Am J Transl Res 2012;4(2):219-228 www.ajtr.org /ISSN:1943-8141/AJTR1202002 Original Article Identification of an interleukin 13-induced epigenetic signature in allergic airway inflammation Aik T Ooi1, Sonal Ram1, Alan Kuo1, Jennifer L Gilbert1, Weihong Yan2, Matteo Pellegrini3, Derek W Nickerson1, Talal A Chatila4, Brigitte N Gomperts1,5,6,7 1David Geffen School of Medicine at UCLA, Department of Pediatrics, Mattel Children's Hospital, Los Angeles, CA 90095, USA; 2Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, 90095 USA; 3Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA, 90095 USA; 4Division of Immunology, The Children's Hospital and Department of Pediatrics, Harvard Medical School, Boston MA 02115; 5David Geffen School of Medicine at UCLA, Department of Medicine, Division of Pulmonary and Critical Care Medicine, Los Angeles, CA 90095, USA; 6Broad Stem Cell Research Center at UCLA, Los Angeles, CA, 90095, USA; 7Jonsson Comprehensive Cancer Center at UCLA, Los Angeles, CA, 90095, USA Received February 12, 2012; accepted March 22, 2012; Epub April 10, 2012; Published April 30, 2012 Abstract: Epigenetic changes have been implicated in the pathogenesis of asthma. We sought to determine if IL13, a key cytokine in airway inflammation and remodeling, induced epigenetic DNA methylation and miRNAs expression changes in the airways in conjunction with its transcriptional gene regulation. Inducible expression of an IL13 trans-gene in the airways resulted in significant changes in DNA methylation in 177 genes, most of which were associated with the IL13 transcriptional signature in the airways. A large number of genes whose expression was induced by IL13 were found to have decreased methylation, including those involved in tissue remodeling (Olr1), leukocyte influx (Cxcl3, Cxcl5, CSFr2b), and the Th2 response (C3ar1, Chi3l4). Reciprocally, some genes whose expression was sup-pressed were found to have increased methylation (e.g. Itga8). In addition, miRNAs were identified with targets for lung development and Wnt signaling, amongst others. These results indicate that IL13 confers an epigenetic methyla-tion and miRNA signature that accompanies its transcriptional program in the airways, which may play a critical role in airway inflammation and remodeling. Keywords: Epigenetics, miRNA, DNA methylation, allergic airway disease asthma-like symptoms in their offspring, sug- gesting that inherited epigenetic changes may Asthma is a common chronic inflammatory air- lead to asthma [5]. This link between environ- way condition with a strong genetic and inherita- mental pollutants and airway inflammation bility component, as siblings and first-degree could result from epigenetic modifications, as relatives of those with the disease are often cells alter their epigenetic profiles following ex- affected [1, 2]. However, the remodeling seen in posure to environmental toxins [6]. asthma likely reflects a combination of genetic predisposition and environmental exposures. Epigenetic changes such as DNA methylation of Studies have shown the importance of environ- CpG dinucleotides within the transcription start mental exposures on the development of sites are common mechanisms for gene expres- asthma. For example, studies have revealed sion regulation [7, 8]. Many studies have re- that children exposed to prenatal polycyclic aro- ported that asthma risk and airway inflamma- matic hydrocarbons and postnatal tobacco tion can be influenced by epigenetic regulation smokers were more likely to have difficulty [4, 9]. It has been shown that methylation of a breathing and probable asthma [3, 4]. Studies highly conserved CpG in the Ifng (interferon on pregnant mice demonstrated that exposure gamma) promoter is associated with polarizing to aerosolized leachate of residual oil fly ash naïve T cells into the pro-allergic T helper type 2 would lead to the development of several (Th2) cells [10]. These Th2 cells play a major DNA methylation in epigenetic regulation in asthma role in the development of asthma due to their this, we performed gene expression arrays, me- production of pro-inflammatory cytokines [11]. thylated DNA immunoprecipitation (MeDIP) ar- Methylation of CpG islands within the promoter rays, and miRNA expression arrays with RNA or region of ADAM33, a gene important in the de- DNA isolated from whole lungs. We found gene velopment of asthma in human, was shown to expression patterns that correlated with DNA regulate the transcription of the gene [12, 13]. methylation status in genes associated with Apart from DNA methylation, microRNAs allergic airway inflammatory processes. We also (miRNAs) can also regulate gene expression in identified miRNAs that were differentially ex- an epigenetic manner by targeting and degrad- pressed with targets for inflammatory genes. ing specific messenger RNAs [14]. It has been These results suggested that IL13 rapidly in- shown that miRNA expression is dysregulated in duced epigenetic responses that could contrib- asthma, and this could be one of the major ute to the regulation of genes involved in aller- mechanisms for the initiation and development gic airway inflammation. of the disease [15, 16]. Other epigenetic mechanisms in inflammatory cells, such as