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Endocrinology Vol. 147, No. 12 6046-6055
Copyright © 2006 by The Endocrine Society

Angiotensin II-Mediated Protein Kinase D Activation Stimulates Aldosterone and Cortisol Secretion in H295R Human Adrenocortical Cells

Damian G. Romero, Bronwyn L. Welsh, Elise P. Gomez-Sanchez, Licy L. Yanes, Silvia Rilli and Celso E. Gomez-Sanchez

Division of Endocrinology (D.G.R., E.P.G.-S., L.L.Y., C.E.G.-S.), G. V. (Sonny) Montgomery Veterans Affairs Medical Center, and Departments of Medicine (D.G.R., B.L.W., E.P.G.-S., L.L.Y., S.R., C.E.G.-S.) and Pharmacology and Toxicology (E.P.G.-S.), The University of Mississippi Medical Center, Jackson, Mississippi 39216

Address all correspondence and requests for reprints to: Damian G. Romero, Ph.D., Division of Endocrinology, Department of Medicine, The University of Mississippi Medical Center, 2500 North State Street, Jackson, Mississippi 39216. E-mail: dromero{at}medicine.umsmed.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Protein kinases are important mediators in intracellular signaling. Angiotensin II is the most important modulator of adrenal zona glomerulosa cell physiology. Angiotensin II regulates steroidogenesis and proliferation among many other metabolic processes. H295R human adrenal cells are a widely used experimental model to study adrenal cell physiology and metabolism. We screened for protein kinase expression levels using the Kinetwork system in H295R cells after 3 h angiotensin II treatment. Protein kinase D (PKD) was the protein kinase that suffers the most dramatic changes. PKD is a member of a new class of serine/threonine protein kinases that is activated by phosphorylation. Our studies indicated that angiotensin II time- and dose-dependently increased PKD phosphorylation, which occurred within 2 min of angiotensin II treatment and at concentrations as low as 1 nM. PKD phosphorylation was also dose-dependently increased by the PKC activator phorbol 12-myristate 13-acetate. Angiotensin II-mediated PKD phosphorylation was blocked by several PKC inhibitors. Furthermore, PKC{epsilon} translocation inhibitor peptide decreased angiotensin II-mediated PKD phosphorylation, and PKC{epsilon} down-regulation by RNA interference also decreased PKD phosphorylation mediated by angiotensin II. Cotransfection of constitutively active PKD mutant constructs up-regulated aldosterone synthase and 11ß-hydroxylase expression in reporter assays. Constitutively active PKD mutants increased aldosterone and cortisol secretion under angiotensin II stimulatory conditions. This study reveals that PKD is an intracellular signaling mediator of angiotensin II regulation of steroidogenesis in human adrenal cells. These data provide new insights into the molecular mechanisms involved in angiotensin II-induced physiological and pathophysiological events in adrenal cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANGIOTENSIN II (Ang II) is the most important regulator of adrenal zona glomerulosa cell physiology. Ang II is an octapeptide that is generated by the renin-angiotensin system (RAS). Beside the well known systemic RAS, there is also an intraadrenal RAS (1, 2, 3, 4). Ang II binds to specific seven-transmembrane G protein-coupled receptors in adrenal cells triggering several intracellular signaling cascades including phospholipase C/diacylglycerol/protein kinase C (PKC) and calcium/calmodulin/calmodulin-dependent kinase (5, 6, 7). Much remains to be understood about the downstream targets of these signaling cascades that amplify Ang II signal. Protein kinases are involved in almost all cellular signaling pathways regulating a large variety of cellular physiological processes involving metabolism, replication, differentiation, etc. (8).

The H295R human adrenocortical cell (9) is a widely used in vitro model for the study of adrenal cell physiology and metabolism because it is the only human adrenal cell line that presents a steroid secretion pattern and regulation similar to primary cultures of adrenal cells (10).

PKD, also known as PKCµ, is a serine/threonine kinase with unique structural, enzymological, and regulatory properties that are different from those of the PKC family members. PKD contains several domains in a unique combination that define, together with PKD2 and PKD3/PKC{nu}, a new protein kinase family (11, 12, 13, 14, 15, 16). The most prominent characteristics of PKD are the presence of a catalytic domain related to myosin light chain and calcium/calmodulin-regulated kinases, a pleckstrin homology domain that regulates PKD activity, and a highly hydrophobic stretch of amino acids in its N-terminal region (11, 12, 13, 14, 15, 16). PKD can be activated by a variety of stimuli, including regulatory peptides (Ang II, bombesin, bradykinin, endothelin, and vasopressin), growth factors, phorbol esters, and T- and B-cell receptor agonists via PKC-dependent pathways (11, 12, 13, 14, 15, 16). PKD activation appears to involve the phosphorylation of Ser744 and Ser748 in the mouse (Ser738 and Ser742 in human) within the activation loop of the catalytic domain (17) as well as the autophosphorylation of Ser916 in the mouse (Ser910 in human) (18). PKD has been implicated in the regulation of a variety of cellular functions, including signal transduction, membrane trafficking, protein transport, and cell survival, migration, differentiation, and proliferation (11, 12, 13, 14, 15, 16).

