ELSEVIER
MCE
Molecular and Cellular Endocrinology
AngII induces transient phospholipase D activity in the H295R glomerulosa cell model
Xiangjian Zhengª, Wendy B. Bollag a,b,c,*
a Institute of Molecular Medicine and Genetics/CB-2803, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912-2630, USA
Department of Medicine, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912-2630, USA
” Department of Cell Biology and Anatomy, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912-2630, USA
Received 17 April 2003; accepted 15 May 2003
Abstract
In this report we demonstrate that in human adrenocortical carcinoma NCI H295R cells, a model for adrenal glomerulosa cells, PLD was activated both by AngII and protein kinase C (PKC)-activating phorbol 12-myristate 13-acetate (PMA). However, while PMA triggered sustained PLD activation, AngII induced transient PLD activation, in contrast to results in bovine glomerulosa cells in primary culture. Despite the transient effect of AngII on PLD activity, PLD-derived lipid signals were required for maximal AngII-elicited aldosterone secretion. AngII-induced PLD activation was inhibited by PKC inhibitors, but not by tyrosine kinase or calcium/calmodulin-dependent kinase inhibitors or a calmodulin antagonist. Both AngII- and PMA-stimulated PLD activity was enhanced by phosphoinositide 3-kinase (PI3K) inhibitors. Akt, a downstream protein kinase activated by the products of PI3K, was constitutively active in H295R cells, and this activity was blocked by PI3K inhibitors. These results suggested that in H295R adrenocortical carcinoma cells, AngII-induced PLD activation was promoted by PKC and inhibited by the constitutively active PI3K pathway.
C) 2003 Elsevier Ireland Ltd. All rights reserved.
Keywords: NCI H295R cells; Aldosterone; Phosphoinositide 3-kinase; Protein kinase C; Phosphatidic acid; Adrenocortical carcinoma
1. Introduction
Aldosterone is a steroid hormone that regulates sodium homeostasis in the body. The primary regulator of its synthesis and secretion is angiotensin II (AngII), which functions through binding to two AngII recep- tors. The type I receptor (AT1) is thought to mediate most, if not all, of the cardiovascular effects of AngII
including aldosterone secretion and smooth muscle contraction (reviewed in Unger, 2002). The function of the type II receptor (AT2) is less clear, but AT2 may mediate brain as well as feedback inhibitory effects of AngII (von Bohlen und Halbach et al., 2001 and reviewed in Unger, 2002). Thus, AT2 receptor homo- zygous null mutant mice have an enhanced vasopressor response to AngII relative to wild-type mice (Hein et al., 1995; Siragy et al., 1999 and reviewed in Stoll and Unger, 2001), as well as greater numbers of cells in certain regions of the brain (von Bohlen und Halbach et al., 2001). Of great interest have been the signal transduction mechanisms by which AngII functions in its various target cells.
In adrenal glomerulosa cells, binding of AngII to the AT1 receptor results in the activation of several signaling pathways. Thus, AngII activates phosphoinositide-spe- cific phospholipase C, which hydrolyzes membrane phosphatidylinositol 4,5-bisphosphate (PIP2) to gener- ate two second messengers-inositol 1,4,5-trispho-
Abbreviations: AngII, angiotensin II; BSA, bovine serum albumin; CaMK, calcium/calmodulin-dependent protein kinase; DAG, diacylglycerol; KRB+, bicarbonate-buffered Kreb’s Ringer solution containing 2.5 mM sodium acetate; PA, phosphatidic acid; PEt, phosphatidylethanol; PI3K, phosphoinositide 3-kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PLD, phospholipase D; PMA, phorbol 12-myristate 13-acetate; RT-PCR, reverse transcription polymerase chain reaction.
* Corresponding author. Tel .: +1-706-721-0698; fax: +1-706-721- 7915.
