• ACI
• Basic science
• Bone marrow stimulation
• Cartilage lesions
• Chondral injury
• Microfracture
• OCD
• Osteochondral lesions

• Humans
• Cartilage, Articular/injuries
• Cartilage Diseases/therapy

• anterior cruciate ligament
• microRNA
• osteoarthritis
• small nuclear RNA
• small nucleolar RNA

• Wellcome Trust
• MR/P020941/1 Medical Research Council
• Wellcome Trust
• Medical Research Centre

• Combination therapy
• Disease modifying OA drugs
• Goat ex vivo OA model
• Human OA cartilage
• Sulfated carboxymethylcellulose
• Tissue inhibitor of metalloproteases 3

• LRP1
• Low-density lipoprotein receptor-related protein 1
• Osteoarthritis
• Suramin
• TIMP-3

• Humans
• Tissue Inhibitor of Metalloproteinase-3/metabolism
• Tissue Inhibitor of Metalloproteinase-3/pharmacology
• Tissue Inhibitor of Metalloproteinase-3/therapeutic use
• Suramin/pharmacology
• Suramin/metabolism
• Suramin/therapeutic use
• Cartilage/metabolism
• Osteoarthritis/drug therapy
• Osteoarthritis/metabolism
• Metalloproteases/metabolism
• Metalloproteases/pharmacology
• Metalloproteases/therapeutic use

• 21776 Versus Arthritis

• articular cartilage
• heparan sulfate proteoglycan
• osteoarthritis
• syndecan-4

• Biomarker
• Dysplasia
• Mechanism
• Osteoarthritis

• cartilage degeneration
• drug delivery systems
• hydrogels
• intra-articular administration
• joint diseases
• osteoarthritis
• polymeric systems
• vesicular systems

• Humans
• Drug Delivery Systems
• Osteoarthritis/drug therapy
• Arthritis, Rheumatoid/drug therapy
• Cartilage, Articular

• Humans
• Procollagen N-Endopeptidase/metabolism
• Aggrecans/metabolism
• ADAMTS5 Protein
• ADAMTS4 Protein
• Zinc
• Disintegrins
• Osteoarthritis/metabolism
• ADAM Proteins/metabolism
• Thrombospondins

• FS/IBSRF/20/25032 British Heart Foundation
• Università di Pisa

• ECM
• TIMP diversity
• TIMPs
• cancer
• inflammation
• protein engineering
• protein evolution

• Bone diseases
• Cell differentiation
• DNA methylation
• Epigenetics
• Histones
• Long noncoding RNA

• Anti-angiogenic factor
• Articular cartilage
• Inflammation
• Phenotype stability
• Tissue engineering
• Vascularization

• Cartilage
• Endocytosis
• Extracellular matrix
• Metalloproteinase
• Serine proteinase
• Sulphated glycosaminoglycan
• TIMP

• Cartilage, Articular
• Collagen
• Extracellular Matrix
• Humans
• Matrix Metalloproteinase 13
• Matrix Metalloproteinases
• Osteoarthritis

• 21447 Versus Arthritis
• 20039 Versus Arthritis
• 17620 Versus Arthritis
• MR/P020941/1 Medical Research Council
• 22418 Versus Arthritis

• aggrecan
• alternative splicing
• arthritis
• cartilage
• extracellular matrix
• posttranslational modifications
• small molecule inhibitor

Ectodomain shedding is a key mechanism of several biological processes, including cell-communication. Disintegrin and metalloproteinases (ADAMs), together with the membrane-type matrix metalloproteinases, play a pivotal role in shedding transmembrane proteins. Aberrant shedding is associated to several pathological conditions, including arthritis. Tissue inhibitor of metalloproteases 3 (TIMP-3), an endogenous inhibitor of ADAMs and matrix metalloproteases (MMPs), has been proven to be beneficial in such diseases. Thus, strategies to increase TIMP-3 bioavailability in the tissue have been sought for development of therapeutics. Nevertheless, high levels of TIMP-3 may lead to mechanism-based side-effects, as its overall effects on cell behavior are still unknown. In this study, we used a high-resolution mass-spectrometry-based workflow to analyze alterations induced by sustained expression of TIMP-3 in the cell surfaceome. In agreement with its multifunctional properties, TIMP-3 induced changes on the protein composition of the cell surface. We found that TIMP-3 had differential effects on metalloproteinase substrates, with several that accumulated in TIMP-3-overexpressing cells. In addition, our study identified potentially novel ADAM substrates, including ADAM15, whose levels at the cell surface are regulated by the inhibitor. In conclusion, our study reveals that high levels of TIMP-3 induce modifications in the cell surfaceome and identifies molecular pathways that can be deregulated via TIMP-3-based therapies.

