Linda Troeberg

Proteases involved in cartilage matrix degradation in osteoarthritis.

Osteoarthritis is a common joint disease for which there are currently no disease-modifying drugs available. Degradation of the cartilage extracellular matrix is a central feature of the disease and is widely thought to be mediated by proteinases that degrade structural components of the matrix, primarily aggrecan and collagen. Studies on transgenic mice have confirmed the central role of Adamalysin with Thrombospondin Motifs 5 (ADAMTS-5) in aggrecan degradation, and the collagenolytic matrix metalloproteinase MMP-13 in collagen degradation. This review discusses recent advances in current understanding of the mechanisms regulating expression of these key enzymes, as well as reviewing the roles of other proteinases in cartilage destruction. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.

Calcium pentosan polysulfate is a multifaceted exosite inhibitor of aggrecanases

Degradation of the cartilage proteoglycan aggrecan is a key early event in the development of osteoarthritis. Adamalysin with thrombospondin motifs (ADAMTS) −4 and ADAMTS‐5 are considered to be the main enzymes responsible for aggrecan breakdown, making them attractive drugs targets. Here we show that calcium pentosan polysulfate (CaPPS), a chemically sulfated xylanopyranose from beechwood, is a multifaceted exosite inhibitor of the aggrecanases and protects cartilage against aggrecan degradation. CaPPS interacts with the noncatalytic spacer domain of ADAMTS‐4 and the cysteine‐rich domain of ADAMTS‐5, blocking activity against their natural substrate aggrecan with inhibitory concentration 50 values of 10–40 nM but only weakly inhibiting hydrolysis of a nonglycosylated recombinant protein substrate. In addition, CaPPS increased cartilage levels of tissue inhibitor of metallo‐proteinases‐3 (TIMP‐3), an endogenous inhibitor of ADAMTS‐4 and −5. This was due to the ability of CaPPS to block endocytosis of TIMP‐3 mediated by low‐density lipoprotein receptor‐related protein. CaPPS also increased the affinity of TIMP‐3 for ADAMTS‐4 and −5 by more than 100‐fold, improving the efficacy of TIMP‐3 as an aggrecanase inhibitor. Studies with TIMP‐3‐null mouse cartilage indicated that CaPPS inhibition of aggrecan degradation is TIMP‐3 dependent. These unique properties make CaPPS a prototypic disease‐modifying agent for osteoarthritis.—Troeberg, L., Fushimi, K., Khokha, R., Emonard, H., Ghosh, P., Nagase, H. Calcium pentosan polysulfate is a multifaceted exosite inhibitor of aggrecanases. FASEB J. 22, 3515–3524 (2008)

Production of cytokines, vascular endothelial growth factor, matrix metalloproteinases, and tissue inhibitor of metalloproteinases 1 by tenosynovium demonstrates its potential for tendon destruction in rheumatoid arthritis.

