Role In Extracellular Matrix Degradation Research Articles

Predictive value of cathepsin-D for cervical lymph node metastasis in head and neck squamous cell carcinoma.

Prognosis of head and neck squamous cell carcinoma (HNSCC) is strongly associated with cervical lymph node metastasis. Cathepsin-D is a lysosomal protease expressed in all cells. Its role in extracellular matrix degradation is postulated to promote tumor invasion and metastasis. Increased cathepsin-D has been demonstrated in cervical lymph node metastasis in HNSCC. Formalin fixed tumor biopsy samples from 34 patients with HNSCC of the oral cavity, oropharynx, or hypopharynx were analyzed for the presence of cathepsin-D by immunohistochemistry (1:8000, Calbiochem, Cambridge, MA). Tumors were considered positive if >50% of cells showed strong cytoplasmic staining. All patients had T1 or T2 lesions ranging in size from 1-4 cm and 19 (56%) had cervical metastasis. Eight (24%) were well differentiated and 26 (76%) were moderately or poorly differentiated. Thirteen tumors (38%) had high cathepsin-D expression that was strongly associated with cervical lymph node metastasis (p = 0.008). When adjusted for tumor stage and grade, cathepsin-D positivity was nearly twice as likely to be associated with node metastasis (p = 0.011). We demonstrated cathepsin-D expression in biopsies from a subset of patients with HNSCC and a strong association between this protease and cervical lymph node metastases. Cathepsin-D is a potential independent predictor of cervical lymph node metastasis in HNSCC and merits additional study.

Serum matrix metalloproteinase-3 (stromelysin-1) concentration in patients with chronic liver disease

Background/Aims: Matrix metalloproteinase (MMP)-3 plays an important role in extracellular matrix degradation, because of its broad substrate specificity and its activation of other proMMPs. Our aims in the present study were to determine whether the measurement of serum MMP-3 is clinically useful for assessing ongoing liver fibrolysis in patients with chronic liver disease. Methods: We measured the serum MMP-3 concentrations with a sandwich enzyme immunoassay in 58 patients with chronic hepatitis, 22 patients with liver cirrhosis, 45 patients with hepatocellular carcinoma and 124 healthy individuals. The liver MMP-3 content was also measured in autopsied livers. Results: Among the healthy controls, the serum levels of MMP-3 were about 2-fold higher in the males than in the females. In this study, the serum MMP-3 results of mainly the male group were analyzed because of the large number of male subjects. Compared to the control level, the mean serum MMP-3 concentration was 55% lower in chronic hepatitis, 53% lower in liver cirrhosis and 46% lower in hepatocellular carcinoma. There was no significant difference in the serum MMP-3 levels among the chronic hepatitis, liver cirrhosis and hepatocellular carcinoma groups. The serum MMP-3 levels were not related to the histological degree of necroinflammation or of liver fibrosis in the patients with chronic hepatitis. No significant difference in serum MMP-3 levels was observed among three Child's subgroups in the group of cirrhotic patients. In the group of patients with hepatocellular carcinoma, the serum MMP-3 levels were not related to the severity of liver function, the HCC tumor size, or the histological differentiation. The serum MMP-3 level was not correlated with serum markers for connective tissue turnover, i.e. procollagen type III peptide, 7S fragment of type IV collagen, hyaluronan and tissue inhibitor of metalloproteinase-1 in the patients with chronic liver disease or hepatocellular carcinoma. Conclusions: The measurement of serum MMP-3 is of little use for assessing fibrolysis in chronically diseased livers.

Degradation of extracellular matrix by metastatic follicular thyroid carcinoma cell lines: role of the plasmin activation system.

