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MARCKS Regulation of Mucin Secretion by Airway Epithelium in Vitro

Interaction with Chaperones

¹ Department of Molecular Biomedical Sciences, North Carolina State University, Raleigh, North Carolina

Correspondence and requests for reprints should be addressed to Kenneth B. Adler, Ph.D., North Carolina State University, College of Veterinary Medicine, 4700 Hillsborough Street, Raleigh, NC 27606. E-mail: Kenneth_Adler{at}ncsu.edu

We have reported previously that myristoylated alanine-rich C kinase substrate (MARCKS) is a key regulatory molecule controlling mucin secretion by airway epithelial cells in vitro and in vivo. The results of those studies supported a mechanism whereby MARCKS, upon phosphorylation by protein kinase C (PKC), translocates from plasma membrane to cytoplasm, where its binding to membranes of intracellular mucin granules is a key component of the secretory pathway. It remains unknown how MARCKS is targeted to and/or preferentially attaches to mucin granule membranes. We hypothesized that the chaperone cysteine string protein (CSP) may play an important role in this process. CSP was shown to associate with membranes of intracellular mucin granules in well-differentiated normal human bronchial epithelial (NHBE) cells in vitro, as determined by ultrastructural immunohistochemistry and Western blotting of isolated granule membranes. CSP in these cells complexed with MARCKS, as shown by co-immunoprecipitation. Given reported associations between CSP and a second chaperone, heat shock protein 70 (HSP70), a role for HSP70 in the MARCKS-dependent secretory mechanism also was investigated. HSP70 appeared to form a trimeric complex with MARCKS and CSP associated with mucin granule membranes within airway epithelial cells. Transfection of the HBE1 human bronchial epithelial cell line with siRNAs targeting sequences of MARCKS, CSP, or HSP70 resulted, in each case, in significant knockdown of expression of these proteins and subsequent attenuation of mucin secretion. The results provide the first evidence that CSP and HSP70, and their interactions with MARCKS, are involved in mucin secretion.

Key Words: MARCKS • cysteine string protein • heat shock protein 70 • airway • mucin

CLINICAL RELEVANCE This research reveals an important role for the chaperone protein, cysteine string protein, in mucin secretion and suggests possible new therapeutic targets for treatment of disease characterized by excess mucus secretion.

 

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