An essential function for autocrine hedgehog signaling in epithelial proliferation and differentiation in the trachea

Abstract

The tracheal epithelium is a primary target for pulmonary diseases as it provides a conduit for air flow between the environment and the lung lobes. The cellular and molecular mechanisms underlying airway epithelial cell proliferation and differentiation remain poorly understood. Hedgehog (HH) signaling orchestrates communication between epithelial and mesenchymal cells in the lung, where it modulates stromal cell proliferation, differentiation and signaling back to the epithelium. Here, we reveal a previously unreported autocrine function of HH signaling in airway epithelial cells. Epithelial cell depletion of the ligand sonic hedgehog (SHH) or its effector smoothened (SMO) causes defects in both epithelial cell proliferation and differentiation. In cultured primary human airway epithelial cells, HH signaling inhibition also hampers cell proliferation and differentiation. Epithelial HH function is mediated, at least in part, through transcriptional activation, as HH signaling inhibition leads to downregulation of cell type-specific transcription factor genes in both the mouse trachea and human airway epithelial cells. These results provide new insights into the role of HH signaling in epithelial cell proliferation and differentiation during airway development.

Keywords: Airway epithelial cells; Smoothened; Sonic hedgehog; Trachea; Tracheomalacia.

Fig. 1.

Nkx2.1^Cre;Shh^flox/flox mice exhibit fracture of the tracheal cartilage, pulmonary hemorrhage and cystic lungs. (A,B) mRNA in situ detection of Cdh1 (dark green), Trp63 (pink), Shh (magenta), Smo (yellow), Ptch1 (cream), Ptch2 (blue), Hhip (pale green), Gli1 (orange), Gli2 (cyan) and Gli3 (khaki) expression and DAPI staining (gray) of longitudinal sections of wild-type tracheae at different developmental stages. The dashed square in the left-hand images indicates the area enlarged and rotated in the right-hand images. Dashed lines in the right-hand images indicate the border between the tracheal epithelium and mesenchyme. (C) Quantification of the percentage of positive cells within each cell state that express HH signaling components. (D) Quantification of the percentage of positive cells that express HH signaling components. Number of cells at E12.5=239, E13.5=455, E14.5=1232, E15.5=931, E16.5=365 and E18.5=286. (E) Representative gross morphology of P0 control (n=6) and ShhCKO mutants (n=6). (F) Quantification of P0 control (n=6) and ShhCKO mutant (n=6) respiratory rates. (G) Representative images of transverse sections of tracheae stained with Alcian Blue and nuclear Fast Red from P0 control (n=5) and ShhCKO mutants (n=5). (H) Quantification of P0 control (n=5) and ShhCKO mutant (n=5) tracheal lumen areas. (I) Representative images of ventral views of whole-mount tracheae stained with Alcian Blue from P0 control (n=6) and ShhCKO mutants (n=6). Arrows indicate tracheal cartilage rings. (J) Quantification of the number of intact tracheal cartilage rings from P0 control (n=6) and ShhCKO mutants (n=6). (K) Representative images of ventral views of the trachea and lungs from P0 control (n=6) and ShhCKO mutants (n=6). White arrow indicates pulmonary cysts. Blue arrow indicates blood spots. (L) Percentage of lungs with hemorrhage. (M) Percentage of lungs with cysts. Scale bars: 2 mm in K; 1 mm in E,I; 400 μm in G; 100 μm in low-magnification tracheal large images in A,B; 10 μm in high-magnification tracheal images in A,B. Unpaired Student's t-test. Data are mean±s.d. (F,H,J,L,M). E, epithelium; M, mesenchyme; Shh, sonic hedgehog; Smo, smoothened; ShhCKO, sonic hedgehog conditional knockout.

Fig. 2.

