RESUMO
BACKGROUND AND AIMS: Hepatic bile acid homeostasis is regulated by negative feedback inhibition of genes involved in the uptake and synthesis of bile acids. Bile acids down-regulate the rate-limiting gene for bile acid synthesis, cholesterol 7alpha-hydroxylase (cyp7a), via bile acid receptor (fxr) activation of an inhibitory nuclear receptor, shp. We hypothesized that shp would also mediate negative feedback regulation of ntcp, the principal hepatic bile acid transporter. METHODS: Primary rat hepatocytes or transfected HepG2 and Cos cells were treated with retinoids with or without bile acids, and effects on bile acid transport and ntcp and shp gene expression and promoter activity were determined. Gel shift assays were performed using synthetic fxr, rxr, and rar proteins. RESULTS: Bile acid treatment of primary rat hepatocytes prevented retinoid activation of ntcp gene expression and function; this corresponded temporally with shp gene activation. Bile acid-mediated down-regulation occurred via fxr-dependent suppression of the ntcp RXR:RAR response element. Moreover, cotransfected shp directly inhibited retinoid activation of the ntcp promoter. CONCLUSIONS: These studies show negative feedback regulation of ntcp by bile acid-activated fxr via induction of shp. This novel regulatory pathway provides a means for coordinated down-regulation of bile acid import and synthesis, thereby protecting the hepatocyte from bile acid-mediated damage in cholestatic conditions.
Assuntos
Proteínas de Bactérias/genética , Ácidos e Sais Biliares/genética , Proteínas de Transporte/genética , Grupo dos Citocromos c/genética , Neoplasias Hepáticas/genética , Proteínas de Membrana Transportadoras , Receptores Citoplasmáticos e Nucleares/genética , Animais , Proteínas de Bactérias/farmacologia , Ácidos e Sais Biliares/biossíntese , Carcinoma Hepatocelular/genética , Grupo dos Citocromos c/farmacologia , Humanos , Masculino , Transportadores de Ânions Orgânicos Dependentes de Sódio , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Simportadores , Células Tumorais CultivadasRESUMO
Nuclear calcium (Ca(2+)) regulates a number of important cellular processes, including gene transcription, growth, and apoptosis. However, it is unclear whether Ca(2+) signaling is regulated differently in the nucleus and cytosol. To investigate this possibility, we examined subcellular mechanisms of Ca(2+) release in the HepG2 liver cell line. The type II isoform of the inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) was expressed to a similar extent in the endoplasmic reticulum and nucleus, whereas the type III InsP(3)R was concentrated in the endoplasmic reticulum, and the type I isoform was not expressed. Ca(2+) signals induced by low InsP(3) concentrations started earlier or were larger in the nucleus than in the cytosol, indicating higher sensitivity of nuclear Ca(2+) stores for InsP(3). Nuclear InsP(3)R channels were active at lower InsP(3) concentrations than InsP(3)R from cytosol. Enriched expression of type II InsP(3)R in the nucleus results in greater sensitivity of the nucleus to InsP(3), thus providing a mechanism for independent regulation of Ca(2+)-dependent processes in this cellular compartment.