The plasmin activation system plays a key role in extracellular matrix degradation in many malignant tumors. Because no data are available on the involvement of the plasmin activation system in matrix degradation by thyroid carcinoma, the present study was performed using follicular thyroid carcinoma cell lines obtained from a primary tumor (FTC-133) and metastases (FTC-236 and FTC-238) of one patient. Matrix degradation by these cell lines was studied assessing the release of radioactivity from S35-methionine labeled extracellular matrix coated onto plastic. The involvement of constituents of the plasmin activation system as well as matrix metalloproteinases (MMPs), another class of proteolytic enzymes, which can be activated by plasmin, were assessed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and zymography. In the matrix degradation experiment, S35 release by FTC-133 was significantly higher than FTC-236 and FTC-238. S35 degradation could be inhibited by the plasmin inhibitor aprotinin and by anti-human urokinase-type plasminogen activator (uPA) antibody, indicating the involvement of the plasmin activation system. Matrix degradation could also be inhibited by the MMP inhibitor marimastat, thus demonstrating the involvement of MMPs in matrix degradation by these cell lines. Zymographic assays revealed activity of uPA in all cell lines. However, in contrast with FTC-236 and FTC-238, no plasminogen activator inhibitor (PAI) or PAI1 mRNA were found in FTC-133. Therefore, the differences in PAI activity as observed between the cell lines may originate from differences in PAI1 gene transcription. Differences in PAI1 expression did not affect the attachment of these cell lines to vitronectin. We conclude that the plasmin activation system is involved in extracellular matrix degradation by these metastatic follicular thyroid carcinoma cell lines. Differences in extracellular matrix degradation between the cell lines correspond with differences in PAI1 gene expression, indicating the significance of PAI1 in extracellular matrix degradation by metastatic follicular thyroid carcinoma.

Expression of matrix metalloproteinase-1, -2 and -3 in squamous cell carcinoma and actinic keratosis.

Matrix metalloproteinase (MMP) plays an important role in extracellular matrix degradation associated with cancer invasion. An expression of MMP-1 (interstitial collagenase), MMP-2 (72-kDa type IV collagenase) and MMP-3 (stromelysin-1) was investigated in squamous cell carcinoma (SCC) and its precancerous condition, actinic keratosis (AK), using in situ hybridization techniques. MMP-1 mRNA was detected in tumour cells and/or in stromal cells in all cases of SCC, four of six AKs adjacent to SCC and four of 16 AKs. MMP-2 and MMP-3 mRNAs were detected in SCC but not in AK. The expression of MMP-3 correlated to that of MMP-1 (P = 0.03) localized at the tumour mass and stroma of the invasive area, while MMP-2 mRNA was detected widely throughout the stroma independent of MMP-1 expression. Our results indicated that the expression of MMP-1, -2 and -3 showed different localization patterns, suggesting a unique role of each MMP in tumour progression. Moreover, MMP-1 expression could be an early event in the development of SCC, and AK demonstrating MMP-1 mRNA, might be in a more advanced dysplastic state, progressing to SCC. © 1999 Cancer Research Campaign

Increase of urokinase-type plasminogen activator receptor expression in human gingival fibroblasts by Porphyromonas gingivalis lipopolysaccharide.

Urokinase-type plasminogen activator receptor (uPAR) presenting on the cell surface with glycosylphosphatidylinositol (GPI) anchor is a key component in the plasminogen activator (PA)-plasmin system and plays a critical role in extracellular matrix degradation. It is believed that uPAR serves to localize and accelerate this generation system. In this study, we examined the levels of both uPA and uPAR in human gingival fibroblasts treated with Porphyromonas gingivalis lipopolysaccharide (LPS). Human gingival fibroblasts from the seventh to tenth doubling passages were plated at 5x10(4) cells per well in 24-well plates. The confluent-stage cells were cultured for 24 hours in alpha-MEM medium containing 2% fetal calf serum, after which they were incubated with P. gingivalis LPS. PA activity was measured using plasminogen and plasmin substrate S2251. PA activity in the cell lysate was increased by LPS and reached maximum at 1 microg/ml LPS after being incubated for 8 hours. PA activity released by phosphatidylinositol-specific phospholipase C, which detaches the GPI anchor, was also increased by LPS. The activity was inhibited by amiloride, which is a specific inhibitor for uPA. LPS increased the protein and mRNA levels of both uPA and uPAR in gingival fibroblasts. These findings suggest that increase of the uPAR level by LPS may play an important role in the progression of periodontal diseases through pericellular proteolysis.

