MMP-9 Synthesis Research Articles

Transforming growth factor-β suppresses tumor necrosis factor α-induced matrix metalloproteinase-9 expression in monocytes

The inflammatory response is marked by the release of several cytokines with multiple roles in regulating leukocyte activities, including the secretion of matrix metalloproteinases (MMPs). Although the effects of individual cytokines on monocyte MMP expression have been studied extensively, few studies have examined the influence of combinations of cytokines, which are likely present at inflammatory sites. Herein, we report our investigation of the combinatorial effects of tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta on MMP-9 synthesis. We found that TGF-beta suppressed TNF-alpha-induced MMP-9 secretion by MonoMac-6 monocytic cells in a dose-dependent manner, with a maximal effect of TGF-beta observed at 1 ng/ml. Such suppression was likely regulated at the pretranslational level, because steady-state mRNA levels of TNF-alpha-induced MMP-9 were reduced by TGF-beta, and pulse-chase radiolabeling also showed a decrease in new MMP-9 protein synthesis. The suppressive effects of TGF-beta were time dependent, because short exposures to TNF-alpha before TGF-beta or simultaneous exposure to both cytokines efficiently reduced MMP-9 secretion. Expression of the tissue inhibitor of metalloproteinases (TIMP)-1 and TNF-alpha receptors was unaffected by either cytokine individually or in combination. Affinity binding with radiolabeled TGF-beta demonstrated that levels of TGF-beta receptors were not increased after preincubation with TGF-beta. Suppression of TNFalpha-induced MMP-9 secretion by TGF-beta correlated with a reduction in prostaglandin E2 (PGE2) secretion. Furthermore, the effect of TGF-beta or indomethacin on blockage of TNF-alpha-stimulated MMP-9 production was reversed by the addition of either exogenous PGE2 or the cyclic AMP (cAMP) analogue Bt2cAMP. Thus, we concluded that TGF-beta acts as a potent suppressor of TNF-alpha-induced monocyte MMP-9 synthesis via a PGE2- and cAMP-dependent mechanism. These results suggest that various combinations of cytokines that are present at inflammatory sites, as well as their balance during different stages of inflammation, may provide the signals necessary for directing MMP-mediated leukocyte activities.

Skin and oral fibroblasts exhibit phenotypic differences in extracellular matrix reorganization and matrix metalloproteinase activity.

Oral mucosal wounds are characterized by rapid re-epithelialization and remodelling. In vitro, oral mucosal fibroblasts exhibit a fetal phenotype with increased extracellular matrix reorganizational ability, migration and experimental wound repopulation when compared with skin fibroblasts. To investigate whether phenotypic differences in the expression and production of matrix metalloproteinase (MMP) -2 and tissue inhibitors of metalloproteinases (TIMPs) could play an important part in mediating these in vitro differences. Skin and oral mucosal fibroblast MMP-2, TIMP-1 and TIMP-2 mRNA expression and protein production were studied in three-dimensional collagen lattices using quantitative competitive reverse transcriptase-polymerase chain reaction (QCRT-PCR), enzyme-linked immunosorbent assay (ELISA), zymography and reverse zymography. Oral mucosal fibroblasts exhibited increased levels of the 62-kDa active form of MMP-2 and lattice contraction when compared with skin fibroblasts. Oral mucosal and skin fibroblast MMP-2 gene expression and synthesis of the 72-kDa pro-MMP-2 was similar as assessed by QCRT-PCR, zymography and ELISA. Differential MMP-2 activation was, however, related to phenotypic differences in TIMP activity between the skin and oral mucosal fibroblasts, as assessed by reverse zymography. These studies propose a mechanism by which fibroblast phenotype may contribute directly to the observed preferential remodelling of oral wounds.

Expression of MMP2, MMP9, MT1-MMP, TIMP1, and TIMP2 mRNA in valvular lesions of the heart.

