In addition to its documented role in the proteolytic processing of Notch and APP, PS1 has been shown to interact with β-catenin. However, the biological consequences of this latter interaction remain controversial. In the present study, we provided evidence that loss of PS1 function results in increased stability of cytosolic β-catenin, leading to enhanced β-catenin/Lef-dependent signaling, among which is elevation of cyclin D1 levels. This, in turn, is associated with increased entry into the cell cycle and cell proliferation, a phenotype that can be reversed by expressing wild-type PS1, but not a PS1 form that does not associate with β-catenin. Conversely, overexpression of PS1 in vitro results in LEF-mediated repression of cyclin D1 transcription in a dose-dependent manner. All these effects on β-catenin turnover by PS1 are independent of APP and notch proteolysis and appear to be mediated at the level of β-catenin phosphorylation/ubiquitination. Finally, FAD mutations in PS1 cannot efficiently restore β-catenin degradation in PS1 null primary fibroblasts.
Using primary mouse embryonic fibroblasts deficient in PS1, we demonstrated that PS1 negatively modulates the cytosolic (and hence signaling) pool of β-catenin. We showed that both cyclin D1 mRNA and protein were increased in PS1−/− cells and this was correlated with higher β-catenin/LEF–dependent transcriptional activity from the cyclin D1 promoter in these cells. Moreover, we observed that, as previously described for colon cancer cells, increased levels of cyclin D1 in PS1−/− cells correlated with accelerated progression through the G1 phase of the cell cycle. It is important to note that increased cyclin D1 levels are unlikely to represent an epiphenomenon of hyperproliferation in PS1 null cells. This is because PS1 expression can specifically modulate LEF-mediated transcription in a dose-dependent manner, consistent with the concept that increase in cyclin D1 levels occurs before hyperproliferation itself and as a consequence of PS1 deficiency. In other words, we have provided compelling evidence that PS1, through modulation of β-catenin signaling, directly affects not only cyclin D1 levels, but also cell growth associated with cyclin D1 activity. Finally, further support of a direct causal effect rather than merely a correlative link was demonstrated by using a mutant PS1 construct where the interaction between PS1 and β-catenin was lost (). In short, accumulation of β-catenin and increased cell proliferation in PS1-deficient cells can be reversed by reintroducing wild-type PS1, but not PS1Δcat, a PS1 mutant that does not bind β-catenin.
Our results also suggested a model in which PS1 functions to regulate β-catenin turnover at a step after β-catenin phosphorylation. In the absence of PS1, the rapid dephosphorylation of β-catenin after Wnt-3a stimulation was retained. Moreover, modulation of axin phosphorylation was also not altered during Wnt stimulation and withdrawal in PS1-deficient cells. However, the elevation of cytosolic β-catenin levels was seen both in basal (i.e., unstimulated) and after Wnt stimulation, and was accompanied by an increase in the levels of phosphorylated β-catenin. Thus, in the absence of PS1, turnover of β-catenin was prolonged in both Wnt-3a–stimulated and at basal conditions. Lastly, the increase in phosphorylated β-catenin, which should normally be at very low levels due to rapid degradation, was correlated with a decrease in ubiquitination. We therefore hypothesize that PS1 modulates β-catenin degradation by facilitating ubiquitination. This mechanism would be consistent with two recent reports examining the interaction of Drosophila
presenilin homologue (DPS) and Armadillo/β-catenin (Arm/βcat). In Drosophila
, loss of endogenous DPS resulted in the accumulation of Arm/β-cat in the cytoplasm (Noll et al. 2000
), leading the authors to suggest a possible role of DPS in proteosomal degradation. In addition, in a genetic screen of candidates that modify an Arm/βcat phenotype, DPS was identified as a negative regulator of Wg/Wnt signaling (Cox et al. 2000
). Therefore, both of these findings in Drosophila
are entirely consistent with our present observations.
