MMP1 bimodal expression and differential response to inflammatory mediators is linked to promoter polymorphisms
- Muna Affara1,
- Benjamin J Dunmore2,
- Deborah A Sanders2,
- Nicola Johnson1,
- Cristin G Print†3, 1 and
- D Stephen Charnock-Jones†1, 2, 4Email author
© Affara et al; licensee BioMed Central Ltd. 2011
Received: 26 August 2010
Accepted: 19 January 2011
Published: 19 January 2011
Identifying the functional importance of the millions of single nucleotide polymorphisms (SNPs) in the human genome is a difficult challenge. Therefore, a reverse strategy, which identifies functionally important SNPs by virtue of the bimodal abundance across the human population of the SNP-related mRNAs will be useful. Those mRNA transcripts that are expressed at two distinct abundances in proportion to SNP allele frequency may warrant further study. Matrix metalloproteinase 1 (MMP1) is important in both normal development and in numerous pathologies. Although much research has been conducted to investigate the expression of MMP1 in many different cell types and conditions, the regulation of its expression is still not fully understood.
In this study, we used a novel but straightforward method based on agglomerative hierarchical clustering to identify bimodally expressed transcripts in human umbilical vein endothelial cell (HUVEC) microarray data from 15 individuals. We found that MMP1 mRNA abundance was bimodally distributed in un-treated HUVECs and showed a bimodal response to inflammatory mediator treatment. RT-PCR and MMP1 activity assays confirmed the bimodal regulation and DNA sequencing of 69 individuals identified an MMP1 gene promoter polymorphism that segregated precisely with the MMP1 bimodal expression. Chromatin immunoprecipation (ChIP) experiments indicated that the transcription factors (TFs) ETS1, ETS2 and GATA3, bind to the MMP1 promoter in the region of this polymorphism and may contribute to the bimodal expression.
We describe a simple method to identify putative bimodally expressed RNAs from transcriptome data that is effective yet easy for non-statisticans to understand and use. This method identified bimodal endothelial cell expression of MMP1, which appears to be biologically significant with implications for inflammatory disease. (271 Words)
Numerous strategies have been used in an attempt to sift through the vast amounts of data produced from microarray expression studies [1–4]. There has been much interest given to the identification of bimodally expressed mRNA transcripts, particularly in the context of cancer, where two distinct populations of patients can be defined by differing levels of a set of specific transcripts. These make excellent candidate biomarkers and often tend to show good correlation between transcript and protein abundance . To this end, statistical approaches using mixture-model based clustering combined with either Akaike information criterion (AIC) or the Bayesian informatics criterion (BIC) have frequently been applied [6–9]. One method based on systematic classification of gene expression profiles has been applied to over 2,000 microarray samples . These methods have a strong theoretical base and have proven successful in identifying bimodality. However, they do depend on the investigator having a relatively high level of statistical understanding . In this study we suggest a simple screening approach based on hierarchical clustering to identify bimodally expressed transcripts from microarray expression data that can be used alongside more complex approaches. While this method is not motivated by statistical theory, it appears to work well and is easily understood by laboratory scientists with only basic statistical training, who are in a good position to immediately follow up their results experimentally.
The matrix metalloproteinase, MMP1, is one of the most abundant proteases in the matrix metalloproteinase family. It is capable of degrading type I, II and III collagens, and is one of only four MMPs able to degrade triple helical collagens. It therefore plays a pivotal role in extracellular matrix (ECM) remodelling in both normal development and pathology . MMP1 is tightly regulated at both the transcriptional and post-translational levels. It is produced as a zymogen that is activated by serine proteases and its activity is regulated by inhibitors such as the tissue inhibitors of metalloproteinase's (TIMPs), which compete with the substrate for the enzyme active site .
