Increased canonical WNT/β-catenin signalling and myxomatous valve disease

This editorial refers to ‘Loss of Axin2 results in impaired heart valve maturation and subsequent myxomatous valve disease’ by A. Hulin et al., doi: 10.1093/cvr/cvw229.

Myxomatous valve disease (MVD) is a common form of valvular heart disease which causes severe valvular regurgitation.¹ Increased accumulation of glycosaminoglycans (GAG), disrupted collagen fiber organization, increased cell proliferation, increased presence of inflammatory cells, and induction of Wingless-type MMTV integration site family (WNT), Transforming Growth Factor-beta (TGFβ), and Bone Morphogenetic Protein (BMP) signalling is reported in late stage human MVD.^2–4

Valve development involves cell–cell and cell–matrix interactions (Figure 1). Recent studies suggest that defects in heart valve development can lead to heart valve disease in adults.⁵ Valve formation starts with an epithelial mesenchymal transition (EMT) (E9.5–E10.5) that gives rise to mesenchymal cushions in both the atrioventricular and outflow tract regions of the developing heart. Cardiac cushions become mature valves through mesenchymal expansion (E10.5–E12.5), differentiation (E12.5–E14.5), and maturation (E14.5-postnatal stages). Although heart valve remodelling can be conceptually or arbitrarily organized into multiple processes based on the molecular and cellular markers, it is very difficult to establish precisely when a developmental event (such as EMT) in valvulogenesis formally ends. In fact, it has been demonstrated that valvulogenesis continues after birth to adjust to the postnatal maturation of the heart.

Figure 1.

Valve development and homeostasis and valve pathogenesis. Elucidation of the WNT/β-catenin mechanisms through which loss of Axin2 affects the developmental transition during valvulogenesis, from cushion formation – cushion remodeling – valve maturation – adult valve homeostasis, will improve basic understanding of the etiology of MVD.

WNT/β-catenin signalling is active during heart valve development⁶ and has been reported in human MVD.^2,7 There are 19 WNT family members which signal through the canonical (via β-catenin) pathway but can also signal through non-canonical pathways.⁸ Since β-catenin can localize in nucleus and at adherens junctions, studies involving genetic deletion of β-catenin to determine the role of canonical WNT/β-catenin in murine model are often difficult to interpret. The consequences of increased WNT/β-catenin signalling due to loss of Axin2, a WNT signaling inhibitor and also a downstream target of WNT/β-catenin signaling, offers significant advantage to study the role of canonical WNT/β-catenin signaling in valve homeostasis and MVD.

In this issue, using Axin2 knockout (Axin2^LacZ/LacZ or Axin2^-/-) mice Hulin et al.⁹ provide the first evidence that Axin2 is required to limit activation of WNT/β-catenin signaling after birth and that loss of Axin2 leads to progressive myxomatous extracellular matrix (ECM), typical of human MVD. Remarkably, SOX9 expression and collagen deposition are decreased, whereas increased inflammation precedes and potentially drives ECM remodeling and BMP signaling in MVD in Axin2^-/- mice. Furthermore, they showed that WNT/β-catenin signaling is activated specifically in valve interstitial cells (VICs) located in the thickened/diseased mitral valve of a murine model (i.e. Fibrillin1^+/C1039G) of Marfan syndrome (MFS), suggesting an involvement of hyper-activated WNT/β-catenin pathway in the pathogenesis of MVD in MFS.

Aortic valve calcification has been previously shown to be associated with increased WNT/β-catenin signaling.¹⁰ An intriguing question is why Axin2^-/- mice do not develop calcification even up to 1 year-of-age, although WNT/β-catenin signaling is increased after birth. Axin has two functionally identical isoforms, Axin1 and Axin2. Axin1^-/- embryos die at E9.5 with forebrain and neural tube defects. Axin2^-/- have skull abnormalities,¹¹ valve malformations⁶ and MVD,⁹ indicating that Axin1 does not fully compensate for the lack of Axin2.The knock-in of Axin2 into the deleted Axin1 gene rescues the embryonic lethal phenotype of Axin1^-/- mice.¹¹ While Axin1^+/- mice are normal, the Axin1^+/-Axin2^-/- mice developed severe brain and craniofacial abnormalities at birth compared to Axin2^-/-mice alone.¹¹ Thus, one Axin1 allele is sufficient in the presence of Axin2 but not in its absence. These findings also suggest that when Axin1 is absent or reduced, Axin2 can partially compensate for its developmental functions. Axin2 does not fully compensate because it is not ubiquitously expressed. Based on the interesting observations of Hulin et al.⁹ one possibility would be that the total level of Axin1 plus Axin2 needs to be elevated in VICs; Axin1 provides a basal level in all valvular cell types including VICs, while Axin2, which is induced by WNT/β-catenin signalling, is regulated to provide elevated Axin levels in VICs. Thus, unique and/or synergistic biological function of both Axin1 and Axin2 in valve pathogenesis should be determined in future investigations which could potentially clarify the role of activated WNT/β-catenin role in MVD and/or calcific aortic valve disease.