PS1 is required for the cleavage of Notch-1 to release the signaling intracellular domain (NICD), a step necessary for initiating downstream gene transcriptional activation (De Strooper et al. 1999
; Struhl and Greenwald 1999
; Ye et al. 1999
). There is evidence to suggest that the Notch and Wnt pathways are mutually inhibitory (Axelrod et al. 1996
). Therefore, it can be argued that enhanced β-catenin/LEF signaling and increased cell proliferation in the absence of PS1 could be secondary to defective Notch signaling. However, Notch-1 from PS1−/− cells expressing PS1Δcat is efficiently cleaved. The fact that this mutant does not restore the β-catenin phenotype strongly suggests that Notch-1 deficiency is not the cause of the abnormal β-catenin phenotype. Thus, modulating β-catenin signaling and facilitating Notch-1 proteolytic cleavage are likely independent and parallel functions of PS1. It remains possible that the β-catenin activity facilitated by PS1 in vivo is required at later stages of development and consequently masked by its early requirement for Notch-1 signaling.
Consistent with the results on Notch-1 proteolysis, the requirement of PS1 in γ-secretase cleavage is independent of β-catenin turnover. PS1Δcat also restores efficiently γ-secretase activity in PS1−/− cells, both in terms of APP COOH-terminal fragments and Aβ generation. These findings, entirely consistent with the recent report of Saura et al. 2000
again strongly suggest that this latter activity and the role in β-catenin homeostasis are likely to be independent functions of PS1. Thus, the association of PS1 with β-catenin is not required in the intracellular compartment where APP and Notch proteolysis takes place.
Taken together, our findings provide a parsimonious interpretation that is consistent with the current model of the Wnt–β-catenin pathway in which stabilization of β-catenin, in this case through loss of PS1, leads to proliferative signal via activation of cyclin D1. These results also argue that the discrepant results reported by different laboratories may reflect measurements of dissimilar pools of β-catenin. While we examined the Wnt-3a–responsive cytosolic pool of endogenous β-catenin defined as the fraction that was saponin extractable, others assessed both cytosolic and membrane-bound β-catenin expressed by transient transfections (Zhang et al. 1998a
). However, the membrane-bound β-catenin pool is not responsive to Wnt stimulation (data not shown). Furthermore, the presence of lower molecular weight β-catenin species was used to imply a stimulation of degradation without formal proof. Although the identity and functional significance of these species remain unknown, we suspect that they accumulate when degradation of full-length β-catenin is defective (see, for example, Schlosshauer et al. 2000
). In short, these discrepancies underscore the importance of defining accurately the β-catenin pool under study, since its biological role is tightly linked to its subcellular localization.
In our proposed scenario, the loss of PS1 would be similar to that postulated in a number of human cancers, including colon, melanoma, hepatocarcinomas, and pilomatricomas of the skin. In these tumors, mutations in APC, axin, or β-catenin result in abnormally high β-catenin levels, and increased β-catenin/LEF signaling is thought to contribute to neoplasic transformation (for reviews, see Polakis 1999
; Roose and Clevers 1999
). In this respect, it is of interest that epithelial hyperplasia and skin tumors have been described in the majority of PS1 null mice rescued with the human PS1 transgene (hPS1) driven by the neuronal-specific human Thy-1 (hThy) promoter (Zheng et al. 2000
). PS1 null mice die either embryonically or perinatally. However, animals rescued by Thy-1–driven hPS1 survive into adulthood. Since the skin of these animals has been shown to be deficient in PS1 and to show elevated β-catenin levels, the tumor phenotype in these rescue animals is consistent with constitutively upregulated transcription of cyclin D1 in PS1-deficient skin, due to impaired attenuation of Wnt signals. However, a definitive causal link between β-catenin levels, PS1 deficiency, and tumor formation remains to be established.
In summary, our results demonstrated that PS1 is part of the β-catenin degradation machinery, thereby adding PS1 to a list of known proteins that regulate β-catenin stability (Orsulic and Peifer 1996
; Hart et al. 1998
; Ikeda et al. 1998
; Seeling et al. 1999
; Zorn et al. 1999
). We propose a model in which PS1 contributes to the rapid turnover of β-catenin after its phosphorylation, possibly by some enhancement of the ubiquitination process. We have shown that FAD mutations in PS1 represent a loss of its function as a β-catenin modulator. Whether this phenotype contributes to Alzheimer's disease is an issue not addressed in this study. Nevertheless, our results further emphasize the importance of maintaining a rapid turnover of cytosolic β-catenin in normal cell growth and differentiation.