his- Materials and methods tone modifications and chromatin remodeling, have also been implicated to play a role in the Transgenic mice development of airway inflammation [17, 18]. Mice were housed and bred under the regula- Interleukin 13 (IL13) is a pleiotropic 12 kDa tion of the Division of Laboratory Animal Medi- cytokine that is produced and secreted in large cine at the University of California, Los Angeles. quantities by Th2 cells [19-21]. A large number The CC10-rtTA-IL13 transgenic (TG) mouse is a of studies have demonstrated that IL13 is over- well-characterized model of allergic airway dis- produced in asthma and have implicated IL13 in ease [29]. Briefly, the Clara cell 10-kDa (CC10) the pathogenesis of Th2 inflammation and air- gene promoter was used to conditionally ex- way remodeling [22, 23]. Lung-specific constitu- press IL13 in the mouse lung in a doxycycline- tive overexpression of IL13 produces airway inducible fashion. For these experiments, mice epithelial cell hypertrophy, macrophage-rich in- were exposed to doxycycline for a time period of flammation, subepithelial airway fibrosis, mucus either 1 week or 4 weeks. The control group metaplasia, airways hyperresponsiveness, and consisted of wild type (WT) mice that were ex- other physiological changes observed in asthma posed to doxycycline. Baseline leakiness of the [24]. IL13 instillation or expression in the air- IL13 transgene expression was found with ways also induces a recognizable set of genes these mice so that even in the absence of doxy- that mediate various aspects of tissue inflam- cycline they have slightly elevated IL13 and mild mation and remodeling [25-28]. Importantly, allergic airway inflammation [29]. Therefore, even after the cessation of IL13 treatment, a doxycycline untreated transgenic mice were not subgroup of induced genes remain expressed, used as controls in our studies. suggesting the possibility that they maintain autonomous expression through epigenetic After exposure to the appropriate time period of modifications [26]. doxycycline, mice were euthanized and the lungs were removed for subsequent studies For our studies, we used a well-characterized described below. transgenic mouse model of allergic airway in- flammation induced by IL13 [29]. In this model, Gene expression analysis IL13 is conditionally overexpressed in the mouse lung when treated with doxycycline. TG and WT mice were treated with doxycycline Upon IL13 induction, these mice showed inflam- for one week. Mice were euthanized and the left matory cell infiltration, pronounced emphysema, upper lobes from all mice were removed for increased pulmonary compliance, lung volume RNA extraction using the TRIzol method. The enlargement, mucus metaplasia, and increased RNA quality was checked using an Agilent 2100 expression of matrix metalloproteinases and Bioanalyzer, prior to amplification of RNA and cathepsins in the lung [29]. We used this mouse subsequent microarray hybridization on the Affy- model to examine genes that were epigeneti- metrix 430 2.0 array, following standard Affy- cally regulated by DNA methylation and miRNAs metrix protocols. Raw gene expression data (.cel in allergic airway inflammation. To accomplish files) were generated by standard Affymetrix 220 Am J Transl Res 2012;4(2):219-228 DNA methylation in epigenetic regulation in asthma protocols and deposited in the Gene Expression averaged, and a z-score was computed by sub- Omnibus of the National Center for Biotechnol- tracting the average value of probes on the en- ogy Information (accession number GSE37085). tire array and dividing by the standard deviation The import and normalization of the raw data of probes in the array. The resulting value was were completed by Bioconductor in the R soft- then multiplied by the square root of the num- ware environment. Two types of statistical tests ber of probes within the window to obtain the (t-test and rank product) were performed for final normalized z-score. We computed the z- each probe set in order to detect differentially scores for both samples, and reported the dif- expressed genes between the TG and WT lung ference in z-scores between them. Each of tissue. For both methods, only genes with an these regions was associated with the nearest adjusted P-value < 0.01 and fold change ≥ 2 gene, whose transcriptional start site was within based on the false discovery rate were consid- 2 kb. We then generated a list of regions that ered as differentially expressed genes and were had significant differences in z-scores (indicating a change in methylation) along with a significant change in gene expression. MeDIP method and data analysis Statistics TG and WT mice were treated with doxycycline for one week. Mice were then euthanized and All experiments were performed with at least lungs were surgically removed. The DNA was three different primary cultures or mice in inde- extracted from the left lower lobes of the lungs. pendent experiments except for the MeDIP ex- Methylated DNA immunoprecipitation (MeDIP) periment, which was performed once as a dis- was performed per the Nimblegen MeDIP array covery experiment for validation in more tissue. protocol. Briefly, an antibody that recognizes 5- Significance was