The aim of this study was to study which protein kinases are regulated by Ang II in H295R human adrenocortical cells. After identifying PKD as the protein kinase most regulated by Ang II in H295R cells, we performed additional studies to determine Ang II-mediated PKD activation regulation, the intracellular signaling mechanisms involved, and the role of PKD in adrenal steroidogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Ang II was obtained from American Peptide Company Inc. (Sunnyvale, CA). Phorbol 12-myristate 13-acetate (PMA), bisindolylmaleimide I (GF 109203X), Gö 6976, Gö 6983, Ro-31-8220, Ro-32-0432 and myristoylated PKC inhibitor peptide (amino acids 20–28), and PKC{epsilon} translocation inhibitor peptide and its scrambled negative control were from EMD Biosciences (San Diego, CA). Antibodies against PKD, p-PKD(Ser744/748), and p-PKD(Ser916) were from Cell Signaling (Danvers, MA). Anti-PKC{epsilon} antibody was from BD Biosciences (San Jose, CA). Anti-actin antibody (catalog no. JLA20, a monoclonal antibody developed by J. J. Lin) was obtained from the Developmental Studies Hybridoma Bank supported by the National Institute of Child Health and Human Development and maintained by The University of Iowa, Department of Biological Sciences (Iowa City, IA).

Cell culture
H295R human adrenocortical cells (9) were cultured in H295R complete media containing DMEM/F12 (1:1) supplemented with 2% Ultroser G (Biosepra, Villeneuve-la-Garenne, France), ITS-Plus (Discovery Labware, Bedford, MA), and an antibiotic/antimycotic mixture (Invitrogen, Carlsbad, CA), as we previously described (19) in six-well plates for 24 h after reaching confluence. Medium was replaced with 3 ml fresh medium containing different agents and cultured for 10 min, unless otherwise indicated. At the end of the incubation period, cells were washed with ice-cold PBS and subjected to protein extraction and analysis as described below. Inhibitors were added 30 min before the other reagents and remained during the incubation period.

Protein kinase screening
H295R cells were treated with or without Ang II (100 nM) for 3 h. Cells were washed with ice-cold PBS and lysed in 20 mM 3-(N-morpholino)propanesulfonic acid (pH 7.0), 5 mM EDTA, 2 mM EGTA, 0.5% Igepal CA-630, protease inhibitors cocktail (Roche, Indianapolis, IN), and phosphatase inhibitors cocktail sets I and II (EMD Biosciences). Lysates were scraped, incubated for 15 min in ice, and cleared by centrifugation for 15 min at 12,000 x g at 4 C. Protein concentration was determined by Bradford reagent (Bio-Rad, Hercules, CA) using BSA as standard. Whole lysate protein samples (600 µg) were analyzed by Kinetworks KPKS-1.2 protein kinase screen (Kinexus Bioinformatics Corp., Vancouver, British Columbia, Canada). The Kinetworks analysis involved resolution of a single lysate sample by SDS-PAGE and subsequent immunoblotting with panels of up to three primary antibodies per channel in a 20-lane Immunetics Multiblotter. The antibody mixtures were carefully selected to avoid overlapping cross-reactivity with target proteins. Normalized trace quantity units (cpm) were arbitrary based on the intensity of ECL fluorescence detection for target immunoreactive proteins recorded with a Fluor-S MultiImager and quantified using Quantity One Software (Bio-Rad). The protein kinases detected by the Kinetworks KPKS 1.2 screen are listed in Fig. 1Go. Detailed information and protocols of the Kinetworks analysis have been published previously (20) and can also be found at the Kinexus Bioinformatics Corp. website (www.kinexus.ca). A cutoff of 30% up- or down-regulation was used to determine expression level of regulated protein kinases.


Figure 1
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  		FIG. 1. Effect of Ang II on protein kinase expression levels. H295R cells were incubated in the absence (left) or presence (right) of 100 nM Ang II for 3 h. Total protein was extracted and analyzed by Kinetworks KPKS-1.2 protein kinase screen analysis. Arrows and numbers indicate the identities of protein targets.

 
Plasmids
pSUPER.retro.gfp.neo was used as the backbone plasmid. pSUPER-PKC{epsilon} was constructed using previously reported short hairpin RNA (shRNA) sequences (21); pSUPER-control contained no insert. Reporter plasmids carrying the human 11ß-hydroxylase and aldosterone synthase (22) were generated by transferring the promoter regions from pGL3-Basic to pGL4.10[luc2] (Promega, Madison, WI), as we have previously described (23). Human PKD mutants PKD(S738E/S742E) and PKD-{Delta}PH in pcDNA3 (Invitrogen) have been previously described (21).

Plasmid transfection for PKD down-regulation
H295R cells were transfected using Nucleofector technology (Amaxa Biosystems, Gaithersburg, MD). Two million log phase cells were resuspended in 100 µl Nucleofector Solution R, mixed with 1 µg plasmid DNA, and electroporated using the proprietary program T-20. Cells were allowed to recover for 15 min in RPMI 1640 medium at 37 C and then plated in six-well plates with 3 ml H295R complete medium per well. Cells were cultured for 60 h. The medium was then removed and the cells incubated with prewarmed medium with or without 10 nM Ang II for 10 min. At the end of the incubation period, cells were washed with ice-cold PBS and subjected to protein extraction and analysis as described below.