Expression of cathepsin K in lung epithelial cells.

Alveolar and bronchial epithelial cells have been shown to have regulatory functions in the maintenance of lung structure and function. Recent evidence supports the premise that these cells can synthesize a variety of extracellular matrix components in vitro, suggesting an active participation in connective tissue remodeling. Their possible role in extracellular matrix degradation, however, is less clear. This study addresses the question of whether alveolar and bronchial epithelial cells express the highly collagenolytic and elastinolytic cysteine proteinase cathepsin K, which has recently been newly described. We provide evidence that the epithelial cell lines A549 and BEAS-2B are capable of expressing cathepsin K messenger RNA. Furthermore, we show that cathepsin K is expressed in normal bronchial epithelial cells. Western blot analyses of human lung-tissue lysates revealed specific immunoreactivity at molecular weights of 46 and 27 kD, corresponding to the procathepsin and the mature cathepsin K. Immunohistochemical analyses showed a pronounced staining of bronchial epithelial cells and in single alveolar epithelial cells. Using a specific fluorogenic cytochemical assay, the intracellular activity of the enzyme was localized. These findings demonstrate that bronchial and alveolar epithelial cells are capable of expressing cathepsin K, which could be of considerable importance for remodeling processes of the extracellular matrix in the lung.

Gelatinases A and B Are Up-Regulated in Rat Lungs by Subacute Hyperoxia: Pathogenetic Implications

Subacute hyperoxia may cause basement membrane disruption and subsequent fibrosis. To test the role of extracellular matrix degradation in hyperoxic damage, we analyzed the expression of gelatinases A and B and tissue inhibitors of metalloproteinases (TIMP)-1 and TIMP-2 in rats exposed to 85% O2. Oxygen-exposed rats were studied at 1, 3, 5, and 7 days, and compared with air-breathing rats. Lung mRNAs assayed by Northern and in situ hybridization showed an up-regulation of lung gelatinases A and B from the 3rd day on. Gelatinase A was localized in alveolar macrophages and in interstitial and alveolar epithelial cells. Gelatinase B mRNA and protein were localized in macrophages and bronchiolar and alveolar epithelial cells. Increased gelatinase A and B activities were demonstrated in bronchoalveolar lavage. TIMP-1 and TIMP-2 were constitutively expressed, and only TIMP-1 displayed a moderate increase with hyperoxia. To elucidate transcriptional mechanisms for increased gelatinase B expression after hyperoxia, nuclear transcription factor-kappabeta activation was explored. Oxidative stress significantly increased the lung expression of nuclear transcription factor-kappabeta (p65) protein, and nuclear transcription factor-kappabeta activation and increased levels of gelatinases A and B were found in isolated type II alveolar cells obtained from hyperoxic rats. Conceivably, subacute hyperoxia induces excessive gelatinase activity, which may contribute to lung damage.

