A 35-year-old woman is referred for right upper quadrant abdominal pain and fever. She had a cholecystectomy for gallstones 4 years previously. An aunt has also had a cholecystectomy. Abdominal ultrasound examination shows numerous hyperechoic foci within the liver and some "comet tail" artifacts. Because of her age (less than 40), the recurrence of biliary symptoms after cholecystectomy, her family history and the intrahepatic hyperechoic foci, the diagnosis of low phospholipid-associated cholelithiasis (LPAC) syndrome is suspected and confirmed by the demonstration of a point mutation of the ABCB4 gene. Therapy by ursodeoxycholic acid (AUDC) is started. Symptoms improve and they disappear completely within a few weeks. The cause, pathophysiology, diagnosis and treatment are reviewed.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can induce drug transporter genes such as the ATP-binding cassette G member 2 (ABCG2), which contributes to multidrug resistance. We investigated the effect of TCDD pretreatment on drug transporters induction from cancer cells of various origins. Cell viabilities after treatment of cisplatin were measured to evaluate acquiring cisplatin resistance by TCDD. Acquring cisplatin resistance was found only in cisplatin senstivie cancer cells including gastric SNU601, colon LS180, brain CRT-MG and lymphoma Jurkat cells which showed a significant increase in cell viability after combined treatment with TCDD and cisplatin. High increase of ABCG2 gene expression was found in SNU601 and LS180 cells with a mild increase in the expression of the ABCC3, ABCC5,and SLC29A2 genes in SNU601 cells, and of major vault protein (MVP) in LS180 cells. The AhR inhibitor kaempferol suppressed the upregulation of ABCG2 expression and reversed the TCDD-induced increase in cell viability in LS180 cells. However, in CRT-MG cells, other transporter genes including ABCC1, ABCC5, ABCA3, ABCA2, ABCB4, ABCG1, and SLC29A1 were up-regulated. These findings suggested the acquiring cisplatin resistance by TCDD associated with cancer cell-type-specific induction of drug transporters.

Multidrug/multixenobiotic resistance (MDR/MXR) confers resistance to a diverse range of potentially toxic pharmaceuticals and environmental contaminants through a cellular response that involves the coordinated induction and activity of the ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp) and the Phase I metabolizing enzyme cytochrome P450 3A (CYP3A). In mammals, ligand-mediated pregnane X receptor (PXR) transcriptional activity regulates the induction of P-gp and CYP3A; however, this mechanism has not been well-characterized in piscine species. Zebrafish (Danio rerio) treated with the Pxr agonist pregnenolone 16α-carbonitrile (PCN) showed decreased P-gp (zebrafish Abcb4) and CYP3A (zebrafish Cyp3a65) mRNA levels after 48h exposure; however, treatment with PCN also resulted in increased hepatic MDR/MXR functional activity (i.e. increased Rhodamine 123 efflux) in vivo. Consistent with mammalian-like MDR/MXR regulated by PXR, the PCN-mediated modulation of hepatic Abcb4 and Cyp3a65 mRNA levels and MDR/MXR functional activity was attenuated by co-treatment with PCN and the mammalian PXR antagonist, ketoconazole (KTC). These results provide evidence that zebrafish Pxr may play a role in MDR/MXR through transcriptional regulation of abcb4 and cyp3a65 gene expression.

BACKGROUND

There are three types of progressive familial intrahepatic cholestasis (PFIC). Type 3 is characterized by elevated gamma-glutamyl transferase (γ-GT) and it can be diagnosed in adolescence/adulthood. The genetic defect of PFIC 3 appears to explain the pathogenesis of intrahepatic cholestasis of pregnancy (ICP).

OBJECTIVE

Draw attention to this rare disease, especially in adulthood, and clarify the association between ICP and PFIC 3.

RESULTS

We describe a series of cases from a Portuguese northern family with two brothers presenting chronic cholestasis since adolescence. Brother 1: since 15-years-old with pruritus and elevated γ-GT ∼6x. Brother 2: pre-term, due to severe maternal pruritus and jaundice, since 13-years-old with pruritus, jaundice and ∼8x γ-GT elevation. Common causes of cholestasis were excluded and liver histologies were nonspecific. Research for mutation on ABCB4 gene showed mutations in both alleles.

