Characterization of OATP1B3-1B7 (LST-3TM12) - a novel transporter of the OATP1B family

The effectiveness of a drug is determined by its pharmacodynamic and pharmacokinetic properties. While pharmacodynamics describes the interaction between the target structure and the drug, pharmacokinetics is an umbrella term for all the processes influencing the entry of a drug into the organism and eventually its elimination from the organism. An important mechanism that affects the pharmacokinetics of a drug is its transport across membranes by uptake and efflux transporters. Two uptake transporters that have been extensively studied for their impact on pharmacokinetics are OATP1B3 and OATP1B1. These two transporters are encoded on chromosome 12 by the genes SLCO1B3 and SLCO1B1, respectively. Between these genes lies another gene locus annotated as SLCO1B7. This gene is deemed to be a pseudogene as no function has been reported for its translational product. In 2005, an mRNA sequence named LST-3TM12 that is highly similar to SLCO1B7 was submitted to the National Center of Biotechnology (NCBI) by Mizutamari, H. and Abe, T. (NCBI#, AY257470). The aim of this thesis was to assess the function and transcriptional regulation of this mRNA. By aligning the transcripts of SLCO1B3, SLCO1B7, and LST-3TM12, we could show that the initial five exons of LST-3TM12 originate from SLCO1B3 and the remaining part of LST-3TM12 is encoded by SLCO1B7. Due to this finding, LST-3TM12 is referred to as OATP1B3-1B7 in this thesis. Because the OATP1B3-1B7 mRNA and OATP1B3 have the same 5’UTR it seemed likely that they share the same promoter, which was corroborated by our finding that silencing the exon 4 of SLCO1B3 significantly inhibited OATP1B3-1B7 mRNA transcription. Given that the gene SLCO1B3 is controlled by, among others, farnesoid X receptor (FXR), we tested and confirmed that FXR also regulates OATP1B3-1B7 transcription. Hence, OATP1B3-1B7 is part of the FXR regulated gene-network. Our functional assessments of OATP1B3-1B7 revealed that OATP1B3-1B7 transports exogenous and endogenous compounds. Endogenous substances were dehydroepiandrosterone sulfate (DHEAS), estradiol β-D-glucuronide (E2G), taurocholic acid (TCA), and lithocholic acid (LCA). Exogenous substances were the drugs ezetimibe and atorvastatin. Real-time PCR assessment of OATP1B3-1B7 mRNA showed that it is highly expressed in the liver and, to a lesser extent, in the small intestine. Consequently, the protein OATP1B3-1B7 is detectable in the liver and intestine. Strikingly, the cellular location of OATP1B3-1B7 is not sinusoidal, as is the case for OATP1B1 and OATP1B3, but it is located in the smooth endoplasmic reticulum (SER). Given that OATP1B3-1B7 has a broad substrate range and is expressed in tissues dedicated to metabolism, we hypothesized that OATP1B3-1B7 could have a function related to the high metabolic activity of these tissues. One enzyme class that is highly expressed in such tissues are uridine 5'-diphospho-glucuronosyltransferases (UGTs). UGTs are anchored in the SER membrane and have their active enzymatic site facing the SER lumen. However, it is still not clearly understood how UGT substrates reach and leave the active enzymatic site. As OATP1B3-1B7 is a SER transporter, we assessed whether it could provide access to or exit from the lumen and thus contribute to the metabolic activity of the UGTs. We have investigated this hypothesis with regard to ezetimibe, which is a substrate of OATP1B3-1B7 and is highly metabolized by UGTs. In this case, we were able to show that inhibition of OATP1B3-1B7 lowers the glucuronidation rate of ezetimibe. Hence, we propose that OATP1B3-1B7 is a drug transporter that is a gateway for the SER lumen

OATP1B3-1B7 (LST-3TM12) Is a Drug Transporter That Affects Endoplasmic Reticulum Access and the Metabolism of Ezetimibe