Plasmid transfection for PKD overexpression
H295R cells were transfected using Nucleofector technology (Amaxa Biosystems). Three million log phase cells were resuspended in 100 µl Nucleofector Solution R, mixed with 3 µg plasmid DNA, and electroporated using the proprietary program P-20. Cells were allowed to recover for 15 min in RPMI 1640 media at 37 C and then plated in 24-well plates with 1 ml H295R complete medium per well. Cells were cultured for 16 h. The medium was then removed and the cells incubated with prewarmed medium with or without 10 nM Ang II for 24 h. At the end of the incubation period, cell culture supernatants were saved for aldosterone and cortisol determination by ELISA as previously reported (24, 25). Cells were lysed with M-Per lysis buffer (Pierce, Rockford, IL) and protein concentration measured by the bicinchoninic acid method (Pierce).

Peptide transfection
PKC{epsilon} translocation inhibitor peptide transfection was performed using the Chariot Reagent (Active Motif, Carlsbad, CA). Briefly, H295R cells were grown in six-well plates until approximately 90% confluent and transfected with 500 ng peptide following the manufacturer’s suggested protocol. Cells were incubated with transfection reagent for 1 h and then supplemented with 1 ml/well fresh medium and incubated for 2 more hours. Medium was removed and cells were incubated with prewarmed medium with or without 10 nM Ang II for 10 min. At the end of the incubation period, cells were washed with ice-cold PBS and subjected to protein extraction and analysis as described below (see Western blot).

PKC{epsilon} translocation
For protein translocation studies to the plasma membrane, cells were lysed in 20 mM Tris-HCl buffer (pH: 7.5) supplemented with 5 mM EDTA, 1 mM EGTA, protease inhibitor cocktail (Roche, Indianapolis, IN) and phosphatase inhibitor cocktail set I and II (EMD Biosciences, San Diego, CA), sonicated and centrifuged at 80,000 x g for 90 min at 4 C. Pellets were resuspended in M-Per lysis buffer (Pierce) supplemented with 5 mM EDTA, 1 mM EGTA, protease inhibitor cocktail (Roche, Indianapolis, IN) and phosphatase inhibitor cocktail set I and II (EMD Biosciences, San Diego, CA), sonicated and subjected to protein analysis as described below.

Western blot
Cells were lysed with M-Per lysis buffer (Pierce) supplemented with 5 mM EDTA, 1 mM EGTA, protease inhibitor cocktail (Roche, Indianapolis, IN), and phosphatase inhibitor cocktail sets I and II (EMD Biosciences, San Diego, CA). Lysates were cleared by centrifugation at 12,000 x g for 15 min at 4 C, and the protein concentration of the supernatant was measured by the bicinchoninic acid method (Pierce) using BSA as standard. Equal aliquots of cell lysates were resolved on 12% SDS-PAGE, transferred to polyvinylidene difluoride membranes using a semidry technique, and incubated with various primary antibodies followed by relevant affinity-purified horseradish-peroxidase-conjugated secondary antibodies. Blots were developed with Super Signal West Pico Chemiluminescent substrate (Pierce, Rockford, IL) and exposed to autoradiography film. Films were scanned and quantified with a Kodak Image Station 440 using the 1D Kodak image analysis software. Membranes were stripped using Restore Western blot stripping buffer (Pierce, Rockford, IL) and reprobed with a different set of antibodies.

Reporter assays
H295R cells were grown in 24-well plates with H295R complete medium without antibiotic/antimycotics until 90–95% confluent. H295R cells were transfected by a combination of cationic lipids (Lipofectamine 2000; Invitrogen) and magnetofection (CombiMag, OzBiosciences, France). Cells were transfected with 3 µg DNA per well (2 µg reporter plasmid plus 1 µg expression plasmid), 2 µl/well Lipofectamine 2000 (Invitrogen) and 4.5 µl/well CombiMag following the manufacturer’s suggested protocols. Cells were cultured overnight, medium replaced with 1 ml/well fresh medium with or without Ang II (10 nM) and cultured for an additional 24 h. Cells were lysed with Glo Lysis buffer (Promega) and luciferase activity quantified with Bright-Glo Luciferase assay kit (Promega). Reporter assays were performed in quadruplicate using three different plasmid DNA maxipreps in each experiment to avoid plasmid DNA preparation-related effects.

