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. 2005 Oct;16(10):4672-83.
doi: 10.1091/mbc.e05-01-0054. Epub 2005 Aug 10.

The endogenous ratio of Smad2 and Smad3 influences the cytostatic function of Smad3

Sang Gyun Kim  1 , Hyun-Ah Kim, Hyun-Soon Jong, Jung-Hyun Park, Noe Kyeong Kim, Seung Hwan Hong, Tae-You Kim, Yung-Jue Bang
Affiliations

The endogenous ratio of Smad2 and Smad3 influences the cytostatic function of Smad3

Sang Gyun Kim et al. Mol Biol Cell. 2005 Oct.

Abstract

Although Smad2 and Smad3, critical transcriptional mediators of transforming growth factor-beta (TGF-beta) signaling, are supposed to play a role in the TGF-beta cytostatic program, it remains unclear whether TGF-beta delivers cytostatic signals through both Smads equally or through either differentially. Here, we report that TGF-beta cytostatic signals rely on a Smad3-, but not a Smad2-, dependent pathway and that the intensity of TGF-beta cytostatic signals can be modulated by changing the endogenous ratio of Smad3 to Smad2. Depleting endogenous Smad3 by RNA interference sufficiently interfered with TGF-beta cytostatic actions in various TGF-beta-sensitive cell lines, whereas raising the relative endogenous ratio of Smad3 to Smad2, by depleting Smad2, markedly enhanced TGF-beta cytostatic response. Consistently, Smad3 activation and its transcriptional activity upon TGF-beta stimulation were facilitated in Smad2-depleted cells relative to controls. Most significantly, a single event of increasing this ratio by Smad2 depletion was sufficient to restore TGF-beta cytostatic action in cells resistant to TGF-beta. These findings suggest a new important determinant of sensitivity to TGF-beta cytostatic signaling.

