Matrix metalloproteinase 17 is necessary for cartilage aggrecan degradation in an inflammatory environment.

OBJECTIVE: Aggrecan is a critical component of cartilage extracellular matrix. Several members of the 'a disintegrin and metalloproteinase with thrombospondin motifs' (ADAMTS) family have been characterised as aggrecanases by their ability to generate fragments containing the NITEGE neoepitope from aggrecan. Increased NITEGE fragments in synovial fluid and articular cartilage are a hallmark of osteoarthritis (OA) and it is hypothesised that the enhanced rate of aggrecan degradation is critical for cartilage destruction in OA. Recently, matrix metalloproteinase 17 (MMP17, also known as MT4-MMP) has been implicated in the activation of one of the key aggrecanases: ADAMTS4. In the present work, the hypothesis that MMP17 mediates the interleukin 1ß (IL-1ß) induced release of NITEGE neoepitope from human and murine articular cartilage is investigated. METHODS: MMP17 was quantified at the protein and RNA level and NITEGE neoepitope generation by immunohistochemistry. Human postmortem articular cartilage explants were treated with recombinant MMP17, or IL-1ß in the presence or absence of an MMP17 inhibitor. Glycosaminoglycan (GAG) loss into the media was quantified using the 1,9-dimethylmethylene blue (DMMB) assay. Intra-articular injection (IAI) of IL-1ß or meniscotibial ligament transaction was carried out in MMP17 null mice. RESULTS: The data reveal an association between increased MMP17 protein and NITEGE staining in areas of OA cartilage damage. Ex vivo treatment of normal human cartilage with recombinant MMP17 protein increased NITEGE generation in the cartilage and GAG loss into the media. In addition, IL-1ß mediated cartilage GAG loss, and increased NITEGE neoepitope expression, were attenuated with an MMP17 inhibitor. IAI of IL-1ß into C57BL6/Jax mice resulted in increased MMP17 expression in articular cartilage and increased GAG content in the synovial fluid. MMP17 null mice were protected against this increase. However, aggrecan loss driven by mechanical stress following medial meniscotibial ligament transection was not dependent on MMP17. CONCLUSION: These data further implicate MMP17 in the control of articular cartilage extracellular matrix aggrecan integrity in an inflammatory environment.

Characterisation of cell-penetrating peptide-mediated peptide delivery.

Cell-penetrating peptides such as antennapedia, TAT, transportan and polyarginine have been extensively employed for in vitro and in vivo delivery of biologically active peptides. However, little is known of the relative efficacy, toxicity and uptake mechanism of individual protein transduction domain-peptide conjugates, factors that will be critical in determining the most effective sequence. In the present study, we show by FACS analysis that unconjugated antennapedia, TAT, transportan and polyarginine demonstrate similar kinetic uptake profiles, being maximal at 1-3 h and independent of cell type (HeLa, A549 and CHO cell lines). A comparison of the magnitude of uptake of cell-penetrating peptide conjugates demonstrated that polyarginine=transportan>antennapedia>TAT. However, examination of cellular toxicity showed that antennapedia

Expression profiling of metalloproteinases and their inhibitors in cartilage.

OBJECTIVE: To profile the expression of all known members of the matrix metalloproteinase (MMP), ADAMTS, and tissue inhibitor of metalloproteinases (TIMP) gene families in normal cartilage and cartilage from patients with osteoarthritis (OA). METHODS: Human cartilage was obtained from femoral heads at joint replacement for OA or following fracture to the femoral neck. Total RNA was purified, and gene expression was assayed using quantitative real-time polymerase chain reaction. RESULTS: Several members of the above gene families were regulated in OA. Genes that showed increased expression in OA were MMP13, MMP28, and ADAMTS16 (all at P < 0.001), MMP9, MMP16, ADAMTS2, and ADAMTS14 (all at P < 0.01), and MMP2, TIMP3, and ADAMTS12 (all at P < 0.05). Genes with decreased expression in OA were MMP1, MMP3, and ADAMTS1 (all at P < 0.001), MMP10, TIMP1, and ADAMTS9 (all at P < 0.01), and TIMP4, ADAMTS5, and ADAMTS15 (all at P < 0.05). Correlation analysis revealed that groups of genes across the gene families were coexpressed in cartilage. CONCLUSION: This is the first comprehensive expression profile of all known MMP, ADAMTS, and TIMP genes in cartilage. Elucidation of patterns of expression provides a foundation with which to understand mechanisms of gene regulation in OA and potentially to refine the specificity of antiproteolytic therapies.

Crystal structure of human MMP9 in complex with a reverse hydroxamate inhibitor.

Matrix metalloproteinases (MMPs) and their inhibitors are important in connective tissue re-modelling in diseases of the cardiovascular system, such as atherosclerosis. Various members of the MMP family have been shown to be expressed in atherosclerotic lesions, but MMP9 is consistently seen in inflammatory atherosclerotic lesions. MMP9 over-expression is implicated in the vascular re-modelling events preceding plaque rupture (the most common cause of acute myocardial infarction). Reduced MMP9 activity, either by genetic manipulation or through pharmacological intervention, has an impact on ventricular re-modelling following infarction. MMP9 activity may therefore represent a key mechanism in the pathogenesis of heart failure. We have determined the crystal structure, at 2.3 A resolution, of the catalytic domain of human MMP9 bound to a peptidic reverse hydroxamate inhibitor as well as the complex of the same inhibitor bound to an active-site mutant (E402Q) at 2.1 A resolution. MMP9 adopts the typical MMP fold. The catalytic centre is composed of the active-site zinc ion, co-ordinated by three histidine residues (401, 405 and 411) and the essential glutamic acid residue (402). The main differences between the catalytic domains of various MMPs occur in the S1' subsite or selectivity pocket. The S1' specificity site in MMP9 is perhaps best described as a tunnel leading toward solvent, as in MMP2 and MMP13, as opposed to the smaller pocket found in fibroblast collagenase and matrilysin. The present structure enables us to aid the design of potent and specific inhibitors for this important cardiovascular disease target.