Statistical analysis
Results are expressed as mean ± SEM. Two groups were compared by Student’s t test, and multiple groups were analyzed by ANOVA followed by Tukey’s post hoc comparisons. Statistical calculations were performed with GraphPad Prism package version 4.03 (GraphPad Software, Inc., San Diego, CA) or Statistica version 7.1 (StatSoft, Inc., Tulsa, OK). Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Protein kinase screening
Protein kinases are crucial regulators in intracellular signaling. Ang II is one of the most important physiological regulators of adrenal cells. To evaluate Ang II effects on protein kinase expression on adrenal cells, we treated H295R human adrenocortical cells with or without Ang II for 3 h and quantified protein kinase expression using the Kinetworks protein profiling system. Figure 1Go shows a blot of a multiblot expression profile of protein kinase expression level screening of control and Ang II-treated H295R cells. As shown in Fig. 1Go, H295R cells expressed 43 of the 75 screened protein kinases. Ang II treatment caused a dramatic change in PKD expression profile (Fig. 1Go, band 8). PKD migrates as a doublet with the upper band representing phosphorylated isoforms of PKD. After Ang II treatment, phosphorylated PKD shows a dramatic 250% increase, whereas nonphosphorylated PKD (lower band) did not show major changes. Ang II treatment also modified the expression level of several kinases including up-regulation of Raf1 (57%), BMX (Etk) (44%), MAPK kinase IV (MEK4) (42%), casein kinase II (CK2{alpha}) (36%), and PKC{epsilon} (34%) and down-regulation of MST1 (–43%) and RhoA kinase (ROK{alpha}) (–42%). Because PKD was the protein kinase that showed the most dramatic changes after Ang II treatment, we focused on this newly described kinase.

Angiotensin II mediated PKD phosphorylation
To expand the findings that Ang II increases PKD phosphorylation, we performed the following experiments, including Ang II time-course and dose-response curves. PKD phosphorylation was determined by Western blot using two commercially available phospho-PKD-specific antibodies against key phosphorylated residues. One of them recognizes the endogenous levels of PKD only when dually phosphorylated at Ser738 and Ser742 (Ser744 and Ser748 in mouse), and the other recognizes PKD only when phosphorylated at Ser910 (Ser916 in mouse). Ang II (100 nM) induced PKD phosphorylation after 1 h stimulation in both phosphorylation sites under study (Fig. 2AGo). Phosphorylated PKD levels slowly declined, reaching basal levels 12 h after hormone stimulation. PKD expression levels were detected using anti-PKD antibody. PKD levels remained constant during the course of Ang II stimulation up to 48 h. These results confirm our previous findings in the protein kinase screening, which indicated that 3 h Ang II treatment significantly increased PKD phosphorylation. To study the rate of Ang II-mediated PKD phosphorylation, H295R cells were treated with Ang II (100 nM) for periods up to 1 h. Ang II rapidly induced PKD phosphorylation at both Ser738/742 and Ser910 phosphorylation sites within 2 min, reaching maximal levels in 5 min and remaining constantly elevated for the first hour of treatment (Fig. 2BGo). These results indicate that PKD phosphorylation is an early intracellular signal triggered by Ang II in adrenal cells. To further confirm Ang II-mediated phosphorylation of PKD, we performed an Ang II dose-response curve. H295R cells were incubated with increasing concentrations of Ang II for 10 min to reach maximal phosphorylation levels, and PKD phosphorylation was analyzed by Western blot. Ang II dose-dependently increased PKD phosphorylation at both Ser738/742 and Ser910 phosphorylation sites (Fig. 3Go). PKD phosphorylation was detectable at Ang II concentrations as low as 1 nM. These results clearly show for the first time that Ang II phosphorylates PKD in a time- and dose-dependent manner in H295R adrenocortical cells.


Figure 2
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  		FIG. 2. Ang II time-dependent regulation of PKD phosphorylation. H295R cells were incubated with Ang II (100 nM) for the time periods indicated. Cell extracts were analyzed by PAGE and probed for pPKD(S738/742), pPKD(S910), PKD, and actin. A, Long-range time curve from 0–48 h Ang II treatment; B, short-range time curve from 0–60 min Ang II treatment.

 

Figure 3
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  		FIG. 3. Ang II dose-dependent regulation of PKD phosphorylation. H295R cells were incubated with increasing concentrations of Ang II for 10 min. Cell extracts were analyzed by PAGE and probed for pPKD(S738/742), pPKD(S910), and PKD.

 
PKD phosphorylation was mediated by PKC
The phospholipase C/diacylglycerol/PKC intracellular signaling cascade is one of the main pathways triggered by Ang II in adrenal cells (5, 6, 7). There are multiple related PKC isoforms, which can be classified into three distinct groups on the basis of structural and regulatory differences: the conventional PKCs ({alpha}, ßI, ßII, and {gamma}), which are regulated by calcium, diacylglycerol, and phospholipids; the novel PKCs ({delta}, {epsilon}, {eta}, and {theta}), which are regulated by diacylglycerol and phospholipids but are calcium insensitive; and the atypical PKCs ({xi} and {lambda}), which are calcium and diacylglycerol insensitive and whose regulation is less well characterized. PKD activation has been reported to be mediated by PKC in several systems (8). To study whether PKC is involved in Ang II-mediated PKD phosphorylation, we performed the following series of studies. H295R cells were treated with increasing concentrations of the PKC activator PMA (1–1000 nM) or vehicle (dimethylsulfoxide) for 10 min, and PKD phosphorylation status was determined by Western blot (Fig. 4Go). PMA dose-dependently increased PKD phosphorylation at both the Ser738/742 and Ser910 phosphorylation sites. PKD phosphorylation at Ser910 was detected at PMA concentrations as low as 10 nM. Although PKD Ser916 (Ser910 in human) is an autophosphorylation site and its autophosphorylation is dramatically up-regulated by PMA treatment in vivo (18), PKD Ser 744/748 (Ser738/742 in human) is a transphosphorylation site independent of PKD catalytic activity (26). PKD Ser738/742 phosphorylation was dose-dependently up-regulated by PMA, indicating that PKC is involved in PKD phosphorylation in H295R cells.