evaluated by Student's t-test. methylcytosine was used to target the methy- lated fraction of the genome resulting in enrich- MicroRNA expression analysis and correlation ment of CpG islands [30]. The MeDIP fractions with gene expression were hybridized to Nimblegen 2.1M Deluxe Pro- moter Arrays that cover 10 kb of all annotated The CC10-rtTA-IL13 TG mice were treated with promoters. The MeDIP data was visualized us- doxycycline for 1 week. The control group con- ing the Affymetrix Integrated Genome Browser sisted of WT mice treated with doxycycline. After exposure, lungs were removed and RNA was extracted from left upper lobes in all mice. RNA extracts were amplified followed by microarray Correlation of MeDIP methylation data and hybridization on Affymetrix GeneChip miRNA gene expression data array (released in 2009) to profile the miRNA expressions in WT and TG mice. The raw expres- To identify regions of significant enrichment in sion data were first processed for background MeDIP, we used a z-score analysis to reflect estimation and correction, normalization, and methylation status for a selected region. For Z- summarization using miRNAQCTools, an Affy- score calculations, the log ratios for each Nim- metrix software specifically developed for the blegen array were normalized by subtracting GeneChip miRNA arrays (http://www. affymetrix. their mean log ratio values. Outlier probes with com/ estore/ browse/level_ seven_ software_ log ratio values greater than two were set to products_ only.jsp?productId= 131558& cate- two. To measure changes in methylation be- goryId = cat50004 & productName = miRNA-QC tween samples we computed the average log -Tool#1_1). The processed expression data ratios of all probes within a 2 kb window cen- were then log2-transformed and analyzed with tered on each probe; only windows with 5 or the significant analysis of microarray (SAM) soft- more probes were considered for analysis. ware [31]. The miRNA genes with q-value less These were then normalized to generate a Z- than 0.05 and fold change above 2 were cho- score for the window using the following equa- sen as significantly regulated and used for fur- tion: z = MeanWindow *SquareRoot (#probes in ther analysis. Matlab was used to analyze window)/ Standard Deviation of Array. mRNA data. Raw data from six microarrays were first normalized followed by a filter to eliminate The probes within these windows were then probes with a maximum intensity less than 100. 221 Am J Transl Res 2012;4(2):219-228 DNA methylation in epigenetic regulation in asthma The gene fold changes between TG and WT MethPrimer website (www. urogene. org/ groups were calculated. Targetscan database methprimer/ index1. html). We searched for was used to predict the targets of miRNAs, and potential primer sites within the 2 kb window only targets with the context+ score less than - where the z-score was calculated for Cxcl3 in 0.35 were considered. The correlations of the MeDIP data analysis. Two sets of primers were miRNAs on gene expression were estimated by designed, one primed for fully methylated DNA computing the z-score, which is the averaged (M primers), while the other primed for fully un- expression fold change of targeted genes be- methylated DNA (U primers). The primer se- tween TG and WT subtracted by the average quences are, methylated forward primer: 5'-AAT expression fold change of all genes, multiplied AGA AAT AAA TGT AC GGG GAT TC-3'; methylated by the square root of the number of targets, and reverse primer: 5'-AAT AGA AAT AAA TGT ACG divided by the standard deviation of the expres- GGG ATT C-3'; unmethylated forward primer: 5'- sion fold change of all genes. DAVID Bioinfor- TAG AAA TAA ATG TAT GGG GAT TTG A-3'; un- matics Resources were used to match microar- methylated reverse primer: 5'- TAGAA ATA AAT ray probe to genes and to obtain gene func- GTA TGG GGA TTT GA-3'. The thermal cycler set- tional annotations. Published data set ting was as follow: 2 minutes at 95°C, 40 cycles (GSE18010) by Tachdjian et al. [27], which of [30 seconds at 95°C, 30 seconds at 53°C, 1 used identical mice treated with the same con- minute at 72°C], 5 minutes at 72°C, hold at 4° ditions, was used to compare and verify our C. Amplified products were visualized on a 2% agarose gel stained with Safe View Classic. Ex- pected sizes for the amplified regions are 185 Quantitative real-time PCR bp for the M primers and 182 bp for the U prim- For the validation of gene expression data by quantitative real-time PCR, TaqMan primers and probes (for Cxcl3) and SYBR Green primers and probes (for Pdgfra, Prkca) were obtained from Identification of methylation patterns that corre- Applied Biosystems. RNA was isolated from left spond with gene expression levels in IL13- upper lobes of the lungs from TG and WT mice induced allergic airway inflammation treated with doxycycline for one week. Standard protocols recommended by Applied Biosystems Gene expression profiling of CC10-raTA-IL13 TG were followed for the experiments. The expres- mouse lung revealed a similar gene signature of sion levels of the candidate genes were com- allergic airway inflammation that has been de- pared to that of Gapdh, which was used as an scribed previously [27]. IL13 induction