Table 1.
Primer sequences used in real time PCR analyses.
Fig 1.
Phospho-JNK immunoreactivity is increased in lung tissues in settings of airways and parenchymal fibrosis.
(A) p-JNK in lung tissue derived from mice with bleomycin or adTGFβ1-induced pulmonary fibrosis. Mice were exposed to agents, and respective vehicle controls (con), and 3 weeks thereafter, lung tissues were homogenized for the assessment of pJNK via Western blot analysis. PBS or control-adenoviral vector exposed mice resulted in similar lack of p-JNK immunoreactivity (see Fig 1B below), and therefore only one representative control group (Con) is shown. (B) Immunolocalization of p-JNK (red) in lung tissue from mice 3 weeks after exposure to a representative vehicle control, bleomycin or adenovirus expressing recombinant active transforming growth factor beta-1 (adTGFβ1). Lower Panels; Co-localization of p-JNK with CCSP in control vehicle, bleomycin or adTGFβ1. Results were evaluated via confocal microscopy. Co-localization of p-JNK and CCSP is indicated by a yellow color. Scale bars: 50 μm. (C) p-JNK (blue) in lung tissue from patients with idiopathic pulmonary fibrosis (IPF) or non-IPF lung (Normal, n = 5 patients/group). Scale bars: 300 μm.
Fig 2.
Depiction of the conditional ablation strategy of JNK1 from lung epithelial cells.
(A) Schematic of JNK1 ablation from airway epithelial cells using transgenic mice expressing CCSP-rtTA, TetO-Cre, and Jnk1loxP/loxP alleles. Upon feeding doxycycline (dox)- containing food, JNK1 is ablated from airway epithelial cells, designated as ΔEpi Jnk1. (B) Confirmation of ablation of JNK1 in airway epithelial cells. 3 weeks after initiation of Dox feeding, single cells lung suspensions were created and cells were sorted by flow cytometry. The EpCAM positive, CD45 negative, Sca1 low fraction of lung cells was isolated for assessment of JNK1 expression via Western Blot analysis. Note that JNK1 was only ablated from the EpCAM positive, CD45 negative, Sca1low fraction of lung cells. (C) Visualization of Cre-recombination in CCSP-rtTA, TetO-Cre-expressing mice which were bred with Tomato red reporter mice. Red: Cre-expressing cells. Scale bars: 50 μm.
Fig 3.
Conditional ablation of JNK1 from lung epithelial cells attenuates lung fibrosis induced by bleomycin.
JNK1 was selectively ablated from the bronchiolar epithelial cells and type II cells using a CCSP promoter driving a reverse tetracycline activator (rtTA), the tetracyclin operon driving CRE recombinase (TetOP-Cre), and mice carrying a LoxP-flanked Jnk1 allele. Assessment of total collagen content in lung tissue 3 weeks following administration of bleomycin via hydroxyproline content (A), Masson’s trichrome staining (B), Sircol assay (C) and semi-quantitative evaluation of Masson’s trichrome reactive material (D). (E) Assessment of tissue elastance in mice exposed to bleomycin, and the impact of ablation of epithelial JNK1. ΔEpi Jnk1 mice or respective control groups, as described in the methods section were fed dox food for one week prior to exposure to bleomycin. 3 weeks post-administration of bleomycin, mice were evaluated via forced oscillation mechanics to assess tissue elastance. (WT- Jnk1: PBS n = 6, Bleo n = 12, ΔEpi Jnk1: PBS n = 5, Bleo n = 8 respectively mice/group from 2 independent experiments). * p< 0.05 compared to PBS control group. † p< 0.05 compared to respective WT groups. (ANOVA). Scale bars: 50 μm.
Fig 4.
Conditional ablation of JNK1 from lung epithelial cells attenuates lung fibrosis induced by adTGFβ1.
JNK1 was selectively ablated from the bronchiolar epithelial cells and type II cells using a CCSP promoter driving a reverse tetracycline activator (rtTA), the tetracyclin operon driving CRE recombinase (TetOP-Cre), and mice carrying a LoxP-flanked Jnk1 allele. Assessment of total collagen content in lung tissue 3 weeks following administration of bleomycin via hydroxyproline content (A), Masson’s trichrome staining (B), Sircol assay (C) and semi-quantitative evaluation of Masson’s trichrome reactive material (D). E: Assessment of pulmonary fibrosis, using the sircol assay in response to AdTGFβ1 in mice containing the CCSP-rtTA, TetO-Cre, and Jnk1 loxP/loxP alleles, or CCSP-rtTA, TetO-Cre alleles, in the absence of doxycycline-containing food. (WT- Jnk1: AdCtr n = 6, adTGFβ1 n = 8, ΔEpi Jnk1: AdCtr n = 5, adTGFβ1 n = 8 respectively mice/group from 2 independent experiments). * p< 0.05 compared to the adCtr control groups. † p< 0.05 compared to respective WT groups. (ANOVA). Scale bars: 50 μm.