Figure 4
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  		FIG. 4. PMA dose-dependent regulation of PKD phosphorylation. H295R cells were incubated with increasing concentrations of PMA for 10 min. Cell extracts were analyzed by PAGE and probed for pPKD(S738/742), pPKD(S910), and PKD.

 
To confirm the role of PKC on Ang II-mediated PKD phosphorylation, H295R cells were preincubated with several PKC inhibitors for 30 min and then stimulated with Ang II (100 nM) for 10 min. Bis I, Gö 6983, Ro-31-8220 and Ro-32-0432 inhibited Ang II-mediated PKD phosphorylation at both Ser738/742 and Ser910 phosphorylation sites (Fig. 5Go). PKC inhibitors are not isozyme specific, but each one has a particular isozyme pattern of inhibition. When PKC inhibitor pattern information is combined with isozyme expression, it can give a very good indication of the specific isozyme involved in a particular signaling pathway. Previous studies in H295R cells indicate that this cell line expresses high levels of PKC{alpha}, -{epsilon}, -{theta}, and -{xi} and low levels of PKC{lambda} (27). Gö 6983 has been reported to inhibit classical ({alpha} and ß), novel ({delta}, {epsilon}, and {gamma}) and atypical ({xi}) PKC isozymes (28, 29). Accordingly, we found that it inhibited Ang II-mediated PKD phosphorylation in H295R cells. Bis I, Ro-31-8220 and Ro-32-0432 inhibit classical and novel PKC isozymes (30, 31, 32, 33) and also dose-dependently decrease Ang II-mediated PKD phosphorylation, suggesting that the classical PKC{alpha} or the novel PKC{epsilon} or PKC{theta} could be involved in PKD phosphorylation in H295R cells. Gö 6976 inhibits classical but not novel or atypical PKCs (31). Gö 6976 did not inhibit Ang II-mediated PKD phosphorylation in H295R cells at concentrations up to 5 µM. This indicates that classical PKCs are not involved in PKD phosphorylation in H295R cells and discards PKC{alpha} as a possible candidate. Use of PKC{alpha} pseudosubstrate peptide (34) and rottlerin (35) further indicated that PKC{alpha} and PKC{delta} isozymes did not have a major role in PKD phosphorylation in H295R cells. These results point to the novel PKC isozymes PKC{epsilon} and PKC{theta} as possible candidates. Recently, it was reported that Ang II treatment for 10 min of H295R cells caused an important increase in PKC{epsilon} phosphorylation, whereas there were no appreciable changes in PKC{theta} phosphorylation status (36). Our findings and those from other groups suggest that PKC{epsilon} could be a suitable candidate to mediate PKD phosphorylation in H295R cells.


Figure 5
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  		FIG. 5. Effect of PKC inhibitors on Ang II-mediated PKD phosphorylation. H295R cells were treated with different PKC inhibitors or vehicle for 30 min and then incubated in the presence or absence of Ang II (100 nM) for 10 min. Cell extracts were analyzed by PAGE and probed for pPKD(S738/742), pPKD(S910), and PKD.

 
PKC{epsilon} mediated Ang II-mediated PKD phosphorylation
Although our results suggest that PKC{epsilon} could mediate PKD phosphorylation in H295R cells, we performed experiments to directly test the role of PCK{epsilon} on PKD phosphorylation. First, we confirmed that H295R cells express PKC{epsilon} that migrates in Western blots with an apparent molecular weight identical to a rat brain homogenate used as positive control (Fig. 6AGo). PKC{epsilon} translocates to the plasma membrane upon activation, where it mediates PKD phosphorylation and concomitant activation (37). To study whether Ang II treatment mediates PKC{epsilon} translocation in our experimental model, H295R cells were incubated with Ang II (100 nM) for increasing periods of time and PKC{epsilon} localization analyzed by Western blot in the membrane fraction. Ang II caused a rapid translocation of PKC{epsilon} to the plasma membrane only 2 min after treatment. PKC{epsilon} remained associated to the membrane fraction for up to 30 min after Ang II treatment. These results indicate that PKC{epsilon} is activated by Ang II treatment in H295R cells and that PKC{epsilon} is translocated to the correct intracellular location to interact with PKD within the same time period as PKD phosphorylation occurs (compare Figs. 2BGo and 6BGo).


Figure 6
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  		FIG. 6. PKC{epsilon} expression and Ang II effect on PKC{epsilon} membrane translocation in H295R cells. A, Total protein extracts from rat brain (10 µg) or H295R cells (50 µg) were analyzed by PAGE and probed with anti-PKC{epsilon}; B, H295R cells were incubated with Ang II (100 nM) for the time periods indicated. Membrane fraction extracts were analyzed by PAGE and probed with anti-PKC{epsilon} antibody.

