Beta-catenin inhibition as a novel therapeutic strategy for porphyria

卟啉症是一类由血红素生物合成途径缺陷引起的代谢性疾病

The porphyrias are a group of metabolic disorders that are caused by defects in heme biosynthesis pathway enzymes, and liver is commonly either a source or target of excess porphyrins. Treatment for most of the porphyrias is limited, and often focused on symptomatic relief and palliative care. Thus, mechanistic-based studies that emphasize therapeutic development are desperately needed. Previous work has identified the Wnt/b-catenin signaling pathway as a modulatable target in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet-induced mouse model of porphyria. Inhibition of b-catenin in mice subjected to DDC results in a significant reduction in liver injury due to decreased porphyrin accumulation. We identified a network of key heme biosynthesis enzymes such as δ-aminolevulinic acid (ALA) synthetase and ALA-dehydratase that are suppressed in the absence of b-catenin, resulting in decreased production of porphyrin intermediates and DDC-associated protein aggregation. Autophagy is also increased in mice lacking b-catenin, which may further contribute to protection from injury. Thus, the overarching hypothesis of the proposal is that inhibiting Wnt/b-catenin signaling in clinically-relevant models of porphyria will alleviate injury and progression of disease through decreased production of porphyrin intermediates and/or increased autophagy. In aim 1, we will determine the most proximal step in the pathway that is affected by b-catenin inhibition by treating hepatocytes with ALA and measuring the appearance of porphyrin intermediates; investigate the mechanism by which b-catenin regulates heme enzymes through site-directed mutagenesis, in silico studies, and chromatin immunoprecipitation; and demonstrate therapeutic relevance of targeting β-catenin in patients by utilizing immunohistochemistry to correlate the extent of b-catenin expression with expression of heme enzymes. In aim 2, we will characterize the role and regulation of autophagy in porphyria after Wnt/b-catenin inhibition. We will use RFP-EGFP-LC3 mice, a pH-dependent fluorescent reporter strain, and a genetic knockout of glutamine synthetase, a component of the b-catenin/mTOR pathway, as well as in vitro assays, to comprehensively address the contribution of this cellular process to the observed protected phenotype. In aim 3, we will determine whether inhibiting b-catenin in genetic mouse models of porphyria decreases porphyrin accumulation and improves liver pathology. Two well-characterized porphyria models: the ferrochelatase (Fechm1Pas) mutant mice, which mimics human erythropoietic protoporphyria with significant liver involvement; and the T1/T2 mouse, which is compound heterozygous for hydroxymethylbilane synthase and mimics acute intermittent porphyria upon stimulation with phenobarbitol, will be utilized to determine whether therapeutic intervention with a b-catenin inhibitor can prevent progression or provide protection during acute attacks. Thus, the proposed studies will further our understanding of the potential for inhibiting b-catenin to treat porphyria, & will provide highly significant information for therapeutic and translational use.