Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It has been suggested that increased collagenase-3 (MMP-13) activity plays a pivotal role in the pathogenesis of osteoarthritis (OA). We have used tetracycline-regulated transcription in conjunction with a cartilage-specific promoter to target a constitutively active human MMP-13 to the hyaline cartilages and joints of transgenic mice. Postnatal expression of this transgene resulted in pathological changes in articular cartilage of the mouse joints similar to those observed in human OA. These included characteristic erosion of the articular cartilage associated with loss of proteoglycan and excessive cleavage of type II collagen by collagenase, as well as synovial hyperplasia. These results demonstrate that excessive MMP-13 activity can result in articular cartilage degradation and joint pathology of the kind observed in OA, suggesting that excessive activity of this proteinase can lead to this disease.
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PMID:Postnatal expression in hyaline cartilage of constitutively active human collagenase-3 (MMP-13) induces osteoarthritis in mice. 1113 76

We have developed monoclonal antibody 5109 against a unique highly acidic sequence in type II collagen. When paired with previously reported monoclonal antibody 9A4, 5109 can be used as the capture antibody in an ELISA assay for the neoepitope generated by collagenase-cleavage of type II collagen. The assay detects the sequence ZGlyGluX(759)GlyAspAspGlyProSerGlyAlaGluGlyProX(771)GlyProGlnGly(775) where Z is a variable length polypeptide, X is proline or hydroxyproline, and Gly(775) corresponds to C-terminal amino acid of the 3/4 piece after collagenase cleavage. Antibody 5109 detects the first and 9A4 the second underlined sequence. Antibody 5109 recognizes its epitope with a K=1.2x10(-8) M independently of hydroxylation of X(759). When X(771) is proline, the sequence is 90x more sensitively detected by this ELISA than when it is hydroxyproline. Type II collagen of human articular cartilage was fragmented by cyanogen bromide (CNBr) and trypsin. The immunoreactive fragment was captured with 5109 and sequenced. Proline(771) averaged 81% hydroxylated. Other 3rd position prolines were >97% hydroxylated. In urine of control individuals of 50-70 years of age, we failed to detect the presence of the collagen fragment in a majority (8/10) of specimens. The two controls with measurable levels averaged 123 pM. In a similar age cohort of osteoarthritic patients, the majority (9/10) showed measurable values of urinary collagen fragments averaging 312 pM. This assay can be used for monitoring type II collagen metabolism in patients with osteoarthritis.
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PMID:Analysis of collagenase-cleavage of type II collagen using a neoepitope ELISA. 1115 May 34

Chondrocytes assemble an extracellular matrix in which the relative composition of type IX versus type II collagen and aggrecan changes during assembly. On maturation and differentiation into hypertrophic cells type IX collagen first loses the NC4 globular domain of the alpha 1(IX) chain that protrudes from the collagen fibril. Subsequently, collagenase 3 (matrix metalloproteinase 13; MMP13) is up-regulated as type X collagen is expressed leading to extensive cleavage and removal of type II collagen and of the remaining COL2 domain of type IX collagen alpha 1(IX) chain. The proteoglycan aggrecan is selectively retained in the extracellular matrix. Inhibition of collagenase leads to arrest of hypertrophy as well as gene expression of MMP13. Thus proteolysis and in particular MMP13 are required for chondrocyte differentiation and for matrix resorption in skeletal development.
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PMID:Cartilage matrix resorption in skeletogenesis. 1127 78

