TNF-α Stimulates Bone Marrow Mesenchymal Stem Cells VCAM-1Production By ERK/JNK—NF-κB Signaling Pathways

Background:Recently, more and more mesenchymal stem cell (mesenchymal stem cells, MSCs)-related researches and clinical trials have developed in different subjects around the world which evoked new hope in a variety of disorders. MSCs are a subset of stromal stem cells that have the ability of self-renewal and multipotency, which can differentiate into cells of the mesodermal lineages and other embryonic lineages, such as osteocytes, chondrocytes, adipocytes, myocardial cells and neurons. Moreover, MSCs could be easily expanded for several passages in vitro without losing their self-renew ability. A increasing number of evidences indicated that MSCs possess potential immunomodulation, anti-inflammation properties and trophic effects There is a growing recognition that mesenchymal stem cells may develop organ-protective effects mainly by a paracrine mechanism, rather than transdifferentiation. Homing to the injured tissue may be the necessary step for MSCs to exert their paracrine function. Preclinical animal experiments demonstrate that MSCs which systemic infused flow into the vascular circulation, firstly accumulated in the lung, and then preferentially engraft into inflamed or ischemic tissues and to the liver, spleen, kidney, and bone marrow, a behavior that is named "homing". However, MSCs are found at low or very low frequencies in most target organs or tissues, as shown by many experimental methods such as fluorescent protein labeling, immunohistochemistry, real-time PCR and fluorescent in situ hybridization. It would limit the effect of cell therapy to diseases in clinical. Therefore, how to enhance the effect of cell therapy and be safe has been the hotpot in medical major around the world. Recently, Tao have found that using high-resolution confocal and dynamic microscopy, like leukocytes, MSCs preferentially adhere to and migrate across tumor necrosis factor-a(TNF-a)-activated endothelium. TNF-a can upregulate vascular cell adhesion molecule-1(VCAM-1) production both of endothelium and MSCs. MSCs adhered to endothelium in VCAM-1and G-protein-coupled receptor signaling-dependent manner. And then there were two modes of transmigration for MSCs:paracellular (between endothelial cells) and transcellular (directly through individual endothelial cells) diapedesis through discrete gaps and pores in the endothelial monolayer and engrafted into target tissues to excert theirs therapeutical effect. TNF-α-induced adhesion could be completely blocked by pretreating with anti-VCAM-1monoclonal antibodies in MSCs. Therefore, TNF-a and VCAM-1play an important roles in MSCs’homing and adhension. MAPK (mitogen activated protein kinase) can be stimulated by a lot of growth factors and inflammatory factors.It involved in cell proliferation, differentiation,transformation and apoptosis. MAPK can be classified as ERK1/2、JNK、p38and ERK5signaling pathways, nuclear factor kappa(NF-KB) which popular in eukaryotic cells is polytropism and multifunction protein. NF-κB signaling pathway involves in body’s immunoreaction, inflammatory response and tumor development. Therefore, we infer that TNF-a may stimulate MSCs produce VCAM-1by NF-κB,ERK and JNK signaling pathway.Objective:Observe the effect of TNF-α on the VCAM-1expression of human bone marrow MSCs and the relationship between them. And explore the relationship among these three signaling pathways and the effect of TNF-a-stimulated VCAM-1expression of human bone marrow MSCs. This study was aimed to explore the mechanisms of MSCs’homing stimulated to enhance the effect of clinical cell therapy.Methods:(1) Bone marrow (10ml) was obtained from the iliac crest of voluntary donors from whom informed consent had been obtained. Bone marrow MSCs were isolated by density gradient centrifugation combined with adherent culture method.(2)When the cells were cultured to the three passages, we use flow cytometry to detect the surface antigen expression on the cells.(3)Put the cells of the three passages into the differential environment to induce their differentiation potential to adipocytes and osteoblasts.(4) Using flow cytometry to analyze expression of VCAM-1on bone marrow MSCs exposed to6.25%12.5、25、50、100ng/ml TNF-α for24h. QPCR was used to detect expression of VCAM-1at mRNA levels in TNF-α-stimulated group. Using Western blotting to detect the the activity of NF-κB、ERK and JNK signaling pathway respective in TNF-α-stimulated group.(5) Flow cytometry was used to test the VCAM-1of PDTC(inhibitor of NF-κB signaling pathway)-stimulated group、U0126(inhibitor of ERK signaling pathway)-stimulated group or SP600125(inhibitor of JNK signaling pathway)-stimulated group. QPCR was used to detect the VCAM-1mRNA of PDTC-stimulated group, U0126-stimulated group and SP600125-stimualted group.Western blotting was used to test the activities of NF-κB、ERK and JNK signaling pathway of PDTC-stimulated group, U0126-stimulated group and SP600125-stimualted group respective.