An Introductory Guide to Protease Sensitive Linker Design Using Matrix Metalloproteinase 13 as an Example.

Proteases play a crucial role, not only in physiological, but also in pathological processes, such as cancer, inflammation, arthritis, Alzheimer's, and infections, to name but a few. Their ability to cleave peptides can be harnessed for a broad range of biotechnological purposes. To do this efficiently, it is essential to find an amino acid sequence that meets the necessary requirements, including interdependent factors like specificity, selectivity, cleavage kinetics, or synthetic accessibility. Cleavage sequences from natural substrates of the protease may not be optimal in terms of specificity and selectivity, which is why these frequently require arduous and sometimes unsuccessful optimization such as by iterative exchange of single amino acids. Hence, here we describe the systematic design of protease sensitive linkers (PSLs)─peptide sequences specifically cleaved by a target protease─guided by the mass spectrometry based determination of target protease specific cleavage sites from a proteome-based peptide library. It includes a procedure for identifying bespoke PSL sequences, their optimization, synthesis, and validation and introduces a program that can indicate potential cleavage sites by hundreds of enzymes in any arbitrary amino acid sequence. Thereby, we provide an introduction to PSL design, illustrated by the example of matrix metalloproteinase 13 (MMP13). This introduction can serve as a guide and help to greatly accelerate the development and use of protease-sensitive linkers in diverse applications.

Development of a putative Zn2+-chelating but highly selective MMP-13 inhibitor.

Starting from an already known MMP-13 inhibitor, 1, we pursued an SAR-approach focusing on optimizing interactions close to the Zn2+ binding site of the enzyme. We found the oxetane containing compound 32 (MMP-13 IC50 = 42 nM), which exhibited complete inhibition of collagenolysis in in vitro studies and an excellent selectivity profile among the MMP family. Interestingly, docking studies propose that the oxetane ring in 32 is oriented towards the Zn2+ ion for chelating the metal ion. Chelating properties of MMP13-inhibitors are often connected with non-selectivity within the enzyme family. Compound 32 demonstrates a rare example where the selectivity can be explained via combinatorial effects of interactions within the S1' loop and a chelating effect of the oxetane moiety. Furthermore, in vivo pharmacokinetic studies were performed demonstrating a concentration of 1.97 µM of 32 within the synovial fluid of the rat knee joint, which makes the compound a promising lead compound for further optimization and development for osteoarthritis.

Development of a selective matrix metalloproteinase 13 (MMP-13) inhibitor for the treatment of Osteoarthritis.

Osteoarthritis (OA) is a chronic disorder that causes damage to the cartilage and surrounding tissues and is characterized by pain, stiffness, and loss of function. Current treatments for OA primarily involve providing only relief of symptoms but does not affect the overall trajectory of the disease. A major goal for treating OA has been to slow down or reverse disease progression. Matrix metalloproteinase-13 (MMP-13) is expressed by chondrocytes and synovial cells in human OA and is thought to play a critical role in cartilage destruction. Herein we report a new, allosteric MMP-13 inhibitor, AQU-019, that has been optimized for potency, metabolic stability, and oral bioavailability through a combination of structure activity relationship (SAR) and deuterium substitution as a potential disease modifying OA drug (DMOAD). The inhibitor was demonstrated to be chondroprotective when injected intraarticular (IA) in the monoiodoacetic acid (MIA) rat model of OA.

TAP2, a peptide antagonist of Toll-like receptor 4, attenuates pain and cartilage degradation in a monoiodoacetate-induced arthritis rat model.