MMP1 plays a clinically important role in inflammatory disease, and has been implicated in numerous pathological processes including wound healing , tumour metastasis  and arthritis . The MMP-1 gene  contains a 1G/2G polymorphism in its promoter at -1607 from the transcriptional start site . This polymorphism has been associated with increased MMP-1 expression in several cell types including; melanoma, stromal fibroblast, MCF-7/ADR breast cancer cells [16–19], and with several pathologies including: tumour metastasis [20, 21], arthritis [22, 23], periodontitis , chronic obstructive pulmonary disease  and cardiovascular disease [26, 27].
Using our simple clustering method to examine RNA transcript abundance in HUVECs isolated from 15 different human individuals, we identified MMP1 as one of a small group of RNAs expressed in a bimodal manner in both un-treated endothelial cells, and in endothelial cells treated by inflammatory mediators. Our results suggest that the regulation of MMP1 expression is a complex process that is modulated by a promoter polymorphism around the binding sites for several TFs including ETS1, ETS2 and GATA3.
Identification of mRNA transcripts with bimodal expression patterns among a set of individuals
These shortlisted transcripts were further assessed using several strategies including; i) visual inspection of histograms on a gene by gene basis (an R script used to generate these is given in Additional File 4); ii) evaluation of the associated bootstrap p-values obtained during clustering and iii) consideration of additional information of biomedical interest. We were especially interested in the presence of promoter SNPs, which may in theory cause bimodal RNA expression patterns, for example using the SNPer or rSNPs databases. Based on all these considerations, subset of transcripts were selected to take forward for further investigation; DDX3Y (a Y-chromosome encoded RNA, which should segregate with gender), MMP1 and SLC2A11 (biologically interesting TNFα and IFNγ targets, which are important in inflammation).
DDX3Y is differentially expressed on the basis of its location on the Y-chromosome
MMP1 is differentially expressed and differentially active in endothelial cells
To investigate whether the bimodal expression of MMP1 mRNA was also evident at the level of MMP1 enzymatic activity, this activity was measured in 20 HUVEC isolates. The activity of MMP1 was low in those HUVEC cultures that had low MMP1 RNA abundance and high in those HUVEC cultures that had high MMP1 RNA abundance (Figure 5c).
MMP-1 gene promoter polymorphisms segregate with MMP1 expression and enzymatic activity
Segregation of genotype for the -1607 MMP-1 promoter polymorphism with basal abundance of MMP-1 transcript
High Basal Expression
Low Basal Expression
Homozygous 1G SNP
Homozygous 2G SNP
Heterozygous 1G/2G SNP
Elevated MMP1 expression induced by inflammatory mediator in 1G homozygous cells is not due to greater overall activity of pro-inflammatory signalling pathways
Ets1, Ets2, Fos and GATA3 are potential mediators of the different expression levels of MMP1 mRNA in 1G -vs- 2G individuals
The significance of non-coding polymorphisms in pathology is being increasingly recognised, with much research being carried out to identify the functional importance of the millions of SNPs mapped to date in the human genome . This study suggests a complementary strategy, whereby we first identified those transcripts that showed bimodal expression levels, and then identified the polymorphism responsible for this differential expression. Clustering methods  and statistical methods [7, 10, 33] have previously been used to identify bimodal expression in large datasets. These methods have the advantage of being motivated by strong theoretical statistical considerations. However, they also require a moderate level of statistical understanding, and in addition some of these methods can only be easily applied to large-scale meta-analysis of several data sets  and may be less suitable for small expression data sets generated in a single laboratory. The approach we suggest is able to be used alongside more complex approaches by laboratory scientists with only basic statistical training, who are in a good position to immediately follow up their results experimentally. We believe that for bioinformatic tasks such as this, providing several complimentary methods that span the continuum of statistical complexity is important in order to bridge the gap between experimental biologists and statisticians.