CONCLUSIONS

Disease and mechanisms that determine cholestasis are complex and their understanding may provide new therapeutics.

The etiology of intrahepatic cholestasis of pregnancy (ICP) involves environmental, hormonal and genetic factors. It is thought that ICP may be related to the polymorphic variants of several genes involved in the metabolism and transport of bile acids (BA). The goal of our study was to evaluate the possible role of genetic polymorphic variants of ABC transporters in patients with ICP.

96 women with ICP (mean age of 30.42 years, mean gestational age of 36.83 gestation weeks) and 211 healthy pregnant women (mean age of 30.68 years, mean gestational age of 39.05 gestation weeks) were enrolled in the study. Genetic analysis was performed using a polymerase chain reaction / restriction fragment length polymorphism (PCR/RFLP) method. The following polymorphisms were analysed: 1331T>> C (V444A) ABCB11 and 1954A>> G (R652G) ABCB4.

Our analysis of frequency of genotypes and alleles of the 1954A>> G ABCB4 polymorphism revealed no significant differences between the ICP and control groups. For the 1331T>> C polymorphism of the ABCB11 gene the results revealed a higher frequency of 1331CC genotypes in the ICP group (39.58% vs. 29.38%. OR = 1.57, p = 0.05). Also, the frequency of the 1331C allele was higher in the ICP group compared to the control group (64.06% vs. 55.69%, OR = 1.42, p = 0.03).

The overrepresentation of mutated variants of the 1331T>> C ABCB11 polymorphism in the ICP group suggests its contribution to the etiology of the intrahepatic cholestasis of pregnancy. Analysis of genotypes' co-existence pointed to the possibility of the mutated variants of polymorphism 1954A>> G ABCB4 and 1331T>> C ABCB11 having a summation effect on the development of ICP.

A 2-year-old female with cirrhosis was found to have a liver copper of 248 μg/g dry weight. She was eventually diagnosed with ABCB4 disease on the basis of heterozygote A546D and R176W mutations. Her liver disease was partially responsive to ursodeoxycholic acid therapy. Copper overload occurs in cholestatic liver disease and this must be considered to avoid misdiagnosis of Wilson disease.

Mithramycin demonstrates preclinical anticancer activity, but its therapeutic dose is limited by the development of hepatotoxicity that remains poorly characterized. A pharmacogenomics characterization of mithramycin-induced transaminitis revealed that hepatotoxicity is associated with germline variants in genes involved in bile disposition: ABCB4 (multidrug resistance 3) rs2302387 and ABCB11 [bile salt export pump (BSEP)] rs4668115 reduce transporter expression (P < 0.05) and were associated with ≥grade 3 transaminitis developing 24 hours after the third infusion of mithramycin (25 mcg/kg, 6 hours/infusion, every day ×7, every 28 days; P < 0.0040). A similar relationship was observed in a pediatric cohort. We therefore undertook to characterize the mechanism of mithramycin-induced acute transaminitis. As mithramycin affects cellular response to bile acid treatment by altering the expression of multiple bile transporters (e.g., ABCB4, ABCB11, sodium/taurocholate cotransporting polypeptide, organic solute transporter α/β) in several cell lines [Huh7, HepaRG, HepaRG BSEP (-/-)] and primary human hepatocytes, we hypothesized that mithramycin inhibited bile-mediated activation of the farnesoid X receptor (FXR). FXR was downregulated in all hepatocyte cell lines and primary human hepatocytes (P < 0.0001), and mithramycin inhibited chenodeoxycholic acid- and GW4046-induced FXR-galactose-induced gene 4 luciferase reporter activity (P < 0.001). Mithramycin promoted glycochenodeoxycholic acid-induced cytotoxicity in ABCB11 (-/-) cells and increased the overall intracellular concentration of bile acids in primary human hepatocytes grown in sandwich culture (P < 0.01). Mithramycin is a FXR expression and FXR transactivation inhibitor that inhibits bile flow and potentiates bile-induced cellular toxicity, particularly in cells with low ABCB11 function. These results suggest that mithramycin causes hepatotoxicity through derangement of bile acid disposition; results also suggest that pharmacogenomic markers may be useful to identify patients who may tolerate higher mithramycin doses. SIGNIFICANCE STATEMENT: The present study characterizes a novel mechanism of drug-induced hepatotoxicity in which mithramycin not only alters farnesoid X receptor (FXR) and small heterodimer partner gene expression but also inhibits bile acid binding to FXR, resulting in deregulation of cellular bile homeostasis. Two novel single-nucleotide polymorphisms in bile flow transporters are associated with mithramycin-induced liver function test elevations, and the present results are the rationale for a genotype-directed clinical trial using mithramycin in patients with thoracic malignancies.