Matrix metalloproteinase (MMP) family members are involved in the physiological remodeling of tissues and embryonic development as well as pathological destruction of extracellular matrix components. To study the mechanisms of MMP action on collagenous substrates, we have constructed homotrimeric, fluorogenic triple-helical peptide (THP) models of the MMP-1 cleavage site in type II collagen. The substrates were designed to incorporate the fluorophore/quencher pair of (7-methoxycoumarin-4-yl)acetyl (Mca) and N-2,4-dinitrophenyl (Dnp) in the P(5) and P(5)' positions, respectively. In addition, Arg was incorporated in the P(2)' and P(8)' positions to enhance enzyme activity and improve substrate solubility. The desired sequences were Gly-Pro-Lys(Mca)-Gly-Pro-Gln-Gly approximately Leu-Arg-Gly-Gln-Lys(Dnp)-Gly-Ile/Val-Arg. Two fluorogenic substrates were prepared, one using a covalent branching protocol (fTHP-1) and one using a peptide self-assembly approach (fTHP-3). An analogous single-stranded substrate (fSSP-3) was also synthesized. Both THPs were hydrolyzed by MMP-1 at the Gly approximately Leu bond, analogous to the bond cleaved in the native collagen. The individual kinetic parameters for MMP-1 hydrolysis of fTHP-3 were k(cat) = 0.080 s(-1) and K(M) = 61.2 microM. Subsequent investigations showed fTHP-3 hydrolysis by MMP-2, MMP-3, MMP-13, a C-terminal domain-deleted MMP-1 [MMP-1(Delta(243-450))], and a C-terminal domain-deleted MMP-3 [MMP-3(Delta(248-460))]. The order of k(cat)/K(M) values was MMP-13 > MMP-1 approximately MMP-1(Delta(243-450)) approximately MMP-2 >> MMP-3 approximately MMP-3(Delta(248-460)). Studies on the effect of temperature on fTHP-3 and fSSP-3 hydrolysis by MMP-1 showed that the activation energies between these two substrates differed by 3.4-fold, similar to the difference in activation energies for MMP-1 hydrolysis of type I collagen and gelatin. This indicates that fluorogenic triple-helical substrates mimic the behavior of the native collagen substrate and may be useful for the investigation of collagenase triple-helical activity.
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PMID:Kinetic analysis of matrix metalloproteinase activity using fluorogenic triple-helical substrates. 1134 45

Hyaluronan (HA) is a component of cartilage matrix with known effects on chondrocytes. We tested the effects of adding HA to 3-dimensional (3-D) collagen. sponges on chondrocyte function in vitro. Bovine articular chondrocytes isolated by collagenase digestion were injected into either collagen or HA/collagen scaffolds comprising different amounts of HA (2, 5, 10, and 14% w/w). Expression of aggrecan and type II collagen genes was measured by gene-specific quantitative competitive reverse transcriptase-polymerase chain reactions, and the extracellular matrix was estimated by histomorphometrical analyses. After 7-day culture, the chondrocytes in 2% (w/w) HA sponges expressed fourfold more mRNA transcripts for type II collagen (p = 0.002) and twofold more mRNA transcripts for aggrecan (p = 0.022) than in control collagen sponges. Furthermore, there was 45% more extracellular matrix in 2% (w/w) HA sponges and 43% less matrix in the 10% (w/w) HA sponges compared with plain collagen sponges (p > 0.05). In sum, a small amount of HA in 3-D collagen scaffolds enhanced chondrogenesis, but a greater amount was inhibitory. This 3-D system represents a novel tool to identify mechanisms by which extracellular matrix molecules influence chondrocyte function. Further, these results show the potential for modifying scaffolds to improve production of engineered cartilage for in vivo applications.
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PMID:Effects of hyaluronan on engineered articular cartilage extracellular matrix gene expression in 3-dimensional collagen scaffolds. 1142 90

To investigate the behavoir of TMJ condylar cartilage cells in vitro, the mandibular condylar cartilage cells were harvested from a 5-month-old human fetus by dissection and sequential digestion with 0.25% trypsin and 0.2% collagenase (type II). The isolated cells were cultured in DMEM medium and identified by histochemical and immunohistochemical methods. Cell proliferation, morphology and ultrastructure were observed by phase-contrast microscope, cytologic staining and electronic microscope. In primarily cultured cells, polygonal chondroblast-like cells dominated and they were confirmed by the positive result of immunohistochemical examination for type II collagen and Toludin blue staining. In conclusion, the TMJ cells in this culture system kept their phenotype in vivo.
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PMID:[The culture of TMJ condylar cartilage cells and study on their biological behaviors in vitro]. 1147 91

The collagenases of the matrix metalloproteinase family are key enzymes in mediating irreversible cartilage collagen loss in arthritis. Inhibition of these enzymes is, therefore, an important therapeutic target. New approaches to collagenase inhibition include active site inhibitors designed for specific enzymes, inhibition of cell signalling molecules and transcription factors involved in collagenase gene expression, prevention of zymogen activation and induction of natural inhibitor production.
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PMID:The inhibition of metalloproteinases as a therapeutic target in rheumatoid arthritis and osteoarthritis. 1171 57

