Please use this identifier to cite or link to this item: https://hdl.handle.net/10316/114766
Title: ECSIT is essential for RANKL-induced stimulation of mitochondria in osteoclasts and a target for the anti-osteoclastogenic effects of estrogens
Authors: Carvalho, Adriana Marques 
Sardão, Vilma A. 
Kim, Ha-Neui
Almeida, Maria S
Keywords: osteoclasts; bone resorption; estrogens; ECSIT; mitochondrial metabolism; complex I
Issue Date: 2023
Publisher: Frontiers Media S.A.
Project: National Institute of Health (R01AR56679, R01AG068449, R01AR080736, P20GM125503) 
UAMS Bone and Joint Initiative 
FCT - project IF/01182/2015 
PhD fellowship SFRH/BD/ 140817/2018 
Serial title, monograph or event: Frontiers in Endocrinology
Volume: 14
Abstract: Introduction: Estrogens 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. 17b-estradiol (E2) 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 E2 on osteoclast progenitors. Methods: Bone marrow-derived macrophages (BMMs) from C57BL/6 mice were cultured with RANKL (30 ng/ml) with or without E2 (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. Results: We found that RANKL promoted ECSIT-TRAF6 interaction and increased the levels of ECSIT in mitochondria. E2 abrogated these effects of RANKL. Silencing of ECSIT decreased osteoclast differentiation and abrogated the inhibitory effects of E2 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 E2, were prevented. Instead, RANKL stimulated apoptosis of osteoclast progenitors. Discussion: These 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.
URI: https://hdl.handle.net/10316/114766
ISSN: 1664-2392
DOI: 10.3389/fendo.2023.1110369
Rights: openAccess
Appears in Collections:IIIUC - Artigos em Revistas Internacionais
I&D CIBB - Artigos em Revistas Internacionais
I&D CNC - Artigos em Revistas Internacionais

Show full item record

Page view(s)

155
checked on May 29, 2024

Download(s)

146
checked on May 29, 2024

Google ScholarTM

Check

Altmetric

Altmetric


This item is licensed under a Creative Commons License Creative Commons