Complement C3a activates osteoclasts by regulating the PI3K/PDK1/SGK3 pathway in patients with multiple myeloma

Abstract

Objective: Myeloma bone disease (MBD) is the most common complication of multiple myeloma (MM). Our previous study showed 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 to screen 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%) of C3a 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, and 4.752, respectively) and 10 μg/mL (median: 5.140, 3.702, 2.250, and 5.172, respectively) of C3a was significantly increased compared to 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 resorption pits 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 significantly increased (33.845 ± 8.331%) (P < 0.001 and P < 0.001, respectively) compared to the control. There was no difference between the C4a and control groups. RNA-seq analysis showed that C3a promoted the proliferation of osteoclasts using the phosphoinositide 3-kinase (PI3K) signaling pathway. The relative expressions of PIK3CA/phosphoinositide dependent kinase-1 (PDK1)/serum and glucocorticoid inducible protein kinases (SGK3) genes and PI3K/PDK1/p-SGK3 protein in the C3a group were significantly higher than 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 a SGK inhibitor. Overall, our results identified potential therapeutic targets and strategies for MBD patients.

Keywords

• multiple myeloma
• complement c3a
• osteoclasts
• pi3k/pdk1/sgk3 pathways
• sgk inhibitor