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play an important role in several diseases. This study was undertaken to investigate the mRNA synthesis of MMP2, MMP9, membrane-type 1 (MT1)-MMP, and matrix metalloproteinase inhibitors TIMP1 and TIMP2 by in situ hybridization in a set of heart mitral and aortic valves operatively removed due to degenerative or inflammatory valvular diseases. The material consisted of 21 valves, eight with endocarditis and 13 with a degenerative valvular disease. The samples were studied by in situ hybridization with specific probes for MMP2, MMP9, MT1-MMP, TIMP1, and TIMP2. Synthesis of MMP2 mRNA was found in seven valves, five with endocarditis and two with degenerative valvular disease. Signals for MMP9 mRNA were found in two cases with endocarditis and five cases with degenerative valvular disease. No signal for MT1-MMP mRNA was found in the lesions. TIMP1 mRNA, on the other hand, was found in 17 cases, both endocarditis and degenerative valvular disease. TIMP2 mRNA was found in three cases of endocarditis. The signals for MMP2, MMP9, TIMP1, and TIMP2 mRNA were localized in endothelial cells and in fibroblast-like cells expressing alpha-smooth muscle actin, thus showing myofibroblast-type differentiation. The results show that matrix metalloproteinases MMP2 and MMP9, and matrix metalloproteinase inhibitors TIMP1 and TIMP2 mRNAs are synthesized in diseased valves and suggest that they may contribute to matrix remodelling in valvular disease.

Cyclic tensile strain suppresses catabolic effects of interleukin-1beta in fibrochondrocytes from the temporomandibular joint.

To discern the effects of continuous passive motion on inflamed temporomandibular joints (TMJ). The effects of continuous passive motion on TMJ were simulated by exposing primary cultures of rabbit TMJ fibrochondrocyte monolayers to cyclic tensile strain (CTS) in the presence of recombinant human interleukin-1beta (rHuIL-1beta) in vitro. The messenger RNA (mRNA) induction of rHuIL-1beta response elements was examined by semiquantitative reverse transcriptase-polymerase chain reaction. The synthesis of nitric oxide was examined by Griess reaction, and the synthesis of prostaglandin E2 (PGE2) was examined by radioimmunoassay. The synthesis of proteins was examined by Western blot analysis of the cell extracts, and synthesis of proteoglycans via incorporation of 35S-sodium sulfate in the culture medium. Exposure of TMJ fibrochondrocytes to rHuIL-1beta resulted in the induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2), which were paralleled by NO and PGE2 production. Additionally, IL-1beta induced significant levels of collagenase (matrix metalloproteinase 1 [MMP-1]) within 4 hours, and this was sustained over a period of 48 hours. Concomitant application of CTS abrogated the catabolic effects of IL-1beta on TMJ chondrocytes by inhibiting iNOS, COX-2, and MMP-1 mRNA production and NO, PGE2, and MMP-1 synthesis. CTS also counteracted cartilage degradation by augmenting expression of mRNA for tissue inhibitor of metalloproteinases 2 that is inhibited by rHuIL-1beta. In parallel, CTS also counteracted rHuIL-1beta-induced suppression of proteoglycan synthesis. Nevertheless, the presence of an inflammatory signal was a prerequisite for the observed CTS actions, because fibrochondrocytes, when exposed to CTS alone, did not exhibit any of the effects described above. CTS acts as an effective antagonist of rHuIL-1beta by potentially diminishing its catabolic actions on TMJ fibrochondrocytes. Furthermore, CTS actions appear to involve disruption/regulation of signal transduction cascade of rHuIL-1beta upstream of mRNA transcription.

KiSS-1 Represses 92-kDa Type IV Collagenase Expression by Down-regulating NF-κB Binding to the Promoter as a Consequence of IκBα-induced Block of p65/p50 Nuclear Translocation

