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Hepatic Tmem30a Deficiency Causes Intrahepatic Cholestasis by Impairing Expression and Localization of Bile Salt Transporters

  • Leiming Liu
    Affiliations
    Laboratory of Cancer Biology, Key Laboratory of Biotherapy in Zhejiang Province, Sir Runrun Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China

    Key Laboratory of Regenerative Medicine of Ministry of Education, Institute of Aging and Regenerative Medicine, Jinan University, Guangzhou, China
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  • Lingling Zhang
    Affiliations
    Institute of Aging Research, Leibniz Link Partner Group on Stem Cell Aging, School of Medicine, Hangzhou Normal University, Hangzhou, China
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  • Lin Zhang
    Affiliations
    Sichuan Provincial Key Laboratory for Human Disease Gene Study and School of Medicine, Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital, Chengdu, China

    Key Laboratory for NeuroInformation of Ministry of Education and Medicine Information Center, School of Medicine, University of Electronic Science and Technology of China, Chengdu, China
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  • Fan Yang
    Affiliations
    Key Laboratory of Regenerative Medicine of Ministry of Education, Institute of Aging and Regenerative Medicine, Jinan University, Guangzhou, China

    Leibniz Institute for Age Research - Fritz Lipmann Institute, Friedrich-Schiller University of Jena, Jena, Germany
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  • Xudong Zhu
    Affiliations
    Institute of Aging Research, Leibniz Link Partner Group on Stem Cell Aging, School of Medicine, Hangzhou Normal University, Hangzhou, China
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  • Zhongjie Lu
    Affiliations
    Key Laboratory of Combined Multi-Organ Transplantation, Ministry of Public Health, Division of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China
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  • Yeming Yang
    Affiliations
    Sichuan Provincial Key Laboratory for Human Disease Gene Study and School of Medicine, Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital, Chengdu, China

    Key Laboratory for NeuroInformation of Ministry of Education and Medicine Information Center, School of Medicine, University of Electronic Science and Technology of China, Chengdu, China
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  • Haiqi Lu
    Affiliations
    Laboratory of Cancer Biology, Key Laboratory of Biotherapy in Zhejiang Province, Sir Runrun Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China
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  • Lifeng Feng
    Affiliations
    Laboratory of Cancer Biology, Key Laboratory of Biotherapy in Zhejiang Province, Sir Runrun Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China
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  • Zhuo Wang
    Affiliations
    Laboratory of Cancer Biology, Key Laboratory of Biotherapy in Zhejiang Province, Sir Runrun Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China
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  • Hui Chen
    Affiliations
    Key Laboratory of Combined Multi-Organ Transplantation, Ministry of Public Health, Division of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China
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  • Sheng Yan
    Affiliations
    Key Laboratory of Combined Multi-Organ Transplantation, Ministry of Public Health, Division of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China
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  • Lin Wang
    Affiliations
    Department of Hepato-Biliary Surgery, Xijing Hospital, The Fourth Military Medical University, Xi'an, China
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  • Zhenyu Ju
    Affiliations
    Key Laboratory of Regenerative Medicine of Ministry of Education, Institute of Aging and Regenerative Medicine, Jinan University, Guangzhou, China

    Institute of Aging Research, Leibniz Link Partner Group on Stem Cell Aging, School of Medicine, Hangzhou Normal University, Hangzhou, China
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  • Hongchuan Jin
    Correspondence
    Address correspondence to Hongchuan Jin, M.D., Ph.D., Laboratory of Cancer Biology, Sir Runrun Shaw Hospital, #401, 3 E Qingchun Rd, Hangzhou, Zhejiang 310016, China.
    Affiliations
    Laboratory of Cancer Biology, Key Laboratory of Biotherapy in Zhejiang Province, Sir Runrun Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China
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  • Xianjun Zhu
    Correspondence
    Xianjun Zhu, Ph.D., Key Laboratory for Human Disease Gene Study, Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital, 32# W Sec 2, 1st Ring Rd, Chengdu, Sichuan 610072, China.
    Affiliations
    Sichuan Provincial Key Laboratory for Human Disease Gene Study and School of Medicine, Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital, Chengdu, China

