Transforming growth factor beta 1 induces apoptosis through cleavage of BAD in a Smad3-dependent mechanism in FaO hepatoma cells

Abstract

Transforming growth factor beta (TGF-beta) induces apoptosis in a variety of cells. We have previously shown that TGF-beta 1 rapidly induces apoptosis in the FaO rat hepatoma cell line. We have now studied the effect of TGF-beta 1 on the expression of different members of the Bcl-2 family in these cells. We observed no detectable changes in the steady-state levels of Bcl-2, Bcl-X(L), and Bax. However, TGF-beta 1 induced caspase-dependent cleavage of BAD at its N terminus to generate a 15-kDa truncated protein. Overexpression of the 15-kDa truncated BAD protein enhanced TGF-beta 1-induced apoptosis, whereas a mutant BAD resistant to caspase 3 cleavage blocked TGF-beta 1-induced apoptosis. Overexpression of Smad3 dramatically enhanced TGF-beta 1-induced cleavage of BAD and apoptosis, whereas antisense Smad3 blocked TGF-beta 1-induced apoptosis and BAD cleavage. These results suggest that TGF-beta 1 induces apoptosis through the cleavage of BAD in a Smad3-dependent mechanism.

FIG. 1.

Expression of Bcl-2 family proteins during TGF-β1-induced apoptosis in FaO rat hepatoma cells. (A) Cellular DNA fragmentation after TGF-β1 treatment. Genomic DNAs were extracted from control and TGF-β1-treated cells at 6, 12, 24, and 36 h. Extracted DNAs were resolved on agarose gel. (B) The effect on Bcl2, Bcl-X_L, Bax, and BAD protein levels in FaO cells was determined. Cells were incubated with 5 ng of TGF-β1/ml. Cell lysates were made, and equivalent amounts of cellular proteins were separated by SDS-15% PAGE, blotted, and then probed with the appropriate antibody. Blots were shown are typical of at least three individual experiments. (C) Effect of Z-VAD fmk (50 μM) on levels of endogenous full-length BAD and 15-kDa cleaved form of BAD in cells treated with 5 ng of TGF-β1/ml for 14 h. Western blot analyses were performed with an anti-BAD (C-20) antibody. Results are representative of three separate experiments. (D) Effect of cycloheximide and puromycin on levels of endogenous full-length BAD and the 15-kDa cleaved form of BAD in cells treated with 5 ng of TGF-β1/ml for 14 h. Western blot analyses were performed with an anti-BAD (C-20) antibody.

FIG. 2.

Differential effects of WT, DM56/61, and truncated BAD on FaO cell viability before and after TGF-β1 treatment. (A) Expression of WT BAD, DM56/61 BAD, and tBAD68 proteins in the FaO-infected cells was determined by a Western blot analysis using anti-HA antibody. (B) Apoptotic progression in FaO cells stably expressing WT BAD, DM56/61 BAD, and tBAD68 treated with TGF-β1. The cells were treated with TGF-β1 for 12 h. Cell lysates were made, and equivalent amounts of cellular proteins were separated by SDS-15% PAGE, blotted, and then probed with the anti-HA antibody. Levels of the C-terminal HA-tagged 15-kDa cleaved form of BAD in full-length BAD (WT-BAD) and DM56/61-BAD expressing cells were examined. Western blot analysis was performed with an anti-BAD (C-20) antibody. Results are representative of three separate experiments. (C) Cellular DNA fragmentation after TGF-β1 treatment. Genomic DNAs were extracted from the WT-BAD and DM56/61-BAD cells after TGF-β1 treatment at 24 h. Extracted DNAs were resolved on agarose gel. (D) TUNEL procedure was carried out and pictures were taken under a light microscope (magnification, ×200). (E) TUNEL-positive apoptotic cells at the respective incubation time were counted, and the percentage of apoptotic cells was graphed. Similar results were achieved in three separate experiments with comparable outcomes.

FIG. 3.