with endogenous control. The data are presented in doxycycline for 7 days significantly modified ∆CT, which is obtained by subtracting the CT DNA methylation patterns in 177 unique genes, values of Gapdh from the CT values of the can- most of which were associated with the IL13 didate genes. A smaller ∆CT value indicates transcriptional signature in the airways. A list of higher expression level. The PCR reactions were selected genes with differential expression level performed on the Applied Biosystems Step One and DNA methylation status is shown in Table 1. Plus Real-Time PCR System. The z-scores reflect methylation status: a nega- tive number indicates a hypomethylated state; Methylation-specific PCR positive number indicates a methylated state. The z-scores were computed within a 2 kb win- For the validation of MeDIP data, we compared dow that contained 5 or more probes, centered the promoter region of Cxcl3 in TG and WT mice on the probe location listed in the table. by methylation-specific PCR (MS-PCR). Mice were treated the same way as those used in the A large number of genes whose expression was MeDIP experiment, and genomic DNA was ex- induced by IL13 were found to have decreased tracted from the left lower lobes of the lungs. methylation, including those involved in tissue Bisulfite conversion was performed on the ex- remodeling (Olr1), leukocyte influx (Cxcl3, Cxcl5, tracted genomic DNA using the Zymo Research CSFr2b), and the Th2 response (C3ar1, Chi3l4). EZ DNA Methylation-Direct Kit. Bisulfite- Reciprocally, some genes whose expression is converted DNA was used as template for MS- suppressed were found to have increased me- PCR. The primers were designed using the thylation. Among them are Itga8, Rbpms, Bex2, 222 Am J Transl Res 2012;4(2):219-228 DNA methylation in epigenetic regulation in asthma Table 1. Gene expression and promoter methylation profiling in TG and WT mice treated with doxycycline z-score difference and Palld (z-score difference: 1.99, 0.93, 4.37, and 0.68, respectively). Validation of gene expression and DNA pro- moter methylation for Cxcl3 We chose Cxcl3 as a candidate for the valida- tion of gene expression from the microarray data and promoter methylation identified by MeDIP. To validate gene expression, we per- formed quantitative real-time PCR for Cxcl3 us- ing RNA isolated from lungs of 3 TG mice and 3 WT mice treated with doxycycline for a week. We found increased expression of the gene in the lungs of TG mice compared to that of WT mice Figure 1. Data validation of Cxcl3 gene expression (p = 0.0034) (Figure 1A). The real-time PCR and DNA methylation status in WT and TG mice. (A) data thus confirmed the gene expression mi- Gene expression data of Cxcl3 from the Affymetrix 430 2.0 array was validated by quantitative real-time PCR. The box-plot presents the data in ∆CT, which is To validate the methylation status of Cxcl3 pro- the difference between the CT values of Cxcl3 and moter that was determined by MeDIP, we per- Gapdh. A higher ∆CT value indicates lower gene ex- formed bisulfite conversion on the genomic DNA pression. Cxcl3 expression was found to be higher in extracted from 4 TG mice and 4 WT mice, fol- the TG lungs compared to the WT lungs (p = lowed by methylation-specific PCR on the pro- 0.0034), which validated our gene expression array data. (B) Methylation-specific PCR was performed to moter region used for z-score calculation in the validate MeDIP array data for Cxcl3. Panel I shows MeDIP array data. There were more methylated PCR-amplified fragments from WT mice while Panel II CpG sites than unmethylated CpG sites in 3 out shows amplified fragments from TG mice. M col- of 4 WT lungs, whereas all TG lungs had more umns represent amplified products by the M primers unmethylated CpG sites in the same promoter while U columns represent amplified products by the region (Figure 1B). These findings verified the U primers. Three out of 4 WT mice harbored methy- results from MeDIP, which indicated that CpG lated CpGs in the investigated Cxcl3 promoter re- methylation of the Cxcl3 promoter was higher in gion, while all 4 of the TG mice did not. These data the WT than in the TG lungs shown in Table 1. correlate with the MeDIP array data that promoter CpG methylation is lost in the transition from WT These results suggested that IL13 induces me- lungs to IL13-induced TG lungs, which explains the thylation modifications in the genome within a increased Cxcl3 expression in the TG lungs. 