Acod1 negatively impacts osteoclastogenesis via GPR91-mediated NFATc1 activation

Abstract

Immune cells undergo metabolic reprogramming upon exposure to inflammatory stimuli. The immuneresponsive gene 1 (Irg1) encodes aconitate decarboxylase (Acod1), an enzyme that generates itaconate from cis-aconitate in the TCA cycle and is one of the most highly induced genes in macrophages during inflammation. Itaconate inhibits succinate dehydrogenase activity leading to the accumulation of succinate. As the adjustment of energy metabolism also plays an important role during the formation of bone-resorbing osteoclasts, we investigated if Irg1 is regulated during osteoclastogenesis. We show that M-CSF/RANKL treatment induces Irg1 at an early time-point in bone marrow-derived macrophages (BMDM) as well as in the RAW264.7 macrophage cell line. Next, we stably overexpressed Acod1 in RAW264.7 cells. The metabolism of these Acod1 cells shifted towards glycolysis, as indicated by an increase in mTOR activation, subsequent 4EB-P1 phosphorylation and reduced ATP levels. When we investigated the ability of Acod1 cells to differentiate into osteoclasts, we observed a remarkable suppression of osteoclast-associated genes and the number of TRAP-positive, multi-nucleated osteoclasts was greatly reduced but not completely abrogated. Surprisingly, NFATc1 was detectable in nuclear extracts in untreated Acod1 cells leading to residual transcriptional NFAT activity in luciferase assays. This is caused by the elevated levels of succinate in Acod1 cells, as succinate can bind extracellularly to its cognate receptor GPR91 leading to Gq-mediated activation of NFATc1. When we investigated the expression of Gpr91, we found RANKL-mediated induction of Gpr91 to be severely reduced in Acod1 cells and we suggest that GPR91 is a target of RANKL-mediated NFATc1 activation. However, on the protein level, the receptor was still expressed at the cell surface. The observed repression of Gpr91 in Acod1 overexpressing cells was also detected by treatment with octyl-itaconate, showing that this is an itaconate-mediated effect. We hypothesize that the itaconate-mediated increase in succinate levels causes activation of NFATc1 signalling, although the transcriptional activity does not lead to osteoclastogenesis. In the presence of RANKL, these pre-activated cells are slow in switching to RANKL-mediated induction of osteoclast genes, which decreases their ability to differentiate into osteoclasts.