Canonical Wnt signals are mediated by the transcriptional effector, β-Catenin. In the absence of Wnt signaling, β-Catenin is phosphorylated by a destruction complex of glycogen synthase kinase-3β (GSK3β), adenomatous polyposis coli (APC), and axin 1
. Wnt signaling disrupts the destruction complex, allowing the unphosphorylated β-Catenin protein at the serine at residue 37 (Ser37) or threonine at residue 41 (Thr41) to accumulate and function as a co-activator for the transcription factor TCF/LEF 1
The TCF/β-Catenin complex targets many genes that promote the cell cycle and simultaneously regulates transcription of some members of the Notch signaling pathway, establishing reciprocal interactions between Wnt and Notch signals 2,3
. The canonical Notch signaling pathway is initiated when transmembrane ligands bind to the extracellular domain of transmembrane Notch receptors. This leads to cytoplasmic cleavage of the Notch intracellular domain (NICD) by γ-secretase and its rapid translocation to the nucleus. In the nucleus, Notch functions as a co-activator for the DNA-binding transcription factor RBP-J to activate Notch target genes, which often promote cellular differentiation 4
We recently demonstrated that Notch1 antagonizes Wnt/β-Catenin signaling by reducing levels of active β-Catenin in cardiac progenitor cells (CPCs) 5
, which represent a multipotent transient amplifying cell population. However, the mechanism of Notch’s negative regulation of β-Catenin protein and the breadth of this event in other stem cell types were unknown.
To determine if Notch negatively regulates β-Catenin protein levels in stem cells, we used a Notch1
siRNA to decrease Notch1 levels in E14 embryonic stem cells (ESCs). We found that reduced Notch1 levels did not noticeably affect the levels of total or N-terminal phosphorylated (Ser37) β-Catenin protein but resulted in an increase in the dephosphorylated, transcriptionally active form of β-Catenin protein (). The active form of β-Catenin normally constitutes a small fraction of total β-Catenin and was detected with antibodies that specifically recognize dephosphorylated β-Catenin at Ser37 and Thr41 6
. In agreement with this finding, knockdown (KD) of Notch1 in ESCs showed significantly more TCF/β-Catenin-dependent luciferase activity than controls (, s1
). Interestingly, knocking down transcripts of all four Notch receptors (Notch1, 2, 3, 4
) by applying Notch1–4
siRNAs further increased β-Catenin activity but the degree of increase was mild (, s1
). This indicates that Notch1 is the predominant Notch receptor for this event in ESCs, consistent with the high level of Notch1 in ESCs 7
. The increase in TCF/β-Catenin-dependent luciferase activity was also observed in Notch
siRNA-treated neural stem cells (NSCs) () and in mouse CPCs lacking Notch1 in vivo and in vitro 5
, suggesting that Notch1 may function to negatively regulate active β-Catenin levels in stem cell populations.
Figure 1 Notch Negatively Regulates Active β-Catenin in Stem Cells Independently of RBP-J. a, Western analysis of ESCs transfected with control or Notch1 (N1) siRNA with active (Act), Phospho (Ser37), or total β-Catenin antibodies that detect N-terminal-dephosphorylated (more ...)
We next sought to determine if the regulation of β-Catenin protein occurs through the canonical Notch signaling pathway involving the transcription factor, RBP-J. We introduced an RBP-J
-specific siRNA into ESCs to reduce RBP-J levels. Despite a ~70% KD of RBP-J
mRNA (), active β-Catenin levels were unchanged (). To determine if RBP-J mediates the Notch regulation of β-Catenin in vivo, we deleted Notch1
in CPCs by inter-crossing Notch1tm2Rko
floxed allele) or RBP-Jflox/flox
with mice containing Cre recombinase in the Isl1
. Isl1 marks an undifferentiated pool of CPCs 11
, whose expansion depends on Wnt/β-Catenin signaling 12,13
. Unlike embryos with a Notch1
deletion, the resulting RBP-J
mutant embryos showed no expansion of CPCs (). These data suggested that Notch-mediated regulation of active β-Catenin protein in ESCs and CPCs did not involve RBP-J-dependent transcriptional regulation.