Our clustering strategy successfully identified the abundance of several mRNA transcripts including MMP1 as bimodally distributed in human endothelial cells in both resting and inflammatory mediator-treated conditions. Up-regulated MMP1 expression has been associated with many pathologies in which endothelial cells are involved [20, 21, 26, 27]. We sequenced the region spanning the well characterised polymorphism at -1607 from the transcription start site in the MMP-1 promoter [16–19] and found that this polymorphism was strongly associated with the bimodal expression observed in the HUVEC dataset. Individuals either homozygous or heterozygous for the 2G polymorphism at -1607 possessed constitutively higher levels of MMP1 (over 100 fold relative to the 1G isolates, P < 0.0001), implying a dominant effect of the 2G allele. This contrasts with previous findings in fibroblasts, where marginal differences in the basal levels of MMP1 between the 1G and 2G isolates were observed [17, 34].
Inflammatory mediator treatment revealed a differential response in MMP1 stimulation between HUVEC isolates homozygous for the 1G allele and those possessing the 2G allele. Whereas MMP1 mRNA levels were increased in all isolates homozygous for the 1G allele; in isolates possessing the 2G allele, MMP1 mRNA levels were essentially unchanged (Figure 5). Regulation at the level of MMP1 enzyme activity mirrored this response to inflammatory mediator treatment. One possibility is that MMP1 expression is at maximum levels in the 2G isolates, even under the basal condition. In leukocytes continuously treated with high doses of inflammatory mediators (50 ng/ml, of TNF-α and Il-1β, 2 or 3 times over 24hrs), MMP1 mRNA levels are genotype independent . Whether this is the case in HUVECs remains to be determined.
Chromatin immunoprecipitation (ChIP) was conducted to investigate the potential involvement of TFs that have putative response elements spanning the polymorphism. We found that three TFs bound to this polymorphic region of the MMP1 promoter in endothelial cells. Using nuclear extracts from fibroblast and A2058 melanoma cells, Rutter et al. previously demonstrated that recombinant ETS1 binds strongly to the 2G promoter and weakly to the 1G promoter and that this binding is dependent on cooperation with an adjacent AP1 site at -1602 . Their study along with others have revealed that several members of the AP1 family, including c-JUN, Fra and Fos, are involved in the heterodimmer complexes bound at this cooperative AP1 site [16, 35, 36]. While the limited ChIP analysis we have performed clearly indicated that GATA3, Fos, Ets1 and Ets2 do indeed bind to this region of the MMP1 promoter, further studies using larger numbers of individuals will be required to identify differential binding between genotypes or cell culture conditions.
Identifying the functional importance of the millions of human SNPs is becoming a major challenge. Simultaneously, the amount of available RNA transcriptome data is rapidly growing, driving scientists to devise new methods to extract the most biologically and clinically useful information from RNA abundance profiles. Therefore, a strategy that identifies functionally important SNPs by virtue of the bimodal abundance across the human population of the associated mRNAs is potentially very useful. Here, we discuss a simple method based on hierarchical clustering to identify bimodally expressed transcripts, which may be used with either microarray or RNAseq data. This method complements more statistically complex approaches and is suitable for use by laboratory scientists with only basic statistical training, who are in a good position to immediately follow up their results experimentally. This strategy identified bimodal endothelial cell expression of several transcripts including MMP1, which appears to be biologically significant with implications for inflammatory disease and for understanding the complex relationships between TFs and polymorphic promoter elements.
Cell culture and IM treatment
Umbilical cords were collected after written informed consent and the study was approved by the Cambridge Research Ethics Committee. The population sampled for this study were of unknown demography, with no information obtained during donor collection relating to parental age, ethnicity or familial history of disease. HUVECs were isolated by collagenase digestion, as previously described . Cells were cultured in fully supplemented media without antibiotics (basal EBM-2 with a propriety mix of heparin, hydrocortisone, vascular endothelial growth factor, epidermal growth factor, fibroblast growth factor, 2% foetal calf serum (FCS, Lonza, Cambridge, UK), at 37°C/5% CO2 until passage 4. To carry out inflammatory mediator treatment for microarray gene expression profiling, passage 4 HUVECs were treated with a cocktail of 10 ng/ml TNF-α, IL-1β and IL-8 for 24 hours prior to RNA extraction.