E-mail address: wbollag@mail.mcg.edu (W.B. Bollag).
sphate and diacylglycerol (DAG) (reviewed in Barrett et al., 1989; Ganguly and Davis, 1994; Rasmussen et al., 1995). Inositol 1,4,5-trisphosphate releases calcium from an intracellular storage site to increase cytosolic calcium levels and activate calcium/calmodulin-dependent pro- tein kinases (CaMK), thereby initiating the aldosterone secretory response (Barrett et al., 1989; Rasmussen et al., 1995). DAG, on the other hand, activates protein kinase C (PKC) isoenzymes, and this kinase family has been suggested to mediate sustained aldosterone pro- duction (reviewed in Barrett et al., 1989; Rasmussen et al., 1995). Through an incompletely determined me- chanism possibly involving both capacitative influx pathways (Burnay et al., 1994) and voltage-dependent channels (Barrett et al., 1995; Chen et al., 1999; Rossier et al., 1996), AngII also increases calcium influx, and this sustained influx is required for maintained aldoster- one secretion (reviewed in Barrett et al., 1989; Ganguly and Davis, 1994). In bovine glomerulosa cells in primary culture, AngII also activates phospholipase D (PLD) (Bollag et al., 1990), an enzyme that hydrolyzes primar- ily phosphatidylcholine to produce phosphatidic acid (PA) (reviewed in Exton, 2000; Frohman et al., 1999). PA can, in turn, be dephosphorylated by lipid phos- phate phosphatases to yield DAG (reviewed in Wagg- oner et al., 1999). AngII-induced PLD activation in bovine glomerulosa cells in primary culture occurs through the AT1 receptor, is sustained, and exhibits a dose-response similar to that for hormone-elicited steroidogenesis (Jung et al., 1998). Although the exact role of PLD in steroidogenesis is not known, inhibition of PLD-generated lipid signaling in these cells inhibits aldosterone secretion (Bollag et al., 2002), suggesting an involvement of PLD in this process. The mechanism by which AngII activates PLD is also unclear, although recent results in bovine glomerulosa cells in primary culture suggest a role for PKC in this process (Bollag et al., 2002).
The human adrenocortical carcinoma cell line NCI H295R has been developed and characterized by Dr Rainey and colleagues (Bird et al., 1993 and reviewed in Rainey and Mrotek, 1999). These cells produce and secrete multiple steroid hormones and precursors (Rainey et al., 1993), including aldosterone (Bird et al., 1993). Indeed, aldosterone secretion can be stimulated by a variety of agonists, such as AngII, elevated extracellular potassium levels, parathyroid hormone and parathyroid hormone-related peptide and dibu- tyryl-cyclic AMP (which mimics adrenocorticotropic hormone-elicited signal transduction) (Bird et al., 1993, 1995; Hanley et al., 1993). Moreover, the H295R cells respond to AngII in a similar manner as freshly- isolated or glomerulosa cells in primary culture. Thus, AngII dose-dependently increases cytosolic calcium concentration and induces phosphoinositide turnover through the AT1 receptor (Bird et al., 1993). On the
other hand, in contrast to freshly-isolated and glomer- ulosa cells in primary culture, initial aldosterone secre- tory rates in response to AngII are low, and a plateau level of aldosterone secretion is not achieved until after 24 h of AngII exposure (Bird et al., 1993). Nevertheless, because of the many similarities between this cell line and glomerulosa cell systems, H295R cells have been utilized as models for glomerulosa cell biology and in studies of the regulation of aldosterone biosynthesis and secretion.
In this report, we investigated the characteristics and role of AngII-induced PLD activation in aldosterone secretion in the H295R cells. Unexpectedly, AngII- elicited PLD activation in these cells was transient, rather than sustained as observed previously in bovine glomerulosa cells in primary culture (Jung et al., 1998). Nevertheless, in these cells, as in cells in primary culture (Bollag et al., 2002), PLD signaling was necessary for a maximal secretory response, since inhibiting PLD- mediated lipid signaling decreased aldosterone secretion. In subsequent experiments we defined both stimulatory and inhibitory mechanisms by which AngII regulates PLD activity.
2. Materials and methods
2.1. Materials
NCI H295R cells were generously provided by Dr William Rainey (University of Texas Southwestern Medical Center, Dallas, TX). UltroSer G was obtained from Biosepra (France) under a permit from the United States Department of Agriculture. DMEM/Ham’s F12, antibiotic/antimycotic and TriZOL were purchased from Gibco BRL (Grand Island, NY). ITS+ [final concentration of 6.25 µg/ml insulin, 6.25 µg/ml trans- ferrin, 6.25 ng/ml selenious acid, 5.35 µg/ml linoleic acid +0.125% (w/v) bovine serum albumin (BSA)] was obtained from Collaborative Biomedical Products (Bed- ford, MA). AngII and BSA were purchased from Sigma Chemical Company (St. Louis, MO) and [3H]oleate from NEN Life Science Products (Boston, MA). Wort- mannin, LY 294002, Ro 31-8220, bisindolylmaleimide I (Bis I), Gö 6976, KN93, calmidazolium, H89 and myristoylated protein kinase A inhibitor (14-22 amide) were obtained from Calbiochem (San Diego, CA). PA and phosphatidylethanol were purchased from Avanti Polar Lipids (Alabaster, AL). Anti-Akt and anti-phos- pho-473serine-Akt were from Cell Signaling Technology (Beverly, MA). Other reagents were obtained from standard suppliers and were of the highest grade available.