    Key Laboratory for NeuroInformation of Ministry of Education and Medicine Information Center, School of Medicine, University of Electronic Science and Technology of China, Chengdu, China
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Open ArchivePublished:September 15, 2017DOI:https://doi.org/10.1016/j.ajpath.2017.08.011
      Mutations in ATP8B1 or ATP11C (members of P4-type ATPases) cause progressive familial intrahepatic cholestasis type 1 in human or intrahepatic cholestasis in mice. Transmembrane protein 30A (TMEM30A), a β-subunit, is essential for the function of ATP8B1 and ATP11C. However, its role in the etiology of cholestasis remains poorly understood. To investigate the function of TMEM30A in bile salt (BS) homeostasis, we developed Tmem30a liver-specific knockout (LKO) mice. Tmem30a LKO mice experienced hyperbilirubinemia, hypercholanemia, inflammatory infiltration, ductular proliferation, and liver fibrosis. The expression and membrane localization of ATP8B1 and ATP11C were significantly reduced in Tmem30a LKO mice, which correlated with the impaired expression and localization of BS transporters, such as OATP1A4, OATP1B2, NTCP, BSEP, and MRP2. The proteasome inhibitor bortezomib partially restored total protein levels of BS transporters but not the localization of BS transporters in the membrane. Furthermore, the expression of nuclear receptors, including FXRα, RXRα, HNF4α, LRH-1, and SHP, was also down-regulated. A cholic acid–supplemented diet exacerbated the liver damage in Tmem30a LKO mice. TMEM30A deficiency led to intrahepatic cholestasis in mice by impairing the expression and localization of BS transporters and the expression of related nuclear receptors. Therefore, TMEM30A may be a novel genetic determinant of intrahepatic cholestasis.
      Bile acids are synthesized in the liver and secreted into the gut, and they have multiple physiological functions, including bile flow, absorption of lipophilic nutrients, clearing toxic molecules, and antimicrobial and metabolic effects.
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      Bile acid transporters and regulatory nuclear receptors in the liver and beyond.
      Disturbances in bile acid homeostasis contribute to cholestasis, gallstone disease, and malabsorption. Cholestasis causes liver damage due to the excessive accumulation of bile salts (BSs) and other toxins in the liver. Hereditary mutations in hepatobiliary transporter genes result in defects in the expression and function of the hepatobiliary transport systems, which are implicated in the pathogenesis of cholestasis. Progressive familial intrahepatic cholestasis (PFIC) types 1 and 2 are caused by different mutations in ATP8B1 and ABCB11 (which encodes the BS export pump, BSEP), respectively.
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      The third type of PFIC (PFIC3) is caused by a genetic defect in ABCB4 (which encodes multidrug resistance 3).
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      Progressive familial intrahepatic cholestasis.
      In addition, mutations in MRP2 (multidrug resistance-associated protein 2) are associated with Dubin-Johnson syndrome, which results in jaundice and intrahepatic cholestasis of pregnancy.
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      Association of the multidrug-resistance-associated protein gene (ABCC2) variants with intrahepatic cholestasis of pregnancy.
      Recently, mutations in TJP2 (tight junction protein 2) and N1RH4 (which encodes the farnesoid X receptor, FXR) were also reported to cause progressive familial intrahepatic cholestasis in humans.
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      University of Washington Center for Mendelian Genomics
      Mutations in TJP2 cause progressive cholestatic liver disease.
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      Mutations in the nuclear bile acid receptor FXR cause progressive familial intrahepatic cholestasis.
      ATP8B1 is a widely expressed P4-type ATPase that functions as a phospholipid flippase to maintain phospholipid asymmetry in the membrane. Compared with patients with PFIC1, Atp8b1G308V/G308V mice showed mild abnormalities, including decreased weight at weaning and elevated serum bile acid levels.
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      • Bull L.N.
      • Elferink R.P.
      • Freimer N.B.
      A mouse genetic model for familial cholestasis caused by ATP8B1 mutations reveals perturbed bile salt homeostasis but no impairment in bile secretion.
      In addition, mice deficient in ATP11C (which encodes a paralogous P4-type ATPase) exhibited impaired localization of BS transporters and plasma conjugated hyperbilirubinemia and unconjugated hypercholanemia.
      • Siggs O.M.
      • Schnabl B.
      • Webb B.
      • Beutler B.
      X-linked cholestasis in mouse due to mutations of the P4-ATPase ATP11C.
      • Matsuzaka Y.
      • Hayashi H.
      • Kusuhara H.
      Impaired hepatic uptake by organic anion-transporting polypeptides is associated with hyperbilirubinemia and hypercholanemia in Atp11c mutant mice.
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      • Naik J.
      • Utsunomiya K.S.
      • Duijst S.
      • Ho-Mok K.
      • Bolier A.R.
      • Hiralall J.
      • Bull L.N.
      • Bosma P.J.
      • Oude Elferink R.P.
      • Paulusma C.C.
      ATP11C targets basolateral bile salt transporter proteins in mouse central hepatocytes.
      • Yabas M.
      • Teh C.E.
      • Frankenreiter S.
      • Lal D.
      • Roots C.M.
      • Whittle B.
      • Andrews D.T.
      • Zhang Y.
      • Teoh N.C.
      • Sprent J.
      • Tze L.E.
      • Kucharska E.M.
      • Kofler J.
      • Farell G.C.
      • Broer S.
      • Goodnow C.C.
      • Enders A.
      ATP11C is critical for the internalization of phosphatidylserine and differentiation of B lymphocytes.
      • Siggs O.M.
      • Arnold C.N.
      • Huber C.
      • Pirie E.
      • Xia Y.
      • Lin P.
      • Nemazee D.
      • Beutler B.
      The P4-type ATPase ATP11C is essential for B lymphopoiesis in adult bone marrow.
      From these studies, the P4-ATPases have important roles in preventing intrahepatic cholestasis. Transmembrane protein 30 (TMEM30) or cell division cycle protein 50 (CDC50) binds to the P4-ATPases to form a heterodimer, and mammals express three members of the TMEM30 family: TMEM30A (CDC50A), TMEM30B (CDC50B), and TMEM30C (CDC50C). TMEM30A is ubiquitously expressed and binds 11 of 14 P4-ATPases.
      • Paulusma C.C.
      • Folmer D.E.
      • Ho-Mok K.S.
      • de Waart D.R.
      • Hilarius P.M.
      • Verhoeven A.J.
      • Oude Elferink R.P.
      ATP8B1 requires an accessory protein for endoplasmic reticulum exit and plasma membrane lipid flippase activity.
      • Bryde S.
      • Hennrich H.
      • Verhulst P.M.
      • Devaux P.F.
      • Lenoir G.
      • Holthuis J.C.
      CDC50 proteins are critical components of the human class-1 P4-ATPase transport machinery.
      • van der Velden L.M.
      • Wichers C.G.
      • van Breevoort A.E.
      • Coleman J.A.
      • Molday R.S.
      • Berger R.
      • Klomp L.W.
      • van de Graaf S.F.
      Heteromeric interactions required for abundance and subcellular localization of human CDC50 proteins and class 1 P4-ATPases.
      • Folmer D.E.
      • Mok K.S.
      • de Wee S.W.
      • Duijst S.
      • Hiralall J.K.
      • Seppen J.
      • Oude Elferink R.P.
      • Paulusma C.C.
      Cellular localization and biochemical analysis of mammalian CDC50A, a glycosylated beta-subunit for P4 ATPases.
      • Takatsu H.
      • Baba K.
      • Shima T.
      • Umino H.
      • Kato U.
      • Umeda M.
      • Nakayama K.
      • Shin H.W.
      ATP9B, a P4-ATPase (a putative aminophospholipid translocase), localizes to the trans-Golgi network in a CDC50 protein-independent manner.
      TMEM30A is required for the flippase activity and localization of these P4-ATPases. Depletion of TMEM30A in cell lines impaired the endoplasmic reticulum (ER) exit of these P4-ATPases and caused a severe defect in the formation of membrane ruffles because of defects in the inward translocation of phosphatidylethanolamine and phosphatidylserine that subsequently inhibited cell migration.
      • Paulusma C.C.
      • Folmer D.E.
      • Ho-Mok K.S.
      • de Waart D.R.
      • Hilarius P.M.
      • Verhoeven A.J.
      • Oude Elferink R.P.
      ATP8B1 requires an accessory protein for endoplasmic reticulum exit and plasma membrane lipid flippase activity.
      • Bryde S.
      • Hennrich H.
      • Verhulst P.M.
      • Devaux P.F.
      • Lenoir G.
      • Holthuis J.C.
      CDC50 proteins are critical components of the human class-1 P4-ATPase transport machinery.
      • van der Velden L.M.
      • Wichers C.G.
      • van Breevoort A.E.
      • Coleman J.A.
      • Molday R.S.
      • Berger R.
      • Klomp L.W.
      • van de Graaf S.F.
      Heteromeric interactions required for abundance and subcellular localization of human CDC50 proteins and class 1 P4-ATPases.
      • Kato U.
      • Inadome H.
      • Yamamoto M.
      • Emoto K.
      • Kobayashi T.
      • Umeda M.
      Role for phospholipid flippase complex of ATP8A1 and CDC50A proteins in cell migration.
      TMEM30A-deficient cells are engulfed by macrophages because of the exposure of phosphatidylserine in the outer leaflet in the plasma membrane, and these cells lost their ability to induce tumor formation in nude mice.
      • Segawa K.
      • Kurata S.
      • Yanagihashi Y.
      • Brummelkamp T.R.
      • Matsuda F.
      • Nagata S.
      Caspase-mediated cleavage of phospholipid flippase for apoptotic phosphatidylserine exposure.
      TMEM30A also plays a key role in the uptake of the anticancer drugs and choline phospholipids in mammalian cells.
      • Munoz-Martinez F.
      • Torres C.
      • Castanys S.
      • Gamarro F.
      CDC50A plays a key role in the uptake of the anticancer drug perifosine in human carcinoma cells.
      • Chen R.
      • Brady E.
      • McIntyre T.M.
      Human TMEM30a promotes uptake of antitumor and bioactive choline phospholipids into mammalian cells.
      Knockdown of Tmem30a in wild-type FVB mice resulted in elevated plasma levels of unconjugated BSs but no liver damage.
      • de Waart D.R.
      • Naik J.
      • Utsunomiya K.S.
      • Duijst S.
      • Ho-Mok K.
      • Bolier A.R.
      • Hiralall J.
      • Bull L.N.
      • Bosma P.J.
      • Oude Elferink R.P.
      • Paulusma C.C.
      ATP11C targets basolateral bile salt transporter proteins in mouse central hepatocytes.
      However, it is still unclear whether TMEM30A plays a role in cholestasis.
      In this study, we investigated the in vivo roles of Temem30a in the liver by generating Tmem30a liver-specific knockout (LKO) mice. Heterozygous Tmem30a KO first mice were crossed with Flp mice to generate Tmem30aflox/+ mice, which were then crossed with Alb-Cre mice to generate Tmem30aflox/flox-Alb-Cre mice. These Tmem30a LKO mice were used to investigate the function of TMEM30A in cholestasis. Tmem30a LKO mice exhibited severe hyperbilirubinemia and hypercholanemia, inflammatory infiltration, ductular proliferation, and liver fibrosis. These pathologic phenotypes in Tmem30a LKO mice were exacerbated on 0.5% cholic acid (CA)-supplemented diet. Mechanistically, the expression and localization of ATP8B1, ATP11C, and several BS transporters, including basolateral uptake transporters [organic anion-transporting polypeptide 1A4 (OATP1A4), OATP1B2, and Na+-taurocholate cotransporting polypeptide (NTCP)] and canalicular efflux transporters (BSEP and MRP2) in hepatocytes, were severely impaired. In addition, the expression of regulatory nuclear receptors related to bile acid synthesis and transport, including FXRα, retinoid X receptor α (RXRα), hepatocyte nuclear factor 4 α (HNF4α), liver receptor homolog-1 (LRH-1), and short heterodimer partner (SHP), was also significantly decreased. These data demonstrated that TMEM30A is essential for the expression and proper localization of BS transporters and thereby is critical for the maintenance of BS homeostasis.

      Materials and Methods

      Generation of Tmem30a LKO Mice

      The original Tmem30a allele is of type KO first by using the FRT-Flp strategy and also is conditional potential by using the Flox-cre system. Mice carrying the two floxed alleles on the third exon of the Tmem30a gene were generated by conventional homologous recombination in embryonic stem cells and blastocyst injection of correctly targeted embryonic stem cell clones. Tmem30a KO first mice were first interbred with Flp mice
      • Farley F.W.
      • Soriano P.
      • Steffen L.S.
      • Dymecki S.M.
      Widespread recombinase expression using FLPeR (flipper) mice.
      (The Jackson Laboratory, Bar Harbor, ME; strain name: B6.129S4-Gt(ROSA)26Sortm1(FLP1)Dym/RainJ; stock number 009086; alias ROSA26::FLPe knock in) to generate Tmem30aflox/+ mice. Tmem30aflox/+ mice were then crossed with Albumin-Cre
      • Postic C.
      • Shiota M.
      • Niswender K.D.
      • Jetton T.L.
      • Chen Y.
      • Moates J.M.
      • Shelton K.D.
      • Lindner J.
      • Cherrington A.D.
      • Magnuson M.A.
      Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase.
      (alias Alb-Cre; The Jackson Laboratory) mice to generate LKO mice with the genotype Tmem30aflox/flox-Alb-Cre mice (Supplemental Figure S1A). Tmem30aflox/flox or Alb-Cre mice were used as littermate WT controls (genetic background: C57BL/6J). To monitor the efficiency of Cre-mediated deletion of the LoxP-flanked exon 3 in the liver, a TdTomato reporter
      • Madisen L.
      • Zwingman T.A.
      • Sunkin S.M.
      • Oh S.W.
      • Zariwala H.A.
      • Gu H.
      • Ng L.L.
      • Palmiter R.D.
      • Hawrylycz M.J.
      • Jones A.R.
      • Lein E.S.
      • Zeng H.
      A robust and high-throughput Cre reporting and characterization system for the whole mouse brain.
      was used [strain name: B6.Cg-Gt(ROSA)26Sortm14(CAG-TdTomato)Hze/J; alias Ai14D]. This reporter line contains a loxP-flanked STOP cassette that prevents transcription of the downstream CAG promoter-driven red fluorescent protein variant TdTomato. In the presence of Alb-Cre recombinase, the STOP cassette in the reporter mice will be removed in the Cre-expressing hepatocyte and TdTomato will be expressed. Because this CAG promoter-driven reporter construct was inserted into the Gt(ROSA)26Sor locus, TdTomato expression pattern was determined by the tissue(s) that expressed Cre recombinase.
      All animals received humane care according to the criteria outlined in the NIH's Guide for the Care and Use of Laboratory Animals,
      Committee for the Update of the Guide for the Care and Use of Laboratory Animals
      National Research Council: Guide for the Care and Use of Laboratory Animals: Eighth Edition.
      and all animal experiments were approved by the Animal Care and Ethics Committee at Hangzhou Normal University and Sichuan Provincial People's Hospital.

      Plasma Biochemistry, BS Species Identification and Quantification, and Bile Formation Assays

      Reagents for serum biochemical analysis were purchased from DiaSys Diagnostic Systems GmbH (Holzheim, Germany). All samples were tested on a Hitachi (Tokyo, Japan) Clinical Analyzer 7180, according to the manufacturer's protocols. BS species were identified and quantified as previously described in detail.
      • Garcia-Canaveras J.C.
      • Donato M.T.
      • Lahoz A.
      Ultra-performance liquid chromatography-mass spectrometry targeted profiling of bile acids: application to serum, liver tissue, and cultured cells of different species.
      The bile formation assays were performed as previously described in detail
      • de Waart D.R.
      • Naik J.
      • Utsunomiya K.S.
      • Duijst S.
      • Ho-Mok K.
      • Bolier A.R.
      • Hiralall J.
      • Bull L.N.
      • Bosma P.J.
      • Oude Elferink R.P.
      • Paulusma C.C.
      ATP11C targets basolateral bile salt transporter proteins in mouse central hepatocytes.
      ; the concentration of phospholipid was tested according to the instruction (catalog number MAK122; Sigma-Aldrich, St. Louis, MO).

