TGF-β and Wnt proteins cooperate in tissue specification during development. Smad proteins, β-catenin and LEF1/TCF can act as transcriptional regulators in the TGF-β or Wnt signaling cascade, regulating specific gene expression depending on the cellular context. Here we report on the physical association and functional cooperation between Smad7 and β-catenin in the regulation of apoptosis in prostate cancer cells. We show that TGF-β treatment of PC-3U and HaCaT cells lead to an increase of the levels of β-catenin and LEF1 in a Smad7-dependent manner and to a TGF-β-dependent association of Smad7 with β-catenin and LEF1. Our data also show that Smad7 is required for the TGF-β induced phosphorylation of p38, GSK-3β on Ser9, and Akt on Ser 473, which correlates with the observed TGF-β-induced accumulation of β-catenin. Smad7, β-catenin, and TCF-4 had synergistic effects on the synthetic TCF-4 promoter TOPFLASH, and β-catenin and LEF1/TCF enhanced transcription by a Gal4-Smad7 fusion protein. These observations suggest that Smad7 can affect gene regulation. Interestingly, PC-3U and HaCaT cells transiently transfected with β-catenin siRNA were protected against apoptosis induced by TGF-β, demonstrating a functional cooperation of Smad7 and β-catenin in this pathway. Our current data are summarized schematically in Fig. .
FIG. 7. Schematic illustration of the molecular mechanisms involved in the TGF-β-Smad7 signaling pathway resulting in activation of Akt and p38, followed by phosphorylation and inactivation of GSK-3β. The inactivated GSK-3β thus cannot (more ...)
Smad3 and Smad4 have previously been found to be directly associated with LEF1, and TGF-β has been shown to enhance the interaction between Smad3 and LEF1 in HEK 293T cells and to promote their activity on a Xtwn
promoter. Furthermore, putative Smad4 binding sites were identified in the Xtwn
). The association between Smad3, Smad4, and β-catenin was found to be indirect, while colocalization of endogenous β-catenin and Smad4 was demonstrated in the nucleus of Xenopus
animal cap cells, injected with Xwnt
). We report here that TGF-β treatment leads to an interaction between endogenous β-catenin, LEF1 and endogenous Smad7 in prostate cancer cells. Our results suggest that Smad7 is required for the TGF-β-induced interaction between β-catenin and LEF1, as we observed in two cell lines that repression of Smad7 by siRNA prevents the TGF-β-induced assembly of the β-catenin-LEF1 complex.
Interestingly, the interaction between Smad7 and β-catenin occurred primarily via the N-terminal part of Smad7. This finding is notable since the N-terminal part of Smad7 is not conserved in the R-Smads and Smad4. In contrast, the C-terminal MH2 domain of Smad7 was found to interact with LEF1 and TCF-4. Moreover, the interactions were stronger with the MH2 domain only than with wild-type Smad7, suggesting that wild-type Smad7 can occur in a latent conformation. We examined whether the colocalization of phospho-Smad3 and β-catenin in the nucleus was affected by Smad7 and we observed colocalization of the proteins in the nucleus of TGF-β-treated PC-3U cells but not in PC-3U/AS-S7 cells with a small amount of Smad7 (see Fig. S4 in the supplemental material). This finding suggests that Smad7 is required for the TGF-β-induced accumulation of β-catenin and its cooperation with the Smad3-Smad4 complex. Our findings thus suggest that the specific effects of β-catenin on different promoters are moderated by alterations in the nuclear amounts of the β-catenin-LEF1/TCF complex as well of R-Smads, Co-Smads, and I-Smads and their transcriptional coregulators or corepressors.
We have earlier reported that expression of Smad7 is required for TGF-β-induced activation of the TAK1-MKK3-p38 MAP kinase pathway and subsequent apoptosis of prostate cancer cells (18
). In the current study, our data indicate that both Smad7 and p38 MAP kinase positively contributed to the accumulation of β-catenin and that high levels of Smad7 cause an increase of c-myc
expression. Our preliminary data suggest that Smad7 has positive effects on a full-length human c-Myc promoter (see Fig. S1 in the supplemental material). However, future studies are required to more precisely investigate the effects of Smad7 alone or in combination with β-catenin and LEF1 on the c-myc
promoter. In addition, the identification of a specific responsive site in the promoter is needed for a proper evaluation of the putative effects of Smad7 on c-Myc.
