Fig 1.
Mediation of GLI-1 and GLI-2 in hypoxia-induced CTGF expression.
A, WI-38 cells were subjected to hypoxia (1% O2) from 0 to 48 h. CTGF and α-tubulin levels in cell lysates were detected using western blotting. The results are expressed as the mean ± SEM of three independent experiments. * p < 0.05, compared with the control group without hypoxia stimulation. B, WI-38 cells were subjected to hypoxia for 0–48 h. The medium CTGF level was detected with ELISA. The results are expressed as the mean ± SEM of four independent experiments. * p < 0.05, compared with the control group without hypoxia stimulation. C, WI-38 cells were transfected with control siRNA (con siRNA), GLI-1 siRNA, and GLI-2 siRNA. After 24 h, the cells were subjected to hypoxia for an additional 6 h. CTGF and α-tubulin levels were detected, as described previously. The results are expressed as the mean ± SEM of three independent experiments. * p < 0.05, compared with hypoxia plus the control siRNA group. D, WI-38 cells were transfected with control siRNA (con siRNA), GLI-1 siRNA, GLI-2 siRNA, 0.5 μg of CTGF-Luc, and 0.1 μg of pBK-CMV-Lac Z. After 24 h, the cells were subjected to hypoxia (1% O2) for an additional 24 h. The luciferase activity assay is described in the “Materials and Methods” section. The results are expressed as the mean ± SEM of three independent experiments performed in duplicate. * p < 0.05, compared with hypoxia plus the control siRNA group. E, WI-38 cells were transfected with control siRNA (con siRNA), GLI-1 siRNA, and GLI-2 siRNA. After 24 h, GLI-1, GLI-2, and α-tubulin levels were detected using western blotting. Typical traces represent three independent experiments with similar results. F, NHLF cells were transfected with control siRNA (con siRNA), GLI-1 siRNA, and GLI-2 siRNA for 24 h. The cells were subjected to hypoxia for an additional 6 h. CTGF and α-tubulin levels were detected, as described previously. The results are expressed as the mean ± SEM of three independent experiments. * p < 0.05, compared with hypoxia plus the control siRNA group.
Fig 2.
Hypoxia-induced translocation of GLI-1 and GLI-2 from the cytosol to the nucleus in WI-38 cells.
A and B, WI-38 cells were subjected to hypoxia (1% O2) for 2 h. In confocal microscopy, the cells were incubated with antibodies specific for GLI-1 (A) and GLI-2 (B), and immunoreactivity was performed by incubating the cells with an FITC-conjugated secondary antibody. All slides were counterstained with DAPI (blue) to distinguish the nuclei, which were visualized under a fluorescence confocal microscope. The traces indicate similar results obtained from three independent experiments.
Fig 3.
Involvement of MEK in hypoxia-induced CTGF expression.
A, WI-38 cells were treated with PD98059 (an MEK inhibitor) for 20 min before they were subjected to hypoxia (1% O2) for an additional 6 h. CTGF and α-tubulin levels in cell lysates were detected using western blotting. The results are expressed as the mean ± SD of three independent experiments. * p < 0.05, compared with the hypoxia group without PD98059 treatment. B, WI-38 cells were transfected with 0.5 μg of CTGF-Luc and 0.1 μg of pBK-CMV-Lac Z for 24 h. The cells were treated with PD98059 before they were subjected to hypoxia (1% O2) for an additional 24 h. The luciferase activity assay is described in the “Materials and Methods” section. The results are expressed as the mean ± SEM of four independent experiments performed in duplicate. * p < 0.05, compared with the hypoxia group without PD98059 treatment.
Fig 4.
Involvement of ERK and p38 MAPK activation in hypoxia-induced CTGF expression.
A, WI-38 cells were transfected with 0.5 μg of CTGF-Luc and 0.1 μg of pBK-CMV-Lac Z for 24 h. The cells were treated with U0126 before they were subjected to hypoxia (1% O2) for an additional 24 h. The luciferase activity assay is described in the “Material and Methods” section. The results are expressed as the mean ± SEM of three independent experiments performed in duplicate. * p < 0.05, compared with the hypoxia group without U0126 treatment. B, WI-38 cells were transfected with control siRNA (con siRNA), MEK1 siRNA, and ERK1 siRNA. After 24 h, the cells were subjected to hypoxia for an additional 24 h. CTGF and α-tubulin levels were detected using western blotting, as described previously. The results are expressed as the mean ± SEM of three independent experiments. * p < 0.05, compared with hypoxia plus the control siRNA group. C, WI-38 cells were transfected with control siRNA (con siRNA), MEK1 siRNA, and ERK1 siRNA. After 24 h of transfection, MEK1, ERK1, and α-tubulin levels were detected using western blotting. Typical traces represent three independent experiments that yield similar results. D, WI-38 cells were pretreated with SB203580 for 20 min before they were subjected to hypoxia (1% O2) for an additional 24 h. CTGF and α-tubulin levels in the cell lysates were detected using western blotting. The results are expressed as the mean ± SEM of five independent experiments. * p < 0.05, compared with the hypoxia group without SB203580 treatment. E, WI-38 cells were subjected to hypoxia (1% O2) for the indicated time intervals, after which the levels of p38 phosphorylation and p38α were detected using western blotting. The results are expressed as the mean ± SEM of three independent experiments. * p < 0.05, compared with the control group without hypoxia treatment.
