Comprehensive analysis of the transcriptional response of human decidual cells to lipopolysaccharide stimulation.

Decidual cells are central to innate immunity at the maternal/fetal interface. We sought to characterize the response of decidual cells to stimulation and then removal of lipopolysaccharide (LPS) using a whole genome approach. Decidual cells were isolated from term unlabored cesarean sections. Cells were stimulated with LPS and RNA isolated both pre-stimulation and 2 and 24 h post-stimulation. Media were changed and RNA extracted 48 h later. Gene expression was measured using Agilent 44K whole genome microarrays. Data were visualized and interpreted using Ingenuity Pathway Analysis (IPA) software and selected (n=5) target gene expression was verified with quantitative real-time PCR. Genes related to immune function were up-regulated at 2 and 24 h after LPS exposure and then generally returned to baseline or were at least substantially reduced after LPS removal. Pathway analysis also revealed that genes involved in lipid metabolism (specifically cholesterol and steroid biosynthesis), iron metabolism, and the plasminogen system were coordinately altered following exposure to LPS. Our novel, preliminary findings provide insight into possible mechanisms via which the host inflammatory response could contribute to preterm birth and warrant further investigation in preterm samples.

High throughput determination of TGFß1/SMAD3 targets in A549 lung epithelial cells.

BACKGROUND: Transforming growth factor beta 1 (TGFß1) plays a major role in many lung diseases including lung cancer, pulmonary hypertension, and pulmonary fibrosis. TGFß1 activates a signal transduction cascade that results in the transcriptional regulation of genes in the nucleus, primarily through the DNA-binding transcription factor SMAD3. The objective of this study is to identify genome-wide scale map of SMAD3 binding targets and the molecular pathways and networks affected by the TGFß1/SMAD3 signaling in lung epithelial cells. METHODOLOGY: We combined chromatin immunoprecipitation with human promoter region microarrays (ChIP-on-chip) along with gene expression microarrays to study global transcriptional regulation of the TGFß1/SMAD3 pathway in human A549 alveolar epithelial cells. The molecular pathways and networks associated with TGFß1/SMAD3 signaling were identified using computational approaches. Validation of selected target gene expression and direct binding of SMAD3 to promoters were performed by quantitative real time RT-PCR and electrophoretic mobility shift assay on A549 and human primary lung epithelial cells. RESULTS AND CONCLUSIONS: Known TGFß1 target genes such as SERPINE1, SMAD6, SMAD7, TGFB1 and LTBP3, were found in both ChIP-on-chip and gene expression analyses as well as some previously unrecognized targets such as FOXA2. SMAD3 binding of FOXA2 promoter and changed expression were confirmed. Computational approaches combining ChIP-on-chip and gene expression microarray revealed multiple target molecular pathways affected by the TGFß1/SMAD3 signaling. Identification of global targets and molecular pathways and networks associated with TGFß1/SMAD3 signaling allow for a better understanding of the mechanisms that determine epithelial cell phenotypes in fibrogenesis and carcinogenesis as does the discovery of the direct effect of TGFß1 on FOXA2.

Structural and regulatory characterization of the placental epigenome at its maternal interface.

Epigenetics can be loosely defined as the study of cellular "traits" that influence biological phenotype in a fashion that is not dependent on the underlying primary DNA sequence. One setting in which epigenetics is likely to have a profound influence on biological phenotype is during intrauterine development. In this context there is a defined and critical window during which balanced homeostasis is essential for normal fetal growth and development. We have carried out a detailed structural and functional analysis of the placental epigenome at its maternal interface. Specifically, we performed genome wide analysis of DNA methylation in samples of chorionic villus (CVS) and maternal blood cells (MBC) using both commercially available and custom designed microarrays. We then compared these data with genome wide transcription data for the same tissues. In addition to the discovery that CVS genomes are significantly more hypomethylated than their MBC counterparts, we identified numerous tissue-specific differentially methylated regions (T-DMRs). We further discovered that these T-DMRs are clustered spatially along the genome and are enriched for genes with tissue-specific biological functions. We identified unique patterns of DNA methylation associated with distinct genomic structures such as gene bodies, promoters and CpG islands and identified both direct and inverse relationships between DNA methylation levels and gene expression levels in gene bodies and promoters respectively. Furthermore, we found that these relationships were significantly associated with CpG content. We conclude that the early gestational placental DNA methylome is highly organized and is significantly and globally associated with transcription. These data provide a unique insight into the structural and regulatory characteristics of the placental epigenome at its maternal interface and will drive future analyses of the role of placental dysfunction in gestational disease.