Fig 5.
Evaluation of epithelial and mesenchymal gene expression profiles in lungs from ΔEpiJnk1 mice, or control groups subjected to bleomycin-induced lung fibrosis.
Analysis of mesenchymal (A) or epithelial (B) mRNA expression in homogenized lung tissue from mice exposed to bleomycin for 3 weeks. Results were normalized to the housekeeping gene cyclophilin, and are expressed as fold expression changes (+/- SEM) compared to the WT vehicle control groups. (WT- Jnk1: PBS n = 6, Bleo n = 12, ΔEpiJnk1: PBS n = 5, Bleo n = 8 respectively mice/group from 2 independent experiments). * p< 0.05 compared to the PBS control group. † p< 0.05 compared to the respective WT group. (ANOVA). C: Evaluation of mesenchymal proteins (α-SMA (Acta2), Col1a1, FSP1 (S100a4)) in lungs from ΔEpiJNK1 mice, or control groups subjected to bleomycin-induced lung fibrosis, and the impact of ablation of epithelial JNK1. Homogenized lung tissues were subjected to Western blot analysis for the indicated proteins. β-actin: Loading control. Shown are results from individual mice.
Fig 6.
Evaluation of epithelial and mesenchymal gene expression profiles in lungs from ΔEpiJNK1 mice, or control groups subjected to adTGFβ1-induced lung fibrosis.
Analysis of mesenchymal (A) or epithelial (B) mRNA expression in homogenized lung tissue from mice exposed to adTGFβ1 for 3 weeks. Results were normalized to the housekeeping gene cyclophilin, and are expressed as fold expression changes (+/- SEM) compared to the WT vehicle control groups. (WT- Jnk1: AdCtr n = 6, adTGFβ1 n = 8, ΔEpi Jnk1: AdCtr n = 5, adTGFβ1 n = 8 respectively mice/group from 2 independent experiments). * p< 0.05 compared to the adCtr control groups. † p< 0.05 compared to respective WT groups. (ANOVA). C: Evaluation of mesenchymal proteins (α-SMA (Acta2) and Col1a1 in lungs from ΔEpi Jnk1 mice or control groups (WT mice) subjected to adTGFβ1-induced lung fibrosis. Homogenized lung tissues were subjected to Western blot analysis for the indicated proteins. β-actin: Loading control. Shown are results from individual mice.
Fig 7.
The impact of delayed ablation of epithelial JNK1 in adTGFβ1-induced lung fibrosis.
Delayed ablation of JNK1 from lung epithelial cells reverses existing increases in lung fibrosis induced by AdTGFβ1. Mice expressing either CCSP-rtTA TetO-CRE transgenes along with the WT Jnk1 allele, or CCSP-rtTA, tetO-CRE, Jnk1LoxP/LoxP transgenes (WT Jnk1) were exposed to AdTGFβ1 or AdCtr vector. As a control, CCSP-rtTA, tetO-CRE, Jnk1LoxP/LoxP transgene expressing mice were kept on regular chow (Jnk1 LoxP). Two weeks thereafter, 3 mice/group were euthanized for assessment of total lung collagen content while the remainder of animals was administered dox food for an additional 4 weeks prior to assessment of total lung collagen content. Lung fibrosis was assessed via the Sircol assay (A) or Masson’s Trichrome staining and analysis (B and C). D: Expression of epithelial and mesenchymal genes in homogenized lung tissue of various groups. (n = WT- Jnk1 and Jnk1 Loxp (no dox) adCtr/adTGFβ1 = 4/4/6/6, WT- Jnk1 and ΔEpi Jnk1 (on dox) adTGFβ1 = 8/11 respectively mice/group from 2 independent experiments). Results were normalized to the housekeeping gene cyclophilin, and are expressed as fold expression changes (+/- SEM) compared to the WT vehicle control groups. * p< 0.05 compared to PBS or AdCtr control groups. † p< 0.05 compared to respective WT groups. (ANOVA). Scale bars: 50 μm.