Runx2通过抑制细胞巨自噬以诱导C2C12细胞向成骨细胞分化

摘要

AIM: To explore the relationship between macroautophagy and Runx2-induced osteogenic differentiation of C2C12 cells. METHODS: The cells of C2C12/Runx2Dox subline were used in the study, which expresses Runx2 in response to doxycycline ( Dox). The cells were treated with Dox (10 mg/L) for 0 d, 1 d, 3 d, and 6 d, and the expression levels of LC3b, Beclin-1, p62, and LAMP-2 were detected at each time point using real-time qPCR. The LC3-I/LC3-II ratio was measured by Western blotting. The cells were treated with different concentrations of 3-methyladenine (3-MA) or rapamycin (Rap) for 14 days in the presence of Dox, and the alkaline phosphatase (ALP) activity was measured. The cells were treated with 3-MA (5 mmol/L) or Rap (10 μmol/L) for 1 d, 3 d, and 6 d in the presence of Dox, and the expression levels of ALP and osteocalcin ( OC) were detected. RESULTS; The expression levels of LC3b and Beclin-1 were dramatically down-regulated during Runx2-induced osteogenic differentiation of C2C12 cells, and the levels of p62 and LAMP-2 were not changed during this process. The conversion of LC3-I into LC3-II was inhibited. The ALP activity was increased in 3-MA (5 mmol/L)-treated cells, but decreased in Rap (10 μmol/L)-treated cells. The expression levels of ALP and OC were up-regulated by 3-MA treatment and were down-regulated by Rap treatment. CONCLUSION: Runx2 induces osteogenic differentiation of C2C12 cells by inhibiting the formation of autophagosome through down-regulating LC3 and Beclin-1 as well as inhibiting the conversion of LC3-I to LC3-II.%目的:探讨细胞巨自噬与Runx2诱导C2C12细胞成骨分化的关系.方法:在强力霉素(doxycycline,Dox)诱导Runx2表达的细胞系C2C12/Runx2Dox中进行研究.Dox (10 mg/L)处理0d、1d、3d及6d后,realtime qPCR检测LC3b、Beclin-1、p62和LAMP-2表达情况,Western blotting分析LC3-Ⅰ/LC3-Ⅱ比值.设置不同的3-甲基腺嘌呤(3-methyladenine,3-MA)或雷帕霉素(rapamycin,Rap)浓度,Dox处理14 d后分析碱性磷酸酶(alkaline phosphatase,ALP)活性.用3-MA(5 mmol/L)或Rap(10 μmol/L)与Dox共同处理1d、3d及6d后检测ALP及骨钙素(osteocalcin,oc)表达情况.结果:(1) C2C12细胞向成骨分化时,LC3b与Beclin-1显著下调,p62与LAMP-2无明显变化；(2) LC3-Ⅰ向LC3-Ⅱ转换的过程被抑制；(3) 3-MA(5mmol/L)可增强ALP活性,而Rap(10 μmol/L)则抑制其活性；(4) 3-MA可上调ALP及OC表达,Rap则下调二者表达.结论:Runx2通过下调LC3和Beclin-1、抑制LC3-Ⅰ向LC3-Ⅱ转换的方式阻碍自噬体形成,以诱导C2C12细胞分化为成骨细胞.