Smad proteins are sufficient and necessary for transcription but only sufficient and not necessary for G₁ arrest induced by TGF-β. (A,B) Smad-induced transcription was modified in EpH4 mammary epithelial cells by expressing activated Smad2 (Smad2^EDME) or dominant-negative Smad2 (Smad2^AAMA) or the Mad Homology domain 1 of Smad4 (Smad4^MH1) using high-titer retroviral vectors. (C) Transcription of transient TGF-β reporter normalized to β-actin control and Western blot for the endogenous p15 (TGF-β activated), cdc25A (TGF-β repressed), and p16 (control). (D) FACS-DNA profiles of EpH4 cells expressing the respective Smad protein. Cells were released from contact inhibition into cell cycle for 22 h, in the presence or absence of TGF-β. The activated Smad2^EDME arrests cells in G₁ under all conditions and activates transcription; dominant-negative Smad2^AAMA or SMAD4^MH1 were unable to prevent the TGF-β-induced G₁ arrest in EpH4 cells despite their ability tototo antagonize TGF-β-induced transcriptional effects. (E) The expression of Smad4^MH1 was sufficient to overcome Smad2^EDME-mediated cell cycle arrest but not TGF-β-mediated arrest, arguing for an additional, not transcription-mediated, mechanism for induction of G₁ arrest by TGF-β.

p70^s6k pathway is rate limiting for TGF-β-induced G₁ arrest. (A,B) Synergistic action of TGF-β with Wortmannin and Rapamycin on G₁ arrest, but not transcriptional activation of a TGF-β responsive promoter. (A) FACS analysis of EpH4 cells 22 h after cell cycle release. Cells were treated with various drugs as indicated during the entire release period. High concentrations of TGF-β Wortmannin or Rapamycin alone induce G₁ arrest in epithelial cells (left panel). Ineffective doses of Wortmannin and Rapamycin complement low, noninhibitory TGF-β concentrations to cause a complete G₁ arrest (central and right panels). (B) Wortmannin, LY 294002, and Rapamycin did not affect TGF-β-induced PAI-1 reporter gene transcription. (C,D) Overexpression of p70^s6k (C, Western blot) and FACS analysis determining proportion of cells in G₁ after TGF-β induction in dependence of p70^s6k and Smad2^AAMA expression (D). Only coexpression of p70^s6k and dominant-negative Smad2 protein release cells from TGF-β-induced G₁ arrest. Neither p70^s6k (D, lane 3) nor Smad2^AAMA (D, lane 2) alone are capable to relieve the TGF-β-mediated cell cycle arrest, whereas both proteins expressed together effectively do so (lane 4). (E) FACS analysis of cells released from G₁ arrest 22 h after TGF-β induction in dependence of p70^s6k and Smad4^MH1 expression. Activation of p70^s6k and inhibition of Smad-induced transcription are sufficient to completely protect cells from TGF-β-induced cell cycle arrest.

TGF-β inhibits p70^s6k and antagonizes EGFR signaling pathways at the level of p70^s6k. (A) p70^s6k kinase activity in EpH4 cells arrested in G₁ and exposed to TGF-β for various times. Sixty-five percent inhibition of p70^s6k activity by TGF-β was observed after 5 min, persisting for more than 1 h. (B) p70^s6k kinase activity was determined in epithelial cells, arrested in G₁ by contact inhibition, released into cell cycle, and treated with TGF-β or left untreated. TGF-β inhibits the increase of p70^s6k activity in early G₁. (♦) Control; (□) plus 5ng/mL TGF-β. (C) p70^s6k activity in response to TGF-β and an EGFR kinase inhibitor in epithelial EpH4 cells after 20 min. The inhibitory effects of a combination of TGF-β and PD 153.035 are synergistic. (D) p70^s6k activity in response to TGF-β and TGF-α in unsynchronized cycling cells. TGF-β inhibits the p70^s6k; TGF-α activates p70^s6k and partially neutralizes the inhibitory action of TGF-β on p70^s6k activity. (Open bars) 5 ng/mL of TGF-α; (gray bars) TGF-α + TGF-β; (black bars) 5ng/mL of TGF-β. (E,F) Immunoprecipitation and Western blot of p70^s6k transiently cotransfected in 293 cells with wild-type TβRI and II or inactive mutants (TβRI +II dn). (E) TGF-β inhibits the hyper-phosphorylation of p70^s6k in dependence on an activatable TGF-β–TβR complex. Serum-induced hyperphosphorylated forms of p70^s6k (p70^s6k pp) disappear only after TGF-β stimulation of the coexpressed wild-type TβRI and TβRII (lanes 6,7), but not by stimulation of kinase inactivated TβRI/II (lane 2,3). (F) Corresponding p70^s6k kinase activities determined in immunoprecipitates shown in E. The decrease of p70^s6k pp (Fig. 4a, lane 7) is accompanied by a reduction of p70^s6k kinase activity by more than 60%.

TGF-β induces de-phosphorylation of p70^s6k by stimulating association of PP2A subunits C, Aβ and Bα and p70^s6k and association of PP2A-Bα and TβRI. (A) Western blot for PP2A-C after immunoprecipitation of p70^s6k TβR expressing cells were treated with TGF-β and/or the PP2A inhibitor okadaic acid or left untreated. Interaction of PP2A with p70^s6k is stimulated in cells expressing the TβRI/TβRII receptor complex on addition of TGF-β (lane 6), but inhibited by the presence of okadaic acid (Okad.A., lane 9). Coexpression of inactive TβRI/TβRII neg. fails to stimulate interaction (lanes 2,5,8) above basal binding activity (lanes 1,4,7). (B) Western blot for the expression of the endogenous regulatory subunit PP2A-Bα EpH4 epithelial cells express high levels of PP2A-Bα, and its expression is not altered by 1 h of TGF-β induction. Endogenous levels of PP2A-Bα in Mv1Lu cells and primary embryonic fibroblasts are below detection limits. (C) Immunoprecipitation for PP2A-Bα and Western blot for TβRI and TβRII. PP2A-Bα interacts with TβRI and TβRII on TGF-β stimulation in EpH4, but similar complexes are not detectable in Mv1Lu cells or primary embryonic mouse fibroblasts (MEF). (D) Western blot for PP2A-Bα after immunoprecipitation of p70^s6k in EpH4 or Mv1Lu cells. PP2A-Bα interaction with p70^s6k is observed specifically in response to TGF-β in EpH4 cells and not in response to other inhibitors of p70^s6k or in Mv1Lu cells. (E) Western blot for p70^s6k after immunoprecipitation of PP2A-Bα (lanes 1,2) or using a control antibody (lanes 3,4) and direct Western blot for Thr389 of p70^s6k from cells stimulated with TGF-β in early G₁. In response to TGF-β, PP2A-Bα is found in complexes with p70^s6k and Thr 389 phosphorylation of p70^s6k decreases. (F) Western blot for PP2A-Aβ before (top panel) and after (bottom panel) immunoprecipitation with p70^s6k. In EpH4 cells, Aβ interacts with p70^s6k specifically in response to TGF-β.

Model for the two independent pathways initiated by TGF-β to induce G₁-arrest. Both, inhibition of p70^s6k and activation of Smad-induced transcription are independently sufficient to induce G₁ arrest.