Remarkably, activated WNT/β-catenin signalling and MVD is associated with decreased SOX9 in the Axin2^-/- mice.⁹ Given that TWIST1 is known for its function in heart valve development and disease and can negatively regulate Sox9 expression in chondrogenesis,¹² it should be determined whether WNT/β-catenin signalling by positively regulating TWIST1 levels can contribute to MVD. Hulin et al.⁹ have not demonstrated if an altered Twist1 expression is associated with MVD in Axin2^-/- mice.

Although the data indicate a potential role of enhanced BMP signalling in MVD of Axin2^-/- mice,⁹ the exact role of TGFβ signalling and how BMP, TGFβ, and WNT signalling integrate in the MVD remains unclear (Figure 2). Future experiments should determine if BMP reduction (e.g. via partial BMPR1 deletion) can block or treat MVD in Axin2^-/- mice. Both Axin1 and Axin2 bind to SMAD3 and regulate its TGFβ-dependent activation (Figure 2). Also, degradation of SMAD7, an inhibitory TGFβ-SMAD and a TGFβ target gene (Figure 2), is accelerated in the presence of Axin, with a subsequent increase in TGFβ/SMAD signaling.¹¹ The authors' conclusion of increased cellular inflammation and adverse VIC remodeling in Axin2^-/- mice is therefore consistent with published data indicating that reduced TGFβ signalling causes tissue inflammatory diseases.¹³ Thus, it is possible that Axin2 deletion results in a shift from TGFβ/SMAD3 toward WNT/β-catenin and BMP signalling which collectively can contribute to MVD (Figure 2).

Figure 2.

Loss of Axin2 can cause MVD by shifting TGFβ/SMAD3 pathway toward WNT/β-catenin and BMP signaling. This hypothetical model is based on published findings. In the absence of Axin2, TGFβ-dependent SMAD3 signaling is expected to decrease (indicated by a minus [-] sign), whereas both WNT-dependent β-catenin and BMP-dependent SMAD1/5 signaling are likely to increase (indicated a plus [+] sign). Without the WNT, the destruction complex remains in the cytoplasm, where it ubiquitinates β-catenin by β-TrCP. WNT induces the association of the intact complex with phosphorylated LRP. After binding to LRP, the destruction complex still captures and phosphorylates β-catenin, but ubiquitination by β-TrCP is blocked. Newly synthesized β-catenin accumulates. TGFβ2 and/or BMP2 both could bind to TGFβR3, suggesting a crosstalk between TGFβ and BMP pathways. Fibrillin-1 is thought to restrict bioavailability of both TGFβ and BMP ligands. Both endoglin and TGFβR3 facilitates ligand binding to the canonical TGFβ-receptor (TGFβR1/TGFβR2) heteromeric complex. Axin which interacts with SMAD3 and TGFβRs promotes phosphorylation of the TGFβ-specific SMADs (i.e. pSMAD2/3). Canonical BMP signaling occurs through BMP receptors and BMP-specific SMADs (i.e. SMAD1/5). WNT, TGFβ and BMP signaling each induces specific target genes such as Smad7, Axin 2, and Periostin, respectively. Based on the individual activities of WNT, TGFβ, and BMPs in MVD, the overall signaling integration may play an important role in MVD.

Hulin et al.⁹ have made another interesting observation that WNT/β-catenin signaling is increased in Fibrillin1^+/C1039G mice which exhibit MVD. As described above, it is possible that activated WNT/β-catenin signalling might correlate with MVD in the MFS mice. Loss of Fibrillin-1 is thought to induce changes in ECM which result in ectopic activation of TGFβ ligands and their downstream SMAD-dependent TGFβ signalling.¹³ Given the paradoxical roles of increased and/or decreased TGFβ signalling in MFS-like disorders,¹³ it would be important to determine how TGFβ and WNT/β-catenin signalling integrate in the pathogenesis of MVD.

Finally, this proposition is also consistent with the findings indicating that loss of TGFβ2 or Axin2 in mice cause thickened valves at birth,^9,14 suggesting that Axin2 deletion by shifting the balance from TGFβ/SMAD3 toward WNT/β-catenin signalling could contribute to MVD (Figure 2). Since Tgfb2 knockout mice die at birth,^14,15 further studies should be done to determine if the loss of TGFβ2 in VICs causes MVD in adult mice and whether MVD in Tgfb2 conditional knockout mice is also associated with decreased expression of Axin2 as well as reduced SMAD3 activation.

In conclusion, the work of Hulin et al.⁹ significantly adds to our knowledge by showing that activated WNT/β-catenin signaling causes MVD. The model displayed by the authors provides hope that we will finally begin to understand the complex integration of underlying molecular cell signaling pathways, such as WNT/β-catenin, BMP, and TGFβ/SMAD3 pathways, in development and pathogenesis of MVD that causes suffering for numerous human adults. The possibility that WNT, BMP, and TGFβ pathways could integrate in MVD merits further study.