Multidrug resistance 3 (MDR3) is expressed on the canalicular membrane of the hepatocytes and plays an important role in protecting the liver from bile acids. Altered ABCB4 gene expression can lead to a rare hepatic disease, low phospholipid-associated cholelithiasis (LPAC). In this study, we characterized 3 ABCB4 mutations in LPAC patients using various in vitro assay systems. We first measured the ability of each mutant to transport paclitaxel and then the mechanisms by which these mutations might change MDR3 transport activity were determined using immunoblotting, cell surface protein biotinylation, and immunofluorescence. Through a membrane vesicular transport assay, we observed that the uptake of paclitaxel was significantly reduced in membrane vesicles expressing 2 ABCB4 mutations, F165I and S320F. Both mutants showed significantly decreased total and cell surface MDR3 expression. These data suggest two missense mutations of ABCB4 may alter function of MDR3 and ultimately can be determined as LPAC-causing mutations.

Identification of the transport systems involved in bile secretion and of the genes codifying these systems has allowed the etiology of familial intrahepatic cholestasis to be determined in most affected children. Mutations in ATP8B1 cause a defect in FIC1, an aminophospholipid flipase, and give rise to a variable spectrum of disease, ranging from progressive intrahepatic cholestasis to benign recurrent cholestasis, due to alterations in the lipid composition of the membranes and decreased expression of the nuclear factor FXR. Mutations in ABCB11 cause a defect of the canalicular bile salt export pump (BSEP), with early clinical manifestations and progression to hepatocellular failure in childhood. Mutations in ABCB4 cause an alteration in the MDR3 phospholipid transporter, and a variable spectrum of disease from progressive ductal injury to cirrhosis in children, and gallstones, cholestasis of pregnancy, or late cirrhosis in adults.

The adenosine triphosphate-binding cassette (ABC) is a large group of proteins involved in material transportation, cellular homeostasis, and closely associated with chemoresistance. ATP-binding cassette protein B4 (ABCB4) is a member of ABCs which has a similar structure to ABCB1, but fewer researches were performed. The present study is aimed to investigate the putative mechanism of ABCB4 in 5-fluorouracil (5-Fu) resistance. Then, we found that ABCB4 was significantly down-regulated in the 5-Fu resistant HCT8 cell lines by polymerase chain reaction (PCR) and Western blot. The knockdown of ABCB4 by small interfering RNA decreased the apoptosis by 5-Fu in resistant HCT8R cell lines without influencing the proliferation. Also, we found a lower expression of cleaved caspase and PARP by Western blot after the knockdown of ABCB4. However, the knockdown of ABCB4 did not influence the proliferation and apoptosis. Furthermore, the histological detection of ABCB4 mRNA level in human colorectal cancer tissues and even in the recurrent tissues after 5-Fu single-agent chemotherapy was employed to provide more concrete evidence that ABCB4 may be a tumor suppressor gene to regulate chemoresistance in colorectal cancer. Moreover, a 109-patient cohort revealed that ABCB4 predicted a poor recurrence-free survival and overall survival. In summary, ABCB4 was down-regulated in the 5-Fu resistant cells and knockdown of ABCB4 alleviated the cell apoptosis and predicts a shorter recurrence-free survival and overall survival.

