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. 2009 Jan;40(1):4-12.
doi: 10.1165/rcmb.2007-0310OC. Epub 2008 Jul 10.

Identification of a cigarette smoke-responsive region in the distal MMP-1 promoter

Becky A Mercer  1 , Alison M Wallace, Constance E Brinckerhoff, Jeanine M D'Armiento
Affiliations

Identification of a cigarette smoke-responsive region in the distal MMP-1 promoter

Becky A Mercer et al. Am J Respir Cell Mol Biol. 2009 Jan.

Abstract

Tobacco-related diseases are leading causes of death worldwide, and many are associated with expression of matrix metalloproteinase-1 (MMP-1). We have reported extracellular signal-regulated kinase (ERK)1/2-dependent induction of MMP-1 by cigarette smoke in lung epithelial cells. Our objectives were to define regions of the human MMP-1 promoter required for activation by smoke, to identify differences in responses of the 1G/2G -1607 polymorphic promoters to smoke, and to identify relevant transcription factors whose activity in airway epithelial cells is increased by smoke. The responses of deletion and mutant promoter constructs were measured in transfected cells during exposure to cigarette smoke extract (CSE). DNA oligonucleotide arrays were used to identify transcription factors activated after smoke exposure. CSE activated the MMP-1 promoter, and this induction was prevented by PD98059 blockade of ERK1/2 phosphorylation. Deletion studies revealed the distal 1kb promoter region (-4438 to -3280 upstream of the transcription start site) is essential for CSE induction of MMP-1, and confers activation of a minimal promoter. Studies of 1G and 2G MMP-1 polymorphic promoter variants revealed higher 2G allele basal and CSE-responsive activities than the 1G allele. Cotransfection, mithramycin, and electrophoretic mobility shift assay studies identified activating and repressive roles for Sp1 and PEA3 transcription factors, respectively. Oligonucleotide DNA arrays confirmed activation of Sp1 and PEA3 by CSE. These data demonstrate that the MMP-1 promoter is a direct target of cigarette smoke in lung epithelial cells. This characterization of a smoke response region in the distal MMP-1 promoter has implications for smoking-related diseases such as cancer, heart disease, and emphysema.

