An immunohistochemical study of extracellular matrix proteins laminin, fibronectin and type IV collagen in paediatric glioblastoma multiforme

Cultures of human epidermal keratinocytes obtained from adult epidermis were initiated using irradiated BALB/3T3 cells as feeder layers. At different stages of confluence of the epidermal islands, feeder cells were removed and the extracellular matrix proteins of both pure component cells and cocultures were analyzed biochemically and by immunochemical methods and compared to those of skin fibroblasts of the same donors. The keratinocytes synthesized and secreted fibronectin and small amounts of laminin and type IV collagen. In addition, a nondisulfide-linked collagenous polypeptide (Mr = 120,000) was synthesized by the keratinocytes and was confined to the cell layers. Collagenous polypeptides with Mr = 120,000 were also synthesized by organ cultures of epidermal tissue and were detected in its acid or detergent extracts but again no secretion to culture medium was found. The Mr = 120,000 collagen had biochemical and immunological properties distinct from those of types I-V collagens. In immunofluorescence of keratinocyte cultures, fibronectin staining was prominent in the lining marginal cells of the expanding periphery of the epidermal cell islands but was not detected in the terminally differentiating cells in the upper layers of stratified colonies. Very little type IV collagen was found deposited in pericellular matrix form by the keratinocytes. In contrast, the mouse 3T3 feeder cells were found to produce both type IV collagen and laminin in addition to the previously identified connective tissue glycoproteins of fibroblasts, interstitial procollagens, and fibronectin. Basement membrane collagen of the 3T3 cells was found deposited as apparently unprocessed procollagen alpha 1(IV) and alpha 2(IV) chains. The production in culture conditions of basal lamina glycoproteins by the fibroblastic feeder cells may promote the attachment and growth of the cocultured keratinocytes.

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Degradation of extracellular-matrix proteins by human cathepsin B from normal and tumour tissues

Biochemical Journal ◽

10.1042/bj2820273 ◽

1992 ◽

Vol 282 (1) ◽

pp. 273-278 ◽

Cited By ~ 261

Author(s):

M R Buck ◽

D G Karustis ◽

N A Day ◽

K V Honn ◽

B F Sloane

Keyword(s):

Extracellular Matrix ◽

Cathepsin B ◽

Type Iv Collagen ◽

Degradation Products ◽

Extracellular Matrix Proteins ◽

Matrix Proteins ◽

Neutral Ph ◽

Type Iv ◽

Reducing Conditions ◽

Extracellular Matrix Components

Our laboratory has previously demonstrated that increased malignancy of several histological types of human and animal tumours is associated with increases in their cathepsin B activity, particularly cathepsin B activity associated with plasma-membrane/endosomal vesicles or shed vesicles. Here we report that cathepsin B from normal or tumour tissues degrades purified extracellular-matrix components, type IV collagen, laminin and fibronectin, at both acid pH and neutral pH. The number and sizes of degradation products were analysed by SDS/PAGE. Cathepsin B from both sources exhibited similar activities towards, and similar patterns of cleavage of, the extracellular-matrix proteins. At neutral pH, cathepsin B from both sources appeared to undergo autodegradation, a process that was decreased in the presence of alternative substrates such as the extracellular-matrix proteins. Cathepsin B readily degraded type IV collagen at 25 degrees C, indicating activity towards native type IV collagen. Fibronectin degradation products of 100-200 kDa and of 18 and 22 kDa were observed. A single 70 kDa fragment was released from laminin under non-reducing conditions and multiple fragments ranging from 45 to 200 kDa under reducing conditions. These results suggest that cathepsin B at or near the surface of malignant tumour cells may play a functional role in the focal dissolution of extracellular matrices.

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Cwp84, a Surface-Associated Protein of Clostridium difficile, Is a Cysteine Protease with Degrading Activity on Extracellular Matrix Proteins

Journal of Bacteriology ◽

10.1128/jb.00578-07 ◽

2007 ◽

Vol 189 (20) ◽

pp. 7174-7180 ◽

Cited By ~ 79

Author(s):

Claire Janoir ◽

Séverine Péchiné ◽

Charlotte Grosdidier ◽

Anne Collignon

Keyword(s):

Extracellular Matrix ◽

Clostridium Difficile ◽

Cysteine Protease ◽

Specific Inhibitor ◽

Extracellular Matrix Proteins ◽

Maximum Activity ◽

Matrix Proteins ◽

Specific Antibodies ◽

Type Iv

ABSTRACT Clostridium difficile pathogenicity is mediated mainly by its A and B toxins, but the colonization process is thought to be a necessary preliminary step in the course of infection. The aim of this study was to characterize the Cwp84 protease of C. difficile, which is highly immunogenic in patients with C. difficile-associated disease and is potentially involved in the pathogenic process. Cwp84 was purified as a recombinant His-tagged protein, and specific antibodies were generated in rabbits. Treatment of multiple-band-containing eluted fractions with a reducing agent or with trypsin led to accumulation of a unique protein species with an estimated molecular mass of 61 kDa, corresponding most likely to mature autoprocessed Cwp84 (mCwp84). mCwp84 showed concentration-dependent caseinolytic activity, with maximum activity at pH 7.5. The Cwp84 activity was inhibited by various cysteine protease inhibitors, such as the specific inhibitor E64, and the anti-Cwp84-specific antibodies. Using fractionation experiments followed by immunoblot detection, the protease was found to be associated with the S-layer proteins, mostly as a nonmature species. Proteolytic assays were performed with extracellular matrix proteins to assess the putative role of Cwp84 in the pathogenicity of C. difficile. No degrading activity was detected with type IV collagen. In contrast, Cwp84 exhibited degrading activity with fibronectin, laminin, and vitronectin, which was neutralized by the E64 inhibitor and specific antibodies. In vivo, this proteolytic activity could contribute to the degradation of the host tissue integrity and to the dissemination of the infection.

