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.     

Oxidative refolding of lysozyme in trifluoroethanol (TFE) and ethylene glycol: interfering role of preexisting alpha-helical structure and intermolecular hydrophobic interactions

We investigated the effect of aldosterone on Src kinase. In the kidney cell line, M-1 aldosterone leads to a >2-fold transient activation of Src kinase seen as early as 2 min after aldosterone administration. Maximal Src kinase activation was measured at an aldosterone concentration of 1 nM. In parallel to activation, autophosphorylation at Tyr-416 of Src kinase increased. Src kinase activation was blocked by spironolactone. Aldosterone led to increased association of Src with HSP84. Furthermore, rapamycin blocked aldosterone-induced Src activation. We conclude that Src activation by aldosterone is mediated through the mineralocorticoid receptor and HSP84.     

Genomic and nongenomic stimulatory effect of aldosterone on H+-ATPase in proximal S3 segments

The genomic and nongenomic effects of aldosterone on the intracellular pH recovery rate (pHirr) via H(+)-ATPase and on cytosolic free calcium concentration ([Ca(2+)](i)) were investigated in isolated proximal S3 segments of rats during superfusion with an Na(+)-free solution, by using the fluorescent probes BCECF-AM and FLUO-4-AM, respectively. The pHirr, after cellular acidification with a NH(4)Cl pulse, was 0.064 ± 0.003 pH units/min (n = 17/74) and was abolished with concanamycin. Aldosterone (10(-12), 10(-10), 10(-8), or 10(-6) M with 1-h or 15- or 2-min preincubation) increased the pHirr. The baseline [Ca(2+)](i) was 103 ± 2 nM (n = 58). After 1 min of aldosterone preincubation, there was a transient and dose-dependent increase in [Ca(2+)](i) and after 6-min preincubation there was a new increase in [Ca(2+)](i) that persisted after 1 h. Spironolactone [mineralocorticoid (MR) antagonist], actinomycin D, or cycloheximide did not affect the effects of aldosterone (15- or 2-min preincubation) on pHirr and on [Ca(2+)](i) but inhibited the effects of aldosterone (1-h preincubation) on these parameters. RU 486 [glucocorticoid (GR) antagonist] and dimethyl-BAPTA (Ca(2+) chelator) prevented the effect of aldosterone on both parameters. The data indicate a genomic (1 h, via MR) and a nongenomic action (15 or 2 min, probably via GR) on the H(+)-ATPase and on [Ca(2+)](i). The results are compatible with stimulation of the H(+)-ATPase by increases in [Ca(2+)](i) (at 10(-12)-10(-6) M aldosterone) and inhibition of the H(+)-ATPase by decreases in [Ca(2+)](i) (at 10(-12) or 10(-6) M aldosterone plus RU 486).     

Aldosterone stimulates proliferation of cardiac fibroblasts by activating Ki-RasA and MAPK1/2 signaling

Aldosterone plays a pathological role in cardiac fibrosis by directly affecting cardiac fibroblasts. Understanding of the cellular mechanisms of aldosterone action in cardiac fibroblasts, however, is rudimentary. One possibility is that aldosterone promotes proliferation of cardiac fibroblasts by activating specific cellular signaling cascades. The current study tests whether aldosterone stimulates proliferation of isolated adult rat cardiac myofibroblasts (RCF) by activating Kirsten Ras (Ki-RasA) and its effector, the MAPK1/2 cascade. Aldosterone (10 nM) significantly increased RCF proliferation. This action was sensitive to the mineralocorticoid receptor (MR) antagonist spironolactone. Expression of MR in RCF and the whole rat heart was confirmed by immunoblotting. Aldosterone significantly increased absolute and active (GTP bound) Ki-RasA levels in RCF. Aldosterone, in addition, significantly increased phospho-c-Raf and phospho-MAPK1/2. The effects of aldosterone on Ki-RasA and phospho-c-Raf proteins were inhibited by spironolactone but not RU-486, suggesting that aldosterone acts via MR. Inhibitors of MEK1/2 and c-Raf prevented aldosterone-induced activation of MAPK1/2 and proliferation. These results show that aldosterone directly increases RCF proliferation through MR-dependent activation of Ki-RasA and its effector, the MAPK1/2 cascade. Activation of cardiac fibroblasts through such a cascade may play a role in the pathological actions exerted by aldosterone on the heart.     

