Fig 2: TGF-ß stimulates SMAD7 expression via FOXO3.(A) Real-time qRT-PCR evaluations on the NHLF cells after stimulation with rTGF-ß (10 ng/ml) and rCHIT1 (250 ng/ml) with (+) and without (-) siRNA silencing of FOXO3. (B) HEK293 cells transfected with luciferase-tagged SMAD7 promoter construct with (ProSMAD7WT; WT) and without (ProSMAD7?(FOXO3); mutant form) FOXO3 consensus sequence were stimulated with rTGF-ß and/or rCHIT1, and then luciferase activity was measured. (C) Western blot evaluations on the pFOXO3 and FOXO3 expression in the lungs of WT (-), TGF-ß Tg (+), CHIT1 Tg (+), and CHIT1/TGF-ß Tg mice. Whole-lung lysates were further separated into cytoplasmic and nuclear fractions and subjected to the immunoblots using anti-pFOXO3 and FOXO3 antibodies. Values in panels A and B represent the mean ± SEM of triplicated evaluations in a minimum of two separate experiments. Panel C is a representative immunoblot of three separate experiments. H3, histone H3, used as internal control of nuclear protein. **P < 0.01, ***P < 0.001, one-way ANOVA. ns, not significant.

Fig 3: Expression levels of CHIT1 and Smad7 in the lung epithelial cells from the patients with IPF and controls.The expression levels of CHIT1 and SMAD7 in alveolar type 1 (AT1) and type 2 (AT2) cells detected by qRT-PCR, respectively. *P < 0.01, t test.

Fig 4: CHIT1 interacts with TGFBRAP1 and FOXO3.(A) Co-immunoprecipitation (IP) and immunoblot (IB) evaluations on the MLE12 epithelial cells transfected with CHIT1, TGFBRAP1, and FOXO3 expressing constructs. (B) Double-labeled fluorescent IHC to localize expression of CHIT1, TGFBRAP1, and FOXO3 in NHLF. Panels A and B are representative Western blots and IHC evaluations of three separate experiments, respectively. Bars in panel B, 50 µm.

Fig 5: Decreased SMAD7 expression in the lungs from patients with SSc-ILD.(A) IHC evaluation on the SMAD7 expression in the lung sections from normal control and patients with SSc-ILD. SMAD7 staining of SSc-ILD lungs (right) reveals decreased number of Samd7-positive cells especially in the stromal region as compared with normal control (left) that showed a number of parenchymal cells and also macrophages are strongly stained. (B) Quantitation of SMAD7-positive (+) cells in the lung sections from controls and SSc-ILD per high-powered field. (C) Serum CHIT1 activity in the SSc-ILD patients with stable and progressive lung function. “Progressive” was defined in the patients with more than 10% of absolute drop of forced vital capacity (FVC). (D) Survival analysis based on the levels of chitinase activity (below and above median value of 53.54 nM/ml/h) in the patients with SSc-ILD. SSc-ILD subjects (n = 56) with values exceeding the median value of 53.54 (dotted line) demonstrate reduced progression-free survival as defined by absolute loss of %FVC within 2 years of enrollment. Scale bars, 30 µm, **P < 0.01, ***P < 0.001, Spearman’s rank test. n, number of individuals subjected to the evaluation.