Open Collections

UBC Faculty Research and Publications

Functional characterization of the matrix metalloproteinase-1 cigarette smoke-responsive region and association… Wallace, Alison M; Mercer, Becky A; He, Jianqing; Foronjy, Robert F; Accili, Domenico; Sandford, Andrew J; Paré, Peter D; D’Armiento, Jeanine M Sep 19, 2012

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata


52383-12931_2012_Article_1277.pdf [ 237.21kB ]
JSON: 52383-1.0223978.json
JSON-LD: 52383-1.0223978-ld.json
RDF/XML (Pretty): 52383-1.0223978-rdf.xml
RDF/JSON: 52383-1.0223978-rdf.json
Turtle: 52383-1.0223978-turtle.txt
N-Triples: 52383-1.0223978-rdf-ntriples.txt
Original Record: 52383-1.0223978-source.json
Full Text

Full Text

RESEARCH Open AccessFunctional characterization of the matrixmetalloproteinase-1 cigarette smoke-responsiveregion and association with the lung health studyAlison M Wallace1,2, Becky A Mercer2, Jianqing He1, Robert F Foronjy3, Domenico Accili4, Andrew J Sandford1,5,Peter D Paré1,5 and Jeanine M D’Armiento2*AbstractBackground: Prior studies have demonstrated that the distal 1.5 kb of the MMP-1 promoter is fundamental indirecting the induction of the MMP-1 gene by cigarette smoke.Methods: To characterize the genetic variants in the MMP-1 cigarette smoke-responsive element, deepre-sequencing of this element was performed on DNA samples from participants in the Lung Health Study.Furthermore, evidence of Sp1 binding to the MMP-1 promoter was assessed using chromatin immunoprecipitationassays and the influence of cigarette smoke exposure on this interaction was evaluated in cultured human smallairway epithelial cells.Results: Ten polymorphisms (four novel) were detected in the cigarette smoke-responsive element. Chromatinimmunoprecipitation assays to assess the protein-DNA interactions at Sp1 sites in the MMP-1 promoter showedincreased binding to the Sp1 sites in the cigarette smoke-responsive element in small airway epithelial cells treatedwith cigarette smoke extract. In contrast, a Sp1 site outside of the element exhibited the opposite effect. None ofthe polymorphisms were more prevalent in the fast decliners versus the slow decliners (fast decliners = mean−4.14% decline in FEV1% predicted per year vs. decline in FEV1% predicted per year).Conclusions: Sequencing analyses identified four novel polymorphisms within the cigarette smoke-responsiveelement of the MMP-1 promoter. This study identifies functional activity within the cigarette smoke-responsiveelement that is influenced by cigarette smoke and examines this region of the promoter within a small patientpopulation.Keywords: Chromatin immunoprecipitation, COPD, Metalloproteinase, Polymorphisms, Transcription factorsBackgroundChronic obstructive pulmonary disease (COPD), whichis characterized by both emphysema and inflammatoryscarring and narrowing of small airways, is a major causeof morbidity and mortality worldwide [1]. Cigarettesmoke is the single most important factor in the devel-opment of COPD. Not all smokers develop COPD, how-ever, smoking is responsible for up to 90% of cases inthe developed world [2]. Current diagnostic and thera-peutic options for this disease are limited.Much attention has been given to the role of matrixmetalloproteinases (MMPs), a family of zinc-dependentproteinases with the capacity to degrade both elastin andcollagen, in the pathogenesis of COPD. Although the ori-ginal protease-antiprotease imbalance theory of COPD fo-cused on destruction of elastin in the lung, there isevidence that collagen degradation is important as well. In1992 D’Armiento and coworkers [3] found that over-expression of human MMP-1 (interstitial collagenase) intransgenic mice led to the development of emphysema.Subsequent studies demonstrated that the important tar-get for MMP-1 was type III collagen and that adult-onsetemphysema developed in strains of mice expressingMMP-1 in the lung [3,4]. In humans, increased levels of* Correspondence: jmd12@columbia.edu2Department of Medicine, Division of Molecular and Pulmonary Medicine,Columbia University College of Physicians and Surgeons, New York, NY, USAFull list of author information is available at the end of the article© 2012 Wallace et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (, which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.Wallace et al. Respiratory Research 2012, 13:79, localized within the resident alveolar epithelialcells, have been reported in the lungs of patients with em-physema but not in normal controls [5,6]. In addition,Type II pneumocytes are