Because inflammation in osteoarthritis (OA) is related to the Toll-like receptor 4 (TLR4) signaling cascades, TLR4 is a reasonable target for developing therapeutics for OA. Thus, we investigated whether TAP2, a peptide antagonist of TLR4, reduces the monoiodoacetate (MIA)-induced arthritic pain and cartilage degradation in rats. TLR4 expression of human OA chondrocytes and synoviocytes and the knee joint tissue of MIA-induced arthritis were evaluated. MIA-induced arthritic model using Sprague-Dawley rats (6 week-old-male) were treated with TAP2, a TLR4 antagonist, and evaluated with behavioral test, immunohistochemistry, and quantitative PCR. TLR4 was highly expressed in the knee joints of patients with OA and the MIA-induced rat model. Further, a single intraarticular injection of TAP2 (25 nmol/rat) molecules targeting TLR4 on day 7 after MIA injection dramatically attenuated pain behavior for about 3 weeks and reduced cartilage loss in the knee joints and microglial activation in the spinal dorsal horns. Likewise, the mRNA levels of TNFα and IL-1ß, reactive oxygen species, and the expression of MMP13 in the knee joints of TAP2-treated rats was significantly decreased by TAP2 treatment compared with the control. Moreover, interestingly, the duration of OA pain relief by TAP2 was much longer than that of chemical TLR4 antagonists, such as C34 and M62812. In conclusion, TAP2 could effectively attenuate MIA-induced arthritis in rats by blocking TLR4 and its successive inflammatory cytokines and MMP13. Therefore, TAP2 could be a prospective therapeutic to treat patients with OA.

Synthesis, Molecular Docking, and In Vitro Boron Neutron Capture Therapy Assay of Carboranyl Sinomenine.

In comparison with pristine sinomenine and carborane precursors, the calculations of molecular docking with matrix metalloproteinases (MMPs) and methylcarboranyl-n-butyl sinomenine showed improved interactions. Accordingly, methylcarboranyl-n-butyl sinomenine shows a high potential in the treatment of rheumatoid arthritis (RA) in the presence of slow neutrons. The reaction of potassium salt of sinomenie, which is generated from the deprotonation of sinomenine (1) using potassium carbonate in a solvent of N,N-dimethyl formamide, with 4-methylcarboranyl-n-butyl iodide, (2) forms methylcarboranyl-n-butyl sinomenine (3) in 54.3% yield as a new product. This new compound was characterized by 1H, 13C, and 11B NMR spectroscopy, FT-IR spectroscopy, and elemental analyses to confirm its molecular composition. In addition to molecular docking interactions with MMPs, the in vitro killing effects of 3, along with its toxicity measurements, exhibited its potential to be the new drug delivery agent for boron neutron capture synovectomy (BNCS) and boron neutron capture therapy (BNCT) for the treatment of rheumatoid arthritis (RA) and cancers in the presence of slow neutrons, respectively.

Artificial Intelligence Approach to Find Lead Compounds for Treating Tumors.

It has been demonstrated that MMP13 enzyme is related to most cancer cell tumors. The world's largest traditional Chinese medicine database was applied to screen for structure-based drug design and ligand-based drug design. To predict drug activity, machine learning models (Random Forest (RF), AdaBoost Regressor (ABR), Gradient Boosting Regressor (GBR)), and Deep Learning models were utilized to validate the Docking results, and we obtained an R2 of 0.922 on the training set and 0.804 on the test set in the RF algorithm. For the Deep Learning algorithm, R2 of the training set is 0.90, and R2 of the test set is 0.810. However, these TCM compounds fly away during the molecular dynamics (MD) simulation. We seek another method: peptide design. All peptide database were screened by the Docking process. Modification peptides were optimized the interaction modes, and the affinities were assessed with ZDOCK protocol and Refine Docked protein protocol. The 300 ns MD simulation evaluated the stability of receptor-peptide complexes. The double-site effect appeared on S2, a designed peptide based on a known inhibitor, when complexed with BCL2. S3, a designed peptide referred from endogenous inhibitor P16, competed against cyclin when binding with CDK6. The MDM2 inhibitors S5 and S6 were derived from the P53 structure and stable binding with MDM2. A flexible region of peptides S5 and S6 may enhance the binding ability by changing its own conformation, which was unforeseen. These peptides (S2, S3, S5, and S6) are potentially interesting to treat cancer; however, these findings need to be affirmed by biological testing, which will be conducted in the near future.

Drug Design Inspired by Nature: Crystallographic Detection of an Auto-Tailored Protease Inhibitor Template.

