The RNA binding protein HuR differentially regulates unique subsets of mRNAs in estrogen receptor negative and estrogen receptor positive breast cancer

<p>Abstract</p> <p>Background</p> <p>The discordance between steady-state levels of mRNAs and protein has been attributed to posttranscriptional control mechanisms affecting mRNA stability and translation. Traditional methods of genome wide microarray analysis, profiling steady-state levels of mRNA, may miss important mRNA targets owing to significant posttranscriptional gene regulation by RNA binding proteins (RBPs).</p> <p>Methods</p> <p>The ribonomic approach, utilizing RNA immunoprecipitation hybridized to microarray (RIP-Chip), provides global identification of putative endogenous mRNA targets of different RBPs. HuR is an RBP that binds to the AU-rich elements (ARE) of labile mRNAs, such as proto-oncogenes, facilitating their translation into protein. HuR has been shown to play a role in cancer progression and elevated levels of cytoplasmic HuR directly correlate with increased invasiveness and poor prognosis for many cancers, including those of the breast. HuR has been described to control genes in several of the acquired capabilities of cancer and has been hypothesized to be a tumor-maintenance gene, allowing for cancers to proliferate once they are established.</p> <p>Results</p> <p>We used HuR RIP-Chip as a comprehensive and systematic method to survey breast cancer target genes in both MCF-7 (estrogen receptor positive, ER+) and MDA-MB-231 (estrogen receptor negative, ER-) breast cancer cell lines. We identified unique subsets of HuR-associated mRNAs found individually or in both cell types. Two novel HuR targets, <it>CD9 </it>and <it>CALM2 </it>mRNAs, were identified and validated by quantitative RT-PCR and biotin pull-down analysis.</p> <p>Conclusion</p> <p>This is the first report of a side-by-side genome-wide comparison of HuR-associated targets in wild type ER+ and ER- breast cancer. We found distinct, differentially expressed subsets of cancer related genes in ER+ and ER- breast cancer cell lines, and noted that the differential regulation of two cancer-related genes by HuR was contingent upon the cellular environment.</p

Selective accumulation of langerhans-type dendritic cells in small airways of patients with COPD

<p>Abstract</p> <p>Background</p> <p>Dendritic cells (DC) linking innate and adaptive immune responses are present in human lungs, but the characterization of different subsets and their role in COPD pathogenesis remain to be elucidated. The aim of this study is to characterize and quantify pulmonary myeloid DC subsets in small airways of current and ex-smokers with or without COPD.</p> <p>Methods</p> <p>Myeloid DC were characterized using flowcytometry on single cell suspensions of digested human lung tissue. Immunohistochemical staining for langerin, BDCA-1, CD1a and DC-SIGN was performed on surgical resection specimens from 85 patients. Expression of factors inducing Langerhans-type DC (LDC) differentiation was evaluated by RT-PCR on total lung RNA.</p> <p>Results</p> <p>Two segregated subsets of tissue resident pulmonary myeloid DC were identified in single cell suspensions by flowcytometry: the langerin+ LDC and the DC-SIGN+ interstitial-type DC (intDC). LDC partially expressed the markers CD1a and BDCA-1, which are also present on their known blood precursors. In contrast, intDC did not express langerin, CD1a or BDCA-1, but were more closely related to monocytes.</p> <p>Quantification of DC in the small airways by immunohistochemistry revealed a higher number of LDC in current smokers without COPD and in COPD patients compared to never smokers and ex-smokers without COPD. Importantly, there was no difference in the number of LDC between current and ex-smoking COPD patients.</p> <p>In contrast, the number of intDC did not differ between study groups. Interestingly, the number of BDCA-1+ DC was significantly lower in COPD patients compared to never smokers and further decreased with the severity of the disease. In addition, the accumulation of LDC in the small airways significantly correlated with the expression of the LDC inducing differentiation factor activin-A.</p> <p>Conclusions</p> <p>Myeloid DC differentiation is altered in small airways of current smokers and COPD patients resulting in a selective accumulation of the LDC subset which correlates with the pulmonary expression of the LDC-inducing differentiation factor activin-A. This study identified the LDC subset as an interesting focus for future research in COPD pathogenesis.</p

