Journal: Cells

Article Title: FOXO1 Couples KGF and PI-3K/AKT Signaling to NKX2.1-Regulated Differentiation of Alveolar Epithelial Cells

doi: 10.3390/cells11071122

Figure Lengend Snippet: FOXO1 attenuates NKX2.1 activation of SFTPC and SFTPB . ( A ) MLE-15 cells were co-transfected with a 3.7 kb SFTPC reporter construct (3.7- SFTPC -Luc) and NKX2.1 or FOXO1 expression constructs alone or in combination. Dual luciferase assays 48 h following transduction showed that FOXO1 inhibited NKX2.1 activation of the SFTPC reporter. Firefly luciferase activity was normalized to Renilla luciferase. n = 4 for each group. One-way ANOVA, asterisk indicates p < 0.05. ( B ) MLE-15 cells were co-transfected with 3.7- SFTPC -Luc, pRC/CMV/ NKX2.1 and increasing concentrations of FOXO1 expression construct. Dual luciferase assays 48 h post-transduction showed that FOXO1 inhibited NKX2.1-mediated induction of the SFTPC reporter in a dose-dependent manner. The data are shown as normalized to the absence of FOXO1. n = 4 for each group. One-way ANOVA, asterisk indicates p < 0.05 compared to absence of FOXO1 (empty vector). ( C ) MLE-15 cells were co-transfected with a 1.4 kb SFTPB reporter construct (1.4- SFTPB -Luc), NKX2.1 and FOXO1 expression constructs alone, or in combination. Dual luciferase assays 48 h post-transduction showed that FOXO1 inhibited NKX2.1 activation of the SFTPB reporter. Firefly luciferase activity was normalized to Renilla luciferase. The data are shown as normalized to the absence of FOXO1 and NKX2.1. n = 3 for each group. One-way ANOVA, asterisk indicates p < 0.05. ( D ) Schematic showing the putative FOXO1 and NKX2.1 binding sites on human SFTPC promoter. ( E ) MLE-15 cells were co-transfected with a 3.7 kb SFTPC reporter construct (3.7- SFTPC C-Luc), NKX2.1 and FOXO1 wild-type (WT), or FOXO1H215R (DNA binding mutant) expression constructs alone or in combination. Dual luciferase assays 48 h post-transduction showed that FOXO1-H215R maintained the ability to repress NKX2.1 activation of the SFTPC reporter. Firefly luciferase activity was normalized to Renilla luciferase. The data are shown as normalized to the absence of FOXO1 and NKX2.1. n = 3 for each group. One-way ANOVA, asterisk indicates p < 0.05. ( F ) MLE-15 cells were co-transfected with a 318 bp Sftpc reporter p318mu sftpc -Luc which lacks a FOXO1 binding site and a NKX2.1 expression construct, together with increasing amounts of FOXO1 expression construct. Dual luciferase assays 48 h post-transduction showed that FOXO1 inhibited NKX2.1-induced activation of the 318 bp Sftpc reporter in a dose-dependent manner. The data are shown as normalized to the absence of FOXO1 (empty vector). n = 3 for each group. One-way ANOVA, asterisk indicates p < 0.05 compared to absence of FOXO1.

Article Snippet: 3.7- SFTPC -Luc contains the 3.7-kb human SFTPC promoter in pGL2Basic (Promega, Madison, WI, USA). pRC/CMV/NKX2.1 contains the 2.3-kb human NKX2.1 cDNA in pRC/CMV (Invitrogen). p318 mu- Sftpc -Luc contains −318 to -118 of the murine Sftpc promoter cloned into SmaI/XhoI sites of pGL2Basic [ ]. pCMV6-XL4/FOXO1 containing the human FOXO1 cDNA was purchased from Origene (Rockville, MD, USA). pCDNA3/FOXO1-AAA and pCDNA3/FOXO1 H215R were purchased from Addgene (Cambridge, MA, USA). pCDNA3/FOXO1, pCDNA3-FOXO1-N (1–257 a.a.), pCDNA3-FOXO1-C (211–416 a.a.), pGEX-KG-FOXO1 and pGEX-KG-FOXO1-N (1–257 a.a.) were generous gifts from Dr. K.L.

