MAPK Pathways Are Invoved In TNFα-regulated GLUT1 Expression And Osteoblast Differentiation

Bone homeostasis is maintained by osteoclasts and osteoblasts, imbalance between the two kinds of cell activity directly led to the imbalance of the role of bone resorption and bone formation,resulting in corresponding bone disease happening. Under normal physiological conditions, the effect of osteoclasts and osteoblasts is closely coordinated so as to avoid the occurrence of bone loss and bone hyperplasia; but in chronic inflammatory bone diseases such as arthritis, osteomyelitis, periodontal bone loss, the imbalance of osteoclasts and osteoblasts led to abnormal bone loss or bone formation. In rheumatoid arthritis(RA), increased levels and activities of proinflammatory cytokines such as tumor necrosis factor(TNF)-α result in joint and/or bone destruction; Diabetes and obese patients also had higher levels of TNFα expression, their bone mineral density and bone mass are lower than those of healthy people, they are more likely to have osteoporosis. Osteoporosis is a skeletal disorder characterized by a systemic impairment of bone mass, strength, and microarchitecture, which increases the propensity of fragility fractures. Most of these diseases belong to chronic inflammatory disease. So it is very necessary and important to research the effect of TNFα on osteoblast differentiation and bone formation.We first investigated the influence of TNFα on osteoblast differentiation using MC3T3-E1 cell line. After TNFα stimulating differentiated MC3T3-E1 cells, we applied the real-time fluorescent quantitative PCR, alkaline phosphatase activity assay and alizarin red S staining experiments to detect the effect of TNFα on osteoblast differentiation and mineralization. Further, we explored the molecular mechanism of TNFα affecting osteogenetic differentiation. MAPKs are well‐studied pathways that are implicated in the regulation of osteoblast differentiation, and different regulation patterns have been observed in response to various factors. We used ERK, JNK and p38 kinase corresponding inhibitors to test the effect of MAPK signaling pathways on regulation of osteoblast differentiation affected by TNFα with western blotting and alkaline phosphatase activity assay methods in MC3T3-E1 cells. In addition, we also explored the glucose metabolism of osteoblast and its preliminary regulation mechanism induced by TNFα according to the expression change of GLUT1 in MC3T3-E1 cells.The main research results are:(1) Real-time fluorescent quantitative PC R results showed that TNFα supressed the expression of osteogenetic marker genes ALP, OC and BSP in MC3T3-E1 cells. Alkaline phosphatase activity assay and alizarin red S staining results showed that TNFα treatment group had lower alkaline phosphatase activity and more shallow dyeing compared with negative control.(2) TNFα activated MAPK kinase activity and its short-term stimulus increased the expression of GLUT1 in MC3T3-E1 cells by the dose and time-dependently.(3) Knockdown of GLUT1 expression downregulated the expression of osteogenetic marker genes ALP and BSP in MC3T3-E1 cells.(4) MAPK(ERK, JNK, p38) inhibitors treatment blocked the activation of MAPK kinase activity and upregulation of GLUT1 expression stimulated by TNF α, among them, ERK and JNK inhibitors reduced the decrease of alkaline phosphatase activity induced by TNFα, and JNK and p38 inhibitors significantly reversed the upregulation effect of GLUT1 expression induced by TNFα.Conclusion: TNFα suppressed osteogenic differentiation in MC3T3-E1 cells; MAPK signaling pathways played an important role in o steogenesis and ERK and JNK pathways were involved in the regulation of osteogenic differentiation by TNFα; TNFα had the short-term and long-term effect on the expression of GLUT1 in osteoblast, and short-term stimulus upregulated the expression of GLUT1, long-term action may decrease GLUT1 expression by inhibiting osteoblast differentiation though NF-kappa B pathway; MAPK signaling pathways, especially the JNK and p38 pathway were related to the upregulation of GLUT1 induced by TNFα. Probably TNFα regulated the expression of GLUT1 and then regulated glucose metabolic process through MAPK and NF-kappa B signal pathways in MC3T3-E1 cells.