The 92-kDa type IV collagenase (MMP-9) plays a critical role in tissue remodeling. We undertook a study to determine whether the KiSS-1 gene, previously shown to suppress cancer spread (metastases), negatively regulates MMP-9 expression. Six cell lines positive for MMP-9 mRNA were deficient in KiSS-1 mRNA. One of these cell lines, HT-1080, stably transfected with a KiSS-1 expression construct, demonstrated substantially lower MMP-9 enzyme activity/protein and in vitro invasiveness. The lower MMP-9 enzyme activity reflected reduced steady-state mRNA levels which, in turn, was due to attenuated transcription. Activation of ERKs and JNKs by phorbol 12-myristate 13-acetate and tumor necrosis factor alpha, respectively, leading to increased MMP-9 amounts was not antagonized by KiSS-1 expression, suggesting that MAPK pathways modulating MMP-9 synthesis are not the target of KiSS-1. Although MMP-9 expression is regulated by AP-1, Sp1, and Ets transcription factors, KiSS-1 did not alter the binding of these factors to the MMP-9 promoter. However, NF-kappaB binding to the MMP-9 promoter required for expression of this collagenase was reduced by KiSS-1 expression. Diminished NF-kappaB binding reflected less p50/p65 in the nucleus secondary to increased IkappaBalpha levels in the cytosols of the KiSS-1 transfectants. Thus, KiSS-1 diminishes MMP-9 expression by effecting reduced NF-kappaB binding to the promoter.

Thrombin Suppresses Matrix Metalloproteinase 2 Activity and Increases Tissue Inhibitor of Metalloproteinase 1 Synthesis in Cultured Human Peritoneal Mesothelial Cells

Recently, high levels of intraperitoneally generated thrombin were found in the effluent of patients treated with continuous ambulatory peritoneal dialysis (CAPD). The aim of the present study was to investigate in human peritoneal mesothelial cells (HMCs) the effect of thrombin on the activity and synthesis of matrix metalloproteinases (MMPs), which regulate the degradation of basement membrane collagen. Cultured HMCs were isolated from omental tissue and used at confluence for the experiments. Conditioned media were obtained by incubating cells with serum-free M199 containing the relevant doses of thrombin. Activity of MMP-2 and MMP-9 were determined by an activity assay system. The antigen levels of MMPs and of the specific tissue inhibitor of metalloproteinases 1 (TIMP-1) were measured by ELISA. Northern blot analysis was applied to analyze mRNA expression of MMP-2 and TIMP-1. Incubation of HMCs with increasing doses of thrombin resulted in a concentration- and time-dependent suppression of MMP-2 activity. No changes in MMP-9 activity were seen. After a 48-hour stimulation period with thrombin (5 U/mL), MMP-2 activity decreased to 53% of that seen in control conditions. Antigen measurements revealed that this decrease was paralleled by a slight reduction in MMP-2 levels, which became significant at a thrombin dose of 5 U/mL [50.65 +/- 7.5 ng/10(5) cells (48 hours, 5 U/mL) vs 64.6 +/- 10.1 ng/10(5) cells (control)]. Under the same conditions, TIMP-1 levels were considerably increased [3.9 +/- 0.46 microg/10(5) cells (48 hours, 5 U/mL) vs 1.2 +/- 0.14 microg/10(5) cells (control)]. Hirudin (10 U/mL) completely inhibited the thrombin-induced effects on MMP-2 and TIMP-1 synthesis. These results were also reflected by Northern blot hybridization, where a slight decrease in MMP-2 and an increase in TIMP-1 mRNA expression were observed in response to thrombin. Our results suggest that high thrombin levels suppress MMP-2 activity through decreased MMP-2 and increased TIMP-1 synthesis. Thus, thrombin may promote the accumulation of basement membrane collagen. In addition to fibrin formation, this mechanism may represent a further contribution by thrombin to peritoneal thickening during CAPD.

The tripeptide-copper complex glycyl-L-histidyl-L- lysine-Cu 2+ stimulates matrix metalloproteinase-2 expression by fibroblast cultures

Glycyl-histidyl-lysine-Cu 2+ (GHK-Cu) is a tripeptide-copper complex known to be a potent wound healing agent. We previously showed its ability to stimulate in vitro and in vivo the synthesis of extracellular matrix components. The aim of this study was to determine the effects of GHK-Cu on MMP-2 synthesis by dermal fibroblasts in culture. We showed that GHK-Cu increased MMP-2 levels in conditioned media of cultured fibroblasts. This effect was reproduced by copper ions but not by the tripeptide GHK alone. This stimulation was accompanied by an increase of MMP-2 mRNA level. We also showed that GHK-Cu increased the secretion of the tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2. Taken together, our results underline that GHK-Cu is not only an activator of connective tissue production but also of the remodeling of the extracellular matrix. It is able to modulate MMP expression by acting directly on wound fibroblasts.