Role of the tachykinin NK1 receptor in a murine model of cigarette smoke-induced pulmonary inflammation

<p>Abstract</p> <p>Background</p> <p>The tachykinins, substance P and neurokinin A, present in sensory nerves and inflammatory cells such as macrophages and dendritic cells, are considered as pro-inflammatory agents. Inflammation of the airways and lung parenchyma plays a major role in the pathogenesis of chronic obstructive pulmonary disease (COPD) and increased tachykinin levels are recovered from the airways of COPD patients. The aim of our study was to clarify the involvement of the tachykinin NK₁receptor, the preferential receptor for substance P, in cigarette smoke (CS)-induced pulmonary inflammation and emphysema in a mouse model of COPD.</p> <p>Methods</p> <p>Tachykinin NK₁receptor knockout (NK₁-R^-/-) mice and their wild type controls (all in a mixed 129/sv-C57BL/6 background) were subjected to sub acute (4 weeks) or chronic (24 weeks) exposure to air or CS. 24 hours after the last exposure, pulmonary inflammation and development of emphysema were evaluated.</p> <p>Results</p> <p>Sub acute and chronic exposure to CS resulted in a substantial accumulation of inflammatory cells in the airways of both WT and NK₁-R^-/-mice. However, the CS-induced increase in macrophages and dendritic cells was significantly impaired in NK₁-R^-/-mice, compared to WT controls, and correlated with an attenuated release of MIP-3α/CCL20 and TGF-β1. Chronic exposure to CS resulted in development of pulmonary emphysema in WT mice. NK₁-R^-/-mice showed already enlarged airspaces upon air-exposure. Upon CS-exposure, the NK₁-R^-/-mice did not develop additional destruction of the lung parenchyma. Moreover, an impaired production of MMP-12 by alveolar macrophages upon CS-exposure was observed in these KO mice. In a pharmacological validation experiment using the NK₁receptor antagonist RP 67580, we confirmed the protective effect of absence of the NK₁receptor on CS-induced pulmonary inflammation.</p> <p>Conclusion</p> <p>These data suggest that the tachykinin NK₁receptor is involved in the accumulation of macrophages and dendritic cells in the airways upon CS-exposure and in the development of smoking-induced emphysema. As both inflammation of the airways and parenchymal destruction are important characteristics of COPD, these findings may have implications in the future treatment of this devastating disease.</p

Alternative polyadenylation variants of the RNA binding protein, HuR: abundance, role of AU-rich elements and auto-Regulation

The RNA-binding protein, HuR, is involved in the stabilization of AU-rich element-containing mRNAs with products that are involved in cell-cycle progression, cell differentiation and inflammation. We show that there are multiple polyadenylation variants of HuR mRNA that differ in their abundance, using both bioinformatics and experimental approaches. A polyadenylation variant with distal poly(A) signal is a rare transcript that harbors functional AU-rich elements (ARE) in the 3′UTR. A minimal 60-nt region, but not a mutant form, fused to reporter-3′UTR constructs was able to downregulate the reporter activity. The most predominant and alternatively polyadenylated mature transcript does not contain the ARE. HuR itself binds HuR mRNA, and upregulated the activity of reporter from constructs fused with ARE-isoform and the HuR ARE. Wild-type tristetraprolin (TTP), but not the zinc finger mutant TTP, competes for HuR binding and upregulation of HuR mRNA. The study shows that the HuR gene codes for several polyadenylation variants differentially regulated by AU-rich elements, and demonstrates an auto-regulatory role of HuR

Epithelial Posttranscriptional Gene Regulatory Networks In Chronic Airway Inflammation: In Silico Mapping Of RNA-Binding Protein Expression

