Manganese-superoxide dismutase (Sod2) removes mitochondrially derived superoxide (O 2 . ) at near-diffusion limiting rates and is the only antioxidant enzyme whose expression is regulated by numerous stimuli. Here it is shown that Sod2 also serves as a source of the intracellular signaling molecule H 2 O 2 . Sod2-dependent increases in the steady-state levels of H 2 O 2 led to ERK1/2 activation and subsequent downstream transcriptional increases in matrix metalloproteinase-1 (MMP-1) expression, which were reversed by expression of the H 2 O 2 -detoxifying enzyme, catalase. In addition, a single nucleotide polymorphism has recently been identified (1G/2G) at base pair ؊1607 that creates an Ets site adjacent to an AP-1 site at base pair ؊1602 and has been shown to dramatically enhance transcription of the MMP-1 promoter. Luciferase promoter constructs containing either the 1G or 2G variation were 25-or 1000-fold more active when transiently transfected into Sod2-overexpressing cell lines, respectively. The levels of MMP-2, -3, and -7 were also increased in the Sod2-overexpressing cell lines, suggesting that Sod2 may function as a "global" redox regulator of MMP expression. In addition, Sod2 ؊/؉ mouse embryonic fibroblasts failed to respond to the cytokine-mediated induction of the murine functional analog of MMP-1, MMP-13. This study provides evidence that the modulation of Sod2 activity by a wide array of pathogenic and inflammatory stimuli may be utilized by the cell as a primary signaling mechanism leading to matrix metalloproteinase expression.

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.

The matrix metalloproteinase (MMP) family degrades the extracellular matrix. One member of this family, MMP-1, initiates the breakdown of interstitial collagens. The expression of MMP-1 is controlled by the mitogen activated protein kinase (MAPK) pathway(s) via the activity of activator protein-1 (AP-1) and polyoma enhancing activity-3/ E26 virus (PEA3/ETS) transcription factors through consensus binding sites present in the promoter. Another ETS site in the MMP-1 promoter is created at )1607 bp by a single nucleotide polymorphism (SNP), which contains two guanines (5¢-GGAT-3¢; Ô2G SNPÕ), rather one guanine (5¢-GAT-3¢; Ô1G SNPÕ), adjacent to an AP-1 binding site at )1602 bp. The 2G SNP displays greater transcriptional activity than the 1G SNP, and AP-1 and Ets families of transcription factors cooperate to increase transcription. The 2G SNP has been linked to the incidence and the progression of several cancers and is also associated with non-neoplastic diseases; although the underlying mechanism(s) has yet to be elucidated. In this study we demonstrate that the expression of Fos-like region antigen (Fra-1), an AP-1 transcription factor component that also correlates strongly with neoplastic disease, is necessary for MMP-1 transcription in A2058 melanoma cells. The inhibition of Fra-1 expression preferentially downregulates transcription from the MMP-1 promoter DNA containing the 2G SNP, compared to DNA containing the 1G SNP. This study provides evidence that, in cooperation with the 2G DNA polymorphism, the AP-1 family member, Fra-1, contributes to the high constitutive expression of MMP-1 in melanoma cells.

Degradation of stromal collagens in the extracellular matrix is mediated largely by matrix metalloproteinase-1 (MMP-1; collagenase-1), and high constitutive levels of MMP-1 in breast cancer correlate with a poor prognosis and invasive disease. MMP-1 expression is, in part, controlled by the mitogen-activated protein kinase (MAPK) pathway(s), which may target several activator protein-1 (AP-1) and polyoma enhancing activity-3/E26 virus (PEA3/ETS) sites within the promoter. An additional ETS site in the MMP-1 promoter is conferred by a single nucleotide polymorphism (SNP) at -1607 bp, when two guanines (5'-GGAT-3'; '2G allele/SNP') are present instead of one guanine (5'-GAT-3'; '1G allele/SNP'). This SNP is adjacent to an AP-1 site at -1602 bp, and in the presence of the 2G allele (ETS site), these sites cooperate to induce higher levels of transcription. ERK 1/2 is one component of the MAPK pathway and is constitutively active in MCF-7/ADR breast cancer cells, which are 1G/2G heterozygotes. This study demonstrates that when these cells are treated with PD098059, an ERK-specific inhibitor, MMP-1 mRNA levels are significantly decreased, suggesting that high constitutive expression of MMP-1 in these cells results from continuous ERK 1/2 activation. Using transient transfection, we determined that this signaling pathway targets different AP-1/ETS sites, depending upon which allele is present. Furthermore, in these cells, the AP-1 site at -1602 bp enhances transcription in the presence of the 2G SNP, but represses transcription from the 1G SNP. Finally, inhibiting ERK signaling and MMP-1 expression blocks type I collagen degradation and reduces the invasive ability of the MCF-7/ADR cells. We conclude that ERK 1/2 signaling and the 2G SNP mediate high levels of MMP-1 expression, which may contribute to the invasive potential of these breast cancer 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.