2.2. Cell culture
NCI H295R cells were cultured as described in Bird et al. (1993). Briefly, cells were grown in DMEM/Ham’s F12 (1:1 vol:vol) containing 1% ITS+, 2% UltroSer G, 100 U/ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ ml fungizone, to approximately 70-75% confluence. The cells were then down-regulated for 20-24 h in serum-free DMEM/Ham’s F12 (containing 0.01% BSA and antibiotics) prior to experimentation. Passage numbers 9-13 were used for all experiments. Glomer- ulosa cells isolated from near-term fetal calves as described in Jung et al. (1998), were placed in primary culture overnight in a serum-containing medium also as in Jung et al. (1998). The cells were then down-regulated in serum-free medium for 20-24 h prior to experimenta- tion, again as described in Jung et al. (1998).
2.3. Measurement of aldosterone secretion
H295R cells were incubated with the appropriate agents in serum-free DMEM/Ham’s F12 medium (con- taining BSA and antibiotics) for 5 h. Supernatants were collected and stored at -20 ℃ until assay. Aldosterone release into the medium was assessed using a radio- immunoassay kit from Diagnostic Products Corpora- tion (Los Angeles, CA) as in Betancourt-Calle et al. (1999).
2.4. PLD activity assay
PLD activity was monitored as the production of radiolabeled phosphatidylethanol in [3H]oleate-prela- beled cells, as described in Bollag (1998). Briefly, cells incubated for 20-24 h in serum-free DMEM/Ham’s F12 containing 5 uCi/ml [3H]oleate were equilibrated in KRB+ and stimulated with the appropriate agents for the indicated times in the presence of ethanol. Cells were solubilized in 0.2% SDS containing 5 mM EDTA and lipids extracted into chloroform/methanol. Phospholi- pids were separated by thin-layer chromatography on silica gel 60, visualized with autofluorography using En3Hance, identified by co-migration with authentic standards and cut out and quantified by liquid scintilla- tion spectrometry. For experiments with the P13K inhibiors, a 5- and 15-minute preincubation was per- formed prior to AngII or PMA stimulation for wort- mannin and LY 294002, respectively.
2.5. Western analysis of Akt activation
H295R cells down-regulated in serum-free medium were exposed to 100 nM AngII or plain medium in the presence and absence of wortmannin or LY 294002 (with pre-incubation as above) at the appropriate concentrations for the indicated times. Cells were
scraped and solubilized in lysis buffer (1% SDS in 50 mM Tris-HC1, pH 6.8). Equal amounts of protein (determined with the Biorad assay using BSA as the standard) were separated on a 10% polyacrylamide gel and transferred to Immobilon P. Blots were probed with anti-Akt or anti-phospho-Akt primary antibodies, fol- lowed by a horseradish peroxidase-coupled anti-rabbit secondary antibody. Immunoreactive proteins were visualized with enhanced chemiluminescence (Pierce, Rockford, IL) using Kodak film. For experiments in bovine glomerulosa cells in primary culture, 10 nM AngII was added to down-regulated cells for the indicated times prior to analysis of the Akt phosphor- ylation status, as described above.
2.6. Reverse-transcription polymerase chain reaction (PCR) analysis of PLD isoform expression
RNA was isolated using TriZOL, as described by the manufacturer, from H295R cells or bovine glomerulosa cells in primary culture and cDNA generated with reverse transcriptase. PCR was then performed using the following primers: human PLD-1: forward 5’- gccattgccttcgtcctgct-3’ and reverse 5’-tgacccgcttca- cactgccca-3’ (700 bp), bovine PLD-1: forward 5’- ccgaattcccattcccacca-3’ and reverse 5’-cctccccaccaccgag- cat-3’ (550 bp) and PLD-2: forward 5’-ggtccaa- gaggtggctggt-3’ and reverse 5’-ccgccttcctcttgagcat-3’ (477 bp). The conditions used were: denaturing at 94 ℃ for 45 s, annealing at 51 ℃ for 60 s and elongation at 68 ℃ for 60 s for a total of 30 cycles. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was also ampli- fied to demonstrate the integrity of the cDNA.