also known to express MMP-1[5]. In vitro, direct exposure of human small airway epi-thelial cells to cigarette smoke induced MMP-1 mRNAand protein expression via the extracellular-signal-regulated kinases (ERK)/mitogen activated protein kinase(MAPK) pathway and human emphysematous lung tissueshowed significantly increased ERK activity compared tocontrol lung [7]. Through deletion studies our laboratoryidentified that the distal 1.5 kb of the MMP-1 promoterwas fundamental to the direct induction of the MMP-1gene by cigarette smoke and that Sp1 is activated bycigarette smoke [8].Only a small subset of smokers develop COPD andonce established, the disease process varies greatly fromperson to person. There is an underlying genetic suscep-tibility determining both the onset and the course of thedisease.The heterogeneity of this disease suggests thatthere may be some genes and/or gene variants that con-tribute to COPD in general, while other genes and/orgene variants may be relevant only for a particularphenotype. It is unknown whether DNA sequence vari-ation in the MMP-1 gene contributes to the individualsusceptibility and/or phenotypic variation. We hypothe-sized that DNA sequence variation in this responsiveelement could be associated with a COPD phenotype,accelerated lung function decline, and if an associationwas found may suggest a functional role of these poly-morphisms. Furthermore, based on our previous work,we speculated that gene variants could modulate Sp1binding and therefore influence smokers’ susceptibilityto COPD through variations in MMP-1 expression.In the present study the genetic variants in the cigarettesmoke-responsive region were characterized, with particu-lar interest in identifying single nucleotide polymorphisms(SNPs) altering Sp1 binding sequences. Chromatin immu-noprecipitation (ChIP) assays were performed to assessthe protein-DNA interactions at Sp1 sites in the MMP-1promoter to define the functional binding sites and deter-mine whether or not these sites were influenced bycigarette smoke exposure in cultured human small airwayepithelial cells. An association study was also performedto test whether SNPs within this region were associatedwith an accelerated rate of decline in lung function in theLung Health Study (LHS) participants.MethodsStudy populationDNA for re-sequencing and genetic association studieswas obtained from subjects selected from participants inPhase I of the National Heart, Lung, and Blood InstituteLHS. Details of the study have been previously published[9]. Briefly, study participants were current smokers,35–60 years of age, who had mild to moderate airflowobstruction (FEV1 55–90% predicted and FEV1/FVC≤ 0.70). The primary outcome variable was rate of de-cline in post-bronchodilator FEV1 over a follow-upperiod of five-years. A subset of 582 individuals wereselected from the 5,887 subjects in the LHS because theyrepresent phenotypic extremes. They include the 277subjects (fast decliners) who continued to smoke duringthe first five-years of follow-up, had yearly measure-ments of FEV1, and showed the fastest decline in lungfunction over that time (FEV1% predicted change =−4.14%/year), and the 305 subjects (slow decliners) whocontinued to smoke and exhibited the slowest declineover the same period of time (FEV1% predicted change= +1.07%/year). Of the subset, 551 non-Hispanic whiteparticipants (261 fast decliners and 290 slow decliners)were selected for the association study. This study wasapproved by the University of British Columbia/Provi-dence Health Care Research Ethics Board.Identification of DNA sequence variantsHigh-throughput sequencing of the MMP-1 cigarettesmoke-responsive element (~1.5 kb) was performed atthe Genome Sciences Centre at the British ColumbiaCancer Agency using Applied Biosystems chemistries(ABI BigDye™ v3.1 Terminator Chemistry) and equip-ment (ABI 3730xl). Sequencing PCR primers weredesigned using Overlapping Primersets ( Theprimer sequences were: Region 1 (−3660 to −4343) sense5’-CTAAAAGCTCTGCAGGCCAC-3’ and antisense 5’-CGCTTAGGCTGGAGTGTAGG-3’ region 2 (−3510 to−4147) sense 5’-GGCATGCAAATCACCAAAA-3’ andantisense 5’-AATCCTCCCCTTCAAGCTGT-3’ region 3(−2890 to −3748) sense 5’-TCTTAGGAGAGTAAAAGTCATGGACA-3’ and antisense 5’-TTCTTGGTTGCTTCATGCTG-3’. For the purpose of sequencing, a M13 forwardlinker was added to the 5’ end of the forward primers(TGTAAAACGACGGCCAGT) and a M13 reverse linkerwas added to the 5’ end of the reverse primers(CAGGAAACAGCTATGAC). Sequencing was performedusing M13 universal sequencing primers. PCR