Recently, clinical studies demonstrated that magnetic resonance relaxometry with determination of relaxation times T1 and T2⁎ may aid in staging and management of liver fibrosis in patients suffering from viral hepatitis and steatohepatitis. In the present study we investigated T1 and T2⁎ in different models of liver fibrosis to compare alternate pathophysiologies in their effects on relaxation times and to further develop noninvasive quantification methods of liver fibrosis. MRI was performed with a fast spin echo sequence for measurement of T1 and a multigradient echo sequence for determination of T2⁎. Toxic liver fibrosis was induced by injections of carbon tetrachloride (1.4 mL CCl4 per kg bodyweight and week, for 3 or 6 weeks) in BALB/cJ mice. Chronic sclerosing cholangitis was mimicked using the ATP-binding cassette transporter B4 knockout (Abcb4 -/-) mouse model. Untreated BALB/cJ mice served as controls. To assess hepatic fibrosis, we ascertained collagen contents and fibrosis scores after Sirius red staining. T1 and T2⁎ correlate differently to disease severity and etiology of liver fibrosis. T2⁎ shows significant decrease correlating with fibrosis in CCl4 treated animals, while demonstrating significant increase with disease severity in Abcb4 -/- mice. Measurements of T1 and T2⁎ may therefore facilitate discrimination between different stages and causes of liver fibrosis.

Cost-effective identification of novel pharmacogenetic variants remains a pressing need in the field. Using data from the Genetics of Lipid Lowering Drugs and Diet Network, we identified genomic regions of relevance to fenofibrate response in a sample of 173 families. Our approach included a multipoint linkage scan, followed by selection of the families showing evidence of linkage. We identified a strong signal for changes in LDL-cholesterol (LDL-C) on chromosome 7 (peak logarithm of odds score = 4.76) in the full sample (n = 821). The signal for LDL-C response remained even after adjusting for baseline LDL-C. Restricting analyses only to the families contributing to the linkage signal for LDL-C (N = 19), we observed a peak logarithm of odds score of 5.17 for chromosome 7. Two genes under this peak (ABCB4 and CD36) were of biological interest. These results suggest that linked family analyses might be a useful approach to gene discovery in the presence of a complex (e.g. multigenic) phenotype.

Chronic cholestasis results in liver injury and eventually liver failure. Although ursodeoxycholic acid (UDCA) showed limited benefits in primary biliary cirrhosis, there is an urgent need to develop alternative therapy for chronic cholestatic disorders. Previous studies from our laboratory demonstrated that all-trans-retinoic acid (atRA) is a potent suppressor of CYP7A1, the rate-limiting enzyme in bile acid synthesis. atRA also repressed the expression of tumor growth factor-β and collagen 1A1 in activated primary human stellate cells and LX2 cells. When administered together with UDCA to bile duct-ligated rats, this combined therapy significantly reduced the bile acid pool size and improved liver conditions. To further examine whether atRA alone or in combination with UDCA has greater beneficial effects than UDCA treatment alone, we assessed this treatment in two additional chronic cholestatic rodent models: α-naphthylisothiocyanate (ANIT)-treated rats and the Mdr2(-/-) (Abcb4(-/-)) knockout mouse. atRA alone significantly reduced bile duct proliferation, inflammation, and hydroxyproline levels in ANIT-treated rats, whereas the combination of atRA and UDCA significantly reduced plasma bile salt level compared with UDCA treatment. atRA alone or in combination with UDCA significantly reduced plasma levels of alkaline phosphatase and bile salts in 12-week-old Mdr2(-/-) mice. Reduced bile duct proliferation and inflammation were also observed in the livers of these mice. Together, atRA alone or in combination with UDCA significantly reduced the severity of liver injury in these two animal models, further supporting the combination treatment of atRA and UDCA as a potential new therapy for patients with chronic cholestatic liver disease who have not responded fully to UDCA.