The Aldosterone-Mineralocorticoid Receptor Pathway Exerts Anti-Inflammatory Effects in Endotoxin-Induced Uveitis

We have previously shown that the eye is a mineralocorticoid-sensitive organ and we now question the role of mineralocorticoid receptor (MR) in ocular inflammation. The endotoxin-induced uveitis (EIU), a rat model of human intraocular inflammation, was induced by systemic administration of lipopolysaccharide (LPS). Evaluations were made 6 and 24 hours after intraocular injection of aldosterone (simultaneous to LPS injection). Three hours after onset of EIU, the MR and the glucocorticoid metabolizing enzyme 11-beta hydroxysteroid dehydrogenase type 2 (11β-HSD2) expression were down-regulated in iris/ciliary body and the corticosterone concentration was increased in aqueous humor, altering the normal MR/glucocorticoid receptor (GR) balance. At 24 hours, the GR expression was also decreased. In EIU, aldosterone reduced the intensity of clinical inflammation in a dose-dependent manner. The clinical benefit of aldosterone was abrogated in the presence of the MR antagonist (RU26752) and only partially with the GR antagonist (RU38486). Aldosterone reduced the release of inflammatory mediators (6 and 24 hours: TNF-α, IFN-γ, MIP-1α) in aqueous humor and the number of activated microglia/macrophages. Aldosterone partly prevented the uveitis-induced MR down-regulation. These results suggest that MR expression and activation in iris/ciliary body could protect the ocular structures against damages induced by EIU.     

Vascular endothelial growth factor induces heat shock protein (HSP) 27 serine 82 phosphorylation and endothelial tubulogenesis via protein kinase D and independent of p38 kinase

Proteomic analysis identified HSP27 phosphorylation as a major change in protein phosphorylation stimulated by Vascular Endothelial Growth Factor (VEGF) in Human Umbilical Vein Endothelial Cells (HUVEC). VEGF-induced HSP27 phosphorylation at serines 15, 78 and 82, but whereas HSP27 phosphorylation induced by H2O2 and TNFalpha was completely blocked by the p38 kinase inhibitor, SB203580, VEGF-stimulated serine 82 phosphorylation was resistant to SB203580 and small interfering(si)RNA-mediated knockdown of p38 kinase and MAPKAPK2. The PKC inhibitor, GF109203X, partially reduced VEGF-induced HSP27 serine 82 phosphorylation, and SB203580 plus GF109203X abolished phosphorylation. VEGF activated Protein Kinase D (PKD) via PKC, and siRNAs targeted to PKD1 and PKD2 inhibited VEGF-induced HSP27 serine 82 phosphorylation. Furthermore recombinant PKD selectively phosphorylated HSP27 at serine 82 in vitro, and PKD2 activated by VEGF in HUVECs also phosphorylated HSP27 selectively at this site. Knockdown of HSP27 and PKDs markedly inhibited VEGF-induced HUVEC migration and tubulogenesis, whereas inhibition of the p38 kinase pathway using either SB203580 or siRNAs against p38alpha or MAPKAPK2, had no significant effect on the chemotactic response to VEGF. These findings identify a novel pathway for VEGF-induced HSP27 serine 82 phosphorylation via PKC-mediated PKD activation and direct phosphorylation of HSP27 by PKD, and show that PKDs and HSP27 play major roles in the angiogenic response to VEGF.     