conditionswere as follows: initial denaturation at 95°C for 15 minutes;35 cycles at 94°C for 30 seconds, 58°C for 30 seconds, and72°C for 50 seconds; and final extension at 72°C for10 minutes. Discovery of sequence variation was per-formed using novoSNP [10], which has higher sensitivityand specificity than PolyPhred [11]. Each SNP had a com-pletion rate of greater than 90% in all subjects and aHardy-Weinberg equilibrium P value of greater than 0.01in non-Hispanic whites.Wallace et al. Respiratory Research 2012, 13:79 Page 2 of 8 immunoprecipitationChIP was performed using the Upstate (Millipore) ChIPKit (Billerica, MA, USA) according to the manufacturer’sinstructions. Chromatin was harvested from humansmall airway epithelial cells (Lonza, Walkersville, MD)that were seeded at 7.5×105 cells per T75 flask (BDFalcon, San Jose, CA) and treated with control media or5% cigarette smoke extract at 80% confluence for24 hours, prior to crosslinking, as previously described[7]. Cells between passages two and six were used in allin vitro experiments. Conditions were established to ob-tain chromatin fragments 200–600 bp in length usingthe Fisher Scientific F 550 Sonic Dismembrator. Immu-noselection of chromatin fragments was performedusing 2 ug of rabbit polyclonal Sp1 antibody (sc-59 X;Santa Cruz Biotechnology, Santa Cruz, CA). An aliquotthat was immunoprecipitated without antibody was usedas a negative control. Detection of specific DNAsequences was performed using real-time quantitativePCR with the use of an ABI Prism 7900HT SequenceDetection System (Applera Corporation, Norwalk, CT,USA) using the following primers: -3987 site (83 bpamplicon) sense 5’-TCTCCAGTAAGGCTGGGTGT-3’and antisense 5’-CTGGCCTCAAGCAGTTCTCT-3’;-3455 site (117 bp amplicon) sense 5’-TGCAGACACC-TACTATGTTGAG-3’ and antisense 5’-ATAATGTCAC-CATGCCACCAC-3’; and-2209 site (68 bp amplicon)sense 5’-TAGAGAAGG GAGGAAAAAGCAG-3’ andantisense 5’-GTTGGAAA TAGAGCCTTGGAGT-3’.Statistical analysisThe associations were analyzed by binary logistic regres-sion to adjust for potential confounding factors. Theoutcome was a dichotomous variable, that is, fast declineor slow decline. Potential confounding factors includedin the analysis were age, sex, smoking history (expressedas pack-years), initial level of lung function (pre-bronchodilator FEV1 percent predicted), and methacho-line responsiveness. The latter variable was expressed asa two-point dose–response slope as previously described[12]. Hardy–Weinberg equilibrium was calculated usingan online calculator ( [13]. Minimal detectable odds ratio atP = 0.05 and 0.80 power for different minor allele fre-quency (MAF) in the case–control association study wasperformed using PGA Power Calculator software [14].All other tests were performed using the JMP Statisticssoftware package (SAS Institute Inc., Cary, NC). Statis-tical significance was defined at the 5% level.ResultsDescriptive statistics and univariate comparisons betweenfast decliners and slow decliners are summarized in Table 1.Age, sex, smoking history (pack-years), baseline FEV1%predicted postbronchodilator, and methacholine responsewere borderline or significantly different between the twogroups. Therefore, the association of genotypes with declineof lung function was analyzed by logistic regression to ad-just for these factors.Re-sequencing of the MMP-1 cigarette smoke-responsiveregion revealed a total of ten polymorphisms (Table 2).Four of these genetic variants have not been previouslyreported in the National Center for Biotechnology Informa-tion SNP database (dbSNP). The presence and frequency ofsix dbSNPs were confirmed; three previously reporteddbSNPs were not identified.Several transcription factors are induced by cigarettesmoke and have been shown to be important for regulat-ing MMP-1 expression [8]. Since not all smokers de-velop COPD, despite smoking being the most importantrisk factor for the development of this disease, gene var-iants in the cigarette smoke-responsive element couldprovide a susceptibility locus for smoke-induced lungdisease. None of the polymorphisms in this region cor-respond specifically to the Sp1 sites at positions −3987and −3455 that through site-directed mutagenesis weknow are important for regulating MMP-1 expression(data not shown). Furthermore, other transcription fac-tor binding sites of interest, such as AP-1 sites at posi-tions −4293 and −4210 and c-Ets-1 sites at positions−3838 and −3344, did not overlap specifically with anypolymorphisms identified in this cohort of smokers, pos-sibly suggesting that the transcription factor bindingTable 1 Characteristics of study subjectsSlow decliners (n = 290) Fast decliners (n = 261) P valueAge 47.4 ± 0.4 49.6 ± 0.4 0.0007Male (%) 193 (66.6) 155 (59.4) 0.082Pack-years 38.3 ± 1.1 43.2 ± 1.2 0.002Baseline FEV1, % predicted* 79.8 ± 0.5 74.6 ± 0.6 <0.0001Methacholine response† −8.0 ± 0.9 −24.2 ± 2.1 <0.0001Data are presented as percent or mean (SE).