De novo drug discovery is still a challenge in the search for potent and selective modulators of therapeutically relevant target proteins. Here, we disclose the unexpected discovery of a peptidic ligand 1 by X-ray crystallography, which was auto-tailored by the therapeutic target MMP-13 through partial self-degradation and subsequent structure-based optimization to a highly potent and selective ß-sheet peptidomimetic inhibitor derived from the endogenous tissue inhibitors of metalloproteinases (TIMPs). The incorporation of non-proteinogenic amino acids in combination with a cyclization strategy proved to be key for the de novo design of TIMP peptidomimetics. The optimized cyclic peptide 4 (ZHAWOC7726) is membrane permeable with an IC50 of 21 nm for MMP-13 and an attractive selectivity profile with respect to a polypharmacology approach including the anticancer targets MMP-2 (IC50 : 170 nm) and MMP-9 (IC50 : 140 nm).

Design, cyclization, and optimization of MMP13-TIMP1 interaction-derived self-inhibitory peptides against chondrocyte senescence in osteoarthritis.

The matrix metallopeptidase 13 (MMP13) is a central regulator of chondrocyte senescence that contributes to the development and progression of osteoarthritis (OA). In the present study, the native inhibitory structure of MMP13 in complex with its natural cognate inhibitor, the tissue inhibitor of metalloproteinases 1 (TIMP1), was modeled at atomic level using a grafting-based structural bioinformatics method with existing crystal structures. The modeled complex structure was then examined in detail, from which a TIMP1 inhibitory site that directly inserts into the active site of MMP13 enzyme was identified. The inhibitory site contains a coiled inhibitory loop (ILP) and a stretched N-terminal tail (NTT); they are highly structured in the intact MMP13-TIMP1 complex interface, but exhibit a large flexibility and intrinsic disorder when split from the interface context. In vitro binding assays demonstrated that the isolated ILP and NTT peptides cannot effectively rebind at the MMP13 active site (Kd > ~100 µM or = n.d.), although they have all key interacting residues in the enzyme inhibition. In silico simulations revealed that splitting of the peptide segments from TIMP1 inhibitory site does not influence the direct intermolecular interaction between MMP13 and the peptides substantially; instead, the large conformational flexibility of these isolated peptides in absence of interface context is primarily responsible for the affinity impairment, which would incur a considerable entropy penalty upon the peptide binding to MMP13. An extended version of ILP peptide, namely eILP (63TPAMESVCGY72), was redesigned with a rational strategy to derive a number of its cyclized counterparts by introducing a disulfide bridge across the peptide two-termini; the redesign reduces the peptide flexibility in free state and constrains the peptide pre-folding to a native-like conformation, which would help the peptide binding with minimized entropy penalty. Binding assays substantiated that the affinity Kd values of four designed cyclic peptides (, , and ) were improved to 23, 67, 42 and 18 µM, respectively, from the 96 µM of linear eILP peptide.

Development of an inflammatory tissue-selective chimeric TNF receptor.

BACKGROUND: Inhibiting TNF-α is an effective therapy for inflammatory diseases such as rheumatoid arthritis. However, systemic, nondiscriminatory neutralization of TNF-α is associated with considerable adverse effects. METHODS: Here, we developed a trimeric chimeric TNF receptor by linking an N-terminal mouse Acrp30 trimerization domain and an MMP-2/9 substrate sequence to the mouse extracellular domain of TNF receptor 2 followed by a C-terminal mouse tetranectin coiled-coil domain (mouse Acrp-MMP-TNFR-Tn). RESULTS: Here, we show that the Acrp30 trimerization domain inhibited the binding activity of TNFR, possibly by closing the binding site of the trimeric receptor. Cleavage of the substrate sequence by MMP-9, an enzyme highly expressed in inflammatory sites, restored the binding activity of the mouse TNF receptor. We also constructed a recombinant human chimeric TNF receptor (human Acrp-MMP-TNFR-Tn) in which an MMP-13 substrate sequence was used to link the human Acrp and the human TNF receptor 2. Human Acrp-MMP-TNFR-Tn showed reduced binding activity, and MMP-13 digestion recovered its binding activity with TNF-α. CONCLUSION: Acrp-masked chimeric TNF receptors may be able to be used for inflammatory tissue-selective neutralization of TNF-α to reduce the adverse effects associated with systemic neutralization of TNF-α.

