Degradation Of Basement Membrane Components Research Articles

EMMPRIN/CD147 as a novel independent prognostic biomarker in melanoma.

e20027 Background: EMMPRIN (Extracellular Matrix MetalloPRoteinase INducer) is a member of the immunoglobulin superfamilyknown tostimulate the degradation of basement membrane components by increas...

Implant of polymer containing pentacyclic triterpenes from Eugenia punicifolia inhibits inflammation and activates skeletal muscle remodeling.

Sustained chronic inflammation induces activation of genes involved in cellular proliferation and apoptosis, thereby causing skeletal muscle degeneration. To investigate in vitro effects of isolated pentacyclic triterpenes from Eugenia punicifolia (Ep-CM) upon signaling pathways involved in the regulation of skeletal muscle cell line proliferation, and in vivo muscular tissue remodeling. C2C12 cells were seeded on eight-well plates and [(3)H]-thymidine incorporation, TUNEL assays, mitochondria viability, zymography for matrix metalloproteases (MMPs), Western blot analysis for MAPKinase signaling pathway, NFκB activation and HMGB1 production subsequently determined under basal conditions and after Ep-CM treatment. A polymer containing Ep-CM was implanted on the volar surface of gastrocnemius muscles subjected to acute injury induced by bupivacaine for local slow and gradual release of bioactive compounds, and mice killed 4days after surgery. Ep-CM inhibited proliferation of C2C12 myoblast cell line in a dose-dependent manner, confirmed by reduction of [(3)H]-thymidine uptake without affecting cell viability or inducing apoptosis. The cytostatic effect of Ep-CM occurred mainly via inhibition of phosphorylated extracellular signal-regulated kinase (pERK) activation and DNA synthesis, possibly inhibiting the G1 phase of the cell cycle, since Ep-CM increased pAkt and p27(kip1) but reduced Cyclin D1. Ep-CM in vitro treatment increased MMP-9 and MMP-2 activities of C2C12 myoblast cells, but reduced in vivo MMP-9 activity and acute muscular inflammation. Besides cytostatic and anti-inflammatory effects, Ep-CM pentacyclic triterpenes also contributed to degradation of basement membrane components by activating mechanisms of skeletal muscle remodeling in response to local injury.

Immunohistochemical expression of basement membrane laminin in histological grades of oral squamous cell carcinoma: A semiquantitative analysis

Background and Objectives:To determine the immunohistochemical (IHC) localization of basement membrane component laminin in histological grades of oral squamous cell carcinoma (OSCC). The purpose of this study was to demonstrate the loss of continuity of the basement membrane in OSCC using an antibody directed against laminin using advanced polymer staining system.Materials and Methods:A total of 30 cases of OSCC: 10 cases of well differentiated squamous cell carcinom (WDSCC), 10 cases of moderately differentiated squamous cell carcinoma (MDSCC), and 10 cases of poorly differentiated squamous cell carcinoma (PDSCC) were subjected to heat-induced antigen retrieval method using ethylene-di-amine-tetraacetic acid buffer in a microwave oven. Then the sections were stained with anti-laminin polyclonal antibody and visualized using super sensitive polymer horseradish peroxidase detection system. In each case, the integrity of the basement membrane laminin was assessed by using statistical analysis.Results:Statistical analysis showed a decreased distribution of laminin from WDSCC to MDSCC to PDSCC (P value 0.0573). The intracytoplasmic staining of laminin gradually increased from WDSCC to MDSCC to PDSCC (P value 0.0198).Interpretation and Conclusion:WDSCC cases showed more laminin expression in basement membrane around the tumor islands and less loss of continuity compared to MDSCC and PDSCC cases suggesting a greater enzymatic degradation of basement membrane components in MDSCC and PDSCC than WDSCC. The loss of structural basement membrane laminin and the presence of laminin in the tumor cells of PDSCC cases suggest that laminin helps in tumor invasion. The expression of laminin in the basement membrane may be a useful parameter to evaluate tumor histologic differentiation and aggressiveness.