2.7. Statistical analysis
Results were analyzed for statistical differences using analysis of variance (ANOVA) followed by a Student- Neumann-Keuls post-hoc test, as performed by the INSTAT program (GraphPad Software, San Diego, CA). A P value of 0.05 or less was taken as significant.
3. Results
3.1. Inhibition of PLD-generated lipid signals inhibited AngII-induced aldosterone secretion from H295R cells
Although there are no selective inhibitors of PLD available (Rizzo and Romero, 2002), this enzyme has the unique characteristic of utilizing primary alcohols for a transphosphatidylation reaction to generate phos- phatidylalcohols in place of PA (reviewed in Klein et al., 1995; Liscovitch et al., 2000). Since these phosphatidy- lalcohols are generally poorly metabolized (reviewed in Liscovitch et al., 2000), incubation with a small amount
of a primary alcohol can inhibit PLD-mediated PA and DAG generation. We previously utilized this strategy to inhibit AngII-induced increases in PA and DAG and demonstrate a requirement for PLD-derived lipid sig- nals for maximal steroidogenesis in primary cultures of bovine adrenal glomerulosa cells (Bollag et al., 2002). To determine whether PLD-derived lipid signals were involved in AngII-induced aldosterone secretion in the H295R cells, cells were stimulated with AngII or phorbol 12-myristate 13-acetate (PMA) in the presence or absence of 1-butanol and aldosterone released into the medium measured by radioimmunoassay (Fig. 1). As previously shown (e.g. Bird et al., 1993), AngII elicited aldosterone secretion from the H295R cells. However, in contrast to previous reports (Bird et al., 1995; Clark et al., 1995), PMA was also found to significantly enhance steroidogenesis in these cells, as has been observed in the bovine glomerulosa cells in primary culture (Betancourt- Calle et al., 1999). 1-Butanol (0.3%) inhibited AngII- but not PMA-induced aldosterone secretion. The same concentration of the related organic alcohol, tert- butanol, which cannot be utilized by PLD in the transphosphatidylation reaction, had no effect on AngII-stimulated aldosterone secretion.
3.2. AngII induced a transient PLD activation
Experiments then focused on the time course of the PLD activation in response to AngII and PMA in the H295R cells (Fig. 2). Unexpectedly and in contrast to our results with bovine adrenal glomerulosa cells in
250
*
Aldosterone secretion (Percentage over control)
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Con AngII AngII 1-But AngII t-But PMA PMA
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PLD activity (Percentage over control)
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primary culture (Jung et al., 1998), the activation in response to AngII was transient, with an initial large increase in activity that declined to a plateau level not significantly different from the control. This pattern was observed with both 10 (Fig. 2B) and 100 nM AngII (Fig. 2A), concentrations which produce sustained PLD activation in the glomerulosa cells in primary culture (Jung et al., 1998). On the other hand, 10 nM PMA induced a sustained PLD activation response (Fig. 2A).
3.3. H295R expressed both PLD-1 and PLD-2
The disparity in the time course of AngII-induced PLD activation in H295R versus glomerulosa cells in primary cultures could be the result of a difference in the
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isoform(s) expressed. To determine whether the H295R cells possessed both cloned PLD isoforms, reverse transcriptase-polymerase chain reaction (RT-PCR) to detect PLD-1 and -2 was performed using the primers detailed in Section 2. As shown in Fig. 3A, both PLD-1 (lane 2) and PLD-2 (lane 3) were expressed in H295R cells. This result was similar to the expression pattern in bovine glomerulosa cells in primary culture, which also expressed both isoforms by RT-PCR (Fig. 3B).
