Despite its well-known role as a mediator of TGFβ1 signaling, a comprehensive list of SMAD3 binding targets is not available. To identify SMAD3 binding targets on a genome-wide scale, we performed chromatin immunoprecipitation for SMAD3 in a human lung alveolar epithelial carcinoma cell line A549 and identified its binding targets using promoter region microarrays (ChIP-on-chip). Additionally, a global gene expression analysis was performed in the same cells before and after stimulation with TGFβ1. Analysis of both ChIP-on-chip and gene expression microarray using computational approaches revealed multiple target molecular pathways affected by the TGFβ1/SMAD3 pathway. We have identified a novel TGFβ1/SMAD3 target gene, FOXA2
, a key regulator of embryonic lung development as well as proper function of the mature lung 
. Identification of global targets and molecular pathways associated with TGFβ1/SMAD3 pathway will provide insights to its function and lead to better understanding of its important roles in multiple cellular processes.
SMAD3 is a well-known mediator of TGFβ induced-fibrosis. Lack of SMAD3 in mice confers resistance to TGFβ, injury, or inflammation mediated renal and lung fibrosis 
as well as chemical-induced liver and pancreatic fibrosis 
. Despite this key role, to the best of our knowledge, this is the first global assessment of SMAD3 targets using ChIP-on-chip technology. Interestingly, genes associated with TGFβ pathway accounted for 10% of directly bounded genes by SMAD3, but many of the pathways affected by TGFβ1/SMAD3 identified using a combination of ChIP-on-Chip and microarray analysis were consistent with the roles of TGFβ in development, fibrosis and cancer. Additionally, multiple known genes associated with EMT and IPF were affected by TGFβ1/SMAD3, including the recently reported S100A2, RRAS, MYO1D (Table S3) 
, SERPINE1 
and TAGLN 
In this study, we identified a novel connection between the TGFβ1/SMAD3 transcriptional regulatory pathway and FOXA2, a transcription factor vitally necessary for lung development and function 
. TGFβ1 is a known regulator of pulmonary surfactant levels and is known to suppress levels of surfactant protein B (SFTPB) and SFTPC specifically through thyroid transcription factor (TTF-1). Pulmonary surfactants are lipoprotein complexes produced by type II alveolar epithelial cells 
and play important roles in lung development and normal lung function. Similarly, TTF-1 is also a critical transcription factor in lung development and it is regulated by FOXA2 
. Previously it was argued that FOXA2 regulates TTF-1 levels and SFTPB/C through protein-protein interactions 
. However, the current data strongly suggests that SMAD3 directly binds the promoter of FOXA2 and regulates its activity at the transcriptional level. TGFβ1 selectively activates or represses specific surfactant genes and these regulations are time dependent (data not shown). The exact transcriptional regulatory mechanisms of surfactants through the TGFβ1/SMAD3/FOXA2 regulatory chain remain to be elucidated.
This study provided a comprehensive list of SMAD3 binding targets and global molecular analysis of TGFβ1/SMAD3 signaling networks in the human A549 lung alveolar epithelial cell line. In this context it is important to mention that A549 cells are human alveolar basal epithelial cells derived originally from an explanted adenocarcinoma of the lung. While A549 cells do not necessarily share all features of alveolar epithelial cell, they are commonly used to study pathways and mechanisms relevant to the lung alveolar epithelium because they express alveolar type II markers such as SFTPA2, ZO1 and SFTPC 
. In our case, we used A549 cells as an in-vitro
screening tool for identifying specific targets of SMAD3 binding in a lung epithelial cell system. While we believe that the majority of identified SMAD3 target genes in A549 cells are likely to be also true for primary epithelial cells it is plausible that binding targets that require SMAD3 and additional co-factors, only expressed in normal epithelial cells, may not be fully represented in our system. Thus validation of specific TGFβ1/SMAD3 targets in human primary cells is probably needed to focus on specific pathways as we did in the case of FOXA2
. Naturally, our comprehensive list of SMAD1 targets in A549 cells will be of interest also to cancer researchers because of the role of TGFβ1/SMAD3 signaling in lung cancer 
and because A549 is also often used in lung cancer research. The analyses of both baseline and after stimulation ChIP-on-chip enhance the mechanistic value of our observations and allow more insights into the pathways recruited in response to TGFβ1/SMAD3 signaling.
In conclusion, the availability of a comprehensive list of SMAD3 signaling targets in response to TGFβ1 stimulation, the analysis of the transcriptional and molecular networks associated with this pathway in lung epithelial cells will improve our understanding of the effects of TGFβ1/SMAD3 signaling in fibrosis and cancer. The discovery of the direct effect of TGFβ1/SMAD3 on FOXA2, a major player in lung development and surfactant production and a key regulator of epithelial cell phenotype, should have significant impact on our understanding of the phenotype of lung alveolar epithelial cells in fibrosis and carcinogenesis and should encourage further research into the role of this molecule in fibrosis.