• ectodomain shedding
• metalloproteinases
• proteomics
• tissue inhibitor of metalloproteases 3 (TIMP-3)

• ADAM Proteins/metabolism
• Cell Membrane/chemistry
• Cell Membrane/metabolism
• HEK293 Cells
• Humans
• Mass Spectrometry
• Membrane Proteins/metabolism
• Proteome/analysis
• Proteome/metabolism
• Proteomics
• Tissue Inhibitor of Metalloproteinase-3/metabolism

For decades, disintegrin and metalloproteinase 17 (ADAM17) has been the object of deep investigation. Since its discovery as the tumor necrosis factor convertase, it has been considered a major drug target, especially in the context of inflammatory diseases and cancer. Nevertheless, the development of drugs targeting ADAM17 has been harder than expected. This has generally been due to its multifunctionality, with over 80 different transmembrane proteins other than tumor necrosis factor α (TNF) being released by ADAM17, and its structural similarity to other metalloproteinases. This review provides an overview of the different roles of ADAM17 in disease and the effects of its ablation in a number of in vivo models of pathological conditions. Furthermore, here, we comprehensively encompass the approaches that have been developed to accomplish ADAM17 selective inhibition, from the newest non-zinc-binding ADAM17 synthetic inhibitors to the exploitation of iRhom2 to specifically target ADAM17 in immune cells.

• ADAM17
• EGFR
• TIMPs
• TNF
• ectodomain shedding
• iRhoms
• metalloproteinases

Osteoarthritis (OA) is a common degenerative disease characterized by the destruction of articular cartilage and chronic inflammation of surrounding tissues. Matrix metalloproteinase-13 (MMP-13) is the primary MMP involved in cartilage degradation through its particular ability to cleave type II collagen. Hence, it is an attractive target for the treatment of OA. However, the detailed molecular mechanisms of OA initiation and progression remain elusive, and, currently, there are no interventions available to restore degraded cartilage. This review fully illustrates the involvement of MMP-13 in the initiation and progression of OA through the regulation of MMP-13 activity at the molecular and epigenetic levels, as well as the strategies that have been employed against MMP-13. The aim of this review is to identify MMP-13 as an attractive target for inhibitor development in the treatment of OA.

• MMP-13
• cartilage
• inhibitor
• matrix metalloproteinases
• osteoarthritis
• regulation
• type II collagen

• Cartilage, Articular/drug effects
• Cartilage, Articular/pathology
• Catalytic Domain
• Collagen Type II/metabolism
• Crystallography, X-Ray
• Disease Progression
• Drug Development
• Epigenesis, Genetic/drug effects
• Humans
• Hydrophobic and Hydrophilic Interactions
• Matrix Metalloproteinase 13/genetics
• Matrix Metalloproteinase 13/metabolism
• Matrix Metalloproteinase 13/ultrastructure
• Matrix Metalloproteinase Inhibitors/pharmacology
• Matrix Metalloproteinase Inhibitors/therapeutic use
• Molecular Targeted Therapy/methods
• Osteoarthritis/drug therapy
• Osteoarthritis/genetics
• Osteoarthritis/pathology