Drug transporters play a crucial role in pharmacokinetics. One subfamily of transporters with proven clinical relevance are the OATP1B transporters. Recently we identified a new member of the OATP1B family named OATP1B3-1B7 (LST-3TM12). This functional transporter is encoded by; SLCO1B3; and; SLCO1B7; OATP1B3-1B7 is expressed in hepatocytes and is located in the membrane of the smooth endoplasmic reticulum (SER). One aim of this study was to test whether OATP1B3-1B7 interacts with commercial drugs. First, we screened a selection of OATP1B substrates for inhibition of OATP1B3-1B7-mediated transport of dehydroepiandrosterone sulfate and identified several inhibitors. One such inhibitor was ezetimibe, which not only inhibited OATP1B3-1B7 but is also a substrate, as its cellular content was significantly increased in cells heterologously expressing the transporter. In humans, ezetimibe is extensively metabolized by hepatic and intestinal uridine-5'-diphospho-glucuronosyltransferases (UGTs), the catalytic site of which is located within the SER lumen. After verification of OATP1B3-1B7 expression in the small intestine, we determined in microsomes whether SER access can be modulated by inhibitors of OATP1B3-1B7. We were able to show that these compounds significantly reduced accumulation in small intestinal and hepatic microsomes, which influenced the rate of ezetimibe; β; -D-glucuronide formation as determined in microsomes treated with bromsulphthalein. Notably, this molecule not only inhibits the herein reported transporter but also other transport systems. In conclusion, we report that multiple drugs interact with OATP1B3-1B7; for ezetimibe, we were able to show that SER access and metabolism is significantly reduced by bromsulphthalein, which is an inhibitor of OATP1B3-1B7. SIGNIFICANCE STATEMENT: OATP1B3-1B3 (LST-3TM12) is a transporter that has yet to be fully characterized. We provide valuable insight into the interaction potential of this transporter with several marketed drugs. Ezetimibe, which interacted with OATP1B3-1B7, is highly metabolized by uridine-5'-diphospho-glucuronosyltransferases (UGTs), whose catalytic site is located within the smooth endoplasmic reticulum (SER) lumen. Through microsomal assays with ezetimibe and the transport inhibitor bromsulphthalein we investigated the interdependence of SER access and the glucuronidation rate of ezetimibe. These findings led us to the hypothesis that access or exit of drugs to the SER is orchestrated by SER transporters such as OATP1B3-1B7

OATP1B3-1B7 a novel Organic Anion Transporting Polypeptide is modulated by FXR ligands and transports bile acids

OATP1B3-1B7 (LST-3TM12) is a member of the OATP1B (; SLCO1B; )-family. This transporter is not only functional, but also expressed in the membrane of the smooth endoplasmic reticulum of hepatocytes and enterocytes. OATP1B3-1B7 is a splice variant of; SLCO1B3; where the initial part is encoded by; SLCO1B3; , whereas the rest of the mRNA originates from the gene locus of; SLCO1B7; . In this study we not only showed that; SLCO1B3; and the mRNA encoding for OATP1B3-1B7 share the 5' untranslated region, but also that silencing of an initial; SLCO1B3; exon lowered the amount of; SLCO1B3; and of; SLCO1B7; mRNA in Huh-7 cells. To validate the assumption that both transcripts are regulated by the same promoter we tested the influence of the bile acid sensor farnesoid X receptor (FXR) on their transcription. Treatment of Huh-7 and HepaRG cells with activators of this known regulator of OATP1B3 not only increased; SLCO1B3,; but also OATP1B3-1B7 mRNA transcription. Applying a heterologous expression system we showed that several bile acids interact with OATP1B3-1B7 and that taurocholic acid and lithocholic acid are OATP1B3-1B7 substrates. As OATP1B3-1B7 is located in the smooth endoplasmic reticulum it may grant access to metabolizing enzymes. In accordance are our findings showing that the OATP1B3-1B7 inhibitor bromsulphthalein significantly reduced uptake of bile acids into human liver microsomes. Taken together we report that OATP1B3-1B7 transcription can be modulated with FXR agonists and antagonists and that OATP1B3-1B7 transports bile acids

LST-3TM12 is a member of the OATP1B family and a functional transporter

Organic anion transporting polypeptides (OATPs) and particularly the two members of the OATP1B family are known for their role in pharmacokinetics. Both SLCO1B3 and SLCO1B1 are located on chromosome 12 encompassing the gene locus SLCO1B7. Hitherto, this particular gene has been assumed to be a pseudogene, even though there are published mRNA sequences linked to this chromosomal area. It was aim of this study to further investigate SLCO1B7 and the associated mRNA LST-3TM12. In a first step, we aligned all mRNAs linked to the chromosomal region of SLCO1B-transporters. This in silico analysis revealed that LST-3TM12 is a product of splicing of SLCO1B3 and SLCO1B7, and encodes for a protein with twelve transmembrane domains. The existence of LST-3TM12 mRNA was verified by polymerase chain reaction showing liver enriched expression. In addition, immunohistological staining showed that LST-3TM12 protein was expressed in the endoplasmic reticulum (ER) of hepatocytes. Localization in the ER was further verified by immunoblot analysis showing high amounts of LST-3TM12 in liver microsomes. Function of LST-3TM12 was assessed by transport studies after heterologous expression in HeLa cells, where the transporter was shown to be expressed not only in the ER but also in the plasma membrane. Overexpression of LST-3TM12 was associated with enhanced cellular accumulation of dehydroepiandrosterone sulfate (Vmax 300.2pmol mg-1 min-1; Km 34.2µm) and estradiol 17β-glucuronide (Vmax 29.9mol mg-1 min-1 and Km 32.8µM). In conclusion, LST-3TM12 is a functional splice variant of SLCO1B3 and SLCO1B7 expressed in the ER of human liver

Steroid profiling in H295R cells to identify chemicals potentially disrupting the production of adrenal steroids