Aldosterone modulates neural vasomotor response in hypertension: role of calcitonin gene-related peptide

Objective: We analyse the effect of aldosterone on vasomotor response induced by electrical field stimulation (EFS) in mesenteric arteries from Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Results: Aldosterone (0.001–1 μM) reduced vasoconstrictor response to EFS in a dose- and time-dependent manner only in SHR. Thus, the rest of experiments were performed only in SHR. Aldosterone did not affect either noradrenaline response or release. Effect of aldosterone (1 μM) on EFS response was not affected by N^G-nitro-arginine-methyl esther (100 μM), and was abolished by capsaicin (0.5 μM) and the calcitonin gene-related peptide antagonist (CGRP 8–37, 0.5 μM). Calcitonin gene-related peptide (0.1 nM–0.1 μM) induced a concentration-dependent relaxation, which was enhanced by aldosterone (1 μM). Incubation with either spironolactone (1 μM), glibenclamide (10 μM), RU 486 10 μM, ODQ (10 μM) or cycloheximide (10 μM) significantly reduced the enhancement of CGRP-relaxation produced by aldosterone, while remained unmodified by SQ 22,536. Conclusions: Aldosterone decreases the vasoconstrictor response to EFS in mesenteric arteries from SHR but not from WKY. This effect is mediated by an increased response to the sensory neurotransmitter CGRP, substantially, through glucocorticoid receptors activation. Furthermore, this effect is mediated by an increase of cGMP synthesis and ATP-dependent potassium channel activation.     

Aldosterone stimulates epidermal growth factor receptor expression

The steroid hormone aldosterone plays an important role during pathological tissue modifications, similar to cardiovascular or renal fibrosis. The underlying mechanisms for the pathological actions are not understood. Interaction of aldosterone with the epidermal growth factor (EGF) receptor is an attractive hypothesis to explain pathological tissue remodeling elicited by aldosterone, because (i) mineralocorticoids can sensitize cells for EGF, (ii) mineralocorticoid receptor (MR)-antagonists reduce EGFR-mRNA expression, (iii) EGFR itself supports the development of cardiovascular or renal fibrosis, and (iv) signaling elements involved in the pathological action of aldosterone (similar to ERK1/2 or NFkB) are typical downstream modules during EGF signaling. In addition, an interaction of aldosterone and EGF with respect to ERK1/2 activation has been described. Here we show that aldosterone stimulates EGFR expression in renal tissue of adrenalectomized rats and in human renal primary cell cultures. Furthermore, Chinese hamster ovary (CHO) cells normally devoid of EGFR or MR express EGFR after transfection with human MR (CHO-MR cells) but not after transfection with human glucocorticoid receptor (CHO-GR cells). In CHO-MR cells, EGFR-expression is up-regulated by aldosterone and inhibited by spironolactone. CHO-MR cells but not CHO-GR cells respond with ERK1/2 phosphorylation to EGF exposure. The responsiveness to other peptide hormones was virtually not affected. These data suggest that EGFR is an aldosterone-induced protein and is involved in the manifold (patho)biological actions of aldosterone.     

Aldosterone increases RAMP1 expression in mesenteric arteries from spontaneously hypertensive rats

OBJECTIVE: We analysed the effect of aldosterone on calcitonin gene-related peptide (CGRP) mediated vasodilation in noradrenaline precontracted endothelium denuded mesenteric arteries segments from Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) and the effect of aldosterone on calcitonin receptor-like receptor (CL receptor) and receptor activity modifying protein 1 (RAMP1) expression in endothelium-denuded mesenteric arteries from SHR rats. RESULTS: CGRP 0.1 nM-0.1 microM induced a concentration-dependent relaxation that was enhanced by aldosterone 1 microM in SHR only. Incubation with RU 486 10 microM significantly reduced the enhancement of CGRP-relaxation produced by aldosterone in SHR. CL receptor expression was not modified in either strain, while RAMP1 expression was enhanced in SHR by aldosterone 1 microM 120 min and 0.1 microM 120 min. This up-regulation of RAMP1 was prevented by RU 486 10 microM. CONCLUSIONS: Aldosterone, through glucocorticoid receptor activation, increases the vasodilatory effect of CGRP in SHR mesenteric arteries, which seems to be mediated by increased RAMP1 expression.     

