Remodeling of collagen matrix by human tumor cells requires activation and cell surface association of matrix metalloproteinase-2

We assessed the functional significance of tumor cell-associated matrix metalloproteinase (MMP)-2 in extracellular matrix remodeling compared with that of the soluble enzyme by evaluating the contraction of three-dimensional collagen lattices by human glioma U251.3 and fibrosarcoma HT-1080 cell lines. In this model, the constitutive synthesis and activation of the MMP-2 proenzyme were modulated by stable transfections of tumor cells with cDNA encoding membrane type 1-MMP (MT1-MMP). The efficiency of transfected cells in contracting collagen lattices was shown to be dependent on the MT1-MMP-mediated activation of MMP-2 accompanied by cell surface association of activated MMP-2, on the cell-matrix interactions controlled by collagen-specific integrins, and on the integrity of actin and microtubule cytoskeletons. Each one of these mechanisms was essential but was not sufficient by itself in accomplishing gel contraction by MT1-MMP-transfected cells. Both MMP-2 activation and gel contraction by transfected glioma cells were inhibited by tissue inhibitor of metalloproteinase (TIMP)-2 and the recombinant COOH-terminal domain of MMP-2. However, the kinetics and mechanisms of their inhibitory effects were different, because TIMP-2 and the COOH-terminal domain of MMP-2 preferentially inhibited the MT1-MMP-dependent and autocatalytic steps of MMP-2 activation, respectively. By contrast, TIMP-1, an efficient inhibitor of soluble MMP-2 activity, failed to affect gel contraction. In addition, soluble MMP-2 activated by either organomercurials or cells was not able to induce the contraction of collagen lattices when added to transfected cells. Therefore, soluble activated MMP-2, sensitive to TIMP-1 inhibition, does not mediate collagen gel contraction by tumor cells, whereas the activity of cell surface-associated MMP-2 plays a critical role in remodeling of the extracellular matrix in vitro. These mechanisms of functional and spatial regulation of MMP-2 may also be applicable to different aspects of tissue reorganization in vivo, including cell migration and invasion, angiogenesis, and wound healing.     

Characterization of the human germ cell nuclear factor gene

The vertex potential (N2, P2) of the laser-evoked potential (LEP) is preceded by a small negativity (N1). The role of the secondary somatosensory cortex (SII) in generation of the N1 is established for the upper but not for the lower limb. We therefore investigated the N1 after painful radiant heat stimulation of hand and foot dorsum in 22 subjects. LEPs were recorded from the scalp with midline and temporal electrodes. After hand stimulation N1 was maximal in the contralateral temporal lead (mean peak latency 156 +/- 23 ms). After foot stimulation N1 was maximal in the same lead (200 +/- 22 ms). In the ipsilateral temporal lead, N1 appeared significantly smaller and later. N2 and P2 were maximal in midline electrodes for both stimulus sites. The latency shift between hand and foot stimulation was identical for all three components. These results suggest a contribution of temporo-parietal cortex (e.g. SII) to the N1 generation for stimulation of upper and lower limb.     

Proteolysis of extracellular matrix by invadopodia facilitates human breast cancer cell invasion and is mediated by matrix metalloproteinases

Breast cancer cell lines vary in invasive behavior and one highly invasive cell line (MDA-MB-231) proteolytically degrades extracellular matrix with invadopodia (Thompson et al. 1992, J Cell Physiol, 150, 534-44; Chen et al 1994, Breast Cancer Res Treat, 31, 217-26). Invadopodial proteolysis of extracellular matrix is thought to be necessary for invasion; however, this has not been demonstrated directly. To obtain such evidence, normal (HBL-100) and malignant (MCF-7, MDA-MB-231) breast cells were evaluated for invadopodial proteolysis of extracellular matrix and invasive behavior. We report that invadopodial proteolysis of immobilized fibronectin is positively correlated with invasion of cells into type I collagen gels. Moreover, reducing the proteolytic activity of invadopodia with the metalloproteinase inhibitor, batimastat (BB-94), also decreases invasion indicating that breast cancer cell invasion is dependent upon proteolytically active invadopodia.     