Fig 5.
Involvement of ERK in hypoxia-induced GLI-1 and GLI-2 translocation.
WI-38 Cells were subjected to hypoxia (1% O2) for a time interval of 0–60 min (A) or 2–24 h (B), after which the levels of ERK phosphorylation and ERK in cell lysates were detected using western blotting. The results are expressed as the mean ± SEM of three and four independent experiments. * p < 0.05, compared with the control group without hypoxia treatment. C, WI-38 cells were subjected to hypoxia (1% O2) for 10 min, before they were immunoprecipitated with a specific ERK antibody and mag sepharose magnetic beads, as described in the “Materials and methods” section. After elusion from the beads, the levels of GLI-1, GLI-2, and ERK in the lysates were detected using western blotting. Typical traces represent three independent experiments that yield similar results. D, WI-38 cells were pretreated with U0126 for 20 min before they were subjected to hypoxia (1% O2) for an additional 2 h, after which nuclear protein was collected. The levels of GLI-1, GLI-2, and lamin A/C in the nuclear extract were detected using western blotting. Typical traces represent three independent experiments that yield similar results.
Fig 6.
Involvement of MEKK1 in hypoxia-induced ERK activation and CTGF expression in WI-38 cells.
A, WI-38 cells were transfected with control siRNA (con siRNA) or MEKK1 siRNA for 24 h before they were subjected to hypoxia (1% O2) for an additional 6 h. The CTGF and α-tubulin levels in the cell lysates were detected using western blotting. The results are expressed as the mean ± SEM of three independent experiments. * p < 0.05, compared with hypoxia plus the control siRNA group. B, WI-38 cells were subjected to hypoxia (1% O2) for 0–60 min. The levels of MEKK1 phosphorylation and MEKK1 expression in the cell lysates were detected using western blotting. The results are expressed as the mean ± SEM of three independent experiments. * p < 0.05, compared with the control group without hypoxia treatment. C, WI-38 cells were transfected with control siRNA (con siRNA) or MEKK1 siRNA for 24 h before they were subjected to hypoxia (1% O2) for an additional 5 min. The levels of ERK phosphorylation and ERK expression in the cell lysates were detected using western blotting. The results are expressed as the mean ± SEM of three independent experiments. * p < 0.05, compared with hypoxia plus the control siRNA group. D, Cells were transfected with MEKK1 siRNA for 24 h. The MEKK1 and α-tubulin levels in the cell lysates were detected using western blotting. The typical traces display similar results obtained from three independent experiments.
Fig 7.
Involvement of AP-1 in hypoxia-induced CTGF expression.
A, WI-38 cells were transfected with 0.5 μg of CTGF wt-Luc, 0.5 μg of AP-1-mt-CTGF-Luc (CTGF AP-1 mt-Luc), and 0.1 μg of pBK-CMV-Lac Z for 24 h after which they were subjected to hypoxia (1% O2) for an additional 24 h. The luciferase activity assay is described in the “Material and Methods” section. The results are expressed as the mean ± SEM of three independent experiments performed in duplicate. * p < 0.05, compared with hypoxia treatment with CTGF wt-Luc transfection. WI-38 Cells were subjected to hypoxia (1% O2) for the indicated time intervals, after which the levels of c-Jun phosphorylation and c-Jun (B) or c-Fos phosphorylation and c-Fos (C) in the cell lysates were detected using western blotting. The results are expressed as the mean ± SEM of three independent experiments. * p < 0.05, compared with the control group without hypoxia treatment. D, Incubated cells subjected to hypoxia (1% O2) for 2 h and detected using the ChIP assay, as described in the “Material and Methods” section. The typical traces indicate the three independent experiments that yield similar results.
Fig 8.
GLI-1 and GLI-2 mediated hypoxia-induced α-SMA and collagen expression.
A, WI-38 cells were transfected with control siRNA (con siRNA), GLI-1 siRNA, and GLI-2 siRNA before they were subjected to hypoxia (1% O2) for an additional 24 h. The α-SMA and α-tubulin levels in cell lysates were detected using western blotting. The results are expressed as the mean ± SEM of three independent experiments. * p < 0.05, compared with hypoxia plus the control siRNA group. B, WI-38 cells were transfected with control siRNA (con siRNA), GLI-1 siRNA, and GLI-2 siRNA before they were subjected to hypoxia (1% O2) for an additional 24 h. Collagen and α-tubulin levels in the cell lysates were detected using western blotting. The results are expressed as the mean ± SD of three independent experiments. * p < 0.05, compared with hypoxia plus the control siRNA group.
Fig 9.
Simplified image displaying the results of hypoxia-induced CTGF expression mediated via the MEKK1/MEK1/ERK1/GLI-1/GLI-2 and AP-1 pathways in human lung fibroblasts.
Hypoxia activates the MEKK1/MEK1/ERK1 pathways, which in turn activate GLI-1/GLI-2 inducing the translocation of both proteins from the cytosol to the nucleus, and then cooperating with AP-1 to induce CTGF expression. Moreover, both GLI-1 and GLI-2 mediate α-SMA and collagen expression through hypoxia stimulation in human lung fibroblasts.