The Regulation of Aggrecanase ADAMTS-4 Expression in Human Achilles Tendon and tendon-Derived Cells

Several members of the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) family have been identified as aggrecanases, whose substrates include versican, the principal large proteoglycan in the tendon extracellular matrix. We have characterized the expression of ADAMTS-4 in human Achilles tendon and tendon-derived cells. ADAMTS-4 mRNA levels were higher in ruptured tendon compared with normal tendon or chronic painful tendinopathy. In tissue extracts probed by Western blotting, mature ADAMTS-4 (68 kDa) was detected only in ruptured tendons, while processed ADAMTS-4 (53 kDa) was detected also in chronic painful tendinopathy and in normal tendon. In cultured Achilles tendon cells, transforming growth factor-ß (TGF-ß) stimulated ADAMTS-4 mRNA expression (typically 20-fold after 24 h), while interleukin-1 induced a smaller, shorter-term stimulation which synergised markedly with that induced by TGF-ß. Increased levels of immunoreactive proteins consistent with mature and processed forms of ADAMTS-4 were detected in TGF-ß-stimulated cells. ADAMTS-4 mRNA was expressed at higher levels by tendon cells in collagen gels than in monolayer cultures. In contrast, the expression of ADAMTS-1 and -5 mRNA was lower in collagen gels compared with monolayers, and these mRNA showed smaller or opposite responses to growth factors and cytokines compared with that of ADAMTS-4 mRNA. We conclude that both ADAMTS-4 mRNA and ADAMTS-4 protein processing may be differentially regulated in normal and damaged tendons and that both the matrix environment and growth factors such as TGF-ß are potentially important factors controlling ADAMTS aggrecanase activities in tendon pathology

c-Maf Transcription Factor Regulates ADAMTS-12 Expression in Human Chondrogenic Cells.

ObjectiveADAMTS (a disintegrin and metalloproteinase with thrombospondin type-1 motif) zinc metalloproteinases are important during the synthesis and breakdown of cartilage extracellular matrix. ADAMTS-12 is up-regulated during in vitro chondrogenesis and embryonic limb development; however, the regulation of ADAMTS-12 expression in cartilage remains unknown. The transcription factor c-Maf is a member of Maf family of basic ZIP (bZIP) transcription factors. Expression of c-Maf is highest in hypertrophic chondrocytes during embryonic development and postnatal growth. We hypothesize that c-Maf and ADAMTS-12 are co-expressed during chondrocyte differentiation and that c-Maf regulates ADAMTS-12 expression during chondrogenesis.DesignPromoter analysis and species alignments identified potential c-Maf binding sites in the ADAMTS-12 promoter. c-Maf and ADAMTS-12 co-expression was monitored during chondrogenesis of stem cell pellet cultures. Luciferase expression driven by ADAMTS-12 promoter segments was measured in the presence and absence of c-Maf, and synthetic oligonucleotides were used to confirm specific binding of c-Maf to ADAMTS-12 promoter sequences.ResultsIn vitro chondrogenesis from human mesenchymal stem cells revealed co-expression of ADAMTS-12 and c-Maf during differentiation. Truncation and point mutations of the ADAMTS-12 promoter evaluated in reporter assays localized the response to the proximal 315 bp of the ADAMTS-12 promoter, which contained a predicted c-Maf recognition element (MARE) at position -61. Electorphoretic mobility shift assay confirmed that c-Maf directly interacted with the MARE at position -61.ConclusionsThese data suggest that c-Maf is involved in chondrocyte differentiation and hypertrophy, at least in part, through the regulation of ADAMTS-12 expression at a newly identified MARE in its proximal promoter

Development of a monoclonal anti-ADAMTS-5 antibody that specifically blocks the interaction with LRP1

