Active epithelial Hippo signaling in idiopathic pulmonary fibrosis (original) (raw)
Activation of YAP-mediated gene expression in IPF. An unbiased analysis of RNAseq data from FACS isolated epithelial cells (CD326+/HTII-280+) from normal and IPF, and primary human bronchiolar epithelial cells (HBECs) expressing activated YAP (S127A) (19), was performed to predict the bioprocesses and pathways shared in these data sets. Genes encoding proteins involved in mTOR, PI3K/AKT, and Hippo/YAP and WNT signaling were predicted to be active by functional classification and network construction using ingenuity pathway analysis (IPA) (Figure 1A). Network analysis predicted extensive interactions among mTOR, PI3K/AKT, planar polarity, and Hippo/YAP signaling. These processes and pathways are involved in the regulation of epithelial cell size, migration, proliferation, differentiation, and cell polarity, supporting the hypothesis that these phenotypic features in IPF are regulated in part by activation of Hippo/YAP–associated signaling (Figure 1A). Predicted pathways and gene expression changes in IPF are shown in Figure 1B, including increased YAP1, JUB, AKT3, AMOTL2, GAS6, and CYP24A1 RNAs. Gene expression profiles of sorted IPF epithelial cells and of primary HBECs expressing activated YAP (S127A) shared enriched bioprocesses, including extracellular matrix organization, cell migration, response to wound, cell size, and epithelial proliferation/ differentiation, as well as increased expression of genes associated with canonical TGF-β, Hippo/YAP, and PI3K/AKT signaling pathways (Figure 1, C and D) essential for processes known to be regulated by the Hippo/YAP pathway (16).
Prediction of signaling interactions in idiopathic pulmonary fibrosis (IPF) epithelial cells. (A) Ingenuity pathway analysis of RNA sequencing data from CD326+/HTII-280+ sorted epithelial cells from healthy donors (n = 3) and IPF (n = 3) was used to predict intensive interactions among mTOR/PI3K/AKT, Hippo/YAP, and polarity signaling pathways. (B) Genes associated with each of the pathways significantly altered in IPF are shown. Each pathway is represented by a distinct color code: mTOR (blue), PI3K/AKT (yellow), Hippo (pink), and polarity (green). (C and D) Functional enrichment analysis predicted that genes induced in CD326+/HTII-280+ IPF epithelial cells (8) and in human airway epithelial cells (HAECs) expressing YAP (20) share (C) commonly activated bioprocesses and (D) signaling pathways including those affecting epithelial cell proliferation, migration, and cell size. The x axis represents the –log10-transformed enrichment P value.
Increased YAP activity in IPF epithelial cells. Immunofluorescence confocal microscopy and in situ hybridization RNA analyses of peripheral lung tissue demonstrated increased nuclear YAP and decreased MST1/2 in IPF epithelial cells that costained with ABCA3 or pan-cytokeratin. Consistent with increased nuclear YAP, staining for Ajuba, a known transcriptional target of YAP, was increased and primarily detected in epithelial cells in IPF lesions (Figure 2, A–C). In sharp contrast, nuclear YAP was rarely detected and MST1/2 was highly expressed in alveolar AT2 cells and bronchial epithelial cells in normal donor lung tissues. Western blot analysis of whole-lung lysates demonstrated increased total YAP in IPF and chronic obstructive pulmonary disease (COPD) tissues, while levels of phosphorylated YAP were similar in normal, IPF, and COPD lung samples (Figure 2D). The ratio of total YAP to phosphorylated YAP was significantly increased in IPF compared with normal lung tissues, consistent with increased active YAP in IPF (Figure 2E).
Hippo/YAP signaling in idiopathic pulmonary fibrosis (IPF). (A–C) Representative immunofluorescence confocal microscopy of donor (n = 4) and IPF (n = 6) lung tissue was used to detect Hippo components. (A) AJUBA (white), ACTA2 (green), and ABCA3 (red). (B) YAP (green) and ABCA3 (red). (C) MST1/2 (red) and ABCA3 (green). White arrows point to cells coexpressing ABCA3 and nuclear YAP in IPF. Scale bars: 100 μm (overviews) and 10 μm (insets). (D) Representative immunoblots of YAP and phosphorylated YAP (p-YAP) from lung tissue lysates of IPF (n = 6), control (n = 4), and COPD (n = 3) patients are shown. (E) Western blots were normalized to β-actin. (F) Multiplexed proximity ligation fluorescence in situ hybridization (PLISH) staining of CTGF (red) and AXL (white) were costained using pan-cytokeratin (PanKRT) (green) in normal donor (n = 6) and IPF (n = 6) lung tissue. Scale bars: 25 μm and 2.5 μm (insets). (G) qPCR analysis of RNA from CD326+ epithelial cells isolated from peripheral lungs of healthy donor (n = 3) and IPF (n = 3) lung tissue was used to quantify SAV1, MST2, and JUB RNAs. ANOVA was used to assess Western blot quantification; Student’s t test was used for qPCR. *P < 0.05.
