Regulation of the cell surface expression and transport capacity of BSEP by small chemical molecules

Transcription

1 Regulation of the cell surface expression and transport capacity of by small chemical molecules Hisamitsu Hayashi and Yuichi Sugiyama Dept. of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Japan

2 Schematic diagram illustrating the enterohepatic circulation of bile acids Liver Bile Acids Biliary excretion via Sinusoidal Membrane Blood :Bile Acids Hepatic uptake via NTCP and OATPs Jejunum NTCP Hepatocyte OATPs Ileum Excretion of bile acids Colon Enterohepatic Circulation Cholestasis Canalicular Membrane Bile flow Bile flow

3 Molecular changes of biliary transport system in patients with PFIC2 Bile acids Normal Anionic compounds PFIC2 (Progressive familial intrahepatic cholestasis type 2) Bile acids Bile acids Anionic compounds MRP2 MDR3 Phospholipid MRP2 MDR3 Phospholipid Bile Duct Biliary Bile acid concentration PFIC2 develops in childhood and leads to end stage liver disease by second decade of life. No medical therapy has been established yet.

4 Predicted topology and frequently found mutations of Extracellular Plasma membrane Intracellular NH 2 * * ATP-binding domain COOH Strautnieks SS et al., Nature Genet 20: (1998) Jansen PL et al., Gastroenterology 117: (1999) Strautnieks SS et al., Gastroenterology 134: (2008)

5 Impaired localization of mutated (MDCK II cells) Plasma membrane Degradation by Proteasome Normal transport activity Golgi complex ER Wild Type Wild Type Z Expression level at cell surface GFP Intracellular localization Apical Y Basolateral MG132 X XZ Proteasome inhibitor treatment Hayashi et al., Hepatology (4): Both and mutations reduce the cell surface expression of due to the impaired trafficking.

6 Analysis of taurocholate transport activity by wild type and mutated (HEK293cells) [ 3 H]TC uptake by membrane vesicles Uptake (pmol / mg protein) GFP GFP 30 expression of 150 due to expression level in membrane vesicles Protein (μg) amount 170kDa Expression level : Problem in PFIC2 patients with and Reduced cell surface Correction by expression level [ 3 H]TC uptake per amount of the impaired trafficking. Therapeutic goal GFP The restoration of Both mutations do not affect expression at the cell the transport function of surface. itself. Hayashi et al., Hepatology (4):916-24

7 Development of the therapeutic method for PFIC2 patients with and Therapy by small chemical molecules Search candidates from the approved drugs for other diseases and low toxic compounds. Immediately applied at the clinical place. 4-Phenylbutyrate (4PBA) FDA approved drug for urea cycle disorder. O OH Increase the cell surface expression of mutated membrane protein. (Rubenstein et al. J Clin Invest ;100(10): )

8 Establishment of screening system for the therapeutic compounds Apical side= canalicular in liver PS apical MDCKII PS trans Basal side = cells sinusoidal in liver PS efflux : Taurocholic acid ( ligand) PS basal : PS apical PS trans = PS basal PS apical + PS efflux The screening system using transcellular transport can evaluate the change of function by candidates without complex procedure, compared to the methods to directly evaluate it.

9 Identification of 4PBA effect using screening system Transcellular transport (pmol/mg protein) Transcellular transport (pmol/mg protein) GFP : ba_ : ba_4pba ab_ ab_4pba Time (min) Time (min) Transcellular transport (pmol/mg protein) Transcellular transport (pmol/mg protein) Time (min) + 4PBA + 4PBA + 4PBA Time (min) ba : basal to apical ab : apical to basal Hayashi H and Sugiyama Y, Hepatology (6):

10 Examination of 4PBA effect on -mediated transport and expression Calculation of PS apical MDCKII cells PS apical Immunoblotting --- cell surface biotinylation mock 4PBA PS apical (μl/min/mg protein) 4PBA, P < 0.05, P < 0.01 GFP 4PBA, P < 0.05, P < PBA treatment induces cell surface expression and transport capacity of and mutated in MDCK II cells.

11 4PBA-mediated prolongation of the half-life life of cell surface-resident resident Biotin-labeling chase study Incubation time (h) 4PBA Plasma membrane Endocytosis Recycling Remaining cell surface (% of 0h) 4PBA Time (h) Degradation Endosome Hayashi H and Sugiyama Y, Hepatology (6): The prolonged half-life of cell surface-resident is responsible for the increased expression.

12 Evaluation of 4PBA effectiveness in vivo Male, SD rat, 8weeks Dose : 600 mg / kg per day in three divided doses by gavage (10 days) BLOOD OATPs Bile acids P-gp BILE NTCP Anionic compounds Hepatocyte MRP2 Canalicular Membrane Immunoblotting Biliary excretion clearance of [ 3 H]TC ---Canalicular membrane vesicles CON 4PBA Bsep P-gp CL bile, liver (ml/min/kg) 1.0 Vehicle 3.2 4PBA CLbile,liver = Biliary excretion DPPIV rate 1.0 / Liver 0.7conc. Hayashi H and Sugiyama Y, Hepatology (6): PBA treatment can increase Bsep expression at the canalicular membrane, and consequently, enhance bile acid transport via the canalicular membrane in SD rats.

13 Conclusion 4-phenylbutyrate treatment induces cell surface expression and transport capacity of and PFIC2-type mutated. The prolonged half-life of cell surface-resident is responsible for the increased expression. BLOOD PFIC2 patients 4PBA treated PFIC2 patients?? BLOOD Bile acids Hepatocyte Bile acids Hepatocyte BILE BILE Hayashi H and Sugiyama Y, Hepatology (6):