Nkx2.1^Cre;Shh^flox/flox mice exhibit defects in tracheal epithelial cell proliferation and differentiation. (A) Immunostaining for FOXJ1 (red), NKX2.1 (green) and DAPI staining (blue) of longitudinal sections of E18.5 control (n=6) and ShhCKO (n=6) tracheae. (B) Quantification of the relative number of FOXJ1⁺ cells in the tracheal epithelium in E18.5 control (n=6) and ShhCKO (n=6) mice. (C) RT-qPCR analysis of Foxj1, Tubb4b, Scgb1a1 and Foxp1 mRNA levels in E18.5 control (n=5) and ShhCKO (n=5) tracheae. (D) Immunostaining for acetylated α-tubulin (red) and DAPI staining (blue) of longitudinal sections of E18.5 control (n=6) and ShhCKO (n=6) tracheae. (E) Quantification of the relative number of acetylated α-tubulin⁺ cells in the tracheal epithelium in E18.5 control (n=6) and ShhCKO (n=6) mice. (F) Immunostaining for SCGB1A1 (red), NKX2.1 (green) and DAPI staining (blue) of longitudinal sections of E18.5 control (n=6) and ShhCKO (n=6) tracheae. (G) Quantification of the relative number of SCGB1A1⁺ cells in the tracheal epithelium in E18.5 control (n=6) and ShhCKO (n=6) mice. (H) Immunostaining for CDH1 (red), Ki67 (green) and DAPI staining (blue) of longitudinal sections of E18.5 control (n=6) and ShhCKO (n=6) tracheae. (I) Ratio of CDH1⁺ cells that are Ki67⁺. (J) EdU fluorescence (red), immunostaining for CDH1 (green) and DAPI staining (blue) of longitudinal sections of E18.5 control (n=6) and ShhCKO (n=6) tracheae. (K) Ratio of CDH1⁺ cells that are EdU⁺. (L) Immunostaining for SCGB1A1 (red), Ki67 (green) and DAPI staining (blue) of longitudinal sections of E18.5 control (n=6) and ShhCKO (n=6) tracheae. (M) Ratio of SCGB1A1⁺ cells that are Ki67⁺. Scale bars: 20 μm. Unpaired Student's t-test. Data are mean±s.d.

Fig. 3.

Smo mediates HH-signaling in tracheal epithelial cells and controls their differentiation and proliferation. (A) Immunostaining for FOXJ1 (red), NKX2.1 (green) and DAPI staining (blue) of longitudinal sections of E18.5 control (n=6) and SmoCKO (n=6) tracheae. (B) Quantification of the relative number of FOXJ1⁺ cells in the tracheal epithelium in E18.5 control (n=6) and SmoCKO (n=6) mice. (C) RT-qPCR analysis of Foxj1, Tubb4b and Scgb1a1 mRNA levels in E18.5 control (n=5) and SmoCKO (n=5) tracheae. (D) Immunostaining for acetylated α-tubulin (red), NKX2.1 (green) and DAPI staining (blue) of longitudinal sections of E18.5 control (n=6) and SmoCKO (n=6) tracheae. (E) Quantification of the relative number of acetylated α-tubulin⁺ cells in the tracheal epithelium in E18.5 control (n=6) and SmoCKO (n=6) mice. (F) Immunostaining for SCGB1A1 (red), NKX2.1 (green) and DAPI staining (blue) of longitudinal sections of E18.5 control (n=6) and SmoCKO (n=6) tracheae. (G) Quantification of the relative number of SCGB1A1⁺ cells in the tracheal epithelium in E18.5 control (n=6) and SmoCKO (n=6) mice. (H) EdU fluorescence (red), immunostaining for CDH1 (green) and DAPI staining (blue) of longitudinal sections of E18.5 control (n=6) and SmoCKO (n=6) tracheae after a 21 h 10 μM EdU treatment. (I) Ratio of CDH1⁺ cells that are EdU⁺. (J) Immunostaining for acetylated α-tubulin (red) and DAPI staining (blue) of longitudinal sections of P9 control (n=4) and Nkx2.1^Cre;R26^SmoM2 (n=4) tracheae. (K) Quantification of the relative number of acetylated α-tubulin⁺ cells in the tracheal epithelium in P9 control (n=4) and Nkx2.1^Cre;R26^SmoM2 (n=4) mice. (L) Immunostaining for SCGB1A1 (red) and DAPI staining (blue) of longitudinal sections of P9 control (n=4) and Nkx2.1^Cre;R26^SmoM2 (n=4) tracheae. (M) Quantification of the relative number of SCGB1A1⁺ cells in the tracheal epithelium in P9 control (n=4) and Nkx2.1^Cre;R26^SmoM2 (n=4) mice. Scale bars: 20 μm. Unpaired Student's t-test. Data are mean±s.d.

Fig. 4.