A key feature of osteoarthritis is the gradual loss of articular cartilage and bone deformation, resulting in the impairment of joint function. The primary cause of cartilage destruction is considered to be the presence of elevated proteases, such as matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs). However, clinically tested global MMP inhibitors have low efficacy that may be due to their lack of selectivity. We previously demonstrated in vitro that a variant of tissue inhibitor of metalloproteinase-3 ([-1A]TIMP3) inhibits ADAMTSs but not MMPs. In this study, we tested whether the selectivity of [-1A]TIMP3 is beneficial compared with that of the wild-type TIMP3 in preventing or delaying the onset of the degenerative effects in a mouse model of osteoarthritis. We generated transgenic mice that overexpressed TIMP3 or [-1A]TIMP3 driven by a chondrocyte-specific type II collagen promoter. TIMP3 transgenic mice showed compromised bone integrity as opposed to [-1A]TIMP3 mice. After surgically induced joint instability, TIMP3 overexpression proved to be less protective in cartilage destruction than [-1A]TIMP3 at late stages of OA. The selective inhibition of ADAMTSs provides the possibility of modifying TIMP3 to specifically target a class of cartilage-degrading proteinases and to minimize adverse effects on bone and possibly other tissues.

Bone is a dynamic organ that undergoes constant remodeling, an energetically costly process by which old bone is replaced and localized bone defects are repaired to renew the skeleton over time, thereby maintaining skeletal health. This review provides a general overview of bone’s main players (bone lining cells, osteocytes, osteoclasts, reversal cells, and osteoblasts) that participate in bone remodeling. Placing emphasis on the family of extracellular matrix metalloproteinases (MMPs), we describe how: (i) Convergence of multiple protease families (including MMPs and cysteine proteinases) ensures complexity and robustness of the bone remodeling process, (ii) Enzymatic activity of MMPs affects bone physiology at the molecular and cellular levels and (iii) Either overexpression or deficiency/insufficiency of individual MMPs impairs healthy bone remodeling and systemic metabolism. Today, it is generally accepted that proteolytic activity is required for the degradation of bone tissue in osteoarthritis and osteoporosis. However, it is increasingly evident that inactivating mutations in MMP genes can also lead to bone pathology including osteolysis and metabolic abnormalities such as delayed growth. We argue that there remains a need to rethink the role played by proteases in bone physiology and pathology.

• bone
• deficiency
• matrix metalloproteinase
• metabolism
• remodeling
• underactivity

• endocytosis
• extracellular matrix
• primary cilium
• secretion
• transcription

• biomaterials
• cleft lip/palate
• extracellular matrix
• extracellular matrix remodeling
• metalloproteinases
• palatal reconstruction
• palatogenesis
• tissue bioengineering

Hypermethylation of gene promoters has been regarded as an epigenetic regulator for gene inactivation in the development of several diseases. In the current study, we aimed to explore how long noncoding RNA X-inactive specific transcript (lncRNA XIST) function in collagen degradation in chondrocytes of osteoarthritis (OA) after tibial plateau fracture by regulating tissue inhibitor of metalloproteinase-3 (TIMP-3) promoter methylation.

In silico analysis was used to screen differentially expressed lncRNAs in cartilage tissues of OA. Chondrocytes were then successfully isolated from normal and OA cartilage tissues and identified, with the expressions of lncRNA XIST and TIMP-3 examined. The methylation levels of TIMP-3 promoter were determined by MS-PCR. The binding of lncRNA XIST to DNA methyltransferase and the binding of TIMP-3 promoter to DNA methyltransferase were determined by a series of experiments, including RIP, RNA pull-down, and ChIP assays.

The differentially expressed lncRNA XIST was determined in OA. In addition, cartilage tissues of OA showed upregulation of lncRNA XIST and downregulation of TIMP-3. LncRNA XIST was primarily localized in the nucleus and was capable of binding to the promoter of TIMP-3. The silencing of lncRNA XIST decreased the methylation levels of TIMP-3 promoter and increased the expressions of TIMP-3, which consequently inhibited collagen degradation in OA chondrocytes. Furthermore, TIMP-3 over-expression reversed the effect of lncRNA XIST on collagen degradation in OA chondrocytes.

Collectively, lncRNA XIST raises collagen degradation in OA chondrocytes after tibial plateau fracture by accelerating the methylation of TIMP-3 promoter by recruiting DNA methyltransferase.

