Dimerization of endogenous MT1-MMP is a regulatory step in the activation of the 72-kDa gelatinase MMP-2 on fibroblasts and fibrosarcoma cells

The secreted gelatinase matrix metalloprotease-2 (MMP-2) and the membrane-anchored matrix metalloprotease MT1-MMP (MMP-14), are central players in pericellular proteolysis in extracellular matrix degradation. In addition to possessing a direct collagenolytic and gelatinolytic activity, these enzymes take part in a cascade pathway in which MT1-MMP activates the MMP-2 proenzyme. This reaction occurs in an interplay with the matrix metalloprotease inhibitor, TIMP-2, and the proposed mechanism involves two molecules of MT1-MMP in complex with one TIMP-2 molecule. We provide positive evidence that proMMP-2 activation is governed by dimerization of MT1-MMP on the surface of fibroblasts and fibrosarcoma cells. Even in the absence of transfection and overexpression, dimerization of MT1-MMP markedly stimulated the formation of active MMP-2 products. The effect demonstrated here was brought about by a monoclonal antibody that binds specifically to MT1-MMP as shown by immunofluorescence experiments. The antibody has no effect on the catalytic activity. The effect on proMMP-2 activation involves MT1-MMP dimerization because it requires the divalent monoclonal antibody, with no effect obtained with monovalent Fab fragments. Since only a negligible level of proMMP-2 activation was obtained with MT1-MMP-expressing cells in the absence of dimerization, our results identify the dimerization event as a critical level of proteolytic cascade regulation.     

Purification and characterization of human 72-kDa gelatinase (type IV collagenase). Use of immunolocalisation to demonstrate the non-coordinate regulation of the 72-kDa and 95-kDa gelatinases by human fibroblasts.

Human gingival fibroblast gelatinase (type IV collagenase) has been purified to homogeneity using a combination of ion exchange chromatography, gel filtration and affinity chromatography. The purified proenzyme electrophoresed under reducing conditions as a single band of 72 kDa which could be activated to a species of 65 kDa. Gelatinase was activated by organomercurials by a process apparently initiated by a conformational change and involving self-cleavage. It was not activated by trypsin or plasmin unlike the other family members, collagenase and stromelysin. Gelatinase otherwise exhibited properties typical of the metalloproteinases: it was inhibited by metal chelating agents and by the specific inhibitor TIMP (tissue inhibitor of metalloproteinases). Its major substrate was shown to be denatured collagen although it was also able to degrade native type IV and V collagens. A polyclonal antibody was raised in a sheep using the purified enzyme as antigen. The antiserum recognised and specifically inhibited the 72-kDa gelatinase but not a 95-kDa gelatinase from pig leukocytes. It was used in immunolocalisation studies on human fibroblasts to investigate the regulation of the production of the two Mr forms of gelatinase. These studies clearly demonstrate that human fibroblasts constitutively synthesize and secrete 72-kDa gelatinase but that 95-kDa gelatinase was inducible by agents such as cytokines. The significance of these results in relation to the likely in vivo r?le of gelatinases is discussed.     

Binding of tissue inhibitor of metalloproteinases 2 to two distinct sites on human 72-kDa gelatinase. Identification of a stabilization site.