Orally Administered Polysaccharide Derived from Blackcurrants (Ribes nigrum L.) Improves Skin Hydration in Ultraviolet-Irradiated Hairless Mice.

Blackcurrants (Ribes nigrum L.) have various benefits for human health. In particular, a polysaccharide derived from blackcurrant was found to be an immunostimulating food ingredient in a mouse model. We named a polysaccharide derived from blackcurrant cassis polysaccharide (CAPS). In a previous clinical study, we reported that CAPS affects skin dehydration, demonstrating its effectiveness against skin inflammation was related to atopic dermatitis; skin inflammation caused skin dehydration. However, there are no studies regarding CAPS effectiveness against skin dehydration. The current study aimed to investigate CAPS effectiveness against skin dehydration. We further demonstrate the effect of oral administration of CAPS on skin dehydration caused by ultraviolet (UV) irradiation-induced inflammation in mice. We found that CAPS administration suppresses skin dehydration caused by UV irradiation. We also found that CAPS decreases interleukin-6 and matrix metalloproteinase transcription levels in the mouse skin. These results show that CAPS improves skin hydration in UV-irradiated mice.

PKR activation causes inflammation and MMP-13 secretion in human degenerated articular chondrocytes.

Osteoarthritis (OA) is a degenerative joint disease affecting a large population of people. Although the elevated expression of PKR (double stranded RNA-dependent protein kinase) and MMP-13 (collagenase-3) have been indicated to play pivotal roles in the pathogenesis of OA, the exact mechanism underlying the regulation of MMP-13 by PKR following inflammatory stimulation was relatively unknown. The purpose of this study was to determine the signaling pathway involved in the PKR-mediated induction of MMP-13 after TNF-α-stimulation. In this study, cartilages of knee joint were obtained from OA subjects who underwent arthroplastic knee surgery. Cartilages were used for tissue analysis or for chondrocytes isolation. In results, the upregulated expression of PKR was observed in damaged OA cartilages as well as in TNF-α-stimulated chondrocytes. Phosphorylation of PKC (protein kinase C) was found after TNF-α administration or PKR activation using poly(I:C), indicating PKC was regulated by PKR. The subsequent increased activity of NADPH oxidase led to oxidative stress accumulation and antioxidant capacity downregulation followed by an exaggerated inflammatory response with elevated levels of COX-2 and IL-8 via ERK/NF-κB pathway. Activated ERK pathway also impeded the inhibition of MMP-13 by PPAR-γ. These findings demonstrated that TNF-α-induced PKR activation triggered oxidative stress-mediated inflammation and MMP-13 in human chondrocytes. Unraveling these deregulated signaling cascades will deepen our knowledge of OA pathophysiology and provide aid in the development of novel therapies.

Downregulation of microRNA-9 increases matrix metalloproteinase-13 expression levels and facilitates osteoarthritis onset.

Matrix metalloproteinase-13 (MMP-13) degrades collagen and other matrix components, thus playing a critical role in the development of osteoarthritis (OA). The expression level of microRNA­9 (miR­9) is significantly depressed in cartilage tissues of OA patients. Furthermore, bioinformatics analysis demonstrated complementary binding sites between miR­9 and MMP­13. The current study, therefore, investigated whether miR­9 is involved in regulating MMP­13 expression levels and OA onset. Cartilage tissues from OA patients and healthy individuals were compared for miR­9, MMP­13 and collagen type II α1 chain (Col2A1) expression levels. A dual luciferase gene reporter assay was performed to evaluate the association between miR­9 and MMP­13. Sodium iodoacetate was injected into the knee joint cartilage tissues to generate the rat OA model. The expression levels of miR­9, MMP­13 and Col2A1 were compared between the model and control rats. In addition, the OA model rats received miR­9 agomir for further expressional assay. Cartilage tissue samples from the OA patients exhibited significantly lower miR­9 and Col2A1 expression levels when compared with the control rats, whilst the expression level of MMP­13 was upregulated. As the target gene of miR­9, MMP­13 is under the targeted regulation of miR­9. The injection of miR­9 agomir into the knee joint cavity significantly depressed MMP­13 expression in the cartilage tissues of OA rats, with reduced collagen degradation and enhanced COL2A1. OA cartilage tissues have lower miR­9 expression and higher MMP­13 expression levels. Thus, miR­9 inhibits the expression level of MMP­13, decreases its inhibitory effects on COL2A1, and therefore contributes to antagonizing OA.

