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


CONCLUSIONS
TRPM8 and phosphorylation of MARCKS-PSD mediates the cold-induced exocytosis of MUC5AC by airway epithelial cells.
Department of Respiratory Medicine, Chongqing Medical University, Chongqing, China.
Minchao
M
Li
Juliy M
JM
Perelman
Xiangdong
X
Zhou
D001483:Base Sequence; D002460:Cell Line; D003080:Cold Temperature; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D005089:Exocytosis / Q000502:physiology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D055271:Mucin 5AC / Q000378:metabolism*; D009154:Mutation; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010766:Phosphorylation; D050053:TRPM Cation Channels / Q000378:metabolism*; D014132:Trachea / Q000166:cytology* / Q000378:metabolism
D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; C524678:MUC5AC protein, human; D008565:Membrane Proteins; D055271:Mucin 5AC; D050053:TRPM Cation Channels; C425966:TRPM8 protein, human; D000076250:Myristoylated Alanine-Rich C Kinase Substrate