Am. J. Respir. Cell Mol. Biol.,
Volume 26, Number 4, April, 2002 506-515
Cell Cycle Regulation of Pulmonary Phosphatidylcholine Synthesis
Irene
Tseu,
Ross
Ridsdale,
Jason
Liu,
Jinxia
Wang,
and
Martin
Post
CIHR Group in Lung Development and the Lung Biology Programme, Hospital for Sick Children Research Institute, Toronto, Ontario,
Canada; and Department of Pediatrics, Institute of Medical Sciences, University of Toronto, Toronto, Ontario, Canada
Pulmonary surfactant phosphatidylcholine (PC) formation increases as alveolar type II cells mature and arrest in G0/G1
state of the cell cycle at late fetal gestation. To determine
whether this G0/G1 arrest is responsible for the increase in PC
synthesis, we investigated the rates of PC synthesis and the
activity, phosphorylation, intracellular distribution, synthesis,
and degradation of a key enzyme of PC synthesis, cytidine
triphosphate (CTP):phosphocholine cytidylyltransferase (CCT ).
In synchronized mouse lung epithelial (MLE)-15 cells, PC production and CCT activity peaked at G0/G1, declined during
transition to G1/S, and remained low during S and G2/M. The
changes in CCT activity were not due to alterations in CCT
gene and protein expression. CCT protein degradation also
did not change during the cell cycle. Indirect immunofluorescence and immunogold electron microscopy revealed that CCT
localized to the cytoplasmic compartment and that its cytosolic localization did not change with the cell cycle. Although
immunoblotting suggested no major redistribution of CCT
mass from cytosol to endoplasmic reticulum, activity measurements revealed that the ratio of particulate/soluble CCT activity
was cell cycle-dependent. The particulate/soluble ratio peaked
at G0/G1 and declined with cell-cycle progression. Furthermore, the decrease in CCT activity during exit from G0/G1 was associated with an increase in CCT phosphorylation.
These data suggest that the cell-cycle changes in PC synthesis
are likely not due to alterations in CCT expression and degradation but are primarily a consequence of changes in CCT
activity, phosphorylation, and membrane affinity.
Abbreviations: bovine serum albumin, BSA; CTP:phosphocholine cytidylyltransferase, CCT; 4',6-diamidino-2-phenylindole, DAPI; enhanced chemiluminescence, ECL; endoplasmic reticulum, ER; fluorescent-activated cell
sorter, FACS; fetal bovine serum, FBS; fetal calf serum, FCS; fluoroisothiocyanate, FITC; green fluorescence protein, GFP; mouse lung epithelial; HA,
haemagglutinin; Ham's F12, insulin, transferin, estradiol, and sodium selenite, HITES; immunoglobulin G, IgG; Eagle's minimum essential medium, MEM; mouse lung epithelial, MLE; normal goat serum, NGS; phosphate-buffered saline, PBS; phosphatidylcholine, PC; respiratory distress
syndrome RDS; reverse transcriptase/polymerase chain reaction, RT-PCR;
SDS-polyacrylamide gel electrophoresis, SDS-PAGE.
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Copyright © 2002 American Thoracic Society.
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