Matrix Metalloproteinase 2 (MMP-2) Degrades Soluble Vasculotropic Amyloid-β E22Q and L34V Mutants, Delaying Their Toxicity for Human Brain Microvascular Endothelial Cells

Patients carrying mutations within the amyloid-beta (Abeta) sequence develop severe early-onset cerebral amyloid angiopathy with some of the related variants manifesting primarily with hemorrhagic phenotypes. Matrix metalloproteases (MMPs) are typically associated with blood brain barrier disruption and hemorrhagic transformations after ischemic stroke. However, their contribution to cerebral amyloid angiopathy-related hemorrhage remains unclear. Human brain endothelial cells challenged with Abeta synthetic homologues containing mutations known to be associated in vivo with hemorrhagic manifestations (AbetaE22Q and AbetaL34V) showed enhanced production and activation of MMP-2, evaluated via Multiplex MMP antibody arrays, gel zymography, and Western blot, which in turn proteolytically cleaved in situ the Abeta peptides. Immunoprecipitation followed by mass spectrometry analysis highlighted the generation of specific C-terminal proteolytic fragments, in particular the accumulation of Abeta-(1-16), a result validated in vitro with recombinant MMP-2 and quantitatively evaluated using deuterium-labeled internal standards. Silencing MMP-2 gene expression resulted in reduced Abeta degradation and enhanced apoptosis. Secretion and activation of MMP-2 as well as susceptibility of the Abeta peptides to MMP-2 degradation were dependent on the peptide conformation, with fibrillar elements of AbetaE22Q exhibiting negligible effects. Our results indicate that MMP-2 release and activation differentially degrades Abeta species, delaying their toxicity for endothelial cells. However, taking into consideration MMP ability to degrade basement membrane components, these protective effects might also undesirably compromise blood brain barrier integrity and precipitate a hemorrhagic phenotype.

The role of IL-18 in the modulation of matrix metalloproteinases and migration of human natural killer (NK) cells

In this study, we examined whether interleukin-18 (IL-18) affects natural killer (NK) cells' migration and matrix metalloproteinases (MMPs) production. We demonstrated that chemotaxis of human NK cells through basement membrane-like Matrigel was augmented by IL-18. As well, IL-18 stimulation induces the production of activated forms of matrix metalloproteinase-2 (MMP-2) as well as the production of pro-MMP-2 from NK cells. We also demonstrated that MT1-MMP expression on human NK cells, which is a major activator of MMP-2, was induced by IL-18 stimulation coordinated with MMP-2 activation. These data suggest that the MT1-MMP/MMP-2 system participates in the degradation of basement membrane components and thus contributes to NK cell migration.

Basement membrane and matrix metalloproteinases in monocrotaline-induced liver injury.

Monocrotaline (MCT) is a pyrrolizidine alkaloid that causes liver injury in animals. In rats, injury is characterized by sinusoidal endothelial cell (SEC) damage and centrilobular parenchymal cell necrosis. Loss of endothelium is a possible outcome of the action of matrix metalloproteinases (MMPs), specifically MMP-9 from neutrophils and SECs and MMP-2 from SECs, on basement membrane collagen. Accordingly, the dynamics of MMPs in MCT-induced SEC damage were studied. Rats were treated with MCT (300 mg/kg, ip), and livers were collected at 8, 12, and 18 h. Immunofluorescence analysis of frozen sections of livers from MCT-treated rats revealed a progressive reduction in basement membrane heparan sulfate proteoglycan and collagen IV. A time-dependent increase in total type IV collagenase activity and MMP-9 content occurred in the livers of MCT-treated rats, as measured by fluorescent collagenase activity assay and gelatin zymography, respectively. Progressive neutrophil accumulation and activation in the liver after MCT treatment were demonstrated by an increased activity of myeloperoxidase and pronounced staining for hypochlorite-modified proteins generated via the myeloperoxidase-hydrogen peroxide-halide system. However, neutrophil depletion did not protect against MCT-induced SEC injury. Treatment of NP-26 cells, a sinusoidal endothelial cell line, with MCT resulted in dose-dependent release of MMP-9 from the cells. The results demonstrate the degradation of basement membrane components with a concurrent increase in the amount and activity of MMP-9, likely originating from sinusoidal endothelial cells, neutrophils, and probably other cell types. This suggests the possibility of a role for MMPs in the SEC detachment and loss that occurs during MCT hepatotoxicity.

Increased membrane type 1 matrix metalloproteinase expression from adenoma to colon cancer: a possible mechanism of neoplastic progression.