An improved collagen zymography approach for evaluating the collagenases MMP-1, MMP-8, and MMP-13.

Collagen zymography is an SDS-PAGE-based method for detecting both the proenzyme and active forms of collagenases. Although collagen zymography is used for assessment of the matrix metalloproteinases MMP-1 and MMP-13, it can be difficult to detect these collagenases due to technical issues. Moreover, it remains unclear whether the collagenase activity of MMP-8 can be detected by this method. Here, we present an improved collagen zymography method that allows quantification of the activities of MMP-1, MMP-8, and MMP-13. Activities of recombinant collagenases could be detected in collagen zymogram gels copolymerized with 0.3 mg/mL type I collagen extracted from rat tail tendon. This improved method is sensitive enough to detect the activity of as little as 1 ng of collagenase. We generated standard curves for the three collagenases to quantify the collagenolytic activity levels of unknown samples. To validate our improved method, we investigated MMP-1 activity levels in human thyroid cancer (8505C) and normal thyroid (Nthy-ori-3-1) cell lines, finding that the proenzyme and active MMP-1 levels were greater in 8505C cells than in Nthy-ori-3-1 cells. Taken together, our data show that collagen zymography can be used in both molecular and clinical investigations to evaluate collagenase activities in various pathological conditions.

Structure-Based Design and Synthesis of Potent and Selective Matrix Metalloproteinase 13 Inhibitors.

We describe the use of comparative structural analysis and structure-guided molecular design to develop potent and selective inhibitors (10d and (S)-17b) of matrix metalloproteinase 13 (MMP-13). We applied a three-step process, starting with a comparative analysis of the X-ray crystallographic structure of compound 5 in complex with MMP-13 with published structures of known MMP-13·inhibitor complexes followed by molecular design and synthesis of potent but nonselective zinc-chelating MMP inhibitors (e.g., 10a and 10b). After demonstrating that the pharmacophores of the chelating inhibitors (S)-10a, (R)-10a, and 10b were binding within the MMP-13 active site, the Zn2+ chelating unit was replaced with nonchelating polar residues that bridged over the Zn2+ binding site and reached into a solvent accessible area. After two rounds of structural optimization, these design approaches led to small molecule MMP-13 inhibitors 10d and (S)-17b, which bind within the substrate-binding site of MMP-13 and surround the catalytically active Zn2+ ion without chelating to the metal. These compounds exhibit at least 500-fold selectivity versus other MMPs.

Virtual High-Throughput Screening for Matrix Metalloproteinase Inhibitors.

Structure-based virtual screening (SBVS) is a common method for the fast identification of hit structures at the beginning of a medicinal chemistry program in drug discovery. The SBVS, described in this manuscript, is focused on finding small molecule hits that can be further utilized as a starting point for the development of inhibitors of matrix metalloproteinase 13 (MMP-13) via structure-based molecular design. We intended to identify a set of structurally diverse hits, which occupy all subsites (S1'-S3', S2, and S3) centering the zinc containing binding site of MMP-13, by the virtual screening of a chemical library comprising more than ten million commercially available compounds. In total, 23 compounds were found as potential MMP-13 inhibitors using Glide docking followed by the analysis of the structural interaction fingerprints (SIFt) of the docked structures.