      Real-Time Quantitative PCR

      Total RNA was isolated from liver samples using Trizol (Invitrogen, Carlsbad, CA) and then reverse-transcribed and analyzed by real-time quantitative PCR with SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA) and specific primers (Table 1) on a Bio-Rad CFX96 Touch.
      Table 1Sequences of Oligonucleotides Used in This Study
      GeneForward primerReverse primer
      Asbt5′-GGAACTGGCTCCAATATCCTG-3′5′-TCCAATCACAGCTATGAGCAC-3′
      Bsep5′-ACTAATGTTGGGATCCAGGGC-3′5′-GGCTTTGTCCAGAGCAAGCTG-3′
      Cyp8b15′-TAGCCCTGTTAGAGTGTGTGTGAC-3′5′-AGTCAGGATCTCTCCCTGAACTTG-3′
      Cyp7a15′-CAATGAAAGCAGCCTCTGAAG-3′5′-AGCCTCCTTGATGATGCTATC-3′
      Cyp27a15′-CCACAAGGGCCTCACCTATG-3′5′-GCACCTGGTCCAGCCGGGTG-3′
      Fgf155′-CCAACTGCTTCCTCCGAATCC-3′5′-TACAGTCTTCCTCCGAGTAGC-3′
      Fgfr45′-CTCGGAAAGCCCCTGGGTGA-3′5′-AGCTTCATCACCTCCATCTCG-3′
      Fxrα5′-GAAAGAGTGGTATCTCTGATGAG-3′5′-ACCGCCTCTCTGTCCTTGATG-3′
      Lrh15′-AACCTCCTGAGTCTCGCACAG-3′5′-ACCTGCTCTTGGACACCTTCC-3′
      Lxrα5′-AATGCCAGGAGTGTCGACTT-3′5′-CTTGCCGCTTCAGTTTCTTC-3′
      Mrp25′-AGCAGGTGTTCGTTGTGTGT-3′5′-AGCCAAGTGCATAGGTAGAGAAT-3′
      Mrp35′-CCCTGCGTATGAACTTAGATC-3′5′-CTGCCTCTGGCCAACACTG-3′
      Mrp45′-GCCACATCCTCATACTCAA-3′5′-CTCTGCTTCCTCGTTTTCT-3′
      Shp5′-TCTGGAGCCTTGAGCTGGGT-3′5′-GCCTTGGCTGGCTGGGTAC-3′
      Ntcp5′-ATCTGACCAGCATTGAGGCTCT-3′5′-CCGTCGTAGATTCCTTTGCTGT-3′
      Atp11c5′-CGAAGAAGAAGTGCCAGGAATCCGA-3′5′-GCTGCAACCACGGTCAATATGCT-3′
      Atp8b15′-GTCTGGGACAGAGTCATTTC-3′5′-CTTATCAGAGAAGATGTAATG-3′
      Oatp1a15′-CAGATAAATGGATTTGCCAG-3′5′-GTCAACAAATAGTTACAGAG-3′
      Oatp1a45′-ATAGCTTCAGGCGCATTTAC-3′5′-ATAGCTTCAGGCGCATTTAC-3′
      Oatp1b25′-TTCACCACAACAATGGCCTA-3′5′-TTTTCCCCACAGACAGGTTC-3′
      Hnf1α5′-CTGATTGAAGAGCCCACA-3′5′-CACTCCGCCCTATTACACT-3′
      Hnf4α5′-AGACAAAGATAAGAGGAACCAG-3′5′-CAGAGATGGGAGAGGTGA-3′
      Mdr25′-CCCCTGTATTGATGCTTTC-3′5′-CCTTTGATGTTGTCTGGTTT-3′
      Rar5′-TGAGCAAGACACAATGACC-3′5′-CGAAGGCAAAGACCAAGT-3′
      Rxr5′-TACGCAAAGACCTGACCTAC-3′5′-CTCCACCTCGTTCTCATTC-3′
      Gr5′-ACAGACAAGCAAGTGGAAAC-3′5′-TGAGGAGAGAAGCAGTAAGG-3′

      Liver Histologic, Immunohistochemistry, Immunofluorescence, and Western Blot Analysis

      Liver tissues were fixed in 0.01 mol/L phosphate-buffered saline (PBS; pH 7.4) containing 10% formalin and then embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) and Masson blue for histologic examinations. Immunohistochemistry was performed with rabbit anti-F4/80 and cytokeratin-19 antibodies. H&E and immunohistochemistry slices were scanned and captured with a Pannoramic MIDI (3DHISTECH Ltd., Budapest, Hungary). For immunofluorescence, frozen tissue sections were fixed in 4% formaldehyde. After washing the samples twice with PBS, they were incubated in PBS/fetal bovine serum (pH 7.4, containing 10% fetal bovine serum) to block nonspecific sites of antibody absorption. Then, these slices were incubated with appropriate primary and secondary antibodies in 0.1% saponin. The images were captured on a laser scanning confocal microscope (Carl Zeiss, Jena, Germany). The plasma membrane proteins were extracted according to the manufacturer's protocol (catalog number 71772; NovaGen, Los Angeles, CA). Liver samples (50 mg) were homogenized in 1 mL of radioimmunoprecipitation lysis buffer (Beyotime, Shanghai, China) containing a protease inhibitor cocktail (Roche, Mannheim, Germany). The VersaDoc Imaging System (Bio-Rad) was used to visualize the proteins and to quantify the band densities. All primary and secondary antibodies are listed in Table 2.
      Table 2The Primary and Secondary Antibodies Used in This Study
      NameCatalogManufacturer
      SHPsc-30169Santa Cruz Biotechnology, Santa Cruz, CA
      ATP8B1sc-134967Santa Cruz Biotechnology
      OATP1A4sc-376424Santa Cruz Biotechnology
      BSEPab112494Abcam, Cambridge, MA
      NTCPab131084Abcam
      LRH-1ab125034Abcam
      HNF4αab199431Abcam
      FXRαab28480Abcam
      RXRαab125001Abcam
      F4/80ab100790Abcam
      PKCζ9368pCell Signaling Technology, Danvers, MA
      Phospho-PKCζ/λ (Thr410/403)9378pCell Signaling Technology
      CK-1912434sCell Signaling Technology
      β-ActinA5316Sigma-Aldrich, St Louis, MO
      Anti-rabbit IgG HRP-linked antibody7074sCell Signaling Technology
      Anti-mouse IgG HRP-linked antibody7076sCell Signaling Technology
      Donkey anti-goat IgG Fab secondary antibody, HRPPA1-29617Thermo Fisher Scientific, Rockford, Illinois
      Donkey anti-goat IgG (H+L) secondary antibody, Alexa Fluor 546A11056Thermo Fisher Scientific
      Donkey anti-goat IgG (H+L) secondary antibody, Alexa Fluor 546A11010Thermo Fisher Scientific
      Goat anti-mouse IgG (H+L) secondary antibody, Alexa Fluor 488A11001Thermo Fisher Scientific
      Rabbit anti-mouse IgG (H+L) secondary antibody, Alexa Fluor 594A21205Thermo Fisher Scientific
      Donkey anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 488A21206Thermo Fisher Scientific
      BSEP, bile salt export pump; CK, cytokeratin; FXR, farnesoid X receptor; HNF, hepatocyte nuclear factor; HRP, horseradish peroxidase; LRH, liver receptor homolog; NTCP, Na+-taurocholate cotransporting polypeptide; OATP, organic anion transporting polypeptide; PKC, protein kinase C; RXR, retinoid X receptor; SHR, short heterodimer partner.

      Proteasome Inhibition and CA-Supplemented Diet

      For proteasome inhibition, mice were intraperitoneally injected with 0.5 mg/kg bortezomib (Selleck, Shanghai, China) or dimethyl sulfoxide and then sacrificed after 6 or 12 hours. The mice were fed a basal 14% protein diet with or without a 0.5% sodium cholate supplement (Supplemental Table S1), and the animals were weighed at 3-day intervals and monitored daily for survival. The mice were euthanized when they were experiencing severe distress, such as dramatic weight loss, back deformations, and slow movement.

      Statistical Analysis

      Statistical analysis was performed using one-way analysis of variance test, followed by t-test. A P value ≤0.05 was considered statistically significant.

