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Updated: Jun 1, 2026
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Published on: October 27, 2020
High throughput determination of TGFβ1/SMAD3 targets in A549 lung epithelial cells
Yingze Zhang1, Daniel Handley, Tommy Kaplan
1Division 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
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.
Insights
Transforming growth factor beta 1 (TGFβ1) signaling regulates lung epithelial cells via SMAD3. This study maps SMAD3 targets, revealing new insights into lung fibrosis and cancer mechanisms, including TGFβ1
Area of Science:
- Molecular Biology
- Cell Biology
- Genomics
Background:
- Transforming growth factor beta 1 (TGFβ1) is implicated in lung diseases like cancer, pulmonary hypertension, and fibrosis.
- TGFβ1 signaling involves the transcription factor SMAD3, regulating gene expression in lung epithelial cells.
Purpose of the Study:
- To create a genome-wide map of SMAD3 binding targets.
- To identify molecular pathways and networks influenced by TGFβ1/SMAD3 signaling in lung epithelial cells.
Main Methods:
- Combined chromatin immunoprecipitation with promoter microarrays (ChIP-on-chip) and gene expression microarrays.
- Utilized computational approaches to identify associated pathways and networks.
- Validated target gene expression and SMAD3 binding using RT-PCR and electrophoretic mobility shift assays.
Main Results:
- Identified known TGFβ1 targets (e.g., SERPINE1, SMAD6, SMAD7) and novel targets like FOXA2.
- Confirmed SMAD3 binding to the FOXA2 promoter and its altered expression.
- Computational analysis revealed multiple molecular pathways affected by TGFβ1/SMAD3 signaling.
Conclusions:
- Global target identification enhances understanding of epithelial cell phenotypes in fibrogenesis and carcinogenesis.
- The direct effect of TGFβ1 on FOXA2 was discovered, providing new mechanistic insights.
