Sox17 and Lung Progenitor Cell Figure 6. Cyclin D1 expression is induced by Sox17. (A–B) Immunohistochemistry for cyclin D1 was performed on lung sections from adult CCSPrtTA (A) and CCSPrtTA/tetO-Sox17 (B) transgenic mice treated with Dox for 2 d. Cyclin D1 staining was increased in bronchioles and type II cells after Sox17 expression. Scale bars, 50 mm. (C) The regulatory region of cyclin D1 contains a conserved Sox binding site (boxed). MLE-15 cells were transiently transfected with human cyclin D1-luciferase reporter constructs and empty vector, t-Sox17, or full length Sox17. While the 247/+187 cyclin D1-luciferase reporter was moderately responsive to Sox17 (2.6-fold), Sox17 strongly activated the cyclin D1 promoter constructs containing the conserved Sox binding site (arrowheads; 296/+187; 6.4-fold and 2944/+187; 5.9-fold). Experiments were performed three times in triplicate and representative results are shown6the standard deviation of the mean. Asterisks indicate statistical significance determined by Student’s t-test (p,0.05). doi:10.1371/journal.pone.0005711.g006 underlying the anti-proliferative effects of TGF-b has been attributed, at least in part, to Smad-dependent transcriptional induction of cyclin-dependent kinase inhibitors including p15 and p21, leading to G1 arrest [21–23]. Since expression of p15 and p21 was decreased in lungs of adult mice following Sox17 expression in respiratory epithelial cells, we sought to determine if Sox17 influences TGF-b-mediated transcriptional activity in vitro using the TGF-b/Smad responsive reporter 3TP-luciferase. In MLE-15 cells, Sox17 markedly inhibited TGF-b1-induced 3TP-luciferase reporter activity in a dose-dependent manner relative to vector control or t-Sox17 (Fig. 7A), demonstrating that Sox17 has an inhibitory effect on the TGF-b pathway. Sox17 interacts with Smad3 and inhibits Smad3- dependent transcriptional activity Since Smad2/3 are the transcriptional effectors of TGF-b signaling, we examined if the inhibition of TGF-b1-induced transcriptional activity by Sox17 was mediated by an interaction with these Smads. GST-pulldown assays revealed a physical interaction between Sox17 and Smad3 (Fig. 7B–C). To identify which domains of Smad3 are required for the interaction, GST- pulldown assays were performed using deletion constructs for Smad3. While both the MAD homology domains (MH1 and MH2) of Smad3 interacted with Sox17 at low stringency binding conditions (data not shown), Smad3 1–145 did not bind to Sox17 under higher stringency conditions (Fig. 7B). Likewise, GST- pulldowns were performed using a series of Sox17 deletions and a point mutant, including a N-terminal deletion that removes 80% of the HMG domain (Sox17 129–419, which is equivalent to the t- Sox17 isoform), a C-terminal truncation that deletes the transactivation domain (Sox17 1–359), and a point mutation in the HMG domain (M76A), which is predicted to disrupt DNA binding but not protein structure [35]. Both of the Sox17 truncations and the point mutant maintained the ability to interact with Smad3 across multiple binding stringencies (Fig. 7C). Together, these data indicate that while multiple domains of Smad3 are capable of interacting with Sox17, the strongest binding is localized to the linker and MH2 regions from amino acids 146–425. Further, the data suggest that amino acids 129–359 of Sox17 mediate the interaction with Smad3. While the interaction observed between Sox17 and Smad3 was corroborated using co-immunoprecipitation assays, an interaction between Sox17 and Smad2 was not observed (data not shown). To examine the functional significance of the interaction between Sox17 and Smad3 on Smad3 transcriptional activity, the 3TP-luciferase reporter was co-transfected with Smad3 in the presence or absence of Sox17 in MLE-15 cells. Similar to its effect on TGF-b1 activity, Sox17 significantly inhibited Smad3-induced reporter activity (Fig. 7D). To identify the domains of Sox17 that mediate the repression of Smad3 transcriptional activity, reporter assays were performed using the Sox17 deletions and point mutant. While Sox17 1–359 maintained the ability to inhibit Smad3 activity, neither Sox17 129–419 nor Sox17 M76A inhibited Smad3 function (Fig. 7D). Immunocytochemistry demonstrated that full length Sox17 as well as the Sox17 deletions and point mutant all localized to the nucleus (Fig. S3). However, PLoS ONE | www.plosone.org 8 May 2009 | Volume 4 | Issue 5 | e5711