Protein kinase D1 modulates aldosterone-induced ENaC activity in a renal cortical collecting duct cell line
Introduction
The steroid hormone aldosterone is a key regulator of electrolyte transport and blood pressure that acts upon multiple tissues and organ systems in the body, where its specific mineralocorticoid receptor (MR) is expressed; such target sites include the distal nephron of the kidney, the distal colon, the heart and the vascular endothelium. The principal effect of aldosterone action in renal and colonic epithelia is in the regulation of membrane transport molecules, such as the epithelial sodium channel (ENaC). ENaC activity plays a crucial role in the maintenance of whole body Na+ homeostasis (Booth et al., 2002, Rogerson and Fuller, 2000); the abundance of ENaC along the aldosterone-sensitive distal nephron (ASDN), coupled with channel trafficking into the apical membrane of the epithelial cells facilitates and determines the rate of Na+ re-absorption from the renal ultra-filtrate. The release of aldosterone as the last phase of the renin/angiotensin cascade maintains tight hormonal control over ENaC expression and membrane insertion. Fully active ENaC is a heterotrimer composed of one each of the α-, β- and γ-subunits (Jasti et al., 2007). Aldosterone regulates ENaC activity through multiple, synergistic mechanisms. For example, MR in complex with aldosterone acts as a ligand-dependent transcription factor that stimulates the tissue-specific transcriptional up-regulation of ENaC subunits. In the distal nephron (and in the lung) it is the ENaCα subunit that is under the direct transcriptional control of ligand-bound MR, while ENaCβ and ENaCγ are constitutively expressed or else only weakly induced by aldosterone (Verrey, 1995). ENaC activity is also subject to indirect genomic regulation by aldosterone through increasing the stability and half-life of pre-expressed ENaC subunits. Aldosterone suppresses the proteasomal degradation of ENaC through the serum glucocorticoid stimulated kinase (SGK-1)-dependent suppression of the ubiquitination of ENaC subunits by the E3 ubiquitin-protein ligase, Nedd4-2 (Snyder et al., 2002, Debonneville et al., 2001). Aldosterone also up-regulates the expression of a ubiquitin-specific protease USP2-45 to de-ubiquitinate and further stabilize pre-expressed ENaC subunits (Fakitsas et al., 2007).
The most rapid reported increase in amiloride-sensitive, ENaC current was detected in isolated principal cortical collecting duct (CCD) cells 2 min after aldosterone treatment (Zhou and Bubien, 2001); the signalling cascade responsible for increased ENaC activity in this model remains unknown. Other experimental systems may provide a clue as to the mechanism behind this rapid increase in ENaC activity; such as the elevation of apical membrane ENaC density that is coupled to the stimulation of RhoA-mediated vesicle trafficking (Pochynyuk et al., 2006). The modulation of the rates of membrane insertion and sub-cellular trafficking may thus be important additional mechanisms for ENaC regulation by aldosterone (Snyder, 2005). While the increase in ENaC activity following aldosterone treatment is more typically detected several hours after hormone treatment and is associated with the direct and indirect transcriptional effects through MR; rapidly activated signalling cascades may serve to potentiate or amplify these effects. The rapid activation of signalling cascades by aldosterone in cells of renal origin has been described by numerous groups [reviewed in Thomas et al. (2007)], and these rapid responses have variously been described as MR-dependent or MR-independent [reviewed in Funder (2005)]. The role of aldosterone-induced rapid signalling responses in modulating the key physiological responses to aldosterone such as renal Na+ re-absorption or K+ secretion is still unclear.
