Stimulation of 92-kDa Gelatinase B Promoter Activity by ras Is Mitogen-activated Protein Kinase Kinase 1-independent and Requires Multiple Transcription Factor Binding Sites Including Closely Spaced PEA3/ets and AP-1 Sequences

Abstract

The 92-kDa type IV collagenase (92-kDa gelatinase B also referred to as MMP-9), which plays a critical role in extracellular matrix degradation, is regulated by growth factors that mediate their effects through the ras proto-oncogene. The current study was undertaken to determine the transcriptional requirements for the induction of 92-kDa gelatinase B expression by an activated ras oncogene. Transfection of OVCAR-3 cells with an expression vector encoding an activated Ha-ras increased 92-kDa gelatinolytic activity and stimulated (over 10-fold) the activity of a CAT reporter driven by 670 nucleotides of 5 flanking sequence of the 92-kDa gelatinase B gene. Transient assays using a CAT reporter driven by 5 deleted fragments of the 92-kDa gelatinase B promoter indicated that a region spanning ؊634 to ؊531 was required for optimal induction of the promoter. The individual deletion, or mutation, of a PEA3/ets (؊540) motif, AP-1 sites (؊533, ؊79), a NF-B (؊600) consensus sequence, and a GT box (؊52) substantially reduced the activation of the promoter by ras. An expression vector encoding the PEA3 transcription factor caused a 3-fold stimulation of the wild type but not the PEA3/ets-deleted 92-kDa gelatinase B promoter. Coexpression of a dominant negative c-jun antagonized the ras-dependent stimulation of the 92-kDa gelatinase B promoter-driven CAT reporter. The signaling pathway mediating the induction of 92-kDa gelatinase B promoter activity by ras was examined. The expression of a phosphatase (CL100) which inactivates multiple mitogen-activated protein kinase members abrogated the stimulation of 92-kDa gelatinase B promoter activity by ras. However, the expression of a kinase-deficient mitogen-activated protein kinase kinase 1 (MEK1) did not prevent the activation of the 92-kDa gelatinase B promoter by ras and a constitutively activated c-raf expression vector was insufficient for 92-kDa gelatinase B promoter activation. Thus, the stimulation of the 92-kDa gelatinase B promoter by ras requires multiple elements including closely spaced PEA3/ets and AP-1 sites and is MEK1-independent.The 92-kDa type IV matrix metalloproteinase (92-kDa gelatinase B also known as MMP-9) plays a major role in cell migration in both physiological and pathological processes (1-3) by facilitating the destruction of the type IV collagencontaining basement membrane which separates the epithelial and stromal compartments (4). The 92-kDa type IV collagenase is secreted as a proenzyme (5) and subsequently activated by multiple enzymes, including cathepsin G, trypsin, stromelysin 1 (6), and 72-kDa gelatinase A (7) by the removal of 73 amino acids from the amino terminus of the protease. The active enzyme, which is capable of digesting native type I, III, IV, and V collagens at nondenaturing temperatures (4, 6), consists of five domains: the aminoterminal and zinc-binding domains shared by all members of the metalloproteinase family, a collagen-binding fibronectinlike domain, a carboxyl-terminal hemopexin-like domain, and a unique 54-amino acid ...

Transcriptional activation of the matrix metalloproteinase-9 gene in an H-ras and v-myc transformed rat embryo cell line

“…Mutation of either TRE-1 or TRE-2 also reduced promoter function. Similar to the data previously shown for both src and ras activation of the MMP-9 gene, disruption of the RBE abrogated promoter function (Gum et al, 1996;. Finally, replacement of the CA dinucleotide repeat sequence in the downstream promoter also markedly reduced reporter expression.…”

“…Coordinate activation of the NFkB, SP-1, and most proximal TRE-1 were required for promoter activation by tumor necrosis factor-a (TNF-a) or by phorbol ester (TPA), while the RBE and most proximal TRE-1 appeared to be su cient for transcriptional activation by the src oncogene . Gum et al showed that several transcription factor binding sites, including the adjoining upstream Ets and TRE-2 sites, were necessary for MMP-9 upregulation mediated by the ras oncogene (Gum et al, 1996).…”

Integrin αvβ6-associated ERK2 mediates MMP-9 secretion in colon cancer cells

“…Induction of MMP-9 promoter activity by oncogenic Ras in squamous carcinoma cells has been shown to be abrogated by blocking the ERK 1/2 pathway (Gum et al, 1997). Increased transcriptional activity of the MMP-9 promoter in Ras-transformed ovarian carcinoma cells has also been shown to be mediated by MAP kinases (Gum et al, 1996). In the present study, we show that down-regulation of constitutive b6 expression using an antisense approach against b6, that is known to suppress MAP kinase activity (Ahmed et al, 2002), dramatically reduced MMP-9 levels in tumour-conditioned medium.…”

Angiogenesis induced by hepatocyte growth factor in non-infarcted myocardium and infarcted myocardium: up-regulation of essential transcription factor for angiogenesis, ets

“…23,24 Thus, previous reports suggest that the ets family may activate the transcription of genes encoding collagenase 1, stromelysine 1 and urokinase plasminogen activator, which are proteases involved in extracellular matrix degradation. [25][26][27] It is believed that the ets family takes part in regulating angiogenesis by controlling the transcription of these genes whose activity is necessary for the migration of endothelial cells from pre-existing capillaries. In this study, we also demonstrated the up-regulation of endogenous HGF expression by exogenously transfected HGF gene, consistent with our previous findings in in vitro experiments.…”