The molecular and morphologic features of herniated human intervertebral disc tissues are of particular importance to clarify the pathogenesis. The present study analyzed the biochemical and morphological features of herniated intervertebral disc tissues to determine the constituent factors responsible for intervertebral disc herniation. A total of 32 herniated disc specimens and 4 control disc samples were analyzed. Collagen subunit composition, collagenase activity, lipid peroxidation level, caspase-3 activity, metal levels, morphologic studies, and genetic analysis were performed on herniated disc tissues of chronic (group A) and acute (group B) group and compared with findings of control group. Nick translation analysis in situ revealed apoptotic-positive stained DNA fragments as black-brown spots in herniated disc tissues. The presence of type II collagen in control disc samples and its absence in herniated samples were confirmed immunohistochemically. The increased caspase-3 activity, the apoptotic-positive stained DNA fragments, and the electron microscopic findings suggest enhanced programmed cell death in herniated discs. The significant increase in lipid peroxidation levels and collagenase activity, and the low metal levels suggest the enhancement of cell death signals in herniated discs, caused by oxygen stress. Linkage analysis of herniated disc tissues in Japanese individuals may suggest ethnic variation. These findings may be helpful in understanding the pathogenesis of herniated disc disease.
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PMID:Biochemical and morphological changes in herniated human intervertebral disc. 1179 73

The recent development of new specific immunoassays has provided an opportunity to study the assembly and resorption of type II and IX collagens of the extracellular matrix in relationship to endochondral calcification in situ. Here, we describe how in the bovine fetal physis prehypertrophic chondrocytes deposit an extensive extracellular matrix that, initially, is rich in both type II and type IX collagens and proteoglycan (PG; principally, aggrecan). The majority of the alpha1(IX)-chains lack the NC4 domain consistent with our previous studies with cultured chondrocytes. During assembly, the molar ratio of type II/COL2 domain of the alpha1(IX)-chain varied from 8:1 to 25:1. An increase in the content of Ca2+ and inorganic phosphate (Pi) was initiated in the prehypertrophic zone when the NC4 domain was removed selectively from the alpha1(IX)-chain. This was followed by the progressive loss of the alpha1(IX) COL2 domain and type II collagen. In the hypertrophic zone, the Ca2+/Pi molar ratio ranged from 1.56 to a maximum of 1.74, closely corresponding to that of mature hydroxyapatite (1.67). The prehypertrophic zone had an average ratio Ca2+/Pi ranging from 0.25 to 1, suggesting a phase transformation. At hypertrophy, when mineral content was maximal, type II collagen was reduced maximally in content coincident with a peak of cleavage of this molecule by collagenase when matrix metalloproteinase 13 (MMP-13) expression was maximal. In contrast, PG (principally aggrecan) was retained when hydroxyapatite was formed consistent with the view that this PG does not inhibit and might promote calcification in vivo. Taken together with earlier studies, these findings show that matrix remodeling after assembly is linked closely to initial changes in Ca2+ and Pi to subsequent cellular hypertrophy and mineralization. These changes involve a progressive and selective removal of types II and IX collagens with the retention of the PG aggrecan.
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PMID:The assembly and remodeling of the extracellular matrix in the growth plate in relationship to mineral deposition and cellular hypertrophy: an in situ study of collagens II and IX and proteoglycan. 1181 58

Fibroblast collagenase (MMP-1), a member of the matrix metalloproteinases family, is believed to be a pathogenesis of arthritis, by cleaving triple-helical type II collagen in cartilage. From the similarity of the active site zinc binding mode with hydroxamate, we designed and synthesized alpha-mercaptocarbonyl possessing compounds (3-5), which incorporated various peptide sequences as enzyme recognition sites. The P4-P1 peptide incorporating compound (3) exhibited as potent inhibition as the hydroxamate (1) and the carboxylate (2) type inhibitors, with an IC50 of 10(-6) M order against MMP-1. But the inhibitor (3) related compounds (6-8) displayed decreased or no inhibitory potencies. These results suggest that the existence of both the carbonyl and thiol groups might be critical for the inhibition, and the distance between the two functional groups is important for inhibitory potency. For Pn' peptide incorporating compounds (4a-k), except for 4h and 4k, all compounds showed IC50 values under sub-nanomolar. Among them, for potent inhibition, Leu was better than Phe and Val as the P1' amino acid, and the P2' position amino acid was necessary, and preferentially Phe. Insertion of the Pn peptide into 4d or 4k, giving compounds 5a-c, did not increase the activities of 4d and 4k. Substitution of the mercapto group with other functional groups lost the activity of compound 4a. The stereochemical preference at the thiol-attached position was also determined by preparation of both isomers of 4a. It was found that the S configuration compound (36b) is approximately 100 times more potent than the corresponding R-isomer (36a).
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PMID:Design and synthesis of sulfur based inhibitors of matrix metalloproteinase-1. 1184 17


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