Mutations in multidrug resistance 3 gene (MDR3 or ABCB4) underlie progressive familial intrahepatic cholestasis type 3 (PFIC3), a severe pediatric liver disease progressing to cirrhosis. Abcb4-/- mice exhibit slowly developing hepatic lesions that can be accelerated by feeding a cholic acid (CA)-supplemented diet. We investigated the beneficial effects of a soybean lecithin (L)-supplemented diet in this model of liver disease. Abcb4-/- mice and wild-type (WT) controls were divided in four groups by the diet they were fed: control (C) diet, L-supplemented diet, CA-supplemented diet, and L- and CA-supplemented (L+CA) diet. After 2 wk on these regimens, liver enzymes and bilirubin were measured in serum with bile flow, total bile acids, and cholesterol (CHOL) and phospholipid (PL) concentrations in bile. Ductular hyperplasia, portal fibroblastic cell proliferation, myofibroblast activation, and hepatic fibrosis were quantified on liver sections. Abcb4-/- mice fed the C diet exhibited mild liver damage. CA produced very high elevations of serum liver enzymes and bilirubin with significant bile duct proliferation, peribiliary fibroblast activation, and fibrosis. The L-supplemented diet dramatically mitigated the hepatic damage in CA-supplemented diet animals. We conclude that L is protective against liver disease in Abcb4-/- mice and suggest that it could offer potential benefit in PFIC3.

The identification of biliary tranporters has enhanced our understanding of bile formation and some liver diseases. In this review, we first describe the main hepatobiliary transporters and their function. Then, some liver diseases related to mutations of biliary tranporters (FIC1/ATP8B1, BSEP/ABCB11, MDR3 /ABCB4 and MRP2/ABCC2) will be described with a focus on the pathological aspects. These diseases include progressive familial intrahepatic cholestasis (PFIC), benign recurrent intrahepatic cholestasis (BRIC), intrahepatic cholestasis of pregnancy, Dubin-Johnson's syndrome and low phospholipid associated cholelithiasis (LPAC).

Progressive familial intrahepatic cholestasis (PFIC) types 1 and 3 are severe cholestatic liver diseases caused by deficiency of ATB8B1 and ABCB4, respectively. Mouse models for PFIC display mild phenotypes compared with human patients, and this can be explained by the difference in bile salt pool composition. Mice, unlike humans, have the ability to detoxify hydrophobic bile salts by cytochrome P450-mediated (re)hydroxylation and thus have a less toxic bile salt pool. We have crossed mouse models for PFIC1 and PFIC3 with Hrn mice that have a reduced capacity to (re)hydroxylate bile salts. Double transgenes were obtained by backcrossing Atp8b1(G308V/G308V) and Abcb4(-/-) mice with Hrn mice that have a liver-specific disruption of the cytochrome P450 reductase gene and therefore have markedly reduced P450 activity. In these mice, a more hydrophobic bile salt pool was instilled by cholic acid supplementation of the diet, and bile formation and liver pathology was studied. As opposed to single transgenes, Atp8b1(G308V/G308V)/Hrn and Abcb4(-/-)/Hrn mice rapidly developed strong cholestasis that was evidenced by increased plasma bilirubin and bile salt levels. The bile salt pool was more toxic in both models; Atp8b1(G308V/G308V)/Hrn mice had a more hydrophobic plasma pool compared with the single transgene, whereas Abcb4(-/-)/Hrn mice had a more hydrophobic biliary pool compared with the single transgene. In line with these findings, liver damage was not aggravated in Atp8b1(G308V/G308V)/Hrn but was more severe in Abcb4(-/-)/Hrn mice. These data indicate that bile salt pool composition is a critical determinant in the initiation and progression of cholestasis and liver pathology in PFIC1 and PFIC3. Most importantly, our data suggest that the hydrophobicity of the plasma bile salt pool is an important determinant of the severity of cholestasis, whereas the hydrophobicity of the biliary bile salt pool is an important determinant of the severity of liver pathology.

BACKGROUND

Progressive familial intrahepatic cholestasis (PFIC) type 3, characterized by high gamma glutamyl transferase (GGT), is an autosomal recessive genetic disease. It often occurs in patients' first years of age. However, high GGT type PFIC is still rare.