*Lung function at the start of the study as measured percent predicted FEV1 (postbronchodilator).†Measurement of the responsiveness of the airways to methacholine expressed as percent decline in FEV1 per final cumulative dose of methacholineadministered (O'Connor et al. 1995).Wallace et al. Respiratory Research 2012, 13:79 Page 3 of 8 are highly conserved. Despite not directly alteringthe above mentioned binding sites, these polymorphismscould affect the binding of other regulatory proteins thatcontrol the activity of the promoter and influencemRNA and protein levels.Our previous studies have shown that in lung epithe-lial cells Sp1 modifies the expression of MMP-1 duringsmoke exposure [8]. Therefore, we next investigated themechanism by which Sp1 regulates MMP-1 expression.To test the hypothesis that Sp1 is a key regulator ofMMP-1 expression in intact cells, we performed ChIPassays on human small airway epithelial cells, in vitro.The DNA corresponding to the Sp1 sites in the cigarettesmoke-responsive element was amplified and chromatinimmunoprecipitated with an antibody to Sp1. The datademonstrated that Sp1 is recruited onto the MMP-1promoter in vitro. The results show an interactionbetween Sp1 and the MMP-1 promoter at baselineand that there is a significant increase in binding in cul-tured human small airway epithelial cells treated withcigarette smoke extract (Figure 1a-b). In contrast, theSp1 site at position −2209, which is outside of thecigarette smoke-responsive element, exhibited the op-posite effect (Figure 1c).Association analyses of the individual SNPs with rateof decline in lung function in the fast decliners and slowdecliners showed no significant associations betweengenetic variants in the MMP-1 cigarette smoke-responsive element and this COPD phenotype in non-Hispanic whites (Table 3). Minimal detectable oddsratios for different MAFs and different genetic modelsare shown in Figure 2. The study design has 80% powerto detect associations with odds ratios of 2.0 for the fiveSNPs with a MAF ≥ 0.05 in co-dominant and dominantgenetic models.DiscussionMMP-1 is important in the pathogenesis of COPD withincreased expression being a common finding in patientswith this disease [5,6,15]. Animal studies have shownthat MMP-1 plays a key role in the initiation of the dis-ease process [3]. Previous studies from our laboratoryhave identified the distal MMP-1 promoter to be funda-mental for the direct activation of the MMP-1 promoterby cigarette smoke [8]. Studies of genetic variants in theMMP-1 promoter are important because identificationof functional polymorphisms will lead to a greaterunderstanding of the regulatory mechanisms involved inboth health and disease, and may provide useful know-ledge for identifying at-risk individuals to allow for earlyinterventions.This present study generated the following threeimportant findings. First, the genetic variants in theMMP-1 cigarette smoke-responsive element were fur-ther characterized and four novel polymorphisms identi-fied, as well as the confirmation of six previouslyreported genetic variants. Second, an important inter-action between Sp1 binding and the MMP-1 cigarettesmoke-responsive element in human small airway epi-thelial cells exposed to cigarette smoke extract wasdocumented. Sp1 protein binding was influenced bycigarette smoke and this DNA-protein interaction oc-curred specifically in the cigarette-smoke responsiveelement and did not appear to occur outside of the coreregulatory region. Third, polymorphisms in the cigarettesmoke-responsive element were not more prevalent insubjects who had an accelerated decline in lung functionover five-years of the Lung Health Study.MMP-1 is tightly regulated at the level of transcrip-tion, post-transcription, and post-translation; it is alsoknown that MMP-1 expression is influenced by geneticTable 2 Identification of polymorphisms in MMP-1 cigarette smoke-responsive elementSNP* SNP ID WW† WM† MM† NC{ Not amplified for this SNP Total N Failed % W% M%17 T/A rs484915 167 265 126 4 20 582 4.1 53.7 46.3352 G/A rs470307 554 9 0 0 19 582 3.3 99.2 0.8412 T/A NA 571 5 0 0 6 582 1.0 99.6 0.4712 G/A rs2408490} 368 149 16 4 45 582 8.4 83.0 17.0724 T/A rs12279710 534 3 0 1 44 582 7.7 99.7 0.3759 T/G rs7107224 391 162 19 5 5 582 1.7 82.5 17.5816A/C rs1155764} 383 157 21 19 2 582 3.6 82.3 17.71164C/T rs34695796 537 19 1 9 16 582 4.3 98.1 1.91227 G/A NA 558 6 0 2 16 582 3.1 99.5 0.51283 G/A NA 554 5 0 7 16 582 4.0 99.6 0.41284 G/A NA 499 49 3 15 16 582 5.3 95.0 5.0* Position in AF023338.