Complement C3a activates osteoclasts by regulating the PI3K/PDK1/SGK3 pathway in patients with multiple myeloma
Fengjuan Jiang, Hui Liu, Fengping Peng, Zhaoyun Liu, Kai Ding, Jia Song, Lijuan Li, Jin Chen, Qing Shao, Siyang Yan, Kim De Veirman, Karin Vanderkerken, Rong Fu
Abstract
Objective: Myeloma bone disease (MBD) is the most common complication of multiple myeloma (MM). Our previous studyshowed that the serum levels of C3/C4 in MM patients were significantly positively correlated with the severity of bone disease.However, the mechanism of C3a/C4a in osteoclasts MM patients remains unclear. Methods: The formation and function of osteoclasts were analyzed after adding C3a/C4a in vitro. RNA-seq analysis was used toscreen the potential pathways affecting osteoclasts, and the results were verified by Western blot, qRT-PCR, and pathway inhibitors. Results: The osteoclast area per view induced by 1 μg/mL (mean ± SD: 50.828 ± 12.984%) and 10 μg/mL (53.663 ± 12.685%) ofC3a was significantly increased compared to the control group (0 μg/mL) (34.635 ± 8.916%) (P < 0.001 and P < 0.001, respectively).The relative mRNA expressions of genes, OSCAR/TRAP/RANKL/cathepsin K, induced by 1 μg/mL (median: 5.041, 3.726, 1.638, and4.752, respectively) and 10 μg/mL (median: 5.140, 3.702, 2.250, and 5.172, respectively) of C3a was significantly increased comparedto the control group (median: 3.137, 2.004, 0.573, and 2.257, respectively) (1 μg/mL P = 0.001, P = 0.003, P < 0.001, and P = 0.008,respectively; 10 μg/mL: P < 0.001, P = 0.019, P < 0.001, and P = 0.002, respectively). The absorption areas of the osteoclast resorptionpits per view induced by 1 μg/mL (mean ± SD: 51.464 ± 11.983%) and 10 μg/mL (50.219 ± 12.067%) of C3a was also significantlyincreased (33.845 ± 8.331%) (P < 0.001 and P < 0.001, respectively) compared to the control. There was no difference between theC4a and control groups. RNA-seq analysis showed that C3a promoted the proliferation of osteoclasts using the phosphoinositide3-kinase (PI3K) signaling pathway. The relative expressions of PIK3CA/phosphoinositide dependent kinase-1 (PDK1)/serum andglucocorticoid inducible protein kinases (SGK3) genes and PI3K/PDK1/p-SGK3 protein in the C3a group were significantly higherthan in the control group. The activation role of C3a in osteoclasts of MM patients was reduced by the SGK inhibitor (EMD638683). Conclusions: C3a activated osteoclasts by regulating the PI3K/PDK1/SGK3 pathways in MM patients, which was reduced using aSGK inhibitor. Overall, our results identified potential therapeutic targets and strategies for MBD patients.