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Interaction of enteropathogenic Yersinia enterocolitica with complex basement membranes and the extracellular matrix proteins collagen type IV, laminin-1 and -2, and nidogen/entactin.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)43942-7 ◽

1994 ◽

Vol 269 (47) ◽

pp. 29732-29738

Author(s):

A Flügel ◽

H Schulze-Koops ◽

J Heesemann ◽

K Kühn ◽

L Sorokin ◽

...

Keyword(s):

Extracellular Matrix ◽

Yersinia Enterocolitica ◽

Collagen Type ◽

Extracellular Matrix Proteins ◽

Basement Membranes ◽

Matrix Proteins ◽

Collagen Type Iv ◽

Type Iv ◽

Enteropathogenic Yersinia ◽

Laminin 1

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The Major Pilin Subunit of the AAF/II Fimbriae from Enteroaggregative Escherichia coli Mediates Binding to Extracellular Matrix Proteins

Infection and Immunity ◽

10.1128/iai.00439-08 ◽

2008 ◽

Vol 76 (10) ◽

pp. 4378-4384 ◽

Cited By ~ 51

Author(s):

Mauricio J. Farfan ◽

Keith G. Inman ◽

James P. Nataro

Keyword(s):

Escherichia Coli ◽

Extracellular Matrix ◽

Extracellular Matrix Proteins ◽

Prototype Strain ◽

Matrix Proteins ◽

Dependent Manner ◽

T84 Cells ◽

Cell Monolayers ◽

Type Iv ◽

Pilin Gene

ABSTRACT Enteroaggregative Escherichia coli (EAEC) adherence to human intestinal tissue is mediated by aggregative adherence fimbriae (AAF); however, the receptors involved in EAEC adherence remain uncharacterized. Adhesion to extracellular matrix proteins is commonly observed among enteric pathogens, so we addressed the hypothesis that EAEC may bind to extracellular matrix proteins commonly found in the intestine. We found that EAEC prototype strain 042 adhered more abundantly to surfaces that were precoated with the extracellular matrix proteins fibronectin, laminin, and type IV collagen. Differences in fibronectin binding of almost 2 orders of magnitude were observed between EAEC 042 and a mutant in the AAF/II major pilin gene, aafA. Purified AafA, refolded as a donor strand complementation construct, bound fibronectin in a dose-dependent manner. Addition of fibronectin to the apical surfaces of polarized T84 cell monolayers augmented EAEC 042 adherence, and this effect required expression of aafA. Finally, increased bacterial adherence was observed when apical secretion of fibronectin was induced by adenosine in polarized T84 cells. Binding to fibronectin may contribute to colonization of the gastrointestinal tract by EAEC.

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Mechanism of saikosaponin-d in the regulation of rat mesangial cell proliferation and synthesis of extracellular matrix proteinsThis paper is one of a selection of papers in this Special Issue, entitled International Symposium on Recent Advances in Molecular, Clinical, and Social Medicine, and has undergone the Journal's usual peer-review process.

Biochemistry and Cell Biology ◽

10.1139/o07-002 ◽

2007 ◽

Vol 85 (2) ◽

pp. 169-174 ◽

Cited By ~ 12

Author(s):

Ning Zu ◽

Ping Li ◽

Ning Li ◽

Patrick Choy ◽

Yuewen Gong

Keyword(s):

Extracellular Matrix ◽

Cell Proliferation ◽

Type Iv Collagen ◽

Mesangial Cells ◽

Kidney Diseases ◽

Peer Review Process ◽

Matrix Proteins ◽

Glomerular Mesangial Cells ◽

Type Iv ◽

Tgf Β1

Glomerulosclerosis is a common disorder in many types of chronic kidney diseases. Previous studies have shown that glomerular mesangial cells (MCs) play an important role in the pathogenesis of glomerulosclerosis. The ability of saikosaponin-d (SSd) to reduce the damage of kidney in progressive glomerulosclerosis has been demonstrated. In this study, the effects of saikosaponin-d on MC proliferation and synthesis of extracellular matrix proteins were investigated. Rat MCs were isolated from Wistar rats and cultured in Dulbecco's modified Eagle's medium. MCs were challenged with lipopolysacchorides and incubated with different concentrations of SSd. Cell proliferation and cytotoxicity were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry, and lactate dehydrogenase assays. Type IV collagen, fibronectin, and TGF-β1 in the conditioned medium were measured. The expression of cyclin-dependent kinase 4, c-Jun, and c-Fos was determined by immunohistochemistry. At a concentration of 4 µg/mL or lower, SSd inhibited MC proliferation but did not cause cell death. SSd also inhibited lipopolysaccharide-induced secretion of type IV collagen, fibronectin, and TGF-β1 in MCs. Additionally, SSd reduced the expression of CDK4, c-Jun, and c-Fos in MCs. We conclude that SSd inhibited MC proliferation and synthesis of extracullular matrix proteins through the downregulation of the CDK4, c-Jun, and c-Fos genes.

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