Sox17 Promotes Cell Cycle Progression and Inhibits TGF-b/Smad3 Signaling to Initiate Progenitor Cell Behavior in the Respiratory Epithelium Alexander W. Lange1, Angela R. Keiser1, James M. Wells2, Aaron M. Zorn2, Jeffrey A. Whitsett1* 1 Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center and the University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America, 2 Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center and the University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America Abstract The Sry-related high mobility group box transcription factor Sox17 is required for diverse developmental processes including endoderm formation, vascular development, and fetal hematopoietic stem cell maintenance. Expression of Sox17 in mature respiratory epithelial cells causes proliferation and lineage respecification, suggesting that Sox17 can alter adult lung progenitor cell fate. In this paper, we identify mechanisms by which Sox17 influences lung epithelial progenitor cell behavior and reprograms cell fate in the mature respiratory epithelium. Conditional expression of Sox17 in epithelial cells of the adult mouse lung demonstrated that cell cluster formation and respecification of alveolar progenitor cells toward proximal airway lineages were rapidly reversible processes. Prolonged expression of Sox17 caused the ectopic formation of bronchiolar-like structures with diverse respiratory epithelial cell characteristics in alveolar regions of lung. During initiation of progenitor cell behavior, Sox17 induced proliferation and increased the expression of the progenitor cell marker Sca-1 and genes involved in cell cycle progression. Notably, Sox17 enhanced cyclin D1 expression in vivo and activated cyclin D1 promoter activity in vitro. Sox17 decreased the expression of transforming growth factor-beta (TGF-b)-responsive cell cycle inhibitors in the adult mouse lung, including p15, p21, and p57, and inhibited TGF-b1-mediated transcriptional responses in vitro. Further, Sox17 interacted with Smad3 and blocked Smad3 DNA binding and transcriptional activity. Together, these data show that a subset of mature respiratory epithelial cells retains remarkable phenotypic plasticity and that Sox17, a gene required for early endoderm formation, activates the cell cycle and reinitiates multipotent progenitor cell behavior in mature lung cells. Citation: Lange AW, Keiser AR, Wells JM, Zorn AM, Whitsett JA (2009) Sox17 Promotes Cell Cycle Progression and Inhibits TGF-b/Smad3 Signaling to Initiate Progenitor Cell Behavior in the Respiratory Epithelium. PLoS ONE 4(5): e5711. doi:10.1371/journal.pone.0005711 Editor: Oliver Eickelberg, Helmholtz Zentrum Mu¨ nchen/Ludwig-Maximilians-University Munich, Germany Received January 23, 2009; Accepted May 4, 2009; Published May 27, 2009 Copyright: 2009 Lange et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: NIH grants HL61646 and HL090156 (J.A.W.); training grant HL007752 (A.W.L.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Jeffrey.Whitsett@chmcc.org Introduction Proteins in the Sry-related high mobility group box (Sox) family of transcription factors regulate developmental processes and cell type specification in various organ systems. Sox proteins share homology in the HMG domain, which binds the consensus DNA sequence (A/ T)(A/T)CAA(A/T)GG to regulate gene expression and mediates protein interactions with transcriptional cofactors [1]. During vertebrate embryogenesis, Sox17 is required for formation of the endoderm, which gives rise to the liver, pancreas, and epithelium of the gastrointestinal and respiratory tracts [2–5]. In addition, constitutive expression of Sox17 in human embryonic stem cells is sufficient to promote differentiation of definitive endoderm progen- itors [6]. Studies on mice with targeted deletion of Sox17 have identified additional roles for Sox17 in the maintenance of fetal hematopoietic stem cells, cardiovascular development, and angio- genesis [7–9]. Sox17 expression is dynamically regulated during endoderm formation and the programming of embryonic stem cells toward respiratory epithelial cell lineages in vitro [10–12]. Although Sox17 is highly expressed in the endoderm and is required for its formation prior to the emergence of the lung primordium, it is not readily detected in the respiratory epithelium thereafter. While conditional misexpression of Sox17 in respiratory epithelial cells of the embryonic mouse lung disrupted branching and differentiation of proximal/distal epithelial cell types, expression of Sox17 in the adult lung epithelium induced the formation of hyperplastic cell clusters in the alveolar region that contained cells expressing markers characteristic of diverse proximal airway epithelial lineages [13]. Together, these findings are consistent with a role for Sox17 in regulating progenitor cell behavior and lineage specification in various tissues. The ability to reprogram mature, differentiated cell types into alternative lineages has been the subject of significant recent interest. Although the mechanisms underlying cell lineage reprogramming remain poorly understood, this phenomenon is generally associated with ectopic expression or reactivation of genes important for embryonic development and organogenesis [14]. While epithelial cells in the mature lung are normally quiescent, subpopulations of endogenous cells within the respira- tory epithelium possess the capacity to reenter the cell cycle, proliferate, and redifferentiate into multiple epithelial cell types with appropriate function and location along the proximal/distal lung axis following injury. This is consistent with the notion that subsets of mature respiratory epithelial cells maintain remarkable PLoS ONE | www.plosone.org 1 May 2009 | Volume 4 | Issue 5 | e5711