Ulrike Benbow

The AP-1 site and MMP gene regulation: what is all the fuss about?

Matrix metalloproteinase (MMP) gene expression occurs under tightly regulated mechanisms that lead to cell and tissue-specific expression of the individual genes. Despite this differential expression, there exists a high degree of similarity among the cis-acting elements in the MMP promoters. The Activator Protein-1 (AP-1) site at approximately -70 bp upstream of the transcriptional start site has long been thought to play a dominant role in the transcriptional activation of the MMP promoters, particularly in response to stimulation with phorbol myristate acetate (PMA). However, more recent data indicate that basal transcription, as well as transactivation by PMA, cytokines, and growth factors requires the specific interaction of AP-1 with other cis-acting elements. Particularly important are PEA3 sites, located either adjacent to this AP-1 site or more distally. On the otherhand, the AP-1 site plays a dominant role in repression of MMPs by transforming growth factor beta (TGF-beta), retinoids and glucocorticoids, although some AP-1 independent mechanisms may also contribute. While the AP-1 site is involved in tissue-specific expression of MMPs, the presence of one or more AP-2 elements appears critical. Thus, the AP-1 site, alone, does not regulate transcription of MMPs. Rather, there is an essential interaction with other cis-acting sequences in the promoters and with certain transcription factors that bind to these sequences. Together, these complex interactions control the transcription of the MMPs in response to particular inducers and repressors.

Cell‐type specific regulation of human interstitial collagenase‐1 gene expression by interleukin‐1β (IL‐1β) in human fibroblasts and BC‐8701 breast cancer cells

Interleukin‐1β (IL‐1β) is a potent cytokine that stimulates interstitial collagenase‐1 (matrix metalloproteinase‐1; MMP‐1). In this study, we compared the mechanism(s) by which IL‐1β induces collagenase gene expression in two very different cells, normal human foreskin fibroblasts (HFFs) and an aggressive breast cancer cell line, BC‐8701 cells. Northern analysis showed that the time course of collagenase induction was distinct in the two cells: although both cells expressed low levels of MMP‐1 constitutively, addition of IL‐1β increased MMP‐1 mRNA in HFFs by 1 h and levels remained high over a 24‐h period. In contrast, MMP‐1 levels in IL‐1β‐treated BC‐8701 cells did not increase until 4 h, peaked by 12 h and then declined. To analyze the transcriptional response, we cloned and sequenced more than 4,300 bp of the human MMP‐1 promoter, and from this promoter clone, we prepared a series of 5′‐deletion constructs linked to the luciferase reporter and transiently transfected these constructs into both cell types to measure both basal and IL‐1β induced transcription. When both cell types were uninduced, promoter fragments containing less than 2,900 bp gave only a minimal transcriptional response, while larger fragments showed increased transcriptional activity. With IL‐1β treatment, significant responsiveness (P < 0.001) in HFFs was seen only with the larger fragments, while in the BC‐8701 cells, all fragments were significantly induced with IL‐1β. Finally, we found that IL‐1β stabilized MMP‐1 mRNA in normal fibroblasts, but not in BC‐8701 breast cancer cells. We conclude that both the transcriptional and post‐transcriptional regulation of MMP‐1 gene expression by IL‐1β is controlled by cell‐type specific mechanisms, and we suggest that IL‐1 induced MMP‐1 expression in tumor cells and in neighboring stromal cells may amplify the invasive ability of tumor cells. J. Cell. Biochem. 66:322–336, 1977.

A Novel Host/Tumor Cell Interaction Activates Matrix Metalloproteinase 1 and Mediates Invasion through Type I Collagen

Along with degradation of type IV collagen in basement membrane, destruction of the stromal collagens, types I and III, is an essential step in the invasive/metastatic behavior of tumor cells, and it is mediated, at least in part, by interstitial collagenase 1 (matrix metalloproteinase 1 (MMP-1)). Because A2058 melanoma cells produce substantial quantities of MMP-1, we used these cells as models for studying invasion of type I collagen. With a sensitive and quantitative in vitro invasion assay, we monitored the ability of these cells to invade a matrix of type I collagen and the ability of a serine proteinase inhibitor and all-trans-retinoic acid to block invasion. Although these cells produce copious amounts of MMP-1, they do not invade collagen unless they are co-cultured with fibroblasts or with conditioned medium derived from fibroblasts. Our studies indicate that a proteolytic cascade that depends on stromal/tumor cell interactions facilitates the ability of A2058 melanoma cells to invade a matrix of type I collagen. This cascade activates latent MMP-1 and involves both serine proteinases and MMPs, particularly stromelysin 1 (MMP-3). All-trans-retinoic acid (10−6 m) suppresses the invasion of tumor cells by several mechanisms that include suppression of MMP synthesis and an increase in levels of tissue inhibitor of metalloproteinases 1 and 2. We conclude that invasion of stromal collagen by A2058 melanoma cells is mediated by a novel host/tumor cell interaction in which a proteolytic cascade culminates in the activation of pro-MMP-1 and tumor cell invasion.