Techniques: Activation Assay, Transfection, Construct, Expressing, Luciferase, Transduction, Activity Assay, Plasmid Preparation, Binding Assay, Mutagenesis

Journal: Cells

Article Title: FOXO1 Couples KGF and PI-3K/AKT Signaling to NKX2.1-Regulated Differentiation of Alveolar Epithelial Cells

doi: 10.3390/cells11071122

Figure Lengend Snippet: FOXO1 physically interacts with the homeodomain of NKX2.1. Co-immunoprecipitation of nuclear extracts that were harvested from primary AT2 cells on day three in culture with anti-NKX2.1 ( A ) or anti-FOXO1 ( B ) Abs shows the association of endogenous FOXO1 and NKX2.1 ( n = 4). IgG was used as a negative control. ( C ) Schematic of GST-tagged FOXO1 fusion proteins (left panel) and GST pull-down assay (right panel). In vitro translated NKX2.1 was incubated with GST-FOXO1 fusion proteins (GST-FOXO1 full-length (FL), GST-FOXO1-N, GST-FOXO1-FK, GST-FOXO1-N + FK, GST-FOXO1-M, GST-FOXO1-C) coupled to glutathione sepharose. Bound NKX2.1 was visualized by Western blotting using an anti-NKX2.1 antibody. The FOXO1 FL and FOXO1 FK domains interact with NKX2.1 (right panel). n = 3. ( D ) Schematic of GST-tagged NKX2.1 fusion proteins (left panel) and GST pull-down assay (right panel). In vitro translated FOXO1 was incubated with GST- NKX2.1 fusion proteins (GST-NKX2.1 FL, GST-NKX2.1-N, GST-NKX2.1-HD, and GST-NKX2.1-C) that were coupled to glutathione sepharose. The bound FOXO1 was visualized by Western blotting using an anti-FOXO1 Ab. FOXO1 interacts with the NKX2.1 homeodomain (right panel). n = 3. ( E , F ) EMSA was performed with nuclear extracts from MLE-15 cells and biotin-labeled oligonucleotides encompassing the NKX2.1 DNA-binding site (−186 to −163 bp) of the SFTPC promoter. The arrow points to the inhibition of the NKX2.1 protein/DNA complexes (lane 2) with increasing amounts of GST-FOXO1 fusion protein (lane 8–10) ( E ) or GST-FOXO1 FK domain fusion protein (lane 8–10) ( F ) but not by GST alone (lane 4–6). Lane 1 is probe only. GST (lane 4) and GST-FOXO1 or GST-FOXO1 FK (lane 7) proteins do not form a complex with the oligonucleotide probe. n = 3. ( G ) Publicly available ChIPseq analysis of NKX2-1 binding surrounding the Sftpc and Sftpb loci in Sftpc + AT2 and Wnt3A + AT1 cells. Peaks were called using MACS2.0 as outlined by Little DR et al. . 0–150 indicates number of Chip-Seq reads overlapping at a given base.

Article Snippet: 3.7- SFTPC -Luc contains the 3.7-kb human SFTPC promoter in pGL2Basic (Promega, Madison, WI, USA). pRC/CMV/NKX2.1 contains the 2.3-kb human NKX2.1 cDNA in pRC/CMV (Invitrogen). p318 mu- Sftpc -Luc contains −318 to -118 of the murine Sftpc promoter cloned into SmaI/XhoI sites of pGL2Basic [ ]. pCMV6-XL4/FOXO1 containing the human FOXO1 cDNA was purchased from Origene (Rockville, MD, USA). pCDNA3/FOXO1-AAA and pCDNA3/FOXO1 H215R were purchased from Addgene (Cambridge, MA, USA). pCDNA3/FOXO1, pCDNA3-FOXO1-N (1–257 a.a.), pCDNA3-FOXO1-C (211–416 a.a.), pGEX-KG-FOXO1 and pGEX-KG-FOXO1-N (1–257 a.a.) were generous gifts from Dr. K.L.

Techniques: Immunoprecipitation, Negative Control, Pull Down Assay, In Vitro, Incubation, Western Blot, Labeling, Binding Assay, Inhibition, ChIP-sequencing

Journal: Cells

Article Title: FOXO1 Couples KGF and PI-3K/AKT Signaling to NKX2.1-Regulated Differentiation of Alveolar Epithelial Cells