3.4. Mechanism of AngII-induced PLD activation in H295R cells
The mechanism of the initial PLD activation in response to AngII in H295R cells was then investigated. Many previous studies have demonstrated that PKC inhibitors can block agonist-induced PLD activation in many cell types (reviewed in Exton, 2000; Frohman et al., 1999). The PKC inhibitors shown in Fig. 4A (Ro 31- 8220, Bis I and Gö 6976) were able to reduce AngII- stimulated PLD activity, as measured by the formation of radiolabeled phosphatidylethanol in [3H]oleate-pre- labeled H295R cells (Fig. 4A). In addition, a myristoy- lated PKC inhibitor inhibited AngII-induced PLD activation slightly, but not significantly (79±6% of the 100% AngII response; data not shown). Bis I appeared to exert the greatest inhibitory effect, inducing almost a complete block of AngII-elicited PLD activation, so this inhibitor was also tested for its ability to inhibit PMA- induced PLD activation. As shown in Fig. 4B, Bis I also completely inhibited PMA-stimulated PLD activity.
We also investigated the potential involvement of cAMP-dependent protein kinase (protein kinase A (PKA)) on AngII-induced PLD activation. The reported
150
PLD activity (Percentage of AngII effect)
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PLD activity (Percentage of PMA effect)
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Bis
PKA inhibitor H89 (20 µM) had a small but significant inhibitory effect on AngII-elicited PLD activation (AngII: 100±4 and AngII+H89: 80±3% of the An- gII-stimulated value; n = 3, P < 0.001 vs. AngII alone). However, the cell-permeant and selective PKA inhibitor myristoylated PKA inhibitor (14-22 amide; 10 µM) had no significant effect on AngII-stimulated PLD activity (data not shown). Together these results suggest a possible non-specific effect of H89 and argue for the lack of involvement of PKA in PLD activation in response to AngII. Similarly, modulators of calcium metabolism, including the calcium channel antagonist nitrendipine, the calmodulin inhibitor calmidazolium and the CaM kinase inhibitor KN93 had no effect on PLD activation either alone or upon stimulation with AngII (Fig. 5). Consistent with the lack of effect of inhibitors of calcium metabolism on PLD activity, the
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PLD activity (Percentage of AngII effect)
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CMZ
KN93
calcium ionophore A23187 (1 µM) also did not induce PLD activation (data not shown). Several tyrosine kinase inhibitors [genistein (a general tyrosine kinase inhibitor) at 50 µM, AG490 (a Janus kinase 2 inhibitor) at 100 uM and PP2 (a src kinase inhibitor) at 10 uM] were also tested for their ability to affect PLD activation in response to AngII, and these agents also did not affect hormone-stimulated PLD activity (data not shown).
3.5. The PI3K pathway inhibited AngII- and PMA- induced PLD activation
The effect of the PI3K pathway inhibitors on agent- elicited PLD activation was also investigated. Interest- ingly, both inhibitors tested, wortmannin and LY
PLD activity (Percentage of agonist effect)
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AngII
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294002, had no effect on PLD activity alone but enhanced the PLD activation response to AngII and PMA (Fig. 6). Thus, wortmannin and LY 294002 induced a 65 and 32% increase in the AngII-stimulated PLD activity, respectively. On the other hand, rapamy- cin (100 ng/ml), an inhibitor of p70S6K, another target of the phosphoinositide-dependent kinase-1 activated through PI3K (reviewed in Chan et al., 1999), did not alter AngII-induced PLD activation (data not shown).
To demonstrate that these compounds in fact inhib- ited the PI3K pathway, their effect on phospho (active)- Akt was determined by western analysis (Fig. 7A). In H295R cells the PI3K pathway appeared to be consti- tutively active, as high levels of phospho-Akt were observed under basal conditions. This result is in contrast to bovine adrenal glomerulosa cells in primary culture, in which basal 473serine-Akt phosphorylation (i.e. activity) was low and was increased in response to AngII (Fig. 7B). However, as expected, wortmannin and LY 294002 at the concentrations used to enhance PLD activity reduced the levels of phospho-Akt measured in the H295R cells.
p-Akt(S473)
Akt —
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4. Discussion
Our finding that AngII induced a transient PLD activation response in H295R cells (Fig. 2) was in contrast to our previous observations in bovine adrenal glomerulosa cells in primary culture, in which PLD was activated in a monotonic and sustained manner by 10 and 100 nM AngII (Jung et al., 1998). The reason for this disparity is unclear, although the transient AngII- elicited PLD activation was not converted to a mono- phasic sustained response by inhibitors of PI3K, PKA or tyrosine kinases (data not shown). This result suggests that none of these kinases functions as a negative feedback mechanism to reduce sustained PLD activation by AngII. It is possible that the divergence between the H295R and bovine glomerulosa cells in primary culture results instead from differences in the PLD isoforms expressed, although by RT-PCR analysis both cell types expressed both known PLD isoenzymes (Fig. 3). However, whether this mRNA is translated into protein is at present unclear.
