Background & Aims:
Bile secretion depends on the delivery and removal of transporter proteins to and from the canalicular membrane. Trafficking of the bile salt export pump (BSEP) to the canalicular ...membrane was investigated in HepG2 cells and rat hepatocytes.
Methods:
Subcellular localization of BSEP was determined by confocal laser scanning microscopy using different BSEP antibodies.
Results:
Ten percent of untreated HepG2 cells developed pseudocanaliculi, but only 15% of these pseudocanaliculi contained BSEP, which largely colocalized with the Golgi marker GM130. Cycloheximide, an inhibitor of protein translation, induced a microtubule- and p38
MAP kinase-dependent decrease of Golgi-associated BSEP, accompanied by a more than 2-fold increase in BSEP-positive pseudocanaliculi. Also, tauroursodeoxycholate (TUDC), which activates p38
MAP kinase (p38
MAPK, increased BSEP-positive pseudocanaliculi by more than 50% in rat sodium taurocholate cotransporting peptide (Ntcp)-transfected but not in untransfected HepG2 cells. The TUDC-dependent increase was sensitive to inhibitors of p38
MAPK and microtubules and involved Ca
2+-independent protein kinase C isoforms as suggested by its sensitivity to Gö6850 but insensitivity to Gö6976. In isolated rat hepatocytes with intact bile secretion, no colocalization of rat isoforms of the bile salt export pump (Bsep) and Golgi was found, but colocalization occurred after inhibition of p38
MAPK and PKC, suggesting that Bsep trafficking to the canalicular membrane depends on the basal activity of these kinases in polarized cells.
Conclusions:
p38
MAPK regulates BSEP trafficking from the Golgi to the canalicular membrane, and the Golgi may serve as a BSEP pool in certain forms of cholestasis or when p38
MAPK activity is inhibited. Activation of p38
MAPK by TUDC can recruit Golgi-associated BSEP in line with its choleretic action.
Bile salt transporters directly or indirectly influence biological processes through physicochemical or signalling properties of bile salts. The coordinated action of uptake and efflux transporters ...in polarized epithelial cells of the liver, biliary tree, small intestine and kidney determine bile salt concentrations in different compartments of the body. Genetic variations of bile salt transporters lead to clinical relevant phenotypes of varying severity ranging from a predisposition for drug-induced liver injury to rapidly progressing end-stage liver disease. This review focuses on the impact of genetic variations of bile salt transporters including BSEP, NTCP, ASBT and OSTα/β and discusses approaches for transporter analysis.
Vascular endothelial growth factor (VEGF) is expressed by many tumors, including hepatocellular carcinoma (HCC) and is involved in tumor angiogenesis. Little is known about its role for HCC ...infiltration into normal liver parenchyma.
The effects of VEGF on the integrity of tight junctions were studied in HepG2 cells and human HCC by means of confocal laser scanning microscopy.
VEGF induced within 45 min a marked loss of pseudocanaliculi and disruption of occludin-delineated tight junctions. This effect of VEGF was mimicked by phorbol-12-myristate-13-acetate (PMA) and was sensitive to protein kinase C (PKC) inhibition by Gö6850. VEGF induced within 15 min the translocation of the PKCα-isoform to the plasma-membrane, but had no effect on the activity of Erks and p38
MAPK. Sections from surgically removed HCC showed expression of VEGF in the tumor and occludin disassembly in normal liver parenchyma next to the tumor.
VEGF induces disruption of tight junctions in a PKC-α dependent manner. In addition to its known angioneogenic properties, VEGF may promote HCC spreading into normal liver parenchyma. The data may provide another rationale for the use of VEGF antagonists for tumor therapy.
Background & Aims: Endotoxin lipopolysaccharide (LPS) induces cholestasis and down-regulates the multidrug resistance protein 2 (MRP2). This study intends to characterize the short-term effects of ...LPS on MRP2.
Methods: The effects of LPS and dexamethasone on excretion of bromosulphalein (BSP), MRP2 messenger RNA (mRNA) levels, and subcellular MRP2 localization were studied by means of liver perfusion, Northern blots, and confocal microscopy.
Results: LPS treatment for 3–12 hours decreased biliary BSP excretion (10 μmol/L) by 40%. Hyposmolarity stimulated BSP excretion to control levels 3 hours after LPS injection, but was ineffective after 12 hours or in saline-treated controls. LPS led to a strong decrease of MRP2 mRNA after 12 hours, but not during the first 6 hours. LPS induced the appearance of MRP2 in intracellular vesicles in the immediate vicinity of the canaliculi within 3 hours, and these vesicles were remote from the canaliculi after 6 and 12 hours. The MRP2-containing vesicles did not stain for dipeptidylpeptidase IV (DPPIV). Dexamethasone counteracted the LPS effects on MRP2 mRNA levels, subcellular distribution, and BSP excretion.
