Literature on vertebrate matrix metalloproteinases and their tissue inhibitors.
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Animals↗
Biomedical subjects
J F Woessner
Publications and source records attributed to J F Woessner.
The small matrix metalloproteinase of the rat uterus.
The uterus of the rat contains a small metalloproteinase that digests Azocoll and proteoglycan. The activity of this enzyme is elevated in the postpartum uterus and parallels the rate of breakdown of matrix proteoglycan (Sellers, A. and Woessner, J.F., Jr., Biochem. J. 189: 521, 1980). The enzyme has been purified to homogeneity. Its molecular weight is 28,000 for the latent form of the enzyme and 19,000 for the active form, as determined by SDS/PAGE. The enzyme has no action on collagens of type I, III, IV or V, but it does digest gelatins of these 4 types. Digestion of type I gelatin is most pronounced for the alpha-2 chain, which is cleaved to two major bands. The B chain of insulin is cleaved at Ala14-Leu15 and Tyr16-Leu17. Proteoglycan is degraded, but no action can be detected against elastin. Both zinc and calcium ions are required for activity. Low levels of phosphoramidon or Zincov are not inhibitory. Detailed comparisons with human gelatinase (matrix metalloproteinase 2) and stromelysin (matrix metalloproteinase 3) show that the uterine proteinase has a distinctive pattern of specificity. The properties match those of human Pump-1 as reported by Quantin et al., Biochemistry 28: 5327, 1989. It is proposed to designate this proteinase as matrix metalloproteinase 7.
Animals↗
Studies on purified neutral proteoglycan-degrading enzyme (stromelysin/matrix metalloproteinase 3) from human articular cartilage.
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Azo Compounds↗
Serum hyaluronan: a status report from the joint.
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Arthritis, Rheumatoid↗
Inhibition of ovulation in the perfused rat ovary by the synthetic collagenase inhibitor SC 44463.
A new, powerful, synthetic inhibitor of mammalian tissue collagenases and related metalloproteinases is inhibitory to ovulation in perfused rat ovaries. Ovaries of immature rats, primed with 20 IU of eCG, were dissected and perfused with 0.1 micrograms/ml LH and 0.2 mM 3-isobutyl-1-methylxanthine (IBMX) for 20 h. Addition of SC 44463 (N4-hydroxy-N1-[1S [(4-methoxphenyl)methyl]-2-(methylamino)-2-oxoethyl]- 2R-(2-methylpropyl)butane-diamide) at a concentration of 25 nM inhibited ovulation by 55% (9.6 +/- 1.7 ovulations per ovary, mean +/- SEM, compared to a control value of 21.7 +/- 1.7); and 250 nM inhibited ovulation by 75% (5.3 +/- 1.1 ovulations per ovary). We previously showed that the related compound SC 40827 inhibited ovulation by 70% when used at a concentration of 25 microM (Brännström et al., Endocrinology 1988; 122:1715-1721). We now show that SC 44463 is 100, 500, and 75 times more powerful than SC 40827 in blocking ovulation, inhibiting action of ovarian interstitial collagenase, and inhibiting action of the small metalloproteinase of the rat uterus, respectively. SC 44463 also inhibits ovarian type IV collagen-digesting activity 50% at a concentration of 18 nM. Ovulation occurs after 9-12 h of perfusion with LH. Compound SC 44463 (25 nM) showed its full inhibitory capacity when added to the medium as late as 7 h after LH, but there was no significant inhibition when it was added at 9 h. This suggests that the major collagenolytic events occur beyond 7 h after stimulation by LH.
1-Methyl-3-isobutylxanthine↗
A tissue inhibitor of metalloproteinases and alpha-macroglobulins in the ovulating rat ovary: possible regulators of collagen matrix breakdown.
