Mitochondria and endoplasmic reticulum interplay at the core of efavirenz-induced hepatic effects

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The non-nucleoside analogue reverse transcriptase inhibitor efavirenz (EFV) is among the most widely used drugs in the combined antiretroviral therapy (cART) employed in the treatment of human immunodeficiency virus (HIV) infection. Although generally considered safe, there is a concern about the side effects induced by EFV-containing therapies. It has been associated with hepatic toxicity and metabolic disturbances and, although the mechanisms involved are not clear, recent evidence has pinpointed a specific mitochondrial action of EFV accompanied by the induction of an endoplasmic reticulum (ER) stress/unfolded protein response in cultured human hepatic cells. In the present work, in order to understand the role of mitochondria in the effects of EFV, firstly, we have assessed the cellular actions of this drug in a model of hepatic cells that lack functional mitochondria (rho⁰ cells generated in Hep3B background). In addition, we have studied mitochondrial dynamics, in wild-type Hep3B cells, which depends among other processes on the interaction between mitochondria and ER. This interaction has also been analysed by evaluation of mitochondrial-associated membranes (MAMs). Finally, in order to further link the two effects of EFV (mitochondria and ER), we have analysed the expression of LONP1, a highly conserved mitochondrial protease whose activation is an adaptive mechanism in both oxidative and ER stress. Throughout the manuscript, the effects of EFV have been compared to those induced by the classic pharmacological inducer of ER stress thapsigargin (TG), the typical mitotoxic agent rotenone (Rot) - a standard complex I inhibitor - and the uncoupler of OxPhos CCCP. On the one hand, EFV-treated rho⁰ cells exhibited a substantial reduction in parameters indicative of mitochondrial interference, such as increased superoxide production, mitochondrial mass/morphology alterations and enhanced expression of LONP1. In line with these results, the cytotoxic effect (cell number, chromatin condensation, cell cycle alterations and induction of apoptosis) of EFV was less pronounced in Hep3B respiration-depleted cells than in wild-type cells. The effect of EFV was both similar and different from those of two distinct mitochondrial stressors, TG and Rot, depending on the parameter studied. On the other hand, markers of mitochondrial dynamics were expressed differentially with the stimuli used (EFV, TG, Rot and CCCP), which points to a specificity of the dual ER/mitochondrial stress induced by EFV. EFV treatment enhanced mitochondria/ER interorganelle interaction, as shown by co-immunoprecipitation experiments of MAMs protein partners. In addition, LONP1 was upregulated at mRNA and protein levels under all conditions. Surprisingly, upon treatment with EFV, its extramitochondrial presence (ER and MAMs) increased. In conclusion, hepatic cells lacking normal mitochondria (rho⁰) are less vulnerable to EFV. This finding may account for the idiosyncratic hepatic reactions triggered by anti-HIV drugs and may also explain the different degrees of susceptibility to liver damage seen in patients undergoing antiretroviral therapy. The specific dual mitochondria-ER effect induced by EFV enhances MAMs content and this is associated with increased extramitochondrial LONP1 expression. This is the first report of this phenomenon in mammalian cells and suggests a novel MAMs-linked function of LONP1.
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