Phosphatidylcholine Secretion Research Articles

Identification of new correctors for traffic-defective ABCB4 variants by a high-content screening approach

ABCB4 is located at the canalicular membrane of hepatocytes and is responsible for the secretion of phosphatidylcholine into bile. Genetic variations of this transporter are correlated with rare cholestatic liver diseases, the most severe being progressive familial intrahepatic cholestasis type 3 (PFIC3). PFIC3 patients most often require liver transplantation. In this context of unmet medical need, we developed a high-content screening approach to identify small molecules able to correct ABCB4 molecular defects. Intracellularly-retained variants of ABCB4 were expressed in cell models and their maturation, cellular localization and function were analyzed after treatment with the molecules identified by high-content screening. In total, six hits were identified by high-content screening. Three of them were able to correct the maturation and canalicular localization of two distinct intracellularly-retained ABCB4 variants; one molecule was able to significantly restore the function of two ABCB4 variants. In addition, in silico molecular docking calculations suggest that the identified hits may interact with wild type ABCB4 residues involved in ATP binding/hydrolysis. Our results pave the way for their optimization in order to provide new drug candidates as potential alternative to liver transplantation for patients with severe forms of ABCB4-related diseases, including PFIC3.

Searching for low phospholipid associated cholelithiasis among patients with post-cholecystectomy biliary pain.

Low phospholipid associated cholelithiasis (LPAC) is associated with variants of the adenosine triphosphate-binding cassette subfamily B, member 4 (ABCB4) gene and is characterized by reduced phosphatidylcholine secretion into bile, impairing the formation of micelles and thus exposing bile ducts to toxic bile acids and increasing cholesterol saturation. LPAC is present in 1% of patients with gallstones and post-cholecystectomy pain is common in this group. LPAC is an under-appreciated cause of post-cholecystectomy pain. The aim of this study is to assess a cohort of patients with post-cholecystectomy pain to identify those with clinical features suggesting that further investigations for LPAC would be beneficial. A retrospective chart review was performed of the first 2 years of post-operative follow-up for all patients under 40 years of age undergoing cholecystectomy for symptomatic gallstones at a tertiary centre between January 2016 and December 2017. 258 patients under the age of 40 underwent a cholecystectomy. 50 patients (19.4%) reported abdominal pain post-cholecystectomy. Five patients (1.9%) fulfilled the criteria for suspected LPAC. Family history of gallstones was documented in 33 of 258 (12.8%) of cases. Obstetric history was obtained in 69 of 197 (35%) female patients. None of the five patients identified above who satisfied the criteria of LPAC had the diagnosis of LPAC considered by their treating clinicians. LPAC is an under-recognized cause of post-cholecystectomy pain. Treatment can avoid long-term symptoms and complications. Clinicians should take a family history and obstetric history to alert them to the diagnosis of LPAC.

Rumen-protected choline and methionine during the periparturient period affect choline metabolites, amino acids, and hepatic expression of genes associated with one-carbon and lipid metabolism

Feeding supplemental choline and Met during the periparturient period can have positive effects on cow performance; however, the mechanisms by which these nutrients affect performance and metabolism are unclear. The objective of this experiment was to determine if providing rumen-protected choline, rumen-protected Met, or both during the periparturient period modifies the choline metabolitic profile of plasma and milk, plasma AA, and hepatic mRNA expression of genes associated with choline, Met, and lipid metabolism. Cows (25 primiparous, 29 multiparous) were blocked by expected calving date and parity and randomly assigned to 1 of 4 treatments: control (no rumen-protected choline or rumen-protected Met); CHO (13 g/d choline ion); MET (9 g/d DL-methionine prepartum; 13.5 g/d DL-methionine, postpartum); or CHO + MET. Treatments were applied daily as a top dress from ~21 d prepartum through 35 d in milk (DIM). On the day of treatment enrollment (d -19 ± 2 relative to calving), blood samples were collected for covariate measurements. At 7 and 14 DIM, samples of blood and milk were collected for analysis of choline metabolites, including 16 species of phosphatidylcholine (PC) and 4 species of lysophosphatidylcholine (LPC). Blood was also analyzed for AA concentrations. Liver samples collected from multiparous cows on the day of treatment enrollment and at 7 DIM were used for gene expression analysis. There was no consistent effect of CHO or MET on milk or plasma free choline, betaine, sphingomyelin, or glycerophosphocholine. However, CHO increased milk secretion of total LPC irrespective of MET for multiparous cows and in absence of MET for primiparous cows. Furthermore, CHO increased or tended to increase milk secretion of LPC 16:0, LPC 18:1, and LPC 18:0 for primi- and multiparous cows, although the response varied with MET supplementation. Feeding CHO also increased plasma concentrations of LPC 16:0 and LPC 18:1 in absence of MET for multiparous cows. Although milk secretion of total PC was unaffected, CHO and MET increased secretion of 6 and 5 individual PC species for multiparous cows, respectively. Plasma concentrations of total PC and individual PC species were unaffected by CHO or MET for multiparous cows, but MET reduced total PC and 11 PC species during wk 2 postpartum for primiparous cows. Feeding MET consistently increased plasma Met concentrations for both primi- and multiparous cows. Additionally, MET decreased plasma serine concentrations during wk 2 postpartum and increased plasma phenylalanine in absence of CHO for multiparous cows. In absence of MET, CHO tended to increase hepatic mRNA levels of betaine-homocysteine methyltransferase and phosphate cytidylyltransferase 1 choline, α, but tended to decrease expression of 3-hydroxy-3-methylglutaryl-coenzyme A synthase 2 and peroxisome proliferator activated receptor α irrespective of MET. Although shifts in the milk and plasma PC profile were subtle and inconsistent between primi- and multiparous cows, gene expression results suggest that supplemental choline plays a probable role in promoting the cytidine diphosphate-choline and betaine-homocysteine S-methyltransferase pathways. However, interactive effects suggest that this response depends on Met availability, which may explain the inconsistent results observed among studies when supplemental choline is fed.

