Aldosterone regulates rapid trafficking of epithelial sodium channel subunits in renal cortical collecting duct cells via protein kinase D activation.
Aldosterone elicits rapid physiological responses in target tissues such as the distal nephron through the stimulation of cell signaling cascades. We identified protein kinase D (PKD1) as an early signaling response to aldosterone treatment in the M1-cortical collecting duct (M1-CCD) cell line. PKD1 activation was blocked by the PKC inhibitor chelerythrine chloride and by rottlerin, a specific inhibitor of PKCdelta. The activation of PKCdelta and PKCepsilon coincided with PKD1 activation and while a complex was formed between PKD1 and PKCepsilon after aldosterone treatment, there was a concurrent reduction in PKD1 association with PKCdelta. A stable PKD1 knockdown M1-CCD-derrived clone was developed in which PKD1 expression was 90% suppressed by gene silencing with a PKD1-specific siRNA. The effect of aldosterone treatment on the subcellular distribution of enhanced cyan fluorescent protein (eCFP)-tagged epithelial sodium channel (ENaC) subunits in wild type (WT) and PKD1 suppressed cells was examined using confocal microscopy. In an untreated confluent monolayer of M1-CCD cells, alpha, beta, and gamma ENaC subunits were evenly distributed throughout the cytoplasm of WT and PKD1-suppressed cells. After 2 min treatment, aldosterone stimulated the localization of each of the ENaC subunits to discrete regions within the cytoplasm of WT cells. The translocation of eCFP-ENaC subunits in WT cells was inhibited by rottlerin and the mineralocorticoid receptor (MR) antagonist spironolactone. No subcellular translocation of eCFP-ENaC subunits was observed in PKD1-suppressed cells treated with aldosterone. These data demonstrate the involvement of a novel MR/PKCdelta /PKD1 signaling cascade in the earliest ENaC subunit intracellular trafficking events that follow aldosterone treatment.
McEneaney
Victoria
V
Department of Molecular Medicine, Royal College of Surgeons in Ireland Education and Research Centre, Smurfit Building, Beaumont Hospital, Dublin 9, Ireland.
Harvey
Brian J
BJ
Thomas
Warren
W
D000098:Acetophenones / Q000494:pharmacology; D000450:Aldosterone / Q000494:pharmacology*; D000818:Animals; D053119:Benzophenanthridines / Q000494:pharmacology; D001578:Benzopyrans / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D053503:Epithelial Sodium Channels / Q000235:genetics / Q000378:metabolism*; D047468:Immunoprecipitation; D007672:Kidney Cortex / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D013148:Spironolactone / Q000494:pharmacology
D000098:Acetophenones; D053119:Benzophenanthridines; D001578:Benzopyrans; D053503:Epithelial Sodium Channels; D013148:Spironolactone; D000450:Aldosterone; C085746:rottlerin; C016299:chelerythrine; C089212:protein kinase D; D011493:Protein Kinase C