      Results

      Hyperbilirubinemia and Hypercholanemia in Tmem30a LKO Mice

      Tmem30a LKO mice were viable and fertile and had normal life spans. The specific depletion of TMEM30A in hepatocytes was confirmed by immunostaining and Western blot analysis with the use of specific TMEM30A antibody
      • Coleman J.A.
      • Molday R.S.
      Critical role of the beta-subunit CDC50A in the stable expression, assembly, subcellular localization, and lipid transport activity of the P4-ATPase ATP8A2.
      (Supplemental Figure S1, B and C), and the expression of TMEM30A in kidney and intestine was similar between littermate controls and Tmem30a LKO mice (Supplemental Figure S1D). Strong yellow pigmentation was observed in the sera from Tmem30a LKO mice compared with littermate controls at 2 months of age (Figure 1A). Serum total bile acid (TBA) levels (168.8 ± 66.6 μmol/L; n = 10) were approximately 20-fold higher in Tmem30a LKO mice than levels in the littermate controls (7.4 ± 1.6 μmol/L; n = 11) (Figure 1B). Serum total bilirubin (TBIL) levels (20.1 ± 6.7 μmol/L; n = 12) were approximately 15-fold higher in Tmem30a LKO mice than levels in littermate controls (1.4 ± 0.2 μmol/L; n = 12) (Figure 1C). TMEM30A deficiency primarily resulted in a dramatic increase in direct bilirubin (DBIL) levels, whereas indirect bilirubin (IBIL) levels were slightly increased (Figure 1C). Serum γ-glutamyl transferase levels were comparable between the two groups (Figure 1D). Next, TBA, TBIL, and DBIL levels were examined at various ages, and it was found that TBA (217.2 ± 53.1 μmol/L; n = 14) and TBIL (23.7 ± 6.3 μmol/L; n = 12) levels peaked at 4 months of age (Figure 1, E–G). According to the assay used to identify and quantify BS species, serum unconjugated and conjugated bile acid levels were dramatically increased (Figure 2A). Only small changes were detected in the hepatic and biliary BS species (Figure 2, B and C). Thus, hepatic TMEM30A deficiency induced intrahepatic cholestasis in mice.
      Figure thumbnail gr1
      Figure 1Hyperbilirubinemia and hypercholanemia in Tmem30a liver-specific knockout (LKO) mice. A: Tmem30a LKO mice showed strong yellow pigmentation in their sera. B: Serum total bile acid (TBA) levels were dramatically elevated in Tmem30a LKO mice compared with the littermate controls. C: Serum total bilirubin (TBIL), direct bilirubin (DBIL), and indirect bilirubin (IBIL) levels were dramatically increased in Tmem30a LKO mice compared with their littermate controls. D: Serum γ-glutamyl transferase (GGT) levels were comparable between the two groups. E–G: Serum TBA (217.2 ± 53.1 μmol/L) (E), DBIL (18.7 ± 5.5 μmol/L) (F), and TBIL (23.7 ± 6.3 μmol/L) (G) levels peaked at 4 months of age, and their increased levels decreased progressively with age in Tmem30a LKO mice. All mice were at 2 months of age except for special indication. Data are expressed as means ± SEM. n = 10 Tmem30a LKO mice (B); n = 11 littermate control mice (B and C); n = 12 Tmem30a LKO mice (C); n = 6 to 8 mice in both groups (D); n = 14 TBA samples (E); n = 13 samples (F), n = 12 samples (G). P < 0.05, ∗∗P < 0.01 versus WT (t-test). WT, wild-type.
      Figure thumbnail gr2
      Figure 2Identification and quantification of bile salt (BS) species in wild-type (WT) and Tmem30a liver-specific knockout (LKO) mice. A: The plasma levels of unconjugated (MCA, CA, DCA, UDCA, CDCA) and conjugated (TMCA, TCA, TCDCA, TDCA, TLCA, GCA) BS species were significantly increased in Tmem30a LKO mice. B: The levels of MCA, cholic acid (CA), and TCA in Tmem30a-deficient livers were slightly increased. C: The levels of muricholate (MCA) and TUDCA in bile were slightly changed in Tmem30a LKO mice. All samples were from mice aged 2 to 3 months. Plasma levels are expressed in μmol/L, biliary levels in nmol/mL, hepatic levels in nmol/g liver, respectively. Error bars represent SEM. n ≥ 6 mice per group (AC). P < 0.05, ∗∗P < 0.01 (t-test). CDCA, chenodeoxycholate; DCA, deoxycholate; G, glycol; LCA, lithocholic acid; T, tauro; UDCA, ursodeoxycholate.

      TMEM30A Deficiency Induces Liver Damage and Impairs Liver Function

      Next, the liver histologic findings and serum biochemistry variables were examined because intrahepatic cholestasis often induces liver damage and impairs liver function. Liver-to-body weight ratios of Tmem30a LKO mice were comparable with the littermate controls (Supplemental Figure S2A). H&E staining and the F4/80 immunohistochemistry analysis revealed significant lymphocyte and macrophage infiltration in Tmem30a-deficient livers (Supplemental Figure S2, B and C). Serum levels of inflammatory cytokines, such as IL-6, IL-18, IL-10, interferon-γ, and tumor necrosis factor-α were consistently increased in Tmem30a LKO mice (Supplemental Figure S2D). Masson blue staining showed that Tmem30a LKO mice exhibited liver fibrosis, which was further confirmed by the increased levels of collagen type 1 α 1 chain (Col1a1), Col3a1, and transforming growth factor-β mRNAs (Supplemental Figure S2E). Immunohistochemistry and real-time PCR of cytokeratin-19 indicated significantly ductular proliferation in Tmem30a LKO mice (Supplemental Figure S2F). Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels were elevated in Tmem30a LKO mice at various ages (Supplemental Figure S3, A–C). However, serum total protein and albumin were slightly reduced (Supplemental Figure S3, D and E). Thus, hepatic deletion of TMEM30A resulted in inflammatory infiltration, ductular proliferation, and liver fibrosis.

      Decreased Expression of ATP11C, ATP8B1, BS Transporters, and Nuclear Receptors in Tmem30a LKO Mice

      Because TMEM30A is required for the localization and functions of P4-ATPases, the expression and localization of ATP11C and ATP8B1 were examined in Tmem30a deficient livers. Although the mRNA levels of ATP8B1 and ATP11C were not changed (Figure 3A), their protein levels were reduced in Tmem30a-deficient livers (Figure 3B). Considering that the protein levels of BS uptake transporters OATPs and NTCP are decreased in Atp11c-deficient mice,
      • Matsuzaka Y.
      • Hayashi H.
      • Kusuhara H.
      Impaired hepatic uptake by organic anion-transporting polypeptides is associated with hyperbilirubinemia and hypercholanemia in Atp11c mutant mice.
      • de Waart D.R.
      • Naik J.
      • Utsunomiya K.S.
      • Duijst S.
      • Ho-Mok K.
      • Bolier A.R.
      • Hiralall J.
      • Bull L.N.
      • Bosma P.J.
      • Oude Elferink R.P.
      • Paulusma C.C.
      ATP11C targets basolateral bile salt transporter proteins in mouse central hepatocytes.
      the expression of BS transporters was analyzed in Tmem30a-deficient livers. The mRNA levels of OATP1B2, NTCP, and BSEP were significantly reduced in Tmem30a-deficient livers (Figure 3A). The protein levels of BS uptake transporters (OATP1A4, OATP1B2, and NTCP) and canalicular efflux proteins (BSEP and MRP2) were significantly reduced in Tmem30a-deficient livers (Figure 3B). These results suggested that the reduced expression of BS transporters was partially mediated by a defect in transcriptional regulation.
      Figure thumbnail gr3
      Figure 3Analysis of mRNA and protein levels of ATP8B1, ATP11C, bile salt (BS) transporters and related nuclear receptors. A: Mean relative mRNA levels of BS transporters (Oatp1b2, Ntcp, Bsep), Atp8b1, Atp11c, related nuclear receptors (Fxrα, Shp, Hnf4α, Lrh, Rxrα, Lxrα, Car, Pxr), and enzymes (Cyp8b1, Cyp27a1) in livers were reduced in Tmem30a LKO mice compared to the littermate controls (relative mRNA levels were normalized to β-actin). B: The protein levels of ATP8B1, ATP11C, and BS transporters in Tmem30a-deficient livers were significantly reduced compared with the littermate controls. C: The protein levels of related nuclear receptors (FXRα, RXRα, HNF4α, LRH-1, SHP) were also significantly reduced in Tmem30a-deficient livers. D: Phosphorylation of protein kinase C (PKC)ζ was also dramatically reduced in Tmem30a liver-specific knockout (LKO) mice. All samples were from mice aged 2 to 3 months. Protein levels were quantified by densitometry and normalized to β-actin. Error bars represent SEM. n ≥ 4 samples (A); n = 5 WT and LKO (B and C); n ≥ 4 samples per group (D). P < 0.05, ∗∗P < 0.01 (t-test). BSEP, bile salt export pump; FXR, farnesoid X receptor; HNF, hepatocyte nuclear factor; LRH, liver receptor homolog; MRP, multidrug resistance-associated protein; NTCP, Na+-taurocholate cotransporting polypeptide; OATP, organic anion transporting polypeptide; RXR, retinoid X receptor; SHP, small heterodimer partner; WT, wild-type.
      The expression of BS transporters and synthetic enzymes is transcriptionally regulated by hepatic nuclear receptors. Real-time PCR analysis demonstrated that the mRNA levels of nuclear receptors, including Fxrα, Shp, Hnf4α, Lrh-1, Rxrα, liver X receptor α, constitutive androstane receptor, pregnane X receptor, and the enzymes cytochrome P450 family 8 subfamily B member 1 and cytochrome P450 family 27 subfamily A member 1 were decreased (Figure 3A). Moreover, the protein levels of FXRα, RXRα, LRH-1, HNF4α, and SHP were also significantly reduced in Tmem30a-deficient livers (Figure 3C). ATP8B1 has been shown to regulate the FXRα pathway by down-regulating the phosphorylation of protein kinase C (PKC)ζ.
      • Alvarez L.
      • Jara P.
      • Sanchez-Sabate E.
      • Hierro L.
      • Larrauri J.
      • Diaz M.C.
      • Camarena C.
      • De la Vega A.
      • Frauca E.
      • Lopez-Collazo E.
      • Lapunzina P.
      Reduced hepatic expression of farnesoid X receptor in hereditary cholestasis associated to mutation in ATP8B1.
      • Frankenberg T.
      • Miloh T.
      • Chen F.Y.
      • Ananthanarayanan M.
      • Sun A.Q.
      • Balasubramaniyan N.
      • Arias I.
      • Setchell K.D.
      • Suchy F.J.
      • Shneider B.L.
      The membrane protein ATPase class I type 8B member 1 signals through protein kinase C zeta to activate the farnesoid X receptor.
      • Chen F.
      • Ananthanarayanan M.
      • Emre S.
      • Neimark E.
      • Bull L.N.
      • Knisely A.S.
      • Strautnieks S.S.
      • Thompson R.J.
      • Magid M.S.
      • Gordon R.
      • Balasubramanian N.
      • Suchy F.J.
      • Shneider B.L.
      Progressive familial intrahepatic cholestasis, type 1, is associated with decreased farnesoid X receptor activity.
      • Koh S.
      • Takada T.
      • Kukuu I.
      • Suzuki H.
      FIC1-mediated stimulation of FXR activity is decreased with PFIC1 mutations in HepG2 cells.
      • Chen F.
      • Ellis E.
      • Strom S.C.
      • Shneider B.L.
      ATPase Class I Type 8B Member 1 and protein kinase C zeta induce the expression of the canalicular bile salt export pump in human hepatocytes.
      Consistent with this result, it was observed that a significant reduction occurred in the levels of phosphorylated PKCζ in Tmem30a-deficient livers (Figure 3D). From these findings, the decreased expression of ATP11C, ATP8B1, BS transporters, and hepatic nuclear receptors may be responsible for hyperbilirubinemia and hypercholanemia in Tmem30a LKO mice.

