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Biomedical subjects

E C Arner

Publications and source records attributed to E C Arner.

At least 19 recordsLinked to original sources
PubMed2001

Design and synthesis of a series of (2R)-N(4)-hydroxy-2-(3-hydroxybenzyl)-N(1)- [(1S,2R)-2-hydroxy-2,3-dihydro-1H-inden-1-yl]butanediamide derivatives as potent, selective, and orally bioavailable aggrecanase inhibitors.

A pharmacophore model of the P1' site, specific for aggrecanase, was defined using the specificity studies of the matrix metalloproteinases and the similar biological activity of aggrecanase and MMP-8. Incorporation of the side chain of a tyrosine residue into compound 1 as the P1' group provided modest selectivity for aggrecanase over MMP-1, -2, and -9. A cis-(1S)(2R)-amino-2-indanol scaffold was incorporated as a tyrosine mimic (P2') to conformationally constrain 2. Further optimization resulted in compound 11, a potent, selective, and orally bioavailable inhibitor of aggrecanase.

Administration, Oral↗
PubMed2001

Host metalloproteinases in Lyme arthritis.

OBJECTIVE: To assess the role of matrix metalloproteinases (MMPs) in cartilage and bone erosions in Lyme arthritis METHODS: We examined synovial fluid from 10 patients with Lyme arthritis for the presence of MMP-2, MMP-3, MMP-9, and "aggrecanase" activity using gelatinolytic zymography and immunoblot analysis. We developed an in vitro model of Lyme arthritis using cartilage explants and observed changes in cartilage degradation in the presence of Borrelia burgdorferi and/or various protease inhibitors. RESULTS: Synovial fluid from patients with Lyme arthritis was found to contain at least 3 MMPs: gelatinase A (MMP-2), stromelysin (MMP-3), and gelatinase B (MMP-9). In addition, there was evidence in 2 patients of "aggrecanase" activity not accounted for by the above enzymes. Infection of cartilage explants with B. burgdorferi resulted in induction of MMP-3, MMP-9, and "aggrecanase" activity. Increased induction of these enzymes by B. burgdorferi alone was not sufficient to cause cartilage destruction in the explants as measured by glycosaminoglycan (GAG) and hydroxyproline release. However, addition of plasminogen, which can act as an MMP activator, to cultures resulted in significant GAG and hydroxyproline release in the presence of B. burgdorferi. The MMP inhibitor batimastat significantly reduced the GAG release and completely inhibited the collagen degradation. CONCLUSION: MMPs are found in synovial fluids from patients with Lyme arthritis and are induced from cartilage tissue by the presence of B. burgdorferi. Inhibition of MMP activity prevents B. burgdorferi-induced cartilage degradation in vitro.

Animals↗
PubMed2000

Age-related changes in aggrecan glycosylation affect cleavage by aggrecanase.

Aggrecan degradation involves proteolytic cleavage of the core protein within the interglobular domain. Because aggrecan is highly glycosylated with chondroitin sulfate (CS) and keratan sulfate (KS), we investigated whether glycosylation affects digestion by aggrecanase at the Glu(373)-Ala(374) bond. Treatment of bovine aggrecan monomers to remove CS and KS resulted in loss of cleavage at this site, suggesting that glycosaminoglycans (GAGs) play a role in cleavage at the Glu(373)-Ala(374) bond. In contrast, MMP-3 cleavage at the Ser(341)-Phe(342) bond was not affected by glycosidase treatment of aggrecan. Removal of KS, but not CS, prevented cleavage at the Glu(373)-Ala(374) bond. Thus, KS residues may be important for recognition of this cleavage site by aggrecanase. KS glycosylation has been observed at sites adjacent to the Glu(373)-Ala(374) bond in steer aggrecan, but not in calf aggrecan (Barry, F. P., Rosenberg, L. C., Gaw, J. U., Gaw, J. U., Koob, T. J., and Neame, P. J. (1995) J. Biol. Chem. 270, 20516-20524). Interestingly, although we found that aggrecanase degraded both calf and steer cartilage aggrecan, the proportion of fragments generated by cleavage at the Glu(373)-Ala(374) bond was higher in steer than in calf, consistent with our observations using aggrecan treated to remove KS. We conclude that the GAG content of aggrecan influences the specificity of aggrecanase for cleavage at the Glu(373)-Ala(374) bond and suggest that age may be a factor in aggrecanase degradation of cartilage.

