No abstract

Matrix metalloproteinase-1 (MMP-1) is one of three collagenases that can degrade the interstitial collagens, types I, II, and III at neutral pH. As these collagens are the most abundant proteins in the body, collagenase plays a critical role in modeling and remodeling the extracellular matrix. Therefore, it is not surprising that MMP-1 gene expression can be regulated at multiple points. Procollagenase can be activated by mechanisms that generate an active enzyme with differing specific activities, and the active enzyme can be inhibited by complexing with either the tissue inhibitor of metalloproteinases (TIMPs) or alpha 2 macroglobulin. The activator protein-1 (AP-1) site in the collagenase promoter plays a prominent role in the transcriptional control of the collagenase gene. It is essential for basal transcription, and contributes to induction by phorbol esters, although other sites in the proximal promoter are essential. In contrast, transactivation by cytokines such as Interleukin-1 depends on sequences in more distal regions of the promoter. Posttranscriptional mechanisms also regulate gene expression, and several cytokines and growth factors increase the stability of the collagenase transcript. Finally, glucocorticoid hormones repress transcription of the collagenase gene by the interaction of glucocorticoid receptors with the AP-1 proteins, Fos and Jun. Retinoids also suppress transcription by mechanisms that involve down-regulation of fos and jun mRNA, sequestration of Fos and Jun proteins, and the formation of complexes of retinoic acid receptors (RAR/RXR heterodimers) and AP-1 proteins on the DNA. These multiple points of regulation assure precise control of collagenolytic activity in a variety of physiologic and pathologic conditions.

Melanoma progresses as a multistep process where the thickness of the lesion and depth of tumor invasion are the best prognostic indicators of clinical outcome. Degradation of the interstitial collagens in the extracellular matrix is an integral component of tumor invasion and metastasis, and much of this degradation is mediated by collagenase-1 (MMP-1), a member of the matrix metalloproteinase (MMP) family. MMP-1 levels increase during melanoma progression where they are associated with shorter disease-free survival. The Ras/Raf/ MEK/ERK mitogen-activated protein kinase (MAPK) pathway is a major regulator of melanoma cell proliferation. Recently, BRAF has been identified as a common site of activating mutations, and, although many reports focus on its growth-promoting effects, this pathway has also been implicated in progression toward metastatic disease. In this study, we describe four melanoma cell lines that produce high levels of MMP-1 constitutively. In each cell line the Ras/Raf/MEK/ERK pathway is constitutively active and is the dominant pathway driving the production of MMP-1. Activation of this pathway arises due to either an activating mutation in BRAF (three cell lines) or autocrine fibroblast growth factor signaling (one cell line). Furthermore, blocking MEK/ ERK activity inhibits melanoma cell proliferation and abrogates collagen degradation, thus decreasing their metastatic potential. Importantly, this inhibition of invasive behavior can occur in the absence of any detectable changes in cell proliferation and survival. Thus, constitutive activation of this MAPK pathway not only promotes the increased proliferation of melanoma cells but is also important for the acquisition of an invasive phenotype.

No abstract

Interleukin-1 beta (IL-1 beta) 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-1 beta 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-1 beta 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-1 beta-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-1 beta 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-1 beta 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-1 beta. Finally, we found that IL-1 beta 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-1 beta 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.

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 cocultured 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.

Matrix metalloproteinase-1 (MMP-1) breaks down interstitial collagens, a major component of stromal tissue and a barrier for invading tumor cells. The degradation of collagen by MMP-1 may, therefore, provide one mechanism for facilitating tumor invasion and metastasis. Because of the potential for excessive matrix degradation, the expression of MMP-1 is tightly regulated, often by the mitogen-activated protein kinase (MAPK) pathway. The MAPK signal cascade consists of three separate pathways, the extracellular response kinase (ERK), p38 and Jun N-terminal kinase, which target proteins of the AP-1 and ETS families transcription of the gene. The MMP-1 promoter contains a single nucleotide polymorphism (SNP) at -1607 bp, which creates an ETS binding site by the addition of a guanine (5'-GGAT-3' or '2G SNP') compared to the 1G SNP (5'-GAT-3'), and enhances MMP-1 transcription. A2058 melanoma cells represent one tumor cell line that is homozygous for the 2G allele and that produces constitutively high levels of MMP-1. Thus, we used these cells to define the mechanism(s) responsible for this high level of expression. We show that inhibition of ERK 1/2 leads to the repression of MMP-1 transcription, and that both the 2G polymorphism and the adjacent AP-1 site at -1602 bp are necessary for high levels of MMP-1 transcription and for the inhibition of MMP-1 expression by PD098059, a specific ERK inhibitor. Furthermore, restoration of MMP-1 levels after ERK 1/2 inhibition requires de novo protein synthesis of a factor necessary for MMP-1 expression. Thus, this study suggests that the ERK 1/2 pathway targets the 2G polymorphism, and that the continuous synthesis of a protein(s) is necessary for the constitutive expression of MMP-1.

Summary Metalloproteinases (MMPs) participate in extracellular matrix remodelling and regulatory signalling during chronic inflammatory states such as atherosclerosis formation. However, the sources and mediators of MMP upregulation need clarification. We investigated whether proinflammatory mouse T helper type 1 (Th1) lymphocytes are more active in MMP secretion than naïve Th0 or anti‐inflammatory Th2 phenotypes, in the absence of specific antigenic stimulation, under baseline conditions and after contact with irradiated macrophages. We also compared the effect of Th0, Th1 or Th2 lymphocyte‐conditioned medium and irradiated lymphocytes on MMP production from macrophages. Finally, we investigated whether CD40–CD40 ligand (CD40L) interactions were involved in T‐cell‐stimulated MMP secretion from macrophages. Under baseline conditions, MMP‐2 messenger RNA (mRNA) and protein levels were greater in Th1 than Th0 or Th2 lymphocytes; MMP‐9 mRNA, but not protein, was also upregulated. In the presence of irradiated macrophages MMP‐2 and MMP‐9 production from Th1 and Th2 was greater than from Th0 lymphocytes. Conditioned media from Th1 but not Th0 or Th2 cells increased MMP‐9 secretion from macrophages. Irradiated Th1 lymphocytes stimulated both MMP‐2 and MMP‐9 secretion from macrophages more than irradiated Th2 or Th0 cells; this activation was independent of CD40–CD40L interaction. These findings demonstrate for the first time greater MMP secretion by Th1 than Th2 or Th0 lymphocytes and their greater ability to upregulate macrophage MMP secretion in the absence of specific antigenic stimulation. These mechanisms could promote matrix turnover in inflammatory states and, for example, promote atherosclerotic plaque rupture.