      Mislocalization of ATP11C, ATP8B1, and BS Transporters in Tmem30a LKO Mice

      The proper membrane localization of ATP8B1, ATP11C, and BS transporters is important for their functions. The membrane proteins were extracted from the livers, and Western blot analysis showed a dramatic reduction in the levels of ATP8B1, ATP11C, OATP1A4, OATP1B2, NTCP, BSEP, and MRP2 in Tmem30a-deficient livers (Figure 4A). Immunofluorescence images confirmed that the localization of these proteins was also significantly reduced on the membrane in Tmem30a-deficient hepatocytes (Figure 4B and Supplemental Figure S4). Confocal images demonstrated that the localization of NTCP, BSEP, and MRP2 was mainly intracellular but not on the membrane (Figure 4, C–E). Interestingly, TMEM30A deficiency resulted in a significant reduction of NTCP levels in both portal and central hepatocyte membrane (Supplemental Figure S4), whereas Atp11c-deficient mice show only a reduction of NTCP levels in the central area.
      • de Waart D.R.
      • Naik J.
      • Utsunomiya K.S.
      • Duijst S.
      • Ho-Mok K.
      • Bolier A.R.
      • Hiralall J.
      • Bull L.N.
      • Bosma P.J.
      • Oude Elferink R.P.
      • Paulusma C.C.
      ATP11C targets basolateral bile salt transporter proteins in mouse central hepatocytes.
      It is plausible that loss of TMEM30A impaired the localization of ATP8B1, ATP11C, and BS transporters in hepatocytes.
      Figure thumbnail gr4
      Figure 4The localization of ATP8B1, ATP11C, and bile salt (BS) transporters was altered in the Tmem30a liver-specific knockout (LKO) mice. A: Western blot analysis of membrane proteins extracted from the livers of the Tmem30a-deficient mice and littermate controls. B: Immunofluorescence images showing the reduced expression and localization of ATP8B1 and ATP11C in Tmem30a-deficient livers. C–E: Confocal images demonstrated that Na+-taurocholate cotransport protein (NTCP) (C), multidrug resistance-associated protein (MRP)2 (D), and BS export pump (BSEP) (E) in Tmem30a LKO mice were mainly localized intracellularly but not on the membrane compared with the littermate controls. White arrows indicate the membrane localization of NTCP, MRP2, and BSEP. All samples were from mice aged 2 to 3 months. Green: ATP8B1 antibody; red: primary antibodies as indicated; blue: DAPI. n ≥ 3 samples per group (C–E). P < 0.05, ∗∗P < 0.01 (t-test). Scale bars: 100 μm (B–E). OATP, organic anion-transporting polypeptide; WT, wild-type.
      From the reduced levels of BSEP and MRP2, it was speculated that bile formation was defective in Tmem30a LKO mice. During BS depletion, the biliary outputs of BSs, cholesterol, and phospholipids were comparable between the two groups (Figure 5, A–C ). On infusion of taurocholate, the biliary outputs of BSs, cholesterol, and phospholipids were decreased in Tmem30a LKO mice (Figure 5, A–C). The biliary outputs of glutathione and ALP were reduced during bile depletion and taurocholate infusion in Tmem30a LKO mice (Figure 5, D and E). Thus, Tmem30a LKO mice exhibited defects in bile formation.
      Figure thumbnail gr5
      Figure 5Bile formation was impaired in Tmem30a liver-specific knockout (LKO) mice. The endogenous bile salt (BS) pools were depleted from Tmem30a LKO mice and littermate controls for 90 minutes. Taurocholate (TC) was infused through the jugular vein, and the infusion rates increased in a stepwise manner every 30 minutes at 400 nmol/min per 100 g for each step (400, 800, 1200, 1600 nmol/min per 100 g). A–C: No difference was found in the biliary outputs of BS (A), cholesterol (B), and phospholipids (C) between Tmem30a LKO mice and littermate controls during BS depletion. However, the biliary outputs of BS, cholesterol, and phospholipids were significantly decreased in Tmem30a LKO mice with the increased infusion rates. D and E: The biliary outputs of glutathione (D) and alkaline phosphatase (ALP) (E) were significantly reduced in Tmem30a LKO mice during both bile depletion and TC infusion. All male mice were tested at 2 to 3 months age. Error bars represent SEM. n ≥ 3 male mice per group (D and E). P < 0.05 (one-way analysis of variance analysis). WT, wild-type.

      Normal Intestinal BS-Related Proteins and Other Organ Function

      Enterohepatic circulation is also important in BS homeostasis. After being released in the gut, BSs are mainly reabsorbed in the terminal ileum by apical sodium-dependent BS transporters (ASBT) and other related proteins. Tmem30a deficiency did not affect the mRNA levels of Abst, fibroblast growth factor-15, Fxrα, and Shp in the intestine (Supplemental Figure S5A), and the protein levels of ABST, ATP8B1, and FXRα were also no different between the two groups (Supplemental Figure S5B). The plasma creatinine, blood urea nitrogen, and uric acid levels in Tmem30a LKO mice were comparable with the littermate controls at various ages (Supplemental Figure S5, C–E). H&E staining of heart, kidney, lung, spleen, muscle, pancreas, and intestine showed no significant differences at 12 months of age between littermate controls and Tmem30a LKO mice (Supplemental Figures S6–S8). Thus, the intestinal BS-related proteins and functions of other organs were not affected in Tmem30a LKO mice.

      Enhanced Proteasomal Degradation of ATP11C, ATP8B1, and BS Transporters in Tmem30a LKO Mice

      TMEM30A-deficient cells exhibited defects in the ER exit,
      • Paulusma C.C.
      • Folmer D.E.
      • Ho-Mok K.S.
      • de Waart D.R.
      • Hilarius P.M.
      • Verhoeven A.J.
      • Oude Elferink R.P.
      ATP8B1 requires an accessory protein for endoplasmic reticulum exit and plasma membrane lipid flippase activity.
      • Bryde S.
      • Hennrich H.
      • Verhulst P.M.
      • Devaux P.F.
      • Lenoir G.
      • Holthuis J.C.
      CDC50 proteins are critical components of the human class-1 P4-ATPase transport machinery.
      • van der Velden L.M.
      • Wichers C.G.
      • van Breevoort A.E.
      • Coleman J.A.
      • Molday R.S.
      • Berger R.
      • Klomp L.W.
      • van de Graaf S.F.
      Heteromeric interactions required for abundance and subcellular localization of human CDC50 proteins and class 1 P4-ATPases.
      and it was speculated that this deficiency may lead to the accumulation of ATP8B1, ATP11C, and BS transporters in the ER and may induce proteasomal degradation. On proteasome inhibition with bortezomib, the protein levels of ATP8B1, ATP11C, OATP1B2, NTCP, BSEP, and MRP2 were partially restored in Tmem30a LKO mice (Figure 6, A and B). However, immunofluorescence staining indicated that the membrane localization of ATP8B1, ATP11C, and BS transporters was not restored. Instead, these proteins were co-localized with protein disulfide isomerase in the ER after the bortezomib treatment, suggesting that they were accumulated in the ER (Supplemental Figure S9). Interestingly, the protein levels of FXRα, RXRα, HNF4α, LRH-1, and SHP were also increased in Tmem30a-deficient livers on proteasome inhibition (Figure 6, C and D). These increased nuclear receptors may partially up-regulate the mRNA levels of BS transporters compared with dimethyl sulfoxide controls in the Tmem30a LKO mice group (Supplemental Figure S10). Therefore, the reduction of BS transporters in Tmem30a LKO mice was not only attributed to the proteasomal degradation induced by the impaired trafficking of ATP8B1, ATP11C, and BS transporters but also the decreased transcriptional activation.
      Figure thumbnail gr6
      Figure 6Protein levels of bile salt (BS) transporters and related nuclear receptors were partially restored in Tmem30a liver-specific knockout (LKO) mice after treatment with the proteasome inhibitor. All mice were sacrificed 6 and 12 hours after treatment with the proteasome inhibitor bortezomib (0.5 mg/kg). A and B: Compared with their dimethyl sulfoxide (DMSO) control groups, wild-type (WT) mice treated with bortezomib showed no difference in the protein levels of ATP8B1, ATP11C and BS transporters (A), whereas Tmem30a LKO mice treated with bortezomib showed dramatically increased levels of these proteins (B). C and D: Compared with their DMSO control groups, WT mice treated with bortezomib showed no difference in the protein levels of farnesoid X receptor (FXR)α, retinoid X receptor (RXR)α, hepatocyte nuclear factor (HNF4)α, liver receptor homolog (LRH)-1, and small heterodimer partner (SHP) (C), whereas Tmem30a LKO mice treated with bortezomib showed dramatically increased levels of these nuclear receptors (D). All mice () were tested at 2 to 3 months age. Protein levels were quantified by densitometry and normalized to β-actin. n ≥ 3 mice per group. P < 0.05, ∗∗P < 0.01 (t-test). BSEP, bile salt export pump; MRP, multidrug resistance-associated protein; NTCP, Na+-taurocholate cotransport protein; OATP, organic anion transporting polypeptide.

