Induction of MMP-1 (collagenase-1) by relaxin in fibrocartilaginous cells requires both the AP-1 and PEA-3 promoter sites

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75676/1/j.1601-6343.2009.01451.x.pd

Expression and regulation of matrilysin in human tumours

Matrilysin is a member of a multigene family of proteolytic enzymes called the matrix metalloproteinases (MMPs). It has previously been determined that matrilysin has a role to play in the development of colon cancers, however, its participation in other tumour types has not been fully explored. The aims of this research were (i) to develop an ELISA for matrilysin; (ii) to examine human breast tumour tissue by a variety of methods for expression of matrilysin; (iii) to investigate the expression of matrilysin in cancers of other tissues using cell lines; and (iv) to examine how the expression of matrilysin could be modulated by polypeptide growth factors and cytokines. A sensitive and specific one-step sandwich ELISA was developed. This was validated and shown to have excellent reproducibility. The limit of detection was 0.45 ng/ml and the linear range was 5 - 5 0 ng/ml. A pilot-scale study of ten breast tumour samples and three normal breast tissue samples demonstrated the presence of matrilysin mRNA and protein in both normal and tumour cells. Immunohistochemistry was found to be the most useful and informative method of detecting matrilysin protein. The intensity of expression varied among tumour specimens but did not correlate with stage of disease. Matrilysin expression was examined in a variety of human tumour cell lines using RTPCR and the matrilysin ELISA. Of the eight lines tested, only four produced the enzyme. Levels of matrilysin could be stimulated with cytokines. Interleukin-6, IGF-I and IGF-II, not previously reported as modulators of MMP activity, were among the most potent stimulators at both the mRNA and protein levels. These results were confirmed using matrilysin promoter-reporter gene constructs transfected into suitable cells

The expression and regulation of matrilysin (MMP-7) in human colon cancer and leukaemia cell lines

Matrilysin (MMP-7, EC 3.4.24.23) is the smallest member o f the matrix metalloproteinase (MMP) family and has been shown to be overexpressed in various tumours including breast and colon cancers. Matrilysin has also been shown to play an important role in several aspects of tumour biology including growth, progression, invasion and metastasis. With respect to colon cancer, matrilysin is unique in that it is the only MMP expressed exclusively by the malignant epithelia o f colonic adenocarcinomas. These facts combine to make matrilysin a promising therapeutic target. However, in order to develop drugs which specifically inhibit matrilysin it is important to understand how matrilysin gene expression is controlled, something which to date remains poorly understood. We have examined a panel of human colon tumour cell lines and have shown that matrilysin expression can be upregulated by a number of cytokines including EGF, IL-6 and bFGF. Analysis of the matrilysin promoter revealed the presence of a number of potential transcription factor binding sites including three ETS sites. We have shown that EGF treatment increased matrilysin gene expression by activation of PEA3 transcription factors using artificial promoter, western blot and EMSA analysis. ‘Supershift’ EMSA analysis showed that other PEA3 subfamily members such as ERM and ER81 may also be involved which is in agreement with other studies. In addition, we have found that EGF increased cellular levels o f [3-catenin through destabilisation of the E-cadherin/catenin complex which resulted in increased binding to the T c f site within the matrilysin promoter. We also examined the expression and regulation of matrilysin in the K562 and HL-60 myeloid leukaemia cell lines. Results showed that only the K562 cell line expressed matrilysin and in vitro invasion assays showed that the K562 cells were up to 4 times more invasive than the HL- 60 cell line. Matrilysin antibody blocking experiments showed a significant decrease in invasion in the K562 cell line suggesting a role for matrilysin in leukaemia invasion. The MMP and TIMP profiles o f these cell lines were also examined. Our data suggests that EGF plays an important role in the regulation of matrilysin gene expression via a number of new mechanisms. Furthermore, we have shown that matrilysin plays an important role in leukaemia cell line invasion. These findings have identified possible new drug targets that will inhibit matrilysin expression which in turn should lead to decreased tumourigenesis and invasion and metastasis

Regulation of protein expression by transforming growth factor beta and other growth modulators