Lipoxin A4 inhibits IL-1 beta-induced IL-6, IL-8, and matrix metalloproteinase-3 production in human synovial fibroblasts and enhances synthesis of tissue inhibitors of metalloproteinases.

Lipoxins are a novel class of endogenous eicosanoid mediators that potently inhibit inflammatory events by signaling via specific receptors expressed on phagocytic cells. Animal models have shown that lipoxin A4 (LXA4) down-regulates inflammation in vivo. Here we demonstrate, for the first time, the expression of LXA4 receptors, and their up-regulation by IL-1 beta, in normal human synovial fibroblasts (SF). We examined whether exogenous LXA4 abrogated IL-1 beta stimulation of SF in vitro. IL-1 beta induced the synthesis of IL-6, IL-8, and matrix metalloproteinases (MMP)-1 and -3. At nanomolar concentrations, LXA4 inhibited these IL-1 beta responses with reduction of IL-6 and IL-8 synthesis, by 45 +/- 7% and 75 +/- 11%, respectively, and prevented IL-1 beta-induced MMP-3 synthesis without significantly affecting MMP-1 levels. Furthermore, LXA4 induced a 2-fold increase of tissue inhibitor of metalloproteinase (TIMP)-1 and a approximately 3-fold increase of TIMP-2 protein levels. LXA4 inhibitory responses were dose dependent and were abrogated by pretreatment with LXA4 receptor antiserum. LXA4-induced changes of IL-6 and TIMP were accompanied by parallel changes in mRNA levels. These results indicate that LXA4 in activated SF inhibits the synthesis of inflammatory cytokines and MMP and stimulates TIMP production in vitro. These findings suggest that LXA4 may be involved in a negative feedback loop opposing inflammatory cytokine-induced activation of SF.

A deficit in collagenase activity contributes to impaired migration of aged microvascular endothelial cells

Angiogenesis is impaired in aging. Delayed neovascularization is due, in part, to slowed endothelial cell migration. Migration requires an optimal level of adhesion to matrix proteins, a process mediated by matrix-degrading metalloproteases (MMPs) such as MMP1. To determine whether impaired angiogenesis in aging is associated with altered synthesis and activity of MMP1, we examined the expression of collagenase and tissue inhibitor of metalloprotease 1 (TIMP1) by immunostain of angiogenic sponge implants from young and aged mice. To characterize the relevance of MMP activity during the movement of aged endothelial cells, the secretion of MMP1 and TIMP1 by late-passage human microvascular endothelial cells (hmEC aged in vitro) and their non-aged (young) counterparts was quantified. The migration of aged human microvascular endothelial cells and the effect of inhibition of TIMP1 on the migration of aged hmEC or collagen I was also measured. Relative to young mice, granulation tissue from aged mice showed less expression of collagenase and increased expression of TIMP1. In vitro, aged hmEC were deficient in MMP1 secretion (55 +/- 13% relative to young cells) and activity but showed increased expression of TIMP1 (280 +/- 109% relative to young cells). Aged hmEC migrated significantly less distance than did young hmEC over a 5-day period (59 +/- 8% relative to young cells). In the presence of a blocking antibody to TIMP1, aged hmEC showed a significant increase in the distance migrated on collagen I over a 5 day period (142 +/- 11% relative to untreated aged hmEC). We propose that deficient MMP1 activity contributes to impaired cellular movement in aged microvascular endothelial cells and that perturbations that enhance collagenase activity increase their migratory ability and angiogenic potential.

Elevated matrix metalloprotease and angiostatin levels in integrin alpha 1 knockout mice cause reduced tumor vascularization.