RNAs associated with YAP activation are increased in IPF epithelial cells. Proximity ligation fluorescence in situ hybridization (PLISH) was used to detect YAP target genes AXL and CTGF in IPF epithelial cells (Figure 2F). AXL RNA was increased throughout the lung and particularly in epithelial cells. SAV1 and MST2, inhibitors of YAP activity, were reduced, while JUB was increased in RNA from CD326+ sorted IPF respiratory epithelial cells (Figure 2G). Loss of the YAP inhibitors, SAV1 and MST2, and increased YAP target gene expression were consistent with increased YAP transcriptional activity in IPF epithelial cells.
Epithelial cell polarity is disrupted in IPF. Since expression of a number of genes related to planar cell polarity, including SCRIB and VANGL1, were increased in CD326+ sorted IPF epithelial cells (Figure 1, A and B), we used immunofluorescence staining of epithelial cell polarity markers scribble (SCRIB) and vang-like (VANGL) to assess epithelial cell polarity in IPF. In contrast to apical localization in normal epithelial cells, SCRIB and VANGL staining was increased and diffuse (Figure 3, A and B), indicating loss of normal apical-basal polarity in IPF epithelial cells. VANGL1, SCRIB, and CELSR1 RNAs were increased in IPF epithelial cells, consistent with disruption of normal cell polarity in IPF (Figure 3C), and consistent with RNAseq data that demonstrated increased expression of cell polarity genes in IPF (Figure 3D).
Vangl and Scribble in idiopathic pulmonary fibrosis (IPF) lung tissue. (A and B) Representative immunofluorescence imaging of healthy donor (n = 4) and IPF (n = 6) lung tissue are shown. (A) Scribble (Scrib) (green) and Krt8 (red) or (B) Vangl (red) and Krt8 (green). Scale bars: 50 μm and 5 μm (insets). (C) CDH1, CELSR1, SCRIB, and VANGL1 RNAs were measured in CD326+ sorted epithelial cells from healthy donors (n = 3) and IPF (n = 3) lungs. (D) RNA sequencing analysis of polarity-associated genes of healthy donors (n = 3) and IPF (n = 3) demonstrating significant increases in epithelial cell polarity genes including WNT9A and WNT7A. *P < 0.05, calculated by Student’s t test. N.S., not significant.
Increased mTOR activity in IPF respiratory epithelial cells and lung tissue. Since the RNA network analysis predicted that YAP and mTOR/PI3K/AKT may interact to regulate gene expression in IPF epithelial cells, immunofluorescence imaging was used to assess levels of phosphorylated S6 kinase (p-S6K), a downstream component of mTOR/PI3K/AKT signaling, in IPF epithelial cells (Figure 4A). p-S6K staining was increased throughout IPF lung tissue and selectively increased in and colocalized primarily with pan-cytokeratin staining in IPF epithelial cells. Immunoblotting demonstrated increased p-PTEN, p-S6, and total S6 in IPF tissues compared with tissue from donors or COPD patients (Figure 4, B and C). Increased expression of mTOR/PI3K/AKT components and inhibition of PTEN in IPF epithelial cells were consistent with increased mTOR activity.
Increased mTOR signaling in idiopathic pulmonary fibrosis (IPF) respiratory epithelium. (A) Representative immunofluorescence imaging of healthy donor (n = 3) and IPF (n = 3) lung for phosphorylated S6K (p-S6K) (red) with pan-cytokeratin (Pan-KRT) (green) is shown. Scale bars: 50 μm and 5 μm (insets). (B) Representative immunoblots were prepared from whole-lung lysates of donor (n = 4), COPD (n = 3), and IPF (n = 6) lung tissue for total S6, phosphorylated S6 (p-S6), and phosphorylated PTEN (p-PTEN). (C) Western blots were normalized to GAPDH. *P < 0.05, determined by ANOVA.