Ivacaftor-Mediated Potentiation of ABCB4 Missense Mutations Affecting Critical Motifs of the NBDs: Repositioning Perspectives for Hepatobiliary Diseases.

ABCB4 (ATP-binding cassette subfamily B member 4) is a hepatocanalicular floppase involved in biliary phosphatidylcholine (PC) secretion. Variations in the ABCB4 gene give rise to several biliary diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3), an autosomal recessive disease that can be lethal in the absence of liver transplantation. In this study, we investigated the effect and potential rescue of ten ABCB4 missense variations in NBD1:NBD2 homologous positions (Y403H/Y1043H, K435M/K1075M, E558K/E1200A, D564G/D1206G and H589Y/H1231Y) all localized at the conserved and functionally critical motifs of ABC transporters, six of which are mutated in patients. By combining structure analysis and in vitro studies, we found that all ten mutants were normally processed and localized at the canalicular membrane of HepG2 cells, but showed dramatically impaired PC transport activity that was significantly rescued by treatment with the clinically approved CFTR potentiator ivacaftor. Our results provide evidence that functional ABCB4 mutations are rescued by ivacaftor, paving the way for the repositioning of this potentiator for the treatment of selected patients with PFIC3 caused by mutations in the ATP-binding sites of ABCB4.

MRCK-Alpha and Its Effector Myosin II Regulatory Light Chain Bind ABCB4 and Regulate Its Membrane Expression.

ABCB4, is an adenosine triphosphate-binding cassette (ABC) transporter localized at the canalicular membrane of hepatocytes, where it mediates phosphatidylcholine secretion into bile. Gene variations of ABCB4 cause different types of liver diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3). The molecular mechanisms underlying the trafficking of ABCB4 to and from the canalicular membrane are still unknown. We identified the serine/threonine kinase Myotonic dystrophy kinase-related Cdc42-binding kinase isoform α (MRCKα) as a novel partner of ABCB4. The role of MRCKα was explored, either by expression of dominant negative mutant or by gene silencing using the specific RNAi and CRISPR-cas9 strategy in cell models. The expression of a dominant-negative mutant of MRCKα and MRCKα inhibition by chelerythrine both caused a significant increase in ABCB4 steady-state expression in primary human hepatocytes and HEK-293 cells. RNA interference and CRISPR-Cas9 knockout of MRCKα also caused a significant increase in the amount of ABCB4 protein expression. We demonstrated that the effect of MRCKα was mediated by its downstream effector, the myosin II regulatory light chain (MRLC), which was shown to also bind ABCB4. Our findings provide evidence that MRCKα and MRLC bind to ABCB4 and regulate its cell surface expression.

