ABSTRACT
The cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) channel in airway epithelial cells, plays an important role in maintaining the volume of the airway surface liquid and therefore mucociliary clearance of respiratory pathogens. A recent study has shown that the E3 ubiquitin ligase Neural precursor cells expressed developmentally downregulated (Nedd4-2) ubiquitinates ΔF508-CFTR in pancreatic epithelial cells and that siRNA-mediated silencing of Nedd4-2 increases plasma membrane ΔF508-CFTR. Because the role of Nedd4-2 in regulating wild-type (wt)-CFTR in airway epithelial cells is unknown, studies were conducted to test the hypothesis that Nedd4-2 also ubiquitinates wt-CFTR and regulates its plasma membrane abundance. We found that Nedd4-2 did not affect wt-CFTR Cl(-) currents in Xenopus oocytes. Likewise, overexpression of Nedd4-2 in human airway epithelial cells did not alter the amount of ubiquitinated wt-CFTR. siRNA knockdown of Nedd4-2 in human airway epithelial cells had no effect on ubiquitination or apical plasma membrane abundance of wt-CFTR. Thus Nedd4-2 does not ubiquitinate and thereby regulate wt-CFTR in human airway epithelial cells.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Respiratory Mucosa/cytology , Respiratory Mucosa/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Antiporters/metabolism , Bronchi/cytology , Cell Membrane/metabolism , Cells, Cultured , Chlorides/metabolism , Cystic Fibrosis/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Endosomal Sorting Complexes Required for Transport/genetics , Humans , Membrane Potentials/physiology , Nedd4 Ubiquitin Protein Ligases , Oocytes/physiology , Phosphoproteins/metabolism , RNA, Small Interfering/genetics , Sodium-Hydrogen Exchangers/metabolism , Sulfate Transporters , Ubiquitin-Protein Ligases/genetics , Ubiquitination/physiology , Xenopus , Xenopus ProteinsABSTRACT
The Atlantic killifish (Fundulus heteroclitus) is an environmental sentinel organism used extensively for studies of environmental toxicants and osmoregulation. Previous research in our laboratory has shown that acute acclimation to seawater is mediated by an increase in SGK1. SGK1 promotes the trafficking of CFTR chloride channels from intracellular vesicles to the plasma membrane of the gill within the first hour in seawater resulting in increased chloride secretion. Although we have shown that the increase in gill SGK1 does not require activation of the glucocorticoid receptor, the mechanisms that mediate the rise SGK1 during acute acclimation is unknown. To test the hypothesis that mitogen activated protein kinase (MAPK14) is responsible for the rise in SGK1 we identified the coding sequence of killifish MAPK14-1 and designed a translational blocking vivo-morpholino targeting MAPK14-1. Injection of the MAPK14-1 vivo-morpholino resulted in a 30% reduction of MAPK14-1 and a 45% reduction in phosphorylated-MAPK14-1 protein in the gill of killifish transitioned from freshwater to seawater. Knock down of phosphorlyated-MAPK14-1 completely blocked the rise in SGK1 mRNA and protein in the killifish gill, providing the first direct and in vivo evidence that MAPK14-1 is necessary for acute seawater acclimation.
Subject(s)
Acclimatization , Fish Proteins/metabolism , Fundulidae/physiology , Mitogen-Activated Protein Kinase 14/physiology , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Animals , Female , Fish Proteins/genetics , Fundulidae/metabolism , Gene Knockdown Techniques , Gills/enzymology , Male , Mitogen-Activated Protein Kinase 14/genetics , Mitogen-Activated Protein Kinase 14/metabolism , Molecular Sequence Data , Morpholinos/genetics , Protein Serine-Threonine Kinases/genetics , Salt Tolerance , Seawater , Sequence Homology, Amino Acid , Sodium-Potassium-Exchanging ATPase/metabolismABSTRACT
Killifish are euryhaline teleosts that adapt to increased salinity by up regulating CFTR mediated Cl(-) secretion in the gill and opercular membrane. Although many studies have examined the mechanisms responsible for long term (days) adaptation to increased salinity, little is known about the mechanisms responsible for acute (hours) adaptation. Thus, studies were conducted to test the hypotheses that the acute homeostatic regulation of NaCl balance in killifish involves a translocation of CFTR to the plasma membrane and that this effect is mediated by serum-and glucocorticoid-inducible kinase (SGK1). Cell surface biotinyation and Ussing chamber studies revealed that freshwater to seawater transfer rapidly (1 hour) increased CFTR Cl(-) secretion and the abundance of CFTR in the plasma membrane of opercular membranes. Q-RT-PCR and Western blot studies demonstrated that the increase in plasma membrane CFTR was preceded by an increase in SGK1 mRNA and protein levels. Seawater rapidly (1 hr) increases cortisol and plasma tonicity, potent stimuli of SGK1 expression, yet RU486, a glucocorticoid receptor antagonist, did not block the increase in SGK1 expression. Thus, in killifish SGK1 does not appear to be regulated by the glucocorticoid receptor. Since SGK1 has been shown to increase the plasma membrane abundance of CFTR in Xenopus oocytes, these observations suggest that acute adaptation (hours) to increased salinity in killifish involves translocation of CFTR from an intracellular pool to the plasma membrane, and that this effect may be mediated by SGK1.