Age Factors↗
PubMed2000

Chondrocyte extracellular matrix synthesis and turnover are influenced by static compression in a new alginate disk culture system.

The goal of this study was to examine the effects of mechanical compression on chondrocyte biosynthesis of extracellular matrix (ECM) components during culture in a new alginate disk culture system. Specifically, we have examined chondrocyte biosynthesis rates, and the structure of aggrecan core protein species present in the cell-associated matrix (CM), in the further removed matrix (FRM) and in the surrounding culture medium. In this alginate disk culture system, chondrocytes can be subjected to mechanical deformations similar to those experienced in vivo. Our results show that over an 8-week culture period, chondrocytes synthesize a functional ECM and can respond to mechanical forces similarly to chondrocytes maintained in native cartilage. In the alginate disk system, static compression was shown to decrease and dynamic compression to increase synthesis of aggrecan of bovine chondrocytes. Western blot analysis of the core proteins of aggrecan molecules identified a number of different species that were present in different relative amounts in the CM, FRM, and medium. Over 21 days of culture, the predominant form of aggrecan found in the ECM was a full-length link-stabilized species. In addition, our data show that the application of 40 h of static compression caused an increase in the proportion of newly synthesized aggrecan molecules released into the medium. However, this was not accompanied by a significant change in the size and composition of aggrecan and aggrecan fragments in the different compartments, suggesting that mechanical compression did not alter the catabolic pathways. Together, these data show that chondrocyte function is maintained in an alginate disk culture system and that this culture system is a useful model to examine chondrocyte ECM assembly and some aspects of catabolism normally found in vivo.

Aggrecans↗
PubMed2000

Brain-enriched hyaluronan binding (BEHAB)/brevican cleavage in a glioma cell line is mediated by a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family member.

Brain-enriched hyaluronan binding (BEHAB)/brevican is a brain-specific extracellular matrix protein containing a cleavage site between Glu(395)-Ser(396), which bears remarkable homology to the "aggrecanase" site in the cartilage proteoglycan aggrecan. Expression of BEHAB/brevican is dramatically increased in human gliomas, notoriously invasive tumors. Recently, we showed that the rat 9L gliosarcoma cell line, which does not express BEHAB/brevican and forms non-invasive tumors when grown as intracranial grafts, can form invasive tumors when transfected with a 5' cDNA fragment of BEHAB/brevican, but not when transfected with the full-length cDNA. In marked contrast, the highly invasive CNS-1 glioma cell line expresses and cleaves BEHAB/brevican protein when grown as an intracranial graft. These results suggest that both synthesis and cleavage of BEHAB/brevican protein may play a role in the invasiveness of gliomas. We report here, using an antibody developed to the neoepitope created by BEHAB/brevican cleavage at the Glu(395)-Ser(396) site, that the CNS-1 cells are able to cleave the protein in vitro. We characterized the CNS-1-derived cleavage activity by assaying its ability to cleave BEHAB/brevican proteoglycan, and determined that the enzyme is a constitutively expressed, secreted activity. Using a variety of protease inhibitors, reverse transcriptase-polymerase chain reaction, and specific antibodies, we determined that this activity is likely to be a member of the ADAMTS family of metalloproteinases, specifically ADAMTS4. These results suggest a novel function for ADAMTS family members in BEHAB/brevican cleavage and glioma and indicate that inhibition of ADAMTS in glioma may provide a novel therapeutic strategy.

