Study On The Function Of KLF4 In Human Odontoblast Differentiation

Objective:KLF4 plays an important role in cyto-differentiation and proliferation. Our previous study showed that KLF4 was specifically expressed in polarizing and elongating odontoblasts. However, the role of KLF4 in odontoblast differentiation was still unknown. The purpose of this study was to investigate the role of KLF4 in odontoblastic differentiation of human dental pulp cells (hDPCs).Methods:PartⅠ:hDPCs were treated with odontoblastic induction medium.Odontoblastic differentiation was determined by detection of alkaline phosphatase (ALPase) activity, expression of mineralization-related genes including ALP, dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1). Also, cell proliferation ability was examined by 5-ethynyl-2’-deoxyuridine (EdU) incorporation assay. Simultaneously, mRNA and protein levels of KLF4 were detected.PartⅡ:pKLF4-IRES2-EGFP plasmid encoding full-length KLF4 was constructed to overexpress KLF4, and biologic effects of KLF4 on hDPCs were investigated by evaluation of ALPase activity, detection of ALP, DSPP and DMP-1 expression and analysis of cell proliferation ability.Part III:Lentiviral vector of RNAi targeting human KLF4 gene was constructed to knockdown KLF4.After package, the virus was used to infect the human dental pulp cells. The biologic effects of knockdown of hDPCs were investigated by evaluation of ALPase activity, detection of DSP and DMP-1 expression.Results: PartⅠ: ALPase activity and expression of odontoblastic differentiation markers progressively increased in hDPCs cultured with odontoblastic induction medium. Meanwhile, the proliferation ability decreased in this procedure. mRNA level of KLF4 increased significantly on day 5 after odontoblastic induction of hDPCs and kept increasing until day 14.While,protein level of KLF4 increased significantly on day 7 and kept increasing until day 14.Immunoflouresence results also confirmed an obvious up-regulation of KLF4 on day 7 of odontoblastic induction.PartⅡ: hDPCs showed upregulated activity of ALPase and expression of mineralization-related genes, including ALP, DMP-1 and DSP, after KLF4 overexpression. Besides, proliferation ability of hDPCs decreased significantly in the KLF4 overexpression group by EdU incorporation assay.PartⅢ: After 14 days odontoblastic induction, expression of KLF4 decreased significantly in shRNA-KLF4 group, and the activity of ALPase was also reduced significantly in contrast to the control group. The expression of DMP1 decreased significantly in shRNA-KLF4 group after 14 days odontoblastic induction, but the expression of DSP was not changed so obviously.Conclusion:Our findings suggest that KLF4 is needed and also able to promote odontoblastic differentiation of hDPCs and inhibit hDPCs’ proliferation.