HH signaling regulates HBE cell differentiation. (A-D) RT-qPCR analysis of mRNA levels of HH-signaling components in ALI-cultured HBE cells (n=6 per stage). (A) SHH, (B) SMO, (C) GLI1 (D) and GLI2. (E) Immunostaining for FOXJ1 (red), MUC5AC (green) and DAPI staining (blue) in HBE cells at the ALI after 9 days of ethanol (n=6) or 10 μM cyclopamine (n=6) treatment. (F) Quantification of the relative number of FOXJ1⁺ cells in HBE cells at the ALI after 9 days of ethanol (n=6) or 10 μM cyclopamine (n=6) treatment. 2943 and 477 FOXJ1⁺ cells were analyzed for controls and 10 μM cyclopamine treatment, respectively. (G) Quantification of the relative number of MUC5AC⁺ cells in HBE cells at the ALI after 9 days of ethanol (n=6) or 10 μM cyclopamine (n=6) treatment. 3039 and 591 MUC5AC⁺ cells were analyzed for controls and 10 μM cyclopamine treatment, respectively. (H) RT-qPCR analysis of FOXJ1, MUC5AC, MUC5B and SPDEF mRNA levels in HBE cells at the ALI after 9 days of ethanol (n=6) or 10 μM cyclopamine (n=6) treatment. (I) Immunostaining for acetylated α-tubulin (red), MUC5B (green) and DAPI staining (blue) in HBE cells at the ALI after 9 days of ethanol (n=6) or 10 μM cyclopamine (n=6) treatment. (J) Quantification of the relative number of acetylated α-tubulin⁺ cells in HBE cells at the ALI after 9 days of ethanol (n=6) or 10 μM cyclopamine (n=6) treatment. 2670 and 312 acetylated α-tubulin⁺ cells were analyzed for controls and 10 μM cyclopamine treatment, respectively. (K) Quantification of the relative number of MUC5B⁺ cells in HBE cells at the ALI after 9 days of ethanol (n=6) or 10 μM cyclopamine (n=6) treatment. 5379 and 1089 MUC5B⁺ cells were analyzed for controls and 10 μM cyclopamine treatment, respectively. (L) Immunostaining for FOXJ1 (red), MUC5AC (green) and DAPI staining (blue) in HBE cells at the ALI after 9 days of double distilled H₂O (n=6) or 100 ng/ml H-SHH (n=6) treatment. (M) Quantification of the relative number of FOXJ1⁺ cells in HBE cells at the ALI after 9 days of double distilled H₂O (n=6) or 100 ng/ml H-SHH (n=6) treatment. 2685 and 5007 FOXJ1⁺ cells were analyzed for controls and 100 ng/ml H-SHH treatment, respectively. (N) Quantification of the relative number of MUC5AC⁺ cells in HBE cells at the ALI after 9 days of double distilled H₂O (n=6) or 100 ng/ml H-SHH (n=6) treatment. 2838 and 3561 MUC5AC⁺ cells were analyzed for controls and 100 ng/ml H-SHH treatment, respectively. (O) RT-qPCR analysis of FOXJ1 and MUC5AC mRNA levels in HBE cells at the ALI after 9 days of double distilled H₂O (n=6) or 100 ng/ml H-SHH (n=6) treatment. Scale bars: 100 μm. Unpaired Student's t-test. Data are mean±s.d. HBE, human bronchial epithelial; H-SHH, recombinant human SHH.

Fig. 5.

HH signaling regulates HBE cell proliferation. (A) Timeline for EdU, BrdU and cyclopamine administration. (B) Immunostaining for Ki67 (violet), BrdU (green) and EdU fluorescence (red) in HBE cells at the ALI after ethanol (n=6) or 10 μM cyclopamine (n=6) treatment at day 3. (C) Immunostaining for Ki67 (violet), BrdU (green) and EdU fluorescence (red) in HBE cells at the ALI after ethanol (n=6) or 10 μM cyclopamine (n=6) treatment at day 5. (D) Immunostaining for Ki67 (violet), BrdU (green) and EdU fluorescence (red) in HBE cells at the ALI after ethanol (n=6) or 10 μM cyclopamine (n=6) treatment at day 7. (E) Ratio of BrdU⁺ cells to EdU⁺ cells at day 3. 1143 and 210 BrdU⁺ cells were analyzed for controls and 10 μM cyclopamine treatment, respectively. (F) Ratio of Ki67⁺ cells to EdU⁺ cells at day 3. 1278 and 225 Ki67⁺ cells were analyzed for controls and 10 μM cyclopamine treatment, respectively. (G) Ratio of BrdU⁺ cells to EdU⁺ cells at day 5. 1491 and 408 BrdU⁺ cells were analyzed for controls and 10 μM cyclopamine treatment, respectively. (H) Ratio of Ki67⁺ cells to EdU⁺ cells at day 5. 1479 and 432 Ki67⁺ cells were analyzed for controls and 10 μM cyclopamine treatment, respectively. (I) Ratio of BrdU⁺ cells to EdU⁺ cells at day 7. 1467 and 375 BrdU⁺ cells were analyzed for controls and 10 μM cyclopamine treatment. (J) Ratio of Ki67⁺ cells to EdU⁺ cells at day 7. 1611 and 429 Ki67⁺ cells were analyzed for controls and 10 μM cyclopamine treatment, respectively. Scale bars: 100 μm. Unpaired Student's t-test. Data mean±s.d.