Effect of matrix metalloproteinases activity on outflow in perfused human organ culture

PURPOSE: To test the hypothesis that extracellular matrix turnover, mediated by the matrix metalloproteinases, modulates aqueous humor outflow facility in a human outflow model. METHODS: Matrix metalloproteinase activity was manipulated and outflow facility evaluated using perfused human anterior segment organ culture. Purified matrix metalloproteinases, tissue inhibitors of metalloproteinases (TIMPs), and several families of synthetic inhibitors of matrix metalloproteinases were added to the perfusion medium. Matrix metalloproteinase expression was increased by adding recombinant interleukin (IL)-1alpha. Kinetic inhibition analysis was conducted for stromelysin, gelatinase A, and gelatinase B with the various inhibitors. Live-dead staining was used to evaluate culture viability. RESULTS: Increasing metalloproteinase activity, by adding purified metalloproteinases or by inducing their expression by IL-1alpha treatment, increased outflow facility. Inhibition of endogenous trabecular metalloproteinase activity using TIMP or several families of synthetic metalloproteinase inhibitors reduced outflow rates. The elevation and the reduction of outflow rates were reversible, with changes requiring 1 to 3 days. Kinetic enzyme inhibition analysis produced 50% inhibitory concentration values for these inhibitors that were compatible with the concentration ranges for outflow inhibition. CONCLUSIONS. The ability of several specific matrix metalloproteinase inhibitors to reduce outflow facility implies that endogenous extracellular matrix turnover by these enzymes was required for the maintenance of trabecular outflow resistance, at least in this human culture model. These observations provide support for the hypothesis that controlled extracellular matrix turnover is important in the regulation of aqueous humor outflow facility.     

Comparative analysis of matrix metalloproteinases by immunocytochemistry, immunohistochemistry and zymography in human primary brain tumours

Patients with chronic fatigue syndrome (CFS) report cognitive difficulties (impaired attention, memory and reasoning). Neuropsychological tests have failed to consistently find cognitive impairments to the degree reported by CFS patients. We tested patients with CFS and sedentary controls in protocols designed to measure sensory reactivity and acquisition of the classically conditioned eyeblink response. Patients with CFS exhibited normal sensitivity and responsivity to acoustic stimuli. However, CFS patients displayed impaired acquisition of the eyeblink response using a delayed-type conditioning paradigm. Sensitivity and responsivity to the airpuff stimulus were normal. In the absence of sensory/motor abnormalities, impaired acquisition of the classically conditioned eyeblink response indicates an associative deficit. These data suggest organic brain dysfunction within a defined neural substrate in CFS patients.     

Thrombin receptor-mediated increase of two matrix metalloproteinases, MMP-1 and MMP-3, in human endothelial cells

Matrix metalloproteinases (MMPs) are responsible for the degradation of extracellular matrix components and are secreted by a variety of cells including human endothelial cells. Because alpha-thrombin is known to interact with matrix components and has been shown to activate latent MMP-2 in human umbilical vein endothelial cells, we investigated whether human alpha-thrombin could also regulate other MMPs secreted by the human saphenous vein or mammary artery endothelial cells (EC). After treatment of EC with increasing concentrations of thrombin for different periods of time, a significantly higher gelatinolytic activity of both MMP-1 and MMP-3 was observed in addition to MMP-2 activation. The effect of thrombin was time and dose-dependent, reaching a maximum at 24 hours. After treatment with 5 NIH U/ml thrombin for 24 hours, Western blotting revealed 9.5- and 4.4-fold increases over control values for MMP-3 and MMP-1, respectively. The synthetic thrombin receptor agonist peptide SFLLRNPNDKYEPF fully reproduced the action of thrombin, whereas chemical inactivation of the catalytic site of thrombin abolished its effect on MMP-1 and MMP-3. Thrombin and SFLLRNPNDKYEPF both induced MMP-3 mRNA synthesis but had no significant influence on constitutive MMP-1 mRNA levels. These results demonstrate that thrombin not only activates latent MMP-2 but also modulates MMP-1 and MMP-3 production in EC, this latter effect being mediated by the G-protein-coupled thrombin receptor. Hence, our present data provide evidence to support the suspected role of thrombin in tissue remodeling and angiogenesis.     