We have previously shown in glomerulosa cells in primary culture that incubation with 0.3% 1-butanol decreased the AngII-elicited increase in PA and DAG formation and aldosterone secretion (Bollag et al., 2002). Despite the transience of AngII-elicited PLD activation, 1-butanol was able to inhibit AngII-induced aldosterone secretion over 5 h in the H295R cells as well (Fig. 1). Nevertheless, this inhibition was not complete nor is it expected to be, since 1-butanol functions to divert some, but not all, PLD-mediated production of PA to phosphatidylbutanol. That the effect was specific to its capacity to inhibit PLD-generated lipid signals, however, was suggested by the inability of tert-butanol, a chemically related organic alcohol that is not utilized by PLD, to significantly inhibit AngII-elicited steroido- genesis. The lack of effect of 1-butanol on PMA-induced secretion suggested also that the alcohol was not simply exerting a non-specific cytotoxicity. This result also suggests that the signaling pathway impacted upon by 1-butanol can be directly activated by (or be substituted for by) the phorbol ester.
In this report we have shown for the first time in theH295R glomerulosa cell model that the phorbol ester PMA can significantly increase aldosterone secretion (Fig. 1). This demonstration is in contrast to previous results from Dr Rainey and colleagues, who indicated no effect of PMA on aldosterone production (Bird et al., 1995; Clark et al., 1995). Note that these investigators showed an approximate 1.4-fold increase in steroidogen- esis by PMA at 48 h, but this change was not significant in comparison with AngII by ANOVA analysis of a representative experiment performed in quadruplicate (Bird et al., 1995). We also observed a much larger effect of AngII on aldosterone secretion relative to PMA (Fig. 1), but such a result is consistent with the idea that
sustained aldosterone production requires both a PKC and a calcium influx signal (reviewed in Rasmussen et al., 1995). Thus, provision of only the PKC signal by PMA induces only a small aldosterone secretory re- sponse, as in Bollag et al. (1990). Alternatively, the capacity of PMA to activate additional signaling enzymes, some of which could have inhibitory effects on secretion, and/or trigger PKC down-regulation (reviewed in Nishizuka, 1995) may underlie the lower secretory response to PMA in comparison with AngII.
As observed in bovine adrenal glomerulosa cells in primary culture (Bollag et al., 2002), PKC appeared to be involved in AngII-stimulated PLD activity in H295R cells (Fig. 4A). Thus, AngII-induced PLD activation was inhibited by all PKC inhibitors tested, including Gö 6976, which is reported to be selective for classical PKC isoforms (Martiny-Baron et al., 1993). Such a result is consistent with a role for a conventional PKC isoform such as PKC-x (Du et al., 2000; Hammond et al., 1997) in this process. Indeed, several groups have shown that PLD-1 is activated by conventional PKC isoenzymes, although whether this requires a phosphorylation event or simply direct binding of the PKC to PLD (Colley et al., 1997; Conricode et al., 1992; Hammond et al., 1997; Houle and Bourgoin, 1999; Lopez et al., 1995; Min et al., 1998; Singer et al., 1996; Zhang et al., 1999) is as yet unclear. Recent evidence also suggests a potential role for PKC in activation of PLD-2 (Han et al., 2002; Siddiqi et al., 2000) as well. Thus, the ability of PKC- activating PMA alone to increase and PKC inhibitors to decrease hormone-stimulated PLD activity does not distinguish between the two PLD isoforms. Also as seen with bovine glomerulosa cells in primary culture (Bollag et al., 2002), modulators of calcium metabolism had no effect on AngII-induced PLD activation, nor did inhibitors of tyrosine kinases affect the response. The PKA inhibitor H89 exerted a small, but significant inhibition, but this inhibition was not reproduced by another selective PKA inhibitor, suggesting little or no role for PKA in PLD activation in response to AngII.