      Exogenous Bile Acids Exacerbated the Phenotypes in Tmem30a LKO Mice

      The mice were challenged with a 0.5% CA-supplemented diet to further substantiate these findings. Jaundice was only observed in Tmem30a LKO mice (Supplemental Figure S11A). The plasma levels of TBA, TBIL, DBIL, ALT, AST, and ALP were dramatically increased in Tmem30a LKO mice after 9 days on the CA-supplemented diet (Figure 7, A–C ). The serum total protein and albumin levels were obviously reduced in Tmem30a LKO mice (Supplemental Figure S11B). No obvious difference was observed in serum creatinine, blood urea nitrogen, and uric acid levels (Supplemental Figure S11C). Male Tmem30a LKO mice lost weight more rapidly than the littermate controls on CA-supplemented diet for 2 weeks (Figure 7D). Moreover, more than one-half of the male Tmem30a LKO mice died within 2 weeks, whereas all male littermate controls survived (Figure 7E). Morbid Tmem30a LKO mice, which showed dramatic weight loss, back deformations, and slow movements, were sacrificed for further analysis. Liver-to-body weight ratios of the morbid mice were dramatically reduced compared with the mice with normal feeding (Supplemental Figures S2A and S11D). H&E staining showed more multinucleate hepatocytes and smaller hepatocyte sizes in morbid Tmem30a LKO mice (Figure 7F). The increased levels of Col1a1, Col3a1, and transforming growth factor-β mRNAs indicated severe liver fibrosis, and Masson blue staining confirmed this result in Tmem30a LKO mice (Supplemental Figure S11, E and F). H&E staining showed that there were no obvious differences in other organs, such as heart, kidney, brain, intestine, and spleen (Supplemental Figures S12 and S13). Interestingly, serum TBA, TBIL, and DBIL levels and serum ALT, AST, and ALP levels were also increased in surviving Tmem30a LKO mice (Supplemental Figure S14, A and B) but not as dramatically as morbid Tmem30a LKO mice (Figure 7, A–C). In addition, H&E staining did not reveal any more difference except for unclear cell boundaries in surviving Tmem30a LKO mice (Supplemental Figure S14C). In summary, the CA-supplemented diet exacerbated the phenotypes and led to more severe liver damage in Tmem30a LKO mice. However, the molecular mechanisms underlying this phenotype are not clear, and further investigation is warranted.
      Figure thumbnail gr7
      Figure 7Exacerbation of the Tmem30a-deficient phenotypes on cholic acid (CA)-supplemented diet. Male Tmem30a-deficient mice and male littermate controls (2 to 3 months) were fed a 0.5% CA-supplemented diet. A–C: Serum total bile acid (TBA) levels (A); serum total bilirubin (TBIL), direct bilirubin (DBIL), and indirect bilirubin (IBIL) levels (B); and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels (C) were dramatically increased in Tmem30a liver-specific knockout (LKO) mice compared with the littermate controls after 9 days on CA-supplemented diet. D: The body weight ratios of male Tmem30a LKO mice were dramatically decreased compared with male littermate controls after 3 weeks on CA-supplemented diet. The body weight ratios were normalized to the weight on day 0 (weight before receiving CA). The mice were weighed at 3-day intervals. E: Death of male Tmem30a LKO mice and male littermate controls after 3 weeks on CA-supplemented diet. Mice were monitored every day for the survival curve. F: Hematoxylin and eosin (H&E) staining showed the presence of more multinucleate hepatocytes and smaller hepatocyte sizes in morbid male Tmem30a LKO mice but not the littermate controls on CA-supplemented diet. Error bars represent SEM. n = 15 male Tmem30a-deficient mice; n = 10 male littermate control mice. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (t-test). Scale bars = 100 μm. Original magnification, ×200.

