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Efavirenz Treatment Activates XBP1 Splicing in Primary Hepatocytes: a Comparison Across Species and an Investigation into the Role of PXR
Author(s) -
Heck Carley J.S.,
Bumpus Namandje N.
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.668.1
Subject(s) - xbp1 , efavirenz , endoplasmic reticulum , unfolded protein response , reverse transcriptase , rna splicing , chemistry , microbiology and biotechnology , reverse transcriptase inhibitor , biology , rna , virology , biochemistry , human immunodeficiency virus (hiv) , gene , antiretroviral therapy , viral load
The non‐nucleoside reverse transcriptase inhibitor efavirenz (EFV) is used to treat human immunodeficiency virus (HIV) despite adverse effects, including hepatoxicity in some individuals. The mechanisms underlying this hepatotoxicity remain largely unknown, although the formation of the primary metabolite, 8OH‐EFV, has been linked to the activation of certain cell death pathways. In light of this, the present study tested whether EFV activates the endoplasmic reticulum (ER) stress response in primary hepatocytes. Splicing of X‐box‐binding protein 1 (XBP1) (a process activated by the ER stress response), was monitored using reverse transcriptase PCR following treatment of primary human and mouse hepatocytes with EFV (10 μM and 50 μM). Incubation of primary hepatocytes isolated from male C57Bl/6 mice with 50 μM EFV resulted in a ratio of spliced to unspliced XBP1 that was 8.8‐fold greater than the vehicle control. In human primary hepatocytes, an increase of 2.6‐fold was observed in response to EFV. In order to gain an understanding of the structural characteristics of EFV that render it a stimulator of XBP1 splicing, treatments were performed using a panel of EFV analogs as well as the 8OH‐EFV. Interestingly, an analog of EFV that differed from EFV in that it contains a trans‐alkene in place of the alkyne exhibited augmented XBP1 splicing response as compared to EFV, with a ratio of spliced to unspliced increased 29.4‐fold over vehicle control in hepatocytes isolated from mice. Several other analogs, including one in which the cyclopropyl ring is broken and the 8OH‐EFV metabolite, did not stimulate splicing of XBP1. Similar patterns of structural dependence were observed between human and mouse primary hepatocytes. Since EFV has been previously reported to activate the nuclear receptor pregnane X receptor (PXR), we tested whether PXR was required for the XBP1 splicing in response to EFV. Interestingly, we observed that XBP1 splicing following EFV treatment was increased in PXR null mice as compared to wild‐type C57Bl/6 mice (76.2‐fold in PXR null versus 8.8‐fold in wild‐type). Similarly, the above mentioned trans‐alkene analog also exhibited enhanced splicing of XBP1 in the PXR null mice as compared to that of wild‐type mice. Viability was measured in primary mouse hepatocytes during the above treatments. All analogs that increased XBP1 splicing also displayed a commensurate decrease in viability, except one in which the oxazinone carbonyl is replaced with a methyl group. In conclusion, EFV stimulates XBP1 splicing in mouse and human primary hepatocytes in a manner that does not appear to be dependent on metabolism to the primary 8OH‐EFV metabolite. Support or Funding Information NIH R01GM103853 and NSF‐GRFP DGE‐1232825