Acute effects of Axin loss in the mouse liver and embryonic development

Hepatocellular carcinomas carrying Axin1 mutations belong to a subset of tumours with an especially poor prognosis. Data obtained from an Axin1 mutant mouse line, challenged the traditional idea of Axin function; as simply a component of the β-Catenin-destruction complex. Axin1 deletion led to the development of highly proliferative HCC in the absence of an obvious Wnt/β-Catenin signature. In order to uncover the mechanism(s) leading to Axin dependent tumourigenesis, this study focused on the role of Axin in two systems. Firstly, we generated an allelic series of Axin mutant ES cell lines to analyse the role of Axin1 and 2 in ES cells. We could show, that single Axin mutants had a largely normal ES cell phenotype. In Axin double mutant ES cells, Wnt target gene expression was slightly upregulated, but cell proliferation stayed at normal levels. By contrast, upon differentiation into embryoid bodies, multiple readouts of the Wnt pathway were increased and a G2/M and cell cycle related gene expression profile was activated, accompanied by severe differentiation defects. In the second system, we developed Axin1 mutant 3D liver cultures, which allow fate tracing of Axin mutant and wt cells in real time. We could show tightly regulatable gene deletion in vitro and produced preliminary evidence that Axin1 loss in culture closely mimics the in vivo situation in respect to G2/M gene expression in the absence of Wnt activation. Overall, the effects of Axin loss on Wnt signalling and cell cycle regulation appeared to be tissue and cell cycle specific. Future use of the 3D culture system, together with the data obtained in Axin mutant ES cells and embryoid bodies, will not only advance our understanding of the involvement of Axin1 in hepatocellular carcinogenesis and cell cycle regulation; but may also be the starting point in the development of new therapeutic strategies

WNT-3A regulates an Axin1/NRF2 complex that regulates antioxidant metabolism in hepatocytes

Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a master regulator of oxidant and xenobiotic metabolism, but it is unknown how it is regulated to provide basal expression of this defense system. Here, we studied the putative connection between NRF2 and the canonical WNT pathway, which modulates hepatocyte metabolism. Results: WNT-3A increased the levels of NRF2 and its transcriptional signature in mouse hepatocytes and HEK293T cells. The use of short interfering RNAs in hepatocytes and mouse embryonic fibroblasts which are deficient in the redox sensor Kelch-like ECH-associated protein 1 (KEAP1) indicated that WNT-3A activates NRF2 in a β-Catenin- and KEAP1-independent manner. WNT-3A stabilized NRF2 by preventing its GSK-3-dependent phosphorylation and subsequent SCF/β-TrCP-dependent ubiquitination and proteasomal degradation. Axin1 and NRF2 were physically associated in a protein complex that was regulated by WNT-3A, involving the central region of Axin1 and the Neh4/Neh5 domains of NRF2. Axin1 knockdown increased NRF2 protein levels, while Axin1 stabilization with Tankyrase inhibitors blocked WNT/NRF2 signaling. The relevance of this novel pathway was assessed in mice with a conditional deletion of Axin1 in the liver, which showed upregulation of the NRF2 signature in hepatocytes and disruption of liver zonation of antioxidant metabolism. Innovation: NRF2 takes part in a protein complex with Axin1 that is regulated by the canonical WNT pathway. This new WNT-NRF2 axis controls the antioxidant metabolism of hepatocytes. Conclusion: These results uncover the participation of NRF2 in a WNT-regulated signalosome that participates in basal maintenance of hepatic antioxidant metabolism

AXIN1 knockout does not alter AMPK/mTORC1 regulation and glucose metabolism in mouse skeletal muscle

AXIN1 is a scaffold protein known to interact with >20 proteins in signal transduction pathways regulating cellular development and function. Recently, AXIN1 was proposed to assemble a protein complex essential to catabolic-anabolic transition by coordinating AMPK activation and inactivation of mTORC1 and to regulate glucose uptake-stimulation by both AMPK and insulin. To investigate whether AXIN1 is permissive for adult skeletal muscle function, a phenotypic in vivo and ex vivo characterization of tamoxifen-inducible skeletal muscle-specific AXIN1 knockout (AXIN1 imKO) mice was conducted. AXIN1 imKO did not influence AMPK/mTORC1 signalling or glucose uptake stimulation at rest or in response to different exercise/contraction protocols, pharmacological AMPK activation, insulin or amino acids stimulation. The only genotypic difference observed was in exercising gastrocnemius muscle, where AXIN1 imKO displayed elevated α2/β2/γ3 AMPK activity and AMP/ATP ratio compared to wild-type mice. Our work shows that AXIN1 imKO generally does not affect skeletal muscle AMPK/mTORC1 signalling and glucose metabolism, probably due to functional redundancy of its homologue AXIN2

AXIN1 knockout does not alter AMPK/mTORC1 regulation and glucose metabolism in mouse skeletal muscle

AXIN1 is a scaffold protein known to interact with >20 proteins in signal transduction pathways regulating cellular development and function. Recently, AXIN1 was proposed to assemble a protein complex essential to catabolic-anabolic transition by coordinating AMPK activation and inactivation of mTORC1 and to regulate glucose uptake-stimulation by both AMPK and insulin. To investigate whether AXIN1 is permissive for adult skeletal muscle function, a phenotypic in vivo and ex vivo characterization of tamoxifen-inducible skeletal muscle-specific AXIN1 knockout (AXIN1 imKO) mice was conducted. AXIN1 imKO did not influence AMPK/mTORC1 signalling or glucose uptake stimulation at rest or in response to different exercise/contraction protocols, pharmacological AMPK activation, insulin or amino acids stimulation. The only genotypic difference observed was in exercising gastrocnemius muscle, where AXIN1 imKO displayed elevated α2/β2/γ3 AMPK activity and AMP/ATP ratio compared to wild-type mice. Our work shows that AXIN1 imKO generally does not affect skeletal muscle AMPK/mTORC1 signalling and glucose metabolism, probably due to functional redundancy of its homologue AXIN2