Altered cav-1 expressions in IPF patients. (A) Microarray analysis of cav-1 mRNA reveals a significant reduction (P = 0.000087, fold 0.5) in IPF patients (n = 13) compared with controls (n = 11). Boxes indicate the middle two quartile values, with the median values shown. Error bars represent 1.5× the interquartile range. (B) Immunohistochemical analysis of cav-1 expression in lung tissue sections (cav-1, green; nucleus, blue; α-SMA, orange). One representative example out of seven for patient sample or control subjects is shown. Bar, 100 μm. Immunohistochemical analysis of cytokeratin 19 (C) and CD31 (D) and cav-1 expression in normal (CTL) and IPF lung tissue sections. One representative example out of three for patient sample or control subjects is shown. Bars: (C) 50 μm; (D) 100 μm. (E) cav-1 protein expressions as detected by immunoblot analysis in lung tissue samples from IPF patients (n = 7) and control subjects (n = 7). Three representative samples of patients and controls are shown. White lines indicate that intervening lanes have been spliced out. (F) cav-1 mRNA expressions were detected by Taqman PCR in pulmonary fibroblasts derived from IPF patients (n = 4) and control subjects (n = 5). (G) cav-1 and β-actin protein expressions were determined by immunoblot analysis in pulmonary fibroblasts derived from IPF patients (n = 4) and control subjects (n = 5). Three representative patients and control samples are shown. The differences in mRNA level of cav-1 expression were compared by the Student's t test. The differences in protein levels of cav-1 expression were compared by the Wilcoxon two-sample test. Differences were considered significant at P < 0.05. Data in E–G represent the mean ± SEM. CTL, control.

cav-1 suppresses BLM-induced pulmonary fibrosis. The C57BL/6 mice infected with ad–cav-1, ad-lacZ, or saline were treated with BLM for 14 d. The lungs were harvested and subjected to (A) H&E staining and (B) Masson trichrome staining of the right lung tissue. One representative example out of four or five is shown for each group in A and B. Bars, 200 μm. (C) Histology grade score analysis of the tissue sections (n = 4 and 5 for groups without and with BLM, respectively). (D) Hydroxyproline content determination of the left lung (n = 4 and 5 for groups without and with BLM, respectively). Data in C and D represent the mean ± SEM. (E) ECM deposition and smad-2 activation in the whole lung as determined by immunoblot analysis. (F) TGF-β1 content determination by ELISA of the homogenized protein of the whole lung (n = 3 for each group). (G) Hydroxyproline content determination of the whole lung 7 d after ad–cav-1 infection and 14 d after BLM instillation (n = 3 for each group). Data in F and G represent the mean ± SEM.

TGF-β1 regulates cav-1 expression. (A) Human primary pulmonary fibroblasts and MRC-5 cells were treated with 4 ng/ml TGF-β1 for 24 h. cav-1 mRNA levels were determined by Taqman PCR. Data represent the mean ± SEM. (B) The same cells were treated with different concentrations of TGF-β1 for 24 h (C) or treated with 4 ng/ml TGF-β1 for 1 and 2 d. cav-1 protein levels were determined by immunoblot analysis. Data are representative of three independent experiments.

cav-1 modulates TGF-β1–induced ECM productions. (A) After adenovirus infection, 4 ng/ml TGF-β1 was added into MRC-5 for 2 d. ECM productions were determined by immunoblot analysis. (B) ECM productions in cav-1 stably transfected and control MRC-5 cells after 2 d of 4-ng/ml TGF-β1 treatment. (C) After siRNA transfection for 24 h, the MRC-5 cells were serum-starved overnight and treated with 4 ng/ml TGF-β1 for 2 d. ECM productions were determined by immunoblot analysis. Three independent experiments were performed, and one representative sample is shown. The differences in densitometric quantification of cav-1 expression were compared by the Student's t test. Differences were considered significant at P < 0.05. Data represent the mean ± SEM.

cav-1 modulates smad activation. Immunoblot analysis of smad-2 phosphorylation; smad-4 and smad-7 expressions in cytosolic extracts; and smad-2/3 and smad-4 existences in nuclear extracts under different conditions in MRC-5. (A) Cells were infected with ad–cav-1 and ad-lacZ and (B) transfected with siRNA targeting cav-1 and control for 24 h. The cells were serum-starved overnight and treated with TGF-β1 for 0, 10, 30, and 60 min. Data are representative of three independent experiments.

MAPK pathways are involved in ECM regulation of cav-1. (A) Immunoblot analysis of TGF-β1–stimulated MAPK activation for 0, 10, 30, and 60 min in MRC-5 cells infected with ad–cav-1 or ad-lacZ. (B) Immunoblot analysis of TGF-β1–stimulated ECM production in MRC-5 cells infected with ad–cav-1 or ad-lacZ. The cells were pretreated with DMSO or PD 98059, or UO 126 or SP 600125 for 1 h, and administrated with TGF-β1 for 2 d. (C) Immunoblot analysis of TGF-β1–stimulated ECM production in pulmonary fibroblasts isolated from JNK1 (+/+) and (−/−) mice infected with ad–cav-1 or ad-lacZ. (D) Immunoblot analysis of smad-2 phosphorylation in cytosolic extracts and smad-2/3 existences in nuclear extracts. (E) Immunoblot analysis of smad-2 phosphorylation in cytosolic extracts and smad-2/3 existences in nuclear extracts in JNK1 wild-type and null fibroblasts infected with ad–cav-1 or ad-lacZ. Data are representative of three independent experiments in A–E. (F) Immunohistochemical analysis of JNK phosphorylation in paraffin-embedded lung tissue slices treated with BLM and infected with ad-lacZ or ad–cav-1 or instilled with saline. Brown indicates phospho-JNK–positive cells. One representative example out of five is shown. Bar, 100 μm. (G) Immunohistochemical analysis of JNK phosphorylation in frozen lung tissue slices from IPF patients and control subjects (phospho-JNK, green; nucleus, blue). One representative example out of seven for patients or controls is shown. Bar, 100 μm.

Summary of the role of cav-1 in pulmonary fibrosis. cav-1 modulates TGF-β1–induced JNK1 and smad-2/3 activity and inhibits α-SMA, fibronectin, and collagen type I production.