PKB在类胰蛋白酶诱导基因表达中的作用

Role of protein kinase B in tryptase-induced gene expression

目的探讨蛋白激酶B(PKB)在类胰蛋白酶(tryptase)诱导基因表达中的作用。方法采用RT-PCR和Western blotting方法,检测tryptase对ECV304细胞PKB(Akt)的表达及其活性和对转录因子AP-1、NF-κB p65亚单位、JNK、p38MAPK、趋化因子IL-8表达的影响。结果在ECV304中1μg/Ltryptase可使PKB蛋白质磷酸化水平增加并促进PKB、转录因子NF-κB P65亚单位、AP-1和趋化因子IL-8的表达,但对JNK、p38MAPK表达影响不大。PI3K特异性抑制剂LY294002可抑制PKB的表达增加,同时可抑制NF-κB P65亚单位和IL-8的表达增加;反义PKB质粒瞬时转染ECV304,可抑制PKB、AP1、NF-κB P65亚单位和IL-8的表达增加;PAR2的抗体可抑制PKB的磷酸化,但不能阻断PKB表达。结论在ECV304细胞tryptase经其膜受体PAR2通过PI3K促进PKB的磷酸化而激活之,通过其下游途径促进趋化因子IL-8、转录因子AP-1、NF-κB P65亚单位和PKB本身的表达增加。

AIM： To study the role of protein kinase B （PKB） in tryptase - induced gene expression on ECV304 cells. METHODS： The expression of PKB, transcript factor AP- 1 and NF - κB P65, IL - 8, JNK, p38MAPK, and the activity of PKB were measured using RT - PCR and Western blotting. RESULTS： Tryptase at concentration of 1μg/L increased the activity of PKB by promoting PKB phosphorylation, promoted the expression of PKB, chemokine IL- 8, transcription factor AP- 1 and NF- κB P65, however, no changes of JNK and p38MAPK was observed. PI3K specific inhibitor （LY294002） abolished the augment of PKB, NF-κB P65 and IL- 8 expression. Antisense PKB cDNA transfection also abolished the augment of PKB, AP- 1, NF- κB P65 and IL - 8 expression. Though PAR2 antibody did not inhibit PKB expression, it did inhibit the phosphorylation by tryptase in ECV304 cells. CONCLUSION： These results indicate that tryptase can activate PKB through PAR2 receptor and subsequently NF - κB, AP1, IL- 8 and PKB exoression.