Figure 1.
Rictor is a target of TGF-β and the effect of mTOR inhibitors on TGF-β signaling in IPF lung fibroblasts.
IPF fibroblasts (<passage 8) isolated from surgical lung biopsy (top panel) or lung transplant patients (middle and lower panels) were serum-starved for 24 hours prior to treatment. In (A) cells were treated with TGF-β (5 ng/ml) for time as shown; (B) cells were treated with TGF-β (5 ng/ml) overnight or left untreated in the presence or absence of indicated inhibitors MLN0128 (0.2 µM), PP242 (2 µM), or rapamycin (Rapa, 0.05 µM), which were added 30 minutes prior to TGF-β. Total cell lysates were prepared and equal amounts of protein were analyzed by Western blot analysis with specific antibodies as indicated. α-tubulin was used as a loading control. Asterisk indicates the carry-over signals between the western blots of α-SMA and SPARC. Band intensity was determined by using Image J software from the NIH. Data was presented as band intensity relative to untreated samples. EDA-FN, extra domain A fibronectin; SPARC, secreted protein acidic and rich in cysteine; α-SMA, α-smooth muscle actin.
Figure 2.
Effect of mTOR inhibitors on TGF-β activation of mTOR and Smad pathways.
Serum-deprived IPF fibroblasts were treated with TGF-β for 60 minutes or left untreated in (A), followed by Western blot analysis with anti-phospho Akt (Ser473 or Thr 308) and anti-total Akt antibodies, or in (B) for 6 hours in the presence or absence of indicated inhibitors MLN0128 (0.2 µM), PP242 (2 µM), or rapamycin (0.02 µM), followed by Western blot analysis with anti-phospho-S6 and anti-α-tubulin antibodies. (C) Serum-deprived IPF fibroblasts were treated with or without TGF-β for 15 minutes in the presence or absence of indicated inhibitors followed by Western blot analysis with an anti-phospho-Smad2 or Smad3 antibody. Expression of total Smad-2, 3, 4 and 7 was analyzed by Western blot. Experiment was done on three lines, which are shown in Figure 1; results were similar between the three lines and results from the IPF fibroblasts isolated from surgical lung biopsy are shown here.
Figure 3.
Rictor but not Raptor regulates Akt phosphorylation (Ser473) and the expression of matrix regulatory proteins.
In (A) IPF fibroblasts isolated from surgical lung biopsy were infected with lentivirus-derived shRNA against raptor or rictor, or control (scramble) as described in Materials and Methods. Western blot analysis was performed with the indicated antibodies. α-tubulin was used as a loading control. (B) Serum-starved IPF fibroblasts were treated with TGF-β for 60 minutes followed by an analysis of Akt phosphorylation by Western blot analysis. Total Akt was used as a loading control. (C). Serum-deprived IPF fibroblasts were treated overnight with TGF-β followed by analysis of matrix-regulatory proteins by Western blot analysis. α-tubulin was used as a loading control. Experiments with the three IPF lines showed similar results and representative results from the surgical lung biopsy fibroblasts are shown.
Figure 4.
Akt inhibition suppresses induction of Rictor by TGF-β.
Serum-starved IPF fibroblasts were pre-treated with Akti (Akt inhibitor VIII/124018) for 30 minutes or left untreated prior to TGF-β (5 ng/ml) treatment for two hours. In (A) cells were pre-treated with Akti at indicated concentration as shown, then followed by TGF-β treatment; (B) cells were pre-treated with Akti at 300 nM prior to TGF-β treatment or left untreated. Total cell lysates were prepared and equal amounts of protein were analyzed by Western blot analysis with specific antibodies as indicated. α-tubulin was used as a loading control.
Figure 5.
(A) Schematic of bleomycin prevention and therapeutic protocols.
(B) Mouse survival rates are from four independent experiments for the prevention model (n = 3 for Saline or MLN groups and n = 6 for Bleo or Bleo + MLN groups) and from five independent experiments for the therapeutic model (n = 3 for Saline or MLN groups, n = 6 for Bleo, and n = 5 for Bleo + MLN groups). (C) Mouse body weights are from bleomycin prevention and therapeutic model experiments (*P<0.05. and **P<0.005) as in (B). Each point represents the mean body weight of mice in the respective treatment group from each experiment.
Figure 6.
MLN0128 inhibits bleomycin-induced lung fibrosis.
Mice were treated according to the schematic shown in Fig. 5A. Mice lungs were harvested at Day 14 (prevention model) or Day 21 (therapeutic model) followed by H&E staining. Scale bar = 100 micron.
Figure 7.
MLN0128 inhibits bleomycin-induced fibrosis.
In (A) mice were treated as described in Fig. 5A followed by harvest of the right lung for a Sircoll collagen assay. The horizontal bar represents the mean value of collagen content (mg/lung) for each sample group. *P<0.05. (B) Analysis of Ashcroft score in left lung of mice from (A); *P<0.001. Data shown is combined from four independent prevention model and five independent therapeutic model experiments.
Figure 8.
MLN0128 blocks TGF-β-mediated attenuation of lung epithelial cell viability.
(A) A transwell culture protocol, as described in detail in Materials and Methods, using IPF fibroblasts co-cultured with A549 cells (*P<0.005) or (B) RLE-6TN cells (*P<0.001), which was followed by analysis of A549 or RLE-6TN viability by an Alamar Blue assay. (C) Downregulation of SPARC or Rictor in A549 (*P<0.05; **P<0.005) or (D) RLE-6TN cells (*P<0.05) by RNA interference in TGF-β-treated IPF fibroblasts was followed by an Alamar Blue assay of A549 or RLE-6TN cells. Data is expressed as mean +/− standard deviation from two IPF fibroblast lines (isolated from surgical lung biopsy and lung transplant) in three independent experiments.
Figure 9.
H2O2 release from IPF fibroblasts is mediated by SPARC and mTORC2.
(A) IPF fibroblasts were treated for 16 h with TGF-β alone or in combination with MLN0128 (0.2 µM) or rapamycin (0.05 µM) followed by measurement of H2O2 (*P<0.05, **P>0.05), as described in detail in Materials and Methods. (B) SPARC or Rictor was downregulated by RNA interference in TGF-β-treated IPF fibroblasts followed by measurement of H2O2; *P<0.05. Data is expressed as mean +/− standard deviation from same two fibroblast lines as in Figure 8, in three independent experiments.