      Discussion

      As a β-subunit, TMEM30A is essential for the function of both ATP8B1 and ATP11C.
      • Paulusma C.C.
      • Folmer D.E.
      • Ho-Mok K.S.
      • de Waart D.R.
      • Hilarius P.M.
      • Verhoeven A.J.
      • Oude Elferink R.P.
      ATP8B1 requires an accessory protein for endoplasmic reticulum exit and plasma membrane lipid flippase activity.
      • Takatsu H.
      • Baba K.
      • Shima T.
      • Umino H.
      • Kato U.
      • Umeda M.
      • Nakayama K.
      • Shin H.W.
      ATP9B, a P4-ATPase (a putative aminophospholipid translocase), localizes to the trans-Golgi network in a CDC50 protein-independent manner.
      Atp8b1 mutant mice exhibited mild abnormalities, and the cholestatic phenotypes were only revealed in Atp8b1 mutant mice challenged with a CA-supplemented diet.
      • Pawlikowska L.
      • Groen A.
      • Eppens E.F.
      • Kunne C.
      • Ottenhoff R.
      • Looije N.
      • Knisely A.S.
      • Killeen N.P.
      • Bull L.N.
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      Atp8b1 deficiency in mice reduces resistance of the canalicular membrane to hydrophobic bile salts and impairs bile salt transport.
      In contrast, Atp11c-deficient mice exhibited conjugated hyperbilirubinemia and unconjugated hypercholanemia. Similar to the Atp11c-deficient mice, Tmem30a-deficient mice showed a dramatic reduction in the levels of OATP1A4 and OATP1B2, which may be responsible for the increased levels of conjugated bilirubin and unconjugated bile acid. Consistently, these phenotypes were also observed in Oatp1a/1b−/− mice.
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      Organic anion transporting polypeptide 1a/1b-knockout mice provide insights into hepatic handling of bilirubin, bile acids, and drugs.
      Furthermore, the distribution of BS species (approximately 80% of the total plasma BS species were unconjugated) in Tmem30a LKO mice was also comparable with the distribution observed in Atp11c-deficient mice. Therefore, the two mouse models have several similar phenotypes in intrahepatic cholestasis. Nevertheless, Tmem30a-deficient mice exhibited more severe defects than Atp11c-deficient mice, as indicated by the 15-fold versus sixfold increase in plasma bilirubin levels as well as the 20-fold versus 10-fold increase in plasma total bile acid levels.
      There are several explanations for the stronger phenotypes observed in Tmem30a LKO mice. First, in addition to the defect in the re-uptake of conjugated bilirubin due to reduced OATP levels,
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      • Keppler D.
      The roles of MRP2, MRP3, OATP1B1, and OATP1B3 in conjugated hyperbilirubinemia.
      Tmem30a LKO mice but not Atp11c-deficient mice showed an extra defect in the expression and localization of MRP2. MRP2 primarily secretes conjugated bilirubin into the bile, and its mutations result in Dubin-Johnson syndrome, which is characterized by conjugated hyperbilirubinemia.
      • Keppler D.
      The roles of MRP2, MRP3, OATP1B1, and OATP1B3 in conjugated hyperbilirubinemia.
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      Multidrug resistance proteins (MRPs/ABCCs) in cancer chemotherapy and genetic diseases.
      Therefore, the reduced MRP2 levels could explain the higher conjugated bilirubin levels in Tmem30a LKO mice. Second, the reduced expression and localization of BSEP were only observed in Tmem30a LKO mice but not in Atp11c-deficient mice, possibly explaining higher total bile acid levels in Tmem30a LKO mice. Third, the localization of NCTP in the portal and central hepatocyte membranes was reduced in Tmem30a LKO mice, whereas Atp11c-deficient mice only lacked NTCP in the central hepatocyte membrane.
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      ATP11C targets basolateral bile salt transporter proteins in mouse central hepatocytes.
      This outcome may contribute to the increased tauro- and glyco-conjugated BS levels in Tmem30a LKO mice. Finally, biliary outputs of cholesterol, ALP, and glutathione were consistent with BSs output, which was decreased in Tmem30a LKO mice, and unchanged in Atp11c-mutant mice, but increased in Atp8b1-deficient mice.
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      ATP11C targets basolateral bile salt transporter proteins in mouse central hepatocytes.
      • Paulusma C.C.
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      Atp8b1 deficiency in mice reduces resistance of the canalicular membrane to hydrophobic bile salts and impairs bile salt transport.
      The reduction of BA synthetic enzymes CYP8B1 and CYP27A1 might be involved in the decrease in BSs output. Both BSEP and MRP2 functions are important for bile formation
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      Contribution of mrp2 in alterations of canalicular bile formation by the endothelin antagonist bosentan.
      ; therefore, the defects in bile formation in Tmem30a KO mice could be due to the reduced expression of BA synthetic enzymes and transporter proteins.
      BS transporters are transcriptionally regulated by a complex interacting network of nuclear receptors.
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      In Tmem30a LKO mice, the mRNA and protein levels of these nuclear receptors, including FXRα, were markedly reduced. FXRα is a critical nuclear receptor for bile acids and regulates the expression of basolateral BS uptake and canalicular efflux transporters, including NTCP, OATPs, BSEP, and MRP2. In addition, FXRα also regulates the activity and/or expression of other nuclear receptors, which subsequently regulate the expression of BS transporters.
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      Role of nuclear receptors for bile acid metabolism, bile secretion, cholestasis, and gallstone disease.
      On proteasome inhibition with bortezomib, the protein levels of hepatic nuclear receptors and BS transporters were partially restored in Tmem30a LKO mice, indicating that the reduction of these proteins was mediated by proteasomal degradation. The mRNA levels of BS transporters were also partially restored, suggesting that the reduced hepatic nuclear receptors were partly responsible for the decreased expression of BS transporters. Nevertheless, serum elevated BSs could also influence the expression of nuclear receptors, BS transporters and synthetic enzymes as a compensatory mechanism. Mutations in ATP8B1 have been suggested to suppress the expression and activity of FXRα by decreasing PKC phosphorylation.
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      ATPase Class I Type 8B Member 1 and protein kinase C zeta induce the expression of the canalicular bile salt export pump in human hepatocytes.
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      Phospholipase D2 mediates signaling by ATPase class I type 8B membrane 1.
      Consistent with this finding, Tmem30a-deficient livers exhibited a marked decrease in the levels of phosphorylated PKC. Thus, the impaired expression and localization of ATP8B1 may be responsible for the decreased activity of the FXRα pathway in Tmem30a-deficient livers. Taken together, the reduced expression of BS transporters was not only attributed to the proteasomal degradation but also to the reduced expression of the nuclear receptors.
      TMEM30A deficiency impaired the ER exit and catalytic phospholipid flippase activity of the P4 ATPase, which maintains an asymmetric distribution of phospholipids on the membrane to ensure membrane fluidity and constitutive signal transduction.
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      ATP8B1 requires an accessory protein for endoplasmic reticulum exit and plasma membrane lipid flippase activity.
      • Kato U.
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      • Begthel H.
      • Knisely A.S.
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      • van de Graaf S.F.
      • Paulusma C.C.
      • Bos J.L.
      ATP8B1-mediated spatial organization of Cdc42 signaling maintains singularity during enterocyte polarization.
      In addition, the phospholipid flippase complex serves as a molecular scaffold to recruit signaling molecules and to provide a docking platform for proteins of the vesicle-generating machinery.
      • de Waart D.R.
      • Naik J.
      • Utsunomiya K.S.
      • Duijst S.
      • Ho-Mok K.
      • Bolier A.R.
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      • Bull L.N.
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      • Oude Elferink R.P.
      • Paulusma C.C.
      ATP11C targets basolateral bile salt transporter proteins in mouse central hepatocytes.
      • Kato U.
      • Inadome H.
      • Yamamoto M.
      • Emoto K.
      • Kobayashi T.
      • Umeda M.
      Role for phospholipid flippase complex of ATP8A1 and CDC50A proteins in cell migration.
      It is plausible that Tmem30a deficiency blocked vesicle assembly and led to the accumulation of cargo proteins. Consequently, cargo proteins, including OATPs, NTCP, MRP2, BSEP, ATP8B1, and ATP11C, were targeted for proteasomal degradation. In line with this, proteasome inhibition by bortezomib led to a partial restoration of the levels of ATP8B1, ATP11C, and BS transporters. Confocal images demonstrated that the restored ATP8B1, ATP11C, and BS transporters were stained with protein disulfide isomerase in the ER, suggesting that the defective trafficking of vesicles containing these proteins was induced by TMEM30A deficiency. In addition, the phosphorylation of PKCζ has been implicated in the transportation of NTCP vesicles and the canalicular localization of BSEP and MRP2
      • McConkey M.
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      Cross-talk between protein kinases Czeta and B in cyclic AMP-mediated sodium taurocholate co-transporting polypeptide translocation in hepatocytes.
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      • Nath S.
      • Anwer M.S.
      • Wolkoff A.W.
      • Murray J.W.
      PKCzeta is required for microtubule-based motility of vesicles containing the ntcp transporter.
      • Stross C.
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      Expression and localization of atypical PKC isoforms in liver parenchymal cells.
      ; therefore, the insufficient phosphorylation of PKCζ in Tmem30a-deficient mice might also contribute to the impaired localization of BS transporters. From these results, the impaired expression and localization of P4-ATPases induced by Tmem30a deficiency may affect the intracellular transport of vesicles containing BS transporters.
      The present Tmem30a LKO mouse model displayed several interesting pathologic features that have not been reported in the previous Atp8b1- and Atp11c-deficient mice. Histologic and biochemical analyses showed dramatically increased inflammatory infiltration and liver damage, which may be attributed to more severe intrahepatic cholestasis in Tmem30a LKO mice. Another possibility is that the exposure of phosphatidylserine on the surface of Tmem30a-deficient hepatocytes elicited an eat me signal for the macrophages.
      • Segawa K.
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      • Brummelkamp T.R.
      • Matsuda F.
      • Nagata S.
      Caspase-mediated cleavage of phospholipid flippase for apoptotic phosphatidylserine exposure.
      The impaired bile formation in Tmem30a-deficient mice also caused the accumulation of BSs in hepatocytes, which then exacerbated the liver damage. With the CA-supplemented diet, the reduction of OATPs and NTCP could reduce the uptake of cholic acid by hepatocytes, but hydrophobic cholic acid could still enter into the hepatocytes by passive diffusion.
      • Hofmann M.
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      • Henzel K.
      • Zimmer G.
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      Small and large unilamellar vesicle membranes as model system for bile acid diffusion in hepatocytes.
      • Fickert P.
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      • Fuchsbichler A.
      • Stumptner C.
      • Pojer C.
      • Zenz R.
      • Lammert F.
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      • Meier P.J.
      • Zatloukal K.
      • Denk H.
      • Trauner M.
      Effects of ursodeoxycholic and cholic acid feeding on hepatocellular transporter expression in mouse liver.
      In contrast to Atp11c-deficient mice, only 60% of Tmem30a-deficient mice died after consuming the CA-supplemented diet. It is speculated that these discrepancies may be due to the differences in gene deletion strategies. In contrast to the whole-body deletion of ATP11C, our mouse model was a LKO. Other organ and tissue in Tmem30a-deficient mice may compensate for the CA-challenge and make a few survivals. Except for intrahepatic cholestasis, Atp11c mutations in mice have been reported to cause macrophage infiltration, B-cell deficiency, dystocia, hepatocellular carcinoma,
      • Siggs O.M.
      • Schnabl B.
      • Webb B.
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      X-linked cholestasis in mouse due to mutations of the P4-ATPase ATP11C.
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      • Whittle B.
      • Andrews D.T.
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      • Broer S.
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      • Enders A.
      ATP11C is critical for the internalization of phosphatidylserine and differentiation of B lymphocytes.
      • Siggs O.M.
      • Arnold C.N.
      • Huber C.
      • Pirie E.
      • Xia Y.
      • Lin P.
      • Nemazee D.
      • Beutler B.
      The P4-type ATPase ATP11C is essential for B lymphopoiesis in adult bone marrow.
      • Segawa K.
      • Kurata S.
      • Yanagihashi Y.
      • Brummelkamp T.R.
      • Matsuda F.
      • Nagata S.
      Caspase-mediated cleavage of phospholipid flippase for apoptotic phosphatidylserine exposure.
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      ATP11C facilitates phospholipid translocation across the plasma membrane of all leukocytes.
      which could promote the deterioration of overall viability, particularly after severe challenges.

      Conclusions

      This study provides a clinically relevant mouse model with phenotypes of intrahepatic cholestasis. Hepatocyte-specific Tmem30a KO mice present many manifestations of human intrahepatic cholestasis. TMEM30A may be a novel genetic determinant of liver disorders characterized by hyperbilirubinemia and hypercholanemia. Mutations in ATP8B1, BSEP, and FXRA have been shown to cause hereditary intrahepatic cholestasis in humans, and screening for TMEM30A mutations in patients with hereditary intrahepatic cholestasis could yield interesting findings. Taken together, this study demonstrated a critical role of TMEM30A in hepatic BS homeostasis and provided new insights into the molecular mechanisms of intrahepatic cholestasis.

      Acknowledgments

      All authors contributed to study conception and design as well as writing the manuscript; L.L., Ling.Z., LinZ., F.Y., Xu.Z., Z.L., Y.Y., H.L., L.F., and Z.W. acquired data; L.L., Ling.Z., H.J., Z.J., and Xi.Z. analyzed and interpreted data; H.C., S.Y., and L.W. provided surgical technical support; H.J., Z.J., Xi.Z., and Ling.Z. obtained funding and supervised the study.

      Supplemental Data

      • Supplemental Figure S1

        Generation of liver-specific knockout (LKO) mice. A: The original Tmem30a allele for this model is of type KO first by using the FRT-Flp strategy and also is conditional potential by using flox-cre system. Mice carrying the two floxed alleles on the third exon of the Tmem30a gene were generated by conventional homologous recombination in embryonic stem (ES) cells and blastocyst injection of correctly targeted ES cell clones. Tmem30afrt/+ flox/+ mice were first interbred with Flp mice. The resulting Tmem30aflox/+ mice were interbred with Alb-cre mice to generate Tmem30aflox/flox-Alb-cre mice. Tmem30aflox/flox mice without Cre expression were used as littermate wild-type (WT) controls (genetic background: C57BL/6J). B: Tmem30a expression was abolished in liver hepatocytes, in which the red fluorescent protein variant TdTomato was expressed, indicating Cre enzyme was expressed. C: The expression of TMEM30A in Tmem30a-deficient livers was almost absence, as revealed by Western blot analysis. D: No obvious difference was seen in the expression of TMEM30A in the kidney and intestine between littermate control and Tmem30a LKO mice. Scale bars = 50 μm.