Characterization of the release of cholecystokinin from a murine neuroendocrine tumor cell line, STC-1

Seven fatty acid derivatives containing one or two butenolide moieties along with ancepsenolide [1], a known bis-butenolide of marine origin, were isolated from the Caribbean gorgonian Pterogorgia citrina. The structures of the new metabolites were elucidated on the basis of spectroscopic data and were confirmed upon chemical conversion to either ancepsenolide or to its homolog, homoancepsenolide [4].     

Characterization of the INT6 mammary tumor gene product

INT6 is a unique gene, highly conserved throughout evolution and associated with mammary tumorigenesis in the mouse. Although it is expressed in all adult tissues of the mouse and early in embryonic development, its function is unknown. To study the normal distribution and the potential function of the Int6 gene products, we produced antibodies against synthetic peptides specific for the Int6 protein. Western blot and immunoprecipitation analysis demonstrated a 43-kD major gene product that is localized in the cytosolic fraction of mammary cell homogenates. This latter observation is supported by immunoperoxidase analysis, which shows a strong staining anti-Int6 peptide in the perinuclear region of the HC11 mammary epithelial cell line, suggesting a possible localization in the Golgi apparatus. Further immunocytochemical studies in the mouse embryo show that Int6 expression is prevalent in migrating neural crest cells, in the notochord, and in condensing cartilage between 9.5 and 14.5 days of development. In these embryonic tissues, Int6 staining co-localizes with the staining of ricinus lectin, and giantin, proteins that are specifically associated with the Golgi apparatus. The restricted expression of the protein within the Golgi apparatus and its strong conservation throughout evolution suggest that Int6 may perform an essential cellular function.     

Characterization of the human dihydropyrimidine dehydrogenase gene

Dihydropyrimidine dehydrogenase (DPD) catabolizes endogenous pyrimidines and pyrimidine-based antimetabolite drugs. A deficiency in human DPD is associated with congenital thymine-uraciluria in pediatric patients and severe 5-fluorouracil toxicity in cancer patients. The dihydropyrimidine dehydrogenase gene (DPYD) was isolated, and its physical map and exon-intron organization were determined by analysis of P1, PAC, BAC, and YAC clones. The DPYD gene was found to contain 23 exons ranging in size from 69 bp (exon 15) to 961 bp (exon 23). A physical map derived from a YAC clone indicated that DPYD is at least 950 kb in length with 3 kb of coding sequence and an average intron size of about 43 kb. The previously reported 5' donor splice site mutation present in pediatric thymine-uraciluria and cancer patients can now be assigned to exon 14. All 23 exons were sequenced from a series of human DNA samples, and three point mutations were identified in three racial groups as G1601A (exon 13, Ser534Asn), A1627G (exon 13, Ile543Val), and G2194A (exon 18, Val732Ile). These studies, which have established that the DPYD gene is unusually large, lay a framework for uncovering new mutations that are responsible for thymine-uraciluria and toxicity to fluoropyrimidine drugs.     

Tumor-suppressive activity of the growth arrest-specific gene GAS1 in human tumor cell lines

The GAS1 gene product induces growth arrest through a p53-dependent mechanism. To investigate whether GAS1 is a tumor suppressor gene, we transfected GAS1-negative human tumor cells with GAS1 plasmids and analyzed growth characteristics of stable transfectants. When a constitutively expressing GAS1 plasmid was transfected into A549 cells, no stable colonies expressing GAS1 were isolated. When A549 cells were transfected with a dexamethasone-inducible GAS1 plasmid, expression of GAS1 inhibited growth in vitro, and fewer slow-growing tumors arose in nude mice. GAS1 also inhibited proliferation of an HT1080 subline with wild-type (wt) p53 and normal MDM2. However, when the HT1080 subline HTD114 was transfected with the constitutive GAS1 plasmid, there was no reduction in colony number. GAS1-transfectant clones had unaltered growth in vitro, were morphologically unchanged and showed no difference in their ability to form tumors in nude mice. Although HTD114 cells contain wt p53, levels of MDM2 were elevated by 10-15 fold. The HT1080-6TGc5 subline with mutant p53 and normal MDM2 was also refractory to GAS1. Our results show that GAS1 suppresses the growth and tumorigenicity of human tumor cells and overexpression of MDM2 or p53 mutation inhibits the GAS1-mediated growth-suppressing pathway.     