Conclusions: LPS induces cholestasis due to an early retrieval of MRP2 from the canalicular membrane, whereas down-regulation of MRP2 mRNA is a later event. LPS-induced MRP2 retrieval from the canalicular membrane is not associated with the retrieval of DPPIV, suggestive for selectivity of the process.
GASTROENTEROLOGY 1999;116:401-410
Bile salts influence signaling and metabolic pathways. In hepatocytes, the sodium taurocholate cotransporting polypeptide (Ntcp) is a major determinant of intracellular bile salt levels. Short-term ...downregulation of Ntcp is not well characterized to date. FLAG and enhanced green fluorescent protein (EGFP) tags were cloned to the extra- and intracellular termini of Ntcp. Endocytosis of Ntcp in transfected HepG2 cells was visualized by fluorescence of EGFP, and membrane surface expression of Ntcp was quantified by flow cytometry with fluorochrome-labeled FLAG antibodies. Activation of protein kinase C (PKC) by phorbolester or thymeleatoxin an activator of Ca(2+)-dependent conventional PKCs (cPKCs), induced endocytosis of Ntcp, whereas the Na(+)-K(+)-ATPase remained in the plasma membrane. The PKC inhibitor BIM I and the cPKC-selective inhibitor Gö6976 abolished PMA-induced endocytosis. Because of this internalization, cell surface expression of Ntcp was reduced by 36 +/- 7%, bile salt uptake was decreased by 25%, and taurolithocholate sulfate-induced cell toxicity was prevented. In conclusion, Ca(2+)-dependent PKCs induce vesicular retrieval of Ntcp, thereby reducing bile salt uptake. This mechanism may protect hepatocytes from toxic intracellular bile salt concentrations.
Homologous recombination in Saccharomyces cerevisiae is a well-studied process. Here, we describe a yeast-recombination-based approach to construct and mutate plasmids containing the cDNA of the ...human bile salt export pump (BSEP) that has been shown to be unstable in E. coli. Using this approach, we constructed the necessary plasmids for a heterologous overexpression of BSEP in the yeast Pichia pastoris. We then applied a new site-directed mutagenesis method, DREAM (Directed REcombination-Assisted Mutagenesis) that completely bypasses E. coli by using S. cerevisiae as the plasmid host with high mutagenesis efficiency. Finally, we show how to apply this strategy to unstable non-yeast plasmids by rapidly turning an existing mammalian BSEP expression construct into a S. cerevisiae-compatible plasmid and analyzing the impact of a BSEP mutation in several mammalian cell lines.
Background & Aims Hepatitis B and D viruses (HBV and HDV) are human pathogens with restricted host ranges and high selectivity for hepatocytes; the HBV L-envelope protein interacts specifically with ...a receptor on these cells. We aimed to identify this receptor and analyze whether it is the recently described sodium-taurocholate co-transporter polypeptide (NTCP), encoded by the SLC10A1 gene. Methods To identify receptor candidates, we compared gene expression patterns between differentiated HepaRG cells, which express the receptor, and naïve cells, which do not. Receptor candidates were evaluated by small hairpin RNA silencing in HepaRG cells; the ability of receptor expression to confer binding and infection were tested in transduced hepatoma cell lines. We used interspecies domain swapping to identify motifs for receptor-mediated host discrimination of HBV and HDV binding and infection. Results Bioinformatic analyses of comparative expression arrays confirmed that NTCP, which was previously identified through a biochemical approach is a bona fide receptor for HBV and HDV. NTCPs from rat, mouse, and human bound Myrcludex B, a peptide ligand derived from the HBV L-protein. Myrcludex B blocked NTCP transport of bile salts; small hairpin RNA-mediated knockdown of NTCP in HepaRG cells prevented their infection by HBV or HDV. Expression of human but not mouse NTCP in HepG2 and HuH7 cells conferred a limited cell-type–related and virus-dependent susceptibility to infection; these limitations were overcome when cells were cultured with dimethyl sulfoxide. We identified 2 short-sequence motifs in human NTCP that were required for species-specific binding and infection by HBV and HDV. Conclusions Human NTCP is a specific receptor for HBV and HDV. NTCP-expressing cell lines can be efficiently infected with these viruses, and might be used in basic research and high-throughput screening studies. Mapping of motifs in NTCPs have increased our understanding of the species specificities of HBV and HDV, and could lead to small animal models for studies of viral infection and replication.
