In the present study, we elucidated a novel mechanism that the interaction of Cav-1 with β-catenin can suppress the phosphorylation of β-catenin at Y333, which is crucial for the nuclear entry of β-catenin during cerebral I/R injury. Blocking this interaction promoted β-catenin nuclear translocation and was protective against cerebral I/R injury (Fig. 6).
Cav-1 performs many functions through its CDC domain consisting of residues 82–101 (82DGIWKASFTTFTVTKYWFYR101). This motif is characterized by the amino acid sequence ΦXXXXΦXXΦ, ΦXΦXXXXΦ, or ΦXΦXXXXΦXXΦ, where Φ represents an aromatic amino acid, e.g., tryptophan, tyrosine, or phenylalanine, and X represents any amino acid (Couet et al. 1997). As a core molecule of the canonical Wnt pathway, β-catenin is controlled by many binding partners, influencing its stability, cellular localization, and transcriptional activity (Gottardi and Peifer 2008). Cav-1 can directly interact with β-catenin in zebrafish (Mo et al. 2010). This interaction occurs between residues 95–98 in the CSD of Cav-1 (including the Phe-Thr-Val-Thr sequence, named FTVT) and residues 330–337 in the armadillo repeats of β-catenin (330YTYEKLLW337) (Mo et al. 2010). This previous study demonstrated that the CSD (95–98) of Cav-1 bound to β-catenin at FTVT, but its biological function was unclear. Here, we synthesized the FTVT peptide to verify how it performs against cerebral I/R injury.
In this study, we first found that the combination of Cav-1 and β-catenin was significantly elevated in ischemic brains at 24 h after reperfusion. Meanwhile, the level of β-catenin in the nucleus decreased significantly (Fig. 2). These results suggest that the binding of Cav-1 to β-catenin might inhibit the nuclear translocation of β-catenin in post-ischemic brains.
As a pivotal component of the Wnt signaling pathway, β-catenin is tightly regulated at three hierarchical levels: stability, spatial separation, and transcriptional activity (Misztal et al. 2011). The nuclear translocation of β-catenin is a hallmark of the activation of the Wnt/β-catenin signaling pathway in neurons (Misztal et al. 2011). However, the regulatory mechanisms of β-catenin nuclear translocation remain to be elucidated. In the present study, we found that ASON (knockdown of Cav-1) and FTVT (interruption of the interaction between β-catenin and Cav-1) inhibited the association of Cav-1 with β-catenin and promoted β-catenin nuclear translocation in cerebral I/R injury (Fig. 2). These results indicate that the interaction of Cav-1 with β-catenin can regulate the nuclear translocation of β-catenin, and dispersing this complex increased β-catenin nuclear translocation in I/R brains.
The stability of β-catenin is inhibited by the β-catenin destruction complex, which is a polyprotein containing the tumor suppressors Axin, adenomatous polyposis coli (APC), the Ser/Thr kinases GSK-3 and CK1, protein phosphatase 2A (PP2A), and the E3-ubiquitin ligase β-TrCP, playing a crucial role in the signal output of the canonical Wnt cascade(Stamos and Weis 2013). The phosphorylated status of Ser33, Ser37, and Thr41 in β-catenin determines its degradation, with higher phosphorylation associated with more degradation(Valenta et al. 2012). To elucidate whether Cav-1 or the Cav-1/β-catenin complex affected β-catenin degradation, we examined Ser33/Ser37/Thr41's phosphorylation of β-catenin using ASON and FTVT. As shown in Fig. 3, the level of the p-β-catenin (Ser33/Ser37/Thr41) was significantly elevated with reperfusion for 24h after transient cerebral ischemia, which implies that β-catenin should be increased in degradation under this treatment, but the results were not as expected. Cav-1 ASON notably attenuated the phosphorylation of β-catenin in the cytoplasm induced by cerebral I/R for 24 h (Figs. 3A, 3B). Unlike Cav-1 ASON, treatment with FTVT did not reverse the level of p-β-catenin (Ser33/Ser37/Thr41) expression (Figs. 3C, 3D) but instead decreased the formation of the Cav-1/β-catenin complex (Fig. 2C). These results demonstrated that Cav-1 plays a crucial role in the stability of β-catenin, but this does not occur via the Cav-1/β-catenin interaction induced by p-β-catenin (Ser33/Ser37/Thr41). Because FTVT is a competitive antagonist that only blocks the interaction of Cav-1 and β-catenin, it did not change the level of p-β-catenin (Ser33/Ser37/Thr41) expression (Figs. 3C, 3D). Thus, our current findings imply that the increased expression of β-catenin in the nucleus and that the protective effects of FTVT may not be related to β-catenin degradation during I/R damage. Moreover, Cav-1 effect β-catenin stability not via the Cav-1 and β-catenin interaction but through other factors involved during cerebral I/R. The factors can block the interaction between β-catenin and GSK3 and recruit β-catenin to the plasma membrane, which stabilizes β-catenin in the cytoplasm(Galbiati et al. 2000).
