The Role And Possible Mechanism Of PTEN Phosphorylation On Cell Proliferation Of MMC Induced By HMGB1

Objective: To investigate whether the PTEN phosphorylation played an important role on the proliferation of MMC induced by HMGB1 and the possible mechanism of PTEN phosphorylation.Methods:1 Mouse mesangial cells?MMC?were used for this study.The cells were collected at 0 h,12 h and 24 h after stimulation with 100 ?g.L^-1 HMGB1 to detect the expression of PCNA by Western blot;and the percentage of Brd U positive cells was detected by flow cytometry technique.2 The MMC were collected at 0 min,10 min,30 min,60 min and 120 min after stimulation with 100 ?g.L^-1 HMGB1 to detect the expression of p-PTEN-Ser380,p-PTEN-Ser370,p-S6K-Thr389 and S6 K proteins by Western blot.3 MMCs were used and divided into five groups?control group,HMGB1 group,HMGB1+EV group,HMGB1+PTEN-S380 A group and HMGB1+PTEN-S380 D group?.BrdU incorporation and immunofluorescence staining were carried out to detect the expression of BrdU;and the expression of Ki67 protein was detected by immunocytochemistrical staining.4 MMCs were divided into three groups?control group,HMGB1 group and HMGB1+PF-4708671 group?.The cells in the HMGB1+PF-4708671 group were pretreated with PF-4708671(25 ?mol.L^-1)for 24 h.Then the cells were collected at 10 min,30 min and 24 h after stimulation with 100 ?g.L^-1 HMGB1 and the expression of p-PTEN-Ser380,p-S6K-Thr389,S6 K and PCNA proteins was detected by Western blot.Results: 1 HMGB1 increased the proliferation of MMCBy flow cytometry,the percentage of BrdU positive cells in HMGB1-24 h group was more significantly higher than those in HMGB1-0 h group and HMGB1-12 h group?P<0.05?.And the expression of PCNA protein in HMGB1-12 h and HMGB1-24 h group was significantly higher than that in 0 min group by Western blot.In addition,the PCNA protein expression in HMGB1-24 h group was the highest in three groups.?P <0.05?.2 HMGB1 induced the phosphorylation of PTEN at Ser-380 in MMCThe result of Western blot showed that the expression of p-PTEN-Ser380 increased at 10 min,30 min and 60 min,peaked at 30 min?P <0.05?.However,there was not significantly different of p-PTEN-Ser370 in these groups.3 Phosphorylation of PTEN at Ser380 mediated the MMC proliferation induced by HMGB1The results of BrdU incorporation and immunofluorescence staining showed that the percentage of BrdU positive cells increased in HMGB1 group compared with the control group?P <0.05?.Compared with HMGB1 group,the percentage of BrdU positive cells furtherly increased in HMGB1+PTEN-Ser380 D group,while the percentage of BrdU positive cells decreased in HMGB1+PTEN-Ser380 A group?P <0.05?.In addition,HMGB1 upregulated the percentage of Ki67-positive cells compared with the control group?P<0.05?;PTEN-Ser380 D significantly upregulated the expression of Ki67 protein,however PTEN-Ser380 A reduced the percentage of Ki67-positive cells in MMC induced by HMGB1?P <0.05?.4 The S6 K protein was phosphorylated by HMGB1 in MMCThe result of Western blot showed that the expression of p-S6K-Thr389 was significantly elevated at 10 min,reduced at 30 min,but the expression of p-S6K-Thr389 was still higher than that in 0 min group and reached to normal level at 60 min and 120 min?P<0.05?.However,the expression of total S6 K among five groups was not significantly different.5 PF-4708671,the special inhibitor of S6 K signal pathway,reduced the expression of p-PTEN-Ser380,p-S6K-Thr389 and decreased the cell proliferation induced by HMGB1The results of Western blot showed that the expression of p-PTEN-Ser380,p-S6K-Thr389 and PCNA protein in the HMGB1 group was higher than that in control group;while PF-4708671 decreased the expression of p-PTEN-Ser380 and p-S6K-Thr389 induced by HMGB1.In addition,PF-4708671 downregulated the PCNA protein expression induced by HMGB1?P <0.05?.Conclusions:1 HMGB1 induced the phosphorylation of PTEN at Ser380 in MMC.2 The phosphorylation of PTEN at Ser380 mediated the cell proliferation of MMC induced by HMGB1.3 The phosphorylation of PTEN at Ser380 was mediated by the activity of S6 K in MMC induced by HMGB1.