We have identified a binding site for tissue inhibitors of metalloproteinases 2 (TIMP-2) on human 72-kDa gelatinase that is distinct from the active site. 72-kDa progelatinase is found in a complex with TIMP-2 in the medium of cultured cells and can be activated with organomercurial compounds to yield a gelatinolytic proteinase that remains bound to TIMP-2. Removal of TIMP-2 from 72-kDa progelatinase by reverse-phase high performance liquid chromatography, followed by reconstitution of the progelatinase in neutral pH buffer, results in autocatalytic activation. When samples of autoactivated gelatinase were blotted onto nitrocellulose, then probed with 125I-TIMP-2, we found a 29-kDa peptide that was capable of binding TIMP-2. We isolated this fragment and identified it as the region of gelatinase from amino acid 414 to the carboxyl terminus in the primary amino acid sequence of progelatinase. This portion of the molecule does not contain the putative zinc- or gelatin-binding sites and is proteolytically inactive. Incubation of 125I-TIMP-2 with 72-kDa progelatinase-TIMP-2 complexes resulted in a concentration-dependent exchange of labeled TIMP-2 with unlabeled TIMP-2, in both the presence and absence of the metalloproteinase inhibitor 1,10-phenanthroline. Saturation binding kinetics for the active site of 72-kDa gelatinase were measured in pools of the 43-kDa active fragment that results from the autoactivation of 72-kDa progelatinase; this fragment has no carboxyl-terminal TIMP-2 binding capability. Binding of 125I-TIMP-2 to the active site was completely inhibited by 1,10-phenanthroline. Binding kinetics for the putative stabilization site were determined with isolated 72-kDa progelatinase. In the presence of 1,10-phenanthroline, 72-kDa progelatinase bound 125I-TIMP-2 but not 125I-TIMP-1. Scatchard analysis yielded an approximate dissociation constant (Kd) of 0.72 nM for the active site and 0.42 nM for the stabilization site.     

Kinetics and physiologic relevance of the inactivation of alpha 1-proteinase inhibitor, alpha 1-antichymotrypsin, and antithrombin III by matrix metalloproteinases-1 (tissue collagenase), -2 (72-kDa gelatinase/type IV collagenase), and -3 (stromelysin).

Serpins encompass a superfamily of proteinase inhibitors that regulate many of the serine proteinases involved in inflammation and hemostasis. In vitro, many serpins are catalytically inactivated by proteinases that they do not inhibit, leading to the concept of proteolytic down-regulation of serpin inhibitory capacity. The extent to which down-regulation of serpin activity occurs in vivo is debated, since little is known of the rates at which the process occurs. To address this debate, we have measured the rates of inactivation of three serpins, alpha 1-proteinase inhibitor (alpha 1PI), alpha 1-antichymotrypsin (alpha 1ACT), and antithrombin III (ATIII), by three human matrix metalloproteinases (MMPs-1, -2, and -3) thought to be involved in tissue destruction and repair. Our object was to establish a working kinetic model which can be used to predict whether serpin inactivation by these proteinases is likely to occur in vivo. We determined the rates of inactivation of these three serpins by each of the MMPs and compared these to rates of inhibition of the MMPs by an endogenous inhibitor, alpha 2-macroglobulin. An equation designed to predict the extent of substrate hydrolyzed by an enzyme in the presence of an enzyme inhibitor gave the following predictions of the inactivation in vivo: (i) ATIII is unlikely to be inactivated by the MMPs. (ii) MMP-2 (72-kDa gelatinase/type IV collagenase) is unlikely to inactivate any of the three serpins. (iii) MMP-1 (tissue collagenase) will inactivate alpha 1PI and alpha 1ACT only when its concentration saturates that of its controlling inhibitors. (iv) MMP-3 (stromelysin) may inactivate small amounts of alpha 1PI and more significant amounts of alpha 1ACT, even in the presence of its controlling inhibitors. Any physiologic or pathologic inactivation of these serpins by these MMPs that occurs in vivo will probably be due to MMP-3, and will likely only take place in tissues and inflammatory loci where the concentration of MMP inhibitors is depressed.     

Calmodulin differentially modulates the interleukin 1-induced biosynthesis of tissue inhibitor of metalloproteinases and matrix metalloproteinases in human uterine cervical fibroblasts.

The effects of a specific calmodulin inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) on the synthesis of tissue inhibitor of metalloproteinases (TIMP) and precursor of matrix metalloproteinase 1/tissue collagenase (proMMP-1) and matrix metalloproteinase 3/stromelysin (proMMP-3), were examined using human uterine cervical fibroblasts in culture. When the cells were treated with human recombinant interleukin 1 alpha, the synthesis of TIMP, proMMP-1, and proMMP-3 was greatly enhanced along with the increase in the steady-state levels of mRNAs for respective proteins. The treatment of the cells with human recombinant interleukin 1 alpha and W-7 further augmented the production of proMMPs-1 and -3 and the accumulation of their mRNAs. In contrast, TIMP production and its steady-state mRNA level were reduced considerably under these conditions. Similar observations were made with another calmodulin inhibitor, trifluoperazine, but not with N-(6-aminohexyl)-1-naphthalenesulfonamide, the weakest inhibitor for calmodulin. These results indicate that calmodulin is required for the interleukin 1-enhanced synthesis of TIMP but it is a suppressor for the synthesis of proMMPs-1 and -3.     