P-82 URSODEOXYCHOLIC ACID AND/OR CIPROFIBRATE FOR TREATING PATIENTS WITH PRESUMPTIVE DIAGNOSIS OF LOW PHOSPHOLIPID CHOLELITHIASIS, A CLINICAL SPECTRUM OF PROGRESSIVE FAMILIAL INTRAHEPATIC CHOLESTASIS TYPE 3

Low Phospholipid-Associated Cholelithiasis (LPAC) is a clinical spectrum of Progressive Familial Intrahepatic Cholestasis type 3 (PFIC3), with mutations in the ABCB4 gene, reduced levels of phosphatidylcholine in bile, formation of cholesterol gallstones, damage of bile ducts epithelium and cholestasis. Ursodeoxycholic acid (UDCA) is effective and fibrates may also be used to activate PPAR - α receptor, inducing bile secretion of phosphatidylcholine. Retrospectively evaluate efficacy and safety of ciprofibrate in LPAC/PFIC3. Diagnosis of PFIC3 was confirmed by detection of mutations of ABCB4 gene. LPAC diagnosis was suggested by 2 out of 5 criteria: biliary symptoms before 40 years; recurrence after cholecystectomy; intrahepatic lithiasis; cholelithiasis in first‐degree relatives; intrahepatic cholestasis of pregnancy or contraceptive‐induced cholestasis. Enzymes, liver function and pruritus were analyzed after 3, 6 and 12 months of UDCA and after ciprofibrate 100mg/day using the Wilcoxon test. 27(93%) patients with clinical diagnosis of LPAC and 2 of PFIC3. 23 (79%) female with mean age at onset of symptoms of 26.7±13.6 years. 23(80%) had family history of biliary disease; 22(76%) cholelithiasis before 40 years; 7(24%) intrahepatic lithiasis. 22/29 (78%) received ciprofibrate after 4.5±4.9 months of UDCA use, in a mean dose of 13.1±2.2mg/kg/day. During UDCA there was a significant decrease in aminotransferases, alkaline phosphatase(AP) and gamma-glutamyltransferase(GGT) levels, without significant improvement in the liver function. After addition of fibrate, pruritus disappeared in all 7 patients, with significant improvement of AP, GGT and albumin in the third month. There was no significant renal dysfunction. Fibrate was discontinued in 8: 1 liver transplantation, 2 irregular use, 5 side effects. 27 patients are still in follow up. Ciprofibrate was beneficial to improve pruritus and laboratory tests in LPAC/PFIC3 after partial response with UDCA. Fibrate therapy was safe and well tolerated.

RAB10 Interacts with ABCB4 and Regulates Its Intracellular Traffic.

ABCB4 (ATP-binding cassette subfamily B member 4) is an ABC transporter expressed at the canalicular membrane of hepatocytes where it ensures phosphatidylcholine secretion into bile. Genetic variations of ABCB4 are associated with several rare cholestatic diseases. The available treatments are not efficient for a significant proportion of patients with ABCB4-related diseases and liver transplantation is often required. The development of novel therapies requires a deep understanding of the molecular mechanisms regulating ABCB4 expression, intracellular traffic, and function. Using an immunoprecipitation approach combined with mass spectrometry analyses, we have identified the small GTPase RAB10 as a novel molecular partner of ABCB4. Our results indicate that the overexpression of wild type RAB10 or its dominant-active mutant significantly increases the amount of ABCB4 at the plasma membrane expression and its phosphatidylcholine floppase function. Contrariwise, RAB10 silencing induces the intracellular retention of ABCB4 and then indirectly diminishes its secretory function. Taken together, our findings suggest that RAB10 regulates the plasma membrane targeting of ABCB4 and consequently its capacity to mediate phosphatidylcholine secretion.

Effect of CFTR correctors on the traffic and the function of intracellularly retained ABCB4 variants.

ABCB4 is expressed at the canalicular membrane of hepatocytes. This ATP-binding cassette (ABC) transporter is responsible for the secretion of phosphatidylcholine into bile canaliculi. Missense genetic variations of ABCB4 are correlated with several rare cholestatic liver diseases, the most severe being progressive familial intrahepatic cholestasis type 3 (PFIC3). In a repurposing strategy to correct intracellularly retained ABCB4 variants, we tested 16 compounds previously validated as cystic fibrosis transmembrane conductance regulator (CFTR) correctors. The maturation, intracellular localization and activity of intracellularly retained ABCB4 variants were analyzed in cell models after treatment with CFTR correctors. In addition, in silico molecular docking calculations were performed to test the potential interaction of CFTR correctors with ABCB4. We observed that the correctors C10, C13, and C17, as well as the combinations of C3+C18 and C4+C18, allowed the rescue of maturation and canalicular localization of four distinct traffic-defective ABCB4 variants. However, such treatments did not permit a rescue of the phosphatidylcholine secretion activity of these defective variants and were also inhibitory of the activity of wild type ABCB4. In silico molecular docking analyses suggest that these CFTR correctors might directly interact with transmembrane domains and/or ATP-binding sites of the transporter. Our results illustrate the uncoupling between the traffic and the activity of ABCB4 because the same molecules can rescue the traffic of defective variants while they inhibit the secretion activity of the transporter. We expect that this study will help to design new pharmacological tools with potential clinical interest.