A synthetic triterpenoid selectively inhibits the induction of matrix metalloproteinases 1 and 13 by inflammatory cytokines

OBJECTIVE To address the effects of a novel synthetic triterpenoid, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), on the induction of matrix metalloproteinases 1 and 13 (MMP-1, MMP-13) by inflammatory cytokines. METHODS Human chondrosarcoma cells stimulated with inflammatory cytokines (interleukin-1beta [IL-1beta], tumor necrosis factor alpha) were used to study the effects of CDDO on the induction of MMPs and the invasion of cells through a collagen matrix. RESULTS CDDO selectively reduced the induction of MMP-1 and MMP-13 at the levels of messenger RNA and protein. Treatment with CDDO prior to cytokine stimulation enhanced this inhibition, and we demonstrated that CDDO functions at the level of transcription. Additionally, CDDO reduced IL-1beta-mediated invasion of cells through a collagen matrix. CONCLUSION This study demonstrates that CDDO is a novel inhibitor of MMP-1 and MMP-13 gene expression mediated by inflammatory cytokines. Thus, CDDO may have therapeutic potential for the inhibition of joint degradation in osteoarthritis.

Consequences of altered TGF-β expression and responsiveness in breast cancer: evidence for autocrine and paracrine effects

To characterize the impact of increased production of TGF-β in a xenograft model of human breast cancer, TGF-β-responsive MDA-231 cells were genetically modified by stable transfection so as to increase their production of active TGF-β1. Compared with control cells, cells that produced increased amounts of TGF-β proliferated in vitro more slowly. In vivo, however, tumors derived from these cells exhibited increased proliferation and grew at an accelerated pace. To evaluate the role of autocrine TGF-β signaling, cells were also transfected with a dominant-negative truncated type II TGF-β receptor (TβRII). Disruption of autocrine TGF-β signaling in the TGF-β-overexpressing cells reduced their in vivo growth rate. Co-inoculation of Matrigel with the TGF-β-overexpressing cells expressing the truncated TβRII compensated for their diminished in vivo growth capacity, compared with the TGF-β-overexpressing cells with an intact autocrine loop. Tissue invasion by the tumor was a distinctive feature of the TGF-β-overexpressing cells, whether or not the autocrine loop was intact. Furthermore, tumors derived from TGF-β-overexpressing cells, irrespective of the status of the autocrine TGF-β-signaling pathway, had a higher incidence of lung metastasis. Consistent with the suggestion that TGF-βs enhancement of invasion and metastasis is paracrine-based, we observed no significant differences among the cell clones in an in vitro invasion assay. Thus, in this experimental model system in vitro assays of cell proliferation and invasion do not accurately reflect in vivo observations, perhaps due to autocrine and paracrine effects of TGF-β that influence the important in vivo-based phenomena of tumor growth, invasion, and metastasis.

High levels of MMP‐1 expression in the absence of the 2G single nucleotide polymorphism is mediated by p38 and ERK1/2 mitogen‐activated protein kinases in VMM5 melanoma cells

Matrix metalloproteinase‐1 (MMP‐1) is one of only a few enzymes with the ability to degrade the stromal collagens (types I and III) at neutral pH, and high expression of MMP‐1 has been associated with aggressive and invasive cancers. We recently reported a single nucleotide insertion/deletion polymorphism (SNP) in the collagenase‐1 (MMP‐1) promoter (Rutter et al. [ 1998 ] Can. Res. 58:5321–5325), where the insertion of an extra guanine (G) at −1607 bp creates the sequence, 5′‐GGAA‐3 (2G allele), compared to the sequence 5′‐GAA‐3′ (1G allele). The presence of 2G constitutes a binding site for the ETS family of transcription factors, and increases MMP‐1 transcription in fibroblasts and A2058 melanoma cells cultured in vitro. In addition, the presence of the 2G allele has been linked to several aggressive malignancies as well as to enhanced expression of MMP‐1. In this study, we describe a melanoma cell line, VMM5, that is 1G homozygous, but that is invasive and expresses high levels of MMP‐1 constitutively. The high level of MMP‐1 expression in VMM5 cells is due to the utilization of both the p38 and ERK1/2 transduction pathways. In contrast, in the A2058 cell line, which also expresses MMP‐1 constitutively and which is 2G homozygous, only the ERK pathway is activated. Thus, our data suggest that in the absence of 2G allele and in the presence of the appropriate transcription factors, tumor cells may use alternative signal/transduction pathways and cis‐acting sequences to achieve high levels of MMP‐1 expression, which contribute to the ability of tumor cells to invade, regardless of their genotype.