Interaction of 92-kDa type IV collagenase with the tissue inhibitor of metalloproteinases prevents dimerization, complex formation with interstitial collagenase, and activation of the proenzyme with stromelysin.

Secreted metalloproteases initiating proteolytic degradation of collagens and proteoglycans play a critical role in remodeling of the connective tissue. Activation of the secreted proenzymes and interaction with their specific inhibitors TIMP and TIMP-2 are responsible for regulation of enzyme activity in extracellular space. We have previously demonstrated that 92- and 72-kDa Type IV procollagenases, in contrast to interstitial collagenase (ClI), form specific complexes with TIMP and the related inhibitor TIMP-2, respectively. The physiologic significance of the proenzyme-inhibitor complex and the mechanism of activation of Type IV collagenases remained unclear. Here, we demonstrate that in the absence of TIMP, 92-kDa Type IV procollagenase (92T4Cl) can form a covalent homodimer and a novel complex with ClI. In the presence of TIMP, the formation of a 92T4Cl proenzyme complex with TIMP prevents dimerization, formation of the complex with ClI, and activation of the 92T4Cl proenzyme by stromelysin, a related metalloprotease. The proenzyme homodimer is unable to form a complex with TIMP. All TIMP-free forms of the proenzyme can be activated by stromelysin. The 92T4Cl-ClI complex can be activated to yield a complex active against both gelatin and fibrillar Type I collagen, suggesting a mechanism for cooperative action of two enzymes in reducing collagen fibrils to small peptides under physiologic conditions.     

The complex between a tissue inhibitor of metalloproteinases (TIMP-2) and 72-kDa progelatinase is a metalloproteinase inhibitor.

Human rheumatoid synovial cells in culture secrete both 72-kDa progelatinase and a complex consisting of 72-kDa progelatinase and a 24-kDa inhibitor of metalloproteinases, TIMP-2. In addition, the culture medium contains TIMP-1, the classical inhibitor of metalloproteinases, with a molecular mass of 30 kDa. TIMP-1 does not form a complex with free 72-kDa progelatinase. Free progelatinase and progelatinase complexed with TIMP-2 can be activated with the organomercury compound p-aminophenylmercury acetate. The activated complex shows less than 10% the enzyme activity of activated free gelatinase. The progelatinase-TIMP-2 complex could be shown to be an inhibitor for other metalloproteinases, such as gelatinase and collagenase secreted by human rheumatoid synovia fibroblasts, as well as for the corresponding enzymes from human neutrophils.     

Domain structure of human 72-kDa gelatinase/type IV collagenase. Characterization of proteolytic activity and identification of the tissue inhibitor of metalloproteinase-2 (TIMP-2) binding regions.

The 72-kDa gelatinase/type IV collagenase, a metalloproteinase thought to play a role in metastasis and in angiogenesis, forms a noncovalent stoichiometric complex with the tissue inhibitor of metalloproteinase-2 (TIMP-2), a potent inhibitor of enzyme activity. To define the regions of the 72-kDa gelatinase responsible for TIMP-2 binding, a series of NH2- and COOH-terminal deletions of the enzyme were constructed using the polymerase chain reaction technique. The full-length and the truncated enzymes were expressed in a recombinant vaccinia virus mammalian cell expression system (Vac/T7). Two truncated enzymes ending at residues 425 (delta 426-631) and 454 (delta 455-631) were purified. Like the full-length recombinant 72-kDa gelatinase, both COOH-terminally truncated enzymes were activated with organomercurial and digested gelatin and native collagen type IV. In contrast to the full-length enzyme, delta 426-631 and delta 455-631 enzymes were less sensitive to TIMP-2 inhibition requiring 10 mol of TIMP-2/mol of enzyme to achieve maximal inhibition of enzymatic activity. The activated but not the latent forms of the delta 426-631 and delta 455-631 proteins formed a complex with TIMP-2 only when excess molar concentrations of inhibitor were used. We also expressed the 205-amino acid COOH-terminal fragment, delta 1-426, and found that it binds TIMP-2. In addition, a truncated version of the 72-kDa gelatinase lacking the NH2-terminal 78 amino acids (delta 1-78) of the proenzyme retained the ability to bind TIMP-2. These studies demonstrate that 72-kDa gelatinases lacking the COOH-terminal domain retain full enzymatic activity but acquire a reduced sensitivity to TIMP-2 inhibition. These data suggest that both the active site and the COOH-terminal tail of the 72-kDa gelatinase independently and cooperatively participate in TIMP-2 binding.     