Mechanism of Taurohyodeoxycholate-induced Biliary Phospholipid Efflux -Understanding the Function of the ABCB4 Enhancer for Developing Therapeutic Agents against Bile Salt-induced Liver Injury

Biliary lipids primarily consist of bile salts, phospholipids, and cholesterol. Bile salts have potent detergent properties and deleterious effects on the cell membrane and are cytotoxic to hepatocytes. We have previously reported that phosphatidylcholine (PC), the predominant bile phospholipid, protects hepatocytes from the cytotoxicity of bile salts, whereas cholesterol reverses the cytoprotective effects of PC against bile salts. ABCB4, a member of the ATP-binding cassette transporter family, secretes biliary phospholipids, especially PC, from the hepatocytes into the bile. Using Abcb4 knockout mice and HEK293 cells that stably expressed ABCB4, we examined the effects of taurine- or glycine-conjugated cholate, ursodeoxycholate, and hyodeoxycholate on the ABCB4-mediated efflux of PC. We observed that the biliary secretion of PC in wild-type mice significantly increased following infusion of all the tested bile salts, especially taurohyodeoxycholate. On the other hand, the biliary secretion of PC in Abcb4 knockout mice was not affected by the bile salt infusions. The results also demonstrated that the efflux of PC from ABCB4-expressing HEK293 cells was significantly stimulated by taurohyodeoxycholate, which has a strong potential to form mixed micelles with PC. Furthermore, the results of our study emphasized the possibility that the specific interactions of bile salts with ABCB4 are necessary for the release of PC molecules from the binding pocket of ABCB4 into the aqueous environment. Further understanding of this mechanism will aid in the development of novel therapeutic agents for cholestatic liver diseases.

Low phospholipid-associated cholelithiasis syndrome: A rare cause of acute pancreatitis that should not be neglected.

BACKGROUNDLow phospholipid-associated cholelithiasis (LPAC) syndrome is a very particular form of biliary lithiasis with no excess of cholesterol secretion into bile, but a decrease in phosphatidylcholine secretion, which is responsible for stones forming not only in the gallbladder, but also in the liver. LPAC syndrome may be underreported due to a lack of testing resulting from insufficient awareness among clinicians.AIMTo describe the clinical and radiological characteristics of patients with LPAC syndrome to better identify and diagnose the disease.METHODSWe prospectively evaluated all patients aged over 18 years old who were consulted or hospitalized in two hospitals in Paris, France (Bichat University Hospital and Croix-Saint-Simon Hospital) between January 1, 2017 and August 31, 2018. All patients whose profiles led to a clinical suspicion of LPAC syndrome underwent a liver ultrasound examination performed by an experienced radiologist to confirm the diagnosis of LPAC syndrome. Twenty-four patients were selected. Data about the patients’ general characteristics, their medical history, their symptoms, and their blood tests results were collected during both their initial hospitalization and follow-up. Cytolysis and cholestasis were expressed compared to the normal values (N) of serum aspartate and alanine transaminase activities, and to the normal value of alkaline phosphatase level, respectively. The subjects were systematically reevaluated and asked about their symptoms 6 mo after inclusion in the study through an in-person medical appointment or phone call. Genetic testing was not performed systematically, but according to the decision of each physician.RESULTSMost patients were young (median age of 37 years), male (58%), and not overweight (median body mass index was 24). Many had a personal history of acute pancreatitis (54%) or cholecystectomy (42%), and a family history of gallstones in first-degree relatives (30%). LPAC syndrome was identified primarily in patients with recurring biliary pain (88%) or after a new episode of acute pancreatitis (38%). When present, cytolysis and cholestasis were not severe (2.8N and 1.7N, respectively) and disappeared quickly. Interestingly, four patients from the same family were diagnosed with LPAC syndrome. At ultrasound examination, the most frequent findings in intrahepatic bile ducts were comet-tail artifacts (96%), microlithiasis (83%), and acoustic shadows (71%). Computed tomography scans and magnetic resonance imaging were performed on 15 and three patients, respectively, but microlithiasis was not detected. Complications of LPAC syndrome required hospitalizing 18 patients (75%) in a conventional care unit for a mean duration of 6.8 d. None of them died. Treatment with ursodeoxycholic acid (UDCA) was effective and well-tolerated in almost all patients (94%) with a rapid onset of action (3.4 wk). Twelve patients’ (67%) adherence to UDCA treatment was considered “good.” Five patients (36%) underwent cholecystectomy (three of them were treated both by UDCA and cholecystectomy). Despite UDCA efficacy, biliary pain recurred in five patients (28%), three of whom adhered well to treatment guidelines.CONCLUSIONLPAC syndrome is easy to diagnose and treat; therefore, it should no longer be overlooked. To increase its detection rate, all patients who experience recurrent biliary symptoms following an episode of acute pancreatitis should undergo an ultrasound examination performed by a radiologist with knowledge of the disease.

