The Mechanism Of JAK2/STAT3 Acetylated Modification In Angiotensin â…¡ Induced Epithelial-to-mesenchymal Transition In Murine Lung Epithelial Cells

PartⅠ:JAK2/STAT3 pathway activation is involved in AngiotensinⅡ induced epithelial-myofibroblast transdifferentiation in murine lung epithelial cellsObjective:To investigate the effect of JAK2/STAT3 signal transduction pathway activation on the transdifferentiation induced by AngiotensinⅡ in murine lung epithelial cells (MLE-12).Methods:(1) Established a model of epithelial-myofibroblast transdifferentiation in MLE-12 induced by Ang Ⅱ. MLE-12 cells were stimulated with Ang Ⅱ at different concentrations for 48h before harvesting, the Ang Ⅱ ranging in concentrations from 0.1 uM to 10uM. Western blotting was used to determine the protein expression levels of a-SMA, E-cadherin.(2) Detect the JAK/STATsignaling pathway of MLE-12 cells induced by AngiotensinⅡ. MLE-12 cells were divided into four groups:control group (lumol/LPBS), Ang Ⅱ group (lumol/LAngⅡ), AngⅡ+DMSO group (lumol/LAngⅡ+10umol/LDMSO) and AngⅡ+AG490group (1umol/L Ang Ⅱ+10 umol/L AG490, a specific inhibitor of JAK2). The cells were cultured in 24-pore plate and 25cm2 plastic culture flask. After treatment for 0.5h, the supematants was collected and cells were harvested for protein extraction. Western blotting were used to determine the protein expression levels of Januskinase2 (JAK2), tyrosine phosphorylated Januskinase2 (p-JAK2), STAT3 and p-STAT3.(3) Detect the EMT of MLE-12 after treatment with AngiotensinⅡ and AG490. MLE-12 were divided into four groups:control group (lumol/L PBS), AngⅡgroup (1umol/L AngⅡ), AngⅡ+DMSOgroup (lumol/LAngⅡ+10umol/LDMSO) and AngⅡ+AG490group (1umol/L Ang Ⅱ+10 umol/L AG490, a specific inhibitor of JAK2). After cultured in 24-pore plate and 25cm2 plastic culture flask for 48h, the supematants was collected and cells were harvested for protein extraction. The expression levels of a-SMA, E-cadherin and TGF-β determined by Western blotting.(4) Detect the proliferation of MLE-12 after treatment with AngiotensinⅡ, DMSO and AG490. MLE-12 were seeded in 96-well plates at 5×103 cells per well in 100 μl of growth medium. After treatment for 48h, CCK-8 kit was used to detect cell proliferation index according to manufacturer’s instructions.The results were presented as the mean±standard deviation (SD). Statistical analyses were performed using the SPSS 16.0 software package. For group comparison, a one-way analysis of variance (ANOVA) was used, followed by Tukey’s test. A p-value less than 0.05 was considered to be statistically significant.Results:(1) Angiotensin Ⅱ can induce epithelial-myofibroblast transdifferentiation in murine lung epithelial cells. Our study revealed that the expression of α-SMA by Ang Ⅱ occurred in a concentration-dependent manner.Maximal expression of α-SMA was observed with luM Ang Ⅱ.(2) Ang Ⅱ induced JAK2/STAT3 activation in cultured murine lung epithelial cells. Our study demonstrated that compared with control group, the expression of JAK2-phosphorylation on Tyr1007 and 1008 and STAT3-phosphorylation on Tyr705 was significantly increased after Ang Ⅱ luom/1 stimulated for 30minutes (P<0.01). While the expression of JAK2 and STAT3 were no significant difference between the four groups.(3) AG490 opposed Ang Ⅱ induced JAK2/STAT3 activation in cultured murine lung epithelial cells. AG490, a tyrosine kinase tyrphostin that inhibits the activity of JAK2. Our study demonstrated that the expression of p-JAK2 and p-STAT3 in AngⅡ+AG490 group were obviously lower than those in Ang Ⅱ group and Ang Ⅱ+DMSO group (P<0.01).(4) Ang Ⅱ induced EMT in MLE-12. E-Cadherin is the marker of epithelial cells, while the a-SMA is the hallmark of fibroblast. We found that compared to control group, the expression of E-Cadherin were significantly decreased after luom/1 Ang Ⅱ stimulated for 48h (P<0.01).While the expression of TGF-β and α-SMA were significantly increased (P<0.01).