Integrin alpha1beta1 is a collagen receptor abundantly expressed on microvascular endothelial cells. As well as being the only collagen receptor able to activate the Ras/Shc/mitogen-activated protein kinase pathway promoting fibroblast cell proliferation, it also acts to inhibit collagen and metalloproteinase (MMP) synthesis. We have observed that in integrin alpha1-null mice synthesis of MMP7 and MMP9 was markedly increased compared with that of their wild-type counterparts. As MMP7 and MMP9 have been shown to generate angiostatin from circulating plasminogen, and angiostatin acts as a potent inhibitor of endothelial cell proliferation, we determined whether tumor vascularization was altered in the alpha1-null mice. Tumors implanted into alpha1-null mice showed markedly decreased vascularization, with a reduction in capillary number and size, which was accompanied by an increase in plasma levels of angiostatin due to the action of MMP7 and MMP9 on circulating plasminogen. In vitro analysis of alpha1-null endothelial cells revealed a marked reduction of their proliferation on both integrin alpha1-dependent (collagenous) and independent (noncollagenous) substrata. This reduction was prevented by culturing alpha1-null cells with plasma derived from plasminogen-null animals, thus omitting the source from which to generate angiostatin. Plasma from tumor-bearing alpha1-null animals uniquely inhibited endothelial cell growth, and this inhibition was relieved by the coaddition of either MMP inhibitors, or antibody to angiostatin. Integrin alpha1-deficient mice thus provide a genetically characterized model for enhanced angiostatin production and serve to reveal an unwanted potential side effect of MMP inhibition, increased tumor angiogenesis.

Balance in asthma between matrix metalloproteinases and their inhibitors

Balance in asthma between matrix metalloproteinases and their inhibitors

Retinoid-mediated suppression of tumor invasion and matrix metalloproteinase synthesis.

Cancer mortality usually results from the tumor invading the local environment and metastasizing to vital organs, e.g. liver, lung, and brain. Degradation of the extracellular matrix is, therefore, the sine qua non of tumor cell invasion. this degradation is mediated mainly by MMPs, and thus, inhibition of MMP synthesis is a target for anticancer agents. Tumor cells must traverse both the basement membrane (type IV collagen) and the interstitial stroma (type I collagen). Therefore, we used scanning electron microscopy to examine the invasive behavior of several aggressive tumor cell lines, A2058 melanoma cells, and SCC and FaDu squamous cell carcinomas through these matrices; and we monitored the ability of all-trans retinoic acid and several RAR-specific ligands to block invasion. We demonstrate that several retinoids, which are specific RAR alpha, beta, or gamma agonists/antagonists, selectively inhibited MMP synthesis in the three tumor cell lines. However, there was not a common pattern of MMP inhibition by a particular retinoid. For instance, a RAR alpha antagonist suppressed MMP-1 and MMP-2 synthesis in the melanoma cell line, but not in the FaDu or SCC-25 cells. On the other hand, synthesis of MMP-1 and MMP-9 by the FaDu cells was affected hardly at all, while a RAR gamma antagonist reduced the levels of MMP-2. Only all-trans retinoic acid reduced MMP-1 synthesis in these cells. We postulate that the differences may be related to a differential pattern of RAR expression in each of these cells, and that the RARs expressed by each cell line may not be targets of these RAR specific compounds. All-trans retinoic acid is a pan ligand, binding to all three RARs and, therefore, may modulate gene expression more generally. We conclude that the power of these new ligands lies in their specificity, which can be directed towards modulating expression of certain RARs and, thus, of certain MMPs. By blocking MMP synthesis, retinoids may be effective in cancer therapy by decreasing tumor invasiveness.

