Erk MAP kinase pathway activation inhibits TGF-β-induced Smad3 phosphorylation
To evaluate the effect of the Erk MAP kinase pathway on TGF-β-induced Smad activation, we treated CHO cells with PMA, a potent activator of Erk MAP kinase signaling (Amaral et al., 1993
). As shown in , treatment with PMA inhibited the TGF-β-induced phosphorylation of Smad3. This inhibition was reversed by treating the cells with U0126, an inhibitor of the MEK1 and MEK2 kinases that activate Erk MAP kinases 1 and 2 (DeSilva et al., 1998
). PMA is known to activate protein kinase C, and Erk MAP kinase pathway activation by PMA depends epistatically on protein kinase C activation (Qiu and Leslie, 1994
). Accordingly, the decrease in TGF-β-induced phosphorylation of Smad3 by PMA treatment was comparably reversed by BMI, a protein kinase C inhibitor, and U0126. In contrast, the PI3-kinase inhibitor LY294002 had no effect ().
Figure 1 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 (more ...)
Erk MAP kinase pathway activation downregulates the cell surface presentation of TβRI, but not TβRII
The inhibition of TGF-β-induced Smad3 phosphorylation raises the possibility that Erk MAP kinase pathway activation affects the levels or activities of TβRII and/or TβRI. We therefore tested whether PMA induced alterations in the cell surface levels of TβRII or TβRI, detected by cell surface protein biotinylation (). PMA induced a time-dependent decrease of cell surface TβRI, but not of TβRII, accompanied by the appearance of a smaller form of TβRI. This form was poorly labeled by cell surface biotinylation, had the size of an extracellularly truncated TβRI and appeared to undergo degradation. In the presence of the proteasomal degradation inhibitor MG-132, the level of truncated TβRI at the cell surface increased and correlated inversely with the reduction of full length TβRI at the cell surface (). The decrease of TβRI at the cell surface and appearance of truncated TβRI were inhibited by the MEK1/2 inhibitor U0126. Consistent with the epistatic dependence of PMA-induced Erk MAP kinase pathway activation on protein kinase C, the protein kinase C inhibitor BMI and MEK1/2 inhibitors U0126 or PD98059 prevented the decrease of cell surface TβRI and the appearance of truncated TβRI in response to PMA, while the PI3-kinase inhibitor LY294002 had no effect (). Similarly to PMA, serum also induced a decrease in cell surface levels of TβRI but not TβRII, resulting from activation of the Erk MAP kinase pathway. The effect of serum was less pronounced than that of PMA, consistent with the stronger Erk MAP kinase activation by PMA ().
The reduction in cell surface TβRI, resulting from Erk MAP kinase pathway activation, is mediated by TACE
To test whether the decrease in cell surface presentation of TβRI resulted from ectodomain shedding, we examined the effect of TAPI-1, a metalloprotease inhibitor. As shown in , TAPI-1 inhibited the decrease in cell surface TβRI levels in response to PMA or serum, indicating that this decrease resulted from metalloprotease-mediated shedding of TβRI at the cell surface.
Figure 2 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. (more ...)
We have shown that activation of the Erk MAP kinase pathway, e.g. in response to PMA or serum, results in increased ectodomain shedding by TACE (Fan and Derynck, 1999
; Fan et al., 2003
). Since TAPI-1 inhibited the decrease in TβRI levels at the cell surface, and TAPI-1 inhibits TACE (Slack et al., 2001
), we examined the effect of knocking down TACE expression using transfected siRNA. As shown in , silencing TACE expression blocked the decrease in TβRI cell surface levels and TβRI shedding in response to PMA or serum. These data indicate that TACE is required for TβRI ectodomain shedding, and strongly suggest that TACE may be the effector of TβRI shedding in response to PMA or serum.
TACE cleaves the type I receptor TβRI, but not the type II receptor TβRII
To examine whether TACE can serve as effector of TβRI ectodomain shedding, we co-expressed TACE with C-terminally Flag-tagged TβRI or TβRII in CHO cells. Under these conditions of overexpression, TACE has proteolytic activity, not requiring activation in response to e.g. PMA (Fan et al., 2003
). TACE co-expression resulted in the appearance of a 40 kd TβRI band, about the size of extracellularly truncated TβRI, concomitantly with a decrease in the level of full size TβRI (). The 40 kd band was expressed at much lower level, and full size TβRI level was at much higher level, in cells without TACE co-expression. Equal expression of the catalytically inactive E406A TACE mutant did not confer TβRI ectodomain shedding. Further, TACE did not affect the TβRII levels or generate a truncated TβRII, suggesting that TβRII is not a substrate of TACE ().
Figure 3 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, (more ...)
