TACE regulates the TβRI cell surface levels. (A, B) TAPI-1 inhibits the downregulation of cell surface TβRI level in response to PMA (A) or serum (B). CHO cells were treated with PMA (A) or serum (B) in the absence or presence of TAPI-1. Cell surface TGF-β receptors were detected by biotinylation, neutravidin bead adsorption and immunoblotting. The second panel from the top is a longer exposure of the top panel, showing the extracellularly truncated TβRI, marked TβRI(TM+C). Other panels show western blots of cell lysates for TβRI, TβRII, pErk1/2, Erk1/2 or α-tubulin (C, F). (C, D) Silencing of TACE expression by transfected TACE siRNA inhibits the decrease in cell surface TβRI induced by Erk MAP kinase activation in response to PMA (C) or serum (D). CHO cells, transfected with TACE siRNA or control siRNA, were treated with PMA (C) or serum (D) and processed as in A and B.

TACE inhibits TGF-β signaling. (A) CHO cells were treated with or without PMA and/or TGF-β for 30 min as in Fig. 1A, in the presence or absence of TAPI-1. (B) CHO cells were transfected with TACE siRNA or control siRNA, 24 h prior to treatments with PMA and/or TGF-β as in A. Cell lysates were assayed by western blotting for phosphoSmad3, Smad3 or TACE. (C) Silencing of TACE expression enhances the long-term TGF-β-induced Smad3 activation. CHO cells, transfected with TACE siRNA or control siRNA, were treated with TGF-β, and lysates were assayed by immunblotting for phosphoSmad3, Smad3 and TACE. (D) Silencing of TACE expression enhances the TGF-β-induced Akt activation. CHO cells, transfected with TACE siRNA or control siRNA, were treated with TGF-β for 90 min, and Akt activation was visualized by immunoblotting for pT308-Akt. Control blots show Akt and TACE.

TACE regulates the epithelial to mesenchymal transition of HaCaT cells in response to TGF-β. (A) HaCaT cells were transfected with TACE siRNA or control siRNA, and treated or not with TGF-β. (B) Effect of the TβRI kinase inhibitor SB431542 on HaCaT cells, transfected with TACE siRNA or control siRNA, and treated or not with TGF-β. Top panels shows the actin organization, and middle and lower panels show E-cadherin and fibronectin detected in the same cells using two color immunofluorescence. Bars, 20 μm.

Activation of the Erk MAP kinase pathway inhibits TGF-β-induced Smad3 phosphorylation and cell surface presentation of TβRI but not TβRII. (A) CHO cells were treated with TGF-β for 30 min. The Erk MAP kinase pathway was activated with PMA for 45 min before adding TGF-β and then during TGF-β treatment, and inhibited using U0126. Smad3 activation was visualized by western blotting for C-terminal Smad3 phosphorylation. (B) Effects of the protein kinase C inhibitor BMI, the Erk MAP kinase pathway inhibitor U0126 and the PI3-kinase inhibitor LY294002 on the PMA-induced decrease of TGF-β-induced Smad3 activation. (C-F) CHO cells were treated with PMA (C-E) or serum (F) for the indicated times (C, D, F) or for 45 min (E), in the absence or presence of the Erk MAP kinase pathway inhibitor U0126 (C-F) or PD98059 (E), the protein kinase C inhibitor BMI (E) or the PI3-kinase inhibitor LY294002 (E), or the proteasome inhibitor MG-132 (D). Cell surface TGF-β receptors were detected by biotinylation, neutravidin bead adsorption and immunoblotting. In C and F, the second panel from the top is a longer exposure of the top panel, showing the extracellularly truncated TβRI, marked TβRI(TM+C), which is also visualized in D, E. Other panels show western blots of cell lysates for TβRI, TβRII, pErk1/2, Erk1/2 or α-tubulin (C, F).

TACE cleaves TβRI but not TβRII. (A) C-terminally Flag-tagged TβRI or TβRII were coexpressed with wild-type TACE or catalytically inactive TACE E406A. Anti-Flag immunoprecipitated proteins were immunoblotted with anti-Flag, and lysates were probed with anti-Flag or anti-TACE. (B) N-terminally HA-tagged TβRI was coexpressed with TACE. Culture media were subjected to immunoprecipitation and western blot with anti-HA antibody. Cell lysates were probed with anti-HA or anti-TACE. (C) Coincubation of immunopurified TβRI and TACE in vitro resulted in cleavage of TβRI by TACE (middle lane). (D) TACE cleaves TβRI, but not the other type I receptors. TACE was coexpressed with the indicated type I receptors. Cell lysates were subjected to anti-Flag immunoprecipitation and immunoblotting with anti-Flag (top panel), or to anti-TACE immunoblotting (lower panel).

Inhibition of TACE expression enhances the TGF-β response and inhibition of cell proliferation by TGF-β in HaCaT epithelial cells. (A) Role of TACE in TβRI cell surface presentation. HaCaT, T4-2, MDA-MB-468, Hela, A549 and HepG2 cells were transfected with TACE siRNA or control siRNA. Cell surface TβRI and TβRII were visualized by immunoblotting of biotinylated proteins. Control panels show TβRI and α-tubulin in cell lysates, as well as TACE in cell lysates (MDA-MB-468, Hela and A549 cells) or cell lysates enriched for TACE by ConA precipitation (HaCaT, T4-2 and HepG2 cells). (B) Cell surface levels of TβRI and TβRII in HaCaT cells transfected with TACE siRNA or control siRNA. (C) HaCaT cells, transfected with TACE siRNA or control siRNA, were treated with TGF-β. Cell lysates were immunoblotted for phospho-Smad3, Smad3 or TACE. (D) Expression of Smad7 and PAI-1 mRNAs in response to TGF-β was quantified using RT-PCR. (E) Cells were cultured in the presence or absence of TGF-β with or without the TβRI inhibitor SB431542 for 12 h or 36 h. BrdU incorporation during a 12 h period is expressed relative to the untreated control. Error bars were generated from triplicate experiments.

TACE downregulates TGF-β signaling in T4-2 breast cancer cells. (A) Treatment of T4-2 cells with PMA or EGF enhances the in vitro proteolytic activity of immunoprecipitated TACE, which is inhibited by treating the cells with the MEK inhibitors PD98059 or U0126. (B) T4-2 cells were treated with PD98059 to inhibit, or with EGF to activate the Erk MAP kinase pathway. Biotinylated cell surface proteins and cell lysates were immunoblotted using the indicated antibodies. (C) T4-2 cells were transfected with TACE siRNA or control siRNA. Cell surface TβRI and TβRII were visualized by immunoblotting of biotinylated cell surface proteins. Cell lysates were immunoblotted for TβRI, TβRII, TACE and α-tubulin. (D) T4-2 cells, transfected with TACE siRNA or control siRNA, were treated with TGF-β. Cell lysates were immunoblotted for phospho-Smad3, Smad3 or TACE. (E) Cells were cultured with or without TGF-β for 24 h or 48 h. The relative proliferation inhibition corresponds to the BrdU incorporation in cells with TGF-β treatment divided by BrdU incorporation in cells without TGF-β treatment. Error bars were generated from triplicate experiments.