Gene expression of matrix metalloproteinase-1 (interstitial collagenase) and matrix metalloproteinase-3 (stromelysin-1) in basal cell carcinoma by in situ hybridization using chondroitin ABC lyase

We studied the relation between the plasma concentration of brain natriuretic peptide (BNP) and echocardiographic findings to determine the sensitivity of BNP as an indicator of left-ventricular dysfunction in elderly patients with various cardiovascular diseases. The plasma concentration of BNP was positively correlated with left-ventricular end-diastolic and end-systolic dimensions (LVEDD and LVESD, respectively) and inversely correlated with the left-ventricular ejection fraction (LVEF) in patients with prior myocardial infarction, dilated cardiomyopathy, and valvular heart disease. The plasma concentration of BNP decreased significantly in association with an increase in LVEF and decreases in LVEDD and LVESD in patients with congestive heart failure following therapy. These observations indicate that the plasma concentration of BNP is a sensitive marker of impaired left-ventricular function in elderly patients with various cardiovascular diseases and may be useful for evaluating the improvement in left-ventricular function and the efficacy of therapy in patients with congestive heart failure.     

Enhancement of fibroblast collagenase (matrix metalloproteinase-1) gene expression by ceramide is mediated by extracellular signal-regulated and stress-activated protein kinase pathways

Inflammatory cytokines tumor necrosis factor-alpha and interleukin-1 trigger the ceramide signaling pathway, initiated by neutral sphingomyelinase-elicited hydrolysis of cell membrane phospholipid sphingomyelin to ceramide, a new lipid second messenger. Here, we show that triggering the ceramide pathway by sphingomyelinase or C2- and C6-ceramide enhances collagenase-1 (matrix metalloproteinase-1; MMP-1) gene expression by fibroblasts. C2-ceramide activates three distinct mitogen-activated protein kinases (MAPKs) in dermal fibroblasts, i.e. extracellular signal-regulated kinase 1/2 (ERK1/2), stress-activated protein kinase/Jun N-terminal-kinase (SAPK/JNK), and p38. Stimulation of MMP-1 promoter activity by C2-ceramide is dependent on the presence of a functional AP-1 cis-element and is entirely inhibited by overexpression of MAPK inhibitor, dual specificity phosphatase CL100 (MAPK phosphatase-1). Activation of MMP-1 promoter by C2-ceramide is also effectively inhibited by kinase-deficient forms of ERK1/2 kinase (MEK1/2) activator Raf-1, ERK1 and ERK2, SAPK/JNK activator SEK1, or SAPKbeta. In addition, ceramide-dependent induction of MMP-1 expression is potently prevented by PD 98059, a selective inhibitor of MEK1 activation, and by specific p38 inhibitor SB 203580. These results show that triggering the ceramide signaling pathway activates MMP-1 gene expression via three distinct MAPK pathways, i.e. ERK1/2, SAPK/JNK, and p38, and suggest that targeted modulation of the ceramide signaling pathway may offer a novel therapeutic approach for inhibiting collagenolytic activity, e.g. in inflammatory disorders.     

An autocrine/paracrine role of human decidual relaxin. I. Interstitial collagenase (matrix metalloproteinase-1) and tissue plasminogen activator

Decidual and placental relaxins have been proposed as autocrine/ paracrine hormones in the remodeling of collagen in the amnion and chorion in the last weeks of pregnancy. The matrix metalloproteinase-1 (MMP-1) is a key enzyme in the degradation of the interstitial collagens which predominate in the fetal membranes. Distribution of the MMP-1 gene and of the MMP-1 protein was shown by in situ hybridization and immunolocalization, respectively, in amnion, chorion, and decidua collected from patients before the onset of spontaneous labor. The distribution of MMP-1 in the chorionic cytotrophoblast and decidua coincided with that of the human relaxin receptor, detected by tissue section autoradiography in tissues collected at the same stage of pregnancy. Fetal membrane explants were used to study the effect of exogenous human relaxin H2. These responded by a dose-dependent increase in expression of the MMP-1 gene, in its secreted protein, and in its enzyme activity in the medium. A similar dose-dependent increase in the tissue plasminogen activator (tPA) gene and protein upon exposure of the explants to relaxin H2 suggested a coordinated cascade system, resulting in increases in secreted activities of MMP-1, MMP-3 (stromelysin), and MMP-9 (gelatinase B). There was no effect on the genes or proteins for MMP-2 (gelatinase A) or tissue inhibitor of metalloproteinase-1 (TIMP-1), showing the specificity of the response. This coordinated regulation by relaxin H2 of tPA, MMP-1, MMP-3, and MMP-9 would result in more complete degradation of the fetal membrane extracellular matrix components.     