 
To determine whether PKC{epsilon} is indeed involved in Ang II-mediated PKD phosphorylation in H295R cells, we used two alternative experimental approaches to specifically down-regulate bioactive PKC{epsilon} by decreasing its protein levels or activity. It has been reported that translocation of several PKC isozymes requires their interaction with isozyme-specific receptors for activated C kinases (RACKs) and that these interactions are blocked by the use of PKC-specific isozyme-derived peptides known as PKC translocation inhibitors (38, 39). It has also been established that isozyme-specific PKC function can be inhibited by the use of PKC translocation inhibitors blocking the interaction between PKC and its endogenous substrates. We used a PKC{epsilon} translocation inhibitor to specifically block PKC{epsilon} translocation and interaction with its substrates; as a control, a scrambled peptide with the same amino acid composition was used. H295R cells were transfected with PKC{epsilon} translocation inhibitor peptide or its scrambled control peptide and then stimulated with Ang II for 10 min. PKC{epsilon} translocation inhibitor peptide decreased PKD phosphorylation at Ser910 (1.00 ± 0.05 vs. 0.63 ± 0.02, P < 0.05) (Fig. 7CGo). PKC{epsilon} translocation inhibitor peptide did not modify total PKD or p-PKD(Ser738/742) levels.


Figure 7
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  		FIG. 7. Effect of PKC{epsilon} translocation inhibitor peptide on Ang II-mediated PKD phosphorylation. A, H295R cells were transfected with PKC{epsilon} translocation inhibitor peptide or a scrambled control peptide, incubated for 2 h, and then treated with or without 10 nM Ang II for 10 min. Cell extracts were analyzed by PAGE and probed for pPKD(S738/742), pPKD(S910), and PKD. Protein bands were quantified and pPKD(S738/742) (B) or pPKD(S910) (C) expressed as arbitrary units (AU) normalized by total PKD. *, P < 0.05 vs. control peptide.

 
To confirm the role of PKC{epsilon} on PKD phosphorylation, we used a different means to decrease PKC{epsilon} levels, RNA interference delivered by shRNA plasmid transfection. Because H295R is a very difficult cell line to transfect, we used the Nucleofector technology (Amaxa Biosystems) that allows us to achieve high transfection efficiency and concomitantly allows us to down-regulate PKC{epsilon} in most of the cell population. PKC{epsilon} shRNA significantly decreased PKC{epsilon} protein levels (1.00 ± 0.10 vs. 0.52 ± 0.09, P < 0.05) by almost 50% 60 h after transfection (Fig. 8Go, D and E). PKC{epsilon} shRNA decreased Ang II-mediated PKD phosphorylation at Ser738/742 (1.00 ± 0.04 vs. 0.75 ± 0.04, P < 0.05) (Fig. 8Go, A–C). PKC{epsilon} shRNA did not modify total PKD or p-PKD(Ser910) levels. PKC{epsilon} down-regulation by the use of two different experimental approaches, PKC{epsilon} translocation inhibitor peptide and RNA interference, indicate that PKC{epsilon} isozyme mediates Ang II-stimulated PKD phosphorylation in H295R cells.


Figure 8
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  		FIG. 8. Effect of PKC{epsilon} down-regulation by shRNA on Ang II-mediated PKD phosphorylation. A, H295R cells were transfected with pSUPER-PKC{epsilon} or pSUPER-control, incubated for 60 h, and then treated with or without 10 nM Ang II for 10 min. Cell extracts were analyzed by PAGE and probed for pPKD(S738/742), pPKD(S910), and PKD. B and C, Protein bands were quantified and pPKD(S738/742) (B) or pPKD(S910) (C) expressed as arbitrary units (AU) normalized by total PKD; D, cell extracts from unstimulated transfected cells were analyzed by PAGE and probed with anti-PKC{epsilon} or anti-actin antibodies; E, protein bands were quantified and PKC{epsilon} expressed as arbitrary units normalized by actin. *, P < 0.05 vs. pSUPER-control.

 
Activated PKD increased steroidogenic enzymes expression
To determine whether PKD activation modulates adrenal steroidogenesis, we performed reporter gene expression studies. We tested the expression of two key steroidogenic enzymes in mineralocorticoid and glucocorticoid synthesis, aldosterone synthase and 11ß-hydroxylase, respectively. To study the role of PKD activation, we used two constitutively active PKD constructs, PKD(S738E/S742E), which carries a double glutamic acid mutation mimicking phosphorylated serines at positions 738/742, and PKD-{Delta}PH, which has a deletion of the autoinhibitory pleckstrin homology domain. Reporter assay studies were performed under basal and suboptimal Ang II stimulatory conditions. All PKD constructs, wild-type-PKD, PKD(S738E/S742E), and PKD-{Delta}PH, caused a 1.35- to 1.65-fold increase in 11ß-hydroxylase reporter gene expression under basal conditions (Fig. 9AGo, white bars). Both wild-type PKD and PKD(S738E/S742E) caused a 2-fold increase in 11ß-hydroxylase expression under Ang II stimulatory conditions compared with Ang II induction caused by the control plasmid (Fig. 9AGo, black bars). Both constitutive active PKD constructs, PKD(S738E/S742E) and PKD-{Delta}PH, increased aldosterone synthase reporter gene expression 3.3- and 3.4-fold under basal conditions, respectively (Fig. 9BGo, white bars). Under Ang II stimulation, wild-type PKD as well as constitutive active PKD constructs caused a remarkable increase in aldosterone synthase expression (Fig. 9BGo, black bars). Wild-type PKD, PKD(S738E/S742E), and PKD-{Delta}PH caused a 5.5-, 5.4-, and 4.2-fold increase in aldosterone synthase expression, respectively, compared with Ang II induction caused by the control plasmid.