ADAM Proteins↗
PubMed1999

Recombinant human aggrecan G1-G2 exhibits native binding properties and substrate specificity for matrix metalloproteinases and aggrecanase.

A recombinant human aggrecan G1-G2 fragment comprising amino acids Val(1)-Arg(656) has been expressed in Sf21 cells using a baculovirus expression system. The recombinant G1-G2 (rG1-G2) was purified to homogeneity by hyaluronan-Sepharose affinity chromatography followed by high performance liquid chromatography gel filtration, and gave a single band of M(r) 90,000-95,000 by silver stain or immunoblotting with monoclonal antibody 1-C-6. The expressed G1-G2 bound to both hyaluronan and link protein indicating that the immunoglobulin-fold motif and proteoglycan tandem repeat loops of the G1 domain were correctly folded. Further analysis of secondary structure by rotary shadowing electron microscopy confirmed a double globe appearance, but revealed that the rG1-G2 was more compact than its native counterpart. The size of rG1-G2 by SDS-polyacrylamide gel electorphoresis was unchanged following digestion with keratanase and keratanase II and reduced by only 2-5 kDa following digestion with either O-glycosidase or N-glycosidase F. Recombinant G1-G2 was digested with purified matrix metalloproteinases (MMP), isolated aggrecanase, purified atrolysin C, or proteinases present in conditioned medium from cartilage explant cultures, and the products analyzed on SDS gels by silver stain and immunoblotting. Neoepitope antibodies recognizing the N-terminal F(342)FGVG or C-terminal DIPEN(341) sequences were used to confirm MMP cleavage at the Asn(341) downward arrow Phe bond, while neoepitope antibodies recognizing the N-terminal A(374)RGSV or C-terminal ITEGE(373) sequences were used to confirm aggrecanase cleavage at the Glu(373) downward arrow Ala bond. Cleavage at the authentic MMP and aggrecanase sites revealed that these proteinases have the same specificity for rG1-G2 as for native aggrecan. Incubation of rG1-G2 with conditioned medium from porcine cartilage cultures revealed that active soluble aggrecanase but no active MMPs, was released following stimulation with interleukin-1alpha or retinoic acid. Atrolysin C, which cleaves native bovine aggrecan at both the aggrecanase and MMP sites, efficiently cleaved rG1-G2 at the aggrecanase site but failed to cleave at the MMP site. In contrast, native glycosylated G1-G2 with or without keratanase treatment was cleaved by atrolysin C at both the aggrecanase and MMP sites. The results suggest that the presence or absence per se of keratan sulfate on native G1-G2 does not affect the activity of atrolysin C toward the two sites.

Aggrecans↗
PubMed1999

Aggrecanase. A target for the design of inhibitors of cartilage degradation.

In arthritic diseases there is a gradual erosion of cartilage that leads to a loss of joint function. Aggrecan, which provides cartilage with its properties of compressibility and elasticity, is the first matrix component to undergo measurable loss in arthritis. This loss of aggrecan appears to be due to an increased rate of degradation, that can be attributed to proteolytic cleavage of the core protein within the interglobular domain (IGD). Two major sites of cleavage have been identified within the IGD. One, between the amino acids Asn341-Phe342, where the matrix metalloproteinases (MMPs) have been shown to clip; and the other, between Glu373-Ala374, which is attributed to a novel protease, "aggrecanase." We have generated aggrecanase in conditioned media from IL-1-stimulated bovine nasal cartilage and have used an enzymatic assay to evaluate this proteinase activity. In these studies we follow the generation of aggrecanase and MMPs in response to IL-1 in this system and examine the contribution of these enzymes in aggrecan degredation. Our data suggest that aggrecanase is a key enzyme in cartilage aggrecan degradation that represents a novel target for cartilage protection therapy in arthritis.

Animals↗
PubMed1999

Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins.