Immunolocalization of interstitial collagenase (MMP-1) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in radicular cysts

To investigate the mechanisms involved in expansion of radicular cysts, monoclonal antibodies against interstitial collagenase (MMP-1) and tissue inhibitor of metalloproteinases-1 (TIMP-1) were used to localize the sites of MMP-1 and TIMP-1 expression in 30 radicular cysts. Positive MMP-1 staining was detected in the lining epithelium and subepithelial fibroblasts, macrophages, endothelial cells and osteoblasts/osteocytes in all specimens. Positive TIMP-1 staining was identified in osteoblasts/osteocytes and endothelial cells of all specimens, and in the lining epithelium and subepithelial fibrous connective tissue wall of five radicular cysts with an intense inflammatory cell infiltrate. The number and distribution of positive cells for MMP-1 or TIMP-1 varied widely among individual specimens, but strong immunostaining was constantly detected at sites with prominent subepithelial inflammation. Results here support the hypothesis that MMP-1 may play an important role in the expansion of radicular cysts. The absence of TIMP-1 expression in lining epithelium and subepithelial fibroblasts and macrophages in most cases studied indicated that an imbalance between MMP-1 and TIMP-1 production may lead to radicular cyst expansion.     

Membrane-type matrix metalloproteinase-1 expression at the site of human placentation

Human trophoblast implantation is a highly regulated process of invasion that requires action of proteolytic enzymes to degrade extracellular matrix components of the endometrium. Among these enzymes, matrix metalloproteinases (MMPs) seem to be particularly important in this degradative process. We previously showed that gelatinase A is extensively expressed in vivo in the human placenta. A new MMP, MT-MMP-1 (membrane-type matrix metalloproteinase-1), which is thought to activate progelatinase A, has recently been described. In this study, we examined the expression of MT-MMP-1, by immunohistochemistry and in situ hybridization, in human placental bed biopsies taken during the first trimester of gestation. Human first trimester intermediate trophoblasts synthesized MT-MMP-1 mRNAs and the protein. The MT-MMP-1 pattern of distribution in placental beds was similar to that of gelatinase A, suggesting a pivotal role for MT-MMP-1 in placentation, perhaps by activating progelatinase A.     

Expression of 72-kDa gelatinase (matrix metalloproteinase-2) in the developing mouse craniofacial complex

Tissue remodelling is an important feature during embryogenesis. Although the matrix metalloproteinases are believed to participate in these processes, the relation between matrix metalloproteinases and tissue remodelling during craniofacial morphogenesis remains unclear. The purpose of the study was to look for the presence of enzymes involved in extracellular matrix degradation during craniofacial morphogenesis. Protein expression of the matrix metalloproteinase, 72-kDa gelatinase (matrix metalloproteinase-2, gelatinase A, 72-kDa type IV collagenase) was studied by gelatine zymography and by indirect immunofluorescence with conventional and confocal microscopy. In the anterior region of the developing mouse face, 72-kDa gelatinase was labelled mainly in the tips and peripheral regions of the nasal and facial prominences. Upon contact and fusion of the prominences, the staining was intensely localized to the zone of the fusion and the tips and peripheral regions of the nasal prominences and the maxilla. The labelling of 72-kDa gelatinase was also present in the peripheral regions of the mandible, second branchial arch, and the face around the developing eye. However, during lens vesicle formation, the staining of 72-kDa gelatinase was absent in the invaginated lens ectoderm. After the lens had completely detached from the surface ectoderm, the staining was resumed in the corneal epithelium and mesenchyme. Gelatine zymography was used to confirm the presence of active and latent 72-kDa gelatinase in the developing mouse craniofacial complex. Collectively, these data indicate that 72-kDa gelatinase may play a significant part in localized tissue remodelling during craniofacial morphogenesis and the aberrant expression or function of the enzyme could be involved in causing facial abnormalities.     