† “W” denotes wild-type allele; “M” denotes mutant allele.{Results are not consistent between forward primer sequencing and reverse primer sequencing.}Rs2408490 and rs1155764 are in complete LD with r2 = 1 in Hapmap database.Wallace et al. Respiratory Research 2012, 13:79 Page 4 of 8 in the promoter [16]. In the cigarette smoke-responsive element spanning approximately 1.5 kb, atotal of ten polymorphisms were identified via re-se-quencing. Four novel polymorphisms were revealed.These data are important because they allow researchersto further analyze the contribution of genetic poly-morphisms to the development and progression of mul-tiple diseases associated with MMP-1 expression. Inaddition to the established role of MMP-1 in COPD, ele-vated expression of MMP-1 has been associated with avariety of pathological conditions such as rheumatoidand osteoarthritis, atherosclerosis, Alzheimer disease,and cancer [17]. There is a functional polymorphism inthe MMP-1 promoter (rs1799750) described by Rutteret al. that has been associated with numerous malignantprocesses [18]. Further understanding of the genetic var-iants in the MMP-1 promoter, and more specifically thecigarette smoke-responsive element, may provide add-itional insights into the molecular mechanisms of thesediseases and help define the role of smoking as a riskfactor.Understanding the molecular mechanisms controllingMMP-1 expression may identify potential targets for theprevention of diseases and degenerative conditions asso-ciated with dysregulation of MMP-1. The first cis-promoter element found to be responsible for regulatingMMP-1 gene expression was AP-1 [19]. Previous studiesby our laboratory have identified a number of transcrip-tion factors, including AP-1, that are important for regu-lating MMP-1 promoter activity [8]. Further analysis inthe present study revealed two Sp1 sites (−3987 and−3455) in the cigarette smoke-responsive element thathave now been analyzed using a ChIP assay to assess thefunctional importance of these sites in vitro. Our datademonstrates that these are functional binding sites andthat binding of Sp1 to the MMP-1 promoter is influ-enced by cigarette smoke. This is in contrast to thedecreased binding of Sp1 to the MMP-1 promoterFigure 1 Sp1 action on the MMP-1 promoter is shown by the fold change in specific DNA sequences detected by qPCR. MMP-1 ChIP assayswere done in cell cultures from human small airway epithelial cells with and without cigarette smoke extract (CSE) treatment using an antibodyto Sp1 or nonimmune serum. We amplified DNA using primers spanning two Sp1 binding sites within the cigarette smoke-responsive element(a,b) and one sequence outside of this element (c). Samples from each experiment were run in triplicate. Data are mean ± SEM. *P < 0.05.Table 3 Association with rate of decline of lung function non-Hispanic whitesSNPpositioninAF023338Rs indbSNPChromosome11 positionSlow decliners Fast decliners P valueWW WM MM WW WM MM17 T/A rs484915 102178458 73 (26%) 136 (49%) 70 (25%) 78 (31%) 117 (47%) 53 (21%) 0.35352 G/A rs470307 102178123 274 (98%) 7 (2%) 0 249 (99%) 2 (1%) 0 0.18412 T/A NA 102178063 283 (99%) 3 (1%) 0 257 (99%) 2 (1%) 0 1.00712 G/A rs2408490 102177763 191 (72%) 64 (24%) 9 (3%) 162 (67%) 73 (30%) 6 (2%) 0.28759 T/G rs7107224 102177716 200 (71%) 72 (25%) 11 (4%) 175 (68%) 77 (30%) 7 (3%) 0.44816 A/C rs1155764 102177659 196 (71%) 69 (25%) 11 (4%) 171 (67%) 76 (30%) 9 (3%) 0.471164 C/T rs34695796 102177311 263 (96%) 12 (4%) 0 244 (97%) 7 (3%) 1 (0%) 0.361227 G/A NA 102177248 277 (99%) 3 (1%) 0 251 (99%) 3 (1%) 0 1.001283 G/A NA 102177192 274 (99%) 3 (1%) 0 251 (99%) 2 (1%) 0 1.001284 G/A NA 102177191 247 (91%) 23 (8%) 2 (1%) 225 (90%) 25 (10%) 1 (0%) 0.74Wallace et al. Respiratory Research 2012, 13:79 Page 5 of 8 of the cigarette smoke responsive-element in re-sponse to cigarette smoke exposure. Not only are thesesites functional, they also occur in a region harbouring anumber of common SNPs. Although the genetic variantsidentified did not directly alter the primary transcriptionfactor binding sites we assessed, they may influence thebinding of chromatin modifying proteins resulting in ameasurable