Serum and knee synovial fluid matrixmetalloproteinase-13 and tumor necrosis factor-alpha levels in patients with late stage osteoarthritis.

OBJECTIVE: To compare the levels of MMP-13 and TNF-α in late stage osteoarthritis, define their predominant pathways and investigate their correlation with McMaster Universities Arthritis Index scores. PATIENTS AND METHODS: A total of 42 patients (mean age 64 ± 8.8) with grade 3 and grade 4 knee osteoarthritis according to Kellegren- Lawrence criteria and who were scheduled for total knee arthroplasty were enrolled in the study. TNF-alpha and MMP-13 levels were measured preoperatively from venous blood samples and intraoperatively from knee synovial fluid via ELISA. Preoperative and 1 month postoperative knee functions were assessed by McMaster Universities Arthritis Index. RESULTS: Grade 4 synovial fluid MMP-13 (4.76 ± 5.82) was elevated compared to grade 3 (3.95 ± 4.45) (p = 0.438), whereas grade 3 serum MMP-13 (1.128 ± 0.308) was found elevated compared to grade 4 (1.038 ± 0.204) (p = 0.430). Grade 4 serum TNF-α (0.253 ± 0.277) was elevated compared to grade 3 (0.206 ± 0.219) whereas grade 3 synovial fluid TNF-α (0.129 ± 0.052) was elevated compared to grade 4 (0.118 ± 0.014). Positive correlation was observed between synovial fluid MMP-13 levels and postoperative WOMAC scores. Mean serum TNF-α level (0.226 ± 0.246 pg/ml) was found higher compared to synovial level (0.124 ± 1.59), synovial MMP-13 level (4.31 ± 1.24) was found higher compared to serum level (1.089 ± 1.519). CONCLUSION: Despite the systemic increase in TNF-α levels concordant with osteoarthritis grade, MMP-13 levels are elevated via local manner with a significant correlation with WOMAC scores. LEVEL OF EVIDENCE: Level IV, Diagnostic study.

Trivalent metal ions based on inorganic compounds with in vitro inhibitory activity of matrix metalloproteinase 13.

Collagenase-3 (MMP-13) inhibitors have attracted considerable attention in recent years and have been developed as a therapeutic target for a variety of diseases, including cancer. Matrix metalloproteinases (MMPs) can be inhibited by a multitude of compounds, including hydroxamic acids. Studies have shown that materials and compounds containing trivalent metal ions, particularly potassium hexacyanoferrate (III) (K3[Fe(CN)6]), exhibit cdMMP-13 inhibitory potential with a half maximal inhibitory concentration (IC50) of 1.3µM. The target protein was obtained by refolding the recombinant histidine-tagged cdMMP-13 using size exclusion chromatography (SEC). The secondary structures of the refolded cdMMP-13 with or without metal ions were further analyzed via circular dichroism and the results indicate that upon binding with metal ions, an altered structure with increased domain stability was obtained. Furthermore, isothermal titration calorimetry (ITC) experiments demonstrated that K3[Fe(CN)6]is able to bind to MMP-13 and endothelial cell tube formation tests provide further evidence for this interaction to exhibit anti-angiogenesis potential. To the best of our knowledge, no previous report of an inorganic compound featuring a MMP-13 inhibitory activity has ever been reported in the literature. Our results demonstrate that K3[Fe(CN)6] is useful as a new effective and specific inhibitor for cdMMP-13 which may be of great potential for future drug screening applications.

Cloning, expression, purification, and characterization of the catalytic domain of sika deer MMP-13.

