Activation of X‐Box Binding Protein 1 Splicing Contributes to Efavirenz‐mediated Hepatocyte Death
Authors: Heck Carley J. S. , Hamlin Allyson N. , Bumpus Namandjé N. 
Published: June 2021

Efavirenz (EFV), a non‐nucleoside reverse transcriptase inhibitor used to treat HIV, has been associated with hepatotoxicity in some patients. Both EFV and 8‐hydroxyEFV (8‐OHEFV), its primary monooxygenated metabolite, have been demonstrated to activate cell death signaling pathways in primary human hepatocytes; however the upstream signaling events that drive this are not well understood. With this work, we investigated whether EFV and 8‐OHEFV activate the inositol‐requiring enzyme 1a (IRE1a)/x‐box binding protein 1 (XBP1) signaling cascade of the endoplasmic reticulum stress response. Using reverse transcription followed by end point PCR, we observed the mRNA splicing of XBP1, a marker for activation, in primary human and mouse (C57Bl6/J) hepatocytes treated with 50 μM of EFV and 8‐OHEFV for 4 hours. EFV treatment resulted in a significant increase in XBP1 splicing that was 45‐(human) and 38‐(mouse) fold higher than vehicle treated cells. Interestingly, 50 μM 8‐OHEFV only significantly increased splicing in primary human hepatocytes, resulting in an 8‐fold increase over vehicle control. Since the single oxygen insertion resulting in the formation of 8‐OHEFV led to significantly lower XBP1 splicing as compared to EFV, we investigated the impact of other structural changes to EFV on its activation of IRE1a‐XBP1 signaling. We treated primary mouse hepatocytes with EFV and sixteen structural analogs of EFV and measured XBP1 splicing. Breaking of the oxazinone ring, breaking of the cyclopropyl ring, removal of the oxazinone carbonyl, or substitution of the oxazinone nitrogen with carbon prevented splicing activation by EFV. On the other hand, changing the EFV alkyne to a trans‐alkene and replacing the oxazinone oxygen resulted in 2‐and 4‐fold greater splicing than that observed with EFV, respectively. Because EFV has been shown previously to activate the xenobiotic sensing nuclear receptor pregnane‐x receptor (PXR), we investigated whether this protein is necessary for XBP1 splicing activation by EFV by treating both PXR‐null and PXR‐humanized primary mouse hepatocytes with EFV. Interestingly, EFV activation of XBP1 splicing in PXR‐null hepatocytes was 7‐fold higher than that in wild‐type C57Bl/6J or PXR‐humanized mice, suggesting that the presence of PXR is not required for EFV to stimulate XBP1 splicing. To investigate whether IRE1a‐XBP1 signaling plays a role in EFV‐mediated cell death, we measured primary mouse hepatocyte viability (using ethidium bromide/acridine orange staining) following either treatment with EFV alone or co‐treatment with the IRE1a endoribonuclease inhibitor STF083010. Incubation of the hepatocytes with EFV resulted in cell death (47.2± 7.6% apoptotic cells) that was elevated as compared to the vehicle control (8.9± 2.6%). Of note, co‐treatment with STF083010 significantly decreased EFV‐activated cell death (23.9± 4.1%). These data demonstrate that EFV, but not 8‐OHEFV, activates IRE1a‐XBP1 signaling, and that this signaling contributes to the hepatocyte cell death in response to EFV.Support or Funding InformationThis work was funded by NSF GRFP DGE‐1232825 awarded to CJSH and NIH R01 GM103853.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.