Improved oxygenation promotes CFTR maturation and trafficking in MDCK monolayers.
Culturing airway epithelial cells with most of the apical media removed (air-liquid interface) has been shown to enhance cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl(-) secretory current. Thus we hypothesized that cellular oxygenation may modulate CFTR expression. We tested this notion using type I Madin-Darby canine kidney cells that endogenously express low levels of CFTR. Growing monolayers of these cells for 4 to 5 days with an air-liquid interface caused a 50-fold increase in forskolin-stimulated Cl(-) current, compared with conventional (submerged) controls. Assaying for possible changes in CFTR by immunoprecipitation and immunocytochemical localization revealed that CFTR appeared as an immature 140-kDa form intracellularly in conventional cultures. In contrast, monolayers grown with an air-liquid interface possessed more CFTR protein, accompanied by increases toward the mature 170-kDa form and apical membrane staining. Culturing submerged monolayers with 95% O(2) produced similar improvements in Cl(-) current and CFTR protein as air-liquid interface culture, while increasing PO(2) from 2.5% to 20% in air-liquid interface cultures yielded graded enhancements. Together, our data indicate that improved cellular oxygenation can increase endogenous CFTR maturation and/or trafficking.
Bebök
Z
Z
Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005, USA.
Tousson
A
A
Schwiebert
L M
LM
Venglarik
C J
CJ
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D016764:Cell Polarity / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005576:Colforsin / Q000378:metabolism / Q000494:pharmacology; D003470:Culture Media / Q000494:pharmacology; D003550:Cystic Fibrosis / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D000860:Hypoxia / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D010100:Oxygen / Q000494:pharmacology*; D021381:Protein Transport / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism
D003470:Culture Media; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D010100:Oxygen