Figure 1.
TGF-β1 induces growth suppression in association with induction of myofibroblast differentiation of human lung fibroblasts.
A, Early passage human lung fibroblasts (IMR-90), seeded at equal density on 35 mm dishes and grown to 50% confluence, were treated with or without TGF-β1 (2 ng/ml) in the presence/absence of the ALK5 inhibitor (SB431542, 0.5 μM) in 10% FBS for 48 h prior to assessments of cell numbers by Coulter counting (n = 6 per group). Results are averages of at least three independent experiments. Data are presented as mean±S.E.M. **indicates p < 0.01 vs. control cells or cells treated with TGF-β and SB431542. B, IMR-90 cells as described in (A) were grown in 96-well plates and labeled with BrdU for 24 h prior to assays for BrdU incorporation (n = 6 per group). Results are averages of at least three independent experiments. Data are presented as mean±S.E.M. **indicates p < 0.01 vs. control cells or cells treated with TGF-β and SB431542. C, IMR-90 cells grown in 10% FBS were stimulated with or without TGF-β1 (2 ng/ml) in the presence/absence of SB431542 (0.5 μM) for 48 h. Cell lysates were subjected to SDS-PAGE and immunoblotted with an antibody against Cyclin D1; the blot was then stripped and probed for α-smooth muscle actin (α-SMA) and β-actin. D, IMR-90 cells as described in (A) were stimulated with or without TGF-β1 (2 ng/ml) for the indicated times. Western immunoblotting was performed with a monoclonal antibody against α-SMA; the blot was stripped and probed for β-actin. E, Immunofluorescent images of IMR-90 cells treated 24h with vehicle only control, TGF-β only (2ng/ml) and TGF-β (2ng/ml) with SMAD inhibitor (SB431542, 0.5 μM). Cells were stained with antibody specific for α-SMA (1:100) with Alexa flour 594 conjugated secondary antibody (red); and 4′,6-diamidino-2-phenylindole (DAPI, 300nM in PBS) staining was used to identify the nucleus (blue). Representative microscopy images are shown. Magnification of ×40, figures were obtained with a Zeiss Axiovert fluorescence microscope. F, α-SMA expression by western blot in IMR-90 cells treated 24h with vehicle only control, TGF-β only (2ng/ml) and TGF-β (2ng/ml) with SMAD inhibitor (SB431542, 0.5 μM). β-actin is used as loading control.
Figure 2.
TGF-β1 induces dose- and time-dependent down-regulation of CAV-1 in human lung fibroblasts.
A and B, Cultured IMR-90 cells were treated with or without TGF-β1 (2 ng/ml) for 24 h and total RNA was isolated. A, cDNA Superarray analysis was performed and mRNA expression levels of Cav-1 and cyclophilin A are shown. Densitometric ratios of Cav-1:cyclophilin A are also shown. B, Histogram of real-time RT-PCR for Cav-1 expression after treated with TGF-β1 (2 ng/ml for 24 h) using triplicate samples from at least three individual experiments, normalized to 18S as mean±S.E.M. *p<0.05 compared to control. C, IMR-90 cells were treated with TGF-β1 (0–5 ng/ml) for 24 h and cell lysates extracted. Cell lysates were subjected to SDS-PAGE and immunoblotted with an antibody against Cav-1; the blot was stripped and probed for β-tubulin. Densitometric ratios of Cav-1:β-tubulin are also shown on the right, as mean±S.E.M. D, IMR-90 cells were stimulated with TGF-β1 (2 ng/ml) for the indicated times. Cell lysates were subjected to SDS-PAGE and immunoblotted with an antibody against Cav-1; the blot was then stripped and probed for β-tubulin. Densitometric ratios of Cav-1:β-tubulin are shown on the right, as mean±S.E.M. Results are averages of at least three independent experiments.
Figure 3.
Down-regulation of Cav-1 by TGF-β1 is mediated by p38 MAPK-dependent and SMAD-independent mechanisms.
