WIF-B cells: an in vitro model for studies of hepatocyte polarity.

ABCG5 (G5) and ABCG8 (G8) are ATP-binding cassette half-transporters that limit intestinal uptake and promote biliary secretion of neutral sterols. Here, we describe the purification of endogenous G5G8 from mouse liver to near homogeneity. We incorporated the native proteins into membrane vesicles and reconstituted sterol transfer. Native gel electrophoresis, density-gradient ultracentrifugation, and chemical cross-linking studies indicated that the functional native complex is a heterodimer. No higher order oligomeric forms were observed at any stage in the catalytic cycle. Sterol transfer activity by purified native G5G8 was stable, stereospecific, and selective. We also report that G5 but not G8 is S-palmitoylated and that palmitoylation is not essential for dimerization, trafficking, or biliary sterol secretion. Both G5 and G8 have short but highly conserved cytoplasmic tails. The functional roles of these C-terminal regions were examined using an in vivo functional assay. ABCG5 (G5) 1 and ABCG8 (G8) are ABC half-transporters that limit the accumulation of neutral sterols in the body (1). The two proteins are expressed primarily in enterocytes and hepatocytes, where they heterodimerize in the endoplasmic reticulum prior to being transported to apical membranes (1-3). Inactivating mutations in either G5 or G8 cause sitosterolemia, a recessive disorder characterized by hypercholesterolemia, phytosterolemia, and premature coronary artery disease (3,4). The role of G5 and G8 in sterol trafficking in vivo has been † This work was supported by Grant HL72304 from the National Institutes of Health (NIH). NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 July 8. Published in final edited form as:Biochemistry. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript examined in detail using genetically modified mice, in which G5 and G8 are either overexpressed or inactivated (5-10). In the intestine, the G5G8 heterodimer limits the absorption of dietary sterols, especially plant-derived sterols (5). G5G8 synthesized in the liver is located in the bile canalicular membrane and is required for efficient secretion of neutral sterols into bile (5).Previously, we developed an in vitro assay using recombinant G5G8 expressed in Sf9 cells to elucidate the mechanism by which G5G8 promotes translocation of sterols across membranes (11). Membrane vesicles prepared from Sf9 cells expressing wild-type G5 and G8 supported the transfer of cholesterol from donor vesicles. G5G8-mediated transfer was stereoselective and specific for neutral sterols. Introduction of mutations predicted to disrupt ATP hydrolysis abolished G5G8-mediated sterol transfer. The recombinant G5G8 transporter was purified to near homogeneity using affinity chromatography, and the purified heterodimer retained ATPdependent sterol transfer activity when incorporated into proteoliposomes (11). These studies provided the first direct evidence that neutral sterols are the primary transport substrate f...

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“…WIF-B cells were a generous gift from Ann Hubbard (Johns Hopkins University, Baltimore, MD) and were cultured as previously described (19).…”

Section: Cell Culturementioning

confidence: 99%

Purification and Reconstitution of Sterol Transfer by Native Mouse ABCG5 and ABCG8

et al. 2008

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“…Data Preparation and Experimental Assays : A549 cells (human pulmonary adenocarcinoma cell) and HEpG2 cells (human hepatocellular carcinoma cell) were usually used in in vitro models for the study of non‐small cell lung cancer and liver cancer, separately 51, 52. A549 and HEpG2 cell lines were purchased from the Chinese Academy of Sciences and maintained in RPMI‐1640/EMEM medium with10% fetal bovine serum (FBS) and 1% penicillin‐streptomycin (Gibco, BRL, Grand Island, NY).…”

Section: Methodsmentioning

confidence: 99%

DeepScreen: An Accurate, Rapid, and Anti‐Interference Screening Approach for Nanoformulated Medication by Deep Learning

et al. 2018

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Accuracy of current efficacy judgment methods for nanoformulated drug remains unstable due to the interference of nanocarriers. Herein, DeepScreen, a drug screening system utilizing convolutional neural network based on flow cytomerty single‐cell images, is introduced. Compared to existing experimental approaches, the high‐throughput system has superior precision, rapidity, and anti‐interference, and is cost‐cutting with high accuracy. First, it can resist most disturbances from manual factors of complicated evaluation progress. In addition, class activation maps generated from DeepScreen indicate that it may identify and locate the tiny variation from cell apoptosis and slight changes of cellular period caused by drug or even nanoformulated drug action at very early stages. More importantly, the excellent performance of assessment on two types of nanoformulations and fluorescent drug proves the fine generality and anti‐interference of this novel system. All these privileged performances make DeepScreen a very smart and promising system for drug detection.