Comparative Proteomics Profiling Reveals Role of Smooth Muscle Progenitors in Extracellular Matrix Production

Objective—Recent studies on cardiovascular progenitors have led to a new appreciation that paracrine factors may support the regeneration of damaged tissues. Methods and Results—We used a shotgun proteomics strategy to compare the secretome of peripheral blood-derived smooth muscle progenitors (SPCs) with human aortic smooth muscle cells. The late-outgrowth SPCs produced fewer proteolytic enzymes and inflammatory cytokines and showed reduced invasive capacity. Similar to smooth muscle cells, SPCs secreted extracellular matrix. However, SPCs produced different matrix proteins, as evidenced by the truncation of proangiogenic domains in collagen &agr;-1 (I) and increased production of periostin. Moreover, SPCs retained serum proteins, including proteoglycans, regulating collagen assembly; and pigment epithelium-derived factor, a potent inhibitor of angiogenesis. As a functional consequence, their conditioned medium was less angiogenic, as demonstrated by endothelial tube formation assays in vitro and implantation of Matrigel plugs into nude, severe combined immunodeficient mice (NOD/SCID). Conclusion—The present study represents an important conceptual development, suggesting that SPCs may contribute to extracellular matrix production.

Confocal assay for invasion: Use of propidium iodide fluorescence and laser reflectance to quantify the rate of migration of cells through a matrix

BACKGROUND Most assays used to measure invasion are based on manual counting of the number of cells that have migrated completely through commercial coated filters. We describe here a confocal fluorescence-imaging method that can assess the relative rates of invasion of cells into a matrix. METHODS After being seeded on the matrix and a period of incubation, the cells are fixed and treated with RNase. Propidium iodide is then added to stain the double-stranded DNA. A confocal microscope system is used to obtain high-resolution images of the red propidium iodide fluorescence and laser reflectance from optical sections at increasing depths in the matrix. The section with high laser reflectance marks the top of the matrix. RESULTS Data were calculated as the total area of red fluorescence above background in each section and were plotted as a percentage of the summed fluorescent areas in all sections. CONCLUSIONS Because the distance into the matrix of the nuclei can be calculated by measuring from the reflective upper surface of the matrix, the method is useful for assessing the rate of cell migration and for comparing the ability of different cells to invade through different matrices under varying conditions.

Selective Inhibition of Collagenase-1, Gelatinase A, and Gelatinase B by Chemotherapeutic Agents

Matrix metalloproteinases (MMPs) play a crucial role in tumor cell invasion and metastasis due to their ability to digest basement membrane and extracellular matrix components and thus facilitate cell movement. MMPs, therefore, are important pharmacological targets, and a number of synthetic inhibitors have been developed. 1 In keeping with this approach we screened a panel of chemotherapeutic agents for their ability to inhibit collagenase-1, gelatinase A, and gelatinase B expression in the highly aggressive human melanoma cell line A2058. We focused on the expression of these enzymes because of their ability to digest collagen types I, III, and IV, the major components of the extracellular matrix and basement membrane. 2 Cells were treated with a variety of cytotoxic drugs such as carboplatin, mitomycin C (MMC), cisplatin, doxorubicin, and N-acetylcysteine (NAC). Among these agents, only doxorubicin specifically repressed collagenase-1 expression at both the protein and mRNA levels, with no effect on gelatinase A and B. 3 Timeand dose-dependency studies revealed that repression of collagenase-1 synthesis could be obtained at non-

Chapter 15 – Transcriptional Regulation of Matrix Metalloproteinases

Publisher Summary Matrix metalloproteinases (MMPs) is a family of enzymes that, collectively, degrade all components of the extracellular matrix. There are at least 26 members of this family—all are active at neutral pH, require Ca2+ for activity, and contain a central zinc atom as part of their structure. Most MMPs are secreted into the extracellular space in a latent form, requiring proteolytic cleavage in order to be enzymatically active. However, one subgroup of MMPs is anchored in the membrane, and these enzymes are activated intracellularly by a furin-like mechanism. MMP-11 (stromelysin 3) is the only nonmembrane-bound MMP that is also activated intracellularly. Most cells in the body can express MMPs, although the expression of certain enzymes is often associated with a particular cell type. Inflammatory cytokines and growth factors and malignant transformation lead to a marked increase in MMP expression. Excess MMP production is, therefore, associated with many diseases, including periodontitis, atherosclerosis, tumor invasion and metastasis, and arthritic disease. Several MMPs may be involved in the pathophysiology of any of these diseases. Mechanisms regulating the expression of these enzymes under normal conditions and in the various diseases are complex, permitting both the coordinate expression of some MMPs and the tissue-specific expression of others. Posttranscriptional mechanisms, i.e., changes in mRNA stability, are used for controlling MMP gene expression. The regulation of gene expression at the level of transcription appears to play a major role in regulating these genes. This chapter focuses on the cis-acting sequences within the MMP promoters and the trans-acting factors that regulate transcription of the MMPs in different cell types and tissues.