• Chondrocytes
• Collagen degradation
• Long noncoding RNA XIST
• Methylation
• Osteoarthritis
• TIMP-3
• Tibial plateau fracture

• TIMP-3
• chondroitin sulfate
• glycosaminoglycans
• isothermal titration calorimetry
• pentosan polysulfate
• suramin

Tissue inhibitor of metalloproteinase-3 (TIMP-3), a secreted glycoprotein, plays an important role in carcinogenesis. It can bind to many proteinases to suppress their activity and thus protect the extracellular matrix from degradation. TIMP-3 may have many anticancer properties, including apoptosis induction and antiproliferative, antiangiogenic, and antimetastatic activities. This review summarizes the structure, proteinase inhibition ability, genetic and epigenetic regulation, cancer therapy potential, and contribution to cancer development of TIMP-3. Furthermore, in this review we discuss its potential as a biomarker for predicting cancer progression and the current state of drugs that target TIMP-3, either alone or in combination with clinical treatment. In conclusion, TIMP-3 can be a biomarker of cancer and a potential target for cancer therapy. This review article can serve as a basis to understand how to modulate TIMP-3 levels as a drug target of cancers.

• cancer therapy
• extracellular matrix
• matrix metalloproteinase
• metastasis
• tissue inhibitors of metalloproteinases-3

• In situ hybridization
• MMPs
• Mandibular condylar cartilage
• Mice
• TIMPs

Metalloproteinases remain important players in arthritic disease, in part because members of this large enzymatic family, namely matrix metalloproteinase-1 (MMP-1) and MMP-13, are responsible for the irreversible degradation of articular cartilage collagen. Although direct inhibition of MMPs fell out of vogue with the initial clinical disappointment of the first generation of compounds, interest in other mechanisms that control these important enzymes has always been maintained. Since these enzymes are critically important for tissue homeostasis, their expression and activity are tightly regulated at many levels, not just by direct inhibition by their endogenous inhibitors the tissue inhibitors of metalloproteinases (TIMPs). Focussing on MMP-13, we discuss recent work that highlights new discoveries in the transcriptional regulation of this enzyme, from defined promoter functional analysis to how more global technologies can provide insight into the enzyme’s regulation, especially by epigenetic mechanisms, including non-coding RNAs. In terms of protein regulation, we highlight recent findings into enzymatic cascades involved in MMP-13 regulation and activation. Importantly, we highlight a series of recent studies that describe how MMP-13 activity, and in fact that of other metalloproteinases, is in part controlled by receptor-mediated endocytosis. Together, these new discoveries provide a plethora of novel regulatory mechanisms, besides direct inhibition, which with renewed vigour could provide further therapeutic opportunities for regulating the activity of this class of important enzymes.

• cartilage
• metalloproteinase
• osteoarthritis
• regulation
• transcription

• Glucocorticoid
• Homeostasis
• Inflammation
• Osteoarthritis
• Receptor
• Signaling

• Animals
• Extracellular Matrix/metabolism
• Glucocorticoids/metabolism
• Humans
• Osteoarthritis/metabolism
• Osteoarthritis/pathology
• Receptors, Glucocorticoid/chemistry
• Receptors, Glucocorticoid/metabolism
• Signal Transduction
• Synovial Membrane/metabolism
• Synovial Membrane/pathology

• Disease models
• In vitro models
• In vivo models
• Osteoarthritis
• Osteochondral defects

