Neutrophil collagenase (matrix metalloproteinase-8 or MMP-8) is regarded as being synthesized exclusively by polymorphonuclear neutrophils (PMN). However, in vivo MMP-8 expression was observed in mononuclear fibroblast-like cells in the rheumatoid synovial membrane. In addition, we detected MMP-8 mRNA expression in cultured rheumatoid synovial fibroblasts and human endothelial cells. Up-regulation of MMP-8 was observed after treatment of the cells with either tumor necrosis factor-␣ (10 ng/ml) or phorbol 12-myristate 13-acetate (10 nM). Western analysis showed a similar regulation at the protein level. The size of secreted MMP-8 was 50 kDa, which is about 30 kDa smaller than MMP-8 from PMN. Conditioned media from rheumatoid synovial fibroblasts contained both type I and II collagen degrading activity. However, degradation of type II collagen, but not that of type I collagen, was completely inhibited by 50 M doxycycline, suggesting specific MMP-8 activity. In addition, doxycycline down-regulated MMP-8 induction, at both the mRNA and protein levels. Thus MMP-8 exerts markedly wider expression in human cells than had been thought previously, implying that PMN are not the only source of cartilage degrading activity at arthritic sites. The inhibition of both MMP-8 activity and synthesis by doxycycline provides an incentive for further studies on the clinical effects of doxycycline in the treatment of rheumatoid arthritis.Extracellular matrix degradation is fundamental to connective tissue remodeling during physiological processes as well as during the progress of several pathological phenomena. Matrix turnover is regulated by a delicate balance among the production, activation, and inhibition of proteolytic enzymes. The matrix metalloproteinases (MMPs) 1 form a gene family of at least 14 enzymes participating in extracellular matrix remodeling. MMPs, together with the factors associated with their regulation, are reported to be highly implicated in various diseases such as rheumatoid arthritis, osteoarthritis, corneal ulceration, atherosclerosis, and tumor invasion and metastasis (for reviews, see Refs. 1-3). Previous studies have demonstrated that neutrophil-derived MMPs such as collagenase (MMP-8) and gelatinase B (MMP-9, 92-kDa type IV collagenase), play a key role in the degradation of extracellular matrix constituents i.e. during the course of inflammatory diseases (4 -7). Collagenases exist as three distinct molecules, namely the fibroblast type (MMP-1, collagenase-1) (8), the neutrophil type (MMP-8) (9), and collagenase-3 (MMP-13) (10). They all are able to degrade specifically the fibrillar collagen types I, II, and III as well as type VII and X collagens (11, 12), serpins (4, 13), ␤-casein, and human ␣ 2 -macroglobulin (14). Among collagenases, MMP-8 most effectively hydrolyzes the native type I and II collagens, whereas MMP-1 prefers type III collagen. MMP-8 is a considerably more efficient enzyme than MMP-1 with respect to almost all substrates except for type III collagen (9). MMP-1 is transcribed and exp...

Carcinoma cell invasion is traditionally studied in three-dimensional organotypic models composed of type I collagen and fibroblasts. However, carcinoma cell behavior is affected by the various cell types and the extracellular matrix (ECM) in the tumor microenvironment. In this study, a novel organotypic model based on human uterine leiomyoma tissue was established and characterized to create a more authentic environment for carcinoma cells. Human tongue squamous cell carcinoma cells (HSC-3) were cultured on top of either collagen or myoma. Organotypic sections were examined by immunohistochemistry and in situ hybridization. The maximal invasion depth of HSC-3 cells was markedly increased in myomas compared with collagen. In myomas, various cell types and ECM components were present, and the HSC-3 cells only expressed ECM molecules in the myoma model. Organotypic media were analyzed by radioimmunoassay, zymography, or Western blotting. During carcinoma cell invasion, matrix metalloprotease-9 production and collagen degradation were enhanced particularly in the myoma model. To evaluate the general applicability of the myoma model , several oral carcinoma , breast carcinoma , and melanoma cell lines were cultured on myomas and found to invade in highly distinct patterns. We conclude that myoma tissue mimics the native tumor microenvironment better than previous organotypic models and possibly enhances epithelial-tomesenchymal transition. Thus, the myoma model provides a promising tool for analyzing the behavior of carcinoma cells.