We purified, cloned, and expressed aggrecanase, a protease that is thought to be responsible for the degradation of cartilage aggrecan in arthritic diseases. Aggrecanase-1 [a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4)] is a member of the ADAMTS protein family that cleaves aggrecan at the glutamic acid-373-alanine-374 bond. The identification of this protease provides a specific target for the development of therapeutics to prevent cartilage degradation in arthritis.

ADAM Proteins↗
PubMed1999

P1, P2'-linked macrocyclic amine derivatives as matrix metalloproteinase inhibitors.

A novel series of 13- and 14-membered macrocyclic amines was developed by linking the P1 and P2' groups. The synthesis entails stereoselective Frater alkylation to install the anti-succinate configuration and macrocyclic amination via nucleophilic displacement. This strategy resulted in a new class of conformationally constrained inhibitors that are potent and selective for MMP-8 and 9 over MMP-1 and 3.

Amines↗
PubMed1999

Macrocyclic hydroxamate inhibitors of matrix metalloproteinases and TNF-alpha production.

Several macrocyclic, hydroxamate derivatives were synthesized and evaluated as inhibitors of matrix metalloproteinases (MMPs) and tumour necrosis factor-alpha (TNF-alpha) production. These macrocycles are anti-succinate based inhibitors linked from P1 to P2'. A variety of functionality was installed at the P1-P2' linkage, which gave inhibitors that displayed excellent MMP inhibition and good TNF-alpha suppression.

Crystallography, X-Ray↗
PubMed1999

Generation and characterization of aggrecanase. A soluble, cartilage-derived aggrecan-degrading activity.

A method was developed for generating soluble, active "aggrecanase" in conditioned media from interleukin-1-stimulated bovine nasal cartilage cultures. Using bovine nasal cartilage conditioned media as a source of the aggrecanase enzyme, an enzymatic assay was established employing purified aggrecan monomers as a substrate and monitoring specific aggrecanase-mediated cleavage products by Western analysis using the monoclonal antibody, BC-3 (which recognizes the new N terminus, ARGS, on fragments produced by cleavage between amino acid residues Glu373 and Ala374). Using this assay we have characterized cartilage aggrecanase with respect to assay kinetics, pH and salt optima, heat sensitivity, and stability upon storage. Aggrecanase activity was inhibited by the metalloprotease inhibitor, EDTA, while a panel of inhibitors of serine, cysteine, and aspartic proteinases had no effect, suggesting that aggrecanase is a metalloproteinase. Sensitivity to known matrix metalloproteinase inhibitors as well as to the endogenous tissue inhibitor of metalloproteinases, TIMP-1, further support the notion that aggrecanase is a metalloproteinase potentially related to the ADAM family or MMP family of proteases previously implicated in the catabolism of the extracellular matrix.

Animals↗
PubMed1998

The interglobular domain of cartilage aggrecan is cleaved by hemorrhagic metalloproteinase HT-d (atrolysin C) at the matrix metalloproteinase and aggrecanase sites.