RBP-J-independent Notch signaling has been described in vertebrates and invertebrates 14
and is thought to involve Notch-mediated transcription through other DNA-binding proteins. However, quantitative PCR (qPCR) revealed that levels of β-Catenin
transcripts were not altered in Notch1
KD ESCs, although Axin2
and Cyclin D1
, direct targets of TCF/β-Catenin 15,16
, were significantly upregulated in Notch1
KD cells (). This raised the possibility that Notch affects β-Catenin protein at the post-translational level. Since the key step in activation of Wnt signaling is regulation of the amount and localization of β-Catenin by GSK3β-dependent phosphorylation of its N-terminus in an APC-based destruction complex, we investigated whether the effects of Notch were mediated by this complex. We first confirmed that a pharmacological GSK3β inhibitor, 6-bromoindirubin-3′-oxime (BIO) specifically inhibits GSK3β activity and inactivates the destruction complex 17
, resulting in the accumulation of active β-Catenin (Fig. s2
). Overexpression of the Notch1 intracellular domain (N1ICD) in ESCs reduced active and total β-Catenin protein levels, but not mRNA, and decreased its activity in the presence of BIO (). The decrease was also evident in ESCs deficient for RBP-J or Mastermind-like (MAML), an essential co-transcriptional regulator for Notch signaling 18
(), providing additional evidence that Notch negatively regulates β-Catenin in a transcription-independent fashion. Furthermore, reduced levels of Notch1 increased β-Catenin activity even beyond that seen in BIO-treated ESCs (). This suggested that Notch-mediated negative regulation of β-Catenin protein in vitro is independent of GSK3β activity.
Figure 2 Notch1 Negatively Regulates Active β-Catenin in ESCs and Physically Interacts with β-Catenin. a, Relative expression of β-Catenin and Cyclin D1 mRNA in ESCs transfected with control or Notch1 siRNA (100 nM), determined by qPCR. (more ...)
To determine if Notch could suppress β-Catenin activity in vivo, independent of GSK3β activity, we expressed a form of β-Catenin that cannot be degraded by the destruction complex, with or without Notch1
, in the domain of precardiac mesoderm. This was done by crossing Isl1Cre
mice with β-Catenin(ex3)loxP
mice. The β-Catenin(ex3)loxP
allele contains loxP
sites surrounding exon 3 of β-catenin
that encodes amino acids required for GSK3β-mediated degradation, generating stabilized β-Catenin upon Cre
, and the Gt(ROSA)26Sortm1(Notch1)Dam/J
allele contains a loxP-stop-loxP
sequence before Notch1
in the Rosa
locus, allowing tissue-specific overexpression of Notch1 upon Cre
. Co-expression of stabilized β-Catenin and Notch1 completely rescued the abnormal expansion of precardiac mesoderm induced by activated β-Catenin alone (). Immunochemical analyses revealed that β-Catenin protein levels were increased in the domains of Isl1Cre
expression (pharyngeal mesoderm, ectoderm and endoderm) in β-Catenin(ex3)loxP
mice, but were reduced close to baseline levels upon Notch1 expression in the same domains (). These findings provide evidence that Notch can negatively titrate β-Catenin protein levels in vivo, independent of GSK3β activity, and thereby regulate expansion of progenitor cells.
Given that Notch expression did not require GSK3β activity to regulate β-Catenin protein, we examined if Notch modulates active β-Catenin protein levels through a direct physical interaction. We expressed N1ICD in ESCs by transfecting cells with a Myc-tagged N1ICD construct and performed co-immunoprecipitation (Co-IP) assays with anti-Myc antibodies with or without BIO. Comparable introduction of control Myc and Myc-N1ICD constructs into ESCs was confirmed by qPCR (Fig. s3
). We did not detect an interaction of endogenous β-Catenin with Notch1 in the absence of BIO (). However, when treated with BIO, which greatly increases active β-Catenin levels by inactivating the destruction complex, Notch1 co-precipitated with endogenous β-Catenin (), but not with APC, Axin, Gsk3β or TrCP (Fig. s4
). This suggested that Notch might selectively interact with active β-Catenin, whose levels are normally very low in ESCs. To investigate this possibility further, we used a human colon cancer cell line, SW480, which contains high levels of active β-Catenin due to an APC mutation that causes colon cancer 21
. When expressed in SW480 cells, Notch strongly associated with endogenous β-Catenin even without BIO treatment (). Further analysis of the precipitated β-Catenin confirmed enrichment of active β-Catenin, but not of N-terminal phosphorylated β-Catenin (). These data suggest that Notch physically associates with the active form of β-Catenin, although we cannot exclude the possibility that interaction with the phosphorylated form is below the level of detection.
Next, we mapped the domains of Notch responsible for β-Catenin association by performing Co-IP experiments with a series of truncated Notch mutants that lacked the extracellular domain 22
(). We found that Notch mutants lacking the RAM domain had limited association with β-Catenin (). To determine if the RAM domain, also required for RBP-J interaction 23
, was necessary for Notch regulation of β-Catenin transcriptional activity, we expressed control and mutant Notch constructs in BIO-treated ESCs with the β-Catenin luciferase reporter. In agreement with the Co-IP result, deleting the RAM domain significantly compromised Notch’s ability to repress β-Catenin activity (). Interestingly, Notch without the transactivation or PEST domains also showed reduced repressive activity, implying these domains also contribute to repression ().
