Tmem30a Liver-specific Knockout Causes Intrahepatic Cholestasis And Hepatocellular Carcinogenesis In Mice

BackgroundNormal fucntions of mammalian liver require the hepatic bile salt secretion and bile formation.The etiology of cholestasis is polymorphic,the impairment or disruption of bile flow and bile production result in the cholestasis.Progressive familial intrahepatic cholestasis(PFIC)is chronic autosomal recessive inherted disease.Mutations in ATP8B1,a member of P4 type ATPase,cause PFIC type 1(PFIC1).Patients with PFIC1 often present symptoms in the first months of life,and cholestasis leads to liver fibrosis and cirrhosis,which eventually results in liver failure or hepatocellular carcinoma.In contrast,ATP8B1 mutant mice have mildly elevated serum bile acid and lighter weaning weight.According their conditions,patients usually leads to death from liver failure and hepatocellular carcinoma at ages ranging from infancy to adolescence.TMEM30A,as a β-subunit,is essential for P4 type ATPase,and ATP8B1 also requires TMEM30A to form the heterodimer.The roles of TMEM30A in the liver are currently unclear.ObjectiveTMEM30A is required for the flippase activity and localization of these P4-ATPases.TMEM30A also plays a key role in the cell migration and the uptake of the anticancer drugs in mammalian cells.The function of TMEM30A in vivo were still unclear,we explored the Tmem30a liver-specific knockout(Tmem30a LKO)mice and aimed to the mechanism of TMEM30A in the liver.MethodsTmem30aflox/flox mice were mated with transgenetic mice with albumin-promoter Cre,and then Tmem30aflox/flox A1bCre+ mice were the Tmem30a LKO mice.All samples were tested on a HITACHI Clinical Analyzer 7180,according to the manufacturer’s protocols.Hematoxylin-eosin(H&E)staining,Masson blue and immunohistochemistry were used to analysed the pathological conditions.Bile salt species in serum,bile,liver and feces were identified and quantified using ultrahigh-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)methods on a Waters Acquity UPLC System and AB Sciex TripleQuad 5500.We tested the mRNA and protein levels of ATP8B1,ATP 11C,bile salt transporter and related nucear receptors by real-time PCR and Western Blotting.Immunofluorescence images showed the localization of ATP8B1,ATP 11C,bile salt transporter in the membrane.The rescued experiment was performed by proteasome inhibition via bortezomin.Cholic acid-supplemented diet was fed to confirm the phenotypes in liver-specific Tmem30a knockout mice.ResultsIn the present study,we explored Tmem30a liver-specific knockout(LKO)mice,liver function tests and histology revealed that Tmem30a LKO mice suffered from hyperbilirubinaemia and hypercholanaemia,inflammatory infiltration,ductular proliferation and liver fibrosis.Serum total bile acid,total bilirubin and direct bilirubin levels in Tmem30a LKO mice were significantly increased compared to the littermate controls.Serum ALT,AST and ALP levels in Tmem30a LKO mice were also significantly increased across varous ages.Western blotting and immunofluorescence analyses indicated that the expression and localization of ATP8B1 and ATP11C were significantly reduced in Tmem30a LKO mice,which correlated with the impaired expression and localization of bile salt transporters,such as organic anion-transporting polypeptide 1A4(OATP1A4),OATP1B2,Na+-taurocholate co-transporting polypeptide(NTCP),bile salt export pump(BSEP)and multidrug resistance-assocaited proteins 2(MRP2).In addition,mRNA levels of OATP1B2,NTCP,BSEP were also reduced.Interestingly,the mRNA and protein levels of related regulatory nuclear receptors,including farnesoid X receptor a(FXRa),retinoid X receptor a(RXRa),hepatocyte nuclear factor 4 a(HNF4a),liver receptor homolog-1(LRH-1),and short heterodimer partner(SHP),were also down-regulated.Proteasome inhibition via bortezomib partially restored the expression of these proteins,however,ATP8B1,ATP11C and BS transporters were localized in endoplasmic reticulum,not in the membrane.A cholic acid-supplemented diet further exacerbated the liver damage in male Tmem30a LKO mice,and more than half of male Tmem30a LKO mice were dead.Interestingly,all Tmem30a LKO mice spontaneously developed hepatocelluar carcinoma from 14 months of age.ConclusionsThe TMEM30A deficiency resulted in intrahepatic cholestasis in mice by impairing the expression and localization of ATP8B1,ATP11C and BS transporters and the expression of related nuclear receptors,and spontaneously developed hepatocellular carcinoma.Therefore,TMEM30A may be a novel genetic determinant of intrahepatic cholestasis,and is involved in hepatocellular carcinogenesis.