Inflammatory Factor TNF-α May Affect N-cadherin Leveland Hematopoietic Support Capacity Of MC3T3-E1 Cell Line

Objective Abnormal hematopoiesis is ofen found in systemic lupus erythematosus(SLE)patients and the underlying mechanism is yet fully elucidated.Both differentiation and self-renewal of hematopoietic stem cells(HSCs)are closely regulated by bone marrow microenvironment,and neural calcium-dependent adhesin protein(N-cadherin)is an adhesion molecule,which can be detected in a variety of hematopoietic supporting cells within bone marrow,was reported to be involved in the interaction between HSCs and bone marrow microenvironment.The levels of some inflammatory factors such as tumor necrosis factor(TNF)-α,which is usually higher in SLE patiants than that in health subjects,were also suggested to have profound effects on the bone marrow mesenchymal stem cells(BMMSCs)and osteoblasts in the bone marrow microenvironment.Once affected by those inflammatory factors,their hematopoiesis-regulated capacity alter in a variety of ways.In this study,we investigated the impact of inflammatory factor TNF-α on both mice pre-osteoblastic cell line MC3T3-E1 and BMMSCs in vitro,and especially explored its influence on N-cadherin expression on both cells and also possible underlying mechanism.In addition,we further intended to investigate the capacity of TNF-α pre-treated MC3T3-E1 to support in vitro expansion of purified mice Sca-1(+)bone marrow cells.With all the experimental work mentioned above,we expected to reveal potential mechanism of hematopoiesis dysregulation in SLE.Methods1、Firstly,MC3T3-E1 cell line was treated with different concentrations(10ng/ml,20 ng/ml and 40 ng/ml)of TNF-α for three days.The expression of N-cadherin was detected by Western blot to decide an optimal concentration of TNF-α.Then,MC3T3-E1 cell line and mice BMMSCs were treated using given concentration of TNF-α for different time.At the end of TNF-α treatment,cells were resuspended in a TNF-α-free medium for a few more days.Western blot was employed to measure expression level of N-cadherin,p-Erk1/2 and p-Akt.2、CCK8 was used to measure proliferation of MC3T3-E1 cell line treated with Erk inhibitor FR180204(0~80 μM)for one to three days and to screen an optimal concentration of Erk inhibitor.Then,Western blot was used to measure expression of N-cadherin and p-Erk1/2 of MC3T3-E1 cell line treated with Erk inhibitor.3、Murine bone marrow cells were flushed from its femurs and tibias.Subsequently,Sca-1(+)bone marrow cells were sorted and enriched by positive magnetic cell sorting(MACS).The rate of positive Sca-1 cells was determined by flow cytometry(FCM).4、MC3T3-E1 cell line was cultured in Stemspan SFEM medium which was specially used for in vitro HSCs culture.The difference in morphology and N-cadherin expression of MC3T3-E1 under either 10% FBS α-MEM or serum-free Stemspan SFEM medium was also assayed by microscope and western blot,respectively.Subsequently,MC3T3-E1 was firstly treated by TNF-α for three days in Stemspan SFEM medium,after that,MC3T3-E1 was moved to TNF-α-free Stemspan SFEM for another few days.Western blot was again employed to measure the expression of N-cadherin.5、MC3T3-E1 cells primed with TNF-α were employed as feeder layer and murine Sca-1(+)bone marrow cells were added to the co-culture system in the presence of SCF,TPO and Flt3-Ligand for a total one week.At the end of co-culture,all the in vitro cultured cells were harvested,the different colony-forming cells was assayed by CFU assay.Results1、We found that N-cadherin expression of MC3T3-E1 cell line was down-regulated after TNF-α treatment and the reduction was more significant when using a higher concentration of TNF-α.According to the change of N-cadherin,we employed the concentration of 20 ng/ml for the following experiment.2、There was no difference on N-cadherin expression of MC3T3-E1 during short time treatment of 20 ng/ml TNF-α.After one and two days of treatment,N-cadherin expression of MC3T3-E1 was down-regulated while the expression level was even higher than basal level after TNF-α was removed for seven days.The expression of p-Erk1/2 was up-regulated during one day treatment of 20 ng/ml TNF-α.On the contrary to N-cadherin,p-Erk1/2 expression was lower than the basic level at the seventh day.There is no difference in p-Akt expression between TNF-α treatment cohort and control one.3、Both N-cadherin and p-Erk1/2 expressions in mice-derived BMMSCs were up-regulated after short(30min and 3h)exposure to TNF-α.However,their expressions were down-regulated during the three days of TNF-α treatment.After TNF-α was removed,N-cadherin level start to rise and even higher than basal level at the following days of continued culture instead.In addition,p-Erk1/2 expression was down-regulated gradually after removal of TNF-α.The The expression of p-Akt in BMMSCs did not change during the TNF-α treatment.4、We performed prolilerative assay of MC3T3-E1 cell line treated with Erk inhibitor FR180204(0~80 μM)by CCK8 and chose 7.5 μM and 10 μM as the optimal concentrations for the following experiment.After treated with 7.5 μM and 10 μM Erk inhibitor,the p-Erk1/2 expression of MC3T3-E1 cell line was down-regulated after treated for one day while higher than basal level after two and three days’ treatment.Instead,the expression of N-cadherin was up-regulated during the whole process of Erk inhibitor treatment.5、The rate of mice bone marrow positive Sca-1 cells enriched by immune magnetic cell sorting(MACS)was determined by FCM.It was showed that the positive percentage was above 90 %. 6、The morphology of MC3T3-E1 cell line cultured with Stemspan SFEM for different time was the same as that cultured with α-MEM containing 10% FBS and N-cadherin expression also had no difference between two medium culture.In addition,after treated with different concentrations of TNF-α liquor which was formulated by Stemspan SFEM for three days,N-cadherin expression of MC3T3-E1 was down-regulated remarkably below basal level.Furthermore,MC3T3-E1 cell line was treated by 20 ng/ml TNF-α for three days and during one week of culture after the removal of TNF-α,N-cadherin expression kept at a higher level than foundation,which implied that when cultured with Stemspan SFEM,the influence of N-cadherin of MC3T3-E1 cell line induce by TNF-α was the same as that cultured with 10% FBSα-MEM.7、MC3T3-E1 cells pre-treated with TNF-α were employed as feeder layer and were in vitro co-cultured with murine Sca-1(+)bone marrow cells in the presence of SCF,TPO and Flt3-Ligand for one week.We found that the total number of CFUs was down-regulated.It was also found that the number of CFU-pre-B was obviously increased and the number of BFU-E and CFU-GM was significantly reduced instead.Conclusions Inflammatory factor TNF-α could affect the expression of N-cadherin and Erk phosphorylation in both mice pre-osteoblastic cell line MC3T3-E1 and BMMSCs.During the process of interacting with murine Sca-1(+)bone marrow hematopoietic cells,MC3T3-E1 cell line which express high level of N-cadherin could alter the CFU capacity of Sca-1 enriched marrow cells.Our research suggested that TNF-α might alter bone marrow hematopoiesis via modulating the N-cadherin expression in hematopoiesis-supporting accessory cells such as osteoblast and BMMSCs.