The overexpression of N1ICD results in excessive cytoplasmic accumulation as well as nuclear localization (Fig. s5
). We therefore investigated whether ligand-dependent cleavage of Notch to free the NICD, which is essential for canonical Notch signaling, was necessary for the Notch regulation of active β-Catenin protein, or if membrane-bound Notch was sufficient.
Notch1 intracellular cleavage occurs between amino acids G1743 and V1744 in a highly conserved manner; mutations of V1744 (V1744K or V1744L) block intracellular cleavage, leaving Notch tethered to the membrane 24
(). We confirmed that the tethered form of Notch (V1744L) remained uncleaved in ESCs and exhibited negligible levels of Notch/RBP-J-dependent luciferase activity (). Surprisingly, expression of the tethered forms of Notch in ESCs decreased β-Catenin transcriptional activity comparable to the repression mediated by the well-known Wnt inhibitor, Dkk1 (). The tethered Notch-mediated repression occurred independent of RBP-J (Fig. s6
). In addition, endogenous active β-Catenin immunoprecipitated with the tethered form of Notch () in the presence of BIO. In agreement with the Co-IP result, active and total β-Catenin protein levels were considerably lower in cells with the tethered form of Notch ().
Figure 3 Membrane-Bound Notch1 Negatively Regulates Active β-Catenin Levels through Numb and Numb-like in Stem Cells. a, Schematic representation of a cleavage site–mutated tethered form of Notch1 (V1774L, top) and Western analysis of ESCs transfected (more ...)
Membrane-bound Notch has no transcriptional activity and would conventionally be considered biologically inert. To determine if negative titration of active β-Catenin protein by membrane-bound Notch has biological consequences, we assayed the effects of tethered Notch on Wnt-dependent differentiation of ESCs to mesoderm 25
. We expressed tethered Notch (V1744) in early embryoid bodies (before induction of mesodermal cells) derived from ESCs containing GFP in the endogenous mesoderm-specific Brachyury
) gene 26
. Fluorescence-activated cell sorting revealed that the number of Bry+
cells was significantly reduced upon expression of tethered Notch. This occurred with or without BIO (). This result indicates that membrane-bound Notch can negatively titrate a cellular response mediated by Wnt/β-Catenin signaling in stem cells.
To determine if endogenous membrane-bound Notch negatively regulates active β-Catenin protein, we blocked Notch endoproteolysis, which is mediated by the presenilin-γ-secretase complex 27
. We found that mouse ESCs treated with the γ-secretase inhibitor (GSI), DAPT 28
, had a significant reduction of active β-Catenin activity and protein levels in a dose-dependent fashion (). This trend was also observed in hESCs, NSCs and bone marrow mesenchymal stem cells () and, importantly, occurred in the absence of any overexpression. The number of Bry+
cells was also decreased in embryoid bodies when treated with DAPT (). Similarly, blocking α-secretase activity, required for ligand-mediated cleavage of the Notch extracellular domain, resulted in a significant reduction of active β-Catenin activity (). To further test the ligand-independent function of Notch, we utilized Notch1lbd/lbd
ESCs where endogenous Notch1 lacks the 11 and 12th EGF repeats required for ligand binding 29
. When stimulated with Wnt3a, we found that Notch1lbd/lbd
ESCs exhibited significantly lower β-Catenin-dependent luciferase activity than controls ().
Membrane-bound Notch is regulated by endosomal sorting pathways, leading to either recycling or lysosomal degradation 30
. In Drosophila
, the conserved endocytic adaptor protein Numb, which is present as two orthologues, Numb and Numb-like (Numbl) in mammals, negatively regulates Notch 31,32
. As one mechanism, Numb inhibits Notch signaling by trafficking membrane-bound Notch into the lysosome for degradation 33
. We found that endogenous Numb was also co-immunoprecipitated with the tethered form of Notch (), suggesting Numb may be involved in Notch-mediated degradation of active β-Catenin.
Figure 4 Notch-Mediated Degradation of β-Catenin Requires Numb and Lysosomal Activity. a, Human colon cancer cells (SW480) transfected with pcDNA-Myc or tethered Notch (V1774L)-Myc constructs, immunoprecipitated (IP) with anti-Myc antibody and immunoblotted (more ...)
To determine if Numb activity was required for degradation of active β-Catenin complexed with membrane-bound Notch, we knocked down Numb and Numbl with siRNAs in ESCs in the presence of the tethered form of Notch (V1744L). Tethered Notch was unable to repress β-Catenin transcriptional activity in Numb and Numbl-deficient ESCs (). In agreement with this finding, knockdown of Numb and Numbl abrogated the ability of tethered Notch to lower active β-Catenin protein levels (). Similarly, Numb and Numbl-deficiency relieved repression of β-Catenin activity observed upon overexpression of N1ICD ().