Matrix Metalloproteinase 2 (MMP2) and MMP9 Are Produced by Kidney Collecting Duct Principal Cells but Are Differentially Regulated by SV40 Large-T, Arginine Vasopressin, and Epidermal Growth Factor

We analyzed the expression and regulation of matrix metalloproteinase 2 (MMP2) and MMP9 gelatinases in a rabbit kidney collecting duct principal cell line (RC.SVtsA58) (Prié, D., Ronco, P. M., Baudouin, B., Géniteau-Legendre, M., Antoine, M., Piedagnel, R., Estrade, S., Lelongt, B., Verroust, P. J., Cassingéna, R., and Vandewalle, A. (1991) J. Cell Biol. 113, 951-962) infected with the temperature-sensitive (ts) SV40 strain tsA58. At the permissive temperature (33 degreesC), cells produced only MMP2. Shifting cells to a nonpermissive temperature (39.5 degreesC) induced a marked increase in total gelatinolytic activity due to an increase of MMP2 and an induction of MMP9 synthesis. This effect was attributed to large-T inactivation at 39.5 degreesC because it was abolished by re-infecting the cells with wild-type SV40 strain LP. Run-on experiments showed that negative regulation of MMP2 and MMP9 by large-T was transcriptional and posttranscriptional, respectively. MMP2 and MMP9 were also produced by primary cultures of collecting duct cells. In rabbit kidney, both MMP2 and MMP9 were almost exclusively expressed in collecting duct cells, where an unexpected apical localization was observed. Arginine vasopressin and epidermal growth factor, which exert opposite hydroosmotic effects in the collecting duct, also exhibited contrasted effects on MMP9 synthesis. Epidermal growth factor increased but arginine vasopressin suppressed MMP9 at a posttranscriptional level, whereas MMP2 was not affected. These results suggest a specific physiological role of MMP2 and MMP9 in principal cells of renal collecting duct.

Inhibition of activator protein 1 activity by paclitaxel suppresses interleukin-1-induced collagenase and stromelysin expression by bovine chondrocytes.

Cytokine-induced collagenase 1 (matrix metalloproteinase 1 [MMP-1]) and stromelysin 1 (MMP-3) expression is dependent on activator protein 1 (AP-1) activation and have a fundamental role in the pathophysiology of arthritic diseases by degrading connective tissues. This study evaluates the effect of paclitaxel on AP-1 activation and examines its effect on the expression of 2 major matrix metalloproteinases, MMP-1 and MMP-3, and its effect on AP-1 activation. MMP-1, MMP-3, c-fos, and c-jun messenger RNA (mRNA) levels were measured in interleukin-1 (IL-1)-induced primary chondrocytes in the presence and absence of paclitaxel. The effect of paclitaxel on AP-1 promoter activity was studied by chloramphenicol acetyltransferase assays in IL-1-stimulated chondrocytes. The same conditions were applied to studies of the effect of paclitaxel on binding at the AP-1 site by gel-shift mobility assays. The cytotoxicity effect of paclitaxel on chondrocytes was studied by examining cell viability and expression of the matrix molecules aggrecan and type II collagen. IL-1-induced MMP-1 and MMP-3 mRNA levels were markedly reduced in paclitaxel-treated chondrocytes. Further, IL-1-induced AP-1 activation and AP-1 binding were inhibited by paclitaxel. However, there was no effect on the expression of c-fos or c-jun mRNA levels. Chondrocyte viability was not affected by paclitaxel, and there was no effect on the expression of housekeeping genes or the major cartilage matrix molecules aggrecan and type II collagen. These studies demonstrate that paclitaxel is a potent inhibitor of MMP-1 and MMP-3 synthesis through the AP-1 site. However, inhibition of AP-1 activity by paclitaxel does not affect the viability of chondrocytes or the expression of matrix molecules.

Small Mechanical Strains Selectively Suppress Matrix Metalloproteinase-1 Expression by Human Vascular Smooth Muscle Cells

Mechanical forces and biochemical stimuli may interact to regulate cellular responses. In this study, we tested the hypothesis that very small mechanical strains interact with growth factors in the regulation of matrix metalloproteinase (MMP)-1. Human vascular smooth muscle cells (VSMCs) were cultured on a precoated silicone membrane in a device that imposes a highly uniform biaxial strain. VSMCs cultured on fibronectin were treated with cyclic 1-Hz strains of 0, 1, or 4%, and MMPs were assayed by Western analysis or gelatin zymography. Small strains did not induce MMP-1 in VSMCs, but strain was a potent inhibitor of platelet-derived growth factor (PDGF)- or tumor necrosis factor-alpha-induced synthesis of MMP-1. In contrast, MMP-2 and TIMP-2 levels were not changed by PDGF and/or mechanical strain. VSMCs strained on the 120-kDa chymotryptic fragment of fibronectin or RGD peptides suppressed PDGF-induced expression of MMP-1, indicating that this effect is not mediated by the heparin-binding domain or connecting segment-1 of fibronectin. Northern analysis of ets-1, a transcriptional activator of MMP-1 expression, showed that strain down-regulated ets-1 expression, whereas c-fos expression was augmented. Thus, small deformations can selectively suppress MMP-1 synthesis by VSMCs, demonstrating the exquisite sensitivity of the cell to mechanical stimuli.

