenase subfamily of matrix metalloproteinases (MMPs) have important roles in the remodeling of collagenous matrices. The proteinase-activated receptor (PAR) family has a unique mechanism of activation requiring proteolysis of an extracellular domain forming a neo-N terminus that acts as a tethered ligand, a process that has been associated with the development of arthritis. Canonical PAR2 activation typically occurs via a serine proteinase at Arg36-Ser37, but other proteinases can cleave PARs downstream of the tethered ligand and "disarm" the receptor. To identify additional cleavage sites within PAR2, we synthesized a 42-amino-acid peptide corresponding to the extracellular region. We observed that all three soluble MMP collagenases, MMP-1, MMP-8, and MMP-13, cleave PAR2 and discovered a novel cleavage site (Ser37-Leu38). Metalloproteinases from resorbing bovine nasal cartilage and recombinant human collagenases could cleave a quenched fluorescent peptide mimicking the canonical PAR2 activation region, and kinetic constants were determined. In PAR2-overexpressing SW1353 chondrocytes, we demonstrated that the activator peptide SLIGKV-NH2 induces rapid calcium flux, inflammatory gene expression (including MMP1 and MMP13), and the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 kinase. The corresponding MMP cleavage-derived peptide (LIGKVD-NH2) exhibited no canonical activation; however, we observed phosphorylation of ERK, providing evidence of biased agonism. Importantly, we demonstrated that preincubation with active MMP-1 reduced downstream PAR2 activation by a canonical activator, matriptase, but not SLIGKV-NH2 These results support a role for collagenases as proteinases capable of disarming PAR2, revealing a mechanism that suppresses PAR2-mediated inflammatory responses.


Assuntos
Matriz Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 8 da Matriz/metabolismo , Receptor PAR-2/antagonistas & inibidores , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Condrossarcoma/genética , Condrossarcoma/metabolismo , Condrossarcoma/patologia , Humanos , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 8 da Matriz/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação , Receptor PAR-2/genética , Receptor PAR-2/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas
3.
PLoS One ; 13(11): e0207240, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30440036

RESUMO

Irreversible cartilage collagen breakdown by the collagenolytic matrix metalloproteinases (MMPs)-1 and MMP-13 represents a key event in pathologies associated with tissue destruction such as arthritis. Inflammation is closely associated with such pathology and occurs in both rheumatoid and osteoarthritis making it highly relevant to the prevailing tissue damage that characterises these diseases. The inflammation-induced activating protein-1 (AP-1) transcription factor is an important regulator of both MMP1 and MMP13 genes with interplay between signalling pathways contributing to their expression. Here, we have examined the regulation of MMP1 expression, and using in vivo chromatin immunoprecipitation analyses we have demonstrated that cFos bound to the AP-1 cis element within the proximal MMP1 promoter only when the gene was transcriptionally silent as previously observed for MMP13. Subsequent small interfering RNA-mediated silencing confirmed however, that cFos significantly contributes to MMP1 expression. In contrast, silencing of ATF3 (a prime MMP13 modulator) did not affect MMP1 expression whilst silencing of the Wnt-associated regulator cysteine- serine-rich nuclear protein-1 (CSRNP1) resulted in substantial repression of MMP1 but not MMP13. Furthermore, following an early transient peak in expression of CSRNP1 at the mRNA and protein levels similar to that seen for cFOS, CSRNP1 expression subsequently persisted unlike cFOS. Finally, DNA binding assays indicated that the binding of CSRNP1 to the AP-1 consensus-like sequences within the proximal promoter regions of MMP1 and MMP13 was preferentially selective for MMP1 whilst activating transcription factor 3 (ATF3) binding was exclusive to MMP13. These data further extend our understanding of the previously reported differential regulation of these MMP genes, and strongly indicate that although cFos modulates the expression of MMP1/13, downstream factors such as CSRNP1 and ATF3 ultimately serve as transcriptional regulators in the context of an inflammatory stimulus for these potent collagenolytic MMPs.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Condrócitos/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Fator 3 Ativador da Transcrição/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Simulação por Computador , Regulação Enzimológica da Expressão Gênica , Humanos , Interleucina-1/administração & dosagem , Interleucina-1/metabolismo , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fatores de Tempo , Transcrição Gênica
4.
PLoS One ; 13(4): e0195864, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29652915

RESUMO

Many catabolic stimuli, including interleukin-1 (IL-1) in combination with oncostatin M (OSM), promote cartilage breakdown via the induction of collagen-degrading collagenases such as matrix metalloproteinase 1 (MMP1) and MMP13 in human articular chondrocytes. Indeed, joint diseases with an inflammatory component are characterised by excessive extracellular matrix (ECM) catabolism. Importantly, protein kinase C (PKC) signalling has a primary role in cytokine-induced MMP1/13 expression, and is known to regulate cellular functions associated with pathologies involving ECM remodelling. At present, substrates downstream of PKC remain undefined. Herein, we show that both IL-1- and OSM-induced phosphorylation of protein kinase D (PKD) in human chondrocytes is strongly associated with signalling via the atypical PKCι isoform. Consequently, inhibiting PKD activation with a pan-PKD inhibitor significantly reduced the expression of MMP1/13. Specific gene silencing of the PKD isoforms revealed that only PKD3 (PRKD3) depletion mirrored the observed MMP repression, indicative of the pharmacological inhibitor specifically affecting only this isoform. PRKD3 silencing was also shown to reduce serine phosphorylation of signal transducer and activator of transcription 3 (STAT3) as well as phosphorylation of all three mitogen-activated protein kinase groups. This altered signalling following PRKD3 silencing led to a significant reduction in the expression of the activator protein-1 (AP-1) genes FOS and JUN, critical for the induction of many MMPs including MMP1/13. Furthermore, the AP-1 factor activating transcription factor 3 (ATF3) was also reduced concomitant with the observed reduction in MMP13 expression. Taken together, we highlight an important role for PKD3 in the pro-inflammatory signalling that promotes cartilage destruction.


Assuntos
Condrócitos/metabolismo , Regulação Enzimológica da Expressão Gênica , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 1 da Matriz/genética , Proteína Quinase C/metabolismo , Condrócitos/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-1/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Modelos Biológicos , Oncostatina M/farmacologia , Fosforilação , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição AP-1/metabolismo
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