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Figures

Figure 1.
Figure 1.
Effects of Smad2 or Smad3 depletion on TGF-β-induced cell cycle arrest and growth inhibition. (A) HaCaT cells were transfected with mock, nonsilencing control (Ctrl), Smad2, Smad3, or Smad2/3 (2 + 3) siRNA and incubated for 24 h. Whole cell lysates were immunoblotted with the indicated antibodies. Anti-α-tubulin was used as an internal loading control. (B) HaCaT cells transfected with the indicated siRNAs were treated with (+) or without (-) TGF-β. Cell proliferation was determined by cell number on day 3 after treating TGF-β. Error bars represent the SDs of three experiments (*p <0.05, **p <0.01). (C) Transfection of the different siRNAs in Hep3B (left), SNU-368 (middle), or Huh7 (right) and cell proliferation measurements were performed as described in B (*p < 0.05, **p < 0.01). (D) HaCaT cells transfected with the indicated siRNAs were incubated in the presence or absence of TGF-β. At the indicated times after treating TGF-β, cells were fixed, stained with propidium iodide, and subjected to flow cytometric analysis. The percentages of cells in the G1 and S phases were determined based on the DNA content histograms (left) and graphed (right). The graphs shown in the right represent means ± SD of three independent experiments.
Figure 2.
Figure 2.
Effects of Smad2 or Smad3 depletion on the TGF-β cytostatic program. (A) HaCaT cells transfected with the indicated siRNAs were incubated with (+) or without (-) TGF-β for 3 h. Total cell lysates were immunoblotted with anti-c-Myc or anti-α-tubulin antibody. (B) Cell lysates were prepared from each siRNA-transfected HaCaT cells treated with TGF-β for the indicated times. Immunoblotting was then performed using the indicated antibodies. (C) The indicated siRNA-transfected HaCaT cells were treated with (+) or without (-) TGF-β for 12 h. Equal amounts of cell extracts (200 μg) were immunoprecipitated with anti-Cdk2 antibody, and its immune complex was used for Cdk2 kinase assay on Histone H1 as a substrate. Fold changes in Cdk2 kinase activity are indicated as numbers. Cdk2 protein levels were determined by immunoblotting. (D) HaCaT cells transfected with the indicated siRNAs were incubated with TGF-β for the indicated times. Whole cell lysates were immunoblotted with anti-pRB antibody. Hyperphosphorylated (pp-RB) and hypophosphorylated (p-RB) pRB are indicated. (E) SNU-620 cells were transfected with the indicated siRNAs and then incubated for 24 h. Whole cell lysates were immunoblotted with the indicated antibodies. (F) SNU-620 cells transfected with the indicated siRNAs were treated with (+) or without (-) TGF-β for 12 h. Cell lysates were then prepared and immunoblotted with the indicated antibodies.
Figure 3.
Figure 3.
Modulation of the endogenous Smad2/Smad3 ratio by depleting individual R-Smads affects their relative signal intensities in response to TGF-β. (A) SNU-368 cells transfected with the indicated siRNAs were incubated with TGF-β for the indicated times. Whole cell extracts were immunoblotted with the indicated antibodies. (B) Smad4 was immunoprecipitated (IP) from whole cell extracts prepared from SNU-368 cells transfected with each siRNA treated with (+) or without (-) TGF-β for 12 h. Levels of phospho-Smad2 (p-Smad2) or phospho-Smad3 (p-Smad3) associated with Smad4 were evaluated by immunoblotting (IB). The amounts of immunoprecipitated Smad4 were determined by blotting with anti-Smad4 antibody. (C) The indicated siRNA-transfected HaCaT cells were incubated for 12 h in the presence (+) or absence (-) of TGF-β. Nuclear fractions were prepared and immunoblotted with the indicated antibodies. Immunoblotting for Lamin B and α-tubulin were performed to confirm nuclear fraction and exclude cytoplasmic contamination, respectively. (D and E) HaCaT cells were transfected with ARE-Luc/FAST-1 or (SBE)4-Luc together with the indicated siRNAs and pSV-β-Gal and then incubated with (dark bars) or without (gray bars) TGF-β. Luciferase activity was measured 18 h after treating TGF-β. Transfection efficiency was normalized versus β-galactosidase activity, and data are presented as means ± SD of four experiments. (F) The indicated siRNA-transfected HaCaT cells were incubated for 12 h in the presence (+) or absence (-) of TGF-β. ChIP analysis was then performed to detect the presence of phospho-Smad3 on the indicated p15INK4b gene (left) and the p21Cip1 gene (right) promoter regions as described in Materials and Methods. Normal rabbit IgG and no addition of antibody were used as the negative controls. To confirm the equal chromatin input, 10% of total lysates before immunoprecipitation were used for purification of total genomic DNA, which were used as templates for PCR reactions (bottom).
Figure 4.
Figure 4.
TGF-β can differentially regulate the endogenous ratio of Smad3 to Smad2 depending on cell type. (A) Whole cell lysates were prepared from the indicated cell lines treated with (+) or without (-) TGF-β for 12 h and then immunoblotted with the indicated antibodies. (B) Indicated cell lines were incubated with (dark bars) or without (gray bars) TGF-β and cell proliferation were determined by cell number on day 2 after treating TGF-β. Percentages of TGF-β-treated cell numbers versus untreated controls were calculated and graphed. The error bars represent the SDs of three experiments. (C) SNU-368 cells were incubated with TGF-β for the indicated times. Whole cell extracts were immunoblotted with the indicated antibodies. (D) Whole cell lysates were prepared from Huh7 (left) or SNU-620 cells (right) treated with TGF-β for the indicated times and then immunoblotted as described in C. (E) Cell lysates were extracted from SNU-620 cells treated with (+) or without (-) TGF-β for 12 h in the presence or absence of proteasomal inhibitor MG132 and then immunoblotted as described in C.
Figure 5.
Figure 5.
Effects of Smad2 or Smad3 overexpression on the TGF-β cytostatic program. (A) The indicated cell lines infected with adenoviruses carrying β-galactosidase (LacZ), FLAG-tagged Smad2, or Smad3 cDNA were incubated with (+) or without (-) TGF-β. Cell proliferation was determined by counting cell numbers on day 2 after treating TGF-β. Data are presented as means ± SD of three experiments (*p < 0.05, **p < 0.01). (B) SNU-368 cells infected as described in A were incubated in the presence or absence of TGF-β and subjected to flow cytometric analysis as described in Figure 1D. The percentages of cells in the G1 and S phases were determined based on the DNA content histograms and graphed. The data shown are representative of four independent experiments. (C) HaCaT cells infected with adenoviruses carrying LacZ or Smad2 were transfected with ARE-Luc/FAST-1 and pSV-β-Gal and then incubated with (dark bars) or without (gray bars) TGF-β. Luciferase activity was measured as described in Figure 3D, and data are presented as means ± SD of four experiments. (D) HaCaT cells infected as described in A were treated with (+) or without (-) TGF-β for 1 h. Total cell lysates were immunoblotted with the indicated antibodies. (E) HaCaT cells were infected with the indicated adenoviruses at 50 MOI (lanes 1–4) or 100 MOI (lane 5) and then incubated for 12 h in the presence (+) or absence (-) of TGF-β. Immunoblotting was performed with the indicated antibodies. (F) HaCaT cells infected as described in E were incubated for 12 h in the presence (+) or absence (-) of TGF-β. A Cdk2 kinase assay was carried out as described in Figure 2C.
Figure 6.
Figure 6.
Restoration of TGF-β-induced cytostasis by Smad2 depletion or Smad3 overexpression in cells resistant to TGF-β. (A) The indicated cell lines were transfected with each siRNA and then incubated with (+) or without (-) TGF-β. Cell proliferation was determined by counting cell numbers on day 2 after treating TGF-β. Data are presented as means ± SD of three experiments (*p < 0.05, **p < 0.01). (B) Indicated cell lines transfected as described in A were treated with or without TGF-β for 24 h and then subjected to flow cytometric analysis. The percentages of cells in the G1 (black bars), S (hatched bars), and G2/M (gray bars) phases were determined based on the DNA content histograms and graphed. (C) SNU-398 (left) or SNU-739 (right) cells infected with adenoviruses carrying β-galactosidase (LacZ), FLAG-tagged Smad2, or Smad3 cDNA were incubated in the presence or absence of TGF-β. At the indicated times after treating TGF-β, cells were analyzed as described in B. The percentages of cells in the G 1 (top) and S (bottom) phases were determined based on their DNA content histograms and graphed. The data shown are representative of four independent experiments.
Figure 7.
Figure 7.
Restoration of TGF-β-induced cytostasis by Smad2 depletion is caused by the facilitation of a Smad3-dependent pathway. (A and B) The indicated cell lines transfected with each siRNA were incubated for 1 h with (+) or without (-) TGF-β. Whole cell lysates were immunoblotted with the indicated antibodies. (C) Lysates were prepared from Panc-1 cells transfected with each siRNA in the presence of TGF-β for the indicated times and immunoblotted with the indicated antibodies. (D) Panc-1 cells transfected with (SBE)4-Luc together with the indicated siRNAs and pSV-β-gal were incubated in the presence or absence of neutralizing anti-TGF-β antibody. Luciferase activity was measured 30 h after transfection. Transfection efficiency was normalized versus β-galactosidase activity, and data are presented as means ± SD of four experiments (left). Whole cell lysates prepared from these cells were immunoblotted with the indicated antibodies (right).
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