Figure 9
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  		FIG. 9. Effect of PKD on 11ß-hydroxylase and aldosterone synthase reporter genes. H295R cells were cotransfected with 11ß-hydroxylase (A) or aldosterone synthase (B) reporter plasmids and wild-type or constitutive active PKD constructs, PKD(S738E/S742E) or PKD-{Delta}PH, or control plasmid (pcDNA3). Cells were allowed to recover overnight and then treated with or without Ang II (10 nM) for 24 h. Data are expressed as percentage of control-basal. *, P < 0.05 vs. control-basal’ #, P < 0.05 vs. control-Ang II.

 
Activated PKD increased steroid secretion
To study whether the stimulatory effect of active PKD on 11ß-hydroxylase and aldosterone synthase expression is translated into an increase of cortisol or aldosterone secretion, we performed transfection studies with the constitutive active PKD-{Delta}PH construct and measured steroid secretion under basal and Ang II stimulatory conditions. Constitutive active PKD construct PKD-{Delta}PH increased Ang II-mediated cortisol (1189 ± 111 vs. 2193 ± 407 pg cortisol/µg protein per day, P < 0.05) (Fig. 10AGo) and aldosterone secretion (23.96 ± 5.21 vs. 46.96 ± 10.62 pg aldosterone/µg protein per day, P < 0.05) (Fig 10BGo).


Figure 10
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  		FIG. 10. Effect of constitutive active PKD on steroid secretion by H295R cells. H295R cells were transfected with PKD-{Delta}PH or control plasmid (pcDNA3), cultured overnight, and then treated with or without Ang II (10 nM) for 24 h. Steroids were measured in cell culture supernatants by ELISA. *, P < 0.05 vs. control-basal; #, P < 0.05 vs. control-Ang II.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major findings of the present study are that 1) PKD is rapidly phosphorylated at Ser738/742 (Ser744/748 in the mouse) and Ser910 (Ser916 in mouse) in adrenal cells in response to Ang II in a time- and dose-dependent manner, 2) PKD phosphorylation is dose-dependently stimulated by the PKC activator PMA and blocked by PKC inhibitors, 3) decreasing bioactive PKC{epsilon} by the use of specific translocation inhibitor peptide or RNA interference decreased Ang II-mediated PKD phosphorylation, 4) constitutively active PKD constructs up-regulated 11ß-hydroxylase and aldosterone synthase expression, and 5) constitutively active PKD constructs increased cortisol and aldosterone secretion under Ang II-stimulated conditions. These results show for the first time that PKD is a downstream intracellular mediator of Ang II action in adrenal cells and regulates adrenal steroidogenesis.

Screening for protein kinases regulated at the protein expression level by Ang II in H295R cells, we found that PKD, which migrates as a doublet corresponding to the phosphorylated and dephosphorylated polypeptides, shows a dramatic change in its phosphorylation status, observed as a significant increase in the phosphorylated polypeptide band in the screening Western blots. We then performed a series of experiments to confirm and further investigate PKD regulation by Ang II.

PKD and the biomolecules that could modulate its activity have been implicated in the regulation of a broad range of biological processes. Several regulatory peptides, including G-protein-coupled receptors ligands (Ang II, bombesin, vasopressin, endothelin, bradykinin, and thrombin) and growth factors (epithelial growth factor and vascular endothelial growth factor), modulate PKD activation in several biological models (11, 12, 13, 14, 15, 16). Ang II has been reported to rapidly and transiently activate PKD in rabbit (40) and rat (41) aortic vascular smooth muscle cells, rat intestinal epithelial cells (42), and neonatal rat cardiomyocytes (43). Our results show that Ang II causes PKD phosphorylation in a time- and dose-dependent manner. It has been shown that PKD phosphorylation at the phosphorylation sites under study showed a perfect correlation with PKD activation (17, 18). Our results show that PKD activation occurs within minutes after Ang II treatment in H295R cells, which indicates that PKD activation is an early signaling event triggered by Ang II in adrenal cells.

Several PKC isozymes have been reported to mediate endogenous PKD activation in vivo. PKC{alpha} mediates PKD activation induced by PMA in BON endocrine cells (44), and by VEGF in bovine aortic endothelial cells and human umbilical vein endothelial cells (45). PKC{delta} mediates thrombin and Ang II-mediated PKD phosphorylation in rat aortic smooth muscle cells (41, 46). PKC{delta} also mediates PKD activation by PMA in BON endocrine cells (44) and oxidative stress in HeLa or HEK-293E cells (21). PKC{epsilon} has been shown to have a critical role in bombesin-mediated PKD activation in Swiss 3T3 cells (37) and norepinephrine-mediated PKD activation in neonatal rat cardiomyocytes (43). Although we cannot exclude the involvement of other PKC isozymes, our results using two different experimental approaches clearly show that PKC{epsilon} has a critical role in PKD activation in human adrenal cells.