A, IMR-90 cells were treated with inhibitors of p38 MAPK (SB203580; 6 μM) or ALK5 (SB431542; 0.5 μM) for 30 min prior to treatment with or without TGF-β1 (2 ng/ml) for a period of 48 h. Cell lysates were extracted and Western immunoblotting performed with an antibody against Cav-1; the blot was then stripped and probed for β-tubulin. B, Densitometric analyses of blots in (A) showed as % inhibition of baseline Cav-1 protein expression levels treated with TGF-β1. *indicates effect of SB203580 to completely block the inhibitory effect of TGF-β1 on Cav-1 expression. Results are averages of at least three independent experiments. Data are presented as mean±S.E.M. C, IMR-90 cells stably transfected with a kinase-deficient p38 MAPK (pcDNA-p38KM) or control vector (pcDNA) were treated with or without TGF-β1 (2 ng/ml) for 24 h. Cell lysates were obtained and subjected to SDS-PAGE and immunoblotted for Cav-1 and α-smooth muscle actin (α-SMA); blots were stripped and probed for β-tubulin. D, IMR-90 cells stably expressing SMAD2 shRNA (pSU6H-shSMAD2) or control vector (pSU6H) were treated with/without TGF-β1 (2 ng/ml) for 24 h. Cell lysates were immunoblotted for SMAD2, Cav-1 and α-SMA. The blots were stripped and probed for β-tubulin.
Figure 4.
Over-expression of Cav-1 inhibits proliferation of human lung fibroblasts/myofibroblasts and abrogates the anti-apoptotic effects of TGF-β1.
A, Human lung fibroblasts (IMR-90) were stably transfected with a plasmid encoding Cav-1 (pRC/CMV2-Cav1) or with empty vector (pRC/CMV2). Localization of Cav-1 protein was then analyzed by immunofluoresence staining with a rabbit polyclonal antibody to Cav-1. B, Stably-transfected cells described in (A) were treated with/without TGF-β1 (2 ng/ml) for 24 h. Cell lysates were obtained and subjected to immunoblotting for Cav-1, α-smooth muscle actin (α-SMA), and β-tubulin. C, Stably-transfected cells described in (A) were serum-deprived for 24 h and then treated with/without TGF-β1 (2 ng/ml) for 48 h followed by stimulation with 10% fetal bovine serum for 24 h. Cell numbers were assessed both prior to and after serum stimulation with an automated Coulter counter (n = 6 per group) as mean±S.E.M. *indicates p < 0.05 vs. control pRC/CMV2 “fibroblasts”. **indicates p < 0.05 vs. pRC/CMV2 “myofibroblasts” (TGF-β1 pre-treated). Similar results were obtained from 3 independent experiments. D, Cells in (C) were labeled with BrdU during the 24 h of serum stimulation (n = 6 per group). BrdU assays were performed as described in “Methods”. *indicates p < 0.05 vs. control pRC/CMV2 “fibroblasts”. **indicates p < 0.05 vs. pRC/CMV2 “myofibroblasts” (TGF-β1 pre-treated). Similar results were obtained from 3 independent experiments. E, Quiescent stably transfected IMR-90 cells described in (A) were treated with/without TGF-β1 (2 ng/ml) for 5 days in serum-free medium. Apoptosis assays using an ELISA for ssDNA (n = 6 for each group) were performed as described in “Methods”. *indicates p < 0.05 vs. control pRC/CMV2 “fibroblasts”. **indicates p < 0.01 vs. pRC/CMV2 “myofibroblasts” (TGF-β1 pre-treated). Similar results were obtained from 3 independent experiments.
Figure 5.
shRNA knock-down of Cav-1 enhances fibroblast/myofibroblast proliferation and protects against serum deprivation-induced apoptosis.
