Sox17 and Lung Progenitor Cell Binding of Smad3 to the p15 promoter was assessed by chromatin immunoprecipitation and quantified by real time PCR. Graph represents average fold enrichment of FLAG immunoprecipitated samples relative to IgG negative control samples. Asterisk indicates statistical significance determined by Student’s t-test (p,0.05). Expression of Smad3 and Sox17 inputs were assessed by immunoblot. doi:10.1371/journal.pone.0005711.g007 none of the Sox17 proteins influenced nuclear translocation of Smad3 in the presence or absence of TGF-b1 (Fig. S3), indicating that Sox17 does not antagonize Smad3 activity by regulating subcellular localization. Together, these data suggest that, although it is not required for binding to Smad3, the N-terminus of Sox17 is important for repression of Smad3 transcriptional activity. To determine if the interaction between Sox17 and TGF-b/ Smad3 signaling influences regulation of cell cycle-related genes, their effects on cell cycle gene promoters was examined. In MLE- 15 cells, Sox17 alleviated repression of cyclin D1 promoter activity by TGF-b1 and Smad3, consistent with an antagonistic effect of Sox17 on the TGF-b/Smad3 pathway (Fig. 7E). Since Sox17 negatively regulated Smad3 transcriptional activity in reporter assays but did not influence Smad3 nuclear import, we sought to determine if the repression was mediated by influencing Smad3 DNA binding. Smad3 regulates p15 gene expression by directly binding to sites located within the first 113 bp of its promoter [21]. Using MLE-15 cells, Smad3 DNA binding to the p15 promoter between 2157 and +103 in the presence or absence of Sox17 was examined by chromatin immunoprecipitation and quantified by real time PCR. Sox17 significantly decreased Smad3 binding to the p15 promoter (Fig. 7F). Together, these experiments show that Sox17 antagonizes Smad3 transcriptional activity by preventing its ability to bind DNA, providing a potential mechanism by which Sox17 decreased the expression of cell cycle inhibitors in the adult mouse lung. In addition, antagonizing TGF-b1/Smad3 repression of the cyclin D1 promoter may also contribute to the Sox17- mediated induction of cyclin D1 expression observed in vivo. Discussion Conditional expression of Sox17 in respiratory epithelial cells of the adult mouse lung induced proliferation and reversibly respecified alveolar type II epithelial cells to express markers characteristic of the differentiated bronchiolar epithelium, sup- porting the concept that a subset of mature respiratory epithelial cells possesses remarkable phenotypic plasticity and progenitor cell capabilities. Activation of this progenitor-like behavior in respira- tory epithelial cells by Sox17 was associated with increased expression of Sca-1 and multiple genes that promote cell cycle reentry/progression. Sox17 decreased expression of cell cycle inhibitors in vivo and interacted with Smad3 to inhibit TGF-b1/ Smad3-mediated transcriptional responses in vitro. Together, these data provide insight into the mechanisms by which Sox17 stimulates respiratory epithelial progenitor cell behavior and lineage respecification in the mature lung. Expression of Sox17 reprogrammed a subset of mature alveolar type II cells to ectopically express markers characteristic of diverse conducting airway cell lineages, including ciliated, non-ciliated secretory cells, and goblet cells that are not normally detected in the alveolar regions of the lung [13], and led to the formation of highly organized bronchiolar-like structures in the peripheral lung. These results indicate that epithelial cells in the adult lung can serve as multipotent progenitors capable of lineage respecification. In support of this concept, conditional expression of SPDEF, an ETS family transcription factor, in respiratory epithelial cells of adult mice converts Clara cells into goblet cells [36]. The notion that Sox proteins have important functions in lineage reprogram- ming is further supported by the contribution of Sox2 toward induced pluripotency in somatic cells [37–40]. While the mechanisms that govern progenitor cell activation and cell fate respecification remain poorly understood, further analysis of Sox17-induced respiratory epithelial progenitors may provide insight into such processes in the mature lung. Lineage reprogramming of adult cell types involves the expression of key developmental regulatory genes [14]. The finding from previous studies that expression of several transcription factors involved in regulating proximal/distal epithelial cell differentiation in the lung, including TTF-1, Foxa1, Foxa2, and b-catenin, is also increased in the Sox17-induced cell clusters suggests that Sox17 can reinitiate a program typical of the developing lung [13]. During endoderm formation in Xenopus, Sox17 regulates expression of Foxa1 and Foxa2, and acts upstream of GATA6 [41]. Foxa1, Foxa2, and GATA6 influence the expression of differentiated respiratory epithelial cell markers, including CCSP and Foxj1 [42–44]. Taken together, these data support the concept that Sox17, which is necessary for endoderm formation, functions upstream of a hierarchy of transcription factors that cooperate in specification of endodermal cells from which respiratory epithelial lineages are derived. Constitutive expression of Sox17 in human embryonic stem cells is sufficient to commit cells to the definitive endoderm lineage, and increased expression of Sox17 is associated with the differentiation of embryonic stem cells toward cells with characteristics of the respiratory epithelium [5,6,10–12]. Together, these findings further support an early role for Sox17 in the establishment of endodermal cells that later serve as precursors of lung epithelial lineages. The induction of proliferation in a subset of bronchiolar cells and alveolar type II cells by Sox17 was associated with increased expression of several cyclin genes known to stimulate cell cycle progression. Among the various cyclin genes induced by Sox17, only cyclin D1 functions in G1 prior to the restriction point of the cell cycle [45,46]. Notably, forced expression of cyclin D1 along with cdk4 is sufficient to reinitiate cell cycle progression in multiple post-mitotic cell types [47,48]. Therefore, the induction of cyclin D1 by Sox17 is likely to play an important role in stimulating mature respiratory epithelial cells to reenter the cell cycle. Our in vitro studies demonstrated that Sox17 directly activated the human cyclin D1 promoter through a conserved site located at 274 bp relative to the first exon. Given the homology between their HMG domains, Sox and TCF/LEF proteins have similar preferences for consensus binding sequences, and previous studies have demon- strated that cyclin D1 is directly regulated by b-catenin/TCF/LEF through this same site [49,50]. In breast cancer cells, Sox2 interacts with b-catenin to cooperatively regulate the cyclin D1 promoter through the 274 bp binding site as well [51]. In addition, Sox17 and Sox4 respectively inhibit and enhance proliferation of colon carcinoma cells through physical interactions with b-catenin and TCF/LEF to modulate protein stability and transcriptional activity, and Sox6 interacts with b-catenin and HDAC1 to repress cyclin D1 promoter activity and proliferation in insulinoma cells [52,53]. Taken together, these findings suggest that Sox proteins and b-catenin/TCF/LEF complexes may compete for common DNA binding sites and that their effects on transcription and cell behavior may depend on relative expression levels, protein interactions, and/or cell type. Although it is unclear whether Sox17 is a transcriptional activator of cyclin D1 in vivo, our data support a potential mechanism by which Sox17 PLoS ONE | www.plosone.org 10 May 2009 | Volume 4 | Issue 5 | e5711