Ambient pressure stimulates immortalized human aortic endothelial cells to increase DNA synthesis and matrix metalloproteinase 1 (tissue collagenase) production

In the present study, we investigated the effect of ambient pressure on [3H]-thymidine incorporation and on the production of matrix metalloproteinase 1 (tissue collagenase/proMMP-1) using human aortic endothelial cells immortalized with simian virus 40 (SE-1). Incubation of cells at ambient pressures of 50 and 100 mmHg for 24 h slightly increased [3H]-thymidine incorporation when directly compared with normal culture conditions. The amount of [3H]-thymidine incorporated in SE-1 reached a maximum at 150 mmHg, while a further increase in pressure to 200 mmHg decreased incorporation. The same ambient pressure slightly stimulated human aortic intimal smooth muscle cells (SMC) to increase [3H]-thymidine incorporation but not medial SMC. Immunoblot analysis also showed that ambient pressure, ranging from 50 to 200 mmHg, like 12-O-tetradecanoyl-phorbol-13-acetate stimulated SE-1 to produce proMMP-1, an effect not seen with either intimal or medial SMC. The amount of proMMP-1 produced also reached a maximum level at 150 mmHg. We postulate that human endothelial cells are ambient pressure sensitive and that relatively lower ambient pressures play an important role in the growth of endothelial cells, while higher pressures injure endothelial cells, resulting in the initiation of atherosclerosis. This cell line may prove useful in the investigation of both the physiological and pathological roles of blood pressure on endothelial cell function.     

Influence of heparin(s) on the interleukin-1-beta-induced expression of collagenase, stromelysin-1, and tissue inhibitor of metalloproteinase-1 in human gingival fibroblasts

Here, we describe the influence of heparin(s) on the interleukin-1-beta (IL-1beta)-induced expression of collagenase (matrix metalloproteinase-1, MMP-1), stromelysin-1 (matrix metalloproteinase-3, MMP-3) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in human gingival fibroblasts (HGF). Amounts of secreted enzymes and inhibitors as well as their mRNA steady-state levels increased significantly following supplementation of HGF culture medium with 2 ng/mL of IL-1 beta1. Addition of heparin to cell culture medium 1 hour following IL-1beta decreased MMP and TIMP-1 expression in a dose-dependent manner. The inhibitory effect of heparin was significant at a concentration as low as 1 microg/mL. These findings could be reproduced with a low Mr heparin fragment devoid of anticoagulant activity. Heparin and fragments might therefore reduce the excessive proteolytic capacity of the gingival fibroblast during inflammation and could be useful as pharmacological agent(s) in gingivitis and periodontitis.     

Presence of matrix metalloproteinase-9 activity in the cerebrospinal fluid of human immunodeficiency virus-infected patients

To determine whether matrix metalloproteinase (MMP)-9 is a potential mediator involved in the frequently detected blood-brain barrier leakage in human immunodeficiency virus (HIV)-infected patients, zymography was used to detect MMP-9 activity in cerebrospinal fluid (CSF) samples of 80 HIV-infected patients and of 10 control patients. CSF MMP-9 activity was detected in 40% of HIV-infected patients (but not in controls) and was significantly more frequent in HIV-infected patients than in those without neurologic deficits (50% vs. 13.6%). The frequency of CSF MMP-9 activity did not significantly differ between neurologically symptomatic HIV-infected patients with or without opportunistic central nervous system disease (51.6% vs. 48.1%). Additionally, the presence of CSF MMP-9 activity in HIV-infected patients was associated with an increased CSF white blood cell count and an elevated CSF-to-serum albumin ratio, suggesting that it may play a role in blood-brain/CSF barrier leakage in HIV-infected patients.     

Increased matrix metalloproteinase-2 activity induced by TGF-beta 1 in duct cells of human salivary gland is associated with the development of cyst formation in vivo

Proteolytic enzyme activity has been shown to be important for cyst formation. In this study, we constructed a cyst-like structure in vivo and analyzed molecular mechanisms involved in the development of the lesion. When SV40-immortalized duct cells of normal human salivary gland (NS-SV-DC) were treated with TGF-beta 1 at a concentration of 1 ng/ml or 5 ng/ml followed by co-inoculation with Matrigel into the backs of nude mice, they formed large cysts containing fluid when 5 ng/ml of TGF-beta 1 was used. Analysis of the fluid demonstrated high MMP activity. Immunohistochemical staining exhibited strong reactivity with anti-MMP-2 antibody in TGF-beta 1 (5 ng/ml)-treated NS-SV-DC. Northern blot analysis indicated that the expression of TGF-beta 1 and MMP-2 mRNAs in cells was greatly enhanced by treatment with 5 ng/ml TGF-beta 1. These findings suggest that the in vivo cyst formation by TGF-beta 1-treated cells is associated with continuous induction of MMP-2 activity.     

