Background Despite the high prevalence of liver dysfunction in systemic lupus erythematosus (SLE), the role of the liver in disease pathogenesis is still widely unknown. Recently, we showed that prior to disease onset, SLE splenocytes overexpress the small GTPase HRES-1/Rab4A and the mechanistic target of rapamycin (mTOR), which regulate endosomal recycling and mitophagy via Drp11. Furthermore, we showed that the inhibition of mTOR or Rab4A resulted in the improvement of SLE in the kidneys and spleen1.
Objectives Here, we characterize the phenotype of liver mitochondria before the onset of SLE in MRL/lpr mice and the rescue of liver dysfunction by inhibition of HRES-1/Rab4A or mTOR with 3-PEHPC or rapamycin, respectively.
Methods 4-week-old MRL/lpr, C57BL/6.lpr, and MRL/MpJ mice were sacrificed with age and gender matched C57BL/6 controls. O2 consumption was measured by a Clark electrode. Reactive oxygen intermediates, reactive nitrogen intermediates, and mitochondrial mass were measured by flow cytometry. Hepatocyte mitotic figures were counted in histopathologic sections of livers at 400x magnification. Protein expression was measured by western blot analysis. Additionally, we treated 4-week-old MRL/lpr mice with either 125μg/kg of the Rab geranylgeranyl transferase inhibitor, 3-PEHPC, or 1mg/kg rapamycin for 10 weeks.
Results Both male and female MRL/lpr liver mitochondria had increased oxygen consumption at complex II of the electron transport chain (ETC) and reduced state 3/state 4 respiration ratios. Male, but not female, MRL/lpr mitochondria had increased mass, nitric oxide production, and peroxynitrite. We found increased mitotic figures in MRL/lpr livers. Expression of HRES-1/Rab4A was significantly increased in all lupus-prone mice. In turn, there was decreased Drp1 expression in MRL/lpr livers. Drp1 phosphorylation at Ser616, an activating modification, was reduced in MRL/lpr livers. MRL/lpr had increased S6K and AKT expression and phosphorylation. 3-PEHPC increased the ratio of phospho-4EBP1 to total 4EBP1. Rapamycin dramatically increased the total and phosphorylated levels of 4EBP1 and AKT while reducing total phospho-S6K. 3-PEHPC increased the expression of complex I while rapamycin increased expression of complex II and IV of the ETC. Rab4A levels were unchanged by 3-PEHPC and were increased by rapamycin compared to vehicle. This was accompanied by an increase in Drp1 expression in the rapamycin cohort. 3-PEHPC blocked the phosphorylation of Ser637, which when phosphorylated prevents mitochondrial fission.
Conclusions Before the onset of SLE, there is mitochondrial dysfunction in the liver mitochondria of MRL/lpr mice. The result of mitochondrial dysfunction is increased oxidative stress that may initiate proliferation of hepatocytes. Rab4A overexpression occurred in the livers of all lupus-prone mice before SLE onset and may be the central regulator of mitochondrial dysfunction, mitophagy, and mTOR activation. We show that these changes are mitigated by inhibition of mTOR or geranylgeranylation of Rab4A in MRL/lpr.
Caza TN, Fernandez DR, Talaber G, et al. HRES-1/Rab4-mediated depletion of Drp1 impairs mitochondrial homeostasis and represents a target for treatment in SLE. Ann Rheum Dis 2013.
Disclosure of Interest None declared
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