Regulation of matrix metalloproteinase expression in human vascular smooth muscle cells by T lymphocytes: a role for CD40 signaling in plaque rupture?

Physical disruption of an atheromatous lesion often underlies acute coronary syndromes. Matrix-degrading enzymes, eg, matrix metalloproteinases (MMPs), may cause loss in mechanical integrity of plaque tissue that favors rupture. T lymphocytes accumulate at sites where atheromata rupture, but the mechanisms by which these immune cells may contribute to plaque destabilization are unknown. This study tested the hypothesis that the T-lymphocyte surface molecule CD40 ligand (CD40L), recently localized in atherosclerotic plaques, regulates the expression of MMPs in human vascular smooth muscle cells (SMCs), the most numerous cell type in arteries. We report here that stimulated human T lymphocytes induced the expression of the matrix-degrading enzymes, ie, interstitial collagenase (MMP-1), stromelysin (MMP-3), gelatinase B (MMP-9), and activated gelatinase A (MMP-2), in human vascular SMCs by cell contact via CD40 ligation, as demonstrated by Western blot analysis, zymography, and antibody neutralization. Recombinant human CD40L (rCD40L) induced de novo synthesis of MMP-1, MMP-3, and MMP-9 on vascular SMCs and stimulated the expression of these enzymes to a greater extent than did maximally effective concentrations of tumor necrosis factor-alpha or interleukin-1beta, established agonists of MMP expression. Interferon gamma, another T-lymphocyte- derived cytokine, inhibited the induction of MMPs by rCD40L. Immunohistochemical analysis of human coronary atheromata colocalized MMP-1 and MMP-3 with CD40-positive SMCs. These results demonstrated that CD40 ligand, expressed on T lymphocytes, promoted the expression of matrix-degrading enzymes in vascular SMCs and thus established a new pathway of immune-modulated destabilization in human atheromata.

The Nuclear Receptor Corepressor SMRT Inhibits Interstitial Collagenase (MMP-1) Transcription through an HRE-Independent Mechanism

Nuclear receptors inhibit synthesis of collagenase-1 (matrix metalloproeinase-1; MMP-1), an enzyme that degrades interstitial collagens and contributes to joint pathology in rheumatoid arthritis. SMRT (Silencing Mediator for Retinoid and Thyroid hormone receptors) mediates the repressive effect of nuclear receptors at hormone responsive elements (HREs), prompting us to investigate whether this co-repressor could also regulate transcription of MMP-1, which lacks any known HREs. We find that primary synovial fibroblasts express SMRT. When over-expressed by transient transfection, SMRT inhibits MMP-1 promoter activity induced by interleukin-1 (IL-1), phorbol phorbol myristate acetate (PMA) or v-Src. SMRT apparently inhibits MMP-1 gene expression by interfering with one or more transcriptional elements clustered in a region between −321 and +63. We conclude that SMRT negatively regulates MMP-1 synthesis through a novel, HRE-independent mechanism that involves proximal regions of the MMP-1 promoter.

Epidermal growth factor and amphiregulin up-regulate matrix metalloproteinase-9 (MMP-9) in human breast cancer cells.