UNASSIGNED

The present study reports a case of liver transplantation for decompensated liver cirrhosis caused by PFIC type 3. An 18-year-old male presented with a history of abdominal distension and jaundice for 2 months. He had abdominal tenderness but no rebounding pain. Moreover, his dullness was felt over the liver and the spleen was palpable 8 cm below the ribs.

UNASSIGNED

Computed tomography and magnetic resonance cholangiopancreato graphy of the upper abdomen revealed cirrhosis, portal hypertension, collateral circulation formation, large spleen, and ascites. Blood biochemistry showed high alanine transaminase, aspartate transaminase, and GGT. The diagnosis of decompensated liver cirrhosis caused by PFIC-3 was finally confirmed by plasma gene detecting.

METHODS

The patient received an open surgery named allogeneic liver transplantation after successful matching of immune types between the recipient and donor. Peritoneal puncture and catheter drainage under B-ultrasound was performed when an encapsulated effusion between the liver and stomach arose.

RESULTS

The patient was discharged without specific discomfort and was almost free of fluid accumulation 51 days after the surgery. At the 6-month follow-up, he had no discomfort and the blood routine, liver functions showed no abnormalities.

CONCLUSIONS

We found a new mutant fragment of ABCB4 gene in the process of diagnosis. Liver transplantation remains the most definitive treatment for PFIC. Current medical therapies and surgical interventions such as biliary diversion have potentially created a synergistic outcome.

Objective: Intrahepatic cholestasis of pregnancy (ICP) is a liver disorder of pregnancy characterized by pruritus, elevated liver enzymes and fasting serum bile acids. Genetic predisposition has been suggested to play a role in its etiology and mutations in the ATP8B1(OMIM ∗602397) (FIC1), ABCB11(OMIM ∗603201) (BSEP), and ABCB4(OMIM ∗171060) (MDR3) genes have been implicated. In the present study, we aimed to investigate the possible role of ATP8B1, ABCB11, and ABCB4 gene mutations in the patients with ICP.

Materials and methods: A total of 25 patients who were diagnosed with ICP were included in the study. Genetic test results and mutation status of the patients as assessed by the next-generation sequencing technology were retrospectively retrieved from the hospital database.

Results: Of all patients, significant alterations in the ATP8B1 (n = 2), ABCB11 (n = 1), and ABCB4 (n = 7) genes were observed in 10 patients using the molecular analysis testing. All these alterations were heterozygous. Of these alterations, four were reported in the literature previously, while six were not. Using the in-silico parameters, there was a pathogenic alteration in the ABCB4 gene in one patient, while there was no clinically relevant alteration in the other gene mutations in the remaining nine patients.

Conclusion: Considering the fact that the alterations were compatible with clinical presentations of the ICP patients and the incidence of these mutations is low in the general population, we believe that our study results are clinically relevant. Further molecular genetic tests in ICP patients and functional studies supporting the results would shed light into the clinical importance of these alterations.

Keywords: ABCB11; ABCB4; ATP8B1; Intrahepatic cholestasis of pregnancy; Mutation.

The transcription and protein activity of defence mechanisms such as ABC transporters, phase I and II of cellular detoxification and antioxidant enzymes can be altered in the presence of emerging contaminants such as pharmaceuticals impacting the overall detoxification mechanism. The present work aimed to characterise the effects of simvastatin on the detoxification mechanisms of embryonic stages of Danio rerio. In a first approach, constitutive transcription of key genes involved in detoxification was determined. Embryos were collected at different developmental stages, and transcription patterns of genes coding for ABC transporters, phase I and II and oxidative stress were analysed. With exception of abcc2, all genes seem to be from maternal transfer (0-2 hpf). Embryos were then exposed to different concentrations of simvastatin (5 and 50 μg/L), verapamil and MK571 (10 μM; ABC protein inhibitors) and a combination of simvastatin and ABC inhibitors. mRNA expression levels of abcb4, abcc1, abcc2, abcg2, cyp1a, cyp3a65, gst, sod, cat was evaluated. Accumulation assays to measure ABC proteins activity and activity of EROD, GST, CAT and Cu/ZnSOD, were also undertaken. Simvastatin acted as a weak inhibitor of ABC proteins and increased EROD and GST activity, whereas Cu/ZnSOD and CAT activity were decreased. Simvastatin up-regulated abcb4 and cyp3a65 transcription (both concentrations), as well as abcc1 and abcc2 at 50 μg/L, and down-regulated gst, sod, cat at 5 μg/L. In conclusion, our data revealed the interaction of simvastatin with detoxification mechanisms highlighting the importance of monitoring the presence of this emerging contaminant in aquatic environments.

