Nrf2 Mitigates RANKL and M-CSF Induced Osteoclast Differentiation via ROS-Dependent Mechanisms

Abstract

Nuclear factor-erythroid 2-related factor 2 (Nrf2) has been shown to be a negative regulator of osteoclast differentiation, but the precise mechanisms have not yet been established. We examined the precise roles of Nrf2 in regulating antioxidants and reactive oxygen species (ROS) levels, especially the cytoplasmic and mitochondrial ROS during osteoclastogenesis in vitro. In the current study, we found that the absence of Nrf2 promotes osteoclast differentiation in bone-marrow-derived macrophages (BMMs) and RAW 264.7 cells. The receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) significantly lowered the levels of Nrf2 and its downstream antioxidant enzymes at mRNA and/or protein levels during osteoclast differentiation in the BMMs of mice and RAW 264.7 mouse leukemic monocytes. Compared to the wild-type cells, Nrf2-deficient cells exhibited heightened sensitivity to both transient RANKL-induced cytoplasmic ROS and prolonged RANKL and M-CSF-induced cytoplasmic and mitochondrial ROS accumulation. Furthermore, exogenous antioxidant agents, including N-acetyl-cysteine (NAC), diphenyleneiodonium chloride (DPI), and mitoquinone mesylate (MitoQ), exhibited substantial capability to suppress the elevation of ROS levels during osteoclast differentiation induced by Nrf2 deficiency, and they consequently inhibited osteoclast differentiation augmented by the lack of Nrf2. The activation of phosphorylated c-FOS resulting from elevated ROS promoted osteoclast differentiation. The inhibition of c-FOS blocked osteoclast differentiation, which was elevated by Nrf2-deficiency. Taken together, these data reveal that Nrf2 effectively decreased the accumulation of intracellular ROS and the phosphorylation of c-FOS during osteoclastic differentiation by regulating antioxidant enzymes and subsequently inhibited RANKL-induced osteoclast differentiation.

The role of N-acetylcysteine in osteogenic microenvironment for bone tissue engineering

“…Further investigation is required to clarify the role of NAC in macrophage polarization regulation. Additionally, —NAC has been observed to scavenge ROS in bone marrow-derived macrophages, RAW264.7 cells, and CD14+ peripheral blood monocytes under conditions promoting osteoclast differentiation induced by RANKL, M-CSF ( Aitken et al, 2004 ; Lee et al, 2005 ; Sakai et al, 2012 ; Cao and Picklo, 2014 ; Kim et al, 2018 ; Soares et al, 2019 ; Guo et al, 2020 ), H 2 O 2 ( Liu et al, 2021 ), Trimethylamine-N-oxide (TMAO) ( Wang et al, 2022 ), LPS ( Yan et al, 2020 ), ferric ammonium citrate (FAC) ( Jia et al, 2012 ), TRP14 knockout ( Hong et al, 2014 ), and NRF2 knockout ( Hyeon et al, 2013 ; Yang et al, 2023 ). By these manners, NAC can mitigate ROS and inhibit osteoclast differentiation via regulating redox signaling pathways, thereby reducing oxidative stress-mediated bone resorption ( Table 3 ).…”

The effects of resveratrol in animal models of primary osteoporosis: a systematic review and meta-analysis

“…Osteoporosis (OP) is a systemic bone disease characterized by damage to the bone microstructure and decreased bone mass, resulting in bone fragility and easy fracture [ 1 , 2 ]. Primary osteoporosis, as a major part of OP, is currently a major public health problem facing patients and medical practitioners globally.…”

Role of Ferroptosis Regulation by Nrf2/NQO1 Pathway in Alcohol-Induced Cardiotoxicity In Vitro and In Vivo

“…It is reported that ENPP2 impacted cardiomyocyte ferroptosis by regulating the expression of GPX4, ACSL4, Nrf2, and AKT . Several studies showed that Nrf2 was translocated from the cytoplasm to nucleus during ferroptosis, inducing the expression of HO-1 and NQO1, both of which eliminated ROS . P53 was the regulation of cell cycle and apoptosis factors, through combining with GPX4 promoter to inhibit the GPX4 activity, causing ferroptosis .…”