223 Am J Transl Res 2012;4(2):219-228 DNA methylation in epigenetic regulation in asthma Table 2. miRNAs with significant differential miRNA in terms of standard deviations away expression (fold change > 2 and q-value < from the mean expression level of all genes on 0.05) and corresponding z-scores for their tar- the array. With the exception of mmu-miR-127, which did not return any target that passed the cutoff of context+ score < -0.35, all 4 of the remaining miRNAs had negative z-scores, indi- mmu-miR-685 2.0244 cating the predicted target genes had lower ex- mmu-miR-379 1.188 pression levels compared to the overall genome mmu-miR-699 1.0953 -wide expression levels (Table 2). Of the target mmu-miR-362 1.0657 genes downregulated by the induced miRNAs, mmu-miR-146b 1.5057 some were involved in translation regulation mmu-miR-127 2.062 (Tnrc6b, El24), transcription regulation (Foxn3, mmu-miR-34a 1.5613 Plagl2), signal transduction (Prkca, Lphn3), and proteolysis (Ube2e3, Dpp8). A list of selected target genes downregulated in TG lungs with q- week of exposure to overexpression of IL13 and values less than 0.05 is shown in Table 3. that this is reflected by a change in the tran- scriptional signature. Validation of miRNA target genes expression Identification of differentially expressed miRNAs We performed quantitative real-time PCR to and their target genes in IL13-induced allergic validate the downregulation of 2 target genes airway inflammation shown in Table 3. Pdgfra and Prkca, both tar- gets of miRNAs induced in TG mice, were cho- Expression of seven miRNAs from the Affymetrix sen as candidates for the validation study. Four GeneChip miRNA array were significantly in- TG mice and four WT mice were sacrificed after duced in the TG lungs compared to WT (Table being treated with doxycycline for 7 days, and 2). Two of the miRNAs examined in the array, RNA was isolated from the upper left lobes of mmu-miR-685 and mmu-miR-699, however, the lungs. From the real-time PCR data, both were discovered to be fragments of RNase P Pdgfra and Prkca showed lower expression lev- RNA and RNase MRP RNA, respectively. These els in the lungs of TG mice compared to WT two miRNAs were since withdrawn from the mice, validating the expression patterns indi- miRBase and were not included in our subse- cated in the Affymetrix 430 2.0 array data quent analysis. Using the Targetscan database, the potential targets of the miRNAs were pre- dicted. Correlating back to the Affymetrix 430 To validate the expression patterns of more tar- 2.0 array for gene expression analysis, we com- get genes, we examined the array dataset pub- puted the z-score for each miRNA, which deter- lished by Tachdjian et al. (GSE18010) [27]. We mined the degree of differential expression of compared the downregulated genes targeted by the predicted target genes for that particular the miRNAs listed in Table 2 with the list gener- Table 3. List of selected genes targeted by miRNAs significantly overexpressed in TG mice. Target gene Fold change (log2) Description of function Cell surface receptor linked signal transduction, neu-ropeptide signaling Wnt signaling pathway, MAPK signaling pathway Lung development, respiratory tube development Translation regulation Posttranslational modification, chaperones Zinc ion binding, transition metal ion binding Cytoskeleton actin filament bundle Proteolysis, catabolic processes Translation regulation 224 Am J Transl Res 2012;4(2):219-228 DNA methylation in epigenetic regulation in asthma Figure 3. Comparison of downregulated genes that were potential targets of overexpressed miRNAs in the TG mice. The blue region shows 2 genes unique to our dataset; the yellow region shows a single gene unique to Tachdjian et al's dataset; the green region shows common genes downregulated in both our datasets that are potential targets of overexpressed miRNAs in the TG mice. Figure 2. Quantitative real-time PCR validating the expression differences between TG and WT lungs for selected genes targeted by miRNAs that were up- on DNA methylation of specific genes in regulated in TG mice. Genes chosen for validation asthma, to our knowledge this is the first ge- study were Pdgfra (targeted by mmu-miR-34a) and nome-wide analysis of DNA methylation status Prkca (targeted by mmu-miR-362). Both of these in experimental asthma, with the results contex- genes were found to be downregulated in the TG tualized by linking them to the pro-asthmatic lungs (p = 0.0006 and 0.0054, respectively), confirm-ing the data from the gene expression array. The ∆CT transcriptional changes induced by IL13 [13, values were calculated by subtracting the CT values of the endogenous control Gapdh from the CT values of the target genes. From our MeDIP array data and the gene ex- pression array data, we found genes with in- creased expression in the TG lungs compared to ated by our dataset (Figure 3). We saw a signifi- the WT lungs that corresponded to the loss of cant overlap of downregulated target genes DNA promoter methylation. A case in point is between the two datasets, with only one the macrophage scavenger receptor 1 (Msr1), uniquely downregulated target gene in which is important in COPD and is upregulated Tachdjian dataset, while 2 uniquely downregu- in smokers [33, 34]. MSR1 is recently shown to lated target genes in our dataset. This analysis be physically interacting with TRAF6 in human showed that in IL13-induced allergic airway in- [35], which is a signal transducer for interleukin- flammation, these genes were consistently 1 (IL1) [36]. IL1 is a pro-inflammatory cytokine downregulated in the lungs, most probably due pivotal in the development of Th2 allergic re- to epigenetic regulation of miRNAs. sponses [37]. Here we showed that Msr1 ex- pression was upregulated during allergic airway inflammation, coordinate with the demethyla- tion of its promoter. We also discovered some In this study, we demonstrate that IL13 expres- genes that were hypermethylated and that cor- sion in the airways