The potent aggrecanase ADAMTS-5 is constitutively secreted by chondrocytes, but it is rapidly endocytosed in normal cartilage via the cell surface endocytic receptor LRP1. Therefore it is difficult to detect the total ADAMTS-5 activity produced. In this study, we isolated a monoclonal anti-ADAMTS-5 antibody 1B7 that blocks LRP1-mediated internalization without affecting the aggrecanolytic activity. Addition of 1B7 to cultured human chondrocytes revealed the full aggrecanolytic activity of ADAMTS-5 generated by the cells. 1B7 is a useful tool to estimate the ADAMTS-5 activity and to identify its potential roles in the tissues

ADAMTS -1 and -4 are up-regulated following transient middle cerebral artery occlusion in the rat and their expression is modulated by TNF in cultured astrocytes

ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) enzymes are a recently described group of metalloproteinases. The substrates degraded by ADAMTS-1, -4 and -5 suggests that they play a role in turnover of extracellular matrix in the central nervous system (CNS). ADAMTS-1 is also known to exhibit anti-angiogenic activity. Their main endogenous inhibitor is tissue inhibitor of metalloproteinases (TIMP)-3. The present study was designed to investigate ADAMTS-1, -4 and -5 and TIMP-3 expression after experimental cerebral ischaemia and to examine whether cytokines known to be up-regulated in stroke could alter their expression by astrocytes in vitro. Focal cerebral ischaemia was induced by transient middle cerebral artery occlusion in the rat using the filament method. Our results demonstrate a significant increase in expression of ADAMTS-1 and -4 in the occluded hemisphere but no significant change in TIMP-3. This was accompanied by an increase in mRNA levels for interleukin (IL)-1, IL-1 receptor antagonist (IL-1ra) and tumour necrosis factor (TNF). ADAMTS-4 mRNA and protein was up-regulated by TNF in primary human astrocyte cultures. The increased ADAMTS-1 and -4 in experimental stroke, together with no change in TIMP-3, may promote ECM breakdown after stroke, enabling infiltration of inflammatory cells and contribute to brain injury. In vitro studies suggest that the in vivo modulation of ADAMTS-1 and -4 may be controlled in part by TNF.</p

Differential expression of ADAMTS -1, -4, -5 and TIMP -3 in rat spinal cord at different stages of acute experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model of inflammatory demyelination, a pathological event common to multiple sclerosis (MS). During CNS inflammation there are alterations in the extracellular matrix (ECM). A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) -1, -4 and -5 are proteases present in the CNS, which are able to cleave the aggregating chondroitin sulphate proteoglycans, aggrecan, phosphacan, neurocan and brevican. It is therefore important to investigate changes in their expression in different stages of EAE induction. We have investigated expression of ADAMTS-1, -4, -5 and Tissue inhibitor of metalloproteinase (TIMP) -3, by real-time RT-PCR. We have also examined protein expression of ADAMTS-1, -4 and -5 by western blotting and immunocytochemistry in spinal cord from animals at different stages of disease progression. Our study demonstrated a decrease in ADAMTS-4 mRNA and protein expression. TIMP-3 was decreased at the mRNA level although protein levels were increased in diseased animals compared to controls. Our study identifies changes in ADAMTS expression during the course of CNS inflammation which may contribute to ECM degradation and disease progression.</p

ADAMTS proteinases: a multi-domain, multi-functional family with roles in extracellular matrix turnover and arthritis

Members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family are known to influence development, angiogenesis, coagulation and progression of arthritis. As proteinases their substrates include the von Willebrand factor precursor and extracellular matrix components such as procollagen, hyalectans (hyaluronan-binding proteoglycans including aggrecan), decorin, fibromodulin and cartilage oligomeric matrix protein. ADAMTS levels and activities are regulated at multiple levels through the control of gene expression, mRNA splicing, protein processing and inhibition by TIMP (tissue inhibitor of metalloproteinases). A recent screen of human cartilage has shown that multiple members of the ADAMTS family may be important in connective tissue homeostasis and pathology

Reactive-site mutants of N-TIMP-3 that selectively inhibit ADAMTS-4 and ADAMTS-5: biological and structural implications.