Immature female Sprague-Dawley rats were primed with 20 IU eCG at 28 days of age and treated with 10 IU hCG 48 h later. Ovulation followed at 12-14 h. Ovaries were extracted at various times after hCG by use of Triton X-100 and 10 mM CaCl2 (Triton extract) followed by heating to 60 degrees C for 6 min with 0.1 M CaCl2 in 50 mM Tris/0.15 M NaCl, pH 7.5 (heat extract). These extracts were tested for their ability to inhibit tissue metalloproteinases by use of the small uterine metalloproteinase (UMP) of the rat. The ovary contains three plasma-derived inhibitors (alpha 1-macroglobulin [alpha 1 M], alpha 2-macroglobulin [alpha 2 M], and alpha 1 inhibitor3 [alpha 1I3]) and one tissue-derived inhibitor of the tissue inhibitor of metalloproteinases (TIMP) family. alpha 1 I3 was shown to inhibit UMP and rat collagenase. The concentration of the tissue inhibitor rose 5-fold from 0.6 micrograms UMP blocked per gram wet tissue in ovaries not primed with eCG to 3.2 micrograms UMP blocked at 8 h after hCG. Over this same time interval, the sum of alpha 1M + alpha 2M per gram of ovary rose 7-fold from 3.2 to 22.4 micrograms UMP inhibited and alpha 1I3 rose 2-fold from 4.4 to 10.7 micrograms UMP inhibited. The increases in the tissue inhibitor are interpreted to be due to increased synthesis by the tissue, whereas the changes in alpha-macroglobulins are postulated to be due to increased vascularity and increased permeability of the vessels.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Effects of hormonal perturbations on the small dermatan sulfate proteoglycan and mechanical properties of the uterine cervix of late pregnant rats.
Rats at 16-18 days of pregnancy were treated with various hormones in attempts to accelerate cervical softening and dilatation. Mechanical properties and biochemical components of the extracellular matrix were quantified at day 19. PGF2 alpha treatment significantly increased cervical wet weight, inner circumference, total amount and concentration of small dermatan sulfate proteoglycan, and the ratio of small proteoglycan to collagen; it decreased the concentration of collagen. Fluprostenol increased the extensibility and the rate of creep and decreased the collagen concentration. The progesterone antagonist ZK 98.734 (11 beta-(4-dimethylaminophenyl)-17 beta-hydroxy-17 alpha-(3-hydroxy-prop-(Z)- enyl)-4,9,(10)-estradiene-3-one) increased the inner circumference and the ratio of small proteoglycan to collagen; it decreased the collagen concentration. Treatment with 17 beta-estradiol increased the amount of medium-sized proteoglycans and decreased the concentration of the small proteoglycan. The results support the hypothesis advanced in our earlier study that the inner circumference of the cervix, a measure of dilatation, is dependent upon the ratio of small dermatan sulfate proteoglycan-II (decorin) to collagen. These studies also suggest that changes in the inner circumference and the extensibility of the cervix involve two distinct processes of connective tissue alteration.
Animals↗
Matrix metalloproteinases and their inhibitors in connective tissue remodeling.
Matrix metalloproteinases are an important group of zinc enzymes responsible for degradation of the extracellular matrix components such as collagen and proteoglycans in normal embryogenesis and remodeling and in many disease processes such as arthritis, cancer, periodontitis, and osteoporosis. A matrixin family is defined, comprising at least seven members that range in size from Mr 28,000 to 92,000 and are related in gene sequence to collagenase. All family members are secreted as zymogens that lose peptides of about 10,000 daltons upon activation. Latency is due to a conserved cysteine that binds to zinc at the active center. Latency is overcome by physical (chaotropic agents), chemical (HOCl, mercurials), and enzymatic (trypsin, plasmin) treatments that separate the cysteine residue from the zinc. Expression of the metalloproteinases is switched on by a variety of agents acting through regulatory elements of the gene, particularly the AP-1 binding site. A family of protein inhibitors of Mr 28,500 or less binds strongly and stoichiometrically in noncovalent fashion to inhibit members of the family. The serum protein alpha 2-macroglobulin and relatives are also strongly inhibitory.
Amino Acid Sequence↗
Role of metalloproteinases in human osteoarthritis.
Extracts of human articular cartilage have been examined for the presence of metalloproteinases that degrade proteoglycans and collagen of the extracellular matrix. Two enzymes (stromelysin and acid metalloproteinase) that degrade proteoglycan are elevated at least 150% in osteoarthritis (OA), whereas the tissue inhibitor of metalloproteinases (TIMP) shows only a small increase. We postulate an imbalance of enzyme over inhibitor that leads to net matrix destruction in OA. Stromelysin is shown to have an acid pH optimum of about 5.5. At this pH it can become spontaneously active and is less susceptible to TIMP inhibition than at pH 7.5. We postulate that the chondrocytes may secrete acid into the pericellular space, leading to localized enzyme activation and proteoglycan digestion.
