Interleukin-17A Facilitates The Osteogenesis Of Mesenchymal Stem Cells In Myeloma

Objective:To study the effects of interleukin-17A on the osteogenesis of mesenchymal stem cells（MSCs）in multiple myeloma（MM）patients,and illustrate the potential related pathways involved in this process.Methods:Bone marrow mononuclear cells（BMMNCs）were obtained from three MM patients via density gradient centrifugation.BMMNCs were cultured in LG-DMEM and the adherent cells were trypsinized,isolated and expanded to produce MSCs.Growth and proliferation of MSCs were observed under microscope.IL-17A was added to the culture system（final concentration 10 ng/ml）,with TNF-α（50ng/ml）,TGF-β（10 ng/ml）and IFN-γ（10 ng/ml）as controls.Blank control was the culture system without the cytokines mentioned above.On day 3,proliferation ratio of each group relative to blank control was assayed using CCK-8 kit with the blank control as 100%.On day 14,the conditioned medium containing dexamethasone(10^-8mol/L),vitamin C（0.05 g/L）andβ-glycerophosphate（10^-22 mol/L）was used to induce the osteoblast formation of MSCs.On day 28,alkaline phosphatase（ALP）staining with color area measurement and Calcium nodules staining with calcium nodule number counting were used to evaluate osteogenesis potential.Western blot（WB）was used to analyse the expressions of proteins Runx2,Wnt5a and TRAF4 which are involved in the process of osteogenesis.Finally,we discussed the effects of IL-17A on the osteogenesis of MSCs and the possible mechanism involved.Results:（1）After the adherent cells were isolated from BMMNCs,trypsinized and subcultured,we harvested the MSCs,which appeared to be slender and homogeneous in size.（2）On day 3 of cytokine treatment,CCK-8 showed that the proliferation ratio of IL-17A,TNF-αand TGF-βgroups were 96.6%±1.7%,93.9%±1.7%and 94.1%±3.0%（P>0.05）,respectively,no significant difference was found from the blank control（P>0.05）.However,in the IFN-γgroup,the proliferation ratio of MSCs was 74.6%±7.2%,which was lower than the blank control（P<0.05）,showing that IFN-γmight inhibit the proliferation of MSCs,but IL-17A,TNF-αand TGF-βhad no significant influence on the proliferation of MM MSCs.（3）The ALP activity of IL-17A group was significantly higher than the blank control group,IOD sum/Area sum were 0.322±0.054 and 0.192±0.031,respectively（P<0.05）.And the ALP activity of TNF-αand TGF-βgroups were also higher than the blank control group,the IOD sum/Area sum were 0.369±0.048 and 0.277±0.041,respectively（compared with blank control,P<0.05）.However,the IOD sum/Area sum of the IFN-γgroup was 0.113±0.031 which was lower than the blank control group（P<0.05）.These rusults showed that IL-17A,followed by TNF-αand TGF-β,could enhance the ALP activity of MSCs and their osteogenic differentiation ability,but IFN-γmight have a inhibitory effect on the osteogenesis of MM MSCs.（4）On day 28of induction culture,the average calcium nodules number of IL-17A,TNF-αand TGF-βgroups were 17.7±1.5,16.8±1.6 and 15.0±1.5,respectively,all were higher than the control group（12.0±1.7）（P<0.05）.But in the IFN-γgroup,the calcium nodule number was 7.3±1.5 which was fewer than the blank control group.（5）Western blot showed that the expression of protein Runx2 was significantly higher in the IL-17A,TNF-αand TGF-βgroup than in blank control group（P<0.05）,but no significant difference was found between IFN-γand blank control group（P>0.05）.Wnt5a expression of IL-17A and TNF-αgroups were significantly decreased.However in TGF-βand IFN-γgroups,the expressions were similar to the blank control.For protein TRAF4 which may be involved in the pathways of osteoblast formation,IL-17A and TNF-αgroups had higher expression compared with the blank control（P<0.05）.TGF-βgroup had similar expression and IFN-γgroup had lower expression compared with the blank control（P<0.05）.Conclusion:（1）IL-17A and TNF-αcould enhance the osteogenesis of MSCs in MM and increase the ALP activity and calcium nodules formation.（2）TGF-βmight also facilitate the differentiation ability of MM MSCs into osteoblast.But IFN-γhad an inhibitory effect on it.（3）The mechanisms of IL-17A and TNF-αstrengthening of osteogenesis might be related to TRAF pathway.