The main goal of this project was to understand the signal transduction pathways through which growth modulators such as TGF-[beta], PMA and retinoic acid mediate protein expression and DNA synthesis. Transforming growth factor [beta] (TGF-[beta]) induces synthesis and secretion of a 48,000 M r protein that we have found to be type I plasminogen activator inhibitor (PAI-1). TGF-[beta] induces PAI-1 in every cell line that we have studied including near term mink lung epithelial CCL64 cells, mouse embryo fibroblast AKR-2B 84A cells, African green monkey kidney epithelial BSC-1 cells, and normal rat kidney fibroblast NRK 49F cells. PAI-1 is induced synergistically by TGF-[beta] and EGF in CCL64 cells. Phorbol 12-myristate 13-acetate induces PAI-1 in CCL64, BSC-1 and NRK cells but not in AKR-2B cells. TGF-[beta] and retinoic acid both induce synthesis and secretion of a 73,000 M r protein by CCL64 cells that we call inhibitor-induced protein, 73 kDa (IIP73). Retinoic acid does not significantly regulate PAI-1 expression. PMA does not induce IIP73. To test whether cAMP was a regulator of the growth modulator effects, we treated cells with agents which increase intracellular cAMP (forskolin, cholera toxin, and dibutyryl cAMP) and measured the effects on expression of PAI-1 and IIP73 and on DNA synthesis. We found that agents that increase intracellular cAMP lowered the basal and induced levels of PAI-1 coordinately and lowered DNA synthesis rates in AKR-2B, BSC-1, CCL64 and NRK cells. However, these same factors had little or no effect on the ability of TGF-[beta] or retinoic acid to induce IIP73. To test for the necessity of activated protein kinase C (PKC) in the induction of PAI-1 and IIP73 expression and to test the role of PKC in stimulation of DNA synthesis, we down-regulated PKC in CCL64 cells by a 48 h incubation with a high concentration of PMA. While PMA could no longer induce PAI-1, there was no effect on the ability of TGF-[beta] to induce PAI-1 and no effect on the ability of TGF-[beta] and retinoic acid to induce IIP73. However, down-regulation of PKC prevented retinoic acid stimulation of DNA synthesis in CCL64 cells. This shows that induction of PAI-1 by TGF-[beta] does not depend on activation of PKC and that retinoic acid may induce proliferation in CCL64 cells through a PKC-dependent mechanism

Investigating the mechanism of mrp/plf gene expression by bFGF

The mitogen-regulated protein/proliferin (mrp/ plf) genes belong to the prolactin/growth hormone gene superfamily and encode at least four closely related proteins. Identified functions of these proteins include stimulation of uterine proliferation and endothelial angiogenesis. In 3T3 cells, basic fibroblast growth factor (bFGF) stimulates the production of mrp/plf mRNAs with a resulting increase in the protein products. Although the three cloned mrp/ plf gene promoters (mrp3, plf42, and plf149 ) are over 97% identical in sequence, only mrp3 is transcriptionally activated by bFGF. We have identified a sequence in the mrp3 promoter, which we have named the bFGF-responsive element (FRE), (-186 to -167), that specifically binds nuclear factors from 3T3, CHO and Hela cells. Analysis of the bFGF-responsiveness of a series of truncated mrp3 promoter sequences combined with footprint analysis, pinpointed a region of the promoter that contains a large variation in sequence between the three promoters and one base in the sequence that is unique to the mrp3. The nuclear factors bound by the FRE are present in the placenta and the fetus in which the gene is expressed. By contrast, the maternal liver does not contain FRE-binding proteins. The FRE is transcriptionally active in a TK fusion promoter and responds to bFGF in this context. Our results show that the FRE is an bFGF-responsive transcriptional element. We demonstrate the sequence between -186 to -167 of the three mrp/plf promoters is differentially regulated by bFGF in a TK fusion promoter. Characterization of the 31--36 kDa protein(s) which bind these sequences suggest that different high affinity binding proteins bind to these different sequences. The core sequence of the FRE is also found in the promoters of genes encoding the interstitial collagenase type I and stromelysin-1, that are also regulated by bFGF as delayed early response genes. The FRE element may be the means by which the expression of mrp3 and other genes are regulated by bFG