Production of membrane-type matrix metalloproteinase-1 (MT-MMP-1) in early human placenta. A possible role in placental implantation?

mRNA for a newly discovered isoform of calpain, termed Lp82, was recently discovered in young rat lens. The purpose of the present experiments was to test for expression of Lp82 protein. Casein zymography after incubation with calcium was used to detect Lp82 proteolytic activity in regions of lenses from young rats. Lp82 protein was detected by immunoblotting or by ELISA after DEAE-5PW chromatography using a polyclonal antibody generated to a peptide sequence in Lp82. Northern blot analysis assessed expression of Lp82 mRNA. Four results demonstrated expression of Lp82 protein; (1) immunoblot reactivity at the predicted molecular mass of 82 kDa, (2) a unique band of calcium-activated lysis in casein zymograms, (3) partial purification and retention of activity from a single Lp82 peak on DEAE-5PW chromatography, and (4) positive immunoblotting and Northern blot analysis only in lens and not in other rat tissues. These results showed that Lp82 protein is lens-preferred, relatively abundant in young rats (especially nucleus), and enzymatically active. Proteolysis of crystallins in the nucleus of young rat lens during normal maturation and cataract formation, formerly attributed solely to m-calpain, may in fact be due to concerted action of both lens Lp82 and ubiquitous m-calpain.     

Kinetic analysis of the binding of human matrix metalloproteinase-2 and -9 to tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2

The dissociation constants (Kd) of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 for the active and latent forms of matrix metalloproteinase (MMP)-2 and MMP-9 were evaluated using surface plasmon resonance (SPR) and enzyme inhibition studies. SPR analysis shows biphasic kinetics with high (nM) and low (microM) affinity binding sites of TIMP-2 and TIMP-1 for MMP-2 (72- and 62-kDa species) and MMP-9 (92- and 82-kDa species), respectively. In contrast, binding data of TIMP-2 to an MMP-2 45-kDa active form lacking the C-terminal domain and to an MMP-2 C-terminal domain (CTD) fragment displays monophasic kinetics with Kd values of 315 and 60 nM, respectively. This suggests that the CTD contains the high affinity binding site, whereas the catalytic domain contains the low affinity site. Also, binding of TIMP-2 to pro-MMP-2 is stronger at both the high and low affinity sites than the corresponding binding of TIMP-2 to the MMP-2 62-kDa form demonstrating the importance of the N-terminal prodomain. In addition, the Kd value of TIMP-1 for the MMP-2 62-kDa species is 28. 6 nM at the high affinity site, yet neither the MMP-2 45-kDa species nor the CTD interacts with TIMP-1. Enzyme inhibition studies demonstrate that TIMPs are slow binding inhibitors with monophasic inhibition kinetics. This suggests that a single binding event results in enzyme inhibition. The kinetic parameters for the onset of inhibition are fast (kon approximately 10(5) M-1 s-1) with slow off rates (koff approximately 10(-3) s-1). The inhibition constants (Ki) are in the 10(-7)-10(-9) M range and correlate with the values determined by SPR.     

Expression of collagen, interstitial collagenase, and tissue inhibitor of metalloproteinases-1 in restenosis after carotid endarterectomy

Extracellular matrix is the principal component of the fibrous caps of atherosclerotic plaques and intimal hyperplastic lesions of reconstructed arteries. Interstitial collagen form an important part of the matrix, and the balance between collagen synthesis and degradation by interstitial collagenase (matrix metalloproteinase-1, MMP-1) may determine whether plaques rupture or vessels develop stenosis. We examined type I procollagen gene expression in human atherosclerotic and restenotic carotid arteries using in situ messenger RNA (mRNA) hybridization and the expression of MMP-1 and its endogenous inhibitor (tissue inhibitor of metalloproteinases-1, TIMP-1) by immunohistochemistry. Compared with normal arteries, atherosclerotic plaques bed increased expression of immunoreactive MMP-1 and TIMP-1 with modest increase of type 1 procollagen mRNA. Early restenotic lesions (< 1.5 years) contained abundant type I procollagen mRNA but little immunoreactive MMP-1 and TIMP-1. Late restenotic lesions (> 4 years) resembled atheroma and exhibited increased immunoreactive MMP-1 and TIMP-1 as well as abundant type I procollagen mRNA. Compared with atherosclerotic plaques, type I procollagen is increased and MMP-1 is decreased in early restenotic lesions. MMP-1 and TIMP-1 expressions are upregulated in lesions with a clear atheroma. These findings suggest that the balance between proteolysis and matrix synthesis may influence both the stability of atheromatous plaques and the development of restenotic lesions.     