Activation of mineralocorticoid agonist and antagonist specific receptors from rat kidney

The kinetics of saturation, as well as of denaturation, confirm the existence of two distinct mineralocorticoid receptor populations one each for the agonist aldosterone (MR2) and the antagonist RU 26752 (MR3) in rat kidney. Receptor activation in vitro was dependent upon the buffer, progressed just as well in the presence of the agonist and the antagonist, and was inhibited by molybdate. These necessitate a reassessment of both the importance of receptor activation in vitro and its possible contribution to hormone action in vivo.     

Mineralocorticoid-induced increase in beta-adrenergic receptors of cultured rat arterial smooth muscle cells

We have investigated the effect of mineralocorticoids on beta-adrenergic receptors in cultured arterial smooth muscle cells. Mineralocorticoid (aldosterone) treatment resulted in a significant increase in beta-adrenergic receptors measured by [3H]dihydroalprenolol (DHA) binding. This effect required at least 20 hours of incubation with aldosterone and was completely blocked by cycloheximide (10 micrograms/ml), indicating protein synthesis was required for this response. Aldosterone at the concentration range of 10(-8)-10(-6) M increased [3H]DHA binding, but was ineffective at 10(-9) M. Scatchard analysis of [3H]DHA binding revealed that the observed significant increase in binding was due to an increased number of binding sites (P less than 0.05), and that the affinity was unchanged. The aldosterone (1 x 10(-8) M) effect was completely blocked by the combination of RU 38486 (10(-6) M) and spironolactone (10(-7) M), but not by the glucocorticoid antagonist RU 38486 alone. While basal c-AMP levels were not changed by aldosterone (10(-6) M) treatment, the isoproterenol (10(-6) M) stimulated level of c-AMP was significantly higher in cells treated with aldosterone (P less than 0.05). We conclude that aldosterone, acting through the mineralocorticoid receptor, has a direct effect on arterial smooth muscle cells mediated through modulation of beta-adrenergic receptors of these cells.     

Involvement of c-Src tyrosine kinase in SHP-1 phosphatase activation by Ang II AT2 receptors in rat fetal tissues

Angiotensin II (Ang II) AT(2) receptors are abundantly expressed in rat fetal tissues where they probably contribute to development. In the present study we examine the effects of Ang II type 2 receptor stimulation on SHP-1 activation. Ang II (10(-7) M) elicits a rapid and transient tyrosine phosphorylation of SHP-1, maximal at 1 min, in a dose-dependent form, blocked by the AT(2) antagonist, PD123319. SHP-1 phosphorylation is followed in time by tyrosine dephosphorylation of different proteins, suggesting a sequence of events. Ang II induces association of SHP-1 to AT(2) receptors as shown by co-immunoprecipitation, Western blot and binding assays. SHP-1 activity was determined in immunocomplexes obtained with either anti-AT(2) or anti-SHP-1 antibodies, after Ang II stimulation (1 min), in correlation with the maximal level of SHP-1 phosphorylation. Interestingly, following receptor stimulation (1 min) c-Src was associated to AT(2) or SHP-1 immunocomplexes. Preincubation with the c-Src inhibitor PP2 inhibited SHP-1 activation and c-Src association, thus confirming the participation of c-Src in this pathway. We demonstrated here for the first time the involvement of c-Src in SHP-1 activation via AT(2) receptors present in an ex vivo model expressing both receptor subtypes. In this model, AT(2) receptors are not constitutively associated to SHP-1 and SHP-1 is not constitutively activated. Thus, we clearly establish that SHP-1 activation, mediated by the AT(2) subtype, involves c-Src and precedes protein tyrosine dephosphorylation, in rat fetal membranes.     

Angiotensin II phosphorylation of extracellular signal-regulated kinases in rat anterior pituitary cells

