H3 (Fig. S2). Since Sox17 induced expression of Sca-1 in a subset of epithelial cells located in putative lung stem/progenitor cell niches in the alveolus and peripheral bronchioles, immunofluorescence was performed to determine if the population of Sca-1 positive cells coexpressed CCSP and/or proSP-C characteristic of BASCs. In lungs from CCSPrtTA/tetO-Sox17 mice maintained on Dox for 5 days, colocalization of Sca-1 and CCSP was observed in cells along the bronchoalveolar duct junctions (Fig. 4F–N). In contrast, CCSP was not detected in the Sca-1-expressing cells detected in the alveolar region. In accordance with this observation, immunohistochemical analysis did not reveal any CCSP-expressing cells in the peripheral lung of CCSPrtTA/tetO-Sox17 mice exposed to Dox for 5 days (data not shown). The Sox17-induced Sca-1 positive cells located in the alveolar region did not coexpress proSP-C (Fig. 4F–N). Further, CCSP+/proSP-C+/Sca-1+ cells were never detected at the broncho- alveolar duct junctions or in the lung periphery (n = 4 mice examining 3 sections from each lobe). Taken together, these studies show that the Sox17 induced Sca-1 expression in cells near the BADJ that coexpressed CCSP but not proSP-C, while the Sca-1 expressing cells induced in the alveolar region expressed neither CCSP nor proSP-C. Thus, while induction of Sca-1 supports the concept of progenitor cell activation, the molecular profile of the Sca-1-expressing population is distinct from that previously describe for BASCs [32], emphasizing the need for a better understanding of the phenotypic and functional characteristics of potential stem/progenitor cells in the lung. Sox17 influences expression of cell cycle regulatory genes Since expression of Sox17 in respiratory epithelial cells in the adult mouse lung induced cell proliferation, the effects on cell cycle-associated gene expression was examined to identify potential downstream targets of Sox17 that regulate this process. Total RNA isolated from whole left lobes of adult CCSPrtTA control and CCSPrtTA/tetO-Sox17 mice maintained on Dox for 2 and 3 days was used to analyze changes in cell cycle-related gene expression with a commercially available oligo SuperArray (data not shown). Subsequently, RT-PCR was used to confirm the changes in expression of a subset of the genes identified in the array. In addition to increased expression of Sox17 (data not shown), mRNAs for the cyclin-dependent kinase inhibitors p15, p21, and p57 were significantly decreased in lungs of CCSPrtTA/ tetO-Sox17 mice relative to CCSPrtTA controls following 1 day of exposure to Dox (Fig. 5). Whereas expression of p57 was also decreased on lungs from CCSPrtTA/tetO-Sox17 mice maintained on Dox for 3 days, expression of p15 and p21 was similar to controls. Further, while expression of p19 was variably decreased in lungs from CCSPrtTA/tetO-Sox17 mice maintained on Dox for 2 days, no differences in the expression of p16 or p27 were observed after expression of Sox17 for 1–3 days (data not shown). Consistent with the induction of proliferation observed by immunohistochemistry (Fig. 3), transcripts for genes that promote cell cycle progression, including Foxm1, cyclin A2, cyclin B1, cyclin D1, and cyclin E1, were increased in lungs of CCSPrtTA/tetO-Sox17 mice after 3 days of Dox treatment (Fig. 5). Together, these results demonstrate that Sox17 expression in adult mouse lung results in decreased levels of cyclin-dependent kinase inhibitors associated with G1 arrest and increased expression of cell cycle-promoting genes, providing insight into the molecular mechanisms that regulate Sox17- induced proliferation associated with the initiation of progenitor cell behavior in respiratory epithelial cells. Sox17 and Lung Progenitor Cell Figure 5. Sox17 regulates genes that control the cell cycle. RT- PCR was used to assess expression of cell cycle-related genes in lung tissue from adult CCSPrtTA and CCSPrtTA/tetO-Sox17 mice treated with Dox for 1 or 3 days. Transcripts for the cyclin-dependent kinase inhibitors p15, p21, and p57 were decreased in Sox17 transgenic lungs after 1 day of Dox and mRNAs for genes associated with cell cycle progression were increased by Sox17 after 3 days Dox exposure. L7 was used as a loading control. doi:10.1371/journal.pone.0005711.g005 tetO-Sox17 mice, we sought to determine if it was a direct downstream target of Sox17 during induction of respiratory epithelial cell proliferation. Quantitative real time RT-PCR performed using total RNA isolated from whole left lobes demonstrated that cyclin D1 mRNA was increased 1.3-fold in lungs from adult CCSPrtTA/tetO-Sox17 mice exposed to Dox for 1 day (data not shown). Staining for cyclin D1 was markedly increased in bronchioles and alveolar type II cells of CCSPrtTA/ tetO-Sox17 mice following 2 days exposure to Dox (Fig. 6A–B). Thus, cyclin D1 mRNA and protein levels were significantly increased in lungs from CCSPrtTA/tetO-Sox17 mice just prior to the onset of proliferation, consistent with the concept that cyclin D1 contributes to the cell cycle reentry of mature respiratory epithelial cells following expression of Sox17. To determine if cyclin D1 is a direct transcriptional target Sox17, the regulatory region of cyclin D1 was examined for putative Sox binding sites. A well-conserved consensus Sox binding sequence was identified in cyclin D1 proximal promoter, located at approximately 274 bp relative to exon 1 in the human and mouse genomic sequences (Fig. 6C). The functional significance of this potential Sox site was assessed by reporter assay using a human cyclin D1 promoter deletion series. While the 247/+187 cyclin D1-luciferase reporter was moderately responsive to Sox17, the cyclin D1-luciferase constructs that contained the consensus Sox binding sequence were markedly activated by Sox17 (Fig. 6C). A truncated Sox17 isoform (t-Sox17), which lacks most of the HMG box and cannot bind DNA [34], did not affect cyclin D1 promoter activity (Fig. 6C), indicating that activation of cyclin D1-luciferase reporters by Sox17 requires DNA binding. Together with the in vivo data, these results support the concept that cyclin D1 is a direct downstream target of Sox17 in respiratory epithelial cells of CCSPrtTA/tetO-Sox17 mice. Sox17 induces cyclin D1 expression Since cyclin D1 is a key regulator of progression through the G1 phase of the cell cycle and was increased in lungs from CCSPrtTA/ Sox17 inhibits TGF-b1-induced transcriptional activity TGF-b is a potent inhibitor of proliferation in multiple epithelial cell types, including alveolar type II cells [28]. The mechanism PLoS ONE | www.plosone.org 7 May 2009 | Volume 4 | Issue 5 | e5711