The validated OECD test guideline 456 based on human adrenal H295R cells promotes measurement of testosterone and estradiol production as read-out to identify potential endocrine disrupting chemicals. This study aimed to establish optimal conditions for using H295R cells to detect chemicals interfering with the production of key adrenal steroids. H295R cells' supernatants were characterized by liquid chromatography-mass spectrometry (LC-MS)-based steroid profiling, and the influence of experimental conditions including time and serum content was assessed. Steroid profiles were determined before and after incubation with reference compounds and chemicals to be tested for potential disruption of adrenal steroidogenesis. The H295R cells cultivated according to the OECD test guideline produced progestins, glucocorticoids, mineralocorticoids and adrenal androgens but only very low amounts of testosterone. However, testosterone contained in Nu-serum was metabolized during the 48h incubation. Thus, inclusion of positive and negative controls and a steroid profile of the complete medium prior to the experiment (t=0h) was necessary to characterize H295R cells' steroid production and indicate alterations caused by exposure to chemicals. Among the tested chemicals, octyl methoxycinnamate and acetyl tributylcitrate resembled the corticosteroid induction pattern of the positive control torcetrapib. Gene expression analysis revealed that octyl methoxycinnamate and acetyl tributylcitrate enhanced CYP11B2 expression, although less pronounced than torcetrapib. Further experiments need to assess the toxicological relevance of octyl methoxycinnamate- and acetyl tributylcitrate-induced corticosteroid production. In conclusion, the extended profiling and appropriate controls allow detecting chemicals that act on steroidogenesis and provide initial mechanistic evidence for prioritizing chemicals for further investigations

High rates of chronic HBV genotype E infection in a group of migrants in Italy from West Africa: Virological characteristics associated with poor immune clearance

<div><p>Hepatitis B virus (HBV) genotype E almost exclusively occurs in African people, and its presence is more commonly associated with the development of chronic HBV (CHB) infection. Moreover, an epidemiological link has been found between the distribution of HBV genotype E infection and African countries with high incidences of hepatocellular carcinoma. As part of a programme for the health assessment of migrants, we evaluated 358 young African subjects for HBV infection; 58.1% (208/358) were positive for an HBV marker, and 54 (25.5%) had CHB. Eighty-one percent of the CHB subjects were infected with HBV genotype E, with a median serum HBV-DNA of 3.2 (IQR: 2.7–3.6) logIU/ml. All patients had high serum HBsAg titres (10,899 [range 5,359–20,272] IU/ml), and no correlation was found between HBsAg titres and HBV-DNA plasma levels. RT sequence analysis showed the presence of a number of immune escape mutations: strains from all of the patients had a serine at HBsAg position 140; 3 also had T116N, Y100C, and P142L+S143L substitutions; and 1 had a G112R substitution. Six (18%) patients had stop-codons at position 216. In 5 of the 9 (26.5%) CHB patients, ultrasound liver biopsy, quantification of total intrahepatic HBV-DNA and cccDNA, and RT/HBsAg sequencing were performed. The median (IQR) total intrahepatic HBV-DNA was 766 (753–1139) copies/1000 cells, and the median (IQR) cccDNA was 17 (10–27) copies/1000 cells. Correlations were observed for both total intrahepatic HBV-DNA and cccDNA with serum HBV-DNA, while no correlation was found for the HBsAg titres. A difference of 2.5/1,000 nucleotides was found in the HBsAg sequences obtained from plasma and from liver tissue, with 3 cases of possible viral anatomical compartmentalization. In conclusion, a high rate of CHB infection due to the E genotype was demonstrated in a group of immigrants from Western Africa. An analysis of the viral strains obtained showed the virological characteristics of immune escape, which may be the cause of viral replication persistence. Moreover, a fair percentage of stop codon mutations were found. The lack of correlation between HBsAg titres and plasma or intrahepatic HBV-DNA found in these subjects suggests a pathway of virus production that is not linked to HBsAg secretion. Studies with a larger number of patients with CHB due to the E genotype are advisable to corroborate these observations.</p></div

Phylogentic tree reporting the clustering of HBsAg sequences from plasma and liver tissue obtained from the 5 HBV genotype E infected patients with available liver biopsies.

<p>Phylogenetic tree was built by using HBsAg sequences from plasma and liver tissue obtained from the 5 HBV genotype E infected patients, and HBsAg sequences from HBV genotype E isolated from Genbank (Accession number:FN594748).</p

Graphs reporting the correlation between cccDNA and HBsAg titer (A) and serum HBV-DNA (B), and between intrahepatic total HBV-DNA and HBsAg titer (c) and serum HBV-DNA (D) in the 5 HBV genotype E infected patients with available liver biopsies.

<p>Graphs reporting the correlation between cccDNA and HBsAg titer (A) and serum HBV-DNA (B), and between intrahepatic total HBV-DNA and HBsAg titer (c) and serum HBV-DNA (D) in the 5 HBV genotype E infected patients with available liver biopsies.</p