TY - JOUR
T1 - Role of exchange protein activated by cAMP 1 in regulating rates of microtubule formation in cystic fibrosis epithelial cells
AU - Rymut, Sharon M.
AU - Ivy, Tracy
AU - Corey, Deborah A.
AU - Cotton, Calvin U.
AU - Burgess, James D.
AU - Kelley, Thomas J.
N1 - Funding Information:
This work was supported by National Institutes of Health (NIH) grants R01EB009481, R01HL109362, and 2P30DK027651, and Cystic Fibrosis Foundation Research Development Program R447-CR11. The Leica microscopes in the Genetics Department Imaging Facility at Case Western Reserve University were made available through NIH-National Center for Research Resources Shared Instrumentation grant S10 RR021228.
Publisher Copyright:
© Copyright 2015 by the American Thoracic Society.
PY - 2015/12
Y1 - 2015/12
N2 - The regulation of microtubule dynamics in cystic fibrosis (CF) epithelial cells and the consequences of reduced rates of microtubule polymerization on downstream CF cellular events, such as cholesterol accumulation, a marker of impaired intracellular transport, are explored here. It is identified that microtubules in both CF cell models and in primary CF nasal epithelial cells repolymerize at a slower rate compared with respective controls. Previous studies suggest a role forcAMPin modulating organelle transport in CF cells, implicating a role for exchange protein activated by cAMP (EPAC) 1, a regulator of microtubule elongation, as a potential mechanism. EPAC1 activity is reduced in CF cell models and in Cftr2/2 mouse lung compared with respective non-CF controls. Stimulation of EPAC1 activity with the selective EPAC1 agonist, 8-cpt-2-O-MecAMP, stimulates microtubule repolymerization to wild-type rates in CF cells. EPAC1 activation also alleviates cholesterol accumulation in CF cells, suggesting a direct link between microtubule regulation and intracellular transport. To verify the relationship between transport and microtubule regulation, expression of the protein, tubulin polymerization-promoting protein, was knocked down in non-CF human tracheal (9/HTEo2) cells to mimic the microtubule dysregulation in CF cells. Transduced cells with short hairpin RNA targeting tubulin polymerization-promoting protein exhibit CF-like perinuclear cholesterol accumulation and other cellular manifestations of CF cells, thus supporting a role for microtubule regulation as a mechanism linking CFTR function to downstream cellular manifestation.
AB - The regulation of microtubule dynamics in cystic fibrosis (CF) epithelial cells and the consequences of reduced rates of microtubule polymerization on downstream CF cellular events, such as cholesterol accumulation, a marker of impaired intracellular transport, are explored here. It is identified that microtubules in both CF cell models and in primary CF nasal epithelial cells repolymerize at a slower rate compared with respective controls. Previous studies suggest a role forcAMPin modulating organelle transport in CF cells, implicating a role for exchange protein activated by cAMP (EPAC) 1, a regulator of microtubule elongation, as a potential mechanism. EPAC1 activity is reduced in CF cell models and in Cftr2/2 mouse lung compared with respective non-CF controls. Stimulation of EPAC1 activity with the selective EPAC1 agonist, 8-cpt-2-O-MecAMP, stimulates microtubule repolymerization to wild-type rates in CF cells. EPAC1 activation also alleviates cholesterol accumulation in CF cells, suggesting a direct link between microtubule regulation and intracellular transport. To verify the relationship between transport and microtubule regulation, expression of the protein, tubulin polymerization-promoting protein, was knocked down in non-CF human tracheal (9/HTEo2) cells to mimic the microtubule dysregulation in CF cells. Transduced cells with short hairpin RNA targeting tubulin polymerization-promoting protein exhibit CF-like perinuclear cholesterol accumulation and other cellular manifestations of CF cells, thus supporting a role for microtubule regulation as a mechanism linking CFTR function to downstream cellular manifestation.
KW - Cholesterol
KW - Cystic fibrosis
KW - Exchange protein activated by camp 1
KW - Microtubules
KW - Rap1
UR - http://www.scopus.com/inward/record.url?scp=84963813335&partnerID=8YFLogxK
U2 - 10.1165/rcmb.2014-0462OC
DO - 10.1165/rcmb.2014-0462OC
M3 - Article
C2 - 25955407
AN - SCOPUS:84963813335
SN - 1044-1549
VL - 53
SP - 853
EP - 862
JO - American Journal of Respiratory Cell and Molecular Biology
JF - American Journal of Respiratory Cell and Molecular Biology
IS - 6
ER -