Two primary cleavage sites have been identified within the interglobular domain of the cartilage aggrecan core protein: one is between amino acid residues Asn 341 and Phe342, where many matrix metalloproteinases (MMP) have been shown to cleave; and the other is between amino acid residues Glu373 and Ala374. Although cleavage at the Glu373-Ala374 site is believed to play a critical role in cartilage aggrecan degradation in arthritic diseases, the enzyme responsible for cleavage at this site, "aggrecanase," has not been identified. Members of the ADAM (a disintegrin and metalloproteinase) family of proteins, which shows structural homology to the snake venom hemorrhagic metalloproteinases (reprolysins), have recently been demonstrated to be expressed in articular chondrocytes. Because many ADAM family members have a putative proteinase function, this raises the possibility that aggrecanase may be a member of this family of proteases. To examine whether reprolysins have the ability to cleave aggrecan at either the aggrecanase site or the MMP site, the snake venom hemorrhagic toxin metalloproteinase HT-d (atrolysin C) was tested for its ability to cleave bovine aggrecan monomer. Cleavage was monitored using the BC-3 antibody, which recognizes aggrecan fragments with the new NH2 terminus ARGSV generated by cleavage at the aggrecanase site, and with the AF-28 antibody, which recognizes aggrecan fragments with the new NH2 terminus FFGVG generated by cleavage at the MMP site. Cleavage at both the aggrecanase and MMP sites occurred in a concentration-dependent manner with 100 nM atrolysin C or greater. AF-28-reactive fragments were generated by 30 min of incubation, and levels were maximal by 8 h; BC-3-reactive fragments were detected at 2 h and continued to increase through 48 h, thus suggesting that atrolysin C can cleave at the MMP and aggrecanase sites. NH2-terminal aggrecan fragments generated by cleavage at the aggrecanase site were also detected using antisera recognizing the new COOH terminus, NITEGE, formed by cleavage at the Glu373-Ala374 bond, indicating that cleavage at this site does not require prior cleavage at the MMP site. These data provide the first demonstration that a reprolysin can cleave the core protein of aggrecan and the first example of a specific protease that can cleave at the aggrecanase site independent of cleavage at the MMP cleavage site.

Aggrecans↗
PubMed1998

Effect of ebselen on IL-1-induced alterations in cartilage metabolism.

OBJECTIVE: To evaluate the effect of the antioxidant-like anti-inflammatory agent, ebselen, on cartilage proteoglycan degradation and to determine whether its cartilage protectant activity is related to its antioxidant activity. MATERIALS AND METHODS: Cartilage in organ culture was stimulated with interleukin-1 (IL-1), and proteoglycan degradation was assessed by measuring the amount of sulfated glycosaminoglycan released into the media, proteoglycan synthesis evaluated by [35S]-sulfate incorporation, and prostaglandin E2 (PGE2) release determined by radioimmunoassay (RIA). Glutathione peroxidase (GSH-Px) activity was evaluated in a coupled test system using NADPH/GSSG reductase as an indicator and cyclooxygenase activity was evaluated using sheep seminal vesicle prostaglandin synthase. RESULTS: Ebselen caused a concentration-dependent inhibition of IL-1-stimulated proteoglycan degradation with an IC50 of 4.7 microM. Cartilage PGE2 release was also reduced in the presence of ebselen (IC50 = 6.2 microM). However, at concentrations up to 100 microM, ebselen had no effect on the inhibition of proteoglycan synthesis by IL-1. Induction of proteoglycan breakdown was also inhibited by a sulfur analog of ebselen. This analog was devoid of GSH-Px activity and was 50-fold less potent in cyclooxygenase inhibitory activity, but was equipotent to ebselen in inhibiting cartilage degradation. CONCLUSIONS: Ebselen, unlike other NSAIDs, blocks cartilage proteoglycan breakdown without inhibiting proteoglycan synthesis. This effect is independent of its GSH-Px activity and its ability to inhibit cyclooxygenase and PGE2 production. Therefore, this compound may provide a new mechanism for protecting cartilage matrix from degradative factors in arthritic joints.

Animals↗
PubMed1998

Cytokine-induced cartilage proteoglycan degradation is mediated by aggrecanase.