Matrix metalloproteinase 13 is one of three mammalian collagenases that are capable of initiating the degradation of interstitial collagens during wound healing. Herein, we report for the first time the molecular cloning of the catalytic domain (CD) of sika deer MMP-13, followed by protein expression in Escherichia coli and purification by affinity chromatography. The final yield was approximately 90.4 mg per liter of growth culture with a purity of 91.6%. The mass recovery during the purification and renaturation were 70.2% and 81.5%, respectively. Using gelatin zymography and a degradation assay, we found that the refolded sika deer MMP-13 (CD) could digest gelatin. The optimal pH and temperature for the enzyme bioactivity was 8.0 and 37 °C, respectively. The Km value for the enzyme-catalyzed digestion of gelatin was 136+/-8 µg/mL, and the Vmax was 4.12 × 10(3) U/µg. sdMMP13 (CD) was able to completely degrade collagen II and gelatin, and partially degrade fibronectin. The sdMMP-13 (CD) activity was significantly inhibited by several chemicals including 1, 10-phenanthroline, EDTA, Fe(2+), Cu(2+), and Mn(2+).

Serum and knee synovial fluid matrix metalloproteinase-13 and tumor necrosis factor-alpha levels in patients with late-stage osteoarthritis.

OBJECTIVE: The aim of this study was to compare serum and synovial fluid levels of matrix metalloproteinase-13 (MMP-13) and tumor necrosis factor-alpha (TNF-α) in 2 stages of osteoarthritis, and investigate their correlation with Western Ontario and McMaster Universities Arthritis Index (WOMAC) scores. METHODS: Forty-two patients (mean age: 64±8.8 years) with grade 3 and grade 4 knee osteoarthritis according to Kellgren-Lawrence criteria were enrolled in the study and underwent total knee arthroplasty. TNF-α and MMP-13 levels were measured preoperatively from venous blood samples and intraoperatively from knee synovial fluid via enzyme-linked immunosorbent assay. Preoperative and 1-month postoperative knee functions were assessed by WOMAC. RESULTS: Grade 4 synovial fluid MMP-13 (4.76±5.82 pg/ml) was elevated compared to grade 3 (3.95±4.45), whereas grade 3 serum MMP-13 (1.128±0.308 pg/ml) was found elevated compared to grade 4 (1.038±0.204) (p=0.438, p=0.430, respectively). Grade 4 serum TNF-α (0.253±0.277) was elevated compared to grade 3 (0.206±0.219), whereas grade 3 synovial fluid TNF-α (0.129±0.052) was elevated compared to grade 4 (0.118±0.014). Positive correlation was observed between synovial fluid MMP-13 levels and postoperative WOMAC scores. Mean serum TNF-α (0.226±0.246 pg/ml) was elevated compared to synovial levels (0.124±1.59), and synovial MMP-13 (4.31±1.24) was elevated compared to serum levels (1.089±1.519). CONCLUSION: Despite the systemic increase in TNF-α levels concordant with osteoarthritis grade, MMP-13 levels are elevated via local manner, with a significant correlation with WOMAC scores.

MMP-13 is constitutively produced in human chondrocytes and co-endocytosed with ADAMTS-5 and TIMP-3 by the endocytic receptor LRP1.

Matrix metalloproteinase 13 (MMP-13) degrades collagenous extracellular matrix and its aberrant activity associates with diseases such as arthritis, cancer, atherosclerosis and fibrosis. The wide range of MMP-13 proteolytic capacity suggests that it is a powerful, potentially destructive proteinase and thus it has been believed that MMP-13 is not produced in most adult human tissues in the steady state. Present study has revealed that human chondrocytes isolated from healthy adults constitutively express and secrete MMP-13, but that it is rapidly endocytosed and degraded by chondrocytes. Both pro- and activated MMP-13 bind to clusters II and III of low-density lipoprotein (LDL) receptor-related protein 1 (LRP1). Domain deletion studies indicated that the hemopexin domain is responsible for this interaction. Binding competition between MMP-13 and ADAMTS-4, -5 or TIMP-3, which also bind to cluster II, further shown that the MMP-13 binding site within cluster II is different from those of ADAMTS-4, -5 or TIMP-3. MMP-13 is therefore co-endocytosed with ADAMTS-5 and TIMP-3 by human chondrocytes. These findings indicate that MMP-13 may play a role on physiological turnover of cartilage extracellular matrix and that LRP1 is a key modulator of extracellular levels of MMP-13 and its internalization is independent of the levels of ADAMTS-4, -5 and TIMP-3.