The tissue inhibitor of metalloproteinases-3 (TIMP-3) is a major regulator of extracellular matrix turnover and protein shedding by inhibiting different classes of metalloproteinases, including disintegrin metalloproteinases (ADAMs). Tissue bioavailability of TIMP-3 is regulated by the endocytic receptor low-density-lipoprotein receptor-related protein-1 (LRP-1). TIMP-3 plays protective roles in disease. Thus, different approaches have been developed aiming to increase TIMP-3 bioavailability, yet overall effects of increased TIMP-3 in vivo have not been investigated. Herein, by using unbiased mass-spectrometry we demonstrate that TIMP-3-overexpression in HEK293 cells has a dual effect on shedding of transmembrane proteins and turnover of soluble proteins. Several membrane proteins showing reduced shedding are known as ADAM10 substrates, suggesting that exogenous TIMP-3 preferentially inhibits ADAM10 in HEK293 cells. Additionally identified shed membrane proteins may be novel ADAM10 substrate candidates. TIMP-3-overexpression also increased extracellular levels of several soluble proteins, including TIMP-1, MIF and SPARC. Levels of these proteins similarly increased upon LRP-1 inactivation, suggesting that TIMP-3 increases soluble protein levels by competing for their binding to LRP-1 and their subsequent internalization. In conclusion, our study reveals that increased levels of TIMP-3 induce substantial modifications in the cellular secretome and that TIMP-3-based therapies may potentially provoke undesired, dysregulated functions of ADAM10 and LRP-1.

The aim of this study is to identify gene expression profiles associated with hyaluronic acid (HA) treatment of normal and osteoarthritis (OA)-like tissue-engineered cartilage. 3D cartilage micromasses were treated with tumour necrosis factor-α (TNF-α) (OA-inducer) and/or HA for 7 days. Viability was examined by PI/FDA staining. To document extracellular matrix (ECM) formation, glycosaminoglycans (GAG) were stained with Safranin-O and cartilage-specific type II collagen was detected immunohistochemically. Genome-wide gene expression was determined using microarray analysis. Normal and OA-like micromasses remained vital and showed a spherical morphology and homogenous cell distribution regardless of the treatment. There was no distinct difference in immunolabeling for type II collagen. Safranin-O staining demonstrated a typical depletion of GAG in TNF-α-treated micromasses (−73%), although the extent was limited in the presence of HA (−39%). The microarray data showed that HA can influence the cartilage metabolism via upregulation of TIMP3 in OA-like condition. The upregulation of VEGFA and ANKRD37 genes implies a supportive role of HA in cartilage maturation and survival. The results of this study validate the feasibility of the in vitro OA model for the investigation of HA. On the cellular level, no inhibiting or activating effect of HA was shown. Microarray data demonstrated a minor impact of HA on gene expression level.

• hyaluronic acid
• in vitro model
• microarray
• osteoarthritis

• Animals
• Cartilage/drug effects
• Cartilage/metabolism
• Cells, Cultured
• Extracellular Matrix
• Hyaluronic Acid/pharmacology
• Osteoarthritis/genetics
• Osteoarthritis/metabolism
• Swine
• Tissue Engineering
• Transcriptome
• Vascular Endothelial Growth Factor A/genetics
• Vascular Endothelial Growth Factor A/metabolism

Osteoarthritis (OA) is the most prevalent joint disease in older people and is characterized by the progressive destruction of articular cartilage, synovial inflammation, changes in subchondral bone and peri-articular muscle, and pain. Because our understanding of the aetiopathogenesis of OA remains incomplete, we haven’t discovered a cure for OA yet. This review appraises novel therapeutics based on recent progress in our understanding of the molecular pathogenesis of OA, including pro-inflammatory and pro-catabolic mediators and the relevant signalling mechanisms. The changes in subchondral bone and peri-articular muscle accompanying cartilage damage are also reviewed.

• articular cartilage
• molecular pathology
• osteoarthritis (OA)
• therapeutics

• Animals
• Anti-Inflammatory Agents/pharmacology
• Anti-Inflammatory Agents/therapeutic use
• Biomarkers
• Bone Remodeling
• Bone and Bones/drug effects
• Bone and Bones/metabolism
• Bone and Bones/pathology
• Cartilage, Articular/drug effects
• Cartilage, Articular/metabolism
• Cartilage, Articular/pathology
• Gene Expression Regulation/drug effects
• Humans
• Molecular Targeted Therapy
• Muscle, Skeletal/metabolism
• Muscle, Skeletal/pathology
• Muscle, Skeletal/physiopathology
• Osteoarthritis/etiology
• Osteoarthritis/metabolism
• Osteoarthritis/pathology
• Osteoarthritis/therapy
• Signal Transduction/drug effects

The present study evaluated the effects of diazine (DZN) on collagenase-induced osteoarthritis (OA) in rats.

