Rescue of defective pancreatic secretion in cystic-fibrosis cells by suppression of a novel isoform of phospholipase C.
BACKGROUND
Cystic fibrosis is caused by mutations in the gene encoding an ion-transport protein, the cystic-fibrosis transmembrane conductance regulator (CFTR). Defective secretion of anions is the primary cause of many of the clinical manifestations of cystic fibrosis, including pancreatic insufficiency. We aimed to identify a molecular mechanism from which a new method to circumvent defective pancreatic secretion could be derived.


METHODS
Multiple-human-tissue RT-PCR and semiquantitative RT-PCR analyses were used to examine gene expression. An antisense technique was used in conjunction with radioimmunoassay, Fura-2 spectrofluorometry, immunohistochemistry, and the short-circuit current technique (Ussing chamber) for elucidation of gene function and its application in rescuing defective pancreatic secretion.


RESULTS
We cloned a newly identified gene, NYD-SP27, which has structural similarity to an isoform of phospholipase C. NYD-SP27 was expressed endogenously in human pancreatic-duct cells and upregulated in cystic fibrosis. Suppression of NYD-SP27, by transfection of its antisense into human cystic-fibrosis pancreatic-duct cells, resulted in augmentation of phospholipase-C-coupled calcium-ion release and protein kinase C activity, improvement in the amount of mutated CFTR reaching the plasma membrane, and restoration of cAMP-activated pancreatic anion secretion.


CONCLUSIONS
NYD-SP27 exerts an inhibitory effect on phospholipase-C-coupled processes that depend on calcium ions and protein kinase C, including CFTR trafficking and function. Its upregulation in pancreatic-duct cells may reveal a previously unsuspected defect in cystic fibrosis contributing to pancreatic insufficiency, and thus represents a new target for pharmacological intervention in cystic fibrosis.
Zhu
Hu
H
Epithelial Cell Biology Research Center, Department of Physiology, Faculty of Medicine, Chinese University of Hong Kong, SAR, Shatin, Hong Kong, China.
Zhu
Jin Xia
JX
Lo
Pui Shan
PS
Li
Jianmin
J
Leung
Ka Man
KM
Rowlands
Dewi Kenneth
DK
Tsang
Lai Ling
LL
Yu
Mei Kuen
MK
Jiang
Jian Li
JL
Lam
Sun Yee
SY
Chung
Yiu Wa
YW
Zhou
Zuomin
Z
Sha
Jiahao
J
Chang Chan
Hsiao
H
D016375:Antisense Elements (Genetics); D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D003550:Cystic Fibrosis / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D010188:Exocrine Pancreatic Insufficiency / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology; D015870:Gene Expression / Q000235:genetics / Q000502:physiology; D006801:Humans; D017136:Ion Transport / Q000235:genetics; D008565:Membrane Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010183:Pancreatic Ducts / Q000166:cytology / Q000502:physiology; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D014162:Transfection / Q000379:methods; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
D016375:Antisense Elements (Genetics); C505032:CFTR protein, human; D008565:Membrane Proteins; D020033:Protein Isoforms; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000242:Cyclic AMP; D011493:Protein Kinase C; D010738:Type C Phospholipases; D002118:Calcium