c-Myc has been shown to potently induce apoptosis, especially under conditions of stress, genotoxic damage or depletion of survival factors (19
); therefore it is possible that β-catenin-mediated induction of c-myc
contributes to Smad7-induced apoptosis. TGF-β is known to repress c-myc
in a Smad3-dependent manner (11
). Smad3 is present in a cytoplasmic complex consisting of the transcription factors E2F4/5 and DP1 and the corepressor p107. In response to TGF-β, this complex associates with Smad4, recognizing a composite Smad-E2F site on c-myc
for repression (11
). Our data propose that Smad7 has a positive effect on the expression of c-Myc, which could be explained by cooperation with β-catenin and LEF1 on the c-myc
promoter. This possibility requires future studies. Alternatively, Smad7-induced activation of the p38 MAP kinase pathway could cause activation of the c
Another possible explanation for the increase in c-Myc expression might be that Smad7, in a TGF-β- and p38 MAP kinase-dependent manner, regulates the localization and levels of β-catenin, through their effect on GSK-3β, as discussed below. Our finding that Smad7, together with β-catenin and LEF1/TCF, induces an increase of c-myc
expression suggests that Smad7 acts as a molecular switch downstream of the activated TGF-β-receptor complex to coordinate responses from both Wnt and TGF-β signals, or alternatively, that TGF-β via Smad7 regulates the localization and activity of β-catenin independently from the canonical Wnt signaling pathway. Oncoproteins like Myc and E2F1 regulate G1
progression and are also potent inducers of apoptosis by their promotion of release of cytochrome c
from mitochondria. The release of cytochrome c
is prevented by survival signals (34
). In this respect, it is interesting that both Smad7 and β-catenin have been shown to inhibit NF-κB activity (3
). Future investigations will more precisely describe the mechanism for the observed positive regulation of Smad7 and p38 MAP kinase on c-Myc expression, and if Smad7 is required for nuclear accumulation of β-catenin in the canonical Wnt signaling pathway.
GSK-3 is a multifunctional serine/threonine kinase, acting as a key regulator in cellular responses evoked by Wnt, receptor tyrosine kinases and G-protein-coupled receptors (16a). GSK-3 has been reported to have important regulatory roles in apoptosis in a tissue specific manner. Thus, overexpression of GSK-3 in mammalian neuronal cells promotes apoptosis, while massive apoptosis is observed in the liver of GSK-3β−/−
embryos, which causes their death around embryonic day 16. GSK-3β was found to be required for the NF-κB-mediated survival responses evoked by tumor necrosis factor alpha (27
We report here that Smad7 is required for TGF-β-induced phosphorylation of Ser9 in GSK-3β. Several growth factors like insulin and epidermal growth factor have previously been shown to induce phosphorylation of GSK-3β on Ser9 and thereby inactivate this kinase. The inhibition of GSK-3β by insulin has been demonstrated to occur via a phosphatidylinositol 3-kinase-dependent pathway via activation of Akt (12a
). The exact mechanism whereby the canonical Wnt signal regulates GSK-3β is not well known, however, it has been reported that GSK-3β is not phosphorylated at Ser9 in response to Wnt signaling (16
). The observed accumulation of β-catenin induced by TGF-β in our study led us to examine the possible role of Akt and GSK-3β in this pathway, since p38 MAP kinase has been demonstrated to be involved in the phosphorylation of Akt on Thr308 and Ser473 (49
). Activated Akt can in turn lead to the phosphorylation on Ser9 in GSK-3β and its inactivation (9
We observed that TGF-β causes phosphorylation of GSK-3β 30 min after stimulation of cells and that phosphorylation of Akt on Ser473 occurs just 15 min after stimulation. Furthermore, TGF-β-induced phosphorylation of Akt required Smad7. These findings indicate that TGF-β-induced activation of p38 MAP kinase preceded the phosphorylation of Akt in PC-3U cells. However, after longer periods of TGF-β stimulation, i.e., 24 and 48 h, when PC-3U cells undergo apoptosis, the amounts of Akt phosphorylated on Ser473 declined, which then could promote an apoptotic response. The requirement of Smad7 for TGF-β-induced activation of p38 MAP kinase is consistent with our previous report that Smad7 facilitates the interaction between MKK3 and p38 MAP kinase in PC-3U cells (18
) and is also in line with the report (58
) showing that the TGF-β-induced activation of p38 MAP kinase is required for TGF-β-induced apoptosis of mouse mammary epithelial cells. As we found that Smad7 is required for the TGF-β-induced activation of p38 and phosphorylation of Akt and GSK-3β, we are currently investigating the possibility that Smad7 could regulate the activity of GSK-3β by acting as a scaffold between p38, Akt, and GSK-3β.