RNA processing and microarray preparation and data processing
RNA was extracted using TRIzol® reagent (Invitrogen, London UK). RNA quality was assessed using the Agilent 2100 bioanalyser. Biotin labelled cRNA was generated and hybridised on the CodeLink Human Uniset 20K microarrays following the manufacturer's instructions (Applied Microarrays, formally supplied by GE Healthcare). CodeLink microarray data was pre-processed to assess array quality using the CodeLink Expression analysis software v4.0. To enable comparable analysis between arrays, normalisation was carried out using the cyclic Loess method [38, 39]. The microarray data has been deposited in NCBI's Gene Expression Omnibus (GEO)  and can be accessed through GEO series accession number GSE23070.
Bimodal analysis of microarray expression data
To identify bimodally expressed RNA transcripts, RNA was prepared from passage 4 HUVECs isolated from 15 different individuals and analysed using CodeLink Human Uniset 20K microarrays (the untreated (UT) data set). In addition, passage 4 HUVECs isolated from 9 different individuals were each treated with 10 ng/ml of each of TNF-α, IL-1β, Il-8, and analysed using microarrays as described above (the inflammatory mediator treated (IM) data set). Unsupervised agglomerative clustering was then applied separately to the UT and IM data sets to enrich for multimodality, using R bioinformatic software (freely available at http://cran.r-project.org/). For each transcript, our algorithm recorded the "height" (Euclidian distance) between the clusters. The height values at either end of the cluster dendrogram were discarded to remove cases where the clustering identified a single outlying individual, and the largest remaining height value was used as an indicator of bimodality/multimodality. For those RNAs with signal intensities that were similar across the set of individuals, the height between clusters is likely to be small. However, where there were two or more distinct clusters of expression values among the set of individuals, the height between clusters is likely to be large. In addition, parametric bootstrapping was carried out during the clustering process to identify the likelihood of identifying the given height value for each gene based on chance alone, as summarised in Figure 1 and in the comments within the R script in Additional File 1. To be strictly statistically correct, the permutation p-values should be adjusted for multiple testing. For example the Benjamini & Hochberg procedure could be used to control the false discovery rate by applying the mt.rawp2adjp function of the 'multtest' R package to the p-values produced from the bootstrap procedure described here. However, this is not included in the current iteration of our method, since it does not alter the ranking of the permutation p-values assigned to each RNA, and it appears to be overly stringent since it masks both of the bimodally-expressed RNAs that were experimentally confirmed in our study. Nevertheless, if larger data sets are analysed, from which the degree of bimodal expression and population distribution parameters for each RNA can be estimated more precisely, it may be worth experimenting with various multiple testing control procedures.
Inflammatory mediator time course and immunoblotting
For the inflammatory mediator time course, passage 4 HUVECs were treated with a cocktail of 10 ng/ml of each of TNF-α, IL-1β, IL-8 for up to 3.5 hours. Whole cell lysates were harvested by scraping in 1X RIPA lysis buffer (Millipore, Watford, UK) with protease inhibitors (Roche, Welwyn Garden City, UK), at time points 0, 0.5, 1.5 and 3.5 hours. Proteins were separated on 12% Tris-glycine SDS-page gels (Invitrogen) and transferred to 0.2 μm nitrocellulose membranes (Invitrogen). All membranes were blocked with 5% skimmed milk in Tris-buffered saline/0.01% Tween®20 at room temperature. Blots were probed with antibodies against ETS1 (sc-350) and ETS2 (sc-351) (both from Santa Cruz Biotechnology) and β-actin (Ambion).
