title: High throughput determination of TGFβ1/SMAD3 targets in A549 lung epithelial cells.
abstract: BACKGROUND
  Transforming growth factor beta 1 (TGFβ1) plays a major role in many lung diseases including lung cancer, pulmonary hypertension, and pulmonary fibrosis. TGFβ1 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 TGFβ1/SMAD3 signaling in lung epithelial cells.
  
  
  METHODS
  We combined chromatin immunoprecipitation with human promoter region microarrays (ChIP-on-chip) along with gene expression microarrays to study global transcriptional regulation of the TGFβ1/SMAD3 pathway in human A549 alveolar epithelial cells. The molecular pathways and networks associated with TGFβ1/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.
  
  
  CONCLUSIONS
  Known TGFβ1 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 TGFβ1/SMAD3 signaling. Identification of global targets and molecular pathways and networks associated with TGFβ1/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 TGFβ1 on FOXA2.
authors.affiliation: Division of Pulmonary, Allergy and Critical Care Medicine, Richard P. and Dorothy P. Simmons Center for Interstitial Lung Disease, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America. zhangy@upmc.edu
authors.forename: Yingze
authors.initials: Y
authors.lastname: Zhang
authors.forename: Daniel
authors.initials: D
authors.lastname: Handley
authors.forename: Tommy
authors.initials: T
authors.lastname: Kaplan
authors.forename: Haiying
authors.initials: H
authors.lastname: Yu
authors.forename: Abha S
authors.initials: AS
authors.lastname: Bais
authors.forename: Thomas
authors.initials: T
authors.lastname: Richards
authors.forename: Kusum V
authors.initials: KV
authors.lastname: Pandit
authors.forename: Qilu
authors.initials: Q
authors.lastname: Zeng
authors.forename: Panayiotis V
authors.initials: PV
authors.lastname: Benos
authors.forename: Nir
authors.initials: N
authors.lastname: Friedman
authors.forename: Oliver
authors.initials: O
authors.lastname: Eickelberg
authors.forename: Naftali
authors.initials: N
authors.lastname: Kaminski
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000166:cytology / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051900:Smad3 Protein / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*
chemical_list: D017931:DNA Primers; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D053773:Transforming Growth Factor beta1