directly induces cyclin D1 to promote proliferation of a subset of mature respiratory epithelial cells. Recent studies have shown that post-mitotic cells can reenter the cell cycle by blocking the expression of cyclin-dependent kinase inhibitors, supporting a role for this protein family in maintaining the balance between quiescence and proliferation [54]. In the present study, Sox17 decreased the expression of p15, p21, and p57 in adult mouse lungs, and inhibited TGF-b1/Smad3 transcrip- tional activity in vitro. In addition, Sox17 physically interacted with Smad3 and abrogated Smad3 binding to the p15 promoter. These findings support a model in which Sox17 antagonizes TGF-b/ Smad-dependent expression of cell cycle inhibitors in mature respiratory epithelial cells, facilitating G1 progression and cell reentry. In addition, Sox17 counteracted repression of the cyclin D1 promoter by TGF-b1 and Smad3. While we favor the notion that Sox17 directly activates the cyclin D1 promoter, it is possible that inhibiting TGF-b/Smad3-mediated repression of cyclin D1 also contributes to its increased expression in CCSPrtTA/tetO-Sox17 mouse lungs. By decreasing expression of inhibitors and increasing expression of positive regulators of the G1 phase of the cell cycle, Sox17 establishes conditions that favor reactivation of proliferation in mature, normally quiescent respiratory epithelial cells. In the present study, Sox17 interacted with Smad3 and repressed TGF-b1/Smad3 transcriptional activity. Although both the MH1 and MH2 domains of Smad3 bound Sox17, the strongest interaction was localized to the linker region and MH2 domains. The linker region of Smad3 contains a transactivation domain and the MH2 domain modulates transcription, whereas the MH1 domain of Smad3 regulates DNA binding and transcription. Thus, Sox17 interaction with these domains is consistent with the antagonistic effects of Sox17 on Smad3 DNA binding and transcriptional activity. While that amino acids 129–359 of Sox17 appear to mediate binding to Smad3, the N-terminus of Sox17 is necessary to antagonize Smad3 activity. Inhibition of b-catenin/ TCF transcriptional activity is also dependent on the N-terminus of Sox17, wherein the HMG domain is required for interaction with TCF factors to influence their protein stability [52,55]. Together, these findings indicate that the N-terminus of Sox17 containing the HMG domain is important for influencing the transcriptional responses of signaling pathways and that there may be multiple regions of Sox17 that contribute to complex protein interactions. Sox17 initiated progenitor cell behavior in respiratory epithelial cells when ectopically expressed at high levels in the adult lung. While these studies provide insight into the effects of Sox17 on reprogramming mature respiratory epithelial cells, it is unclear whether they reflect a physiological role during development or repair. Since Sox17 is highly expressed in the endoderm, which gives rise to the respiratory epithelium, Sox17 likely plays an early role in specification of endodermal precursor cells prior to emergence of the lung primordium. In the lung, Sox17 is expressed in mesenchymal cells during branching morphogenesis and in pulmonary endothelial cells later in development and in the adult [13]. However, Sox17 mRNA was not detected in respiratory epithelial cells and immunohistochemical staining for endogenous Sox17 was not observed in the lung epithelium in the present study [13]. A number of Sox family members are expressed at high levels in various cell types in the developing and mature lung, including Sox2, 4, 7, 9, 11, 17, and 18 [56–60]. However, whether Sox proteins have distinct or redundant functions during lung formation and repair remains to be elucidated. Our data shows that the ability of Sox17 to induce progenitor cell behavior is mediated, at least in part, by activating cyclin D1 and decreasing TGF-b-responsive cell cycle inhibitor expression to promote proliferation. As the transcriptional Sox17 and Lung Progenitor Cell pathways that regulate cell proliferation and differentiation during lung formation are reactivated during regeneration of pulmonary cell lineages following injury, determining how Sox proteins and other transcription factors integrate signals from multiple path- ways is important toward understanding the regulatory mecha- nisms that control lung development and homeostasis. Materials and Methods Mice rCCSPrtTA and (tetO)7CMV-Sox17-IRES-NucGFP transgenic mice used for conditional expression of Sox17 in respiratory epithelial cells have been previously described [13,33,61]. Adult rCCSPrtTA/ (tetO)7CMV-Sox17-IRES-NucGFP double transgenic and rCCSPrtTA single transgenic control mice, referred to as CCSPrtTA/tetO-Sox17 and CCSPrtTA, respectively, were maintained on doxycycline- containing food (625 mg/kg; Harlan Teklad, Madison, WI) as described for specific experiments. Animals were housed in pathogen- free conditions according to protocols approved by the Institutional Animal Care and Use Committee at Cincinnati Children’s Hospital Research Foundation. Mice were sacrificed by anesthesia using a mixture of ketamine, aceprozamine, and xylazine and exsanguination by severing the inferior vena cava and descending aorta. All experiments were performed using at least 3 animals per group. Immunohistochemistry and Immunofluorescence Lungs of adult mice were inflated with 4% paraformaldehyde/ phosphate-buffered saline (PBS), fixed by immersion overnight at 4uC, and processed according to standard protocols for paraffin embedding. Immunohistochemistry was performed using primary antibodies for guinea pig-anti Sox17 (1:10,000), rabbit anti- phospho-histone H3 (1:1000; Santa Cruz), rabbit anti-CCSP (1:5000), rabbit anti-Foxj1 (1:14,000), rabbit anti-cyclin D1 (1:400; Abcam), and rat anti-Sca-1 (1:250; BD Pharmingen). Briefly, sections (5 mm) were deparaffinized, rehydrated through a graded ethanol series, and endogenous peroxidase activity was inactivated in 1.5% H2O2 in methanol. Microwave antigen retrieval was performed (except for Sca-1) using 10 mM citrate buffer, pH 6.0 and sections were blocked for 1–2 h in 4% normal goat or donkey serum in PBS-0.1% Triton X-100 (PBST) followed by primary antibody incubation overnight at 4uC. Sections were then washed and incubated with biotinylated secondary antibodies (1:200; Vector Labs) followed by incubation in ABC reagent (Vectastain Elite ABC kit; Vector Labs). Antigen localization was detected with nickel-diaminobenzidine and enhanced with Tris-Cobalt. Sections were counterstained with 0.1% of Nuclear Fast Red and coverslipped using Permount (Fisher Scientific). Immunofluorescence was performed as described above with the omission of peroxidase treatment. For immunofluorescent double and triple labeling, primary antibodies for guinea pig anti- Sox17 (1:1000), rabbit anti-phospho-histone H3 (1:500), rat anti- Sca-1 (1:50), rabbit anti-proSP-C (1:500), and guinea pig anti- CCSP (1:10,000) were used with fluorophore-conjugated second- ary antibodies (Alexa Fluor-488, Alexa Fluor-568, and Alexa Fluor-688; Molecular Probes). Sections were mounted with Vectashield anti-fade reagent containing DAPI (Vector Labs). Brightfield and fluorescent images were obtained using a Zeiss Axioplan2 microscope equipped with AxioVision Software. RNA analysis Total RNA was extracted from whole left lobes of adult mice using TRIzol reagent (Invitrogen) and RNeasy Mini Kit (Qiagen) according to the manufacturer’s recommendations. Mouse Oligo GEArrays (SuperArray Bioscience Corporation) were used per the PLoS ONE | www.plosone.org 11 May 2009 | Volume 4 | Issue 5 | e5711