Background : Altered messenger RNA (mRNA) turnover and transla-tion rates are important mechanisms by which post- transcriptional gene regulation (PTR) contributes to inflammation. RNA- binding proteins (RBPs) chiefly coordinate these processes but their path-ogenic role in chronic lung inflammatory diseases is only partially characterized. We aimed at evaluating the expression of a curated list of mRNA- binding RBPs (mRBPs) [Nature 2014;15:829] in selected transcriptomic GEO databases of primary airway epithelium isolated in lung inflammatory diseases. We hypothesized that global changes in mRBP expression can be used to infer their putative pathogenetic roles and identify novel disease- related regulatory networks. Method : We evaluated the expression of 692 mRBPs in a microarray database generated from epithelial cells obtained by bronchial brush-ings of stable COPD patients (C), smokers (S) and non- smokers (NS) as controls with normal lung function (n = 6/12/12 each, respectively) [Cancer Res. 2006;66:10729] deposited in the Gene Expression Omnibus (GEO) repository (GEO ID: GSE5058). Fluorescence inten-sity data from individual datasets were extracted and normalized by the medians for fold change (FC) expression among groups. FCs were set at ≥ |2.0|0.0 with a false discovery rate (FDR) of ≤ 0.05. Pearson correlation matrices for correlated expression changes and heatmaps were generated using tMEV tools v4_9_0.45. Gene Ontology (GO) was performed with Ingenuity Pathway Analysis (IPA) software. Results : Significant mRBP gene expression changes were detected in S vs NS, COPD vs NS and COPD vs S comparisons (n genes = 249, 464 and 445, respectively). Genes with FC ≥ |2.0| constituted 16% of those detected in S vs NS and more than 40% in COPD vs NS and COPD vs S (n = 40, 214 and 186, respectively). Interestingly, the ma-jority of these genes were downregulated in COPD vs NS (n = 137, 64%) and COPD vs S (n = 150, 80%) while only 17% were down-regulated in S vs NS (n = 7). Correlation analysis identified discrete clusters of co- expressed genes. GO analysis revealed significant en-richments in canonical pathways both specific and shared across the comparisons. Conclusion : The novel characterization of mRBPs expression in air-way epithelium and further definition of their functional impact is necessary to understand how PTR contributes to chronic inflamma-tory lung disease and whether it can be targeted therapeutically

Epithelial posttranscriptional gene regulatory networks in chronic airway inflammation: In silico mapping of RNA-binding protein expression

Background: Altered messenger RNA (mRNA) turnover and translation rates are important mechanisms by which post‐transcriptional gene regulation (PTR) contributes to inflammation. RNA‐binding proteins (RBPs) chiefly coordinate these processes but their pathogenic role in chronic lung inflammatory diseases is only partially characterized. We aimed at evaluating the expression of a curated list of mRNA‐binding RBPs (mRBPs) [Nature 2014;15:829] in selected transcriptomic GEO databases of primary airway epithelium isolated in lung inflammatory diseases. We hypothesized that global changes in mRBP expression can be used to infer their putative pathogenetic roles and identify novel disease‐related regulatory networks. Method: We evaluated the expression of 692 mRBPs in a microarray database generated from epithelial cells obtained by bronchial brushings of stable COPD patients (C), smokers (S) and non‐smokers (NS) as controls with normal lung function (n = 6/12/12 each, respectively) [Cancer Res. 2006;66:10729] deposited in the Gene Expression Omnibus (GEO) repository (GEO ID: GSE5058). Fluorescence intensity data from individual datasets were extracted and normalized by the medians for fold change (FC) expression among groups. FCs were set at ≥ |2.0|0.0 with a false discovery rate (FDR) of ≤ 0.05. Pearson correlation matrices for correlated expression changes and heatmaps were generated using tMEV tools v4_9_0.45. Gene Ontology (GO) was performed with Ingenuity Pathway Analysis (IPA) software. Results: Significant mRBP gene expression changes were detected in S vs NS, COPD vs NS and COPD vs S comparisons (n genes = 249, 464 and 445, respectively). Genes with FC ≥ |2.0| constituted 16% of those detected in S vs NS and more than 40% in COPD vs NS and COPD vs S (n = 40, 214 and 186, respectively). Interestingly, the majority of these genes were downregulated in COPD vs NS (n = 137, 64%) and COPD vs S (n = 150, 80%) while only 17% were downregulated in S vs NS (n = 7). Correlation analysis identified discrete clusters of co‐expressed genes. GO analysis revealed significant enrichments in canonical pathways both specific and shared across the comparisons. Conclusion: The novel characterization of mRBPs expression in airway epithelium and further definition of their functional impact is necessary to understand how PTR contributes to chronic inflammatory lung disease and whether it can be targeted therapeutically