OA was produced via intra-articular injections of collagenase type II into the knee joint. The rats were then treated with DZN (25, 50, or 100 mg/kg, p.o.) for three weeks. At the end of the protocol, all rats were evaluated for paw latency, paw edema, and knee swelling. Additionally, serum concentrations of glycosaminoglycan (GAG), alkaline phosphatase (ALP), and C-reactive protein (CRP) were determined. X-rays were performed to estimate radiological and histopathological changes in the knee joint. The expressions of antioxidant enzymes, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs) were estimated in the synovial tissues.

DZN treatment attenuated inflammation in osteoarthritic rats, as evidenced by decreases in paw edema and knee swelling and enhanced paw latency compared to the negative control group. Additionally, there were significant decreases in the serum levels of CRP and GAG and increases in ALP in the DZN-treated groups compared to the negative control group. The radiological and histopathological results showed that DZN protected against cartilage damage in the knee joint. Additionally, MMP levels decreased and there were significant reductions in the expressions of antioxidant enzymes and TIPMs in the DZN-treated groups compared to the negative control group.

The present findings demonstrated the chondroprotective effects of DZN via its modulation of the expressions of TIMP-1 and MMPs in the synovial tissues of osteoarthritic rats.

• chondrocytes
• osteoarthritis
• treatment

• Animals
• Cartilage/drug effects
• Cartilage/metabolism
• Cartilage/pathology
• Cell Line, Tumor
• Chondrocytes/drug effects
• Chondrocytes/metabolism
• Chondrocytes/pathology
• Dose-Response Relationship, Drug
• Extracellular Space/drug effects
• Extracellular Space/metabolism
• HEK293 Cells
• Humans
• Organ Culture Techniques
• Osteoarthritis/drug therapy
• Osteoarthritis/metabolism
• Osteoarthritis/pathology
• Protein Binding/physiology
• Suramin/pharmacology
• Suramin/therapeutic use
• Swine
• Tissue Inhibitor of Metalloproteinase-3/metabolism

• 20887 Arthritis Research UK
• R01 AR040994 NIAMS NIH HHS
• 19466 Versus Arthritis
• 19466 Arthritis Research UK
• 20205 Arthritis Research UK
• 20887 Versus Arthritis

Following inflammatory stimuli, GSK3 inhibition functions as a hub with pleiotropic effects leading to cartilage degradation. However, little is known about the effects triggered by its direct inhibition as well as the effects on mitochondrial pathology, that contributes to osteoarthritis pathogenesis. To this aim we assessed the molecular mechanisms triggered by GSK3β inactivating stimuli on 3-D (micromass) cultures of human articular chondrocytes. Stimuli were delivered either at micromass seeding (long term) or after maturation (short term) to explore “late” effects on terminal differentiation or “early” mitochondrial effects, respectively. GSK3β inhibition significantly enhanced mitochondrial oxidative stress and damage and endochondral ossification based on increased nuclear translocation of Runx-2 and β-catenin, calcium deposition, cell death and enhanced remodelling of the extracellular matrix as demonstrated by the increased collagenolytic activity of supernatants, despite unmodified (MMP-1) or even reduced (MMP-13) collagenase gene/protein expression. Molecular dissection of the underlying mechanisms showed that GSK3β inhibition achieved with pharmacological/silencing strategies impacted on the control of collagenolytic activity, via both decreased inhibition (reduced TIMP-3) and increased activation (increased MMP-10 and MMP-14). To conclude, the inhibition of GSK3β enhances terminal differentiation via concerted effects on ECM and therefore its activity represents a tool to keep articular cartilage homeostasis.