Characterization of a relatively rare class B, type 2 trypanosome variant surface glycoprotein gene

We have examined the capacity of peripheral blood T cells from RA patients to be polarized in vitro towards a type 1 (T1) or a type 2 (T2) phenotype. Peripheral blood T cells from RA patients and from healthy donors were primed by 1 week of culture with soluble OKT3 in the presence of polarizing cytokines. The recovered T cells were restimulated and their cytokine secretion profile determined. Priming of T cells from RA patients in the presence of recombinant (r)IL-2 plus rIL-12 induced a shift towards a TI pattern, characterized by increased production of interferon-gamma, that was more pronounced than in the case of healthy donors. Conversely, priming of T cells from RA patients in the presence of IL-4 failed to induce a shift towards a T2 profile after 1 week, whereas it induced T cells from healthy donors to acquire such a profile characterized by heightened production of IL-4, IL-5 and IL-13. However, a T2 polarization profile emerged in T cells from RA patients that were primed in the presence of rIL-4 and subsequently maintained in culture in rIL-2 alone for 1 or 2 additional weeks. We conclude that in vitro differentiation of peripheral T cells towards a type 2 phenotype is impaired in RA. Nevertheless, conditions required to drive peripheral T cells towards a type 2 phenotype were established. Administration of autologous polyclonal T cells expressing a type 2 cytokine secretion profile is proposed as a therapeutic strategy in RA.     

Polarized distribution of metalloproteinases in the bovine interphotoreceptor matrix

Previous studies from this laboratory had indicated that metalloproteolytic activity was present in the interphotoreceptor matrix. This report extends those observations by providing evidence for the presence of multiple forms of metalloproteinase and for their polarized distribution within the interphotoreceptor matrix as determined by zymogram analysis on substrate-loaded gels. Retinal pigment epithelium-associated interphotoreceptor matrix, both unfractionated as well as fractions separated by gel filtration, exhibited bands of proteolytic activity on gelatin-loaded gels at about 70-75 kDa and 90 kDa, possibly due to gelatinases A and B (MMP-2 and MMP-9, respectively). In contrast, no neutral proteolytic activity was seen in retina-associated interphotoreceptor matrix unless it was first fractionated by gel filtration, whereupon a diversity of forms was exhibited, including additional major bands of proteolytic activity at about 150 kDa and 100 kDa, on gelatin-loaded gels, and at about 180 kDa, on casein-loaded gels, along with many minor species. In all cases, all proteinase activity was inhibited by chelating agents. Since these enzymes may be involved in the turnover and remodeling of components present in the interphotoreceptor matrix, many of which are distributed in a compartmentalized fashion, this unequal distribution of metalloproteinases may be correlated with substrate specificity.     

Immunohistochemical study of matrix metalloproteinases and their tissue inhibitors in pulmonary Langerhans' cell granulomatosis

OBJECTIVE: To evaluate the role of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in the pathogenesis of the lesions of pulmonary Langerhans' cell granulomatosis. DESIGN: Immunohistochemical and confocal microscopic studies were made of lung biopsy specimens from five patients with pulmonary Langerhans' cell granulomatosis. RESULTS: The reactivity of Langerhans' cells was moderate to intense for MMP-2, weaker for MMP-9, and faint for TIMP-1 and TIMP-2. Type IV collagen colocalized with MMP-2 in areas of damage to epithelial basement membranes, a finding that emphasizes the potential importance of this enzyme in the pathogenesis of the destructive lesions of pulmonary Langerhans' cell granulomatosis. In the more advanced fibrotic lesions, TIMP-2 colocalized with basement membranes and with fibrillar collagen, suggesting that it contributes to the permanence of the fibrosis. CONCLUSION: These results indicate an important role for MMPs and TIMPs in pulmonary Langerhans' cell granulomatosis.     