Collagenase-3 (matrix metalloproteinase-13) expression is induced in oral mucosal epithelium during chronic inflammation

Increased proliferation of mucosal epithelium during inflammation is associated with degradation of subepithelial connective tissue matrix and local invasion of the epithelial cells. Here we have studied, whether collagenase-3 (MMP-13), a collagenolytic matrix metalloproteinase with an exceptionally wide substrate specificity, is expressed in the epithelium of chronically inflamed mucosa. Examination of human gingival tissue sections from subjects with chronic adult periodontitis with in situ hybridization revealed marked expression of MMP-13 in basal cells of some epithelial rete ridges expanding into connective tissue. Immunohistochemical staining demonstrated that these cells also expressed strongly laminin-5, suggesting that they are actively migrating cells. A strong signal for MMP-13 mRNA was occasionally also noted in the suprabasal epithelial cells facing the gingival pocket, whereas no collagenase-1 (MMP-1) mRNA was detected in any areas of the epithelium. MMP-13 expression was also detected in fibroblast-like cells associated with collagen fibers of the inflamed subepithelial connective tissue. In organ culture of human oral mucosa, MMP-13 mRNA expression was observed in epithelial cells growing into connective tissue of the specimens. Regulation of MMP-13 expression was examined in cultured normal nonkeratinizing epithelial cells isolated from porcine periodontal ligament. In these cells, MMP-13 expression at the mRNA and protein level was potently enhanced (up to sixfold) by tumor necrosis factor-alpha, transforming growth factor-beta(1), and transforming growth factor-alpha and by keratinocyte growth factor in the presence of heparin. In addition, plating periodontal ligament epithelial cells on type I collagen stimulated MMP-13 expression (sevenfold) as compared with cells grown on tissue culture plastic. The results of this study show, that expression of MMP-13 is specifically induced in undifferentiated epithelial cells during chronic inflammation due to exposure to cytokines and collagen. Thus, it is likely that MMP-13 expression is instrumental in the subepithelial collagenolysis and local invasion of the activated mucosal epithelium into the connective tissue.     

Integrin alpha 2 beta 1 is a positive regulator of collagenase (MMP-1) and collagen alpha 1(I) gene expression

A classical model for studying the effects of extracellular matrix is to culture cells inside a three-dimensional collagen gel. When surrounded by fibrillar collagen, many cell types decrease the production of type I collagen, and the expression of interstitial collagenase (matrix metalloproteinase-1; MMP-1) is simultaneously induced. To study the role of the collagen-binding integrins alpha 1 beta 1 and alpha 2 beta 1 in this process, we used three different osteogenic cell lines with distinct patterns of putative collagen receptors: HOS cells, which express only alpha 1 beta 1 integrin, MG-63 cells, which express only alpha 2 beta 1 integrin, and KHOS-240 cells, which express both. Inside collagen gels, alpha 1 (I) collagen mRNA levels were decreased in HOS and KHOS-240 cells but not in MG-63 cells. In contrast, MMP-1 expression was induced in KHOS-240 and MG-63 cells but not in HOS cells. Transfection of MG-63 cells with alpha 2 integrin cDNA in an antisense orientation reduced the expression level of alpha 2 integrin. These cell clones showed induction and reduction of mRNA levels for MMP-1, respectively. HOS cells normally lacking alpha 2 beta 1 integrin were forced to express it, and this prevented the down-regulation in the levels of alpha 1 (I) collagen mRNA when cells were grown inside collagen gels. The data indicate that the level of MMP-1 expression is regulated by the collagen receptor alpha 2 beta 1 integrin. The down-regulation of collagen alpha 1 (I) is mediated by another receptor. Integrin alpha 2 beta 1 may compete with it and thus be a positive regulator of collagen synthesis.     

Mitogenic activity of peptides related to the sequence of human fibroblast growth factor-1

Linear synthetic peptides related to the human Fibroblast Growth Factor-1 (hFGF-1) segment [112-147] were tested for their capacity of mimicking FGF mitogenic activity, binding to heparin-Sepharose columns, stimulating DNA synthesis and competing with hFGF-1 for the cellular receptors. The results obtained indicated that the activity of these compounds is dependent on the presence of the sequence WFVGLK in their structures. The affinity for the cellular receptors increased when this sequence was elongated in order to incorporate amino acid residues that are important for FGF-heparin binding.