Enhancing effect of taurohyodeoxycholate on ABCB4-mediated phospholipid efflux

Hydrophilic bile salts, ursodeoxycholate and hyodeoxycholate, have choleretic effects. ABCB4, a member of the ABC transporter family, is essential for the secretion of phospholipids from hepatocytes into bile. In this study, we assessed the effects of taurine- or glycine-conjugated cholate, ursodeoxycholate and hyodeoxycholate on the ABCB4-mediated phosphatidylcholine (PC) efflux using Abcb4 knockout mice and HEK293 cells stably expressing ABCB4. To evaluate the effects of bile salts on bile formation in Abcb4+/+ or Abcb4−/− mice, the bile was collected during intravenous infusion of saline or bile salts. The biliary PC secretion in Abcb4+/+ mice was significantly increased by the infusions of all tested bile salts, especially taurohyodeoxycholate. On the other hand, Abcb4−/− mice exhibited extremely low secretion of PC into bile, which was not altered by bile salt infusions. We also showed that the PC efflux from ABCB4-expressing HEK293 cells was stimulated by taurohyodeoxycholate much more strongly than the other tested bile salts. However, taurohyodeoxycholate did not restore the activities of ABCB4 mutants. Furthermore, light scattering measurements demonstrated a remarkable ability of taurohyodeoxycholate to form mixed micelles with PC. Therefore, the enhancing effect of taurohyodeoxycholate on the ABCB4-mediated PC efflux may be due to the strong mixed micelle formation ability.

Thyroid hormone receptor β1 stimulates ABCB4 to increase biliary phosphatidylcholine excretion in mice

The ATP-binding cassette transporter ABCB4/MDR3 is critical for biliary phosphatidylcholine (PC) excretion at the canalicular membrane of hepatocytes. Defective ABCB4 gene expression and protein function result in various cholestatic liver and bile duct injuries. Thyroid hormone receptor (THR) is a major regulator of hepatic lipid metabolism; we explored its potential role in ABCB4 regulation. Thyroid hormone T3 stimulation to human hepatocyte models showed direct transcriptional activation of ABCB4 in a dose- and time-dependent manner. To determine whether THRβ1 (the main THR isoform of the liver) is involved in regulation, we tested THRβ1-specific agonists (e.g., GC-1, KB-141); these agonists resulted in greater stimulation than the native hormone. KB-141 activated hepatic ABCB44 expression in mice, which enhanced biliary PC secretion in vivo. We also identified THR response elements 6 kb upstream of the ABCB4 locus that were conserved in humans and mice. Thus, T3-via THRβ1 as a novel transcriptional activator regulates ABCB4 to increase ABCB4 protein levels at the canalicular membrane and promote PC secretion into bile. These findings may have important implications for understanding thyroid hormone function as a potential modifier of bile duct homeostasis and provide pharmacologic opportunities to improve liver function in hepatobiliary diseases caused by low ABCB4 expression.

Intestinal de novo phosphatidylcholine synthesis is required for dietary lipid absorption and metabolic homeostasis

De novo phosphatidylcholine (PC) synthesis via CTP:phosphocholine cytidylyltransferase-α (CTα) is required for VLDL secretion. To determine the precise role of de novo PC synthesis in intestinal lipid metabolism, we deleted CTα exclusively in the intestinal epithelium of mice (CTαIKO mice). When fed a chow diet, CTαIKO mice showed normal fat absorption despite a ∼30% decrease in intestinal PC concentrations relative to control mice, suggesting that biliary PC can fully support chylomicron secretion under these conditions. However, when fed a high-fat diet, CTαIKO mice showed impaired passage of FAs and cholesterol from the intestinal lumen into enterocytes. Impaired intestinal lipid uptake in CTαIKO mice was associated with lower plasma triglyceride concentrations, higher plasma glucagon-like peptide 1 and peptide YY, and disruption of intestinal membrane lipid transporters after a high-fat meal relative to control mice. Unexpectedly, biliary bile acid and PC secretion was enhanced in CTαIKO mice due to a shift in expression of bile-acid transporters to the proximal intestine, indicative of accelerated enterohepatic cycling. These data show that intestinal de novo PC synthesis is required for dietary lipid absorption during high-fat feeding and that the reacylation of biliary lyso-PC cannot compensate for loss of CTα under these conditions.