A, IMR-90 cells stably transfected with a plasmid encoding shRNA targeted against Cav-1 (pSU6H-shCav1) or with control plasmid (pSU6H) were treated with/without TGF-β1 (2 ng/ml) for 24 h. Cell lysates were obtained and Western blots for Cav-1, α-smooth muscle actin (α-SMA) and β-tubulin performed. B, Stably-transfected cells described in (A) were serum-deprived for 24 h and treated with/without TGF-β1 (2 ng/ml) for 48 h followed by stimulation with 10% FBS for 24 h. Cell counts were assessed both prior to and after serum stimulation with an automated Coulter counter (n = 6 per group) shown as mean±S.E.M. *indicates p < 0.05 vs. control pSU6H “fibroblasts”. **indicates p < 0.05 vs. pSU6H “myofibroblasts” (TGF-β1 pre-treated). Similar results were obtained from 3 independent experiments. C, Stably transfected cells described in (A) were serum-deprived for 24 h and treated with/without TGF-β1 (2 ng/ml) for 48 h followed by BrdU labeling for 24 h in the presence of 10% FBS (n = 6 per group) shown as mean±S.E.M. *indicates p < 0.05 vs. control pSU6H “fibroblasts”. Similar results were obtained from 3 independent experiments. D, Quiescent stably-transfected cells described in (A) were treated with/without TGF-β1 (2 ng/ml) for 5 days and apoptotic assay for ssDNA performed as described in “Methods” (n = 6 per group) shown as mean±S.E.M. *indicates p < 0.05 vs. control pSU6H “fibroblasts”. **indicates p < 0.05 vs. pSU6H “myofibroblasts” (TGF-β1 pre-treated). Similar results were obtained from 3 independent experiments.
Figure 6.
Blocking p38 MAPK inhibits myofibroblast proliferation.
A, IMR-90 cells stably expressing a p38 MAPK dominant negative (pcDNA-p38KM) and cells stably transfected with an empty vector (pcDNA) were serum-starved for 24 h and treated with/without TGF- β1 (2 ng/ml) for 48 h followed by stimulation with 10% fetal bovine serum for 24 h (n = 6 per group), data shown as mean±S.E.M. *indicates p < 0.05 vs. control pcDNA “fibroblasts”. **indicates p < 0.01 vs. control pcDNA “myofibroblasts” (TGF-β1 pre-treated). Similar results were obtained from 3 independent experiments. B, Cells described in (A) were grown in 96-well plates and serum-starved for 24 h and treated with/without TGF- β1 (2 ng/ml) for 48 h followed by BrdU labeling for 24 h in the presence of 10% fetal bovine serum (n = 6 per group), data shown as mean±S.E.M. *indicates p < 0.05 vs. control pcDNA “fibroblasts”. **indicates p < 0.01 vs. control pcDNA “myofibroblasts” (TGF-β1 pre-treated). Similar results were obtained from 3 independent experiments. C, IMR-90 cells stably expressing SMAD2 shRNA and cells stably transfected with an empty vector (pSU6H) were serum-starved for 24 h and treated with/without TGF- β1 (2 ng/ml) for 48 h followed by stimulation with 10% fetal bovine serum for 24 h (n = 6 per group) data shown as mean±S.E.M. *indicates p < 0.05 vs. control pSU6H “fibroblasts” (no TGF-β1 pre-treatment). **indicates p < 0.05 vs. control pcDNA “myofibroblasts” (TGF-β1 pre-treated). D, Cells described in (C) were grown in 96-well plates and serum-starved for 24 h and treated with/without TGF- β1 (2 ng/ml) for 48 h followed by BrdU labeling for 24 h in the presence of 10% fetal bovine serum (n = 6 per group) data shown as mean±S.E.M. *indicates p < 0.05 vs. control pSU6H “fibroblasts” (no TGF-β1 pre-treatment). **indicates p < 0.05 vs. control pcDNA “myofibroblasts” (TGF-β1 pre-treated).
Figure 7.
Schematic representation of TGF-β1-activated signaling pathways mediating mesenchymal cell growth -suppressive and -promoting effects.
TGF-β1 activates the cell surface TGF-β receptor(s) complex that leads to rapid activation of the canonical SMAD pathway as well as the SMAD-independent p38 MAPK pathway. Activation of the SMAD pathway is required for the induction of a cellular program of growth-arrest and myofibroblast differentiation. In contrast, activation of the p38 MAPK pathway, independently of SMAD2/3, is required the down-regulation of Cav-1 by TGF-β1. Down-regulation of Cav-1 by TGF-β1 “primes” differentiated myofibroblasts for enhanced proliferative responses to mitogens and resistance to apoptosis. These divergent TGF-β signaling pathways may explain, in part, the contextual effects of TGF-β1 as both a growth-inhibitor and –promoter on the same target (mesenchymal) cells.