functional effect on promoter activity. Un-fortunately, this study is limited by the fact that suscepti-bility to COPD (i.e. accelerated decline in lung functionwith smoking) and Sp1 binding to the MMP-1 genewere not measured in the same individuals since the ap-propriate cells were not available from the LHS partici-pants. However, the findings that the SNPs identified donot directly alter the Sp1 bindings sites at the DNA leveland are not associated with rapid decline in lung func-tion are interesting and potentially important. It may bethat since Sp1 plays a significant role regulating MMP-1expression these sites are conserved. These negativeresults do suggest that the SNPs are not a risk for rapiddecline in lung function among smokers.COPD is a chronic disease of complex phenotype[20,21]. There is even speculation that COPD representsa syndrome comprised of many rare diseases [22]. In thepresent association study the relationship between poly-morphisms in the cigarette smoke-responsive elementand one COPD phenotype, rate of decline in lung func-tion, was tested. No significant associations were founddespite the sufficient power of the study. Such a findinghowever is not surprising since the results of the basicstudies suggest that MMP-1 expression is critical in thepathogenesis of lung destruction and generation of theemphysema phenotype [3]. It will be necessary in futuredetailed human studies to test for an association of thecigarette smoke-responsive element polymorphisms withCT evidence of lung destruction. Unfortunately, CT datawas not available on the LHS participants and thereforewe were unable to investigate this further in our popula-tion. Additionally, our lab has demonstrated thatcigarette smoke directly induces the expression ofMMP-1 highlighting the importance of MMP-1 in thesusceptibility to lung destruction in smokers [3]. AllLHS participants exhibit some degree of airflow limita-tion, indicating that they are already responsive to thedamaging effects of cigarette smoke. Therefore, to testthe importance of this element in disease susceptibility itwill be necessary to assess these genetic variants in alter-native well-characterized cohorts of smokers with andwithout COPD, as well as normal non-smokers.We acknowledge several limitations to the present studyin addition to the issues outlined above. Although an asso-ciation between the MMP-1 polymorphisms and rate ofdecline in lung function was not identified, these poly-morphisms may influence other COPD-related pheno-types. We know that lung function normally declines aspart of the aging process, while accelerated lung functiondecline is a hallmark of COPD [23,24], therefore, the useof this phenotype seems reasonable for genetic associationstudies and decline in lung function over time has been acommon end point in clinical trials of COPD therapies[25,26]. However despite the rigor of the lung functionphenotyping in the LHS, the five-year period of follow-upmay not be sufficient to clearly separate groups whose de-cline in lung function varied significantly over time. In-deed, the use of this single COPD phenotype and a shortfollow-up period are limitations of this study. On the otherhand our group has previously identified susceptibility var-iants in candidate genes using this approach [27,28]. Theassociation study was limited to non-Hispanic white sub-jects but the genetic basis of COPD may differ between ra-cial/ethnic groups as several COPD association studieshave shown distinct results within different races. Indeed,it is known that MMP-1 polymorphisms are associatedwith diseases such as cancer in other ethnic groups[29,30]. Therefore, it will be of interest to further investi-gate the role of these polymorphisms in alternative racial/ethnic populations. Future studies may also include haplo-type analysis to fully characterize the genetic architectureof this region.ConclusionsIn conclusion, the genetic variants in the MMP-1cigarette smoke-responsive element have been furthercharacterized revealing four novel polymorphisms. Evi-dence has been generated providing insight into thetranscriptional regulation of the MMP-1 promoter inFigure 2 The detectable relative risk given the number ofsubjects is shown for a variety of marker allele frequenciesusing different genetic models.Wallace et al. Respiratory Research 2012, 13:79 Page 6 of 8 to cigarette smoke exposure, which couldhave a significant impact on the understanding ofmany age-associated degenerative disease processes.COPD association studies are challenging