HtrA1 Regulate Odontoblastic Differentiation Of Dental Pulp Cells Via TGF-?1/Smad Pathway

Objective: hDPCs were differentiated into odontoblast after induced by mineralizing medium.Mineralized nodules formation,ALP activity and the expressions of mineral-associated genes were investigated.Then,the expressions of HtrA1 and factors of TGF-?1/Smad pathway during the odontoblastic differentiation of hDPCs were investigated.In addition,the mineralized nodules,the expressions of HtrA1 and factors of TGF-?1/Smad pathway during mineralization induction of hDPCs also were detected after HtrA1 gene suppressed by RNA interference.The relationship between HtrA1 and TGF-?1/Smad pathway in the odontoblastic differentiation of hDPCs was analyzed.The concrete mechanism of dental pulp repair and dentin formation was probed.Our study may provide a novel theory for pulp protection in clinical.Metheds: 1.After cultured by mineralizing medium 28 days,hDPCs were divided into 5 groups: day 0,7,14,21 and 28.Mineralized nodules formation,ALP activity and the expressions of mineral-associated genes,including ALP,COL?and DSPP were evaluated respectively.Simultaneously,the expressions of HtrA1 and factors of TGF-?1/Smad pathway also were detected by RT-PCR.2.Lentiviral vector of RNAi targeting human HtrA1 gene was constructed for knocking down the expression of HtrA1.Resistant clones of hDPCs knockdown were selected by puromycin after the virus infection.The efficiency of RNA interference was detected.Growth potentialities were evaluated by drawing the growth curves of infected and normal hDPCs.The infected hDPCs were treated with mineralization medium for 21 days.Next,mineralized nodules formation,ALP activity and the expressions of the mineral-associated genes were detected.Simultaneously,the expressions of HtrA1 and factors of TGF-?1/Smad pathway also were detected by RT-PCR.Results: 1.The third generation hDPCs showed fibroblast-like morphology and spindle-shape.hDPCs showed positive expression of vimentin but negative for keratin by immunocytochemical staining,which revealed its mesenchymal origin-derived and odontoblast characteristic.During the process of hDPCs differentiated into odontoblasts,the mineralized nodules formation were increased significantly,the ALP activity was enhanced and the expressions of the mineral-associated genes were progressively increased.The mRNA expression of HtrA1 got the peak on day 7,and then progressively declined.The mRNA expression of TGF-?1 began to increase on day 7 and got the peak on day 21.The mRNA expressions of T?R?and T?R? were increased on day 7and got the peak on day 21.The mRNA expressions of Smad2 and Smad4 began to increase on day 14 and got the peak on day 21.Simultaneously,the mRNA expressions of Smad6 and Smad7 were dramatically increased on day 21 and remained high level on day 28.2.After 4 days' infection,about 60 percent cells of hDPCs showed green fluorescence in NC group and H group but not in the blank group.After selected by puromycin,the green fluorescence positive rate of hDPCs was increased tomore than 95%.There was no significant difference on the HtrA1 mRNA expression between blank group and NC group.Compared with NC group,however,both mRNA and protein expressions of HtrA1 were dramatically decreased in H group.The growth curves of normal and infected h DPCs showed the same trend of growth potentiality.Compared with NC group,the mineralized nodules formation,the ALP activity and expressions of mineral-associated genes were decreased significantly in H group during odontoblastic differentiation of hDPCs.Meanwhile,the mRNA expressions of TGF-?1 and downstream factors of TGF-?1/Smad pathway,including T?R?,T?R?,Smad2,Smad4,Smad6 and Smad7,were significantly decreased after HtrA1 knockdown during odontoblastic differentiation of hDPCs.Conclusion:The mRNA expressions of HtrA1,T?R ? and T?R ? were increased during odontoblastic differentiation of h DPCs.Meanwhile,the downstream factors of TGF-? 1/Smad pathway,including Smad2,Smad4,Smad6 and Smad7 were also increased.HtrA1 knockdown inhibited the process of hDPCs differentiated into odontoblast,also inhibited the expression of TGF-?1/Smad pathway simultaneously.We speculated that HtrA1 might promote odontoblastic differentiation of hDPCs through activation of TGF-?1/Smad pathway.