Intra-cellular and inter-cellular protein translocation can be observed by microscopic imaging of tissue sections prepared immunohistochemically. A manual densitometric analysis is time-consuming, ...subjective and error-prone. An automated quantification is faster, more reproducible, and should yield results comparable to manual evaluation. The automated method presented here was developed on rat liver tissue sections to study the translocation of bile salt transport proteins in hepatocytes. For validation, the cholestatic liver state was compared to the normal biological state.
An automated quantification method was developed to analyze the translocation of membrane proteins and evaluated in comparison to an established manual method. Firstly, regions of interest (membrane fragments) are identified in confocal microscopy images. Further, densitometric intensity profiles are extracted orthogonally to membrane fragments, following the direction from the plasma membrane to cytoplasm. Finally, several different quantitative descriptors were derived from the densitometric profiles and were compared regarding their statistical significance with respect to the transport protein distribution. Stable performance, robustness and reproducibility were tested using several independent experimental datasets. A fully automated workflow for the information extraction and statistical evaluation has been developed and produces robust results.
New descriptors for the intensity distribution profiles were found to be more discriminative, i.e. more significant, than those used in previous research publications for the translocation quantification. The slow manual calculation can be substituted by the fast and unbiased automated method.
Bile secretion is regulated by different signaling transduction pathways including protein kinase C (PKC). However, the role of different PKC isoforms for bile formation is still controversial. This ...study investigates the effects of PKC isoform selective activators and inhibitors on PKC translocation, bile secretion, bile acid uptake, and subcellular transporter localization in rat liver, isolated rat hepatocytes and in HepG2 cells. In rat liver activation of Ca2+-dependent cPKCα and Ca2+-independent PKC∈ by phorbol 12-myristate 13-acetate (PMA, 10nmol/liter) is associated with their translocation to the plasma membrane. PMA also induced translocation of the cloned rat PKC∈ fused to a yellow fluorescent protein (YFP), which was transfected into HepG2 cells. In the perfused liver, PMA induced marked cholestasis. The PKC inhibitors Gö6850 (1 μmol/liter) and Gö6976 (0.2 μmol/liter), a selective inhibitor of Ca2+-dependent PKC isoforms, diminished the PMA effect by 50 and 60%, respectively. Thymeleatoxin (Ttx,) a selective activator of Ca2+-dependent cPKCs, did not translocate rat PKC∈-YFP transfected in HepG2 cells. However, Ttx (0.5–10 nmol/liter) induced cholestasis similar to PMA and led to a retrieval of Bsep from the canalicular membrane in rat liver while taurocholate-uptake in isolated hepatocytes was not affected. Gö6976 completely blocked the cholestatic effect of Ttx but had no effect on tauroursodeoxycholate-induced choleresis. The data identify Ca2+-dependent PKC isoforms as inducers of cholestasis. This is mainly due to inhibition of taurocholate excretion involving transporter retrieval from the canalicular membrane.
Cholestatic liver diseases in humans as well as bile acid (BA)-feeding and common bile duct ligation (CBDL) in rodents trigger hyperplasia of cholangiocytes within the portal fields. Furthermore, ...elevation of BA levels enhances proliferation and invasiveness of cholangiocarcinoma (CCA) cells in animal models, thus promoting tumour progression. TGR5 is a G-protein coupled BA receptor, which is highly expressed in cholangiocytes and postulated to mediate the proliferative effects of BA.
BA-dependent cholangiocyte proliferation was examined in TGR5-knockout and wild type mice following cholic acid (CA)-feeding and CBDL. TGR5-dependent proliferation and protection from apoptosis was studied in isolated cholangiocytes and CCA cell lines following stimulation with TGR5 ligands and kinase inhibitors. TGR5 expression was analysed in human CCA tissue.
Cholangiocyte proliferation was significantly reduced in TGR5-knockout mice in response to CA-feeding and CBDL. Taurolithocholic acid and TGR5-selective agonists induced cholangiocyte proliferation through elevation of reactive oxygen species and cSrc mediated epidermal growth factor receptor transactivation and subsequent Erk1/2 phosphorylation only in wild type but not in TGR5-knockout-derived cells. In human CCA tissue TGR5 was overexpressed and the pathway of TGR5-dependent proliferation via epidermal growth factor receptor and extracellular signal-regulated kinase (ERK)1/2 activation also translated to CCA cell lines. Furthermore, apoptosis was inhibited by TGR5-dependent CD95 receptor serine phosphorylation.
TGR5 is an important mediator of BA-induced cholangiocyte proliferation in vivo and in vitro. Furthermore, TGR5 protects cholangiocytes from death receptor-mediated apoptosis. These mechanisms may protect cholangiocytes from BA toxicity under cholestatic conditions, however, they may trigger proliferation and apoptosis resistance in malignantly transformed cholangiocytes, thus promoting CCA progression.
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