[MARCKS-PSD磷酸化在低温诱导人呼吸道上皮细胞分泌MUC5AC中的作用]

[Role of phosphorylation of MARCKS-PSD in the secretion of MUC5AC induced by cold temperatures in human airway epithelial cells].

作者信息

Li Minchao, Perelman Juliy M, Zhou Xiangdong

机构信息

Department of Respiratory Medicine, Chongqing Medical University, Chongqing, China.

出版信息

Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2012 May;37(5):447-52. doi: 10.3969/j.issn.1672-7347.2012.05.003.

DOI:10.3969/j.issn.1672-7347.2012.05.003

PMID:22659655

Abstract

OBJECTIVE

To construct phosphorylation sites domain (PSD) mutant of myristoylated alaninerich C kinase substrate (MARCKS) and explore the role of transient receptor potential melastatin 8 cation channels (TRPM8) and MARCKS in cold-induced synthesis and exocytosis of mucin (MUC) 5AC.

METHODS

Human placental cDNA was used as a template to amplify the full coding region of MARCKS cDNA by PCR. Ser159, Ser 163, Ser 167, Ser 170 in the PSD were mutated to aspartic acids by an overlap PCR method. The resultant PSD mutant cDNA and the wild-type MARCKS cDNA were each subcloned into a mammalian expression vector pcDNA3.0. Recombinant constructs were confirmed by restriction enzyme digestion analysis and DNA sequencing. In intervention experiments, cells were pretreated with the TRPM8 channel antagonist BCTC and transfected with MARCKS-PSD mutant cDNA, and thereafter cold stimulation was applied. The levels of MUC5AC were measured by immunofluorescence and ELISA to clarify the roles of TRPM8 and PSD mutant on the synthesis and secretion of MUC5AC induced by cold, respectively.

RESULTS

Restriction enzyme digestion analysis and DNA sequencing revealed that the pcDNA3.0- MARCKS and pcDNA3.0-MARCKS-PSD mutants were successfully constructed. The levels of intracellular and secreted MUC5AC of cold treated group were significantly higher than those of control group (P<0.05). BCTC attenuated the cold-induced synthesis and secretion of MUC5AC when compared with cold treated group (P<0.05). Transfection of 16HBE cells with the MARCKS-PSD mutant cDNA resulted in significant inhibition of mucin secretion in response to cold, and significantly higher level of intracellular MUC5AC than that of control group (P<0.01), whereas transfection with the vector DNA or the wild-type MARCKS cDNA had no effect on the mucin synthesis and secretion in response to cold (P>0.05).

CONCLUSION

TRPM8 and phosphorylation of MARCKS-PSD mediates the cold-induced exocytosis of MUC5AC by airway epithelial cells.

摘要

目的

构建肉豆蔻酰化富含丙氨酸的C激酶底物（MARCKS）的磷酸化位点结构域（PSD）突变体，探讨瞬时受体电位香草酸亚型8阳离子通道（TRPM8）和MARCKS在冷诱导的黏蛋白（MUC）5AC合成及胞吐中的作用。

方法

以人胎盘cDNA为模板，通过聚合酶链反应（PCR）扩增MARCKS cDNA的完整编码区。采用重叠PCR法将PSD中的丝氨酸159、丝氨酸163、丝氨酸167、丝氨酸170突变为天冬氨酸。将所得的PSD突变体cDNA和野生型MARCKS cDNA分别亚克隆到哺乳动物表达载体pcDNA3.0中。通过限制性内切酶消化分析和DNA测序确认重组构建体。在干预实验中，细胞先用TRPM8通道拮抗剂BCTC预处理，然后用MARCKS - PSD突变体cDNA转染，之后进行冷刺激。通过免疫荧光和酶联免疫吸附测定（ELISA）检测MUC5AC水平，分别阐明TRPM8和PSD突变体在冷诱导的MUC5AC合成和分泌中的作用。

结果

限制性内切酶消化分析和DNA测序显示成功构建了pcDNA3.0 - MARCKS和pcDNA3.0 - MARCKS - PSD突变体。冷处理组细胞内和分泌的MUC5AC水平显著高于对照组（P<0.05）。与冷处理组相比，BCTC减弱了冷诱导的MUC5AC合成和分泌（P<0.05）。用MARCKS - PSD突变体cDNA转染16HBE细胞导致对冷刺激的黏蛋白分泌显著抑制，且细胞内MUC5AC水平显著高于对照组（P<0.01），而用载体DNA或野生型MARCKS cDNA转染对冷刺激的黏蛋白合成和分泌无影响（P>0.05）。