induces epigenetic changes, related with decreased gene expression in the involving coordinate changes in the DNA methy- TG lungs. One example is the Itga8 gene, which lation profile of target gene promoters and the is highly expressed in rat alveolar interstitial induction of a miRNA transcriptome, that may fibroblasts under normal conditions [38, 39]. Its play a critical role in the establishment of aller- expression was repressed in the bacterial en- gic airway inflammation and remodeling. Al- dotoxin mouse model of bronchopulmonary dys- though there have been some studies focusing plasia [40]. ITGA8 was also shown to regulate 225 Am J Transl Res 2012;4(2):219-228 DNA methylation in epigenetic regulation in asthma lung morphogenesis by controlling the migration surface catalytic tyrosine kinase receptor that and adhesion of mesenchymal cells [40]. Fur- has intracellular activity. The platelet-derived thermore, CpG methylation of the Itga8 pro- growth factor (PDGF) signaling pathway is im- moter has been described in some cases of portant in lung development, especially lung human ovarian cancer [41]. Our data suggests growth and the formation of alveoli [47, 48]. that Itga8 expression is repressed in asthma via SNPs in the promoter region of human PDGFRA DNA promoter methylation, leading to dysregu- have been linked to the severity of childhood lated lung morphogenesis. Overall, our results asthma [49]. We showed here that Prkca and implicate epigenetic methylation changes in the Pdgfra expression were decreased in IL13- coordination and consolidation of IL13- induced allergic airway inflammation via miRNA regulated transcriptional circuits involving both regulation. Thus, our results further implicate induced and repressed genes. miRNA induction as yet another epigenetic mechanism by which IL13 controls gene expres- Another aspect of epigenetic regulation, the sion circuitries. expression of miRNAs, was also examined in this study. There are a few published studies on In summary, we have found that IL13 expres- miRNAs in asthma using different methodolo- sion in the airways is associated with coordinate gies and producing different results. In one changes in the methylation status of promoters study in which a real-time PCR approach was of a large number of gene components of the employed to investigate the expression of 227 IL13 transcriptome, consistent with an epige- miRNAs in airway biopsies procured from nor- netic regulatory function of gene methylation in mal subjects and patients with mild asthma, no allergic airway inflammation. We similarly found differential miRNA expression was found [42]. miRNAs with predicted targets for genes that Lu et al. used the same doxycycline inducible are important in maintaining expression of tran- IL13 transgenic mice but examined miRNA ex- scriptional circuitries associated with chronic pression after 28 days of doxycycline on a differ- airway disease. Other studies have also impli- ent microarray platform. This study found 21 cated additional mechanisms, such as histone miRNAs that were differentially expressed in the modifications, in promoting gene expression doxycycline treated versus untreated mice [43]. changes in asthma [18, 50]. These findings In our study, we compared miRNA expression point to the involvement of multiple mecha- levels in doxycycline-treated inducible IL13 nisms of epigenetic regulation in the establish- transgenic mice as compared to wild type syn- ment of allergic airway inflammation and in the geneic mice due to leakiness of the rtTA-CMV evolution of its chronic phenotypes and mani- cassette driving IL13 expression. After correlat- festations. Overall, our studies and those from ing miRNA expression with mRNA expression, other groups, suggest that interventions that we found that only one miRNA, mir146b, over- manipulate the epigenome of the asthmatic lapped with the miRNAs identified by Lu et al. lung may provide highly effective therapies in We examined miRNAs whose expression was induced in the TG lungs and identified their pre- dicted target genes that were downregulated in the disease state. From these data, we com- piled a list of miRNA-regulated genes that play a We thank Dr. Jack Elias for the provision of the role in the development of allergic airway in- CC10-rtTA-IL13 mice. This work was supported by NIH grant R01 HL094561-01 (to BG) and 2R01AI065617-11A1 and U19 AI070453 (to One example of a gene regulated by an IL13- induced miRNA is Prkca, encoding protein kinase C alpha. This protein kinase is involved Address correspondence to: Dr. Brigitte N. Gomperts, in various biological pathways within the cell, David Geffen School of Medicine at UCLA, Depart- including apoptosis, cell cycle regulation, prolif- ment of Pediatrics, 10833 Le Conte Ave., A2- eration, cell motility, and cell morphology [44, 410MDCC, Los Angeles, CA 90095 Tel: (310) 825- 45]. Single-nucleotide polymorphisms (SNPs) 6708; E-mail: [email protected] within the PRKCA gene in humans is signifi- cantly associated with asthma [46]. Another miRNA-targeted gene is Pdgfra, encoding plate- [1] Cantani A, Micera M. A study on 300 asthmatic let-derived growth factor receptor alpha, a cell children, 300 controls and their parents con- 