phenotypic plasticity despite their differentiation status. In support of this concept, several lung stem/progenitor cell candidates have been identified in distinct niches along the airway epithelium, including basal cells, toxicant-resistant ‘‘variant’’ Clara cells, and ciliated cells in the conducting airway, bronchoalveolar stem cells (BASCs) located at the bronchoalveolar duct junction (BADJ), and type II cells in the peripheral lung [15–17]. During embryogenesis, respiratory epithelial cells are derived from progenitors in the foregut endoderm and are specified prior to the morphological appearance of the lung buds [18]. Several families of transcription factors influence lung morphogenesis and differentiation of the diverse respiratory epithelial cell types, including TTF-1, GATA6, b-catenin/TCF/LEF, Forkhead (Fox), and Sox family members [19]. However, whether transcription factors important for lung development can also influence progenitor cell behavior in mature respiratory epithelial cells is not known. The TGF-b pathway regulates diverse biological processes in multiple cell types, including cell proliferation, differentiation, apoptosis, and migration. Following activation of heteromeric type II and type I kinase receptor complexes by TGF-b ligands, intracellular signaling is initiated by phosphorylation of receptor- activated Smad proteins. Phosphorylated Smad2 and Smad3, the effectors of the TGF-b pathway, interact with Smad4 and translocate to the nucleus to regulate transcription of downstream target genes [20]. The cellular responses to TGF-b signaling are further influenced by the interaction of Smad proteins with cofactors to modulate transcriptional activity. TGF-b signaling inhibits proliferation in multiple epithelial cell types by influencing the expression of cell cycle regulatory proteins to induce arrest prior to the restriction point in G1. For example, Smad2, 3, and 4 directly activate the promoters of the cyclin-dependent kinase inhibitors p15 and p21, which in turn inhibit cdk2/4/6-cyclin complex activity [21–23]. In addition to negative regulation of branching morphogenesis in the embryonic lung, TGF-b/Smad signaling and expression of cyclin-dependent kinase inhibitors blocks proliferation of mature alveolar type II cells in culture [24–28]. Further, alveolar epithelial cells from p212/2 mice have an increased proliferation rate and lung tumorigenesis is enhanced in adult mice heterozygous for a mutation in TGF-b1, supporting a role for this pathway in maintaining quiescence in the normal respiratory epithelium in vivo [29–31]. Whether TGF-b/ Smad signaling and cyclin-dependent kinase inhibitors contribute to negative regulation of progenitor cell activation and cell cycle reentry in the mature respiratory epithelium has not been determined. The present study was undertaken to determine the mechanisms by which expression of Sox17 in mature respiratory epithelial cells influences progenitor cell behavior. Conditional expression of Sox17 in the adult mouse lung induced proliferation and reversibly reprogrammed alveolar cells to form structures with phenotypic and morphological characteristics of the proximal airway. During the induction of progenitor cell behavior, Sox17 increased expression of Sca-1, a progenitor cell marker, and stimulated cell proliferation in association with altered expression of cell cycle regulatory genes. In addition, Sox17 physically interacted with Smad3 and negatively regulated Smad3 DNA binding and TGF- b/Smad3 transcriptional responses. These findings provide insight into the ability to reactivate multipotent progenitor cell behavior and reprogram epithelial cell lineages in the lung. Results Sox17 reversibly induces progenitor cell behavior in the alveolar epithelium in vivo Expression of Sox17 in peripheral respiratory epithelial cells of the adult mouse lung reprogrammed mature alveolar type II Sox17 and Lung Progenitor Cell cells toward multiple proximal airway epithelial cell lineages, supporting a role in regulating plasticity/progenitor cell behavior [13]. To determine if alveolar cells become perma- nently respecified following ectopic Sox17 expression, Sox17 was conditionally expressed in respiratory epithelial cells of adult mice. Lungs from CCSPrtTA/tetO-Sox17 and CCSPrtTA single transgenic control mice were harvested after 4 weeks exposure to doxycycline (Dox) or following removal of Dox for 1 week, and were examined for expression of Sox17 and the proximal airway epithelial cell markers CCSP (Clara cells) and Foxj1 (ciliated cells). Expression of Sox17 in peripheral respiratory epithelial cells induced the formation of atypical alveolar cell clusters which contained cells that expressed conducting airway epithelial cell markers CCSP and Foxj1 (Fig. 1B,E,H). In lungs from CCSPrtTA/tetO-Sox17 mice in which Dox treatment was discontinued for 1 week prior to harvest, immunostaining for Sox17 was only detected in endothelial cells in the peripheral lung and was indistinguishable from controls (Fig. 1A,C). More notable, neither the alveolar cell clusters nor peripheral expression of CCSP or Foxj1 were observed in lungs from these mice (Fig. 1C,F,I). Together, these data demonstrate that the Sox17-induced alveolar cell cluster formation and lineage respecification are reversible processes and that continued expression of Sox17 is required to maintain progenitor cell behavior in adult CCSPrtTA/tetO-Sox17 mice, revealing remarkable plasticity within a subset of mature respiratory epithelial cells. Bronchiolar-like lesions are induced in the alveoli following prolonged Sox17 expression To determine the effects of prolonged expression of Sox17 in respiratory epithelial cells, adult CCSPrtTA/tetO-Sox17 mice were maintained on Dox for 12 months. Long-term expression of Sox17 caused the formation of organized sheets of epithelial cells in the peripheral lung with morphological similarities to the bronchiolar epithelium (Fig. 2). The bronchiolar-like structures expressed Sox17 (Fig. 2B) and contained subsets of cells that expressed proximal airway epithelial markers CCSP and Foxj1 (Fig. 2C–D), consistent with bronchiolar cell differentiation. While CCSP+ cells were detected in most of the bronchiolar-like structures, Foxj1+ cells were less frequently observed. Since the ability of Sox17 to reprogram mature alveolar type II cells suggests the induction of progenitor cell behavior, we examined the bronchiolar-like structures for coexpression of CCSP, proSP- C, and Sca-1, a property attributed to bronchoalveolar stem cells (BASCs), a potential lung stem/progenitor population [32]. Expression of Sca-1, a progenitor cell marker in several tissues, was detected in cells within the bronchiolar-like structures and colocalized with CCSP-expressing cells (Fig. 2E–H). While a rare subset of bronchiolar-like lesions contained cells that co-expressed CCSP and proSP-C (data not shown), CCSP+/proSP-C+/Sca-1+ cells were never observed. Thus, the Sox17-induced bronchiolar- like structures contained a mixed population of cells that expressed CCSP, Foxj1, Sca-1, CCSP+/Sca-1+, and CCSP+/ proSP-C+, consistent with reprogramming of progenitor cells along several differentiated pathways. Such bronchiolar-like epithelial sheets were never detected in lungs from CCSPrtTA control mice maintained on Dox for 12 months (data not shown). Together these data show that prolonged expression of Sox17 in the adult mouse lung dramatically influences respiratory epithelial cell differentiation, generating ectopic structures in the peripheral lung with characteristics of the more proximal bronchiolar epithelium. PLoS ONE | www.plosone.org 2 May 2009 | Volume 4 | Issue 5 | e5711