Gelatinase B in proliferative vitreoretinal disorders

Purpose: To investigate whether gelatinases A and B are involved in the pathogenesis of proliferative vitreoretinal disorders. Methods: In a prospective study of 101 consecutive patients, vitreous and paired serum samples were obtained from 38 patients with rhegmatogenous retinal detachment complicated by proliferative vitreoretinopathy, 25 patients with rhegmatogenous retinal detachment with no proliferative vitreoretinopathy, and 38 patients with proliferative diabetic retinopathy. Gelatinase activities were determined by quantitative zymography. Results: All vitreous samples contained comparable levels of the constitutive gelatinase A. Inducible gelatinase B was detected in eight (32%) of 25 vitreous samples from patients with rhegmatogenous retinal detachment with no proliferative vitreoretinopathy (mean ± SD, 319.5 ± 521.0 scanning units), in 17 (44.7%) of 38 vitreous samples from patients with proliferative vitreoretinopathy (560.6 ± 718.9 scanning units), and in 34 (89.5%) of 38 vitreous samples from patients with proliferative diabetic retinopathy (1,707.2 ± 1,220.3 scanning units). The incidence of detection of gelatinase B in proliferative diabetic retinopathy cases was significantly higher than it was in rhegmatogenous retinal detachment with no proliferative vitreoretinopathy and proliferative vitreoretinopathy cases ( P < .001). Gelatinase B levels in the vitreous samples of patients with proliferative diabetic retinopathy were higher than the levels found in patients with rhegmatogenous retinal detachment with no proliferative vitreoretinopathy and in patients with proliferative vitreoretinopathy ( P = .0152). Gelatinase A was detected in all the tested sera, whereas none of the tested paired serum samples contained detectable gelatinase B activity. Conclusions: Gelatinase B may play an important role in extracellular matrix degradation associated with neovascularization in proliferative diabetic retinopathy.

Lung alveolar epithelial cells synthesize interstitial collagenase and gelatinases A and B in vitro

Type II pneumocytes are multifunctional alveolar epithelial cells that play a major role in the maintenance of lung structure and function. Recent evidence supports that these cells can synthesize a variety of extracellular matrix components in vitro, suggesting an active participation in connective tissue remodeling. However, their possible role in extracellular matrix degradation is unknown. In this study the production of matrix metalloproteinases (MMPs) was examined in primary cultures of rat alveolar type II pneumocytes after 2 and 7 days in culture. Under basal conditions, at both periods type II cells expressed interstitial collagenase mRNA. The immunoreactive protein was detected both in the cells and in conditioned media, and collagenolytic activity was revealed after trypsin activation. Gelatinolytic activity was detected by zymography showing a relative molecular mass of ∼72 and 92 kDa (gelatinases A and B). Phorbol treatment increased collagenase and gelatinase activities. In addition, three alveolar epithelial cell lines were analysed for MMP production: MLE-12 (mice), L2 (rat), and A549 (human). The cell lines A549 and MLE-12 revealed collagenase and gelatinase A and B activities whereas the L2 cell line only exhibited gelatinase A activity, even after PMA induction. These findings demonstrate that alveolar epithelial cells synthesize in vitro several MMPs that confer on them the ability to degrade extracellular matrix and basement membrane components, a capacity of considerable importance for the remodeling of the stromal/epithelial interface.

High Glucose Reduces Generation of Plasmin Activity by Mesangial Cells

Mesangium enlargement is a central feature of diabetic nephropathy and almost certainly plays a pathogenic role in this condition. Previous studies have shown that mesangium degradation is reduced in a high glucose mileau. Plasmin has been shown to play an important role in extracellular matrix degradation, both directly and through its ability to activate the matrix metalloproteinases. We therefore investigated how high glucose concentration may affect the various components of the plasminogen cascade on mesangial cells and whether it impairs the ability of the mesangial cell to generate plasmin activity. Results showed decreased binding of plasminogen and the urokinase type plasminogen activator to the mesangial cell surface while the tissue type plasminogen activator and the plasminogen activator-1 associated with mesangial cells were increased. The net effect of these changes was a reduced capacity of mesangial cell layers to generate plasmin activity in a high glucose environment. We postulate that this may be of importance in the reduced mesangium degradation which occurs in diabetes.

