Abstract
In the normal liver, hepatocytes form a uniquely polarised cell layer that enables movement of solutes from sinusoidal blood to canalicular bile. Whilst several cholestatic liver diseases with defects of hepatocyte polarity have been identified, the molecular mechanisms of pathogenesis are not well defined. One example is arthrogryposis, renal dysfunction and cholestasis syndrome, which in most patients is caused by VPS33B mutations. VPS33B is a protein involved in membrane trafficking that interacts with RAB11A at recycling endosomes. To understand the pathways that regulate hepatocyte polarity better, we investigated VPS33B deficiency using a novel mouse model with a liver-specific Vps33b deletion. To assess functional polarity, plasma and bile samples were collected from Vps33b liver knockout (Vps33bfl/fl-AlfpCre) and control (Vps33bfl/fl) mice; bile components or injected substrates were quantitated by mass spectrometry or fluorometry. For structural analysis, livers underwent light and transmission electron microscopy. Apical membrane and tight junction protein localisation was assessed by immunostaining. Adeno-associated virus vectors were used for in vivo gene rescue experiments. Like patients, Vps33bfl/fl-AlfpCre mice showed mislocalisation of ATP-binding cassette proteins that are specifically trafficked to the apical membrane via Rab11a-positive recycling endosomes. This was associated with retention of bile components in blood. Loss of functional tight junction integrity and depletion of apical microvilli were seen in knockout animals. Gene transfer partially rescued these defects. Vps33b has a key role in establishing structural and functional aspects of hepatocyte polarity and may be a target for gene replacement therapy. Hepatocytes are liver cells with tops and bottoms; that is, they are polarised. At their bottoms they absorb substances from blood. They then, at their tops, secrete these substances and their metabolites into bile. When polarity is lost, this directional flow of substances from blood to bile is disrupted and liver disease follows. In this study, using a new mouse model with a liver-specific mutation of Vps33b, the mouse version of a gene that is mutated in most patients with arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome, we investigated how the Vps33b gene product contributes to establishing hepatocyte polarity. We identified in these mice abnormalities similar to those in children with ARC syndrome. Gene transfer could partly reverse the mouse abnormalities. Our work contributes to the understanding of VPS33B disease and hepatocyte polarity in general, and may point towards gene transfer mediated treatment of ARC liver disease.
Highlights
In metazoans, the development of three-dimensional body structures depends on the generation of polarised epithelial layers
Loss of Vps33b expression in hepatocytes of Vps33bfl/fl-AlfpCre animals was confirmed by fluorescence in situ hybridisation and Western blotting techniques (Supplementary Fig. 1A and B)
To assess hepatobiliary injury associated with Vps33b mutation, levels of plasma alkaline phosphatase (ALP) activity; alanine aminotransferase (ALT) activity and bilirubin concentrations were measured in 14week-old animals fed normal chow or a 0.5% cholic acid (CA) diet (n = 4–6)
Summary
The development of three-dimensional body structures depends on the generation of polarised epithelial layers. Polarisation of epithelial cells is a complex process that requires the cooperation of multiple factors including cell junction formation, extracellular matrix interactions and intracellular protein trafficking [1] Correct interaction of these factors enables the establishment of discrete apical and basolateral membrane domains. Genetic disorders of hepatic tight junction formation and protein trafficking entail a loss of hepatocyte polarity This impaired hepatocyte polarity causes pathophysiological features similar to those of cholestatic liver diseases caused by mutations in genes encoding apical ATPbinding cassette transporters [4,5,6,7]. Results: Like patients, Vps33bfl/fl-AlfpCre mice showed mislocalisation of ATP-binding cassette proteins that are trafficked to the apical membrane via Rab11a-positive recycling endosomes. This was associated with retention of bile components in blood.
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