IL-17A Affects The Function Of Osteoblastic MC3T3-E1Cells And Its Underlying Mechanism May Depend On AKT2

Objective:IL-17A plays an important role in alveolar bone remodeling in periodontitis, and our pilot study showed that PI3K/AKT2pathway is actively invovled in bone remodeling process. The present study aimed to investigate the effects of IL-17A on the function of osteoblastic MC3T3-E1cells and the preliminary study of its dependence of AKT2.Methods:The AKT2gene had been knockdown in the osteoblast precursor cell line MC3T3-E1cells following the RNA inference in our previous study. Cells were incubated medium with or without IL-17A respectively. Methyl thiazolyl tetrazolium (MTT) method were adopted to investigate the proliferation of MC3T3-E1cells, also, cell cycle was analyzed by flow cytometry.After adding the osteogenic induction treatment, the involvement of PI3K and p-PI3K was tested by western-blot. The direct effects of IL-17A on the related markers of the osteogenesis including Runt-related transcription factor-2(Runx2),Alkaline phosphatase(ALP),osteocalcin(OCN)and IL-17A receptor were evaluated by real time PCR.ALP and hydroxyproline (HYP) activity analysis and Alizarin Red S staining of matrix mineralization were also used to tested the differentiation and calcification functions.Results:The results showed that p-PI3K and IL-17RA expression levels were increased in both wide-type and AKT2knockdown MC3T3-E1cells when treated with IL-17A,P<0.05. MTT results indicated that the knockdown of AKT2gene inhibited the proliferation of MC3T3-E1cells,P<0.05,but IL-17A didn’t obviously affected it,cell cycle assay showed that AKT2knockdown induced a significant increase in cells arrested in the G0/G1phase (81.793±1.227,62.043±1.317)(p<0.05) and a decrease in cells arrested in the S and G2/M phases (p<0.05);While IL-17A didn’t show significant effects.Furthermore, the gene expression levles of,Runx2, ALP and OCN were enhanced under the induced condition,followed by the increased activity of ALP and HYP,enchanced calcification areas in both wide-type and AKT2knockdown MC3T3-E1cells,P<0.05,the promoting effect in wild-type cells seemed more apparent after the combination of IL-17A, contrasting results of wide-type and AKT2knockdown MC3T3-E1cells with the treatment of IL-17A showed Runx2mRNA expression of day7(2.238±0.091,1.537±0.096);ALP mRNA expression of day7(4.258±0.276,1.260±0.070); OCN mRNA expression of dayl4(2.521±0.279,1.104±0.150)(P<0.05). Relative ALP activity (67.137±5.105,37.557±2.820)(P<0.05), quantitation of mineralization by integrated optical density (IOD)/105(19.638±0.960,5.533±0.251)(P<0.05),hydroxyproline contents in cell culture supernatant (3.527±0.672,1.861±0.400)(P<0.05).Conclusions:AKT2is required in the proliferation,differentiation and calcification activity of osteoblastic MC3T3-E1cells,IL-17A may play a crucial role on the mature process of MC3T3-E1cells in an AKT2-dependent manner.