Jiang Fengjuan, Liu Hui, Peng Fengping, Liu Zhaoyun, Ding Kai, Song Jia, Li Lijuan, Chen Jin, Shao Qing, Yan Siyang, De Veirman Kim, Vanderkerken Karin, Fu Rong
Department of Hematology, Tianjin Medical University General Hospital, Tianjin 300052, China.
Department of Hematology and Immunology-Myeloma Center Brussels, Vrije Universiteit Brussel, Brussels 1090, Belgium.
Cancer Biol Med. 2021 May 7;18(3):721-33. doi: 10.20892/j.issn.2095-3941.2020.0430.
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.
The formation and function of osteoclasts were analyzed after adding C3a/C4a . 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.
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%) ( < 0.001 and < 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 = 0.001, = 0.003, < 0.001, and = 0.008, respectively; 10 μg/mL: < 0.001, = 0.019, < 0.001, and = 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%) ( < 0.001 and < 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).
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.
骨髓瘤骨病（MBD）是多发性骨髓瘤（MM）最常见的并发症。我们之前的研究表明，MM患者血清C3/C4水平与骨病严重程度显著正相关。然而，C3a/C4a在MM患者破骨细胞中的作用机制仍不清楚。
加入C3a/C4a后分析破骨细胞的形成和功能。采用RNA测序分析筛选影响破骨细胞的潜在信号通路，并通过蛋白质免疫印迹法、实时荧光定量聚合酶链反应和信号通路抑制剂进行验证。
与对照组（0 μg/mL）（34.635±8.916%）相比，1 μg/mL（平均值±标准差：50.828±12.984%）和10 μg/mL（53.663±12.685%）C3a诱导的每个视野破骨细胞面积显著增加（分别为P<0.001和P<0.001）。与对照组（中位数分别为3.137、2.004、0.573和2.257）相比，1 μg/mL（中位数分别为5.041、3.726、1.638和4.752）和10 μg/mL（中位数分别为5.140、3.702、2.250和5.172）C3a诱导的破骨细胞相关基因OSCAR/TRAP/RANKL/组织蛋白酶K的相对mRNA表达显著增加（1 μg/mL时分别为P = 0.001、P = 0.003、P<0.001和P = 0.008；10 μg/mL时分别为P<0.001、P = 0.019、P<0.001和P = 0.002）。与对照组（33.845±8.331%）相比，1 μg/mL（平均值±标准差：51.464±11.983%）和10 μg/mL（50.219±12.067%）C3a诱导的每个视野破骨细胞吸收面积也显著增加（分别为P<0.001和P<0.001）。C4a组与对照组之间无差异。RNA测序分析表明，C3a通过磷酸肌醇3激酶（PI3K）信号通路促进破骨细胞增殖。C3a组PIK3CA/磷酸肌醇依赖性激酶-1（PDK1）/血清和糖皮质激素诱导蛋白激酶（SGK3）基因及PI3K/PDK1/p-SGK3蛋白的相对表达显著高于对照组。SGK抑制剂（EMD638683）可降低C3a对MM患者破骨细胞的激活作用。
C3a通过调节MM患者的PI3K/PDK1/SGK3信号通路激活破骨细胞，SGK抑制剂可降低其激活作用。总体而言，我们的研究结果为MBD患者确定了潜在的治疗靶点和策略。