Membrane type 1 matrix metalloproteinase is a membrane-associated matrix metalloproteinase central to the degradation of basement membrane components via the activation of matrix metalloproteinase-2. Although membrane type 1 matrix metalloproteinase is overexpressed in invasive colon cancer, its expression in colonic polyps and carcinoma in situ has not been defined. In addition, the association of membrane type 1 matrix metalloproteinase expression by a primary tumor and recurrence of colon cancers has not been examined. Immunoperoxidase staining was performed on randomly selected specimens containing adenoma (n = 17), carcinoma in situ (n = 9), or metastatic colon carcinoma (n = 8) with mouse monoclonal antibody to human membrane type 1 matrix metalloproteinase. Similar staining was also performed on randomly selected node-negative colon cancers that recurred within five years of resection (n = 17), matched for age, gender, stage, grade, and vascular, lymphatic, and perineural invasion, and node-negative colon cancers that did not recur within five years of resection (n = 17). Staining for membrane type 1 matrix metalloproteinase was graded. Mean scores for the groups were compared by Wilcoxon test. We found a progressive and significant increase in the mean score of membrane type 1 matrix metalloproteinase from normal mucosa to adenoma (P < 0.001), carcinoma in situ (P < 0.006), and invasive cancer (P < 0.009). However, there was no difference in membrane type 1 matrix metalloproteinase expression between the recurrent and nonrecurrent groups of node-negative colon cancer (P = not significant). These data suggest that membrane type 1 matrix metalloproteinase expression increases with progression from normal mucosa to invasive adenocarcinoma; however, it cannot be used as a prognostic indicator on which adjuvant therapy is based in node-negative colon cancer because of its failure to predict recurrence in this patient group.

Importance of Balance between Extracellular Matrix Synthesis and Degradation in Basement Membrane Formation

The epidermal basement membrane (BM) plays important roles in adhesion between epidermis and dermis and in controlling epidermal differentiation. In a skin-equivalent (SE), components of the epidermal BM such as laminin 5 and type IV and VII collagens were detected in conditioned media and in basal keratinocytes. Despite production of these BM components, however, BM was rarely observed at the dermal–epidermal junction. One possible explanation for the absence of BM in SEs is that matrix metalloproteinases (MMPs) degrade newly synthesized extracellular matrices. In fact, several MMPs, such as MMPs-1, 2, 3, and 9, were observed to be present in conditioned media and some of them were in active forms. Tissue inhibitor of metalloproteinase (TIMP)-2 was not detected, although TIMP-1 was present. BM degradation activity presumably exceeds BM formation activity in the SE, resulting in the absence of lamina densa at the dermal–epidermal junction. Synthetic MMP inhibitors CGS27023A and MMP inhibitor I, which inhibit MMPs 1, 2, 3, and 9, markedly augmented deposition of laminin 5 and type IV and VII collagens at the dermal–epidermal junction, resulting in formation of continuous epidermal BM. These results suggest that the balance between synthesis and degradation of BM components is important for BM formation.

Gelatinase expression in pulmonary arteries during experimental pulmonary hypertension.

Structural remodelling of pulmonary vessels is an important feature of pulmonary hypertension (PH), which reflects distal artery muscularization and matrix remodelling. The matrix metalloproteinases (MMPs) are involved in extracellular matrix turnover and hence, in smooth muscle cell migration and endothelial cell migration and proliferation. Among the MMPs, gelatinases (MMP-2 and MMP-9) can degrade basement membrane components and promote cell proliferation and migration. This study evaluated gelatinases in pulmonary vessels during progressive PH in two rat models: exposure to hypoxia or monocrotaline. Zymography of tissue homogenates revealed an association of progression of hypoxic PH with a time-dependent increase in gelatinase MMP-2 activity, specific to pulmonary vessels. Increased MMP-2 activity was also found 30 days postmonocrotaline. Reverse transcription polymerase chain reaction demonstrated upregulation of MMP-2 messenger ribonucleic acid. Immunolocalization showed MMP-2 throughout the pulmonary vasculature, from the trunk to the distal vessels, with strong staining of the intima, media and adventitia. MMP-2 was found in its active form and gelatinolytic activity was correlated to PH severity. Activity localization by in situ zymography corroborated with the immunolocalization findings. In conclusion, the authors demonstrated that matrix metalloproteinase-2 activity is increased in pulmonary vessels during progression of pulmonary hypertension, probably as a result of involvement in the matrix turnover associated with vascular remodelling during pulmonary hypertension.