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“…Rabbit antibody to aminopeptidase N (APN) was described previously (3,24,25). Rat monoclonal anti-ZO-1 hybridoma cells (R40.76) were provided by B. Stevenson (University of Alberta, Edmonton, AB, Canada).…”

Section: Methodsmentioning

confidence: 99%

“…WIF-B cells were grown as previously described (25,58) except that the medium components glucose, ascorbic acid, and linoleic acid (542 ml/l, 15 mg/l, and 0.08 mg/l, respectively) were added to F-12 Kaign's modification (Invitrogen) to achieve a formulation nearly identical to that reported for F-12 Coon's modification. Cells (0.5 ϫ 10 6 ) were plated either onto dishes (10 cm) directly or onto dishes containing six glass coverslips (22 ϫ 22 mm) and fed every other day until use.…”

Section: Cell Culture Infection and Immunofluorescencementioning

confidence: 99%

“…We used the well-characterized WIF-B hepatic cell line (4,25,58), because these cells recapitulate a two-step polarization process reminiscent of that in vivo (13) and express only the rat JAM-A isoform. We found that expression of lentivirus encoding a short hairpin RNA (shRNA) to rat JAM-A knocked down 50% of the endogenous protein in WIF-B cells, with an accompanying loss of hepatic polarity and TJs.…”

mentioning

confidence: 99%

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JAM-A is both essential and inhibitory to development of hepatic polarity in WIF-B cells

Braiterman¹,

Heffernan²,

Nyasae³

et al. 2008

American Journal of Physiology-Gastrointestinal and Liver Physi

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Junctional adhesion molecule (JAM) is involved in tight junction (TJ) formation in epithelial cells. Three JAMs (A, B, and C) are expressed in rat hepatocytes, but only rat JAM-A is present in polarized WIF-B cells, a rat-human hepatic line. We used knockdown (KD) and overexpression in WIF-B cells to determine the role of JAM-A in the development of hepatic polarity. Expression of rat JAM-A short hairpin RNA resulted in ϳ50% KD of JAM-A and substantial loss of hepatic polarity, as measured by the absence of apical cysts formed by adjacent cells and sealed by TJ belts. When inhibitory RNA-resistant human JAM-A (huWT) was expressed in KD cells, hepatic polarity was restored. In contrast, expression of JAM-A that either lacked its PDZ-binding motif (hu⌬C-term) or harbored a point mutation (T273A) did not complement, indicating that multiple sites within JAM-A's cytoplasmic tail are required for the development of hepatic polarity. Overexpression of huWT in normal WIF-B cells unexpectedly blocked WIF-B maturation to the hepatic phenotype, as did expression of three huJAM-A constructs with single point mutations in putative phosphorylation sites. In contrast, hu⌬C-term was without effect, and the T273A mutant only partially blocked maturation. Our results show that JAM-A is essential for the development of polarity in cultured hepatic cells via its possible phosphorylation and recruitment of relevant PDZ proteins and that hepatic polarity is achieved within a narrow range of JAM-A expression levels. Importantly, formation/ maintenance of TJs and the apical domain in hepatic cells are linked, unlike simple epithelia.partitioning-defective polarity protein/atypical protein kinase C complex

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WIF-B cells: an in vitro model for studies of hepatocyte polarity.

Cited by 204 publications

References 68 publications

Purification and Reconstitution of Sterol Transfer by Native Mouse ABCG5 and ABCG8

Purification and Reconstitution of Sterol Transfer by Native Mouse ABCG5 and ABCG8

DeepScreen: An Accurate, Rapid, and Anti‐Interference Screening Approach for Nanoformulated Medication by Deep Learning

JAM-A is both essential and inhibitory to development of hepatic polarity in WIF-B cells

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