since COPDis a heterogeneous condition and many phenotypesexist. Although a significant association between theseMMP-1 polymorphisms and an accelerated rate of de-cline in lung function in subjects with some degree ofairflow limitation was not identified, future studiesperformed on a cohort of smokers with and withoutdisease needs to be carried out to determine if theSNPs in the MMP-1 promoter associate with diseasesusceptibility as would be predicted from the basicbench research. Furthermore, future studies areplanned to further elucidate the mechanisms regulat-ing MMP-1 expression and the functional conse-quences of this altered expression, which includesmapping out which transcription factor binding sites,if any, are altered by SNPs in this region and determin-ing the functionality. As we found with Sp1, AP-1, andc-Ets-1 binding sites, we speculate that other tran-scription factor binding sites in this region may behighly conserved and therefore unaltered. Despite this,although not directly altering the binding of transcrip-tion factors, gene variants in this region could influ-ence the multi-protein complexes that are formedduring transcription and modulate MMP-1 expression.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsAW participated in the study design, prepared the samples for sequencing,carried out the chromatin immunoprecipitation assays, participated in thestatistical analysis, and drafted the manuscript. BM participated in the designof the study. JH performed the sequencing analysis and association studies.RF participated in the design of the study and helped draft the manuscript.DA provided his expertise with the chromatin immunoprecipitation assays.AS participated in the design of the study, participated in the statisticalanalysis, and helped draft the manuscript. PP and JD conceived of the study,participated in its design and coordination, and helped draft the manuscript.All authors read and approved the final manuscript.AcknowledgementsThe authors thank all participants for their contribution to the study.Supported by NIH grant R01:HL086936 (J.D.). A.W. received funding from theBritish Columbia Clinical Genomics Network.Author details1University of British Columbia James Hogg Research Centre, St. Paul’sHospital, Vancouver, BC, Canada. 2Department of Medicine, Division ofMolecular and Pulmonary Medicine, Columbia University College ofPhysicians and Surgeons, New York, NY, USA. 3Department of Medicine,Division of Pulmonary and Critical Care Medicine, St. Luke’s Roosevelt HealthSciences Center, New York, NY, USA. 4Naomi Berrie Diabetes Center andDepartment of Medicine, Columbia University, New York, New York, USA.5Department of Medicine, Division of Respiratory Medicine, University ofBritish Columbia, Vancouver, BC, Canada.Received: 21 June 2012 Accepted: 21 August 2012Published: 19 September 2012References1. Global Strategy for the Diagnosis, Management and Prevention of COPD:Global Initiative for Chronic Obstructive Lung Disease (GOLD): 2010. Young RP, Hopkins RJ, Christmas T, Black PN, Metcalf P, Gamble GD: COPDprevalence is increased in lung cancer, independent of age, sex andsmoking history. Eur Respir J 2009, 34:380–386.3. D'Armiento J, Dalal SS, Okada Y, Berg RA, Chada K: Collagenase expressionin the lungs of transgenic mice causes pulmonary emphysema. Cell 1992,71:955–961.4. Foronjy RF, Okada Y, Cole R, D'Armiento J: Progressive adult-onsetemphysema in transgenic mice expressing human MMP-1 in the lung.Am J Physiol Lung Cell Mol Physiol 2003, 284:L727–L737.5. Imai K, Dalal S, Chen E, Downey R, Schulman L, Ginsburg M, D'Armiento J:Human collagnease (matrix metalloproteinase-1) expression in the lungsof patients with emphysema. Am J Respir Crit Care Med 2001, 163:786–791.6. Segura-Valdez L, Pardo A, Gaxiola M, Uhal BD, Becerril C, Selman M:Upregulation of gelatinases A and B, collagenases 1 and 2, andincreased parenchymal cell death in COPD. Chest 2000, 117:684–694.7. Mercer BA, Kolesnikova N, Sonett J, D'Armiento J: Extracellular regulatedkinase/mitogen activated protein kinase is up-regulated in pulmonaryemphysema and mediates matrix metalloproteinase-1 induction bycigarette smoke. J Biol Chem 2004, 279:17690–17696.8. Mercer BA, Wallace AM, Brinckerhoff CE, D'Armiento JM: Identification of acigarette smoke-responsive region in the distal MMP-1 promoter. Am JRespir Cell Mol Biol 2009, 40:4–12.9. Anthonisen NR, Connett JE, Kiley JP, Altose MD, Bailey WC, Buist AS,Conway WAJ, Enright PL, Kanner RE, O'Hara P: Effects of smokingintervention and the use of an