蛋白激酶Cα-胞外调节蛋白激酶1／2与人气道平滑肌细胞中周期蛋白D1及P21^cip1表达的关系

PKCα-ERK1/2 cascade is involved in up-regulation of cyclinD1 and P21eip1 in human airway smooth muscle cells sensitized by sera from atopic asthmatics

目的探究蛋白激酶C（PKC）α-胞外调节蛋白激酶（ERK）1／2级联对支气管哮喘（简称哮喘）患者血清被动致敏的人气道平滑肌细胞（HASMC）周期蛋白D1（cyclinD1）、周期蛋白依赖性激酶（CDK）抑制剂P21^cip1的调节作用及细胞增殖的影响。方法用含10％哮喘患者血清的DMEM培养基被动致敏HASMC后，按随机数字表法分为对照组、12-肉豆蔻酰-13-乙酸佛波酯（PMA）处理组、PMA±PKCα错配寡核苷酸组、PMA±PKCα反义寡核苷酸组以及PMA±U0126组，每组4个样本。用Westernblot方法检测各组细胞磷酸化PKCα（p-PKCα）、ERK1／2、磷酸化ERK1／2（p-ERK1／2）、cyclinDl和P21eipI的蛋白表达水平，用流式细胞术和四甲基偶氮唑盐（MTT）法检测HASMC增殖。结果PMA处理组p-PKCα水平、ERK1／2和p-ERKl／2蛋白水平高于对照组，cyclinD1、P21^rip1表达明显强于对照组（相对于各对照组的A值分别为2．10±0．29、1．67±0．19、2．20±0．27、1．99±0．22和3．11±0．29，均P〈0．05），HASMC的增殖[S期细胞比例为（30．3±2．4）％，A490值为0．80±0．06]高于对照组[S期细胞比例为（13．9±2．6）％，A490值为0．41±0．04]，均P〈0．05；PMA±PKCct反义寡核苷酸组中p-PKCot水平低于PMA处理组，ERK1／2和p-ERK1／2表达明显弱于PMA处理组，cyclinD1和P21cipl表达也明显低于PMA处理组（相对于各对照组的A值分别为1．23±0．19、1．34±0．18、1．52±0．20、1．45±0．18和1．49±0．18，均P〈0．05），HASMC的增殖显著下降[S期细胞比例为（21．2±2．8）％，A490值为0．51±0．04；g值分别为6．07，12．63；均P〈0．05]；同样与PMA处理组相比，PMA±U0126组中P-PKCα水平无明显改变（A值为1．99±0．18，g=0．94，P〉0．05），但ERK1／2、P—ERKI／2的表达，cyclinD1和P21^eip1的表达明显低于PMA处理组（A值分别为0．95±0．21、1．15±0．19、1．37±0．15和1．96±0．21，均P〈0．05），HASMC的增殖显著下降[S期细胞比例为（22．0±3．2）％，A490值为0．49±0．03；g值分别为5．51、13．45，均P〈0．05]。结论ERK1／2是PKCα的下游信号分子，PKCα-ERK1／2级联参与了PMA所诱导的哮喘患者血清被动致敏的人气道平滑细胞cyclinD1、P21cip1的表达上调及细胞增殖。

Objective To explore the role of PKCα-ERK1/2 cascade in PMA induced up-regulation of cyclinD1 and P21^eip1 in human airway smooth muscle cells （HASMCs） sensitized by sera from atopic asthmatics. Methods HASMCs in cultures were passively sensitized by 10% serum from asthmatic patients and were randomly divided into five groups： the control group, PMA treated group, PMA and PKCα mismatched Oligodeoxynucleotides （PKCα-mmODN） treated group, PMA and PKCα antisense Oligodeoxynucleotides （PKCα-asODN） treated group, PMA and U0126 （ MAP Kinase Kinase inhibitor ） treated group. The expression of p-PKCα, ERK1/2, p-ERK1/2, cyclinD1 and P21^eip1 protein were determined by western blotting. The proliferation of HASMC was examined by cell cycle analysis and MTI＇ colorimetric assay. Results Compared with the control group, the expression of p-PKCα and ERK1/2, p-ERK1/2 protein increased, the expression of cyclinD1, P21^eip1 protein increased correspondingly （the A value % control was 2. 10 ± 0.29, 1.67 ± 0. 19, 2. 20 ± 0. 27, 1.99 ± 0. 22 and 3.11 ± 0. 29 respectively ; q value was 9. 87, 7.06, 10. 57, 11.10 and 20. 33 respectively; all P 〈 0. 05） in PMA treated group, and ceils proliferation [ the percentage of cells in S phase was （ 30. 3 ± 2.4 ） % , A490 value was 0. 80 ± 0. 06 ] enhanced significantly compared with those [ the percentage of cells in S phase was （ 13.9 ± 2. 6） %, A490 value was 0. 41 ±0. 04]of the control group（q =6.07, 12. 63; all P 〈0. 05）. In PMA and PKCα-asODN treated group, the level of p-PKCα decreased, the expression of ERK1/2, p-ERK1/2 and the expression of cyclinD1, P21 eipl decreased correspondingly（the A value % control was 1.23 ± 0. 19, 1.34 ± 0. 18, 1.52 ± 0.20, 1.45 ±0.18 and 1.49 ±0.18 respectively; q value was 7.49, 3.58, 5.97, 6.06 and 15.65 respectively; all P 〈 0. 05 ）, and cells proliferation reduced significantly [ the percentage of cells in S phase was （21.2 ± 2. 8） %, A49o value was 0. 51± 0. 04 ; q = 6. 07, 12. 63 ; all P 〈 0. 05 ], as compared with those of the PMA treated group. In PMA and U0126 treated group , the level of p-PKCα had no significant change（A value wasl. 99 ±0. 18, q =0. 94, P 〉0. 05）, but the levels of ERK1/2, p-ERK1/2 decreased, the expression of cyclinD1, P21^eipl reduced （the A value % control was 0. 95 ±0. 21, 1.15 ±0. 19, 1.37 ± 0. 15 and 1.96 ±0. 21 respectively; q value was 7.79, 9. 16, 6. 92 and 11.16 respectively; all P 〈0. 05）, and cells proliferation reduced significantly [ the percentage of cells in S phase was （22.0 ± 3.2） %, A490 value was 0.49 ±0. 03 ; q = 5.51, 13.45 ; all P 〈 0.05 ], as compared with those of the PMA treated group. Conclusion ERK1/2 is one of the downstream regulators of PKCα, and PKCα-ERK1/2 cascade is involved in PMA induced up-regulation of cyclinD1 and P21^eipl and proliferation in HASMC sensitized by sera from atopic asthmatics.