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High throughput determination of TGFβ1/SMAD3 targets in A549 lung epithelial cells

Zhang, Y and Handley, D and Kaplan, T and Yu, H and Bais, AS and Richards, T and Pandit, KV and Zeng, Q and Benos, PV and Friedman, N and Eickelberg, O and Kaminski, N (2011) High throughput determination of TGFβ1/SMAD3 targets in A549 lung epithelial cells. PLoS ONE, 6 (5).

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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. 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 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. Results and 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. © 2011 Zhang et al.


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Item Type: Article
Status: Published
CreatorsEmailPitt UsernameORCID
Zhang, Yzhang3@pitt.eduZHANG3
Handley, D
Kaplan, T
Yu, H
Bais, AS
Richards, T
Pandit, KV
Zeng, Qqiz30@pitt.eduQIZ30
Benos, PVbenos@pitt.eduGSBCPLRC
Friedman, N
Eickelberg, O
Kaminski, N
ContributionContributors NameEmailPitt UsernameORCID
Date: 26 May 2011
Date Type: Publication
Journal or Publication Title: PLoS ONE
Volume: 6
Number: 5
DOI or Unique Handle: 10.1371/journal.pone.0020319
Schools and Programs: School of Medicine > Critical Care Medicine
Refereed: Yes
MeSH Headings: Base Sequence; Cell Line; Chromatin Immunoprecipitation; DNA Primers; Electrophoretic Mobility Shift Assay; Epithelial Cells--metabolism; Humans; Lung--cytology; Lung--metabolism; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; Smad3 Protein--metabolism; Transforming Growth Factor beta1--metabolism
Other ID: NLM PMC3098871
PubMed Central ID: PMC3098871
PubMed ID: 21625455
Date Deposited: 29 Aug 2012 21:08
Last Modified: 29 Oct 2022 11:55


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