Published versio

The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) family

The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) enzymes are secreted, multi-domain matrix-associated zinc metalloendopeptidases that have diverse roles in tissue morphogenesis and patho-physiological remodeling, in inflammation and in vascular biology. The human family includes 19 members that can be sub-grouped on the basis of their known substrates, namely the aggrecanases or proteoglycanases (ADAMTS1, 4, 5, 8, 9, 15 and 20), the procollagen N-propeptidases (ADAMTS2, 3 and 14), the cartilage oligomeric matrix protein-cleaving enzymes (ADAMTS7 and 12), the von-Willebrand Factor proteinase (ADAMTS13) and a group of orphan enzymes (ADAMTS6, 10, 16, 17, 18 and 19). Control of the structure and function of the extracellular matrix (ECM) is a central theme of the biology of the ADAMTS, as exemplified by the actions of the procollagen-N-propeptidases in collagen fibril assembly and of the aggrecanases in the cleavage or modification of ECM proteoglycans. Defects in certain family members give rise to inherited genetic disorders, while the aberrant expression or function of others is associated with arthritis, cancer and cardiovascular disease. In particular, ADAMTS4 and 5 have emerged as therapeutic targets in arthritis. Multiple ADAMTSs from different sub-groupings exert either positive or negative effects on tumorigenesis and metastasis, with both metalloproteinase-dependent and -independent actions known to occur. The basic ADAMTS structure comprises a metalloproteinase catalytic domain and a carboxy-terminal ancillary domain, the latter determining substrate specificity and the localization of the protease and its interaction partners; ancillary domains probably also have independent biological functions. Focusing primarily on the aggrecanases and proteoglycanases, this review provides a perspective on the evolution of the ADAMTS family, their links with developmental and disease mechanisms, and key questions for the future

Primary cilia disassembly down-regulates mechanosensitive hedgehog signalling: a feedback mechanism controlling ADAMTS-5 expression in chondrocytes

SummaryObjectiveHedgehog signalling is mediated by the primary cilium and promotes cartilage degeneration in osteoarthritis. Primary cilia are influenced by pathological stimuli and cilia length and prevalence are increased in osteoarthritic cartilage. This study aims to investigate the relationship between mechanical loading, hedgehog signalling and cilia disassembly in articular chondrocytes.MethodsPrimary bovine articular chondrocytes were subjected to cyclic tensile strain (CTS; 0.33 Hz, 10% or 20% strain). Hedgehog pathway activation (Ptch1, Gli1) and A Disintegrin And Metalloproteinase with Thrombospondin Motifs 5 (ADAMTS-5) expression were assessed by real-time PCR. A chondrocyte cell line generated from the Tg737ORPK mouse was used to investigate the role of the cilium in this response. Cilia length and prevalence were quantified by immunocytochemistry and confocal microscopy.ResultsMechanical strain upregulates Indian hedgehog expression and activates hedgehog signalling. Ptch1, Gli1 and ADAMTS-5 expression were increased following 10% CTS, but not 20% CTS. Pathway activation requires a functioning primary cilium and is not observed in Tg737ORPK cells lacking cilia. Mechanical loading significantly reduced cilium length such that cilia became progressively shorter with increasing strain magnitude. Inhibition of histone deacetylase 6 (HDAC6), a tubulin deacetylase, prevented cilia disassembly and restored mechanosensitive hedgehog signalling and ADAMTS-5 expression at 20% CTS.ConclusionsThis study demonstrates for the first time that mechanical loading activates primary cilia-mediated hedgehog signalling and ADAMTS-5 expression in adult articular chondrocytes, but that this response is lost at high strains due to HDAC6-mediated cilia disassembly. The study provides new mechanistic insight into the role of primary cilia and mechanical loading in articular cartilage