STAR‐RELATED LIPID TRANSFER PROTEIN 10 AS NOVEL KEY PLAYER IN ETHANOL‐INDUCED ERBB2 BREAST CANCER PROGRESSION

ProposeEpidemiological studies demonstrated that ethanol administration promotes breast cancer development and metastasis. Human breast cancer cells with a high expression of Erb‐B2 Receptor Tyrosine Kinase 2 (ErbB2) exhibited an enhanced response to ethanol‐stimulated cell invasion in vitro triggering the phosphorylation status of mitogen‐activated protein kinase (MAPKs). ErbB2 amplification and/or overexpression has been described in 20–30% of human breast cancers and correlates with poor prognosis. StAR‐related lipid transfer protein 10 (StarD10) is a member of the StarD protein family and lipid transfer protein with selective binding site to phosphatidylcholine (PC) and phosphatidylethanolamine (PE), two potential precursors for lipid metabolism and a major constituent of cell membranes. Choline metabolism and its derived metabolites can produce extensive alterations on phospholipid membrane composition that usually occurs before the morphological tumorigenic changes. StarD10 is highly expressed in 35% of ErbB2‐positive breast cancer suggesting a selective growth advantage and cellular transformation for tumor expressing both proteins. The goal of this study is to investigate the role of StarD10 and ErbB2 cross‐talk in breast cancer as consequence of ethanol administration and elucidate the molecular mechanisms.MethodsThe experiments were performed using MCF‐7 and SKBR‐3 human carcinoma cell lines. mRNA and protein levels were analyzed by Real‐Time PCR and Western Blotting, respectively. StarD10 promoter activity and cell proliferation were performed by promoter reporter and MTT assay, respectively. Phosphatidylcholine level was measured using an enzyme‐coupled assay.ResultsCells treated with 100 mM ethanol for 48 hours showed increased expression of StarD10 and ErbB2. Consistently, ErbB2 overexpression caused an increase of StarD10 expression. Using PROMO program to predict transcription factor binding sites in DNA sequences, we found out that p65, c‐MYC, c‐FOS and c‐JUN, well known to be transcription factors (TFs) ErbB2 downstream targets, are highly predicted to bind StardD10 promoter sequence. Overexpression of all predicted TFs induced StarD10 promoter activity mimicking ErbB2 trigger function. Also, we demonstrated that StarD10 and ErbB2 positively regulate each other's expression. Moreover, ethanol increased phosphatidylcholine level supporting the proposed role of StarD10 as player on tumorigenesis via its lipid transporter function. Interesting, we found that both Stard10 overexpression and silencing induced cell growth and migration in MCF‐7 and SKBR‐3 cells. This data validated the hypothesis of StarD10 is a key protein on induced‐ethanol breast cancer development accordingly with its pathophysiological level. High level of secreted PC was found in ethanol‐treated cells media, while StarD10 silencing completely prevented it. In contrast, StarD10 overexpression promoted PC secretion and induced it further in co‐treatment with ethanol. This finding could help us to explain how potentially StarD10 controls the changes in cell membrane properties during malignancy may be modulating the membrane fluidity.ConclusionsThe ability of Stard10 to influence ErbB2 expression and activity may be involve both dependent and independent lipid binding function. This is the first report demonstrating that ethanol can modulate in dynamic manner the ErbB2 role through StarD10 involvement in breast cancer.Support or Funding InformationNIAAA 5 K01 AA022372‐06This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Comparison of in silico prediction and experimental assessment of ABCB4 variants identified in patients with biliary diseases

