Impact of ATP8B1 Mutation and Maturation on CFTR Functionality and Gene Expression

Background/Objectives: Progressive familial intrahepatic cholestasis type 1 (PFIC1) is a rare autosomal recessive liver disorder caused by mutations in the ATP8B1 gene, leading to defective bile acid secretion and severe clinical outcomes. A milder, episodic form of the disease, benign recurrent intrahepatic cholestasis (BRIC), is also associated with ATP8B1 mutations. PFIC1 patients frequently present with extra-hepatic symptoms. Interestingly, cystic fibrosis (CF) patients also develop liver dysfunction, and we previously showed that CFTR expression is significantly reduced in PFIC1 patients. Methods: In this study, we investigated the relationship between ATP8B1 and CFTR by comparing gene expression and protein maturation in PFIC1 (Byler) and BRIC cell lines to wild-type controls. Results: Both ATP8B1 and CFTR transcripts were markedly decreased in mutant cells, resulting in lower protein synthesis. Distinct ATP8B1 isoforms were identified: a 140 kDa mature form in wild-type cells, a 145 kDa variant in BRIC cells, and a 180 kDa form in PFIC1 cells. Using antibodies targeting the N- and C-terminal domains, we demonstrated that wild-type ATP8B1 undergoes sequential maturation—first at Gly308 in the endoplasmic reticulum, then at Gly556, a region containing the D554N PFIC1 mutation site—yielding a 74 kDa nuclear peptide with transcriptional cofactor characteristics. This peptide contains an NR box flanked by STAT5 binding motifs and a potential PKA phosphorylation site, suggesting interaction with the Small Heterodimer Partner (SHP) and regulation of FXR and HNF1α expression, which are both known to modulate CFTR transcription. Immunoprecipitation experiments revealed a physical association between ATP8B1 and CFTR, supporting a chaperone role for ATP8B1 in CFTR trafficking and membrane insertion. PDZK1 was also found to interact with ATP8B1, potentially stabilizing CFTR at the apical membrane through PDZ-domain interactions. Conclusions: Collectively, our results identify ATP8B1 as a multifunctional protein that not only ensures the correct trafficking and membrane localization of CFTR but should also regulate its transcription via a nuclear signaling pathway. These findings provide new insights into the molecular link between PFIC1 and cystic fibrosis-associated liver disease.