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Figures

<b>Figure 1.</b>
Figure 1.
Matrix metalloproteinase (MMP)-1 promoter activity is enhanced by cigarette smoke. Primary human small airway epithelial cells (SAECs) were transfected with 1 μg of luciferase reporter plasmid containing either no promoter (pGL3) or the 4372-bp MMP-1 promoter (MMP-1). Forty-eight hours later, the cells were treated with SAGM (control media) or SAGM containing 5% cigarette smoke extract (CSE). Twenty-four hours later, the cells were lysed and the luciferase activity determined. Transfections and treatments were performed in triplicate. *P < 0.05 versus control pGL3. **P < 0.01 versus control MMP-1.
<b>Figure 2.</b>
Figure 2.
The G-1607GG SNP increases the response of the MMP-1 promoter to CSE. (A) Diagram of the MMP-1 promoter portion of the full-length 1G (deletion) and 2G (insertion) MMP-1 promoter constructs (vector backbone not shown). (B) Activation of the MMP-1 promoter is extracellular signal–regulated kinase (ERK)1/2 dependent. Lung epithelial cells were transfected with full-length MMP-1 promoter, and the response was measured as Relative Light Units (RLU) under control (open bars) or CSE (solid bars) conditions after 24 hours. Pretreatment with PD98059 (hatched bars) before CSE prevents the induction of both promoters, suggesting that cigarette smoke activates ERK1/2 transcription factor targets, which bind to regions other than the −1607 polymorphic site. *P < 0.05 and ***P < 0.001 compared with control; #P < 0.05 2G versus 1G control activity.
<b>Figure 3.</b>
Figure 3.
Proximal binding sites for the transcription factor activator protein (AP)-1 are required for baseline and CSE-mediated activities of the MMP-1 promoter. Deletion (Δ) of either the −73 or the 1,602 AP-1 site results in loss of promoter activity, demonstrating that these sites are required for basal expression and CSE-mediated induction of MMP-1. Both mutants fail to be activated by CSE. “2G” indicates full-length 2G MMP-1 promoter.
<b>Figure 4.</b>
Figure 4.
The distal 1kb of the MMP-1 promoter is required for induction by CSE. MMP1 promoter deletion constructs used to identify regulatory elements involved in the induction by smoke. Activity of the deletion constructs in SAECs before (open bars) and after (solid bars) 24 hours of exposure to 5% CSE. The full-length 2G MMP-1 promoter is activated by CSE treatment. All other proximal deletion constructs are not induced significantly by CSE. These data suggest that induction by smoke requires the distal 1 kb region of the promoter, and that the 2G (ETS) element alone is not sufficient for induction by CSE. ***P < 0.001 versus control activity. Data shown are RLU.
<b>Figure 5.</b>
Figure 5.
The distal 1 kb of the MMP-1 promoter is activated by cigarette smoke. (A) Diagram of the cloning strategy used to generate the −1700/distal 1 kb plasmid. The distal 1 kb region (dashed line) of the MMP-1 promoter, containing putative cigarette smoke response elements, was removed from the 2G full-length plasmid using SacI/PstI restriction digestion. (B) Transfection of each plasmid revealed that the distal 1 kb confers CSE-responsiveness to the truncated −1700 promoter. This induction is blocked by PD98059. *P < 0.05 compared with −1700. Data shown are fold induction by CSE normalized to control activity.
<b>Figure 6.</b>
Figure 6.
Transcription factor–binding sites in the distal MMP-1 promoter. The full-length (4,438-bp) human MMP-1 promoter sequence (AF023338) was analyzed for predicted transcription factor–binding sites using TESS-TRANSFAC software. The table shows only the distal 2 kb. Binding sites of interest are highlighted, in particular sites for those factors found to be up-regulated by CSE using the DNA arrays (AP-1, Ets, Sp1). In the “Factor” column, the number in front of the factor name is a unique factor identifier assigned by the software. In the “Site” column, the number indicates the position upstream of the TSS (transcription start site). Factors are sorted according to their position along the promoter in the 5′ to 3′ direction. Shading indicates transcription factors examined in the current study.
<b>Figure 7.</b>
Figure 7.
Identification of transcription factors induced by CSE. (A) Representative images of a DNA array pair, showing membranes from Control (left) and 5% CSE–treated cells (right). DNA consensus oligonucleotides are spotted by the manufacturer (Panomics) at a minimum of two locations on the array. Each spot represents a transcription factor. The higher number and intensity of spots on the CSE array indicates that CSE induces expression and/or the DNA-binding activity of numerous transcription factors. (B) Histogram of the CSE-mediated fold-induction of selected transcription factors that were induced by CSE, as determined by comparative densitometric analysis of DNA array pairs. The dashed line indicates the level at which no induction occurred (average ratio of spot intensities of CSE arrays/CON arrays = 1).
<b>Figure 8.</b>
Figure 8.
Mutation of distal PEA3 sites enhances 2G promoter activity. (A) Diagram of the PEA3 mutant MMP-1 promoter construct (mutPEA3), with the mutated distal PEA3 sites marked with an “X”. The proximal Ets site at −1607 was not changed. (B) Mutation of two of these sites in the distal 1 kb enhances basal and CSE-inducible activity, suggesting that these sites may repress transcription. (C) PEA3 overexpression inhibits MMP-1 promoter induction by CSE. Inhibition is dose dependent. SAECs were co-transfected with the 2G and pcDNA3-PEA3 overexpression plasmids. DNA amounts were kept equal among all groups by the addition of pGL3 DNA. All data are CSE data.*P < 0.05. The amounts of co-transfected DNA are higher than typical transfection studies, but are in the range of the amounts used by the providers of the PEA3 plasmid (Mien-Chie Hung, M.D. Anderson Cancer Center).
<b>Figure 9.</b>
Figure 9.
The transcription factor Sp1, an ERK1/2 nuclear target, is induced by CSE and is required for CSE-mediated MMP-1 promoter activation. (A) Electrophoretic mobility shift assay using nuclear lysates from control or CSE-treated lung epithelial cells. No specific band is detected in the Sp1 mutant oligonucleotide. Bottom panel, immunoblot for nucleophosmin. (B) Sp1 immunoblot of nuclear lysates from SAECs treated with 5% CSE (top panel). Nuclear levels of the protein nucleophosmin (bottom panel) is shown as a control. (C) CSE-mediated activation of the full-length 2G MMP-1 promoter under control conditions or after pretreatment with mithramycin, a chemical Sp1 DNA-binding inhibitor.
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