Genetic variations of the phosphatidylcholine transporter, ABCB4 cause several biliary diseases. The large number of reported variations makes it difficult to foresee a comprehensive study of each variation. To appreciate the reliability of in silico prediction programs, 1) we confronted them with the assessment in cell models of two ABCB4 variations (E528D and P1161S) identified in patients with low phospholipid-associated cholelithiasis (LPAC); 2) we extended the confrontation to 19 variations that we had previously characterized in cellulo. Four programs (Provean, Polyphen-2, PhD-SNP and MutPred) were used to predict the degree of pathogenicity. The E528D and P1161S variants were studied in transfected HEK293 and HepG2 cells by immunofluorescence, immunoblotting and measurement of phosphatidylcholine secretion. All prediction tools qualified the P1161S variation as deleterious, but provided conflicting results for E528D. In cell models, both mutants were expressed and localized as the wild type but their activity was significantly reduced, by 48% (P1161S) and 33% (E528D). These functional defects best correlated with MutPred predictions. MutPred program also proved the most accurate to predict the pathogenicity of the 19 ABCB4 variants that we previously characterized in cell models, and the most sensitive to predict the pathogenicity of 65 additional mutations of the Human Gene Mutation Database. These results confirm the pathogenicity of E528D and P1161S variations and suggest that even a moderate decrease (by less than 50%) of phosphatidylcholine secretion can cause LPAC syndrome. They highlight the reliability of in silico prediction tools, most notably MutPred, as a first approach to predict the pathogenicity of ABCB4 variants.

8-Methoxypsoralen disrupts MDR3-mediated phospholipids efflux and bile acid homeostasis and its relevance to hepatotoxicity.

Since its discovery in 1987, multidrug resistance 3 P-glycoprotein (MDR3) had recognized to play a crucial role in the translocation of phospholipids from the inner to outer leaflets of bile canalicular membranes. An increasing number of reports suggest that drug-mediated functional disruption of MDR3 is responsible for drug-induced cholestasis. 8-Methoxypsoralen (8-MOP) is used clinically to treat psoriasis, vitiligo and other skin disorders. However, psoralens safety for long-term use is a concern. In the current study, we evaluate 8-MOP's potential hepatotoxicity and effects on bile formation. Sprague Dawley (SD) rats were treated daily 200mg/kg or 400mg/kg of 8-MOP orally for 28 days. The result showed a prominent decrease in biliary phospholipids output, which associated with the down-regulation of MDR3. Elevated bile acid serum level and increased biliary bile acid outputs were observed in 8-MOP-treated groups. The disturbance of bile acid homeostasis was associated with changes in enzymes and proteins involved in bile acid synthesis, regulation and transport. Human liver cell line L02 was used to determine on the mRNA and protein levels of MDR3. Cells treated with 8-MOP reveled a decrease in fluorescent PC (phosphatidylcholine) secretion into the pseudocanaliculi (formed between adjacent cells) compared with untreated cells. Our investigation represent the first evidence that 8-MOP can induce cholestatic liver injury by disturbing MDR3-mediated phospholipids efflux and bile acid homeostasis.

Exogenous gene transfer of Rab38 small GTPase ameliorates aberrant lung surfactant homeostasis in Ruby rats

BackgroundRab38 small GTPase regulates intracellular transport in melanocytes and alveolar type II epithelial cells. Ruby rats carrying Rab38 and other gene mutations exhibit oculocutaneous albinism, bleeding diathesis, and hence, are a rat model of human Hermansky-Pudlak syndrome (HPS). We previously showed that Long Evans Cinnamon (LEC) rats, one strain of the Ruby rats, developed aberrant lung surfactant homeostasis with remarkably enlarged lamellar bodies in alveolar type II cells.MethodsA replication-deficient recombinant adenovirus expressing rat Rab38 (Ad-Rab38) was constructed. Alveolar type II cells were isolated from the LEC rats and tested for lung surfactant phosphatidylcholine secretion. The rats were also examined whether exogenous expression of Ad- Rab38 could rescue the altered lung surfactant homeostasis in the lungs.ResultsIsolated type II cells infected with Ad-Rab38 exhibited improved secretion patterns of [3H]phosphatidylcholine, i.e. increased basal hyposecretion and decreased agonist-induced hypersecretion. Endobronchial administration of Ad-Rab38 improved the morphology of type II cells and lamellar bodies, reducing their sizes close to those of wild-type rats. The increased amounts of phosphatidylcholine and surfactant protein B in the lamellar body fractions were decreased in the Ad-Rab38 infected lungs.ConclusionsThese results provide strong evidence that the aberrant lung surfactant homeostasis in the LEC rats is caused by Rab38 deficit, and suggest that endobronchial delivery of the responsive transgene could be an effective method to ameliorate the abnormal lung phenotype in the animal model of HPS.