Biosynthesis of tissue inhibitor of metalloproteinases by human fibroblasts in culture. Stimulation by 12-O-tetradecanoylphorbol 13-acetate and interleukin 1 in parallel with collagenase

Biosynthesis of the glycoprotein tissue inhibitor of metalloproteinases (TIMP) by human fibroblasts in culture has been characterized by functional assays, immunoprecipitation, and immunocytochemistry with a monospecific antiserum. As determined by radiolabeling with [35S]methionine, immunoprecipitation, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the secreted form of TIMP had an Mr of 29,000, whereas the form associated with the cell layer had an Mr of 24,000. Unstimulated human lung fibroblasts (HFL-1) secreted TIMP at the rate of approximately 2 micrograms/10(6) cells/24 h, and normal foreskin fibroblasts (HS 27) and skin fibroblasts from a patient with Hurler's disease (GM 1391) secreted TIMP at 0.3 and 0.2 micrograms/10(6) cells/24 h, respectively. Secretion of TIMP was stimulated up to 10-fold by treating the cells with 20-100 ng/ml of 12-O-tetradecanoylphorbol 13-acetate or 10 units/ml of human interleukin 1. In the stimulated HFL-1 cells, TIMP accounted for 0.03-0.09% of the total [35S]methionine incorporated into protein, and 0.3-0.8% of the [35S]methionine in secreted protein. Although TIMP accounted for a relatively small proportion of total protein synthesis of the fibroblasts, greater than 80% of untreated and greater than 95% of stimulated fibroblasts synthesized TIMP, as determined by indirect immunofluorescence. The treatments of the human fibroblasts that increased TIMP secretion also induced synthesis and secretion of proenzyme forms of collagenase, indicating that degradative enzymes and their controlling inhibitors may be synthesized in parallel under certain conditions.     

Cyclic adenosine 3',5'-monophosphate suppresses interleukin 1-induced synthesis of matrix metalloproteinases but not of tissue inhibitor of metalloproteinases in human uterine cervical fibroblasts.

Interleukin 1 (IL-1) mediates many cellular functions, but the signal transduction mechanisms of its actions are not clearly understood. Here, we have examined the exact participation of cAMP in the IL-1-induced production of the precursors of matrix metalloproteinase (MMPs) and their specific inhibitor, tissue inhibitor of metalloproteinases (TIMP) in human uterine cervical fibroblasts. IL-1 significantly augmented the production of proMMP-1 (vertebrate procollagenase), proMMP-3 (prostromelysin), and TIMP without detectable changes in the intracellular level of cAMP. Dibutyryl cAMP (Bt2cAMP) and the cAMP elevating agent (forskolin) did not replace IL-1 as MMP inducers. On the contrary, the IL-1-mediated induction of proMMP-1 and proMMP-3 was significantly suppressed by treatment of the cells with Bt2cAMP, forskolin, or theophylline. The suppressive effect of Bt2cAMP on the IL-1-induced production of proMMP-1 and -3 was not due to the inhibition of zymogen secretion, but resulted from the decrease in the steady-state levels of proMMP-1 and proMMP-3 mRNAs. In contrast, Bt2cAMP slightly enhanced the IL-1-induced production of TIMP. The synthesis of proMMP-2 (72-kDa progelatinase/type IV procollagenase) was not altered by IL-1 and/or Bt2cAMP. These results suggest, first, that induction of proMMP-1 and -3 synthesis may share similar transduction pathways but they are distinct from those for proMMP-2 and TIMP synthesis and, second, that cAMP does not function as a second messenger in the MMPs' induction upon IL-1 stimulation in human uterine cervical fibroblasts. Thus, it is further suggested that the system that increases the intracellular cAMP level may be involved in negative regulation of proMMP-1 and -3 production.     

