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MMP1 bimodal expression and differential response to inflammatory mediators is linked to promoter polymorphisms.

Affara M, Dunmore BJ, Sanders DA, Johnson N, Print CG, Charnock-Jones DS - BMC Genomics (2011)

Bottom Line: Identifying the functional importance of the millions of single nucleotide polymorphisms (SNPs) in the human genome is a difficult challenge.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 describe a simple method to identify putative bimodally expressed RNAs from transcriptome data that is effective yet easy for non-statisticians to understand and use.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK.

ABSTRACT

Background: 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.

Results: 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 immunoprecipitation (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.

Conclusions: We describe a simple method to identify putative bimodally expressed RNAs from transcriptome data that is effective yet easy for non-statisticians 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).

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Immunoblot illustrating inflammatory signalling responses in 3 isolates with low and high basal expression of MMP-1 (labelled isolates 1, 2, 3 and 4, 5, 6 respectively). Low = low basal expressers and High = high basal expressers. Time in hours for each group of low and high basal expressers is given at the top of the figure. (a) IκBα signalling over 3.5 hours after the addition of an inflammatory mediator cocktail containing 10 ng/ml each of TNF-α, Il-1β and Il-8. (b) Phosphorylated IκBα signalling over the time course. (c) ICAM signalling over the time course. Both the high expressers and low expressers show similar levels of IκBα, phospho- IκBα and ICAM over the time course.
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Figure 7: Immunoblot illustrating inflammatory signalling responses in 3 isolates with low and high basal expression of MMP-1 (labelled isolates 1, 2, 3 and 4, 5, 6 respectively). Low = low basal expressers and High = high basal expressers. Time in hours for each group of low and high basal expressers is given at the top of the figure. (a) IκBα signalling over 3.5 hours after the addition of an inflammatory mediator cocktail containing 10 ng/ml each of TNF-α, Il-1β and Il-8. (b) Phosphorylated IκBα signalling over the time course. (c) ICAM signalling over the time course. Both the high expressers and low expressers show similar levels of IκBα, phospho- IκBα and ICAM over the time course.

Mentions: It was possible that the differential responses to IM treatment we observed between individuals with low MMP1 basal expression (homozygous 1G individuals) and high MMP1 basal expression (heterozygotes and homozygous 2G individuals) were simply due to differential activity of the signalling pathways that mediate inflammation. Therefore, molecules known to be downstream of inflammatory mediator signalling were assessed as biomarkers of inflammatory pathway activity in individuals with low and high MMP1 basal expression. Protein expression levels in ICAM1, IкBα and phospho-IкBα were measured in HUVEC lysates from three individuals with low basal MMP1 mRNA and three with high basal MMP1 mRNA, after treatment with 10 ng/ml IL-1β, TNF-α and IL-8 for up to 3.5hrs. Figure 7 shows abundance of ICAM-1, total and phospho-IкBα over the 3.5hr period. Two-way analysis of variance (ANOVA) revealed that there was no significant difference in ICAM1 or total and phospho-IкBα signal between the high and low expressers at all time points (P = 0.8, 0.7 and 0.2 respectively). These results suggest that there is not a large systematic difference between the inflammatory signalling pathways related to this polymorphism.


MMP1 bimodal expression and differential response to inflammatory mediators is linked to promoter polymorphisms.

Affara M, Dunmore BJ, Sanders DA, Johnson N, Print CG, Charnock-Jones DS - BMC Genomics (2011)

Immunoblot illustrating inflammatory signalling responses in 3 isolates with low and high basal expression of MMP-1 (labelled isolates 1, 2, 3 and 4, 5, 6 respectively). Low = low basal expressers and High = high basal expressers. Time in hours for each group of low and high basal expressers is given at the top of the figure. (a) IκBα signalling over 3.5 hours after the addition of an inflammatory mediator cocktail containing 10 ng/ml each of TNF-α, Il-1β and Il-8. (b) Phosphorylated IκBα signalling over the time course. (c) ICAM signalling over the time course. Both the high expressers and low expressers show similar levels of IκBα, phospho- IκBα and ICAM over the time course.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3040839&req=5

Figure 7: Immunoblot illustrating inflammatory signalling responses in 3 isolates with low and high basal expression of MMP-1 (labelled isolates 1, 2, 3 and 4, 5, 6 respectively). Low = low basal expressers and High = high basal expressers. Time in hours for each group of low and high basal expressers is given at the top of the figure. (a) IκBα signalling over 3.5 hours after the addition of an inflammatory mediator cocktail containing 10 ng/ml each of TNF-α, Il-1β and Il-8. (b) Phosphorylated IκBα signalling over the time course. (c) ICAM signalling over the time course. Both the high expressers and low expressers show similar levels of IκBα, phospho- IκBα and ICAM over the time course.
Mentions: It was possible that the differential responses to IM treatment we observed between individuals with low MMP1 basal expression (homozygous 1G individuals) and high MMP1 basal expression (heterozygotes and homozygous 2G individuals) were simply due to differential activity of the signalling pathways that mediate inflammation. Therefore, molecules known to be downstream of inflammatory mediator signalling were assessed as biomarkers of inflammatory pathway activity in individuals with low and high MMP1 basal expression. Protein expression levels in ICAM1, IкBα and phospho-IкBα were measured in HUVEC lysates from three individuals with low basal MMP1 mRNA and three with high basal MMP1 mRNA, after treatment with 10 ng/ml IL-1β, TNF-α and IL-8 for up to 3.5hrs. Figure 7 shows abundance of ICAM-1, total and phospho-IкBα over the 3.5hr period. Two-way analysis of variance (ANOVA) revealed that there was no significant difference in ICAM1 or total and phospho-IкBα signal between the high and low expressers at all time points (P = 0.8, 0.7 and 0.2 respectively). These results suggest that there is not a large systematic difference between the inflammatory signalling pathways related to this polymorphism.

Bottom Line: Identifying the functional importance of the millions of single nucleotide polymorphisms (SNPs) in the human genome is a difficult challenge.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 describe a simple method to identify putative bimodally expressed RNAs from transcriptome data that is effective yet easy for non-statisticians to understand and use.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK.

ABSTRACT

Background: 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.

Results: 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 immunoprecipitation (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.

Conclusions: We describe a simple method to identify putative bimodally expressed RNAs from transcriptome data that is effective yet easy for non-statisticians 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).

Show MeSH
Related in: MedlinePlus