PKC{epsilon} has been suggested to be an upstream kinase of PKD by several experimental approaches. It has been reported that PKD associates with PKC{epsilon}, using biochemical techniques such as in vitro pull-down assays using glutathione S-transferase fusion proteins and coimmunoprecipitations (47). Brandlin et al. (48) reported that PKC{epsilon} phosphorylated PKD in in vitro kinase assays and that both kinases colocalize in vivo in HEK293 and MCF7 cells. Rey et al. (37) showed that PKC{epsilon} translocation and activity are required for PKD phosphorylation in Swiss 3T3 cells and that interfering with PKC{epsilon} activity by the use of translocation inhibitor peptide or RNA interference prevents bombesin-mediated PKD phosphorylation. More recently, it was reported that in neonatal rat cardiomyocytes, PKD coimmunoprecipitates with PKC{epsilon}, and dominant-negative PKC{epsilon} or PKD inhibits both PKD phosphorylation and norepinephrine-mediated atrial natriuretic factor expression (43). Our results and those from others (36) indicate that PKC{epsilon} is activated by Ang II in H295R cells. These results agree with previous observations in rat adrenal cells where it was reported that PKC{epsilon} translocates to the plasma membrane by Ang II or 12- or 15-hydroxyeicosatetraenoic acids (49). Because PKC{epsilon} is expressed across species in adrenal cells, it could be a general mediator of PKD activation. Surprisingly, the PKD phosphorylation pattern inhibition obtained with the PKC{epsilon} translocation inhibitor peptide and shRNA knockdown treatment was different. The shRNA treatment effectively decreased PKC{epsilon} protein levels, whereas the translocation inhibitor peptide blocks PKC{epsilon} translocation and activation but did not decrease PKC{epsilon} total protein levels, allowing inactive PKC{epsilon} to still have protein-protein interactions that may account for the differential phosphorylation pattern with the two experimental approaches used in H295R cells.

PKD activation caused a remarkable up-regulation of aldosterone synthase gene expression in H295R cells. Aldosterone synthase is the last enzyme in the aldosterone biosynthetic pathway and the only one exclusively required for aldosterone synthesis. LeHoux and Lefebvre (36) reported that Ang II activates PKC{epsilon} and that infection with an adenoviral vector containing a constitutively active PKC{epsilon} construct decreases aldosterone synthase gene expression in H295R cells. Our results also indicated that Ang II activated PKC{epsilon}, which then activated PKD and PKD-up-regulated aldosterone synthase expression. Although apparently contradictory, both results may be correct because active PKC{epsilon} may have more than one target protein affecting different intracellular signaling pathways that regulate adrenal steroidogenesis at different time points. The PKD stimulatory effect on aldosterone synthase expression is greatly exacerbated under Ang II stimulatory conditions probably because of the requirement of other molecules generated or up-regulated under Ang II stimulation. Wild-type PKD increased aldosterone synthase expression to levels similar to constitutive active PKD under Ang II stimulatory conditions, probably reflecting that wild-type PKD may be subjected to phosphorylation and consequently activation by endogenous kinases under mineralocorticoid secretion stimulatory conditions. Because we studied the role of only PKD activation on 11ß-hydroxylase and aldosterone synthase expression regulation, we cannot exclude that PKD modulates expression or activity of other steroidogenic proteins. This may account for the discrepancy observed with PKD-{Delta}PH, which caused a significant increased in Ang II-mediated cortisol secretion, whereas it caused a slight increase in 11ß-hydroxylase gene expression that did not reach significance.

In the present report, we demonstrate that Ang II phosphorylates PKD through PKC{epsilon} and that active PKD up-regulates steroidogenic enzyme expression and steroid secretion in human adrenal cells. These findings describe a new intracellular signaling pathway by which Ang II also modulates adrenal cell physiology. Increasing our knowledge of adrenal cell regulatory physiology has important clinical relevance, because primary aldosteronism is the most frequent cause of secondary hypertension with a prevalence close to 15% in hypertensive patients refractory to treatment, yet it could be the most curable cause of hypertension (50, 51, 52). In addition, primary aldosteronism is associated with cardiac fibrosis disproportionate with the elevation of blood pressure (53, 54). PKD may be an important intracellular modulator of aldosterone secretion in adrenal cells, and we speculate that alterations in the levels or activation status of PKD could mediate pathological conditions associated with abnormal adrenal aldosterone secretion.


   Acknowledgments
 
We thank Dr. W. E. Rainey (Medical College of Georgia, Augusta, GA) for generously providing reporter plasmids and H295R cells and A. Toker (Harvard Medical School, Boston, MA) for PKD plasmid constructs. We thank Maria W. Plonczynski for critical reading of the manuscript and Tanganika R. Washington for her excellent technical assistance.


   Footnotes
 
This work was supported by Medical Research funds from the Department of Veterans Affairs and National Institutes of Health Grants HL27255 and HL75321.

Disclosure Statement: The authors have nothing to disclose.

First Published Online September 14, 2006

Abbreviations: Ang II, Angiotensin II; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; RAS, renin-angiotensin system; shRNA, short hairpin RNA.

Received June 14, 2006.

Accepted for publication September 5, 2006.


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 Discussion
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