Role of vascular basement membrane components in diabetic microangiopathy.

The complications of diabetic microangiopathy have been well documented clinically for many years; however, the specific cellular mechanisms that lead to the dysfunction of small vessels in diabetes are unknown. Histopathological studies on diabetic microangiopathy have established thickened vascular basement membrane as one of the first and foremost structural abnormalities. Evidence suggests that this fundamental structural lesion develops, at least in part, from excess accumulation of basement membrane material in the vessel walls. Several vascular basement membrane components have been identified whose expression is up-regulated in diabetes. Enzymes responsible for the degradation of basement membrane components have also been reported to exhibit reduced activity. Strategies are being developed to regulate the abnormal accumulation of extracellular matrix (ECM) components in diabetic microangiopathy. In experimental diabetes, aldose reductase inhibitors have been found to be effective in preventing vascular basement membrane thickening when applied soon after the onset of hyperhexosemia. Clinical trials have been initiated with inhibitors of protein kinase C, an enzyme that up-regulates the synthesis of ECM components in diabetes. Currently, gene therapy strategies are also being developed to inhibit specific gene expression responsible for excess ECM synthesis in diabetes. Recent studies in the authors' laboratory have shown the applicability and efficacy of antisense oligonucleotides toward reducing vascular basement membrane component synthesis. Several gene regulatory approaches are currently being developed to modulate and prevent vascular basement membrane component synthesis. Such strategies may help identify key events that underlie the complex developmental stages of diabetic microangiopathy.

Zymographic analysis of equine laminitis.

To investigate the role of matrix metalloproteinase (MMP) activity in the pathophysiology of equine laminitis, the techniques of in situ zymography and quantitative SDS-PAGE zymography were used to analyse the lamellae and plasma and serum of horses with carbohydrate overload-induced laminitis. The gelatinase activity localised within the epidermal lamellae of laminitic hooves did not differ significantly from normal hooves. In laminitis sections there was an increase in vascular gelatinase activity, possibly associated with the perivascular cuffing of polymorphonucleocytes. Both plasma and serum samples from horses developing laminitis showed a rapid increase in the concentration of circulating latent MMP-9, while MMP-2 remained relatively constant. These results support the hypothesis that laminitis histopathology results from an inadequate regulation of gelatinase activity, resulting in selective degradation of basement membrane components, leading to laminitis due to failure of the basement membrane-epidermis attachment.

Regulation by thyroid hormone of the expression of basement membrane components in rat prepubertal Sertoli cells.

The present study reports the modulation of basement membrane (BM) components, laminin, entactin, and type IV collagen, expression in prepubertal rat Sertoli cell by the thyroid hormone T3. Immunocytochemical studies of permeabilized Sertoli cells in culture showed that T3 treatment (10[-7] M for 24 h) increased the number of cells staining positive for laminin and/or entactin (from 58 +/- 5.3% to 86.4 +/- 6.5%, P < 0.01). In contrast, a strong inhibition of type IV collagen immunopositivity was observed. Western blot analysis of Sertoli cell-conditioned media indicated that T3 treatment significantly (P < 0.01) increased the level of secreted entactin by 60-65% without affecting the levels of laminin A and B1/B2 chains. Moreover, thyroid hormone treatment of Sertoli cells significantly reduced type IV collagen secretion by 62% (P < 0.05). Slot blot analysis of poly-A RNA demonstrated a significant (P < 0.01) increase in the level of entactin messenger RNA (mRNA) by 140% (P < 0.01) and a 50% reduction of type IV collagen alpha1 chain mRNA after thyroid hormone treatment. No effect of the hormone was observed on the accumulation of the laminin B1 and B2 chain mRNAs in Sertoli cell cultures. These effects cannot be ascribed to changes in the degradation of BM components, because no effect of thyroid hormone was observed on plasminogen activators or metalloproteinase secretion by Sertoli cells. These observations indicate the Sertoli cell as a source of entactin within the testis, demonstrate the ability of T3 to differentially regulate the expression of BM components, and can be regarded as a part of the integrated mechanism by which thyroid hormone affects testicular development and differentiation.