226 Am J Transl Res 2012;4(2):219-228 DNA methylation in epigenetic regulation in asthma firms the genetic transmission of allergy and bam B, Harris RA, Coarfa C, Zariff A, asthma. Eur Rev Med Pharmacol Sci 2011; 15: Milosavljevic A, Batts LM, Kheradmand F, Gunaratne PH and Corry DB. Proinflammatory [2] Kurzius-Spencer M, Guerra S, Sherrill DL, Ha- role for let-7 microRNAS in experimental lonen M, Elston RC and Martinez FD. Familial asthma. J Biol Chem 2010; 285: 30139- aggregation of allergen-specific sensitization and asthma. Pediatr Allergy Immunol 2012; 23: [17] Agarwal S, Rao A. Modulation of chromatin structure regulates cytokine gene expression [3] Miller RL, Garfinkel R, Horton M, Camann D, during T cell differentiation. Immunity 1998; 9: Perera FP, Whyatt RM and Kinney PL. Polycyclic aromatic hydrocarbons, environmental tobacco [18] Adcock IM, Tsaprouni L, Bhavsar P and Ito K. smoke, and respiratory symptoms in an inner- Epigenetic regulation of airway inflammation. city birth cohort. Chest 2004; 126: 1071-1078. Curr Opin Immunol 2007; 19: 694-700. [4] Miller RL, Ho SM. Environmental epigenetics [19] McKenzie AN, Culpepper JA, de Waal Malefyt R, and asthma: current concepts and call for stud- Briere F, Punnonen J, Aversa G, Sato A, Dang ies. Am J Respir Crit Care Med 2008; 177: 567- W, Cocks BG, Menon S, de Vries JE, Banchereau J, Zurawski G. Interleukin 13, a T- [5] Hamada K, Suzaki Y, Leme A, Ito T, Miyamoto cell-derived cytokine that regulates human K, Kobzik L and Kimura H. Exposure of preg- monocyte and B-cell function. Proc Natl Acad nant mice to an air pollutant aerosol increases Sci USA 1993; 90: 3735-3739. asthma susceptibility in offspring. J Toxicol [20] Minty A, Chalon P, Derocq JM, Dumont X, Guille- Environ Health A 2007; 70: 688-695. mot JC, Kaghad M, Labit C, Leplatois P, Liauzun [6] Bollati V, Baccarelli A. Environmental epigenet- P, Miloux B, Casellas P, Loison G, Lupker J, ics. Heredity (Edinb) 2010; 105: 105-112. Shire D, Ferrara P, Caput D. Interleukin-13 is a [7] Weber M, Hellmann I, Stadler MB, Ramos L, new human lymphokine regulating inflamma- Paabo S, Rebhan M and Schubeler D. Distribu- tory and immune responses. Nature 1993; tion, silencing potential and evolutionary im- pact of promoter DNA methylation in the hu- [21] Wynn TA. IL-13 effector functions. Annu Rev man genome. Nat Genet 2007; 39: 457-466. Immunol 2003; 21: 425-456. [8] Reik W, Dean W. DNA methylation and mam- [22] Kotsimbos TC, Ernst P and Hamid QA. Inter- malian epigenetics. Electrophoresis 2001; 22: leukin-13 and interleukin-4 are coexpressed in atopic asthma. Proc Assoc Am Physicians [9] Kabesch M, Michel S and Tost J. Epigenetic 1996; 108: 368-373. mechanisms and the relationship to childhood [23] Wills-Karp M, Luyimbazi J, Xu X, Schofield B, asthma. Eur Respir J 2010; 36: 950-961. Neben TY, Karp CL and Donaldson DD. Inter- [10] Jones B, Chen J. Inhibition of IFN-gamma tran- leukin-13: central mediator of allergic asthma. scription by site-specific methylation during T Science 1998; 282: 2258-2261. helper cell development. EMBO J 2006; 25: [24] Zhu Z, Homer RJ, Wang Z, Chen Q, Geba GP, Wang J, Zhang Y and Elias JA. Pulmonary ex- [11] Barnes PJ. Th2 cytokines and asthma: an intro- pression of interleukin-13 causes inflamma- duction. Respir Res 2001; 2: 64-65. tion, mucus hypersecretion, subepithelial fibro- [12] Shapiro SD, Owen CA. ADAM-33 surfaces as an sis, physiologic abnormalities, and eotaxin pro- asthma gene. N Engl J Med 2002; 347: 936- duction. J Clin Invest 1999; 103: 779-788. [25] Follettie MT, Ellis DK, Donaldson DD, Hill AA, [13] Yang Y, Haitchi HM, Cakebread J, Sammut D, Diesl V, DeClercq C, Sypek JP, Dorner AJ and Harvey A, Powell RM, Holloway JW, Howarth P, Wills-Karp M. Gene expression analysis in a Holgate ST and Davies DE. Epigenetic mecha- murine model of allergic asthma reveals over- nisms silence a disintegrin and metalloprote- lapping disease and therapy dependent path- ase 33 expression in bronchial epithelial cells. J ways in the lung. Pharmacogenomics J 2006; Allergy Clin Immunol 2008; 121: 1393-1399, 1399 e1391-1314. [26] Fulkerson PC, Fischetti CA, Hassman LM, Niko- [14] Bartel DP. MicroRNAs: genomics, biogenesis, laidis NM and Rothenberg ME. Persistent ef- mechanism, and function. Cell 2004; 116: 281 fects induced by IL-13 in the lung. Am J Respir Cell Mol Biol 2006; 35: 337-346. [15] Garbacki N, Di Valentin E, Huynh-Thu VA, [27] Tachdjian R, Mathias C, Al Khatib S, Bryce PJ, Geurts P, Irrthum A, Crahay C, Arnould T, Kim HS, Blaeser F, O'Connor BD, Rzymkiewicz Deroanne C, Piette J, Cataldo D and Colige A. D, Chen A, Holtzman MJ, Hershey GK, Garn H, MicroRNAs profiling in murine models of acute Harb H, Renz H, Oettgen HC and Chatila TA. and chronic asthma: a relationship with mRNAs Pathogenicity of a disease-associated human IL targets. PLoS One 2011; 6: e16509. -4 receptor allele in experimental asthma. J Exp [16] Polikepahad S, Knight JM, Naghavi AO, Oplt T, Med 2009; 206: 2191-2204. Creighton CJ, Shaw C, Benham AL, Kim J, Soi- [28] Zimmermann N, Doepker MP, Witte DP, 227 Am J Transl Res 2012;4(2):219-228 DNA methylation in epigenetic regulation in asthma Stringer KF, Fulkerson PC, Pope SM, Brandt EB, smooth muscle cells. J Cell Sci 1995; 108: 537 