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.

Background: Dysfunction in survival and differentiation of osteoblasts commonly occurs in patients with osteoporosis. Cannabinoid receptor type 2 (CNR2) is a major receptor of endocannabinoid system that is crucial for bone mass homeostasis. Our group prior demonstrated that activation of CNR2 signaling promoted osteogenic differentiation of bone marrow derived mesenchymal stem cells in vitro. Autophagy is reported to participate in osteoblastic differentiation. Whether autophagy is regulated by CNR2-mediated cannabinoid signaling is unknown, and how the autophagy-CNR2 interaction affects osteoblastic differentiation requires further elucidation. Methods: hFOB 1.19 osteoblasts were treated with CNR2 agonists HU308 (5, 10, 25, 50 or 100 nM) and JWH133 (1, 2, 5, 10 or 20 μM) in presence or absence of autophagy inhibitor 3-Methyladenine (3-MA). The differentiation of hFOB 1.19 cells was determined via evaluating their alkaline phosphatase (ALP) activity and mineralization ability (Alizarin red staining). Alterations in autophagy-related molecules and osteogenic markers were analyzed via real-time PCR and/or immunoblotting assays. Results: hFOB 1.19 cells spontaneously differentiated towards mature osteoblasts under 39 °C, during which CNR2 expression increased, and autophagy was activated. The strongest autophagy flux was observed at 192 h post differentiation─LC3I to LC3II conversion was enhanced and Beclin 1 expression was upregulated considerably, while p62 expression was downregulated. Treatment of HU308 and JWH133 promoted autophagy in a dose-dependent manner, and suppressed mTOR signaling pathway in hFOB 1.19 cells. In CNR2-silenced cells, HU308’s and JWH133’s effects on autophagy were weakened. HU308 and JWH133 enhanced the ALP activity and mineralization, and upregulated the expression of osteogenic markers, osteopontin and osteocalcin, in hFOB 1.19 cells. Intriguingly, such pro-osteogenic effects induced by CNR2 activation were markedly mitigated by 3-MA. In addition to provoking autophagy, CNR2 agonists also reduced nuclear Nrf2 accumulation and increased Keap1 expression. Further, re-expression of p62 inhibited CNR2 agonists-induced Nrf2 degradation. Conclusions: Osteogenic differentiation induced by CNR2 signaling activation involves autophagy induction and p62-mediated Nrf2 deactivation.