Cartilage, Articular↗
Changes in active and latent collagenase in human placenta around the time of parturition.
High levels of collagenase are present in cervical extracts and in the circulation in women at parturition. This study examines the content of collagenase in human placentas at term, the possible contribution to circulating enzyme, and the changes that occur at parturition. Active and latent forms of collagenase are detectable in placentas with apparent relative molecular mass of 60,000 and 65,000 d, respectively. Gel-filtration chromatography was used to identify the presence of excess tissue inhibitor of metalloproteinase as the major collagenase inhibitor in the extracellular matrix of human placentas. After the onset of labor, there was a significant increase in total collagenase activity. Inactivation of the tissue inhibitor of metalloproteinase by reduction of placental extracts with dithiothreitol and alkylation with iodoacetamide resulted in a twelvefold to seventeenfold increase in collagenase activity. Umbilical cord collagenase levels were significantly lower than those in maternal circulation. The possibility of circulating collagenase originating from placenta in labor is discussed.
Chromatography, Gel↗
Production of collagenase and tissue inhibitor of metalloproteinases (TIMP) by rat growth plates in culture.
Growth plate cartilage from normal and vitamin D-phosphate deficient (-VDP) rats was cultured to study the production of collagenase and tissue inhibitor of metalloproteinases (TIMP) in vitro. All tissues secreted latent collagenase into the medium at a constant rate during the 5 days in culture. Microdissected-VDP growth plates, containing predominatly hypertrophic cells, released up to 8-fold more collagenase into the medium than either intact-VDP or normal growth plates. TIMP was also secreted during the culture, but its rate of production was not as dependent on tissue type as collagenase. The tissue level of collagenase and TIMP before culture was compared with that found in conditioned medium and remnant tissue after culture. During the 5 day culture period microdissected-VDP growth plates, containing predominatly hypertrophic cells, produced 3-times more collagenase/microgram DNA over the starting level than either intact-VDP or normal growth plates. TIMP was never found in tissues after they had been cultured, but was present in all tissues before culture except those containing predominatly hypertrophic cells. The amount of TIMP required to block collagenase was calculated. Growth plates in culture produced enough TIMP to block all collagenase found in the medium and remnant tissue, while extracts of uncultured intact -VDP growth plates, and those divided to contain hypertrophic cells, had an excess of collagenase over TIMP. The results suggest that hypertrophic cells produce far more collagenase than other cells in the growth plate, but all cell types have about the same capacity to synthesize TIMP. As a result, increased collagenase synthesis by hypertrophic cells may surpass increases in TIMP synthesis and lead to collagen removal. This would allow for thinning of the longitudinal septa and expansion of the hypertrophic cells.
Animals↗
Relationship between dilatation of the rat uterine cervix and a small dermatan sulfate proteoglycan.
Cervical linear circumference (lo), extensibility and rate of creep, and the content and concentration of collagen and proteoglycans were determined on uterine cervices of rats at different reproductive stages. The inner circumference increased from 9 +/- 3 (SD) mm at the nongravid stage to a maximum of 41 +/- 5 mm at term; a significant drop to 23 +/- 2 mm occurred by 4 h postpartum with a further drop to 18 +/- 4 mm by 1 day postpartum. The extensibility and rate of creep reached their maxima 1 day before term and returned to the nongravid value by 1 day postpartum. The small (Mr = 95,000) type II dermatan sulfate proteoglycan, the major cervical proteoglycan, increased from 43 +/- 6 micrograms per cervix at the nongravid stage to 196 +/- 33 micrograms at term. The amount of this proteoglycan decreased significantly by 35% to 126 +/- 5 micrograms within 4 h postpartum and declined further to 79 +/- 16 micrograms by 1 day postpartum. The total cervical collagen content increased less than 2-fold during pregnancy, from 3.5 +/- 0.5 to 6.3 +/- 0.7 mg; a decline to 5.8 mg by 1 day postpartum was not significant. The ratio of small proteoglycan: collagen increased 2.5-fold between the nongravid state and term, then returned to the nongravid value by 1 day postpartum. Significant correlations were found between the lo and the amount of small proteoglycan per cervix (r = 0.86; n = 69) and between lo and the ratio of small proteoglycan:collagen (r = 0.83; n = 50) when data from every reproductive stage were combined. A mechanism is proposed whereby the interaction of the proteoglycan with collagen fibers might alter mechanical properties and contribute to cervical dilatation and its rapid reversal.