OBJECTIVE: To evaluate the relationship between specific cleavage of aggrecan at the Glu373-Ala374 'aggrecanase' site and degradation and release of proteoglycan catabolites from cartilage in explant cultures. DESIGN: The monoclonal antibody, BC-3, which specifically recognizes the new N-terminus, ARGSVIL, generated by cleavage of aggrecan at the Glu373-Ala374 'aggrecanase' site, was used to follow the generation of fragments produced by cleavage at this site as compared to degradation of proteoglycan as assessed by glycosaminoglycan (GAG) release from cartilage in response to cytokines and the ability of inhibitors to block this cleavage. RESULTS: (1) There was a strong correlation between specific cleavage at the Glu373-Ala374 bond and the release of aggrecan catabolites in response to interleukin-1 (IL-1) or tumour necrosis factor (TNF) stimulation. (2) This cleavage in the interglobular domain of aggrecan was inhibited by the inclusion of cycloheximide, thus indicating a requirement for de novo protein synthesis in the induction of 'aggrecanase' activity. (3) The inhibitors, indomethacin, naproxen, tenidap, dexamethasone and doxycycline were ineffective in blocking either specific cleavage at the 'aggrecanase' site or aggrecan degradation as measured by GAG release from cartilage. (4) In contrast, compounds which act through two different mechanisms to inhibit MMPs were effective in blocking both specific cleavage at the 'aggrecanase' site and proteoglycan degradation. CONCLUSIONS: Our data suggest that 'aggrecanase' is primarily responsible for proteoglycan cleavage in these experimental systems and that this protease has properties in common with metalloproteases including members of the MMP and ADAM family. Inhibition of 'aggrecanase' may have utility in preventing cartilage loss in arthritis.

Aggrecans↗
PubMed1997

Cleavage of native cartilage aggrecan by neutrophil collagenase (MMP-8) is distinct from endogenous cleavage by aggrecanase.

Cleavage of aggrecan core protein at the Glu373-Ala374 site by the unidentified enzyme, "aggrecanase," is thought to play an important role in cartilage degradation. To examine aggrecan cleavage by MMP-8 at this aggrecanase site, we evaluated the release of fragments with the N terminus ARGSVIL from freeze-thawed bovine nasal cartilage using the monoclonal antibody BC-3. Recombinant human MMP-8 catalytic domain cleaved native aggrecan in a concentration-related manner between 0.2 and 2 microg/ml, with complete release of glycosaminoglycan at 2 microg/ml or greater. Cleavage at the aggrecanase site was observed only at MMP-8 concentrations resulting in complete release of glycosaminoglycan from the cartilage, suggesting that preferential cleavage occurs at a different site. Time course studies indicated that only following depletion of substrate containing the preferred clip site did MMP-8 rapidly cleave at the aggrecanase site. Finally, MMP-8 resulted in a different pattern of BC-3-reactive fragments from that produced by endogenous aggrecanase in live cartilage, and SA751(N-(1(R)-carboxyethyl) -alpha-(S)-(4-phenyl-3-butynyl)glycyl-L-O-methyltyrosine, N-methylamide), a potent inhibitor of MMP-8 (Ki = 2 nM) which was effective in blocking cleavage by MMP-8 at the aggrecanase site with an IC50 in the nanomolar range, did not prevent aggrecan degradation or specific cleavage at this site by endogenously generated aggrecanase at concentrations up to 100 microM. Taken together these data suggest that MMP-8 does not represent cartilage aggrecanase.

Aggrecans↗
PubMed1997

The effects of IL-1 on mitogen-activated protein kinases in rabbit articular chondrocytes.

IL-1-activated chondrocytes express a large number of genes which contribute to cartilage degradation. The signaling pathways activated in response to IL-1 in these cells are not well-defined. We examined the effects of IL-1 and other stimuli on the mitogen activated protein kinase (MAPK) pathways in rabbit articular chondrocytes. We demonstrate that IL-1 activates three MAPKs, ERK, JNK and p38, in a time and dose-dependent manner. Activation is maximal by 15 minutes and returns to baseline levels by 1 hour. Maximal activation of ERK and p38 occurs with 1 ng/ml IL-1 whereas activation of JNK requires 10-fold higher levels. In contrast to IL-1, the PKC activator, PDBu preferentially activates ERK while TNF alpha preferentially activates JNK. LPS and TGF beta fail to stimulate any of the kinases examined. These results suggest that activation of the various MAPK pathways is important in the response of chondrocytes to IL-1, cytokines and growth factors.

Animals↗