YAP activates mTOR/PI3K/AKT signaling in HBECs. To directly test the hypothesis that YAP and mTOR interact to influence cellular functions in IPF, hTERT/CDK4–immortalized human bronchiolar epithelial cells (HBEC3KT, referred to as HBEC3) were transduced with lentivirus expressing YAP (WT) or constitutively active YAP (S127A) to test whether YAP influences mTOR/PI3K/AKT signaling. Since the characteristics of the abnormal epithelial cells in IPF are consistent with conducting airway cells, we utilized HBEC3s that maintain basal characteristics in vitro and are readily transfected with lentiviral constructs, to study the effects of YAP and mTOR interaction. Immunofluorescence confocal microscopy demonstrated increased nuclear YAP after lentiviral transduction (Figure 5A). Western blotting and quantitative PCR (qPCR) analyses demonstrated increased YAP and YAP transcriptional target RNAs, i.e., JUB, AXL, and CTGF, consistent with activation of YAP (Figure 5B). YAP induced planar polarity genes SCRIB and VANGL1 (Figure 5C). Activated YAP (S127A) increased the phosphorylation of S6, PI3K, AKT, and PTEN, indicating that YAP activates the mTOR/PI3K/AKT pathway in HBEC3s (Figure 5D). Expression of YAP (WT) increased p-PI3K but did not alter p-S6, p-AKT, or p-PTEN (Figure 5E). YAP-mediated induction of p-S6, p-PI3K, and inhibition of PTEN support the predicted interactions between YAP and mTOR/PI3K/AKT activity in IPF.
YAP (S127A) activates mTOR/PI3K/AKT. (A) HBEC3s were transduced with lentiviruses expressing YAP (WT), YAP (S127A), or empty GFP vector. YAP (red) was identified by immunofluorescence. Images are representative of (n = 3) transductions. Scale bars: 10 μm. (B) qPCR analysis of cells 48 hours after transduction assessing genes regulated by YAP activity. (C) Analysis of polarity genes CELSR1, SCRIB, and VANGL1 in HBEC3s expressing YAP (WT) and YAP (MUT) for 48 hours. (D) Forty-eight hours after transduction, lysates were prepared and immunoblotted for YAP, p-YAP, p-PTEN, S6, p-S6, PI3K, p-PI3K, AKT, and p-AKT. (E) Western blot quantification normalized to GAPDH. *P < 0.05, assessed by ANOVA. HBEC3s, hTERT/CDK4–immortalized human bronchiolar epithelial cells.
Verteporfin inhibits YAP-induced targets, p-S6, cell proliferation, and migration. Verteporfin inhibits YAP transcriptional activity by influencing YAP-TEAD interactions (27). In HBEC3s, verteporfin inhibits YAP and YAP-induced cell proliferation, without affecting cell viability (Supplemental Figure 1, C and D; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.98738DS1). Verteporfin reduced nuclear YAP staining, total YAP expression, and inhibited p-S6 in HBEC3s (Figure 6A and Supplemental Figure 1A). Since verteporfin is known to be photo activated (27), we assessed YAP and its transcriptional targets AXL, JUB, and CTGF RNAs in the presence (Figure 6) and absence of ambient light (Figure 7). Total YAP was reduced following verteporfin treatment in darkness (Figure 7A). YAP targets were significantly reduced regardless of light exposure (Figures 6B and 7B); however, in the absence of light, higher concentrations of verteporfin were required (Supplemental Figure 4). Consistent with verteporfin regulating YAP activity, PLAU and WNT7B were reduced following YAP (WT) and YAP (S127A) expression, and were significantly increased by verteporfin treatment (Figure 7B). Expression of YAP (WT) and YAP (S127A) increased cell migration, which was blocked by verteporfin in the presence (Figure 6C and Supplemental Figure 1B) or absence of ambient light (Figure 7C and Supplemental Figure 2). Verteporfin inhibited nuclear localization of YAP, total YAP mRNA, YAP target gene expression, and YAP-mediated cell migration. To test whether verteporfin regulates YAP through proteasomal degradation, YAP-expressing cells were treated with verteporfin in the presence of proteasome inhibitor MG132. MG132 protected YAP from verteporfin-induced degradation. To test if mTOR activation rescued the loss of YAP by verteporfin, cells were treated with verteporfin in the presence of mTOR activator MHY1485, whereby expression of YAP and p-S6 were increased; however, MHY1485 failed to block loss of YAP following verteporfin treatment (Figure 7B). Thus, activated mTOR was not sufficient to prevent destabilization and inhibition of YAP by verteporfin.