We studied the effects of ANG II on extracellular signal-regulated kinase (ERK)1/2 phosphorylation in rat pituitary cells. ANG II increased ERK phosphorylation in a time- and concentration-dependent way. Maximum effect was obtained at 5 min at a concentration of 10-100 nM. The effect of 100 nM ANG II was blocked by the AT1 antagonist DUP-753, by the phospholipase C (PLC) inhibitor U-73122, and by the MAPK kinase (MEK) antagonist PD-98059. The ANG II-induced increase in phosphorylated (p)ERK was insensitive to pertussis toxin blockade and PKC depletion or inhibition. The effect was also abrogated by chelating intracellular calcium with BAPTA-AM or TMB-8 by depleting intracellular calcium stores with a 30-min pretreatment with EGTA and by pretreatment with herbimycin A and PP1, two c-Src tyrosine kinase inhibitors. It was attenuated by AG-1478, an inhibitor of epidermal growth factor receptor (EGFR) activation. Therefore, in the rat pituitary, the increase of pERK is a Gq- and PLC-dependent process, which involves an increase in intracellular calcium and activation of a c-Src tyrosine kinase, transactivation of the EGFR, and the activation of MEK. Finally, the response of ERK activation by ANG II is altered in hyperplastic pituitary cells, in which calcium mobilization evoked by ANG II is also modified.     

Transactivation of the epidermal growth factor receptor by heat shock protein 90 via Toll-like receptor 4 contributes to the migration of glioblastoma cells

Extracellular heat shock protein HSP90α was reported to participate in tumor cell growth, invasion, and metastasis formation through poorly understood signaling pathways. Herein, we show that extracellular HSP90α favors cell migration of glioblastoma U87 cells. More specifically, externally applied HSP90α rapidly induced endocytosis of EGFR. This response was accompanied by a transient increase in cytosolic Ca(2+) appearing after 1-3 min of treatment. In the presence of EGF, U87 cells showed HSP90α-induced Ca(2+) oscillations, which were reduced by the ATP/ADPase, apyrase, and inhibited by the purinergic P(2) inhibitor, suramin, suggesting that ATP release is requested. Disruption of lipid rafts with methyl β-cyclodextrin impaired the Ca(2+) rise induced by extracellular HSP90α combined with EGF. Specific inhibition of TLR4 expression by blocking antibodies suppressed extracellular HSP90α-induced Ca(2+) signaling and the associated cell migration. HSPs are known to bind lipopolysaccharides (LPSs). Preincubating cells with Polymyxin B, a potent LPS inhibitor, partially abrogated the effects of HSP90α without affecting Ca(2+) oscillations observed with EGF. Extracellular HSP90α induced EGFR phosphorylation at Tyr-1068, and this event was prevented by both the protein kinase Cδ inhibitor, rottlerin, and the c-Src inhibitor, PP2. Altogether, our results suggest that extracellular HSP90α transactivates EGFR/ErbB1 through TLR4 and a PKCδ/c-Src pathway, which induces ATP release and cytosolic Ca(2+) increase and finally favors cell migration. This mechanism could account for the deleterious effects of HSPs on high grade glioma when released into the tumor cell microenvironment.     

Integrin-mediated RON growth factor receptor phosphorylation requires tyrosine kinase activity of both the receptor and c-Src

Cooperation between integrins and growth factor receptors plays an important role in the regulation of cell growth, differentiation, and survival. The function of growth factor receptor tyrosine kinases (RTKs) can be regulated by cell adhesion to extracellular matrix (ECM) even in the absence of ligand. We investigated the pathway involved in integrin-mediated RTK activation, using RON, the receptor for macrophage-stimulating protein. Adhesion of RON-expressing epithelial cells to ECM caused phosphorylation of RON, which depended on the kinase activity of both RON itself and c-Src. This conclusion is based on these observations: 1) ECM-induced RON phosphorylation was inhibited in cells expressing kinase-inactive c-Src; 2) active c-Src could phosphorylate immunoprecipitated RON from ECM-stimulated cells but not from unstimulated cells; and 3) ECM did not cause RON phosphorylation in cells expressing kinase-dead RON, nor could active c-Src phosphorylate RON immunoprecipitated from these cells. The data fit a pathway in which ECM-induced integrin aggregation causes both c-Src activation and RON oligomerization followed by RON kinase-dependent autophosphorylation; this results in RON becoming a target for activated c-Src, which phosphorylates additional tyrosines on RON. Integrin-induced epidermal growth factor receptor (EGFR) phosphorylation also depended on both EGFR and c-Src kinase activities. This sequence appears to be a general pathway for integrin-dependent growth factor RTK activation.