Yu Hong-Mei, Li Qi, Perelman Juliy M, Kolosov Victor P, Zhou Xiang-Dong
Department of Respiratory Medicine, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China.
Zhonghua Yi Xue Za Zhi. 2011 Feb 15;91(6):391-5.
To explore the effects of sphingosine kinase 1 (SphK1) on mucin (MUC)5AC overexpression under the induction of tumor necrosis factor-α (TNF-α).
The HBE16 airway epithelial cells were cultured, pretreated with SphK1-specific inhibitor DMS or transfected with SphK1 siRNA. Then each group was stimulated by a certain concentration of TNF-α. The relative contents of SphK1, COX-2, p-P38, p-ERK and IκBα were detected by Western blot while the levels of MUC5AC, PGE2 and cAMP analyzed by enzyme linked immunoassay. The transcription activities of cAMP-response-element-binding protein and nuclear factor κB (NF-κB) were detected by luciferase reporter gene detection system. The mRNA expressions of SphK1 and MUC5AC were detected by RT-PCR (reverse transcription-polymerase chain reaction). And the intracellular MUC5AC protein was detected by immunofluorescence.
The levels of protein and mRNA expression of SphK1 and MUC5AC were obviously elevated [(0.69 ± 0.12, 0.86 ± 0.07), (0.60 ± 0.09, 0.79 ± 0.05)]. They were all significantly higher than those in the control group (0.26 ± 0.03, 0.33 ± 0.04, 0.18 ± 0.06, 0.22 ± 0.10). There was an elevation of COX-2, PGE2, cAMP, p-P38, p-ERK, CREB and NF-κB activity (all P < 0.01); the production of IκBα protein was lower than that in the control group (P = 0.003). DMS decreased the levels of MUC5AC mRNA expression (0.37 ± 0.06) and protein production (0.27 ± 0.04), lowered the production of COX-2, PGE2, cAMP, p-P38 and p-ERK, inhibited the activity of CREB and NF-κB and increased the IκBα production (all P < 0.01). Transfection of SphK1 siRNA showed the similar effects as DMS (all P < 0.01).
SphK1 plays an important role in regulating the MUC5AC expression under the induction of TNF-α. And the activation of critical signal factors in that process is dependent on SphK1.
探讨鞘氨醇激酶1（SphK1）在肿瘤坏死因子-α（TNF-α）诱导下对黏蛋白（MUC）5AC过表达的影响。
培养人支气管上皮细胞系HBE16，用SphK1特异性抑制剂二甲基鞘氨醇（DMS）预处理或转染SphK1小干扰RNA（siRNA）。然后用一定浓度的TNF-α刺激各组细胞。采用蛋白质免疫印迹法检测SphK1、环氧化酶-2（COX-2）、磷酸化P38（p-P38）、磷酸化细胞外信号调节激酶（p-ERK）和核因子κB抑制蛋白α（IκBα）的相对含量，采用酶联免疫吸附测定法分析MUC5AC、前列腺素E2（PGE2）和环磷腺苷（cAMP）水平。用荧光素酶报告基因检测系统检测cAMP反应元件结合蛋白（CREB）和核因子κB（NF-κB）的转录活性。采用逆转录-聚合酶链反应（RT-PCR）检测SphK1和MUC5AC的mRNA表达。用免疫荧光法检测细胞内MUC5AC蛋白。
SphK1和MUC5AC的蛋白和mRNA表达水平均明显升高[（0.69±0.12，0.86±0.07），（0.60±0.09，0.79±0.05）]，均显著高于对照组（0.26±0.03，0.33±0.04，0.18±0.06，0.22±0.10）。COX-2、PGE2、cAMP、p-P38、p-ERK、CREB和NF-κB活性均升高（均P<0.01）；IκBα蛋白产生低于对照组（P=0.003）。DMS降低了MUC5AC mRNA表达水平（0.37±0.06）和蛋白产生量（0.27±0.04），降低了COX-2、PGE2、cAMP、p-P38和p-ERK的产生，抑制了CREB和NF-κB的活性，增加了IκBα的产生（均P<0.01）。转染SphK1 siRNA显示出与DMS相似的作用（均P<0.01）。
SphK1在TNF-α诱导下调节MUC5AC表达中起重要作用，该过程中关键信号因子的激活依赖于SphK1。
