DU Chun-Ling, Xu Yong-Jian, Liu Xian-Sheng, Xie Jun-Gang, Zhang Zhen-Xiang, Zhang Jian, Qiao Li-Fen, Ni Wang, Chen Shi-Xin
Pulmonary Disease Laboratory of Ministry of Health of China, Department of Respiratory Medicine, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Zhonghua Jie He He Hu Xi Za Zhi. 2008 Dec;31(12):915-20.
To explore the role of PKCalpha-ERK1/2 cascade in PMA induced up-regulation of cyclinD1 and P21(cip1) in human airway smooth muscle cells (HASMCs) sensitized by sera from atopic asthmatics.
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 PKCalpha mismatched Oligodeoxynucleotides (PKCalpha-mmODN) treated group, PMA and PKCalpha antisense Oligodeoxynucleotides (PKCalpha-asODN) treated group, PMA and U0126 (MAP Kinase Kinase inhibitor)treated group. The expression of p-PKCalpha, ERK1/2, p-ERK1/2, cyclinD1 and P21(cip1) protein were determined by western blotting. The proliferation of HASMC was examined by cell cycle analysis and MTT colorimetric assay.
Compared with the control group, the expression of p-PKCalpha and ERK1/2, p-ERK1/2 protein increased, the expression of cyclinD1, P21(cip1) 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 cells proliferation [the percentage of cells in S phase was (30.3 +/- 2.4)%, A(490) value was 0.80 +/- 0.06] enhanced significantly compared with those [the percentage of cells in S phase was (13.9 +/- 2.6)%, A(490) value was 0.41 +/- 0.04] of the control group (q = 6.07, 12.63; all P < 0.05). In PMA and PKCalpha-asODN treated group, the level of p-PKCalpha decreased, the expression of ERK1/2, p-ERK1/2 and the expression of cyclinD1, P21(cip1) 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)%, A(490) 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-PKCalpha had no significant change (A value was1.99 +/- 0.18, q = 0.94, P > 0.05), but the levels of ERK1/2, p-ERK1/2 decreased, the expression of cyclinD1, P21(cip1) 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)%, A(490) value was 0.49 +/- 0.03; q = 5.51, 13.45; all P < 0.05], as compared with those of the PMA treated group.
ERK1/2 is one of the downstream regulators of PKCalpha, and PKCalpha-ERK1/2 cascade is involved in PMA induced up-regulation of cyclinD1 and P21(cip1) and proliferation in HASMC sensitized by sera from atopic asthmatics.
探讨蛋白激酶Cα（PKCalpha）-细胞外信号调节激酶1/2（ERK1/2）级联反应在特应性哮喘患者血清致敏的人气道平滑肌细胞（HASMCs）中，佛波酯（PMA）诱导细胞周期蛋白D1（cyclinD1）和P21（cip1）上调中的作用。
用哮喘患者的10%血清对培养的HASMCs进行被动致敏，随机分为五组：对照组、PMA处理组、PMA与PKCalpha错配寡脱氧核苷酸（PKCalpha-mmODN）处理组、PMA与PKCalpha反义寡脱氧核苷酸（PKCalpha-asODN）处理组、PMA与U0126（丝裂原活化蛋白激酶激酶抑制剂）处理组。采用蛋白质免疫印迹法检测磷酸化PKCalpha（p-PKCalpha）、ERK1/2、磷酸化ERK1/2（p-ERK1/2）、cyclinD1和P21（cip1）蛋白的表达。通过细胞周期分析和MTT比色法检测HASMC的增殖情况。
与对照组相比，PMA处理组中p-PKCalpha、ERK1/2、p-ERK1/2蛋白表达增加，cyclinD1、P21（cip1）蛋白表达相应增加（A值/对照%分别为2.10±0.29、1.67±0.19、2.20±0.27、1.99±0.22和3.11±0.29；q值分别为9.87、7.06、10.57、11.10和20.33；均P<0.05），细胞增殖[处于S期的细胞百分比为（30.3±2.4）%，A（490）值为0.80±0.06]较对照组[处于S期的细胞百分比为（13.9±2.6）%，A（490）值为0.41±0.04]显著增强（q=6.07、12.63；均P<0.05）。在PMA与PKCalpha-asODN处理组中，p-PKCalpha水平降低，ERK1/2、p-ERK1/2表达及cyclinD1、P21（cip1）表达相应降低（A值/对照%分别为1.23±0.19、1.34±0.18、1.52±0.20、1.45±0.18和1.49±0.18；q值分别为7.49、3.58、5.97、6.06和15.65；均P<0.05），细胞增殖较PMA处理组显著降低[处于S期的细胞百分比为（21.2±2.8）%，A（490）值为0.51±0.04；q=6.07、12.63；均P<0.05]。在PMA与U0126处理组中，p-PKCalpha水平无显著变化（A值为1.99±0.18，q=0.94，P>0.05），但ERK1/2、p-ERK1/2水平降低，cyclinD1、P21（cip1）表达减少（A值/对照%分别为0.95±0.21、1.15±0.19、1.37±0.15和1.96±0.21；q值分别为7.79、9.16、6.92和11.16；均P<0.05），细胞增殖较PMA处理组显著降低[处于S期的细胞百分比为（22.0±3.2）%，A（490）值为0.49±0.03；q=5.51、13.45；均P<0.05]。
ERK1/2是PKCalpha的下游调节因子之一，PKCalpha-ERK1/2级联反应参与了PMA诱导的特应性哮喘患者血清致敏的HASMC中cyclinD1和P21（cip1）的上调及细胞增殖。