OBJECTIVE To investigate the role of proinflammatory cytokines, vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs), and tissue inhibitor of metalloproteinases 1 (TIMP-1) in the destruction of tendons by tenosynovium in rheumatoid arthritis (RA). METHODS Synovial specimens were obtained from encapsulating tenosynovium (n = 17), invasive tenosynovium (n = 13), and wrist joints (n = 17) in 18 RA patients undergoing wrist extensor tenosynovectomy. Synovial membrane cells were dissociated from connective tissue by enzyme digestion and cultured in vitro for 48 hours, and harvested supernatants were assayed for the cytokines tumor necrosis factor alpha (TNFalpha) and interleukin-6 (IL-6), VEGF, MMPs 1, 2, 3, and 13, and TIMP-1 by enzyme-linked immunosorbent assay. Gelatin zymography was performed to demonstrate enzyme activity. Statistical analysis was performed using Students paired 2-tailed t-tests for parametric data and the Wilcoxon signed rank test for nonparametric data. RESULTS MMP-1 and MMP-13 levels were approximately 2.5-fold higher in invasive tenosynovium compared with encapsulating tenosynovium. Levels of MMP-2 were approximately 1.5-fold higher in invasive tenosynovium compared with both encapsulating tenosynovium and wrist joint synovium. MMP-13 (P = 0.009) and IL-6 (P = 0.03) levels were significantly lower in encapsulating tenosynovium compared with wrist joint synovium. Levels of VEGF, TIMP-1, TNFalpha, and MMP-3 were similar in all synovial sample groups. Zymography demonstrated enzyme activity in all synovium samples from all 9 patients assessed. CONCLUSION Tenosynovium produces proinflammatory cytokines and proteolytic enzymes that are important in the tissue degradation seen in RA. Increased production of the enzymes MMP-1, MMP-2, and MMP-13 by invasive tenosynovium suggests a possible explanation for the worse prognosis and increased rupture rate associated with invasive tenosynovitis in RA. Production of VEGF by tenosynovium suggests that angiogenesis may have a role in tenosynovial proliferation and invasion of tendons.

Membrane type 1 matrix metalloproteinase is a crucial promoter of synovial invasion in human rheumatoid arthritis

OBJECTIVE A hallmark of rheumatoid arthritis (RA) is invasion of the synovial pannus into cartilage, and this process requires degradation of the collagen matrix. The aim of this study was to explore the role of one of the collagen-degrading matrix metalloproteinases (MMPs), membrane type 1 MMP (MT1-MMP), in synovial pannus invasiveness. METHODS The expression and localization of MT1-MMP in human RA pannus were investigated by Western blot analysis of primary synovial cells and immunohistochemical analysis of RA joint specimens. The functional role of MT1-MMP was analyzed by 3-dimensional (3-D) collagen invasion assays and a cartilage invasion assay in the presence or absence of tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, or GM6001. The effect of adenoviral expression of a dominant-negative MT1-MMP construct lacking a catalytic domain was also examined. RESULTS MT1-MMP was highly expressed at the pannus-cartilage junction in RA joints. Freshly isolated rheumatoid synovial tissue and isolated RA synovial fibroblasts invaded into a 3-D collagen matrix in an MT1-MMP-dependent manner. Invasion was blocked by TIMP-2 and GM6001 but not by TIMP-1. Invasion was also inhibited by the overexpression of a dominant-negative MT1-MMP, which inhibits collagenolytic activity and proMMP-2 activation by MT1-MMP on the cell surface. Synovial fibroblasts also invaded into cartilage in an MT1-MMP-dependent manner. This process was further enhanced by removing aggrecan from the cartilage matrix. CONCLUSION MT1-MMP serves as an essential collagen-degrading proteinase during pannus invasion in human RA. Specific inhibition of MT1-MMP-dependent invasion may represent a novel therapeutic strategy for RA.

A trypanosome oligopeptidase as a target for the trypanocidal agents pentamidine, diminazene and suramin.

African trypanosomes contain a cytosolic serine oligopeptidase, called OP‐Tb, that is reversibly inhibited by the active principles of three of the five most commonly used trypanocidal drugs: pentamidine, diminazene and suramin. OP‐Tb was inhibited by pentamidine in a competitive manner, and by suramin in a partial, non‐competitive manner. The inhibition of OP‐Tb by a variety of suramin analogues correlated with the trypanocidal efficacy of these analogues (P = 0.03; by paired Students t‐test). Since intracellular (therapeutic) concentrations of pentamidine and suramin are reported to reach approximately 206K i and 15K i respectively, we suggest that these drugs may exert part of their trypanocidal activity through the inhibition of OP‐Tb.