These data suggested that Numb and Numbl were involved in lysosomal trafficking of the Notch-β-Catenin complex for degradation. In agreement with this, inhibition of lysosomal activity with Bafilomycin A1, a potent and specific inhibitor of vacuolar proton ATPases 34
, abrogated the DAPT-induced decrease in active β-Catenin protein in ESCs (). Furthermore, immunocytochemistry revealed that tethered Notch1 (or NICD) and active β-Catenin co-localized with the lysosomal protein, Lamp1 (Fig. s7
). These findings indicate that the Notch-β-Catenin complex is present in the lysosome and that lysosomal activity is important for the Notch-mediated decrease in active β-Catenin.
Extrapolating our results from stem/progenitor cells, we hypothesized that membrane-bound Notch could affect β-Catenin levels in APC-mutated human cancer cells containing elevated active β-Catenin protein. We knocked down Notch 1–4 in SW480 human colorectal cancer cells and found a prominent increase in active β-Catenin protein levels (). This result provided additional evidence for regulation of β-Catenin by Notch, independent of the destruction complex. Conversely, treatment of two human colorectal cancer cell lines, SW480 and HT-29, with DAPT, which chemically prevents NICD cleavage, resulted in a paradoxical dose-dependent decrease in TCF/β-Catenin-dependent transcriptional activity, β-Catenin protein, and a decrease in cell expansion (). Proteasome inhibitors that block the destruction complex-mediated degradation of β-Catenin resulted in increased active β-Catenin levels but failed to prevent the Notch-mediated decrease in β-Catenin protein (). This indicates that Notch regulation of β-Catenin protein is unlikely proteasome-mediated and supports the earlier evidence showing Numb-dependence and potential involvement of the lysosome.
Figure 5 γ-Secretase Inhibitors (GSIs) Suppress Expansion of Human Colon Cancer Cells by Blocking Notch Cleavage. a, Western analysis of active β-Catenin in SW480 human colon cancer cells transfected with control or siRNA against Notch1–4 (more ...)
Chronic use of non-steroidal-anti-inflammatory drugs (NSAIDs) in humans has frequently been reported to lower the risk of developing primary and recurrent colorectal cancer 35,36
. A subset of NSAIDs also has significant γ-secretase inhibitory (GSI) activity 37
, and we correspondingly found that ibuprofen induced a dose-dependent decrease of canonical Notch transcriptional activity, determined by Notch/RBP-J-dependent luciferase activity in SW480 cells (). Ibuprofen treatment also lowered levels of active β-Catenin transcriptional activity and protein (). Importantly, the reduction of β-Catenin protein levels upon Ibuprofen treatment of cancer cells was not observed after knockdown of Notch1-4
(). This suggests that NSAIDs act, at least in part, through Notch to decrease active β-Catenin protein levels, and this regulation may contribute to the overall protective effects of NSAIDs on colorectal cancers. This result is consistent with the observation that GSI treatment in APC mutant mice reduces proliferating adenomas in the intestine 38,39
In the present study, we show that Notch negatively regulates protein levels of active β-Catenin in a post-translational manner and thereby serves to titrate Wnt/β-Catenin signaling in stem and progenitor cells (). In our experiments, the interaction between these two critical regulatory proteins did not require ligand-dependent cleavage of Notch, and membrane-bound Notch could form a complex with the active form of β-Catenin. This was observed in cells with active Wnt signaling, where inactivation of the destruction complex resulted in higher levels of active β-Catenin. Thus, in the presence of Wnt signaling, Notch might serve to titrate active β-Catenin levels to temper the proliferative state of expanding cells and affect cellular decisions.
While NICD can interact with β-Catenin in the cytosol and co-localizes with the lysosomal marker upon overexpression, this is unlikely to be its normal function given its low cytoplasmic levels under physiologic conditions. Instead, it may be the membrane-bound Notch that serves to titrate the active form of β-Catenin in cells responding to Wnt signaling. The evolutionary conservation of this process is striking, as Notch also interacts with Armadillo in endocytic vesicles in Drosophila and negatively regulates Wnt signaling 40
Generally, GSIs, such as DAPT, mimic canonical Notch loss-of-function mutations. However, our findings suggest that GSI treatment paradoxically decreases Wnt/β-Catenin signals through membrane-bound Notch, which effectively reduces active β-Catenin levels and activity. Biochemical approaches to purify the Notch-β-Catenin complex may reveal the more precise mechanism by which Notch affects active β-Catenin accumulation/degradation and provide more specific approaches to disrupt or promote the Notch-β-Catenin interaction.