doi: 10.3390/cells11071122

Figure Lengend Snippet: FOXO1 repressor function is negatively regulated by PI-3K-dependent phosphorylation. ( A ) MLE-15 cells were co-transfected with the 3.7-SFTPC-Luc reporter construct, NKX2.1 expression construct and FOXO1 or FOXO1-AAA, a dephosphorylated constitutively active form of FOXO1. Dual luciferase assays were performed 48 h after transfection. Firefly luciferase activity was normalized to Renilla luciferase. The data are shown as normalized to the absence of FOXO1 and NKX2.1. n = 3 for each group. One-way ANOVA, asterisk indicates p < 0.05. ( B ) Western blotting for p -AKT (ser473) and p-FOXO1 in MLE-15 cells shows that Ly294002 treatment (7 h) decreases p-AKT and p-FOXO1. n = 2. ( C ) MLE-15 cells were co-transfected with a 3.7-SFTPC-Luc reporter construct and NKX2.1 expression construct for 24 h, followed by treatment with Ly294002 (1 µM) for an additional 24 h. Dual luciferase assay showed that Ly294002 inhibited SFTPC reporter activity. The data are shown as normalized to the absence of both NKX2 and Ly294002. n = 3 for each group. One-way ANOVA, asterisk indicates p < 0.05. ( D ) Co-IP was performed with cell lysates that were harvested from MLE-15 cells that were cultured in the presence or absence of PI-3K inhibitor Ly294002 (6 μM) for 48 h. Increased association of NKX2.1 with FOXO1 was detected in the Ly294002-treated samples. n = 3.

Article Snippet: 3.7- SFTPC -Luc contains the 3.7-kb human SFTPC promoter in pGL2Basic (Promega, Madison, WI, USA). pRC/CMV/NKX2.1 contains the 2.3-kb human NKX2.1 cDNA in pRC/CMV (Invitrogen). p318 mu- Sftpc -Luc contains −318 to -118 of the murine Sftpc promoter cloned into SmaI/XhoI sites of pGL2Basic [ ]. pCMV6-XL4/FOXO1 containing the human FOXO1 cDNA was purchased from Origene (Rockville, MD, USA). pCDNA3/FOXO1-AAA and pCDNA3/FOXO1 H215R were purchased from Addgene (Cambridge, MA, USA). pCDNA3/FOXO1, pCDNA3-FOXO1-N (1–257 a.a.), pCDNA3-FOXO1-C (211–416 a.a.), pGEX-KG-FOXO1 and pGEX-KG-FOXO1-N (1–257 a.a.) were generous gifts from Dr. K.L.

Techniques: Transfection, Construct, Expressing, Luciferase, Activity Assay, Western Blot, Co-Immunoprecipitation Assay, Cell Culture

Journal: Cells

Article Title: FOXO1 Couples KGF and PI-3K/AKT Signaling to NKX2.1-Regulated Differentiation of Alveolar Epithelial Cells

doi: 10.3390/cells11071122

Figure Lengend Snippet: Model of mechanisms underlying FOXO1 regulation of lung-specific NKX2.1-activated genes via PI-3K/AKT-dependent phosphorylation. In AT2 and AT2-like cells in the presence of KGF, the PI-3K/AKT pathway is activated, leading to downstream phosphorylation of FOXO1 and subsequent cytoplasmic extrusion and degradation. The absence of nuclear FOXO1 allows active NKX2.1 to remain at the SFTPC and SFTPB promoters, increasing the expression of these AT2 cell-specific target genes. During the transition from AT2 to AT1-like cells (AT1.5 intermediate cells), or in the absence of KGF, the PI-3K/AKT pathway becomes inactivated. This in turn prevents the phosphorylation of FOXO1, allowing it to remain in the nucleus and interact with and pull NKX2.1 from its target promoters. This process is completed in differentiated AT1-like cells, where NKX2.1 removal from target genes by FOXO1 leads to the inhibition of gene expression. The figure was created with BioRender (BioRender Academic License https://biorender.com/ (accessed on 14 March 2022)).

Article Snippet: 3.7- SFTPC -Luc contains the 3.7-kb human SFTPC promoter in pGL2Basic (Promega, Madison, WI, USA). pRC/CMV/NKX2.1 contains the 2.3-kb human NKX2.1 cDNA in pRC/CMV (Invitrogen). p318 mu- Sftpc -Luc contains −318 to -118 of the murine Sftpc promoter cloned into SmaI/XhoI sites of pGL2Basic [ ]. pCMV6-XL4/FOXO1 containing the human FOXO1 cDNA was purchased from Origene (Rockville, MD, USA). pCDNA3/FOXO1-AAA and pCDNA3/FOXO1 H215R were purchased from Addgene (Cambridge, MA, USA). pCDNA3/FOXO1, pCDNA3-FOXO1-N (1–257 a.a.), pCDNA3-FOXO1-C (211–416 a.a.), pGEX-KG-FOXO1 and pGEX-KG-FOXO1-N (1–257 a.a.) were generous gifts from Dr. K.L.

Techniques: Expressing, Inhibition