• ACL reconstruction
• remnant muscle tissue
• tendon graft healing

• Bone composition
• Bone fracture
• Bone strength
• Bone structure
• Bone tissue mineral density
• Tissue inhibitor of metalloproteinase-3

• Animals
• Bone Density/physiology
• Bone and Bones/metabolism
• Female
• Fractures, Bone/metabolism
• Mice, Inbred C57BL
• Mice, Knockout
• Tissue Inhibitor of Metalloproteinase-3/deficiency
• Tissue Inhibitor of Metalloproteinase-3/metabolism

• 20039 Arthritis Research UK
• BB/J003727/1 Biotechnology and Biological Sciences Research Council
• 20581 Arthritis Research UK
• MR/P020941/1 Medical Research Council
• 20039 Versus Arthritis
• 20581 Versus Arthritis

• Endothelial barrier function
• Inflammation
• Metalloproteinase
• TIMP

Bone development and length relies on the growth plate formation, which is dependent on degradative enzymes such as MMPs. Indeed, deletion of specific members of this enzyme family in mice results in important joint and bone abnormalities, suggesting a role in skeletal development. As such, the control of MMP activity is vital in the complex process of bone formation and growth. We generated a transgenic mouse line to overexpress TIMP3 in mouse chondrocytes using the Col2a1-chondrocyte promoter. This overexpression in cartilage resulted in a transient shortening of growth plate in homozygote mice but bone length was restored at eight weeks of age. However, tibial bone structure and mechanical properties remained compromised. Despite no transgene expression in adult osteoblasts from transgenic mice in vitro, their differentiation capacity was decreased. Neonates, however, did show transgene expression in a subset of bone cells. Our data demonstrate for the first time that transgene function persists in the chondro-osseous lineage continuum and exert influence upon bone quantity and quality.

• Animals
• Bone and Bones/pathology
• Bone and Bones/physiology
• Cartilage/metabolism
• Cells, Cultured
• Collagen Type II/genetics
• Femur/diagnostic imaging
• Femur/physiology
• Growth Plate/pathology
• Growth Plate/physiology
• Humans
• Male
• Mice
• Mice, Inbred C57BL
• Mice, Inbred CBA
• Mice, Transgenic
• Osteoblasts/cytology
• Osteoblasts/metabolism
• Osteogenesis
• Promoter Regions, Genetic
• Tensile Strength
• Tibia/diagnostic imaging
• Tibia/physiology
• Tissue Inhibitor of Metalloproteinase-3/genetics
• Tissue Inhibitor of Metalloproteinase-3/metabolism

• 18768 Versus Arthritis
• 20039 Versus Arthritis
• 20581 Versus Arthritis
• 20859 Versus Arthritis
• Arthritis Research UK
• Biotechnology and Biological Sciences Research Council
• Orthopaedic Research UK

• Cryopreservation/methods
• Female
• Gene Expression Regulation
• Humans
• Male
• Osteoarthritis/metabolism
• Osteoarthritis/pathology
• Synovial Membrane/metabolism
• Synovial Membrane/pathology

• ZonMw

Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a central inhibitor of matrix-degrading and sheddase families of metalloproteinases. Extracellular levels of the inhibitor are regulated by the balance between its retention on the extracellular matrix and its endocytic clearance by the scavenger receptor low density lipoprotein receptor-related protein 1 (LRP1). Here, we used molecular modeling to predict TIMP-3 residues potentially involved in binding to LRP1 based on the proposed LRP1 binding motif of 2 lysine residues separated by about 21 Å and mutated the candidate lysine residues to alanine individually and in pairs. Of the 22 mutants generated, 13 displayed a reduced rate of uptake by HTB94 chondrosarcoma cells. The two mutants (TIMP-3 K26A/K45A and K42A/K110A) with lowest rates of uptake were further evaluated and found to display reduced binding to LRP1 and unaltered inhibitory activity against prototypic metalloproteinases. TIMP-3 K26A/K45A retained higher affinity for sulfated glycosaminoglycans than K42A/K110A and exhibited increased affinity for ADAMTS-5 in the presence of heparin. Both mutants inhibited metalloproteinase-mediated degradation of cartilage at lower concentrations and for longer than wild-type TIMP-3, indicating that their increased half-lives improved their ability to protect cartilage. These mutants may be useful in treating connective tissue diseases associated with increased metalloproteinase activity.

• ADAM
• ADAMTS
• extracellular matrix
• glycosaminoglycan
• low-density lipoprotein receptor-related protein 1
• osteoarthritis
• receptor
• tissue inhibitor of metalloproteinase (TIMP)