Expression of matrix proteins in uterine cervical neoplasia using immunohistochemistry

Objective: Neoplastic cells of various tumors are capable of modifying extracellular matrix, and can reduce basement membrane components and express matrix proteins and mRNA. Findings in cervical cancer cells have been consistent concerning their ability to reduce basement membrane, but there is a lack of consensus concerning the expression of matrix proteins in the neoplastic cells. We have investigated the expression of matrix proteins in the subepithelial basement membrane, in blood vessels and in the epithelial cells using immunohistochemistry. Study design: A total of 49 cases, including normal cervix, Cervical Intraepithelial Neoplastia (CIN) grades I, II and III, and invasive squamous cell carcinoma, were stained for fibronectin (FN), laminin (LN) and collagen IV (C IV) and analyzed by immunohistochemistry. Subepithelial and peritumoral basement membrane staining for C IV was scored as 0, 1, 2 or 3 (0, absent; 1, weak and discontinuous; 2, weak or discontinuous; 3, strong and diffuse). Vascular basement membrane staining by all three markers was interpreted as positive or negative. Staining of normal epithelium, CIN lesions and invasive carcinoma cells was evaluated with all three markers. Results: Subepithelial basement membrane staining was graded as score=3 in all normal cervices, in contrast to 9/10, 2/10, 1/10 and 0/9 cases of CIN I, CIN II, CIN III and invasive carcinoma, respectively. Remaining cases showed variable degrees of basement membrane loss. In 6/10 CIN III cases and 8/9 carcinomas, staining was absent (score=0). Blood vessels stained positive for FN, LN and C IV in all cases studied. Epithelial cells were LN-positive in only one case of CIN II and FN-positive in only two cases of CIN III. Conversely, 3/9 carcinomas were LN-positive, 6/9 were FN-positive and 2/9 were C IV-positive, including two cases positive for all three markers. Staining pattern was cytoplasmic, mainly in the periphery of tumor islands. Conclusions: Invasive squamous cell carcinomas of the cervix are capable of expressing matrix proteins that are usually absent in normal squamous cervical cells or preinvasive lesions. Secretion or degradation of these proteins could facilitate tumor invasion of stroma and vessels. In addition, basement membrane dissolution becomes pronounced in CIN II-III lesions, being complete in most invasive carcinomas, probably reflecting the ability of pre-invasive neoplastic cells to degrade basement membrane components with metalloproteases.

Major co-localization of the extracellular-matrix degradative enzymes heparanase and gelatinase in tertiary granules of human neutrophils.

The expression of cell-surface adhesion proteins and the release of extracellular-matrix degradative enzymes constitute crucial processes for the attachment of neutrophils to the endothelium and for the subsequent extravasation of these cells through the endothelial layer. We have analysed in resting human neutrophils the subcellular localization of heparanase, a heparan-sulphate-degrading endoglycosidase that can degrade basement-membrane components, thereby facilitating neutrophil passage into the tissue during an inflammatory reaction. By subcellular fractionation of postnuclear supernatants from resting human neutrophils on continuous sucrose gradients, we have found that heparanase activity was mainly located in gelatinase-containing tertiary granules. Using a specific antibody, the 96-kDa heparanase protein was further located in the gelatinase-rich subcellular fractions. Following immunoblotting and immunoprecipitation analysis in the distinct subcellular fractions, we also found co-localization of heparanase and Mo1 (CD11b/CD18), a leucocyte integrin involved in the attachment of neutrophils to the endothelium, in the fractions enriched in gelatinase-containing tertiary granules. Treatment of human neutrophils with tumour necrosis factor or granulocyte/macrophage colony-stimulating factor induced an increase in the CD11b/CD18 cell-surface expression, as well as the release of both gelatinase (matrix metalloproteinase-9) and heparanase, but not of other granule markers, indicating a major co-localization of gelatinase, heparanase and CD11b/CD18 in the same organelle. Furthermore, confocal laser scanning microscopy using specific antibodies against gelatinase and heparanase revealed a major co-localization of both enzymes in intracellular cytoplasmic granules. The major localization of heparanase and CD11b/CD18 in the gelatinase-containing tertiary granule supports the notion that mobilization of this organelle can regulate extravasation of human neutrophils.

Alveolar epithelial cells in vitro produce gelatinases and tissue inhibitor of matrix metalloproteinase-2.