MMP1 activity assay
Total active MMP1 protein abundance was measured using the Fluorokine Human Active MMP1 Fluorescent Assay (R&D Systems). Supernatants were collected from the cell culture of 20 different passage 4 HUVEC isolates, treated with and without an inflammatory mediator cocktail of 10 ng/ml TNF-α, IL-1β, IL-8 for 24hrs. P-Aminophenylmercuric Acetate (APMA) was added to all samples to activate any inactive MMP1. Measurement of MMP1 activity was carried out according to the manufacturer's instructions.
Sequencing of the MMP1 promoter polymorphism
To characterise the -1607 MMP1 promoter polymorphism, DNA was extracted from HUVEC cell pellets using the DNeasy blood and tissue kit (Qiagen, West Sussex, UK), following the manufacturer's instructions. Genomic DNA (50 ng) was amplified with the following primers: 5'-AACCTATTAACTCACCCTTGT-3' 5'-CCTCCATTCAAAAGATCTTATTATTTAGCATCTCCT-3' . The cycling conditions were as follows: pre-incubation at 94°C for 5 minutes, followed by 35 cycles at 94°C for 30 seconds, 56°C for 30 seconds and 72°C for 1 minute, followed by a final extension at 72°C for 10 minutes. PCR products were diluted 1 in 10 in nuclease free water and directly sequenced using the forward primer at GeneService (Cambridge Science Park, Milton, UK). Amplification of the MMP1 promoter region spanning the -930 and -519 polymorphisms was achieved using the same conditions described above using the following primers: 5'-TTCCAGCCTTTTCATCATCC-3' and 5'-CGGCACCTGTACTGACTGAA-3'. Again the forward primer was used for sequencing.
cDNA was made from 1 μg of total RNA using the Quantitect reverse transcription kit (Qiagen), following the manufacturers protocol. Quantitative PCR was carried out using the the ABI 7700 sequence analyser (Applied Biosystems, Calafornia, USA). Reactions were carried out using the Applied Biosystems universal master mix according to the manufacturers instructions. The Taqman probe primers used were: MMP1 (Hs00233958_m1), DDX3Y (Hs00190539_m1), ETS1 (Hs00901425_m1), ETS2 (Hs00232009_m1), GATA3 (Hs00231122_m1), SLC2A11 (Hs00368843_m1), DERP6 (Hs00209768_m1) and internal control 18S (Hs99999901_s1), all from Applied Biosystems.
Passage 4 HUVECs were either treated with vehicle or an inflammatory cocktail of 10 ng/ml TNF-α, IL-1β, IL-8 for 24 hours. Chromatin was cross-linked by the addition of formaldehyde to a final concentration of 1% for 10 minutes at 37°C. Cells were washed in ice cold phosphate-buffered saline containing 125 mM glycine, 1 mg/ml Pefabloc, 1 μg/ml aprotinin and 1 μg/ml pepstatin A. Chromatin was sonicated and immunoprecipitated using specific antibodies, as described in the ChIP protocol from Upstate Inc. (Charlottesville, VA). The following antibodies were used: ETS1 (sc-350), ETS2 (sc-351), c-Fos (sc-52) and GATA3 (sc-268). All antibodies were from Santa Cruz Biotechnologies. To quantify enrichment of binding, quantitative PCR was carried out on the immunoprecipitated DNA using SYBR Green on the iCycler (Roche). 25 μl reactions with 1 X SensiMixPlus SYBR and fluorescein (Quantace) were carried out according to the manufacturer's instructions. Primers around the MMP1 polymorphism were 5'-TCTTTGTCTGTGCTGGAGTA-3' and 5'-CAATTTCCTCATCTAAGTGGCATA-3'. The primers for the region 5600 bases upstream of the promoter were 5'-TGCTTATGTTAGCTGACCAGAC-3' and 5'-AGTATGCGTTGCCTTGTCCT-3'.
We thank Marie Joquine for here assistance in identification of the bimodally expressed transcripts. This study was part-funded by the Biotechnology and Biological Sciences Research Council (BBSRC) and Gene Networks International (GNI, Shinjuku Park Tower, Tokyo, Japan).
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