Uncoordinate regulation of collagenase,stromelysin, and tissue inhibitor of metalloproteinases genes by prostaglandin E2: Selective enhancement of collagenase gene expression in human dermal fibroblasts in culture

The degradative effects of interleukin-1 (IL-1) on the extracellular matrix of connective tissue are mediated primarily by metalloproteinases and prostaglandins. Clinical observations suggest that these effects can be prevented, to some extent, by the use of non-steroidal anti-inflammatory drugs. We have examined the role of prostaglandin E₂ (PGE₂) in IL-1-induced gene expression by human skin fibroblasts in culture. Incubation of confluent fibroblast cultures with varying concentrations (0.01–1.0 μg/ml) of PGE₂ led to a dose-dependent elevation of collagenase mRNA steady-state levels, the promoter activity, and the secretion of the protein, whereas relatively little effect was observed on stromelysin and TIMP gene expression. Exogenous PGE₂ had no additive or synergistic effect with IL-1 on collagenase gene expression. Furthermore, commonly used non-steroidal anti-inflammatory drugs (indomethacin, acetyl salicylic acid and ibuprofen), at doses which block prostaglandin synthesis in cultured fibroblasts, failed to counteract IL-1-induced collagenase and stromelysin gene expression, nor did they affect TIMP expression. Although the effects of PGE₂ did not potentiate those of IL-1 on collagenase gene expression in vitro, one could speculate that massive production of PGE₂ by connective tissue cells in vivo in response to inflammatory mediators such as IL-1 or tumor necrosis factor-α, could lead to sustained expression of collagenase in connective tissue cells after clearance of the growth factors.     

Regulation of the autoactivation of human 72-kDa progelatinase by tissue inhibitor of metalloproteinases-2

To study the activation of human 72-kDa gelatinase, and its relation to tissue inhibitor of metalloproteinases 2 (TIMP-2), we purified human 72-kDa progelatinase both as a complex with TIMP-2 and as a free proteinase. Activation of progelatinase-TIMP-2 complexes with 4-aminophenylmercuric acetate yielded gelatinolytically active enzyme migrating at 62 kDa. TIMP-2 remained bound to the active enzyme. Removal of TIMP-2 from progelatinase by reverse-phase high performance liquid chromatography in the presence of trifluoroacetic acid, followed by complete dialysis in neutral pH buffer, resulted in multiple fragments. These fragments were formed as a result of the cleavage of 72-kDa progelatinase at several locations. Cleavage at the amino terminus was restricted to the removal of the propeptide, except in the case of degradation leading to inactive fragments. Two active species autocatalytically evolved upon removal of TIMP-2 from progelatinase. The 62 kDa-activated gelatinase lacked the amino-terminal propeptide, which is known to be removed upon treatment with 4-aminophenylmercuric acetate. In addition, an active 42.5-kDa fragment lacking both the propeptide and a portion of the carboxyl terminus was formed. This low-molecular-weight active form of 72-kDa progelatinase retained its ability to bind and degrade gelatin. Self-activation and degradation of 72-kDa progelatinase can be prevented by agents that inhibit metalloproteinases, including 1,10-phenanthroline. Evidence presented here suggests that TIMP-2 binds to a stabilization site that is independent of the active site. This stabilization site does not bind TIMP-1 (TIMP). Occupation of this site by TIMP-2 prevents autocatalytic activation and degradation but does not prevent gelatinolysis by the enzyme-inhibitor complex.     