Phosphatidylcholine passes through lateral tight junctions for paracellular transport to the apical side of the polarized intestinal tumor cell-line CaCo2

Phosphatidylcholine (PC) is the most abundant phospholipid in intestinal mucus, indicative of a specific transport system across the mucosal epithelium to the intestinal lumen. To elucidate this transport mechanism, we employed a transwell tissue culture system with polarized CaCo2 cells. It was shown that PC could not substantially be internalized by the cells. However, after basal application of increasing PC concentrations, an apical transport of 47.1±6.3nmolh−1mMPC−1 was observed. Equilibrium distribution studies with PC applied in equal concentrations to the basal and apical compartments showed a 1.5-fold accumulation on the expense of basal PC. Disruption of tight junctions (TJ) by acetaldehyde or PPARγ inhibitors or by treatment with siRNA to TJ proteins suppressed paracellular transport by at least 50%. Transport was specific for the choline containing the phospholipids PC, lysoPC and sphingomyelin. We showed that translocation is driven by an electrochemical gradient generated by apical accumulation of Cl− and HCO3− through CFTR. Pretreatment with siRNA to mucin 3 which anchors in the apical plasma membrane of mucosal cells inhibited the final step of luminal PC secretion. PC accumulates in intestinal mucus using a paracellular, apically directed transport route across TJs.

Role of Multidrug Resistance Protein 3 in Antifungal-Induced Cholestasis.

Drug-induced liver injury is an important clinical entity resulting in a considerable number of hospitalizations. While drug-induced cholestasis due to the inhibition of the bile salt export pump (BSEP) is well investigated, only limited information on the interaction of drugs with multidrug resistance protein 3 (MDR3) exists and its role in the pathogenesis of drug-induced cholestasis is poorly understood. Therefore, we aimed to study the interaction of drugs with MDR3 and the effect of drugs on canalicular lipid secretion in a newly established polarized cell line system that serves as a model of canalicular lipid secretion. LLC-PK1 cells were stably transfected with human Na(+)-taurocholate cotransporting polypeptide, BSEP, MDR3, and ABCG5/G8 and grown in the Transwell system. Apical phospholipid secretion and taurocholate transport were assayed to investigate the effect of selected drugs on MDR3-mediated phospholipid secretion as well as inhibition of BSEP. The established cell line displayed vectorial bile salt transport and specific phosphatidylcholine secretion into the apical compartment. The antifungal azoles, posaconazole, itraconazole, and ketoconazole, significantly inhibited MDR3-mediated phosphatidylcholine secretion. In contrast, amoxicillin clavulanate and troglitazone did not interfere with MDR3 activity. Drugs interfering with MDR3 activity did not display a parallel inhibition of BSEP. Our in vitro model for MDR3-mediated phospholipid secretion facilitates parallel screening for MDR3 and BSEP inhibitors. Our data demonstrate that the cholestatic potential of certain drugs may be aggravated by simultaneous inhibition of BSEP and MDR3.

Functional Rescue of Trafficking-Impaired ABCB4 Mutants by Chemical Chaperones.

Multidrug resistance protein 3 (MDR3, ABCB4) is a hepatocellular membrane protein that mediates biliary secretion of phosphatidylcholine. Null mutations in ABCB4 gene give rise to severe early-onset cholestatic liver disease. We have previously shown that the disease-associated mutations p.G68R, p.G228R, p.D459H, and p.A934T resulted in retention of ABCB4 in the endoplasmic reticulum, thus failing to target the plasma membrane. In the present study, we tested the ability of two compounds with chaperone-like activity, 4-phenylbutyrate and curcumin, to rescue these ABCB4 mutants by assessing their effects on subcellular localization, protein maturation, and phospholipid efflux capability. Incubation of transfected cells at a reduced temperature (30°C) or exposure to pharmacological doses of either 4-PBA or curcumin restored cell surface expression of mutants G228R and A934T. The delivery of these mutants to the plasma membrane was accompanied by a switch in the ratio of mature to inmature protein forms, leading to a predominant expression of the mature protein. This effect was due to an improvement in the maturation rate and not to the stabilization of the mature forms. Both mutants were also functionally rescued, displaying bile salt-dependent phospholipid efflux activity after addition of 4-PBA or curcumin. Drug-induced rescue was mutant specific, given neither 4-PBA nor curcumin had an effect on the ABCB4 mutants G68R and A934T. Collectively, these data indicate that the functionality of selected trafficking-defective ABCB4 mutants can be recovered by chemical chaperones through restoration of membrane localization, suggesting a potential treatment for patients carrying such mutations.