(5) AG490 opposed angiotensin Ⅱ induced EMT in MLE-12. We found that the pretreatment of cells with AG490 also abolished downstream α-SMA and TGF-β expression (P<0.05). Furthermore, AG490 attenuated Ang Ⅱ induced down-regulation of E-Cadherin (P<0.01).(6) There was no significant difference in proliferation of MLE-12 after treatment with Angiotensin Ⅱ, DMSO and AG490.Conclusions:Activation of JAK2/STAT3 signaling path way may be involved in the Ang Ⅱ induced epithelial-myofibroblast transdifferentiation in murine lung epithelial cells.Part Ⅱ:The role of STAT3 acetylation in angiotensin Ⅱ induced epithelial-myofibroblast transdifferentiation in murine lung epithelial cellsObjective:To explore the role of STAT3 acetylation in angiotensin Ⅱ (Ang Ⅱ)-induced epithelial-myofibroblast transdifferentiation in murine lung epithelial cells (MLE-12).Methods:(1) Detection of JAK/STAT signaling pathway of MLE-12 induced by Angiotensin Ⅱ. Four groups of MLE-12 including control group (1umol/LPBS), Ang Ⅱ group (lumol/LAngⅡ), AngⅡ+DMSO group (1umol/LAngⅡ+10umol/LDMSO) and AngⅡ+SAHA group (1umol/L Ang Ⅱ+3umol/L SAHA, an inhibitor of histone-deacetylase) were cultured in 24-pore plate and 25cm2 plastic culture flask. After treatment for 0.5h, the supematants was collected and cells were harvested for determine the protein expression levels of STAT3, p-STAT3 and acetylation-STAT3 by western blotting.(2) Detection of EMT of MLE-12 after treatment with Angiotensin Ⅱ and SAHA. MLE-12 were divided into four groups:control group (1umol/L PBS), Ang Ⅱ group (lumol/L AngⅡ), AngⅡ+DMSO group(lumol/LAngⅡ+10umol/LDMSO) and AngⅡ+SAHA group (lumol/L Ang Ⅱ+3umol/L SAHA, an inhibitor of histone-deacetylase). The cells were cultured in 24-pore plate and 25cm2 plastic culture flask. After treatment for 48h, the supematants was collected and cells were harvested for protein extraction. Western blotting were used to determine the protein expression levels of a-SMA, E-cadherin and TGF-β.(3) Detection of proliferation of MLE-12 after treatment with Angiotensin Ⅱ, DMSO and SAHA. MLE-12 were seeded in 96-well plates at 5×103 cells per well in 100 μl of growth medium. After treatment for 48h, CCK-8 kit was used to detect cell proliferation index according to manufacturer’s instructions.The results were presented as the mean±standard deviation (SD). Statistical analyses were performed using the SPSS 16.0 software package. For group comparison, a one-way analysis of variance (ANOVA) was used, followed by Tukey’s test. A p-value less than 0.05 was considered to be statistically significant.Results:(1) Ang Ⅱ induced JAK2/STAT3 activation in cultured murine lung epithelial cells. Our study demonstrated that compared with control group, STAT3 phosphorylation on Tyr705 was significantly increased after Ang Ⅱ 1uom/l stimulated for 30 minutes (P<0.01).(2)SAHA opposed Ang Ⅱ induced JAK2/STAT3 activation in cultured murine lung epithelial cells. treatment of the cells with SAHA(3umol/l) significantly increased STAT3 acetylation on Lys685 (P<0.05), the expression of p-STAT3 in AngⅡ+SAHA group were obviously lower than those in Ang Ⅱ group and Ang Ⅱ+DMSO group (P<0.01).(3)Ang Ⅱ induced EMT in MLE-12. E-Cadherin is the marker of epithelial cells, while the β-SMA is the hallmark of fibroblast. We found that compared to control group, the expression of E-Cadherin were significantly decreased after 1uom/1 Ang Ⅱ stimulated for 48h (P<0.01).While the expression of TGF-β and α-SMA were significantly increased (P<0.01).(4)SAHA opposed angiotensin Ⅱ induced EMT in MLE-12. We found that compared with Ang Ⅱ group, the expressions of a-SMA and TGF-P were also decreased in AngⅡ+SAHA group (P<0.05).While the expressions of E-cadherin were increased in Ang Ⅱ+SAHA group (P<0.05).(5)There was no significant difference of proliferation of MLE-12 after treatment with Angiotensin Ⅱ, DMSO and SAHA.Conclusion:STAT3 acetylation attenuates epithelial-myofibroblast transdifferentiation in murine lung epithelial cells in vitro.