Mishra A, King NE, Nikolaidis NM, Wills-Karp M, Finkelman FD and Rothenberg ME. Expression [40] Benjamin JT, Gaston DC, Halloran BA, Schnapp and regulation of small proline-rich protein 2 in LM, Zent R and Prince LS. The role of integrin allergic inflammation. Am J Respir Cell Mol Biol alpha8beta1 in fetal lung morphogenesis and 2005; 32: 428-435. injury. Dev Biol 2009; 335: 407-417. [29] Zheng T, Zhu Z, Wang Z, Homer RJ, Ma B, Riese [41] Cai LY, Abe M, Izumi S, Imura M, Yasugi T and RJ Jr, Chapman HA Jr, Shapiro SD and Elias JA. Ushijima T. Identification of PRTFDC1 silencing Inducible targeting of IL-13 to the adult lung and aberrant promoter methylation of GPR150, causes matrix metalloproteinase- and cathep- ITGA8 and HOXD11 in ovarian cancers. Life Sci sin-dependent emphysema. J Clin Invest 2000; 2007; 80: 1458-1465. [42] Williams AE, Larner-Svensson H, Perry MM, [30] Li N, Ye M, Li Y, Yan Z, Butcher LM, Sun J, Han Campbell GA, Herrick SE, Adcock IM, Erjefalt JS, X, Chen Q, Zhang X and Wang J. Whole genome Chung KF and Lindsay MA. MicroRNA expres- DNA methylation analysis based on high sion profiling in mild asthmatic human airways throughput sequencing technology. Methods and effect of corticosteroid therapy. PLoS One 2010; 52: 203-212. [31] Tusher VG, Tibshirani R and Chu G. Significance [43] Lu TX, Munitz A and Rothenberg ME. MicroRNA- analysis of microarrays applied to the ionizing 21 is up-regulated in allergic airway inflamma- radiation response. Proc Natl Acad Sci USA tion and regulates IL-12p35 expression. J Im- 2001; 98: 5116-5121. munol 2009; 182: 4994-5002. [32] Koppelman GH, Nawijn MC. Recent advances [44] Nishizuka Y. Protein kinase C and lipid signaling in the epigenetics and genomics of asthma. for sustained cellular responses. FASEB J Curr Opin Allergy Clin Immunol 2011; 11: 414- 1995; 9: 484-496. [45] Michie AM, Nakagawa R. The link between [33] Heguy A, O'Connor TP, Luettich K, Worgall S, PKCalpha regulation and cellular transforma- Cieciuch A, Harvey BG, Hackett NR and Crystal tion. Immunol Lett 2005; 96: 155-162. RG. Gene expression profiling of human alveo- [46] Murphy A, Tantisira KG, Soto-Quiros ME, Avila L, lar macrophages of phenotypically normal Klanderman BJ, Lake S, Weiss ST and Celedon smokers and nonsmokers reveals a previously JC. PRKCA: a positional candidate gene for unrecognized subset of genes modulated by body mass index and asthma. Am J Hum Genet cigarette smoking. J Mol Med (Berl) 2006; 84: 2009; 85: 87-96. [47] Bostrom H, Gritli-Linde A and Betsholtz C. PDGF [34] Ohar JA, Hamilton RF Jr, Zheng S, Sadeghnejad -A/PDGF alpha-receptor signaling is required for A, Sterling DA, Xu J, Meyers DA, Bleecker ER lung growth and the formation of alveoli but not and Holian A. COPD is associated with a macro- for early lung branching morphogenesis. Dev phage scavenger receptor-1 gene sequence Dyn 2002; 223: 155-162. variation. Chest 2010; 137: 1098-1107. [48] Souza P, Kuliszewski M, Wang J, Tseu I, Tan- [35] Yu X, Yi H, Guo C, Zuo D, Wang Y, Kim HL, Sub- swell AK and Post M. PDGF-AA and its receptor jeck JR and Wang XY. Pattern recognition scav- influence early lung branching via an epithelial- enger receptor CD204 attenuates Toll-like re- mesenchymal interaction. Development 1995; ceptor 4-induced NF-kappaB activation by di- rectly inhibiting ubiquitination of tumor necrosis [49] Wu LS, Tan CY, Wang LM, Lin CG and Wang JY. factor (TNF) receptor-associated factor 6. J Biol Variant in promoter region of platelet-derived Chem 2011; 286: 18795-18806. growth factor receptor-alpha (PDGFRalpha) [36] Cao Z, Xiong J, Takeuchi M, Kurama T and gene is associated with the severity and allergic Goeddel DV. TRAF6 is a signal transducer for status of childhood asthma. Int Arch Allergy interleukin-1. Nature 1996; 383: 443-446. Immunol 2006; 141: 37-46. [37] Schmitz N, Kurrer M and Kopf M. The IL-1 re- [50] Durham AL, Wiegman C and Adcock IM. Epige- ceptor 1 is critical for Th2 cell type airway im- netics of asthma. Biochim Biophys Acta 2011; mune responses in a mild but not in a more 1810: 1103-1109. severe asthma model. Eur J Immunol 2003; 33: 991-1000. [38] Levine D, Rockey DC, Milner TA, Breuss JM, Fallon JT and Schnapp LM. Expression of the integrin alpha8beta1 during pulmonary and hepatic fibrosis. Am J Pathol 2000; 156: 1927-1935. [39] Schnapp LM, Breuss JM, Ramos DM, Sheppard D and Pytela R. Sequence and tissue distribu-tion of the human integrin alpha 8 subunit: a beta 1-associated alpha subunit expressed in 228 Am J Transl Res 2012;4(2):219-228

Source: http://www.ajtr.us/files/AJTR1202002.pdf


1.name five causes of distended uterus. 11.what is the active management of third stage of labor? i.v. ergometrine is given following the birth of anterior shoulder. the placenta is delivered by the controlled cord traction soon after the endometrial polyp delivery of the baby availing first uterine contraction. if the first attempt fails, another attempt is made after 2-3 minutes


Diagnostic criteria for cervical dystonia: Can botulinum neurotoxin manage, as well as, cure the problem? Jill L. Ostrem, MD Professor of Neurology UCSF Department of Neurology Movement Disorder and Neuromodulation Center Bachmann Strauss Dystonia and Parkinson's Disease Center of Excellence