Stimulation of 92-kDa Gelatinase B Promoter Activity by ras Is Mitogen-activated Protein Kinase Kinase 1-independent and Requires Multiple Transcription Factor Binding Sites Including Closely Spaced PEA3/ets and AP-1 Sequences

The 92-kDa type IV collagenase (92-kDa gelatinase B also referred to as MMP-9), which plays a critical role in extracellular matrix degradation, is regulated by growth factors that mediate their effects through the ras proto-oncogene. The current study was undertaken to determine the transcriptional requirements for the induction of 92-kDa gelatinase B expression by an activated ras oncogene. Transfection of OVCAR-3 cells with an expression vector encoding an activated Ha-ras increased 92-kDa gelatinolytic activity and stimulated (over 10-fold) the activity of a CAT reporter driven by 670 nucleotides of 5' flanking sequence of the 92-kDa gelatinase B gene. Transient assays using a CAT reporter driven by 5' deleted fragments of the 92-kDa gelatinase B promoter indicated that a region spanning -634 to -531 was required for optimal induction of the promoter. The individual deletion, or mutation, of a PEA3/ets (-540) motif, AP-1 sites (-533, -79), a NF-kappa B (-600) consensus sequence, and a GT box (-52) substantially reduced the activation of the promoter by ras. An expression vector encoding the PEA3 transcription factor caused a 3-fold stimulation of the wild type but not the PEA3/ets-deleted 92-kDa gelatinase B promoter. Coexpression of a dominant negative c-jun antagonized the ras-dependent stimulation of the 92-kDa gelatinase B promoter-driven CAT reporter. The signaling pathway mediating the induction of 92-kDa gelatinase B promoter activity by ras was examined. The expression of a phosphatase (CL100) which inactivates multiple mitogen-activate protein kinase members abrogated the stimulation of 92-kDa gelatinase B promoter activity by ras. However, the expression of a kinase-deficient mitogen-activated protein kinase kinase 1 (MEK1) did not prevent activation of the 92-kDa gelatinase B promoter by ras and a constitutively activated c-raf expression vector was insufficient for 92-kDa gelatinase B promoter activation. Thus, the stimulation of the 92-kDa gelatinase B promoter by ras requires multiple elements including closely spaced PEA3/est and AP-1 sites and is MEK1-independent.

In vivo and in vitro invasion in relation to phenotypic characteristics of human colorectal carcinoma cells

In this study we investigated the tumorigenicity, growth pattern and spontaneous metastatic ability of a series of nine human colorectal carcinoma cell lines after subcutaneous and intracaecal xenografting in nude mice. CaCo2 cells were found to be poorly tumorigenic to non-tumorigenic in either site; the other cell lines were tumorigenic in both sites. SW1116, SW480 and SW620 did not show local invasive in the NCI-H716 and LS174T cells were both invasive in the caecum, but only NCI-H716 was invasive in the subcutis. HT29 and 5583 (S and E) cells were invasive in the caecum and from that site metastatic to the lungs and/or the liver. HT29 and 5583S cells were both invasive in the subcutis, but 5583E cells were not. Of each category of in vivo behaviour in the caecum, one cell line was further investigated with regard to invasion in vitro (into embryonic chick heart fragments), E-cadherin expression in vivo and in vitro and in vitro production of u-PA and t-PA. These parameters were chosen in view of their purported role in extracellular matrix degradation and intercellular adhesion, which are all involved in the invasive and metastatic cascade. Invasion in vitro was not predictive for invasion or metastasis in vivo. In the cell line which showed invasion in embryonic chick heart tissue, heterogeneous E-cadherin expression was observed in vitro together with a relatively high production of u-PA. The non-invasive cell lines showed in vitro homogeneous expression of E-cadherin with a relatively low production of u-PA. In vivo expression of E-cadherin was either absent or heterogeneous. We conclude that: (1) colorectal carcinoma xenografts show site-specific modification of in vivo invasive and metastatic behaviour; (2) invasion in vitro does not correlate with invasion and metastasis in vivo; (3) in vitro non-invasion might be associated with homogeneous E-cadherin expression and low production of u-PA; (4) E-cadherin expression in vitro differs from E-cadherin expression in vivo. The results support the notion that the microenvironment in which cancer cells grow is one of the factors involved in the regulation of invasive and metastatic behaviour.