Inhibition and role of let-7d in idiopathic pulmonary fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal fibrotic lung disease characterized by profound changes in epithelial cell phenotype and fibroblast proliferation. OBJECTIVES: To determine changes in expression and role of microRNAs in IPF. METHODS: RNA from 10 control and 10 IPF tissues was hybridized on Agilent microRNA microarrays and results were confirmed by quantitative real-time polymerase chain reaction and in situ hybridization. SMAD3 binding to the let-7d promoter was confirmed by chromatin immunoprecipitation, electrophoretic mobility shift assay, luciferase assays, and reduced expression of let-7d in response to transforming growth factor-beta. HMGA2, a let-7d target, was localized by immunohistochemistry. In mice, let-7d was inhibited by intratracheal administration of a let-7d antagomir and its effects were determined by immunohistochemistry, immunofluorescence, quantitative real-time polymerase chain reaction, and morphometry. MEASUREMENTS AND MAIN RESULTS: Eighteen microRNAs including let-7d were significantly decreased in IPF. Transforming growth factor-beta down-regulated let-7d expression, and SMAD3 binding to the let-7d promoter was demonstrated. Inhibition of let-7d caused increases in mesenchymal markers N-cadherin-2, vimentin, and alpha-smooth muscle actin (ACTA2) as well as HMGA2 in multiple epithelial cell lines. let-7d was significantly reduced in IPF lungs and the number of epithelial cells expressing let-7d correlated with pulmonary functions. HMGA2 was increased in alveolar epithelial cells of IPF lungs. let-7d inhibition in vivo caused alveolar septal thickening and increases in collagen, ACTA2, and S100A4 expression in SFTPC (pulmonary-associated surfactant protein C) expressing alveolar epithelial cells. CONCLUSIONS: Our results indicate a role for microRNAs in IPF. The down-regulation of let-7d in IPF and the profibrotic effects of this down-regulation in vitro and in vivo suggest a key regulatory role for this microRNA in preventing lung fibrosis. Clinical trial registered with www.clinicaltrials.gov (NCT 00258544).

Evidence of systematic expressed sequence tag IMAGE clone cross-hybridization on cDNA microarrays.

We present evidence of a potentially serious source of error intrinsic to all spotted cDNA microarrays that use IMAGE clones of expressed sequence tags (ESTs). We found that a high proportion of these EST sequences contain 5'-end poly(dT) sequences that are remnants from the oligo(dT)-primed reverse transcription of polyadenylated mRNA templates used to generate EST cDNA for sequence clone libraries. Analysis of expression data from two single-dye cDNA microarray experiments showed that ESTs whose sequences contain repeats of consecutive 5'-end dT residues appeared to be strongly coexpressed, while expression data of all other sequences exhibited no such pattern. Our analysis suggests that expression data from sequences containing 5' poly(dT) tracts are more likely to be due to systematic cross-hybridization of these poly(dT) tracts than to true mRNA coexpression. This indicates that existing data generated by cDNA microarrays containing IMAGE clone ESTs should be filtered to remove expression data containing significant 5' poly(dT) tracts.

Concerns about unreliable data from spotted cDNA microarrays due to cross-hybridization and sequence errors.

We discuss our concerns regarding the reliability of data generated by spotted cDNA microarrays. Two types of error we highlight are cross-hybridization artifact due to sequence homologies and sequence errors in the cDNA used for spotting on microarrays. We feel that statisticians who analyze microarray data should be aware of these sources of unreliability intrinsic to cDNA microarray design and use.