Abstract
OATP1B1 and OATP1B3 mediate hepatic uptake of many drugs (e.g., statins) and can mediate transporter-mediated drug-drug-interactions (DDIs). Bortezomib is the first-in-class proteasome inhibitor drug approved by the U. S. Food and Drug Administration for the treatment of multiple myeloma. The potential of bortezomib to cause OATP-mediated DDIs has not been assessed. The current study investigated the involvement of the ubiquitin-proteasome system (UPS) in OATP1B1 and OATP1B3 degradation and determined the effects of proteasome inhibitors on OATP1B1- and OATP1B3-mediated transport. Co-immunoprecipitation of FLAG-OATP1B1/1B3 and HA-ubiquitin was observed in human embryonic kidney (HEK) 293 cells co-transfected with FLAG-tagged OATP1B1/OATP1B3 and hemagglutinin (HA)-tagged ubiquitin, suggesting that OATP1B1 and OATP1B3 can be ubiquitin-modified. Although blocking proteasome activity by bortezomib treatment (50 nM, 7 h) increased the endogenous ubiquitin-conjugated FLAG-OATP1B1 and FLAG-OATP1B3 in HEK293-FLAG-OATP1B1 and–OATP1B3 cells, such treatment did not affect the total protein levels of OATP1B1 and OATP1B3, suggesting that the UPS plays a minor role in degradation of OATP1B1 and OATP1B3 under current constitutive conditions. Pretreatment with bortezomib (50–250 nM, 2–7 h) significantly decreased transport of [3H]CCK-8, a specific OATP1B3 substrate, in HEK293-OATP1B3 and human sandwich-cultured hepatocytes (SCH). However, bortezomib pretreatment had negligible effects on the transport of [3H]E217βG and [3H]pitavastatin, dual substrates of OATP1B1 and OATP1B3, in HEK293-OATP1B1/1B3 cells and/or human SCH. Compared with vehicle control treatment, bortezomib pretreatment significantly decreased the maximal transport velocity (Vmax) of OATP1B3-mediated transport of CCK-8 (92.25 ± 14.2 vs. 133.95 ± 15.5 pmol/mg protein/min) without affecting the affinity constant (Km) values. Treatment with other proteasome inhibitors MG132, epoxomicin, and carfilzomib also significantly decreased OATP1B3-mediated [3H]CCK-8 transport. In summary, the current studies for the first time report ubiquitination of OATP1B1 and OATP1B3 and the apparent substrate-dependent inhibitory effect of bortezomib on OATP1B3-mediated transport. The data suggest that bortezomib has a low risk of causing OATP-mediated DDIs.
Highlights
OATP1B1 and OATP1B3 are predominantly expressed on the basolateral membrane of hepatocytes under physiological conditions and mediate the hepatic uptake of many clinically important drugs, e.g., lipid-lowering statins, antibiotics, and anti-cancer drugs [1]
Unlabeled cholecystokinin-8 (CCK-8), estradiol 17β-D-glucuronide (E217βG), IGEPAL (NP40), Hanks’ Balanced Salt Solution (HBSS), dexamethasone, dimethyl sulfoxide (DMSO), Triton X-100, Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), N-ethylmaleimide, trypsin-EDTA solution, antibiotic antimycotic solution, Dulbecco’s PhosphateBuffered Saline (DPBS), bromosulfophthalein (BSP), bovine serum albumin (BSA), and epoxomicin were purchased from Sigma-Aldrich
To determine whether OATP1B1 and OATP1B3 could be modified by ubiquitination, coimmunoprecipitation (IP) of FLAG-tagged OATP1B1/OATP1B3 with HA-tagged ubiquitin (HA-Ub) was performed in human embryonic kidney 293 (HEK293) cells that were transiently transfected with vectors expressing FLAG-OATP1B1 or FLAG-OATP1B3, either alone or in combination with HAubiquitin, similar to what has been published previously [39]
Summary
OATP1B1 and OATP1B3 are predominantly expressed on the basolateral membrane of hepatocytes under physiological conditions and mediate the hepatic uptake of many clinically important drugs, e.g., lipid-lowering statins, antibiotics, and anti-cancer drugs [1]. The lysosome pathway and ubiquitin-proteasome system (UPS) are the major mechanisms through which proteins are degraded intracellularly [9]. We previously reported that the lysosome pathway is involved in OATP1B1 degradation, and that treatment with the lysosome inhibitor chloroquine decreases OATP1B1-mediated transport in vitro and is associated with increased statin-related myopathy in patients treated concurrently with chloroquine and metabolically stable statins [10]. In addition to being degraded through the lysosome [11], membrane proteins, including some membrane-transport proteins, can be degraded via the ubiquitin-proteasome system [12,13,14]. The ubiquitination of OATP1B1 and OATP1B3 and the effects of proteasome inhibition on their transport function have not been elucidated
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