The Effects Of Protein Phasphatase-1 On The Phosphorylation Of IKK In NF-κB Signal Transduction Pathway In Osteoblast Cells

Protein phosphorylation and dephosphorylation are a major mechanism for the mechanical signaling transduction, leading to specific biological effects. Osteoblast cells are the effectors of mechanical strain stimulation, and also are the sensors of mechanical and biochemical signals. The balance of bone remodeling caused by mechanical strain is mainly maintained by osteoblast cells. It is found that NF-κB signal transduction pathway is activated by mechanical strain, and regulates the gene expression in osteoblast cells. Data have demonstrated that the phosphorylation of IKK complex is mediated by protein kinase in the NF-κB signal transduction pathway. However, the mechanism of dephosphorylation of IKK complex induced by protein phosphatase is unclear. ObjectiveIn this study, MC3T3-E1 osteoblast-like cell line with stable high PP-1 protein expression was to be established. And we would observe the effects of highly expressed PP-1 gene on the phosphorylation of IKK in NF-κB pathway in mouse osteoblast-like MC3T3-E1 cells under mechanical strain. Materials and methodsPart 1 The primer was designed and synthesized according to the gene order in the GenBank. PP-1 gene was augmented from the MC3T3-E1 cells using RT-PCR, and was inserted directly in the pCI-neo vector.The expression vector pCI-neo-PP-1 was transfected into MC3T3-E1 cell lines with Liposomes after identification by PCR and double digestion, and control cells were transfected with empty vetor. The positive cell clones were selected with G418 48h after transfection.Then, the stable transfection and expression of PP-1 in MC3T3-E1 cells were determined by Western blot.Part 2 The stable PP-1 overexpressed osteoblast-like MC3T3-E1 cells or control cells were subjected to 8% elongation using a Flexercell Strain Unit. After 5, 15, 30 and 60 minutes, the levels of pIKKαand pIKKβwere determined by western blot analysis respectively.ResultsPart 1 The results of RT-PCR and enzyme digestion demonstrated that the expression vector pCI-neo-PP-1 was constructed correctly. Western blot test indicated that PP-1 was transcripted and expressed in the transfected cells.Part 2 At 5, 15, 30 and 60 minutes after subjection to 8% elongation, the lever of pIKKαand pIKKβin MC3T3-E1 cells with stable high expression of PP-1 gene were both significant decreased compared with the control cells. ConclusionMC3T3-E1 cells with stable high expression of PP-1 gene were successfully constructed. Furthermore, we demonstrated that highly expressed PP-1 gene inhibited the phosphorylation of IKK in NF-κB pathway in mouse osteoblast-like MC3T3-E1 cells under mechanical strain.