Ectodomain shedding of membrane-bound proteins releases extracellular domains into the medium. Accordingly, the extracellular domain of TβRI, HA-tagged between amino acids 27 and 28, was detected in the medium of cells expressing wild-type TACE, but not inactive TACE (). Further evidence that TβRI can serve as TACE substrate was provided by the cleavage of TβRI by TACE in vitro, using immunopurified proteins (). The low cleavage efficiency was not surprising since we do not know how to activate TACE in vitro. The 40 kd in vitro cleavage product had the same size as the extracellularly truncated TβRI in .
These results, together with the results using TAPI-1 and TACE siRNA, indicate that shedding of TβRI by TACE mediates the decrease in cell surface TβRI levels in response to Erk MAP kinase activation. Cleavage of a TGF-β family type I receptor by TACE was specific for TβRI, since it was not observed with other type I receptors (). Accordingly, BMP-2-induced Smad1 activation was not affected when TACE expression was silenced (data not shown).
TACE expression defines the level of Smad activation by TGF-β
We next evaluated whether the decrease in TGF-β-induced Smad3 activation upon Erk MAP kinase pathway activation was due to the activity of TACE. As shown in , TAPI-1 inhibited the PMA-induced decrease of Smad3 phosphorylation, indicating that inhibition of metalloprotease activity restored Smad3 activation by TGF-β to a level similar to that in the absence of Erk MAP kinase activation. To specifically address whether TACE mediated the decrease in Smad3 activation, cells were transfected with TACE siRNA or control siRNA. Silencing TACE expression reversed the inhibition of TGF-β-induced Smad3 phosphorylation by Erk MAP kinase pathway activation (). In addition, the long-term activation of Smad3 in the absence of PMA was enhanced when TACE expression was silenced (). Finally, the effect of TACE on TGF-β signaling extended to non-Smad signaling. Indeed, also the level of TGF-β-induced Akt activation was enhanced when TACE expression was silenced ().
Figure 4 TACE inhibits TGF-β signaling. (A) CHO cells were treated with or without PMA and/or TGF-β for 30 min as in , in the presence or absence of TAPI-1. (B) CHO cells were transfected with TACE siRNA or control siRNA, 24 h prior to treatments (more ...)
These data indicate that activation of the Erk MAP kinase pathway inhibits the TGF-β-induced Smad3 activation through TACE, and that TACE expression and activity define the Smad and non-Smad signaling activity in response to TGF-β.
TACE expression defines the cell surface TβRI levels and TGF-β responses in HaCaT epithelial cells
We next evaluated whether TACE regulates the cell surface TβRI levels and TGF-β responsiveness in other epithelial cell systems. As shown in , silencing TACE expression enhanced the TβRI cell surface level in HaCaT epithelial cells, T4-2 and MDA-MB-468 breast cancer cells, Hela squamous carcinoma cells, A549 lung carcinoma cells and HepG2 hepatoma cells. The cell surface TβRI levels were not increased in MCF-7 and MCF-10A breast cancer cells upon transfection with TACE siRNA (data not shown). These data indicate that TACE expression regulates the cell surface level of TβRI in a variety of cells.
Figure 5 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 (more ...)
HaCaT cells are seen as a model for how non-transformed, epithelial cells respond to autocrine and paracrine TGF-β signaling, but the role of TACE in these cells has not been explored. To evaluate the regulation of TGF-β signaling by TACE, we compared the TGF-β responses in HaCaT cells, in which TACE expression was silenced, with HaCaT cells transfected with a control siRNA. As shown in , silencing TACE expression increased the cell surface levels of TβRI, without affecting the total TβRI levels, and did not affect the cell surface or total TβRII levels. Further, these cells showed a higher level of Smad3 activation in response to TGF-β, which was still apparent after long exposure to TGF-β (). We also examined the effect of TACE downregulation on the TGF-β target genes encoding Smad7 and PAI-1. As shown in , cells with silenced TACE expression showed increased Smad7 and PAI-1 mRNA levels in response to TGF-β, when compared to control cells. The basal level of Smad7 mRNA in the absence of added TGF-β was also increased, reflecting autocrine TGF-β signaling. These data demonstrate that TACE negatively regulates TGF-β-induced Smad activation and transcription.
TACE regulates the control of cell proliferation by TGF-β
Considering the effect of TACE on TGF-β-induced transcription, we compared the proliferation of HaCaT cells with downregulated TACE expression and control HaCaT cells, in the absence or presence of TGF-β. Cell proliferation was assessed by BrdU incorporation, and examined after 12 or 36 h in the presence of TGF-β. As expected, the antiproliferative effect of TGF-β was more pronounced after 36 h ().