Type II pneumocytes are key cells of the alveolar epithelium. They lie on the alveolar basement membrane, which influences their phenotype and functions. We hypothesized that type II pneumocytes degrade basement membrane components by producing gelatinases, members of the matrix metalloproteinase family. To investigate this hypothesis, we used primary cultures of rat type II pneumocytes and cultures of the human A549 cell line. We found by zymography that 70-kDa gelatinase was present in media conditioned by these cells. This 70-kDa gelatinase was identified as gelatinase A by a Western blot, and the presence of its mRNA was demonstrated by reverse transcription-polymerase chain reaction. A 95-kDa gelatinase could be induced under certain conditions. Production of gelatinases may take place during the turnover of basement membranes, in physiological and in pathophysiological processes. This was suggested by the increase in production of both gelatinases that we observed after in vitro exposure to LPS or interleukin-1. The presence of tissue inhibitors of matrix metalloproteinase-1 and -2 was also demonstrated, suggesting that degradation of extracellular matrix by type II pneumocytes is tightly regulated.

Section Review Biologicals &amp; Immunologicals: Matrix metalloproteinases and malignant disease: Recent developments

One important proteolytic event during metastasis and angiogenesis appears to be the degradation of basement membrane components. A specific class of extracellular matrix (ECM) degrading metallo-enzymes, the matrix metalloproteinases (MMPs), also known as the matrixins, are thought to have a critical role in the creation of the proteolytic defect in basement membrane type IV collagen that allows tumour cells to escape from their primary site. The expression of matrixin proteases has been linked with many physiologic and pathologic processes involving ECM turnover. A number of correlative and functional studies suggest that the action of MMPs is required during tumour invasion and progression, as well as angiogenesis. The correlative studies have demonstrated overexpression of MMPs in many human cancer tissues at both the protein (immunoperoxidase) and mRNA (in situ hybridisation) levels. Functional studies have used endogenous inhibitors (Tissue Inhibitors of Metalloproteinases, TIMPs) or synthetic MMP in...

Production of recombinant human matrix metalloproteinase 7 (Matrilysin) with potential role in tumor invasion by refolding from Escherichia coli inclusion bodies and development of sandwich ELISA of MMP-7

Metastatic potential of prostate cancer is thought to correlate with the degradation of basement membrane components by matrix metalloproteinases (MMPs). The MMP-7 (matrilysin) gene is overexpressed in prostate cancer as well as colorectum and brain cancer. In order to clarify the relation of MMP-7 to clinical stages of prostate cancer, recombinant human MMP-7 was produced to prepare antibodies for immunohistochemistry and immunoassay. Preproform of human MMP-7 was produced in Escherichia coli as inclusion bodies that could be solubilized and refolded to yield an activatable proenzyme. PreproMMP-7 (Mr 31,000) solubilized from inclusion bodies was converted to proMMP-7 (Mr 30,000) during the refolding steps. The refolded proMMP-7 was purified to about 80% homogeneity as MMP-7 by sequential ion-exchange and molecular-sieve chromatography. The active, mature form of MMP-7 (Mr 20,000) could be produced from proforms of MMP-7 by treatment with p-aminophenylmercuric acetate. Activated MMP-7 was shown to have proteolytic activity to fibronectin, casein, and diazotized, denatured collagen (Azocoll). Specific activity, as assayed with the denatured collagen as substrate, was measured to be about 3,100 units/mg protein of mature enzyme. Using recombinant proMMP-7 as antigen, monoclonal and polyclonal antibodies were prepared. A sandwich ELISA was developed using monoclonal antibody as the capture antibody and rabbit anti-proMMP-7 polyclonal IgG conjugated with biotin as the detection antibody; MMP-7 at 10 ng/ml was significantly detectable. The assay system is applicable on the measurement of MMP-7 levels in the clinical and pathologic specimens including serum from patients with different stages in malignancy of prostate cancer. These antibodies are useful for the retrospective analyses of prostate cancer on the basis of immunohistochemical evaluation.

