Hypoxia Promotes Osteoclast Differentiation by Weakening USP18-Mediated Suppression on the NF-κB Signaling Pathway

Abstract

Osteoporosis, a prevalent metabolic bone disorder, is characterized by reduced bone density and increased fracture risk. The pathogenesis of osteoporosis is closely associated with an imbalance in bone remodeling, in which the resorption function of osteoclasts exceeds the formation function of osteoblasts. Hypoxia has been implicated in the promotion of osteoclast differentiation and the subsequent development of osteoporosis. The ubiquitin-proteasome system (UPS) and its regulatory enzymes, deubiquitinating enzymes (DUBs), play a significant role in bone homeostasis. In this study, we investigated the contribution and mechanism of Ubiquitin-specific protease 18 (USP18), a DUB, in osteoclast differentiation under hypoxic conditions. BMDMs and RAW264.7 cells were treated with RANKL to induce osteoclastogenesis and were subjected to overexpression or knockdown of USP18 under normoxic or hypoxia conditions. Osteoclast formation was assessed using TRAP staining, and the expression of osteoclast marker genes was determined using qRT-PCR. The activation of the NF-κB signaling pathway was evaluated using immunoblotting. We found that hypoxia significantly enhanced the differentiation of BMDMs and RAW264.7 cells into osteoclasts, accompanied by a notable downregulation of USP18 expression. The overexpression of USP18 inhibited RANKL-induced osteoclast differentiation, while the knockdown of USP18 promoted that process, unveiling the inhibitory effect of USP18 in osteoclastogenesis. Furthermore, the overexpression of USP18 rescued the hypoxia-induced increase in osteoclast differentiation. Mechanistic insights revealed that USP18 inhibits osteoclastogenesis by suppressing the NF-κB signaling pathway, with a potential target on TAK1 or its upstream molecules. This study indicates that hypoxia promotes osteoclast differentiation through the downregulation of USP18, which, in turn, relieves the suppression of the activation of the NF-κB signaling pathway. The USP18 emerges as a potential therapeutic target for osteoporosis treatment, highlighting the importance of the hypoxia-DUB axis in the pathogenesis of the disease.

Keywords: NF-κB signaling pathway; USP18; hypoxia; osteoclasts; osteoporosis.

Figure 1

Hypoxia significantly enhances the osteoclast differentiation of RAW264.7. (A–C) RAW264.7 cells were treated with 75 ng/mL RANKL for 0–4 days under normoxic (21% O₂) or hypoxic (1% O₂) conditions. Osteoclastogenesis was measured using tartrate-resistant acid phosphatase (TRAP) staining, and TRAP-positive multinucleated cells were considered mature osteoclasts. Scale bar, 200 μm. n = 5. (D–G) qRT-PCR detected the mRNA expression of osteoclast markers using RNA isolated from RAW264.7 cells cultured with or without 75 ng/mL RANKL for 2 days under normoxic (21% O₂) or hypoxic (1% O₂) conditions. The relative mRNA level of individual genes was expressed as the fold induction compared with no treatment (n = 3). Data are presented as mean ± SD. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant).

Figure 2

Hypoxia dramatically suppresses the USP18 expression during osteoclast differentiation. RAW264.7 cells were treated with 75 ng/mL RANKL for 0 h, 24 h, 48 h, and 72 h under normoxic (21% O₂) or hypoxic (1% O₂) conditions. (A,B) A qRT-PCR analysis was performed for USP18 expression. (C) The protein level of USP18 was detected using Western blot. Data are presented as mean ± SD (n = 3) (** p < 0.01, **** p < 0.0001, ns: not significant).

Figure 3

Overexpression of USP18 obviously inhibits RANKL-induced osteoclast differentiation. RAW264.7 cells were transfected with USP18 plasmid for overexpression, followed by treatment with 75 ng/mL RANKL for 0–4 days. (A–C) Osteoclastogenesis was measured using TRAP staining. Scale bar, 200 μm. n = 5. (D–G) qRT-PCR detected the mRNA expression of osteoclast markers using RNA isolated from RAW264.7 cells cultured in the presence of 75 ng/mL RANKL for 2 days (n = 3). Data are presented as mean ± SD (*** p < 0.001, **** p < 0.0001).

Figure 4

Knockdown of USP18 remarkably promotes RANKL-induced osteoclast differentiation. RAW264.7 cells were transfected with siRNA for silencing USP18, followed by treatment with 75 ng/mL RANKL for 0–4 days. (A) Whole-cell lysates were immunoblotted with the indicated antibodies (n = 3). (B) qRT-PCR analysis was performed for USP18 expression (n = 3). (C–E) Osteoclastogenesis was measured using TRAP staining. Scale bar, 200 μm. n = 5. (F–I) qRT-PCR detected the mRNA expression of osteoclast markers using RNA isolated from RAW264.7 cells cultured in the presence of RANKL for 2 days (n = 3). Data are presented as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 5

USP18 rescues hypoxia-induced promotion of osteoclast differentiation. RAW264.7 cells were transfected with USP18 plasmid for overexpression and then treated with RANKL for 2 (qRT-PCR) or 3 (TRAP staining) days under normoxic (21% O₂) or hypoxic (1% O₂) conditions. (A–C) Osteoclastogenesis was measured using TRAP staining. Scale bar, 200 μm. n = 5. (D–G) qRT-PCR detected the mRNA expression of osteoclast markers (n = 3). Data are presented as mean ± SD (*** p < 0.001, **** p < 0.0001, ns: not significant).

Figure 6

USP18 inhibits osteoclast differentiation by suppressing the NF-κB signaling pathway. (A–C) RAW264.7 cells were transfected with USP18 plasmid and siRNA for overexpression and silencing, respectively, followed by treatment with 75 ng/mL RANKL for 2 (A), 24 (B), and 48 (C) hours. Whole-cell lysates were immunoblotted with the indicated antibodies. Data are presented as mean ± SD, (n = 3) (* p < 0.05, ** p < 0.01, **** p < 0.0001).