Summary Although matrix metalloproteinases (MMPs) are among the potential key mediators of cancer invasion, their involvement in premalignant lesions and conditions is not clarified. Therefore, we studied, using in situ hybridization, immunohistochemistry and zymography the expression and distribution of MMP-1 and -2, and their tissue inhibitors (TIMPs -1, -2 and -3) in oral squamous cell carcinomas (SCC) and lymph node metastases as well as in oral lichen planus, epithelial dysplasias and normal buccal mucosa. In oral SCC and lymph node metastasis, MMP-1 mRNA was detected in fibroblastic cells of tumoral stroma. In two out of ten carcinomas studied, the peripheral cells of neoplastic islands were also positive. MMP-2 mRNA expression was noted in fibroblasts surrounding the carcinoma cells, and no signal in carcinoma cells was detected. A clear TIMP-3 mRNA expression was seen in stromal cells surrounding the neoplastic islands in all SCCs and lymph node metastases studied. TIMP-1 mRNA was detected in some stromal cells surrounding the neoplastic islands, whereas the mRNA expression for TIMP-2 was negligible. On the other hand, expression of MMPs and TIMPs was consistently low in oral epithelial dysplasias, lichen planus and normal mucosa. In certain epithelial dysplasias and lichen planus, MMP-1 and -2 mRNA expressions were detected in few fibroblasts under the basement membrane zone, but normal mucosa was completely negative. In SCC and lymph node metastasis, a detectable immunostaining for MMP-1 in stromal cells and in some carcinoma cells was observed. MMP-2 immunoreactivity was detected in the peripheral cell layer in neoplastic islands and in some fibroblast-like cells of tumoral stroma. Immunostaining for TIMP-3 was detected in stromal cells surrounding the neoplastic islands. A weak positive staining for TIMP-1 was located in tumoral stroma, whereas the immunostaining for TIMP-2 was negative. Using zymography, elevated levels of MMP-2 and MMP-9 were observed in carcinoma samples in comparison with lichen planus or normal oral mucosa. Our results indicate that the studied MMPs and TIMPs are clearly up-regulated during invasion in oral SCC. However, there was also a clear, although weak, up-regulation of the expression of the MMPs but not TIMPs in some of the lichen planus and dysplastic lesions.Keywords: oral squamous cell carcinoma; matrix metalloproteinase; tissue inhibitor of metalloproteinase; mouth neoplasm Oral squamous cell carcinoma (SCC) has a high potential for invasiveness associated with a high rate of fatality. Distant-organ metastasis and regional lymph node metastasis are the major cause of mortality from the oral SCC. Oral lichen planus is regarded as a potential condition for malignant transformation, and thus dysplasia or carcinoma could arise from oral lichen planus (WHO, 1997).Matrix metalloproteinases (MMPs) are a highly regulated superfamily of enzymes that degrade almost all extracellular matrix and basement membrane components, processes which are essential for invasion and subseque...

Matrix metalloproteinases (MMPs) collectively degrade extracellular matrix and basement membrane proteins in chronic inflammation and bone-destructive lesions. This study examined the ability of immunoglobulin-producing plasma cells, typically present in sites of chronic inflammation, to express collagenases (MMP-8 and -13) in vivo and in vitro. Phorbol-12-myristate-13-acetate, interleukin-6, and tumour necrosis factor-alpha and heparin with the tumour promoter or cytokines potently enhanced (up to nine-fold) MMP-8 and -13 expression by the RPMI 8226 myeloma cell line, as evidenced by western blotting and semi-quantitative reverse transcriptase-polymerase chain reaction. Immunohistochemical analysis and in situ hybridization revealed that plasma cells expressed MMP-8 and -13 focally in periapical granulomas, odontogenic cysts, and malignant plasmacytomas. MMP-8 and MMP-13 from plasma cells can participate in bone organic matrix destruction at sites of chronic inflammation and neoplastic growth. Since MMP-13 was more frequently expressed than MMP-8 in plasma cells of strongly recurring keratocysts and malignant plasmacytomas, it is concluded that plasma cell MMP-13 has a particularly important role in benign and malignant bone-destructive lesions.

BackgroundThe composition of the matrix molecules is important in in vitro cell culture experiments of e.g. human cancer invasion and vessel formation. Currently, the mouse Engelbreth-Holm-Swarm (EHS) sarcoma -derived products, such as Matrigel®, are the most commonly used tumor microenvironment (TME) mimicking matrices for experimental studies. However, since Matrigel® is non-human in origin, its molecular composition does not accurately simulate human TME. We have previously described a solid 3D organotypic myoma disc invasion assay, which is derived from human uterus benign leiomyoma tumor. Here, we describe the preparation and analyses of a processed, gelatinous leiomyoma matrix, named Myogel.MethodsA total protein extract, Myogel, was formulated from myoma. The protein contents of Myogel were characterized and its composition and properties compared with a commercial mouse Matrigel®. Myogel was tested and compared to Matrigel® in human cell adhesion, migration, invasion, colony formation, spheroid culture and vessel formation experiments, as well as in a 3D hanging drop video image analysis.ResultsWe demonstrated that only 34 % of Myogel’s molecular content was similar to Matrigel®. All test results showed that Myogel was comparable with Matrigel®, and when mixed with low-melting agarose (Myogel-LMA) it was superior to Matrigel® in in vitro Transwell® invasion and capillary formation assays.ConclusionsIn conclusion, we have developed a novel Myogel TME matrix, which is recommended for in vitro human cell culture experiments since it closely mimics the human tumor microenvironment of solid cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1944-z) contains supplementary material, which is available to authorized users.

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The effects of topical betamethasone-17-valerate on collagen propeptide levels, collagen mRNA level, lysyl oxidase mRNA and matrix metalloproteinase (MMP)-1 and MMP-2 mRNA levels were studied in human skin. Three days' treatment of healthy skin with topical betamethasone caused a 70-80% decrease in type I and III collagen propeptides in suction blister fluid. A similar decrease was found in type I collagen mRNA when assayed by either slot-blot hybridization or a quantitative polymerase chain reaction method, indicating that the decrease in collagen synthesis after topical glucocorticoid treatment is apparently due to a decrease in corresponding mRNA. mRNA of lysyl oxidase, which is an important enzyme catalysing the cross-linking of collagen chains, and collagen-degrading enzyme MMP-1 and MMP-2 mRNAs were not decreased in the same skin samples. This suggests that in vivo, glucocorticoids modulate variably the genes involved in collagen synthesis and degradation. Our study provides a solid molecular basis for glucocorticoid-induced dermal atrophy, which results from the decrease in functional collagen mRNA in the skin.

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