The role of matrix metalloproteases and their inhibitors in tumour invasion, metastasis and angiogenesis

One critical event of tumour invasion that signals the initiation of the metastatic cascade is thought to be interaction of the tumour cell with the basement membrane. Basement membranes may also pose as barriers to tumour cell invasion at multiple points later in the metastatic cascade, including during the processes of vascular infiltration and extravasation. Thus, an important proteolytic event in the metastatic cascade, and also angiogenesis, appears to be degradation of basement membrane components. A specific class of extracellular matrix degrading metalloenzymes, the matrix metalloproteases, and their endogenous inhibitors, the tissue inhibitors of metalloproteases, are thought to have a role in the creation of the proteolytic defect in basement membrane type IV collagen. We will review the evidence which indicates that matrix metalloproteases and tissue inhibitors of metalloproteases are essential for tumour cell invasion and angiogenesis. The regulation of matrix metalloproteases will be discussed, including gene activation and transcription, messenger ribonucleic acid (mRNA) stability, binding of proenzymes to cell membranes and/or matrix components, proenzyme activation, and inactivation by endogenous inhibitors. We will also discuss the mechanism for tissue inhibitor of metalloproteases-mediated inhibition of tumour invasion and angiogenesis. This appears, at least in part, to be through inhibition of protease activity required for cellular invasion, although recent observations suggest that tissue inhibitors of metalloproteases affect other distinct groups of biological activities through mechanisms other than matrix metalloprotease inhibition.

Cathepsin B expression in tumour cells and laminin distribution in pulmonary adenocarcinoma.

To determine the correlation between cathepsin B expression and laminin distribution in pulmonary adenocarcinoma tissue. The distribution of cathepsin B and laminin was examined in 28 formalin fixed, paraffin wax embedded specimens of pulmonary adenocarcinoma tissue, using a double immunostaining technique with commercially available antibodies to cathepsin B and laminin, respectively. Tumour cells in 23 (82%) cases reacted to cathepsin B: 13 cases were weakly positive and 10 were strongly positive. Laminin in tumour associated basement membrane produced various staining patterns: two cases had an almost continuous distribution of laminin in tumour associated basement membrane in the tumour tissues, while a moderately discontinuous laminin distribution pattern was found in 12 cases, and a highly fragmented pattern was found in 14 cases. The degree of cathepsin B expression in tumour cells was significantly correlated with the break up of laminin staining. In some cases a discontinuous pattern of tumour associated laminin was frequently observed adjacent to cathepsin B positive tumour cell nests. Considering that cathepsin B has the capacity to degrade basement membrane components, including laminin, the inverse correlation shown in this study between the increase in cathepsin B expression by tumour cells and the diminution of laminin in tumour associated basement membrane could reflect local progression and spread by pulmonary adenocarcinoma.

Ha-ras oncogene induction of invasion and metastasis is associated with the activation and redistribution of protease(s) in rat-kidney cells.

To investigate the role of oncogenes in the development of metastatic ability, the Ha-ras oncogene was transfected into murine kidney cell line NRK and an alternative murine cell line C127 cells. The resulting transfectants, NRK-Ha and HC127, were assayed for their invasive capability in an in vitro invasion assay and compared with their parental cell lines. All transfected cells were demonstrated to express a 0.6 kb mRNA for the ras oncogene by Northern blot analysis. Using a modified Boyden chamber, we showed that the motile ability of the cell lines, transfected versus non-transfected, were not altered by the introduction of the Ha-ras oncogene. However, ras transfected NRK-Ha cells were able to penetrate and invade through an artificial basement membrane coated filter when compared with the non-transfected parental cell line. Neither the C127 cells nor its ras transfected HC127 cells were able to migrate or penetrate through an artificial basement membrane coated filter. Using immunoblot analyses, we determined that invasive NRK-Ha cells expressed a mature form of protease cathepsin B with a molecular weight between 25-28 kD. Furthermore, using subcellular fractionation and immunoblot, we localized this mature form of cathepsin B to the plasma membrane fraction of the invasive NRK-Ha cells. The plasma membrane fraction of invasive NRK-Ha cells was able to degrade basement membrane components such as fibronectin and the patterns seen were similar to those of purified cathepsin B degradation. Non-invasive NRK, C127 and HC127 cells had only the precursor form of cathepsin B with a molecular weight between 38-45 kD and were unable to degrade basement membrane components. We suggest that the presence and expression of the Ha-ras oncogene alone does not confer metastatic capability, but rather that the ras oncogene must signal specific intracellular redistribution of proteases such as cathepsin B in order to promote tumor cell invasiveness.