Molecular cloning of human cathepsin O, a novel endoproteinase and homologue of rabbit OC2

A 1670-bp cDNA coding for a novel human cysteine protease has been isolated from a monocyte-derived macrophage cDNA library. This cDNA predicts a 329-amino acid preprocathepsin with more than 50% identity to both human cathepsin S and cathepsin L and 94% identity to a rabbit cDNA, termed OC2, recently isolated from osteoclasts. Based on its high homology to OC2, we have named the human enzyme cathepsin O. Cathepsin O mRNA was identified as a single ∼1.7 kb transcript in cultures of 15-day-old monocyte-derived macrophages, but was not expressed in human monocytes or alveolar macrophages. When transfected into COS-7 cells, cathepsin O displayed potent endoprotease activity against fibrinogen at acid pH. This novel endoprotease may play an important role in extracellular matrix degradation.

The extracellular matrix of normal chick embryo fibroblasts: its effect on transformed chick fibroblasts and its proteolytic degradation by the transformants.

Extracellular matrix (ECM), prepared from chick embryo fibroblasts, contains fibronectin as the major structural protein along with collagen and other polypeptides as less abundant protein components. When Rous sarcoma virus-transformed chick embryo fibroblasts are cultured on the ECM in the presence of the tumor promoter tetradecanoyl phorbol acetate, the transformed cells lose their characteristic rounded morphology and align on and within the ECM fibrillar network. This restrictive aspect of ECM is only temporary, however, and with time (24-72 h) the transformed cells progressively degrade the ECM fibers and resume their rounded appearance. The matrix degradation can be monitored by employing biosynthetically radiolabeled ECM. The addition of purified chicken plasminogen to the Rous sarcoma virus-transformed chick embryo fibroblast cultures enhances the rate and extent of ECM degradation, due to the elevated levels in the transformed cultures of plasminogen activator. Plasminogen-dependent and -independent degradation of ECM has been characterized with regard to sensitivity to various natural and synthetic protease inhibitors and to the requirement of cell/ECM contact. Plasminogen-dependent degradation of ECM occurs rapidly when ECM and cells are in contact or separated, whereas plasminogen-independent degradation is greatly reduced when ECM and cells are separated, which suggests that cell surface-associated proteolytic enzymes are involved. A possible role in ECM degradation has been indicated for cysteine proteases, metallo enzymes, and plasminogen activator, the latter as both a zymogen activator and a direct catalytic mediator.

Epithelium-mesenchyme interaction in the hormone response of the embryonic mammary gland: [formula omitted], Institute of Molecular Biology, Billrothstr. 11, A-5020 Salzburg, Austria

Mesangium enlargement is a central feature of diabetic nephropathy and almost certainly plays a pathogenic role in this condition. Previous studies have shown that mesangium degradation is reduced in a high glucose mileau. Plasmin has been shown to play an important role in extracellular matrix degradation, both directly and through its ability to activate the matrix metalloproteinases. We therefore investigated how high glucose concentration may affect the various components of the plasminogen cascade on mesangial cells and whether it impairs the ability of the mesangial cell to generate plasmin activity. Results showed decreased binding of plasminogen and the urokinase type plasminogen activator to the mesangial cell surface while the tissue type plasminogen activator and the plasminogen activator-1 associated with mesangial cells were increased. The net effect of these changes was a reduced capacity of mesangial cell layers to generate plasmin activity in a high glucose environment. We postulate that this may be of importance in the reduced mesangium degradation which occurs in diabetes.

Neutral Proteinases from Human Inflammatory Cells: A Critical Review of Their Role in Extracellular Matrix Degradation

The cell biology and in vitro activity of neutral proteinases derived from inflammatory cells and the mechanisms by which these enzymes might injure connective tissue are outlined. Current work directed toward understanding local control of tissue degradation may lead to effective strategies for assessing and reducing connective tissue injury in human disease.