Effect of heparin on the production of matrix metalloproteinases and tissue inhibitors of metalloproteinases by human dermal fibroblasts

The influence of heparin and a heparin fragment devoid of anticoagulant activity on the production of matrix metalloproteinases and tissue inhibitors of metalloproteinases by human dermal fibroblasts was studied. Doses (0.1-400 microg/ml) responses were performed and data obtained were similar whatever heparin or fragment was used. The basal expression of collagenase by fibroblasts decreased quasi-linearly with increasing doses of heparins from 1 to 400 microg/ml. TIMP-1 levels were not affected by supplementing serum free culture medium with heparins. On the contrary, at low concentration, i.e. 1-10 microg/ml, heparins stimulated the secretion of both 72-kDa gelatinase (1.4-1.6-fold) and particularly TIMP-2 (>4-fold). At high doses, MMP-2 and TIMP-2 production by fibroblasts returned to basal levels. These results suggested that the local concentration of heparin released by mast cells could be instrumental in modulating fibroblast growth and proteolytic phenotype.     

Uncoordinate expressions of type I and III collagens,collagenase and tissue inhibitor of matrix metalloproteinase 1 along in vitro proliferative life span of human skin fibroblasts

In vitro human skin fibroblasts aging was characterized by a continuous increase of collagenase mRNA levels. On the contrary, TIMP-1 mRNA level decreased only at late passages (> 65% of proliferative life span). Type I and III mRNA levels showed a high variability depending on cell strains studied. However, type I and III collagen expressions varied parallely. All-trans retinoic acid (RA) decreased collagenase expression and stimulated TIMP-1 expression. Under RA action, high variability in mRNAs levels encoding type I and III collagens was observed with HSF passages. However, RA tended to correct variations in collagens expressions observed along HSF life span.     

Application of N-carboxyalkyl peptides to the inhibition and affinity purification of the porcine matrix metalloproteinases collagenase, gelatinase, and stromelysin.

Several N-carboxyalkyl peptides were synthesized and tested as inhibitors of pig synovial collagenase, 72-kDa gelatinase and stromelysin (matrix metalloproteinases MMP-1, MMP-2, and MMP-3). The most potent of the series, CH3CH2CH2(R,S)CH(COOH)-NH-Leu-Phe-Ala-NH2, competitively inhibited cleavage of dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond by MMP-1 and MMP-2 (KI = 30 and 40 microM, respectively). A similar inhibitory potency was found for MMP-1 with soluble Type I collagen and MMP-3 with substance P as substrate. The inhibitor was coupled to EAH-Sepharose 4B through a C-terminal amide. In the presence of 2 M NaCl at pH 7.2, this matrix bound MMP-1, MMP-2, and MMP-3 from concentrated culture medium of pig synovial membranes. The enzymes coeluted at pH 4.1 and subsequently were resolved by chromatography on DEAE-Sephacel and heparin-Sepharose. Purified MMP-1 catalyzed the o-phenanthroline-sensitive cleavage of collagen into TCA and TCB fragments as well as slower hydrolysis of the alpha 2 chain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of MMP-1 indicated a predominant polypeptide of approximately 44 kDa and minor species of approximately 24 and 21 kDa. The 44-kDa species and one of the smaller polypeptides reacted with an antiserum to residues 195-207 of human fibroblast MMP-1, indicating that porcine MMP-1 contains a similar sequence and that the smaller components were probably derived from MMP-1. Neither MMP-2 nor MMP-3 reacted with this antiserum. Purified porcine MMP-2 degraded gelatin but not collagen and exhibited an apparent Mr of approximately 71 kDa. Additional smaller polypeptides were present, one of which may correspond to tissue inhibitor of metalloproteinases. MMP-3 showed doublets of approximately 47/46 and 26/25 kDa and cleaved substance P at the Gly6-Phe7 bond. This procedure provides a rapid means of obtaining all three MMPs from one source in approximately 15% yield each.     

