Cellular consequences of ATP8B1 deficiency

Mutations in the ATP8B1 gene cause a spectrum of familial intrahepatic cholestasis syndromes which we collectively refer to as ATP8B1 deficiency. Patients with ATP8B1 deficiency present with intrahepatic cholestasis (impairment of bile flow) as primary complication. These patients may also present with extrahepatic symptoms, such as diarrhea, pancreatitis and hearing loss. ATP8B1 is next to liver, also expressed in intestine, pancreas and cochlear hair cells. Aberrant ATP8B1 protein function in these tissues probably accounts for the observed extrahepatic symptoms. After more than a decade following the initial cloning of ATP8B1, we still do not fully understand how mutations in ATP8B1 mechanistically result in intrahepatic cholestasis and the extraheptic symptoms reported. ATP8B1 belongs to the family of P4 P-type ATPases (P4 ATPases), which are putative (amino-) phospholipid translocators. It is believed that P4 ATPases maintain the asymmetrical distribution of phospholipids in the plasma membrane, which is important for signal transduction, vesicular trafficking and membrane stability. These P4 ATPase-specific functions, together with the expression of ATP8B1 in tissues unrelated to bile flow, suggest that ATP8B1 plays a general role in cell physiology. The aim of this thesis was therefore to study the cellular consequences of ATP8B1 deficiency. First the question was addressed why different mutations cause ATP8B1 deficiency at the molecular level. Seven distinct ATP8B1 mutations were systematically characterized. The results indicated that most mutations resulted in protein misfolfing. Treatment with 4-phenylbutyrate, a clinically approved pharmacological chaperone, partially restored defects in expression and localization of some ATP8B1 substitutions. We propose that treatment with pharmacological chaperones may represent an effective therapeutic strategy to ameliorate cholestasis in some patients with ATP8B1 deficiency. ATP8B1 protein expression in the intestine is much more pronounced than in liver. Therefore, the cellular consequences of ATP8B1 deficiency were studied in Caco-2 cells in vitro. Caco-2 cells can be differentiated into polarized monolayers that highly resemble the intestinal epithelium. A loss of function model in Caco-2 cells was constructed by stable expression of short hairpin RNAs (shRNA) complementary to ATP8B1 mRNA. This thesis describes the recent novel insights in the cellular consequences of ATP8B1 deficiency in a model for human enterocytes. The most prominent cellular defect of ATP8B1 depletion is loss of microvilli and the reduction of expression of apically localized plasma membrane proteins. Bile salt import in ATP8B1-depleted Caco-2 cells is severely compromised, but unrelated to decreased FXR expression and function. Together, these results may clarify the etiology of diarrhea in patients with ATP8B1 deficiency. Importantly, shortening and loss of microvilli are also described in other cells with aberrant ATP8B1 protein function, such as hepatocytes and cochlear hair cells. Collectively, these data suggest that the cellular defects of ATP8B1 deficiency described in this thesis are broadly present in all epithelial cells that normally express ATP8B1. These cellular defects provide a rationale why ATP8B1 deficiency results in cholestasis and a variety of extrahepatic symptoms