inhaled anticholinergic bronchodilator onthe rate of decline of FEV1. The Lung Health Study. JAMA 1994,272:1497–1505.10. Weckx S, Del-Favero J, Rademakers R, Claes L, Cruts M, De Jonghe P, VanBroeckhoven C, De Rijk P: novoSNP, a novel computational tool forsequence variation discovery. Genome Res 2005, 15:436–442.11. Rijk PD, Del-Favero J: novoSNP3: variant detection and sequenceannotation in resequencing projects. Methods Mol Biol 2007, 396:331–344.12. O'Connor GT, Sparrow D, Weiss ST: A prospective longitudinal study ofmethacholine airway responsiveness as a predictor of pulmonary-function decline: the Normative Aging Study. Am J Respir Crit Care Med1995, 152:87–92.13. Rodriguez S, Gaunt TR, Day INM: Hardy-Weinberg equilibrium testing ofbiological ascertainment for Mendelian randomization studies. Am JEpidemiol 2009, 169:505–514.14. Menashe I, Rosenberg PS, Chen BE: PGA: power calculator for case–controlgenetic association analyses. BMC Genet 2008, 9:36.15. Wallace AM, Sandford AJ, English JC, Burkett KM, Li H, Finley RJ, Müller NL,Coxson HO, Paré PD, Abboud RT: Matrix metalloproteinase expression byhuman alveolar macrophages in relation to emphysema. COPD 2008,5:13–23.16. Rutter JL, Mitchell TI, Buttice G, Meyers J, Gusella JF, Ozelius LJ, BrinckerhoffCE: A single nucleotide polymorphism in the matrix metalloproteinase-1promoter creates an Ets binding site and augments transcription. CancerRes 1998, 58:5321–5325.17. Kar S, Subbaram S, Carrico PM, Melendez JA: Redox-control of matrixmetalloproteinase-1: a critical link between free radicals, matrixremodeling and degenerative disease. Respir Physiol Neurobiol 2010,174:299–306.18. Chaudhary AK, Singh M, Bharti AC, Asotra K, Sundaram S, Mehrotra R:Genetic polymorphisms of matrix metalloproteinases and theirinhibitors in potentially malignant and malignant lesions of the headand neck. J Biomed Sci 2010, 17:10.19. Angel P, Baumann I, Stein B, Delius H, Rahmsdorf HJ, Herrlich P: 12-O-tetradecanoyl-phorbol-13-acetate induction of the human collagenasegene is mediated by an inducible enhancer element located in the 50-flanking region. Mol Cell Biol 1987, 7:2256–2266.20. Reilly JJ: COPD and declining FEV1 – time to divide and conquer? N EnglJ Med 2008, 359:1616–1618.21. Beasley R, Weatherall M, Travers J, Shirtcliffe P: Time to define the disordersof the syndrome of COPD. Lancet 2009, 374:670–672.22. Rennard SI, Vestbo J: The many ‘small COPDs’: COPD should be anorphan disease. Chest 2008, 134:623–627.Wallace et al. Respiratory Research 2012, 13:79 Page 7 of 8 Kohansal R, Martinez-Camblor P, Agusti A, Buist AS, Mannino DM, SorianoJB: The natural history of chronic airflow obstruction revisited: ananalysis of the Framingham offspring cohort. Am J Respir Crit Care Med2009, 180:3–10.24. Mannino DM, Watt G, Hole D, et al: The natural history of chronicobstructive pulmonary disease. Eur Respir J 2006, 27:627–643.25. Celli BR, Thomas NE, Anderson JA, et al: Effect of pharmacotherapy on rateof decline of lung function in chronic obstructive pulmonary disease:results from the TORCH study. Am J Respir Crit Care Med 2008, 178:332–338.26. Tashkin DP, Celli B, Senn S, et al: A 4-year trial of tiotropium in chronicobstructive pulmonary disease. N Engl J Med 2008, 359:1543–1554.27. He JQ, Foreman MG, Shumansky K, et al: Associations of IL6polymorphisms with lung function decline and COPD. Thorax 2009,64:698–704.28. Sandford AJ, Chagani T, Weir TD, Connett JE, Anthonisen NR, Pare PD:Susceptibility genes for rapid decline of lung function in the LungHealth Study. Am J Respir Crit Care Med 2001, 163:469–473.29. Cao ZG, Li CZ: A single nucleotide polymorphism in the matrixmetalloproteinase-1 promoter enhances oral squamous cell carcinomasusceptibility in a Chinese population. Oral Oncol 2006, 42:32–38.30. Hashimoto T, Uchida K, Okayama N, Imate Y, Suehiro Y, Hamanaka Y:Association of matrix metalloproteinase (MMP)-1 promoterpolymorphism with head and neck squamous cell carcinoma. Cancer Lett2004, 211:19–24.doi:10.1186/1465-9921-13-79Cite this article as: Wallace et al.: Functional characterization of thematrix metalloproteinase-1 cigarette smoke-responsive region andassociation with the lung health study. Respiratory Research 2012 13:79.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at et al. Respiratory Research 2012, 13:79 Page 8 of 8


Citation Scheme:


Citations by CSL (citeproc-js)

Usage Statistics



Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            async >
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:


Related Items