Fusion and apoptosis share a breakdown of the membrane phospholipids asymmetry, modes of which are largely unknown in osteoclastogenesis. Here, we investigated the externalization of phosphatidylserine (PS) and its receptors, and their biological functions in osteoclastogenesis. Strong immunoreactivities in vivo for the PS receptors TIM4, BAI1, and STAB2 were observed in the TRAP-positive multinucleated cells in the alveolar bone that was being remodeled around the developing dental follicles in rats. These receptors were significantly upregulated during M-CSF/RANKL-induced in vitro osteoclastogenesis using mouse bone marrow-derived cells. PS externalization in preosteoclasts was increased by the M-CSF/RANKL treatment. Multinucleation of preosteoclasts was markedly inhibited by antibodies against PS and its receptors. Among the investigated lipid transporter proteins, floppases (Abcb4, Abcc5, and Abcg1) were upregulated, whereas flippases (Atp11c and Atp8a1) downregulated during osteoclastogenesis. Preosteoclast fusion was markedly blocked by the ATPase inhibitor Na₃VO₄ and siRNAs against Abcc5 and Abcg1, revealing the importance of these lipid transporters in PS externalization. Further, the levels of Cd47 and Cd31, don't-eat-me signal inducers, were increased or sustained in the early phase of osteoclastogenesis, whereas those of AnnexinI and Mfg-e8, eat-me signals inducers, were increased in the late apoptotic phase. In addition, Z-VAD-FMK, a pan caspase inhibitor, had no effect on preosteoclast fusion in the early phase of osteoclastogenesis, whereas Abs against PS, TIM4, and BAI1 decreased osteoclast apoptosis during the late phase. These results suggest that PS externalization is essential for the whole process of osteoclastogenesis and share PS receptors and transporters in the early stage fusion and late stage apoptosis. Therefore, modulation of PS and its receptors could be a useful strategy to develop anti-bone resorptive agents.

Understanding of the pathophysiology of cholestasis associated carcinogenesis could challenge the development of new personalized therapeutic approaches and thus improve prognosis. Simultaneous damage might aggravate hepatic injury, induce chronic liver disease and even promote carcinogenesis. We aimed to study the effect of Hepatitis B virus surface protein (HBsAg) on cholestatic liver disease and associated carcinogenesis in a mouse model combining both impairments. Hybrids of Abcb4-/- and HBsAg transgenic mice were bred on fibrosis susceptible background BALB/c. Liver injury, serum bile acid concentration, hepatic fibrosis, and carcinogenesis were enhanced by the combination of simultaneous damage in line with activation of c-Jun N-terminal kinase (JNK), proto-oncogene c-Jun, and Signal transducer and activator of transcription 3 (STAT3). Activation of Protein Kinase RNA-like Endoplasmic Reticulum Kinase (PERK) and Eukaryotic translation initiation factor 2A (eIF2α) indicated unfolded protein response (UPR) in HBsAg-expressing mice and even in Abcb4-/- without HBsAg-expression.

CONCLUSIONS

Cholestasis-induced STAT3- and JNK-pathways may predispose HBsAg-associated tumorigenesis. Since STAT3- and JNK-activation are well characterized critical regulators for tumor promotion, the potentiation of their activation in hybrids suggests an additive mechanism enhancing tumor incidence.