(6)Using flow cytometry to detect VCAM-1expression of PDTC+TNF-α-stimulated group、 U0126+TNF-α-stimulated group and SP600125+TNF-α-stimulated group.QPCR was used to test the VCAM-1mRNA of PDTC+TNF-α-stimulated group、U0126+ TNF-a-stimulated group and SP600125+TNF-α-stimulated group.Using Western blotting to detect the activities of NF-κB,ERK and JNK signaling pathway in PDTC+TNF-a-stimulated group.. U0126+TNF-α-stimulated group and SP600125+TNF-a-stimulated group respective.Statistical methods:use SPSS16.0statistical software to analyze, measurement data are expressed in(x±s), multiple factors analysis using linear regression; More comparison using single factor analysis of variance between groups, when the variance is approximate F inspection Welch method. Together using Turkey multiple comparison when variance method, the variance is not using Dunnett’T3. Inspection level ofa=0.05, double side inspection.Results:(1) The bone marrow MSCs were isolated by density gradient centrifugation combined with adherent separation and cultured to three passages to obtain an ample amount of the bone marrow MSCs with a uniform, fibroblast-like and adherent appearance.(2)Flow cytometry analyzed that the cells positively expressed CD29(99.60%)、CD69(7.93%)、CD44(99.95%)、CD105(99.89%), and negatively expressed CD34(0.51%)、CD45(1.67%).(3) Little lipid drops were observed in the cytoplasm of certain cells on day4. Gradually, lipid drops have increased and numerous cells containing abundant big and round lipid drops were observed following14days of adipogenic differentiation. Positive staining with Oil Red O was observed. This means that cells could be differentiate into adipocytes. After osteogenic differentiation for21days, there were abundant red deposition of calcium by Alizarin Red S staining indicated cells could differentiate into osteoblasts.(4) After the stimulation of TNF-a at different concentrations for24h, flow cytometry analysis showed that VCAM-1expression was markedly increased by TNF-a treatment in a dose-dependent manner. At0-100ng/ml TNF-a, the expression of VCAM-1was increased strongly. In addition, when stimulated with50ng/ml TNF-a, VCAM-1expression levels reached at the peak.50ng/ml TNF-a-stimulated group has no statistical difference between100ng/ml TNF-a-stimulated group. QPCR analysis showed that the VCAM-1mRNA of TNF-a-stimulated group is28.39times as much as control, and the difference has statistical meaning (p<0.05). Western blotting results showed that TNF-a-stimulated group has higher activities of NF-JB,ERK and JNK signaling pathway than control,and the difference has statistical meaning(p<0.05).(5) In flow cytometry results, VCAM-1expression of PDTC-stimulated group、 U0126-stimulated group and SP600125-stimulated group are6.40%,5.22%,and6.21%respective. There were no obvious difference between inhibitors-stimulated groups and control (p>0.05).There were no obvious difference between the three inhibitors stimulated groups and control at VCAM-1mRNA level in QPCR results (p>0.05).There were no statistical difference between PDTC-stimulated group,U0126-stimulated group, SP600125-stimulated group and control in activity of NF-κB,ERK or JNK signaling pathway respective (p>0.05) in Western blotting.(6) The VCAM-1expression of PDTC+TNF-a-stimulated group,U0126+TNF-a-stimulated group and SP600125+TNF-a-stimulated group is19.56%,20.20%and19.59%respective which are have statistical difference compared with control (p<0.05).PDTC+TNF-a-stimulated group, U0126+TNF-a and SP600125+TNF-a are11.30times,14.92times and20.66times as much as control at VCAM-1mRNA level. There were statistical difference between them.(p<0.05).The NF-κB signaling pathway phosphorylation value of PDTC+TNF-a-stimulated group is0.77has a statistical difference compared with TNF-a group which phosphorylation value is1.03(p<0.05); The ERK signaling pathway phosphorylation value of U0126+TNF-a-stimulated group is0.93has a statistical difference compared with TNF-a group which phosphorylation value is1.40(p<0.05); The JNK signaling pathway phosphorylation value of SP600125+TNF-α-stimulated group is2.01has no statistical difference compared with TNF-α group which phosphorylation value is3.70(p>0.05).Conclusion:TNF-α can increase VCAM-1expression markedly in a dose-dependent manner. In addition, when stimulated with50ng/ml TNF-α, VCAM-1expression levels reached at the peak. And TNF-α can increase VCAM-1mRNA in MSCs.Moreover, TNF-α increased apparently protein phosphorylation of NF-κB、 ERK、JNK signaling pathway. NF-κB、ERK、JNK signaling pathway inhibitors could suppressed respective the increase induced by TNF-α both at VCAM-1expression and activities of three signaling pathways. All of this leads to the conclusion that TNF-a interacted with its specific receptors in human bone marrow mesenchymal stem cells, and then activate NF-κB、ERK and JNK signaling pathways to induce VCAM-1expression.