      Figure thumbnail figs1
      Supplemental Figure S2Severe inflammatory infiltration and liver damage in Tmem30a-deficient livers. A: The liver-to-body weight ratios of 2-month-old Tmem30a liver-specific knockout (LKO) mice and littermate controls were similar. B: As shown in the F4/80 immunohistochemistry analysis, macrophage infiltration was increased (red arrow) in the livers of the 2-month-old Tmem30a LKO mice. C: Hematoxylin and eosin (H&E) staining show obvious lymphocyte infiltration at 9 months of age. D: The levels of several inflammatory factors, including IL-6, IL-18, IL-10, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, were increased in Tmem30a LKO mice. E: Masson blue staining was more apparent (black arrow), and mRNA levels of collagen type 1 (Col1)a1, collagen type 3 (Col3)a1, and transforming growth factor (Tgf)-β were increased in Tmem30a-deficient livers, F: Immunohistochemistry and mRNA levels of cytokine-19 (CK-19) indicated significant bile-duct proliferation in Tmem30a-deficient livers (black arrow). All mice were tested at 2 to 3 months age. Data are expressed as means ± SEM. n ≥ 3 mice per group. P < 0.05 (t-test). Scale bars: 200 μm (B, D, and E); 500 μm (F). Original magnification: ×200 (B and C); ×100 (C). GM-CSF, granulocyte macrophage colony-stimulating factor; GRO, growth-related oncogene; MCP, macrophage chemotactic protein; MIP, macrophage inflammatory protein; RANTES, regulated on activation normal T cell expressed and secreted; VEGF, vascular endothelial growth factor.
      Figure thumbnail figs2
      Supplemental Figure S3Biochemical analysis of liver function in Tmem30a liver-specific knockout (LKO) mice and their littermate controls at various ages. A–C: Plasma levels of alanine aminotransferase (ALT) (A), aspartate aminotransferase (AST) (B), and alkaline phosphatase (ALP) (C) in Tmem30a LKO mice were increased compared with littermate controls at various ages. D and E: Serum total protein (TP) (D) and albumin (ALB) (E) levels were similar in Tmem30a LKO mice and littermate controls. Data are expressed as means ± SEM. n ≥ 6 mice (A–C); n ≥ 5 mice (D and E). P < 0.05 (t-test). WT, wild-type.
      Figure thumbnail figs3
      Supplemental Figure S4Localization of bile acid (BA) transporter proteins were reduced on the membrane. A: The expression and localization of organic anion transporting polypeptide (OATP)1A4, OATP1B2, and Na+-taurocholate cotransport protein (NTCP) were decreased in Tmem30a-deficient livers. White arrow indicates portal area; red arrow, central area. B: The expression and localization of bile salt export pump (BSEP) and multidrug resistance-associated protein (MRP)2 were also impaired in Tmem30a liver-specific knockout (LKO) mice. All mice were tested at 2 to 3 months age. Red: primary antibodies as indicated; blue: DAPI. n ≥ 4 mice per group. Scale bars: 100 μm (A and B). WT, wild-type.
      Figure thumbnail figs4
      Supplemental Figure S5No significant changes in expression levels of bile-salt (BS)-related proteins and markers of kidney function in the Tmem30a liver-specific knockout (LKO) mice. A and B: The mRNA (A) and protein (B) levels in the intestines of Tmem30a LKO mice were similar to that of their littermate controls. C–E: Serum levels of creatinine (CREA) (C), blood urea nitrogen (BUN) (D), and uric acid (UA) (E) in Tmem30a LKO mice were similar to that of their littermate controls at various age. All mice were tested at the indicated age. Data are expressed as means ± SEM (t-test). n ≥ 4 mice. ABST, apical sodium-dependent bile salt transporters; FGF, fibroblast growth factor; FXR, farnesoid X receptor; MRP, multidrug resistance-associated protein; SHP, short heterodimer partner; WT, wild-type.
      Figure thumbnail figs5
      Supplemental Figure S6Hematoxylin and eosin (H&E) staining of heart and kidney in littermate control and Tmem30a liver-specific knockout (LKO) mice at 12 months of age. A: The hearts in littermate controls and Tmem30a LKO mice showed similar morphologic characteristics both in global scan (left) or in cardiocytes (right). B: The global scan of kidneys showed normal morphologic characteristics in littermate controls and Tmem30a LKO mice. The medulla and renal cortex also showed normal morphologic characteristics between the two groups. Scale bars: 100 μm (A); 200 μm (B). Original magnification, ×200. WT, wild-type.
      • Supplemental Figure S7

        Hematoxylin and eosin (H&E) staining of lung and spleen in littermate control and Tmem30a liver-specific knockout (LKO) mice at 12 months of age. A: H&E staining revealed similar morphologic characteristics in the lungs of littermate controls and Tmem30a LKO mice. B: The morphologic characteristics of spleens of littermate controls and Tmem30a LKO were similar. Scale bars: 100 μm (A and B). WT, wild-type.

      Figure thumbnail figs6
      Supplemental Figure S8Hematoxylin and eosin (H&E) staining of muscle, pancreas, and intestine in littermate control and Tmem30a liver-specific knockout (LKO) mice at 12 months of age. A: H&E staining revealed similar morphologic characteristics in the skeletal muscle cells of littermate controls and Tmem30a LKO mice. B: H&E staining revealed similar morphologic characteristics in the pancreas cells of littermate controls and Tmem30a LKO mice. C: No differences was found in the morphologic characteristics of intestinal villus and crypts of littermate controls and Tmem30a LKO mice. Scale bars: 200 μm (A and B); 500 μm (C and D). WT, wild-type.
      Figure thumbnail figs7
      Supplemental Figure S9ATP8B1, ATP11C, and bile salt (BS) transporters were co-localized with protein disulfide isomerase (PDI) in the endoplasmic reticulum (ER) on proteasome inhibition by bortezomib. All mice were sacrificed at 12 hours on the proteasome inhibition by bortezomib (0.5 mg/kg). Confocal microscopy images showing the co-localization of ATP8B1 (A), ATP11C (B), organic anion-transporting polypeptide (OATP)1B2 (C), bile salt export pump BSEP (D), and Na+-taurocholate cotransporting polypeptide (NTCP) (E) with PDI in ER in Tmem30a liver-specific knockout (LKO) mice. White arrows indicate the membrane-bound proteins. n ≥ 4 mice. Scale bars = 10 μm. NTCP, Na+-taurocholate cotransporting polypeptide; WT, wild-type.
      Figure thumbnail figs8
      Supplemental Figure S10Analysis of bile salt (BS) transporter mRNAs after treatment with the proteasome inhibitor bortezomib. The mRNA levels of BS transporters in Tmem30a liver-specific knockout (LKO) mice were partially restored compared with the dimethyl sulfoxide (DMSO) controls. Mice were sacrificed at 12 hours after proteasome inhibition by bortezomib (0.5 mg/kg). Data are expressed as means ± SEM. n ≥ 4 mice. P < 0.05 (t-test). Bsep, bile salt export pump; Mdr, multidrug resistance; Mrp, multidrug resistance-associated protein; Ntcp, Na+-taurocholate cotransporting polypeptide; Oatp, organic anion-transporting polypeptide; WT, wild-type.
      Figure thumbnail figs9
      Supplemental Figure S11Deterioration of the Tmem30a-deficient phenotype after feeding a cholic acid (CA)-supplement diet. A: Jaundice was observed in Tmem30a liver-specific knockout (LKO) mice but not their littermate controls. B: Serum total protein (TP) and albumin (ALB) levels were significantly reduced in Tmem30a LKO mice compared with their littermate controls after a 9-day CA-supplemented diet. C: The serum creatinine (CREA), blood urea nitrogen (BUN), and uric acid (UA) levels in the morbid Tmem30a LKO mice were comparable with the littermate controls after a 9-day CA-supplemented diet. D: The liver-to-body weight ratios of morbid male Tmem30a LKO mice were dramatically decreased compared with the littermate controls. E: The mRNAs levels of collagen type 1 α 1 chain (Col1a1), Col3a1 and transforming growth factor (Tgf)-β mRNAs were obviously increased in Tmem30a-deficient livers. F: Masson blue staining indicated severe liver fibrosis in Tmem30a-deficient livers. All male mice were tested at 2 to 3 months age. Data are expressed as means ± SEM. n ≥ 9 male mice. P < 0.05, ∗∗P < 0.01 (t-test). Scale bar = 200 μm. WT, wild-type.
      Figure thumbnail figs10
      Supplemental Figure S12Hematoxylin and eosin (H&E) staining of heart and kidney in littermate control and Tmem30a liver-specific knockout (LKO) mice on cholic acid (CA)-supplemented diet. Male Tmem30a-deficient mice and male littermate controls (2 to 3 months) were fed a 0.5% CA-supplemented diet. A: The global scan (left) and cardiocytes (right) of the hearts indicated that there was no morphologic difference between littermate control and Tmem30a LKO mice. B: H&E staining revealed that there was no morphologic difference in the medulla and renal cortex of kidneys of littermate control and Tmem30a LKO mice. Scale bars: 100 μm (A); 200 μm (B). WT, wild-type.
      Figure thumbnail figs11
      Supplemental Figure S13Hematoxylin and eosin (H&E) staining of brain, intestine, and spleen in littermate control and Tmem30a liver-specific knockout (LKO) mice on cholic acid (CA)-supplemented diet. Male Tmem30a-deficient mice and male littermate controls (2 to 3 months) were fed a 0.5% CA-supplemented diet. A: H&E staining of the brains revealed similar morphologic characteristics in both groups. B: H&E staining of intestinal villi revealed similar morphologic characteristics in both groups. C: H&E staining of the spleens also revealed similar morphologic characteristics in both groups. Scale bars: 5000 μm (A); 500 μm (B); 200 μm (C). WT, wild-type.
      Figure thumbnail figs12
      Supplemental Figure S14Biochemical analysis of surviving Tmem30a liver-specific knockout (LKO) mice. A and B: (A) Plasma total bile acid (TBA), total bilirubin (TBIL) and direct bilirubin (DBIL) levels (A) and plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels (B) were increased in surviving Tmem30a LKO mice, but the increases were not as dramatic as in morbid Tmem30a LKO mice. C: Hematoxylin and eosin (H&E) staining showing the presence of unclear cell boundaries in the surviving Tmem30a LKO mice. All mice were tested at 2 to 3 months age. Data are expressed as means ± SEM. n ≥ 9 mice. P < 0.05, ∗∗P < 0.01 (t-test). Scale bars = 100 μm. WT, wild-type.

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      • Correction
        The American Journal of PathologyVol. 188Issue 8
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          In the article entitled, “Hepatic Tmem30a Deficiency Causes Intrahepatic Cholestasis by Impairing Expression and Localization of Bile Salt Transporters” (Volume 187, pages 2775–2787 of the December 2017 issue of The American Journal of Pathology; https://doi.org/10.1016/j.ajpath.2017.08.011), the authors have noted that the affiliation for Fan Yang is incorrect as published, the Leibniz Institute for Age Research - Fritz Lipmann Institute, Friedrich-Schiller University of Jena, Jena should not have been listed.
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