Cholestasis-induced accumulation of
bile acids in the liver leads to farnesoid X receptor (FXR)-mediated transcriptional down-regulation of the
bile acid importer Na+-
taurocholate cotransporting
protein (NTCP) and to induction of endoplasmic reticulum (ER) stress. However, whether ER stress affects
bile acid uptake is largely unknown. Here, we investigated the role of ER stress on the regulation and function of the
bile acid transporter NTCP. ER stress was induced using
thapsigargin or subtilase
cytotoxin in human
osteosarcoma (U2OS) and human
hepatocellular carcinoma (HepG2) cells stably expressing NTCP. Cellular
bile acid uptake was determined using radiolabeled
taurocholate (TCA). NTCP plasma membrane expression was determined by cell surface biotinylation. Mice received a single injection of
thapsigargin, and effects of ER stress on NTCP
messenger RNA (
mRNA) and
protein were measured by reverse-transcription polymerase chain reaction (RT-PCR) and western blot analysis. Effects of
cholestasis on NTCP and ER stress were assessed in response to 3, 5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) feeding or bile duct
ligation in FXR-/- mice after 7 or 3 days, respectively. Novel NTCP-interacting
proteins were identified by mass spectrometry (MS), interaction verified, and assessed by co-immunoprecipitation and TCA uptake for functional relevance in relation to ER stress. ER stress induction strongly reduced NTCP
protein expression, plasma membrane abundance, and NTCP-mediated
bile acid uptake. This was not controlled by FXR or through a single unfolded protein response (UPR) pathway but mainly depended on the interaction of NTCP with
calnexin, an ER chaperone. In mice, expression of both NTCP and
calnexin was reduced by
thapsigargin or
cholestasis-induced ER stress.
Calnexin down-regulation in vitro recapitulated the effect of ER stress on NTCP. Conclusion: ER stress-induced down-regulation of
calnexin provides an additional mechanism to dampen NTCP-mediated
bile acid uptake and protect hepatocytes against
bile acid overload during
cholestasis.