Functional Differences of the Catalytic and Non-catalytic Domains in Human ADAMTS-4 and ADAMTS-5 in Aggrecanolytic Activity

ADAMTS-4 (aggrecanase-1) and ADAMTS-5 (aggrecanase-2) are multidomain metalloproteinases belonging to the ADAMTS family. We have previously reported that human ADAMTS-5 has much higher aggrecanolytic activity than human ADAMTS-4. To investigate the different proteolytic activity of the two enzymes, we generated a series of chimeras by exchanging various non-catalytic domains of the two proteinases. We found that the catalytic domain of ADAMTS-5 has higher intrinsic catalytic ability than that of ADAMTS-4. The studies also demonstrated that the non-catalytic domains of ADAMTS-5 are more effective modifiers than those of ADAMTS-4, making both catalytic domains more active against aggrecan, an Escherichia coli-expressed interglobular domain of aggrecan and fibromodulin. Addition of the C-terminal thrombospondin type I motif of ADAMTS-5 to the C terminus of ADAMTS-4 increased the activity of ADAMTS-4 against aggrecan and fibromodulin severalfold. In contrast to previous reports (Kashiwagi, M., Enghild, J. J., Gendron, C., Hughes, C., Caterson, B., Itoh, Y., and Nagase, H. (2004) J. Biol. Chem. 279, 10109–10119 and Gao, G., Plaas, A., Thompson, V. P., Jin, S., Zuo, F., and Sandy, J. D. (2004) J. Biol. Chem. 279, 10042–10051), our detailed investigation of the role of the C-terminal spacer domain of ADAMTS-4 indicated that full-length ADAMTS-4 is ∼20-times more active against aggrecan than its spacer domain deletion mutant, even at the Glu373-Ala374 site of the interglobular domain. This discrepancy is most likely due to selective inhibition of full-length ADAMTS-4 by heparin, particularly for cleavage at the Glu373-Ala374 bond. However, removal of the spacer domain from ADAMTS-4 greatly enhanced more general proteolytic activity against non-aggrecan substrates, e.g. E. coli-expressed interglobular domain, fibromodulin, and carboxymethylated transferrin.

Extracellular regulation of metalloproteinases

Matrix metalloproteinases (MMPs) and adamalysin-like metalloproteinase with thrombospondin motifs (ADAMTSs) belong to the metzincin superfamily of metalloproteinases and they play key roles in extracellular matrix catabolism, activation and inactivation of cytokines, chemokines, growth factors, and other proteinases at the cell surface and within the extracellular matrix. Their activities are tightly regulated in a number of ways, such as transcriptional regulation, proteolytic activation and interaction with tissue inhibitors of metalloproteinases (TIMPs). Here, we highlight recent studies that have illustrated novel mechanisms regulating the extracellular activity of these enzymes. These include allosteric activation of metalloproteinases by molecules that bind outside the active site, modulation of location and activity by interaction with cell surface and extracellular matrix molecules, and endocytic clearance from the extracellular milieu by low-density lipoprotein receptor-related protein 1 (LRP1).

Chalcone, acyl hydrazide, and related amides kill cultured Trypanosoma brucei brucei.

BackgroundProtozoan parasites of the genus Trypanosoma cause disease in a wide range of mammalian hosts. Trypanosoma brucei brucei, transmitted by tsetse fly to cattle, causes a disease (Nagana) of great economic importance in parts of Africa. T. b. brucei also serves as a model for related Trypanosoma species, which cause human sleeping sickness.Materials and MethodsChalcone and acyl hydrazide derivatives are known to retard the growth of Plasmodium falciparum in vitro and inhibit the malarial cysteine proteinase, falcipain. We tested the effects of these compounds on the growth of bloodstream forms of T. b. brucei in cell culture and in a murine trypanosomiasis model, and investigated their ability to inhibit trypanopain-Tb, the major cysteine proteinase of T. b. brucei.ResultsSeveral related chalcones, acyl hydrazides, and amides killed cultured bloodstream forms of T. b. brucei, with the most effective compound reducing parasite numbers by 50% relative to control populations at a concentration of 240 nM. The most effective inhibitors protected mice from an otherwise lethal T. b. brucei infection in an in vivo model of acute parasite infection. Many of the compounds also inhibited trypanopain-Tb, with the most effective inhibitor having a Ki value of 27 nM. Ki values for trypanopain-Tb inhibition were up to 50- to 100-fold lower than for inhibition of mammalian cathepsin L, suggesting the possibility of selective inhibition of the parasite enzyme.ConclusionsChalcones, acyl hydrazides, and amides show promise as antitrypanosomal chemotherapeutic agents, with trypanopain-Tb possibly being one of their in vivo targets.