Background Progressive familial intrahepatic cholestasis type 3 (PFIC3) is an uncommon cholestatic liver disease caused by mutations in the ATP binding cassette subfamily B member 4 (ABCB4) gene. Although PFIC3 is frequently identified in childhood, ABCB4 disease-causing alleles have been described in adults affected by intrahepatic cholestasis of pregnancy, hormone-induced cholestasis, low-phospholipid-associated cholelithiasis syndrome or juvenile cholelithiasis, cholangiocarcinoma and in sporadic forms of primary biliary cirrhosis. Cholestanol is a biomarker which is elevated especially in cerebrotendinous xanthomatosis and rarely in primary biliary cirrhosis (PBC) and Niemann Pick type C. Case presentation Here we report a Turkish patient with compound heterozygous mutations in the ABCB4 gene, who has hepatosplenomegaly, low level of high-density lipoprotein, cholestasis and high level of cholestanol. Conclusion This is the first PFIC3 case with a high cholestanol level described in the literature. There are very few diseases linked to increased cholestanol levels, two of which are CTX and PBC. From this case, we can conclude that a high cholestanol level might be another indicator of PFIC type 3.

Background&aims: Primary sclerosing cholangitis (PSC) is characterized by chronic inflammation and progressive fibrosis of the biliary tree. The bile acid receptor TGR5 is found on biliary epithelial cells (BECs), where it promotes secretion, proliferation and tight junction integrity. Thus, we speculated that changes in TGR5-expression in BECs may contribute to PSC pathogenesis.

Methods: TGR5-expression and -localization were analyzed in PSC livers and liver tissue, isolated bile ducts and BECs from Abcb4^-/-, Abcb4^-/-/Tgr5^Tg and UDCA-as well as norUDCA-fed Abcb4^-/- mice. Effects of IL8/IL8 homologues on TGR5 mRNA and protein levels were studied. BEC gene expression was analyzed by single-cell transcriptomics (scRNA-seq) from distinct mouse models.

Results: TGR5 mRNA expression and immunofluorescence staining intensity were reduced in BECs of PSC and Abcb4^-/- livers, in Abcb4^-/- extrahepatic bile ducts, but not in intrahepatic macrophages. No changes in TGR5 BEC fluorescence intensity were detected in liver tissue of other liver diseases, including primary biliary cholangitis (PBC). Incubation of BECs with IL8/IL8 homologues, but not with other cytokines, reduced TGR5 mRNA and protein levels. BECs from Abcb4^-/- mice had lower levels of phosphorylated Erk and higher expression levels of Icam1, Vcam1 and Tgfβ2. Overexpression of Tgr5 abolished the activated inflammatory phenotype characteristic of Abcb4^-/- BECs. NorUDCA-feeding restored TGR5-expression levels in BECs in Abcb4^-/- livers.

Conclusions: Reduced TGR5 levels in BECs of PSC and Abcb4^-/- deficiency promote development of a reactive BEC phenotype, aggravate biliary injury and thus contribute to sclerosing cholangitis pathogenesis. Restoration of biliary TGR5-expression levels represents a previously unknown mechanism of action of norUDCA.

Lay summary: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease-associated with progressive inflammation of the bile duct, leading to fibrosis and end-stage liver disease. Bile acid (BA) toxicity may contribute to the development and disease progression of PSC. TGR5 is a membrane-bound receptor for BAs, which is found on bile ducts and protects bile ducts from BA toxicity. In this study, we investigated whether changes in TGR5 levels occur on bile duct cells. TGR5 levels were reduced in bile ducts from PSC livers and also in bile ducts from a genetic mouse model of PSC. Genetically modified mice with high quantities of TGR5 in their biliary epithelial cells only developed a milder form of sclerosing cholangitis as compared to their control littermates. A similar beneficial effect could be obtained by norUDCA-feeding to the PSC mice. These findings indicate that lower levels of TGR5 in bile ducts may contribute to PSC development and progression. Furthermore, treatment with norUDCA, a drug currently being tested in a phase III trial for PSC, restored TGR5-levels in biliary epithelial cells.

Keywords: Bile acid receptor; Biliary damage; Biliary organoids; Interleukin-8; norUDCA; scRNA-seq.