Verteporfin inhibits YAP transcriptional activity and phosphorylation of S6 in ambient light. HBEC3s were transduced with GFP, YAP (WT), or YAP (S127A) lentiviral vectors and treated with either vehicle (DMSO) or 0.25 μg/ml verteporfin for 48 hours (n = 3). (A) Immunofluorescence of p-S6 (green) and YAP (red) was assessed following verteporfin (0.25 μg/ml) or DMSO. Scale bars: 10 μm. (B) YAP, JUB, AXL, CTGF, WNT7B, and PLAU RNAs were quantified following 0.25 μg/ml verteporfin (VP) or vehicle exposed to ambient light (n = 3). Expression is normalized to DMSO controls. (C) Scratch assay of YAP (S127A)–transduced HBEC3s following DMSO or verteporfin treatment (0.25 μg/ml) in ambient light at T = 0, 8, and 16 hours of assay (n = 3). *P < 0.05, determined by ANOVA. HBEC3s, hTERT/CDK4–immortalized human bronchiolar epithelial cells.
Verteporfin reduces YAP transcriptional activity and phosphorylation of S6 in absence of light. HBEC3s transduced with GFP, YAP (WT), or YAP (S127A) lentiviral vectors were treated with either vehicle (DMSO) or verteporfin (2.0 or 10.0 μg/ml) for 48 hours (n = 3). (A) Western blot for YAP and p-S6 following a verteporfin dose curve in complete darkness show reduced total YAP and p-S6. (B) Western blot analysis of YAP and p-S6 following verteporfin treatment for 48 hours with MHY1485 treatment for the final 24 hours shows increased YAP after mTOR activation. Addition of MG132 for the final 24 hours of verteporfin treatment protects YAP from degradation caused by verteporfin. (C) Yap, JUB, CTGF, AXL, PLAU, and WNT7B RNAs were assessed in experiments performed in complete darkness (n = 4 transfections). RNA expression is normalized to DMSO-treated cells. *P < 0.05, determined by ANOVA. HBEC3s, hTERT/CDK4–immortalized human bronchiolar epithelial cells.
Temsirolimus blocks nuclear YAP, YAP-induced gene targets, cell proliferation and migration. To test if mTOR influences YAP activity, HBEC3s expressing YAP (WT) or YAP (S127A) were treated with temsirolimus, an inhibitor of the mTOR pathway. Temsirolimus reduced YAP-mediated cell proliferation but did not affect cell viability (Supplemental Figure 3, B and C). Temsirolimus inhibited phosphorylation of S6 and remarkably, reduced nuclear YAP staining (Figure 8, A and B). Proteasome blocking with MG132 or activation of mTOR with MHY1485 did not prevent the loss of YAP by temsirolimus (Supplemental Figure 3D). Consistent with the inhibitory effect of temsirolimus on YAP activity, CTGF, JUB, and AXL were reduced (Figure 8C and Supplemental Figure 5). To further assess the interaction of YAP/mTOR in regulating cell behavior, migration scratch assays were performed in HBEC3s expressing YAP (WT) and YAP (S127A). Temsirolimus prevented YAP-induced cell migration in scratch assays (Figure 8D and Supplemental Figure 3A), supporting a network in which mTOR signaling cooperates with YAP to regulate epithelial cell migration and proliferation.
Temsirolimus inhibits phosphorylation of S6, YAP transcriptional activity, and YAP-induced migration. (A) Representative immunofluorescence microscopy was performed in HBEC3s transfected with GFP, YAP (WT), and YAP (S127A) and stained for p-S6 (green) and YAP (red) in the presence of temsirolimus (25 μg/ml) or DMSO (n = 5). Scale bars: 10 μm. (B) Western blot analysis of YAP following temsirolimus treatments shows reduced total YAP expression. (C) YAP, JUB, AXL, and CTGF RNAs were assessed after 48 hours (n = 3). RNA expression is normalized to DMSO-treated cells. (D) Time-lapse imaging of HBEC3s transduced with YAP (S127A) following treatment with temsirolimus at T = 0, 8, and 16 hours of a scratch assay (n = 3). *P < 0.05, assessed by ANOVA. HBEC3s, hTERT/CDK4–immortalized human bronchiolar epithelial cells.