Treatment of HaCaT cells with TGF-β reduced proliferation with ~60 % after 36 h. In cells with silenced TACE expression, the proliferation of the TGF-β-treated cells was only 8% of that of the untreated cells. In the absence of added TGF-β, their proliferation rate was only half of that of the control cells, reflecting a high sensitivity to autocrine control of proliferation by TGF-β, as confirmed by the increased proliferation in the presence of the TβRI inhibitor SB431542 (). These data illustrate that the activity of TACE is a determinant of the antiproliferative effect of TGF-β and of cell proliferation, which is under autocrine control of TGF-β. Thus, increased TACE activity decreases the growth inhibition by autocrine or paracrine TGF-β, and may in this way contribute to cancer progression.
TACE regulates epithelial to mesenchymal transition in response to TGF-β
Epithelial cells can undergo EMT in response to TGF-β. This response combines Smad and non-Smad signaling and results in a loss of epithelial characteristics, such as cortical actin organization and localization of E-cadherin at cell junctions, and acquisition of mesenchymal characteristics, such as reorganization of the actin cytoskeleton, increased fibronectin expression, migration and invasion (Zavadil and Bottinger, 2005). Considering the role of TACE in defining the TGF-β responsiveness, we evaluated whether silencing TACE expression affects the TGF-β-induced EMT response of HaCaT cells ().
Figure 6 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 (more ...)
HaCaT cells transfected with control siRNA have, in the absence of added TGF-β, an epithelial cobblestone morphology with cortical actin staining, localization of E-cadherin at cell contacts, and lack of fibronectin staining. In the presence of increasing TGF-β levels, starting at 0.25 ng/ml of added TGF-β, the cells lost their epithelial phenotype, reorganized their actin cytoskeleton, lost junctional E-cadherin localization and showed increased fibronectin staining (). In contrast, cells with silenced TACE expression (), had already in the absence of added TGF-β a disorganized actin pattern, lacked E-cadherin staining at cell contacts and showed a low level fibronectin staining. Further, the cells acquired a more pronounced fibroblast phenotype at much lower concentrations of added TGF-β, compared with HaCaT cells transfected with control siRNA, reflecting an increased sensitivity to TGF-β ().
The loss of epithelial and acquisition of mesenchymal properties in cells with silenced TACE expression, in the absence of added TGF-β, may result from autocrine TGF-β signaling. As shown in , adding the TβRI inhibitor SB431542 to HaCaT cells with downregulated TACE expression reversed the cells to an epithelial morphology with cortical actin staining, E-cadherin staining at cell-cell contacts, and lack of fibronectin staining. These results illustrate that the EMT response to endogenous or added TGF-β is regulated by TACE.
TACE downregulates TGF-β signaling in T4-2 breast cancer cells
Increased TACE expression has been implicated in the control of signaling by TGF-α family growth factors through the EGFR in breast cancer cells. Indeed, increased TACE activity confers increased TGF-α release, resulting in increased, EGFR-mediated growth stimulation (Borrell-Pages et al., 2003
; Kenny and Bissell, 2007
; Zhou et al., 2006
). We examined the regulation of TGF-β signaling by TACE in T4-2 breast cancer cells, which show autocrine, EGFR-dependent parameters of malignant transformation, resulting from TACE-mediated ectodomain shedding of TGF-α and amphiregulin. Downregulation of TACE in these cells results in decreased EGFR activation and reverses the transformed phenotype (Kenny and Bissell, 2007
Consistent with the regulation of TACE activity by the Erk MAP kinase pathway (Fan and Derynck, 1999
; Diaz-Rodriguez et al., 2002
) (), addition of EGF, which enhances Erk MAP kinase signaling, decreased the level of cell surface TβRI, whereas inhibition of Erk MAP kinase signaling using the MEK inhibitor PD98059 enhanced the TβRI cell surface level. Consequently, EGF signaling decreased, and addition of PD98059 enhanced the level of Smad3 activation ().
Figure 7 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 (more ...)
Using TACE siRNA, we generated T4-2 cells with silenced TACE expression and compared these with T4-2 cells transfected with control siRNA (). As in HaCaT cells, silencing TACE expression resulted in increased cell surface presentation of TβRI, without an effect on the cell surface TβRII level (). Consistent with this difference in cell surface TβRI levels, the decrease in TACE expression resulted in a modestly increased Smad3 activation in response to TGF-β ().
We also examined the proliferation of these cells in the absence or presence of TGF-β for 24 or 48 h. Since these cells show EGFR-dependent signaling that is regulated by TACE’s effects on EGFR ligand production, the T4-2 cells were cultured in the presence of EGF, thus providing maximal EGFR activation. Under these conditions, TGF-β induced a modest growth inhibition, which was enhanced when TACE expression was silenced. As with HaCaT cells, the antiproliferative effect of TGF-β was more pronounced after 48 h, compared to 24 h (). Thus, also in these breast cancer cells with maximized EGFR signaling, did TACE regulate the growth inhibition response to TGF-β, with increased TACE levels making the cells less prone to growth inhibition by TGF-β.