β-catenin nuclear translocation is a decisive step in its transcriptional activity. However, despite extensive investigation, the exact mechanism by which β-catenin enters the nucleus remains to be further elucidated. It has been reported that Cav-1 can affect the canonical Wnt signaling pathway through its binding to β-catenin. Moreover, it has been demonstrated that residues 330 to 337 (330YTYEKLLW337) in the armadillo repeats of β-catenin are responsible for its binding to Cav-1 in vitro and in vivo. Within residues 330–337 of β-catenin, site 333 is a tyrosine (Y) residue based on biochemical and structural analysis (Mo et al. 2010). Previous studies have proven that the phosphorylation of β-catenin at this site (Y333) increased its entry into the nucleus (Yang et al. 2011). To prove whether the binding of Cav-1/β-catenin blocks the Y333 phosphorylation site, thereby lowering β-catenin nuclear translocation during I/R brain injury, we used a competitive antagonist, FTVT, to disrupt the interaction of Cav-1 and β-catenin. We found that the phosphorylation (Y333) of β-catenin was decreased in I/R, and FTVT reversed this (Figs. 4A-4C). To further confirm the relationship between Tyr333-phosphorylated β-catenin and its nuclear translocation, we used site-directed mutagenesis to change the tyrosine at the β-catenin 333 site to phenylalanine (Y333F) in the HEK293 cell line. After transfection of the mutant plasmid into HEK293 cells, the results showed that p-β-catenin (Y333) was decreased significantly compared with cells expressing the wild-type protein (Fig. 4D). Additionally, β-catenin nuclear translocation was also downregulated (Figs. 4D – 4G). During I/R damage, the phosphorylation of β-catenin at Tyr333 may control its entry into the nucleus. These results indicate that cerebral I/R injury can mediate the binding of Cav-1 to β-catenin, which reduces the phosphorylation of β-catenin at Tyr333, thereby disrupting β-catenin entry into the nucleus. In this experiment, the phosphorylation of the Y333 site may be activated by other kinases such as Src and PKM2. Yang et al. reported that β-catenin could be phosphorylated at Tyr333 by the RTK-downstream kinase Src in human glioblastoma cells (Yang et al. 2011). Src-mediated β-catenin phosphorylation at Tyr333 is required for the pyruvate kinase M2 (PKM2)/β-catenin interaction, which triggers the translocation of the PKM2/β-catenin complex into the nuclei of cells (Yang et al. 2011). Previous reports demonstrate that phosphorylation of the Y333 site is activated by other kinases such as Src and PKM2.
In the present study, the knockdown of Cav-1 by treatment with ASON or the disruption of the interaction between Cav-1 and β-catenin by FTVT prevented cerebral I/R-induced neuronal death (Figs. 5A, 5B). This phenomenon may likely be due to the nuclear translocation of β-catenin. A previous study reported that remote ischemic preconditioning exerts myocardial protection by activating the PI3K/Akt/GSK3S signaling pathway associated with the nuclear accumulation of β-catenin and the upregulation of E-cadherin and PPARδ for cell survival (Li et al. 2011). Once located in the nucleus, β-catenin will exert its transcriptional activity. FTVT increased the I/R-induced reduction in c-Myc expression (Figs. 5C, 5D). On the one hand, since β-catenin has no DNA binding ability, it functions as a transcriptional activator in the nucleus by forming a transcriptional complex with TCF/LEF, p300/CBP, and other proteins to initiate the transcription of its various target genes, including c-Myc, Cyclin D1, and c-Jun (Clevers 2006; Staal and Clevers 2005). On the other hand, FTVT can reverse the expression of c-myc induced by I/R injury. Thus, the immunocomplex formed between Cav-1 and β-catenin might play an important role in neuronal survival during cerebral I/R injury. Impairment of this complex protected against cerebral I/R injury. This neuroprotection may work by promoting β-catenin nuclear translocation.
In summary, we concluded that cerebral I/R injury promotes the binding of Cav-1 to β-catenin and decreases the nuclear translocation of β-catenin (Fig. 6). The molecular mechanism demonstrated here is that the binding of Cav-1 to β-catenin after I/R injury promotes phosphorylated β-catenin at Ser33, Ser37, and Thr41 but decreases phosphorylated β-catenin at Tyr333. In contrast, disruption of Cav-1/β-catenin complex by FTVT can increase phosphorylated β-catenin at Tyr333, which can promote nuclear translocation of β-catenin, leading to the activation of Wnt/ β-catenin signaling and reducing the death of neurons after I/R injury. Our study collectively suggests that targeting the interaction between Cav-1 and β-catenin could be a novel therapeutic strategy to protect against neuronal damage during cerebral injury.