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
