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Role of phosphorylation of MARCKS-PSD in the secretion of MUC5AC induced by cold temperatures in human airway epithelial cells / 中南大学学报(医学版)
Journal of Central South University(Medical Sciences) ; (12): 447-452, 2012.
Article in Chinese | WPRIM | ID: wpr-814652
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 (P0.05).@*CONCLUSION@#TRPM8 and phosphorylation of MARCKS-PSD mediates the cold-induced exocytosis of MUC5AC by airway epithelial cells.
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Full text: Available Index: WPRIM (Western Pacific) Main subject: Phosphorylation / Physiology / Trachea / Molecular Sequence Data / Base Sequence / Cell Line / Cold Temperature / Cell Biology / Intracellular Signaling Peptides and Proteins / Epithelial Cells Limits: Humans Language: Chinese Journal: Journal of Central South University(Medical Sciences) Year: 2012 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Main subject: Phosphorylation / Physiology / Trachea / Molecular Sequence Data / Base Sequence / Cell Line / Cold Temperature / Cell Biology / Intracellular Signaling Peptides and Proteins / Epithelial Cells Limits: Humans Language: Chinese Journal: Journal of Central South University(Medical Sciences) Year: 2012 Type: Article