Changes in mitochondrial transmembrane potential (Δψ_m) and the release of cytochrome c induced by TGF-β1 in cells expressing WT, DM56/61, and truncated BAD. (A) Cells incubated for 12 h in the absence (control) or presence of 5 ng of TGF-β1/ml were detached by trypsinization. After 30 min of incubation with Rh123 (5 μM), the intracellular fluorescence intensity was measured in a FACScan flow cytometer. Results representative of experiments run more than three times are shown. For a positive control, CCCP, an uncoupling agent, was incubated with cells for 10 min. (B) Release of cytochrome c from mitochondria to cytosol in the control and DM56/61 BAD cells. After incubation of cells for 12 h in the absence (−) or presence (+) of 5 ng of TGF-β1/ml, mitochondria were separated from the cytosol and cytochrome c content was analyzed by Western blotting as described in Materials and Methods. The ratio between mitochondria and cytosol is shown at the bottom.

FIG. 4.

Effect of overexpression of Smad's on TGF-β1-dependent apoptosis. FaO cells were infected with adenoviruses carrying Smad1, Smad2, Smad3, Smad4, and Smad5 at an MOI of 300 and treated with 5 ng of TGF-β1/ml. Adenovirus carrying β-galatosidase (MOI of 800) was used as a control. (A) A TUNEL procedure was carried out and pictures were taken under a light microscope. Magnification, ×200. (B) TUNEL-positive apoptotic cells at the respective incubation time were counted, and the percentage of apoptotic cells was graphed. (C) Expression of Smad's was confirmed by anti-FLAG immunoblotting. Similar results were achieved in three separate experiments with comparable outcomes.

FIG. 5.

Effect of Smad's on generation of ∼15-kDa cleaved form of BAD induced by TGF-β1. (A) FaO cells were infected with adenoviruses carrying Smad2, Smad3, and Smad7 at an MOI of 300 and treated with 5 ng of TGF-β1/ml for 24 h. Adenovirus carrying β-galatosidase (MOI of 800) was used as a control. Cell lysates were made and equivalent amounts of cellular proteins were separated by SDS-15% PAGE, blotted, and then probed with an anti-BAD (C-20) antibody. Expression of Smad's was confirmed by anti-FLAG immunoblotting (bottom). Results are representative of three separate experiments. Expression of Smad's was confirmed by anti-FLAG immunoblotting (bottom). Results are representative of three separate experiments. (B) Level of Smad3 in FaO cells infected with adenoviruses carrying β-galactosidase and Smad3. Cell lysates were made 24 h after adenovirus infection, and equivalent amounts of cellular proteins were separated by SDS-15% PAGE, blotted, and then probed with either anti-Smd3 antibody or anti-flag antibody.

FIG. 6.

Effect of antisense-Smad3 on SBE4-luc reporter activity, apoptosis, and BAD cleavage induced by TGF-β1. (A) Expression of endogenous Smad3 in control and antisense-Smad3 transfectant clones. Total lysates were analyzed by SDS-PAGE. Western blotting was performed using anti-Smad3 or anti-Smad2 antibodies. Anti-β-actin was used as a control. (B) Either pGL3-basic (control) or SBE4-luc (47) was transfected into either control cells (vector) or antisense Smad3 cells (As-S3#4). Luciferase activity was measured 24 h after TGF-β1 stimulation. Data shown are means of triplicate measurements from one representative transfection. (C) Generation of 15-kDa cleaved form of BAD induced by TGF-β1 was examined in control and antisense-Smad3-expressing cells. Cell lysates were made, and equivalent amounts of cellular proteins were separated by SDS-15% PAGE, blotted, and then probed with an anti-BAD (C-20) antibody. Results are representative of three separate experiments. (D) The TUNEL assay was performed with the cells treated with 5 ng of TGF-β1/ml for 24 h and TUNEL-positive apoptotic cells at the respective incubation time were counted, and the percentage of apoptotic cells was graphed. Similar results were achieved in three separate experiments with comparable outcomes.

FIG. 7.

Changes in mitochondrial transmembrane potential (Δψ_m) and release of cytochrome c induced by TGF-β1 in cells expressing antisense-Smad3. (A) The cells incubated for 12 h in the absence (control) or presence of 5 ng of TGF-β1/ml were detached by trypsinization. After 30 min of incubation with Rh123 (5 μM), the intracellular fluorescence intensity was measured in a FACScan flow cytometer. Results representative of experiments run more than three times are shown. (B) Release of cytochrome c from mitochondria to cytosol in the control and antisense-Smad3 cells. After incubating cells for 12 h in the absence (−) or presence (+) of 5 ng of TGF-β1/ml, mitochondria were separated from cytosol and cytochrome c content was analyzed by Western blotting as described in Materials and Methods. The ratio between mitochondria and cytosol is shown at the bottom.