Involvement of matrix metalloproteinases 2 and 9, tissue inhibitor of metalloproteinases and apoptosis in tissue remodelling in the sheep placenta

Placental growth and development is crucial for successful pregnancy. The aim of this study was to characterize the activity and localization of the matrix metalloproteinase 2 (MMP-2) and MMP-9, which are capable of degrading basement membrane collagen (predominantly collagen type IV), and their endogenous tissue inhibitor of matrix metalloproteinases (TIMPs), in amniotic fluid and in the developing ovine placenta. Cell deletion by apoptosis during placental development was also examined. Zymography with gelatin as substrate indicated that MMP-2 (72 kDa gelatinase A; predominantly latent form) was present in increasing amounts in amniotic fluid from day 70 of gestation to labour (days 140-145), and MMP-9 (92 kDa gelatinase B; predominantly latent form) was detectable from day 125 to labour; there was no increase in MMP-2 or -9 in labour. A broad range of TIMPs was detected in amniotic fluid; the molecular masses corresponded to TIMP-1, -2 and -3. Immunohistochemical techniques localized MMP-2, MMP-9 and TIMP-3 in the sheep placenta, predominantly in the trophoblast layer in uninucleate, but not binucleate, cells. However, MMP-2 and -9 activated proteins in placental homogenates were low throughout pregnancy. Apoptosis was identified by morphological criteria and also by TdT-mediated dUTP nick end labelling. Apoptosis was present in discrete regions in the placenta, predominantly in trophoblast cells near the tips and the basal regions of the fetomaternal interdigitations. During pregnancy the sheep placenta becomes more complex and the area of the fetomaternal interface increases. MMP-2 and -9 are likely to be involved in breaking down basement membranes to allow cell migration during this process. It is suggested that digestion of supporting extracellular matrix may trigger apoptosis and in some way increase the branching pattern in the villi.     

Neutral proteinases of human mononuclear phagocytes. Cellular differentiation markedly alters cell phenotype for serine proteinases, metalloproteinases, and tissue inhibitor of metalloproteinases

Mononuclear phagocytes have the capacity to directly participate in extracellular matrix turnover via secretion of neutral proteinases. We have studied the effects of in vivo and in vitro differentiation upon cellular content or secretion of a spectrum of neutral proteinases, along with a counter-regulatory metalloproteinase inhibitor (TIMP). We found 1) matrix-degradative serine proteinases (leukocyte elastase and cathepsin G) were lost during cellular maturation and/or differentiation; 2) the 92-kDa type IV/type V collagenase and TIMP were secreted earliest in mononuclear phagocyte differentiation, whereas stromelysin secretion was observed only by LPS-stimulated alveolar macrophages; 3) exposure of alveolar macrophages, but not monocytes, to phorbol esters and LPS resulted in markedly augmented secretion of all studied metalloproteinases and TIMP; 4) monocyte-derived macrophages partially (but not completely) mimicked the metalloproteinase secretory phenotype of alveolar macrophages; and 5) the secretory phenotype of alveolar macrophages for interstitial collagenase (but not TIMP) was largely lost during in vitro culture. These results underscore the complexity of the process of differentiation in human mononuclear phagocytes, and provide insights into the variable capacity of mononuclear phagocytes to degrade extracellular matrix components. Moreover, we anticipate that human mononuclear phagocytes at various stages of differentiation will provide a useful model system for study of the variable regulation of secretion of human matrix-degrading metalloproteinases.     

The secretion of the tissue inhibitor of metalloproteinases (TIMP) by human synovial fibroblasts is modulated by all-trans-retinoic acid.

The matrix metalloproteinases are a family of enzymes involved in the turnover of the connective tissues. The regulation of these enzymes is complex, involving the control of synthesis, the activation of proenzyme forms and the presence of specific inhibitors. Retinoids have been reported to inhibit the production of metalloproteinases by human and rabbit synovial fibroblasts and by human skin fibroblasts. The production of the highly specific tissue inhibitor of metalloproteinases (TIMP) by connective tissue cells may be crucial in the regulation of connective tissue breakdown and this present study was undertaken to determine if retinoic acid (RA) could modulate TIMP and collagenase production by synovial fibroblasts. The results show that RA at concentrations from 10(-7) to 10(-5) M significantly stimulated the secretion of TIMP by two of three human synovial cell lines. The effect of mononuclear cell factor (MCF) on TIMP and collagenase levels was also investigated. The apparent reduction of collagenase levels in the presence of RA, could result from a failure to accurately measure this enzyme in the presence of increased levels of TIMP.