ECSIT is essential for RANKL-induced stimulation of mitochondria in osteoclasts and a target for the anti-osteoclastogenic effects of estrogens

IntroductionEstrogens inhibit bone resorption and preserve bone mass, at least in part, via direct effects on osteoclasts. The binding of RANKL, the critical cytokine for osteoclast differentiation, to its receptor in osteoclast precursor cells of the monocyte lineage recruits the adaptor protein TRAF6 and activates multiple signaling pathways. Early effects of RANKL include stimulation of mitochondria. 17 beta-estradiol (E-2) prevents the effects of RANKL on mitochondria and promotes mitochondria mediated apoptotic cell death. However, the molecular mechanisms responsible for the actions of RANKL and estrogens on mitochondria remain unknown. Evolutionarily Conserved Signaling Intermediate in Toll Pathway (ECSIT) is a complex I-associated protein that regulates immune responses in macrophages following the engagement of Toll-like receptors, which also recruit TRAF6. Here, we examined whether ECSIT could be implicated in the rapid effects of RANKL and E-2 on osteoclast progenitors. MethodsBone marrow-derived macrophages (BMMs) from C57BL/6 mice were cultured with RANKL (30 ng/ml) with or without E-2 (10(-8) M). ECSIT-TRAF6 interaction was evaluated by co-immunoprecipitation and ECSIT levels in mitochondria and cytosolic fractions by Western blot. ShRNA lentivirus particles were used to knockdown ECSIT. Osteoclasts were enumerated after tartrate-resistant acid phosphatase staining. Oxygen consumption and extracellular acidification rates were measured with Seahorse XFe96 Analyzer. ATP, lactate, and NAD/NADH were measured with commercial assay kits. NADH oxidation to NAD was used to evaluate Complex I activity. Total and mitochondrial ROS, and mitochondrial membrane potential were measured with H2DCFDA, MitoSOX, and TMRM probes, respectively. Degradation of DEVD-AFC was used to measure Caspase-3 activity. ResultsWe found that RANKL promoted ECSIT-TRAF6 interaction and increased the levels of ECSIT in mitochondria. E-2 abrogated these effects of RANKL. Silencing of ECSIT decreased osteoclast differentiation and abrogated the inhibitory effects of E-2 on osteoclastogenesis. Loss of ECSIT decreased complex I activity, oxygen consumption, NAD(+)/NADH redox ratio, and ATP production and increased mitochondrial ROS. In the absence of ECSIT, the stimulatory actions of RANKL on complex I activity and all other markers of oxidative phosphorylation, as well as their inhibition by E-2, were prevented. Instead, RANKL stimulated apoptosis of osteoclast progenitors. DiscussionThese findings suggest that dysregulated mitochondria cause a switch in RANKL signaling from pro-survival to pro-apoptotic. In addition, our results indicate that ECSIT represents a central node for the early effects of RANKL on mitochondria and that inhibition of ECSIT-mediated mitochondria stimulation might contribute to the bone protective actions of estrogens.