Animals↗
Extracellular collagenase, proteoglycanase and products of their activity, released in organ culture by intact dermal inflammatory lesions produced by sulfur mustard.
Peak (1 and 2 d) and healing (3, 6, and 10 d) inflammatory lesions were produced in rabbits by the topical application of the military vesicant, bis(2-chloroethyl)sulfide, commonly called sulfur mustard (SM). SM produces an acute sterile dermal inflammatory reaction with little or no necrosis, except in the epidermis, which dies during the first day. After an animal was killed, its lesions were excised intact, as full-thickness 1.0-cm2 explants. They were then organ-cultured for 3 d in order to maintain the viability of both local and infiltrating cells. The extracellular fluid in each lesion equilibrated with the culture fluid, which was collected daily and analyzed for collagenase and proteoglycanase activities. These metalloproteinase activities were measured after we had i) destroyed the alpha-macroglobulin inhibitors with KSCN, ii) destroyed the tissue inhibitor of metalloproteinases (TIMP) by reduction and alkylation, and iii) activated the latent proteinase activity with aminophenylmercuric acetate (APMA). Hydroxyproline-containing peptides and glycosaminoglycans (GAG) released into the culture fluids were also measured as indicators of local collagenase and proteoglycanase activity within the inflammatory lesions. In general, the levels of both the metalloproteinases and the products of their activity were higher in second- and third-day culture fluids than in first-day culture fluids, and higher in fluids from SM lesions than in those from normal skin. The activated fibroblast was apparently the major cell type producing the collagenase and proteoglycanase. The hydrolysis of collagen and ground substance occurs pericellularly. An excess of inhibitors exists outside the pericellular region. The daily change in culture fluids apparently decreased such inhibitors, so that by the second and third day of culture we could detect the changes in pericellular enzyme activity that were not detectable on the first day of culture. As the inflammatory lesions healed, the extracellular enzyme products (hydroxyproline and GAG) increased more than the enzymes that produced these products. With healing, a decrease occurs in the extravasation of all serum components, especially the large ones such as the alpha-macroglobulin inhibitors. We propose that during healing, the decrease in these inhibitors allows the metalloproteinases to begin the remodeling process, and that during the peak phase of inflammation, these same inhibitors protect extracellular matrix against hydrolysis by such proteinases.
Animals↗
Purification and characterization of a small dermatan sulphate proteoglycan implicated in the dilatation of the rat uterine cervix.
A small dermatan sulphate proteoglycan (DSPG) was extracted from rat cervices and was purified by using DEAE-Sephacel ion-exchange chromatography, gel filtration on Sepharose CL-2B and CsCl-density-gradient centrifugation. Sedimentation-equilibrium centrifugation gave a weight-average Mr of 95,000. Amino acid analysis showed a high content of aspartic acid, glutamic acid, glycine and leucine. The glycosaminoglycan chains had Mr 50,000 as determined by gel filtration. Chondroitin AC lyase and chondroitin ABC lyase digestions of these chains showed that they were composed of 75% dermatan sulphate and 25% chondroitin sulphate. Chondroitin ABC lyase digestion produced a core protein of Mr 45,000. The core protein, after treatment with HF, had Mr 37,000. Amino acid sequences of the N-terminus and a CNBr-cleavage peptide showed similarity to the sequences of core proteins of small proteoglycans of bovine and human origin, but the N-terminal glycosaminoglycan-attachment site (Ser-Gly-Ile-Ile) differed from the consensus sequence (Ser-Gly-Xaa-Gly) [Bourdon, Krusius, Campbell, Schwartz & Ruoslahti (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 3194-3198]. A polyclonal antibody against the rat cervical DSPG reacted with small proteoglycans from cervices of human, mouse, dog, cow and sheep. DSPG is the major proteoglycan species present in the cervix. The amount of DSPG per cervix increases 4-fold during pregnancy, then falls precipitously within 1 day post partum. A role in cervical dilatation is postulated for this proteoglycan.
Amino Acid Sequence↗
Purification of the neutral proteoglycan-degrading metalloproteinase from human articular cartilage tissue and its identification as stromelysin matrix metalloproteinase-3.