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in the mouse uterus during the peri-implantation period

In this study the SEB-activated LAK cytotoxicity was identified and characterized in human peripheral blood lymphocytes (PBMC). After 3 days of SEB stimulation, the PBMC acquired a cytotoxicity against traditional LAK targets, K-562 and Daudi, beside that human glomerular endothelial cells (HGEC) were effectively lysed. The magnetic separation of SEB-stimulated CD5+ T cells revealed that the dominant LAK cytotoxicity remained in the CD5- lymphocyte fraction. The major part of the SEB-generated cytotoxicity of CD5- cells could be blocked with specific antibodies to IL-2 and IFN-gamma. The IFN-gamma pretreatment of HGEC reduced the target sensitivity, but because of the upregulation of MHC class II on HGEC surface, these cells were able to present SEB to CD5+ cells. These results suggested that in bacterial superantigen-mediated infection, the non-T (NK cells-derived) LAK cells might have a primary pathogenic role, and the adverse effect of IFN-gamma, that was massively secreted from superantigen-stimulated cells, requires greater consideration.     

Plasma membrane-bound tissue inhibitor of metalloproteinases (TIMP)-2 specifically inhibits matrix metalloproteinase 2 (gelatinase A) activated on the cell surface

The cell-surface activation of pro-matrix metalloproteinase 2 (pro-MMP-2) is considered to be critical for cell migration and invasion. Treatment of human uterine cervical fibroblasts with concanavalin A activates pro-MMP-2 on the cell surface by converting it to the 65-kDa form with a minor form of 45 kDa. However, the 65-kDa MMP-2 was inactivated by tissue inhibitor of metalloproteinases (TIMP)-2 that was bound to the plasma membrane upon concanavalin A treatment. TIMP-2 binds to the plasma membrane through its N-terminal domain by two different modes of interaction as follows: one is sensitive to a hydroxamate (HXM) inhibitor of MMPs and the other is HXM-insensitive. TIMP-2 bound to the membrane in a HXM-insensitive manner, comprising about 40-50% of TIMP-2 on the membrane, is the inhibitor of the cell surface-activated MMP-2. It, however, does not inhibit MMP-3, MMP-9, and the 45-kDa MMP-2 lacking the C-terminal domain. The inhibition of the 65-kDa MMP-2 by TIMP-2 is initiated by the interaction of their C-terminal domains. Subsequently, the MMP-2.TIMP-2 complex is released from the membrane, and the activity of MMP-2 is blocked by TIMP-2. In the presence of collagen types I, II, III, V, or gelatin, the rate of inhibition of the 65-kDa MMP-2 by the membrane-bound TIMP-2 decreased considerably. These results suggest that the pericellular activity of MMP-2 is tightly regulated by membrane-bound TIMP-2 and surrounding extracellular matrix components.     

Characterization of the human aldose reductase gene promoter

The promoter region of the human aldose reductase gene has been identified upstream of the translation start ATG codon. The promoter contains a TATA box, a CCAAT promoter element, and three Sp1 protein binding consensus sequences upstream of the capsite. A 640-base pair insert spanning +31 to -609 directs expression of the reporter gene chloramphenicol acetyltransferase in an orientation-specific manner in transfected Hep G2 cells. The promoter activity remained constant with deletions from base pairs -609 to -186. The TATA and the CCAAT consensus sequences show significant promoter activity, whereas the three Sp1 binding consensus sequences, individually, have no significant promoter activity. A GA-rich region (-186 to -146) contains two CGGAAA/G motifs, which show promoter activity and interaction with Hep G2 nuclear extract and GA-binding proteins (GABP alpha and GABP beta 1) as shown by mobility shift assays and DNase I footprinting. Similar cis-elements in herpes simplex virus type 1 interact with rat liver GABP and the viral VP16 protein to mediate the induction of immediate early viral genes. A GC-rich region (-87 to -31) is identified by mobility shift assay, and a consensus sequence of an androgen response element is present at -396 to -382. The human aldose reductase promoter, thus, has regulatory response elements that may be important during early development and puberty. These regulatory elements may play a significant role in the development of certain diabetic complications.