Of particular interest was our finding that inhibition of the PI3K pathway resulted in an enhancement of AngII-stimulated PLD activity (Fig. 6). This result is unique in its contrast to most published accounts in which PI3K inhibitors inhibit PLD activation in re- sponse to multiple agonists (e.g. Gillooly et al., 1999; Mamoon et al., 2001; Powner et al., 2002; Lucas et al., 2002). However, a recent paper by Dr Romero and colleagues (Andresen et al., 2001) also showed a stimulatory effect of LY 294002 (but not wortmannin) on AngII-induced PLD activity in normal vascular smooth muscle cells and of wortmannin (but not LY 294002) in smooth muscle cells from spontaneously hypertensive rats, although the authors did not address their findings. Our observation that the p70S6K inhi- bitor rapamycin did not enhance AngII-elicited PLD
activation suggests that the effects of the PI3K inhibi- tors are not mediated by this enzyme downstream of the PI3K target PDK-1, but by another PI3K-activated pathway, such as Akt (reviewed in Toker, 2000). Alternatively, PI3K is known to phosphorylate various phosphoinositide-containing phospholipids, including PIP2 (reviewed in Chan et al., 1999; Vanhaesebroeck et al., 2001). Since PIP2 is required for PLD activity (reviewed in Exton, 2000; Frohman et al., 1999), it is possible that PI3K may inhibit PLD by decreasing the levels of PIP2 available for interaction with the enzyme. Clearly, this hypothesis requires further testing.
Also interesting was the constitutive activation of the PI3K pathway observed in H295R cells (Fig. 7A), but not bovine adrenal glomerulosa cells in primary culture (Fig. 7B). This finding may be related to the derivation of the H295R cells from human carcinoma tissue, and in fact, constitutive and/or hyper-activation of Akt has been observed in association with several human malignancies (reviewed in Vivanco and Sawyers, 2002). Excessive activity of the synthetic enzyme PI3K and/or reduced function of the degradative enzyme, PTEN, give rise to elevated levels of phosphatidylinositol 3,4,5- trisphosphate and persistent Akt activation (Nicholson and Anderson, 2002). Indeed, many tumors, including glioblastoma, melanoma, and renal and hepatic-cell carcinoma, as well as ovarian, lung, lymphoid, endo- metrial, breast and thyroid cancers, have been found to exhibit mutations in or silencing of PTEN and/or amplification or excessive activation of PI3K (reviewed in Vivanco and Sawyers, 2002). Since Akt is thought to mediate proliferation and/or survival signals (reviewed in Cantley and Neel, 1999; Vanhaesebroeck et al., 2001), maintained Akt activity as a consequence of these alterations likely contributes to the malignant pheno- type of cancer cells, such as the H295R human adrenocortical carcinoma cells. On the other hand, AngII was shown to activate the PI3K pathway in primary cultures of glomerulosa cells, as monitored by the phosphorylation status of Akt (Fig. 7B), in agree- ment with a previous report in which indirect evidence for AngII-induced PI3K activation was provided by the ability of PI3K inhibitors to inhibit AngII signal transduction events (Smith et al., 1999). This activation of the PI3K pathway in response to AngII suggests a possible role for this system in hormone-elicited changes in glomerulosa cell function.
In this report we demonstrated that AngII-induced PLD activation and lipid signal generation was required for maximal aldosterone secretion in H295R cells. Interestingly, H295R cells demonstrated a transient PLD activation in response to AngII but a sustained, monotonic activation induced by PMA. PKC appeared to mediate, at least in part, AngII-stimulated PLD activity. In contrast, changes in calcium metabolism, PKA and tyrosine kinases were not involved in AngII-
induced PLD activation. On the other hand, the PI3K pathway seemed to exert an inhibitory effect on PLD activity in these cells. Thus, the constitutive Akt activa- tion observed in H295R cells inhibited AngII-induced PLD activation. This constitutive Akt activity also differed from the low basal levels seen in bovine glomerulosa cells in primary culture (Fig. 7B). Still unknown is the reason for the biphasic PLD activation in response to AngII in these cells, as well as the identity of the PLD isoform(s) involved. Clearly, additional studies to address these issues are necessary.
Acknowledgements
We thank Dr Bill Rainey (University of Texas Southwestern Medical Center, Dallas, TX) for his generous gift of H295R cells and Mrs Patricia Kent for her assistance with their culture. This work was supported by American Heart Association/Southeastern affiliate award 0051579B and American Heart Asso- ciation National Grant-in-Aid award 0350166N (to WBB), and was completed in partial fulfillment of the requirements for a Doctor of Philosophy (XZ).
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