The 'neutral' proteoglycan-degrading metalloproteinase of human articular cartilage was purified 3,500-fold by use of an anti-(matrix metalloproteinase-3) immunoglobulin G affinity column. Molecular masses of the latent and multiple active forms and specificity of action on casein, transferrin, gelatin and fibronectin were identical with those of authentic stromelysin (matrix metalloproteinase-3) from cultured human rheumatoid synovial fibroblasts. The optimum pH of this proteinase on proteoglycan monomer was pH 5.5, and on Azocoll, 6.2; digestion of fibronectin and gelatin was more extensive at pH 5.5 than at 7.5.
Cartilage, Articular↗
Connective tissue breakdown in ovulation.
A meshwork of collagen over the apical region of the follicle must be breached to permit the ovum to escape. We propose that specific collagenase activity is responsible for collagen breakdown in this region. Immature rats are primed with pregnant mare serum gonadotropin (PMSG), followed at 48 h by hCG. At 8 h after hCG, collagenase activity, measured in extracts of ovarian tissue, is elevated about five-fold. Ovulation follows at 10-12 h. Ovaries from PMSG-primed rats are dissected at 48 h, placed in a perfusion apparatus, and perfused with luteinizing hormone and 3-isobutyl-1-methyl xanthine. The ovulations induced by this treatment can be blocked to the extent of 70% with a synthetic collagenase inhibitor. The activation of procollagenase is believed to involve plasminogen activator and plasmin. In support of this, we find that tranexamic acid at 1 mM inhibits ovulation about 70%. The inhibitor must be added within 3-4 h of LH to be effective. A specific plasmin inhibitor, D-Val-Phe-Lys-chloromethyl ketone, is similarly effective.
Animals↗
Changes in desmosine and pyridinoline crosslinks during rapid synthesis and degradation of elastin and collagen in the rat uterus.
The wet weight of the rat uterus increased 8-fold during pregnancy and fell by 70% within 5 days postpartum. Uterine collagen increased about 5-fold during pregnancy and also fell by 70% within 5 days. The crosslink pyridinoline remained constant at 0.28 mole/mole collagen at every time point, with the possible exception of 11-12 days of pregnancy. The pyridinoline link can therefore form within the short time span of a few days, a feature presumed to be necessary to maintain the full mechanical strength of the uterus during labor. Uterine elastin increased about 8-fold during pregnancy, but the desmosines did not keep pace and fell from a normal value of 1.43 mole/mole elastin to a low of 0.89 at term. Moreover, elastin content reached a maximum several days prior to parturition and then declined continuously to 5 days postpartum. During this decline there was a selective loss of the poorly crosslinked elastin. The desmosines cannot be used as a direct measure of uterine elastin content, because of their continuously changing levels. Desmosines and pyridinoline were measured both by ELISA and by the amino acid analyzer. The two methods gave almost identical results when elastin and collagen were first separated from each other.
Amino Acids↗
Evidence for metalloproteinase and metalloproteinase inhibitor imbalance in human osteoarthritic cartilage.
Cartilage specimens from tibial plateaus, obtained from 13 osteoarthritic (OA) patients and seven controls, were selected from three regions: zone A, center of fibrillated area; zone B, area adjacent to fibrillation, and zone C, remote region of plateau. Acid and neutral metalloproteinases and tissue inhibitor of metalloproteinase (TIMP) were extracted with 2 M guanidine. Methods were developed to selectively destroy either proteinases or TIMP to prevent cross-reaction during assay. Acid and neutral proteinases were elevated approximately 150% in OA; TIMP was elevated approximately 50%. A positive correlation (r = 0.50) was found between acid and neutral proteinase activities in OA, but not in controls. Both proteinases were elevated two-to threefold in zones A, B, and C. However, the self-active form of the acid metalloproteinase was elevated only in zones A and B (200%); it correlated well with the Mankin scores, whereas the total activities did not. TIMP was elevated (50%) only in zones A and B. Both the proteinase levels and the Mankin score were elevated to a greater extent in the medial, than in the lateral, compartment. Titration of TIMP against the two metalloproteinases indicates that there is a small excess of inhibitor over enzymes in normal cartilage. In OA, TIMP does not increase to the same extent as the proteinases; the resultant excess of proteinases over TIMP may contribute to cartilage breakdown.
Adult↗