The EGF family of proteins encompasses several polypeptides such as epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), amphiregulin (AR) and heregulin (HRG-beta 1). These polypeptides regulate proliferation in breast cancer cells through interaction with membrane receptors. It has been previously shown that high EGF receptor number correlates with aggressive behavior and increased metastasis in human breast cancer. In the present study, we investigated the association between EGF and EGF-like ligand-induced DNA synthesis and secretion of MMP-9 and MMP-2 in metastatic SKBR-3 and non-metastatic MCF-7 breast cancer cells. Exposure of SKBR-3 cells to EGF or AR induces expression of MMP-9 but has no effect on MMP-2 secretion. In contrast to EGF and AR, HRG had no effect on gelatinase induction. None of the EGF polypeptides had any effect on gelatinase induction in MCF-7 non-metastatic breast cancer cells. While a relatively specific inhibitor of EGF receptor tyrosine kinase, PD 153035, inhibited EGF-, AR- and HRG-induced cell proliferation, it had no effect on MMP-9 induced by EGF and AR. Experimental evidence suggests that signaling mechanisms for cell proliferation and MMP-9 induction are mediated by different pathways down-stream of EGF receptor autophosphorylation or that low levels of EGF-induced signal that escape inhibition are sufficient to induce MMP-9 but unable to support cell proliferation. In addition, our results suggest that EGF and AR may modulate invasion of metastatic breast cancer cells by increasing the expression of MMPs.

Phenotypes of human esophageal squamous cell carcinoma based on matrix metalloproteinase production

We investigated the effects of epidermal growth factor (EGF) and transforming growth factor-beta (TGF-beta) on cell growth and on regulation of matrix metalloproteinases (MMPs) in four cell lines of human esophageal squamous cell carcinomas (TE8, TE9, TE10, and TE11). EGF stimulated the production of proforms of gelatinase B (MMP-9) by three cell lines that could synthesize EGF by themselves, with TE9 being the exception. Particularly, both the production of MMP-9 and DNA synthesis in TE10 were stimulated significantly by EGF. TGF-beta slightly stimulated DNA synthesis in two cell lines, TE9 and TE11, and TGF-beta secretion by TE9 was detected. The production of proforms of gelatinases A (MMP-2) and MMP-9 was gradually induced by TGF-beta in a concentration-dependent manner in all the cell lines except for TE9. Flow cytometric analysis revealed that all the lines expressed both EGF- and TGF-beta-receptors. In conclusion, our present results indicate that at least there are possibly two distinct phenotypes in human esophageal squamous cell carcinoma: one (TE10) depends on autocrine EGF production that enhances DNA synthesis and MMP-9 production; and the other (TE9) on autocrine TGF-beta that stimulates DNA synthesis but not in relation to gelatinase production.

Expression of messenger RNAs for metalloproteinases 2 and 9, type IV collagen, and laminin in nonneoplastic and neoplastic endometrium

In this study, we analyzed the messenger RNA (mRNA) synthesis of matrix metalloproteinases (MMPs) 2 and 9, and their main substrates laminin and type IV collagen by in situ hybridization in nonneoplastic, hyperplastic, and neoplastic endometrial tissues. In nonneoplastic endometrium stromal synthesis of MMP2, laminin and type IV collagen messenger RNA (mRNA) could be detected throughout the cycle. Their synthesis was strongest in the late secretory and decidualized endometrium. In epithelial cells, only laminin mRNA synthesis was observed. MMP9 mRNA was expressed in only stromal and epithelial cells of the decidualized endometrium. In hyperplastic and neoplastic endometrium, the expression of MMP2, MMP9, laminin, and type IV collagen mRNA was variably present in stromal cells, whereas epithelial cells expressed only laminin mRNA, except for one case of a grade III carcinoma, which also showed signals for MMP2 and MMP9 mRNAs in the carcinoma cells. The presence of MMP2, laminin, and IV collagen mRNA in normal endometrium reflects their importance in the tissue response and matrix remodeling during the proliferative and secretory phases of the menstrual cycle. Their mRNA synthesis follows a similar pattern, suggesting that it is associated with hormonal changes during the menstrual cycle. According to the results, MMP9 does not participate in tissue remodeling during the secretory or proliferative phases, but like MMP2, it is found in the neoplastic endometrial stroma, suggesting that it contributes to the invasion of malignant endometrial cells.