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 ?-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 ?-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.
Porphyrias are rare disorders caused by deficiencies in heme biosynthesis pathway enzymes, and there are few effective medical therapies available. In this proposal, we will assess the mechanism by which inhibition of the Wnt/b-catenin signaling pathway provides protection from liver injury in a chemically-induced mouse model of porphyria, and whether b-catenin inhibition can prevent progression or provide protection in genetic mouse models of erythropoietic and acute intermittent porphyria.