We recently demonstrated that aldosterone stimulated the activation of protein kinase D (PKD)1 in the M1-CCD cell line through an EGFR-coupled signalling cascade that was initiated by the interaction of aldosterone with MR (McEneaney et al., 2007). PKD1 is the prototypic member of the PKD family of serine/threonine protein kinases, and is activated in response to diverse extra-cellular stimuli (Wang, 2006) to regulate crucial cellular processes including cell growth and apoptosis [reviewed in Rozengurt et al. (2005)]. Each of the PKD family isoforms have also been implicated in different aspects of the regulation of sub-cellular trafficking, either through the maintenance of Golgi structure or by regulating TGN fission and Golgi to membrane vesicle trafficking through the activation of PI4K-IIIβ (Hausser et al., 2005, Hausser et al., 2002, Maeda et al., 2001). The RhoA-mediated translocation of ENaC to the cell membrane is coupled to vesicle trafficking through the activation of PI4P5K (Pochynyuk et al., 2006). Interestingly, the ENaC subunits are also known to interact directly with phosphatidylinositide signalling intermediates (Pochynyuk et al., 2007), and PKD1 itself can be activated as a consequence of stimulating RhoA-coupled signalling cascades (Yuan et al., 2001). We have shown that PKD1 activation by aldosterone in M1-CCD cells played a role in early, sub-cellular trafficking events that resulted in the redistribution of eCFP-tagged ENaC subunits within a few minutes of aldosterone treatment (McEneaney et al., 2008). These findings are advanced in this present study through the investigation of the role of aldosterone-induced PKD1 activation in regulating aldosterone-induced ENaC activity, and the sub-cellular distribution of ENaC subunits in M1-CCD cells. The time frame analysed in this present study is coincident with detectable increases in ENaC-dependent trans-epithelial electrical current (ITE), and we found that suppression of PKD1 expression inhibited both the apical translocation of ENaC subunits and the increase in amiloride-sensitive ITE stimulated by aldosterone treatment.
Section snippets
Cell culture and reagents
The M1-CCD cell line (ATCC, CRL-2038) was derived from renal CCD micro-dissected from a mouse transgenic for the early region of SV40 virus (strain Tg(SV40E) Bri7) (Stoos et al., 1991). M1-CCD cells were routinely grown in 75 cm2 polystyrene culture flasks containing 1:1 Dulbecco's modified Eagle's medium and Ham's F-12 medium (DMEM:F-12) with fetal bovine serum (10%); supplemented with l-glutamine (2 mM), penicillin (100 units ml−1), streptomycin (100 μg ml−1) and dexamethasone (5 μM). Cultures were
Aldosterone-induced ENaCα subunit expression in M1-CCD cells is PKD1-dependent
ENaC activity is subject to regulation by aldosterone through different mechanisms including the transcriptional regulation of subunit expression, and the modulation of subunit ubiquitination, which affects ENaC abundance at sub-apical, cytoplasmic pools. These inactive pools of ENaC can be recruited to the apical membrane in response to specific agonists and the activation of their associated secondary messenger systems (Pochynyuk et al., 2006). We have previously shown that aldosterone
Discussion
Aldosterone plays a key role in regulating whole body electrolyte homeostasis, and the distal nephron is the pre-eminent site for endocrine regulation of Na+ efflux from the body. Under the influence of circulatory aldosterone levels, Na+ is reabsorbed at the luminal or apical surface of the ASDN principal epithelial cells from the renal ultra-filtrate through the ENaC and the Na+/Cl− co-transporter. The membrane transporters implicated directly and indirectly at each stage of the Na+
Acknowledgements
The MR-specific monoclonal antibody used in this investigation (MRN 4E4) was a gift from Dr. C.E. Gomez-Sanchez, University of Mississippi, MS, U.S.A. The work was supported by programme grant 060809/Z/00 from the Wellcome Trust and by the Higher Education Authority of Ireland under the Programme for Research in Third Level Institutions (PRTLI) Cycles 3 and 4 (to BJH) and by a Science Foundation of Ireland Research Frontiers Programme grant BMT/1521 (to WT).
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2014, SteroidsCitation Excerpt :Our previous studies have shown that a significant increase in the activity of ENaC in M1-CCD cells is detected between 2 and 4 h following treatment with aldosterone that peaked between 16 and 24 h, an effect that correlated with the sub-cellular redistribution of ENaCβ [5]. Aldosterone treatment stimulated the apical translocation of ENaCα in wild-type M1-CCD, but in PKD1 knock-down cells ENaCα was not introduced to the apical membrane, but rather remained localized in the cytoplasm [5]. This indicates that the trafficking process of ENaCα to the apical membrane is defective in cells suppressed in PKD1 expression and raises a question about the role of protein kinase regulated sub-cellular trafficking in the modulation of ENaC activity by aldosterone.
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