Characterisation of the antitrypanosomal activity of peptidyl alpha-aminoalkyl phosphonate diphenyl esters.

Two groups of irreversible serine peptidase inhibitors, peptidyl chloromethyl ketones and peptidyl phosphonate diphenyl esters, were examined for antitrypanosomal activity against the bloodstream form of Trypanosoma brucei brucei. Both peptidyl chloromethyl ketones and peptidyl phosphonate diphenyl esters inhibited trypsin-like peptidases of the parasites and exhibited antitrypanosomal activity at micromolar concentrations. In live T. b. brucei, labelled analogues of both of these groups of inhibitors primarily targeted an 80-kDa peptidase, possibly a serine oligopeptidase known as oligopeptidase B. In an in vivo mouse model of infection, one of these inhibitors, carbobenzyloxyglycyl-4-amidinophenylglycine phosphonate diphenyl ester, was curative at 5 mg kg(-1) day(-1) but appeared toxic at higher doses. There was no significant correlation between the inhibitory potency (as evaluated against purified T. b. brucei oligopeptidase B) and the in vitro antitrypanosomal efficacy of either group of inhibitors, suggesting that these inhibitors were acting on multiple targets within the parasites, or had different cell permeability properties. These findings suggest that serine peptidases may represent novel chemotherapeutic targets in African trypanosomes.

LRP-1-mediated endocytosis regulates extracellular activity of ADAMTS-5 in articular cartilage

Aggrecan is a major matrix component of articular cartilage, and its degradation is a crucial event in the development of osteoarthritis (OA). Adamalysin‐like metalloproteinase with thrombospondin motifs 5 (ADAMTS‐5) is a major aggrecan‐degrading enzyme in cartilage, but there is no clear correlation between ADAMTS‐5 mRNA levels and OA progression. Here, we report that post‐translational endocytosis of ADAMTS‐5 by chondrocytes regulates its extracellular activity. We found 2‐ to 3‐fold reduced aggrecanase activity when ADAMTS‐5 was incubated with live porcine cartilage, resulting from its rapid endocytic clearance. Studies using receptor‐associated protein (RAP), a ligand‐binding antagonist for the low‐density lipoprotein receptor‐related proteins (LRPs), and siRNA‐mediated gene silencing revealed that the receptor responsible for ADAMTS‐5 clearance is LRP‐1. Domain‐deletion mutagenesis of ADAMTS‐5 identified that the noncatalytic first thrombospondin and spacer domains mediate its endocytosis. The addition of RAP to porcine cartilage explants in culture increased the basal level of aggrecan degradation, as well as ADAMTS‐5‐induced aggrecan degradation. Notably, LRP‐1‐mediated endocytosis of ADAMTS‐5 is impaired in chondrocytes of OA cartilage, with ~90% reduction in protein levels of LRP‐1 without changes in its mRNA levels. Thus, LRP‐1 dictates physiological and pathological catabolism of aggrecan in cartilage as a key modulator of the extracellular activity of ADAMTS‐5.—Yamamoto, K., Troeberg, L., Scilabra, S. D., Pelosi, M., Murphy, C. L., Strickland, D. K., Nagase, H. LRP‐1‐mediated endocytosis regulates extracellular activity of ADAMTS‐5 in articular cartilage. FASEB J. 27, 511–521 (2013). www.fasebj.org