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Endocrinology Vol. 139, No. 4 1801-1809
Copyright © 1998 by The Endocrine Society

ARTICLES

Angiotensin II Activates Mitogen-Activated Protein Kinase Via Protein Kinase C and Ras/Raf-1 Kinase in Bovine Adrenal Glomerulosa Cells

Ying Tian, Roger D. Smith1, Tamas Balla and Kevin J. Catt

Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Dr. K. J. Catt, ERRB, NICHD, National Institutes of Health, Building 49, Room 6A-36, 9000, Rockville Pike, Bethesda, Maryland 20892. E-mail: catt{at}helix.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Angiotensin II (Ang II) stimulates growth and mitogenesis in bovine adrenal glomerulosa cells, but little is known about the signaling pathways that mediate these responses. An analysis of the growth-promoting pathways in cultured bovine adrenal glomerulosa cells revealed that Ang II, acting via the AT1 receptor, caused rapid but transient activation of mitogen-activated protein kinase (MAPK), with an ED50 of 10–50 pM. Although neither Ca2+ influx nor Ca2+ release from intracellular stores was sufficient to activate MAPK, Ca2+ appeared to play a permissive role in this response. A major component of Ang II-induced MAPK activation was insensitive to pertussis toxin (PTX), although a minor PTX-sensitive component could not be excluded. Ang II also induced the rapid activation of ras and raf-1 kinase with time-courses that correlated with that of MAPK. Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate was sufficient to activate both MAPK and raf-1 kinase. However, whereas PKC depletion had no effect on Ang II-induced raf-1 kinase activation, it attenuated Ang II-induced MAPK activation. Ang II also stimulated a mobility shift of raf-1, reflecting hyperphosphorylation of the kinase. However, unlike its activation, raf-1 hyperphosphorylation was dependent on PKC and its time-course correlated not with activation, but rather with deactivation of the kinase. Taken together, these findings indicate that Ang II stimulates multiple pathways to MAPK activation via PKC and ras/raf-1 kinase in bovine adrenal glomerulosa cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE OCTAPEPTIDE hormone, angiotensin II (Ang II), the active component of the renin-angiotensin system, plays a major role in the physiology of the cardiovascular system by acting on targets such as vascular smooth muscle cells (to stimulate vasoconstriction), the hypothalamus (to stimulate vasopressin secretion), and the adrenal cortex (to stimulate aldosterone secretion) (reviewed in Ref.1). In addition to these effects, Ang II also acts as a cellular growth factor by promoting vascular smooth muscle proliferation and cardiac hypertrophy and stimulating neointimal proliferation following arterial injury (2, 3, 4). The peptide also plays a major role in the adrenal glomerulosa cell hypertrophy and hyperplasia that occur during dietary sodium restriction (5). In previous studies from this laboratory, Ang II was found to act as a mitogen in primary cultures of bovine adrenal glomerulosa (BAG) cells (6). Acting via the G protein-coupled AT1 angiotensin receptor, the peptide increased thymidine incorporation into DNA, increased the proportion of cells in S phase, and stimulated the proliferation of BAG cells.

Ang II has been shown to influence several intracellular signaling pathways in its numerous target cells. The peptide can inhibit adenylate cyclase, activate guanylate cyclase, release prostaglandins and leukotrienes, regulate calcium channels, and induce the expression of transcription factors including c-fos, c-jun, junB, and Krox 24 (7, 8). However, the major signaling event activated by the AT1 receptor appears to be phospholipase C-dependent hydrolysis of phosphatidylinositol 4,5 bisphosphate (PtdInsP2). The cleavage of PtdInsP2 results in the generation of inositol 1,4,5-trisphosphate (which mobilizes Ca2+ from intracellular stores) and diacylglycerol [which activates protein kinase C (PKC)] (7). Ang II also stimulates the phosphorylation on tyrosine residues of several proteins in various target cells (9, 10, 11). However, despite detailed studies of these pathways in BAG and other cells, the signaling events that mediate Ang II-stimulated mitogenesis are not well defined.

Mitogen-activated protein kinases (MAPKs, p42MAPK and p44MAPK) are ubiquitous signaling intermediates that, when activated by phosphorylation, play a critical role in cellular proliferation (reviewed in Refs. 12–14). The best understood pathway to MAPK activation is that initiated by receptor tyrosine kinases, exemplified by the EGF receptor (EGF-R). Following activation, the EGF-R is autophosphorylated on tyrosine residues to create docking sites for the SH2 domains of adapter molecules such as shc and Grb2. After binding to the EGF-R, shc is tyrosine phosphorylated by the receptor, thereby creating a binding site for the SH2 domain of Grb2. The latter (which therefore binds to the EGF-R via both shc-dependent, and -independent mechanisms) recruits to the plasma membrane the guanine nucleotide exchange factor, m-sos, which promotes the exchange of GDP for GTP on membrane-anchored ras. GTP-bound ras recruits to the plasma membrane raf-1 kinase, which is then activated by a poorly understood mechanism. Raf-1, in turn, phosphorylates and activates MAPK kinase (Mek), which subsequently phosphorylates and activates MAPK. Following its activation, MAPK translocates to the nucleus where it phosphorylates various targets including transcription factors critical to the control of cellular proliferation.

In addition to this pathway of MAPK activation via receptor tyrosine kinases, MAPK can also be activated via certain G protein-coupled receptors (which lack intrinsic kinase activity). The pathway from Gi-coupled receptors appears to be mediated by the release of Gß{gamma} complexes from pertussis toxin-sensitive G proteins (15, 16). In contrast, the mechanisms leading to MAPK activation via Gq-coupled receptors are poorly defined. Because the mitogenic action of Ang II is mediated via the Gq-coupled AT1 receptor, we have investigated the effects of the peptide on MAPK, and the upstream signaling pathways leading to its activation, in BAG cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
DMEM, Medium 199, donor horse serum (DHS), FBS, and antibiotic/antimycotic solutions were from Biofluids (Rockville, MD). Angiotensin II was from Peninsula Laboratories (Belmont, CA). DuP753 and PD 123177 were generous gifts from Dr. P. C. Wong (Dupont, Wilmington, DE). Phorbol 12-myristate 13-acetate (PMA) was from Sigma Chemical Co. (St. Louis, MO); thapsigargin, BayK and nifedipine were from Calbiochem (San Diego, CA). PTX was from List Biologicals (Campbell, CA).

Bovine adrenal glomerulosa cell preparation
Primary cultures of glomerulosa cells were prepared from bovine adrenal glands as previously described (6). Cells were plated at 1.5 x 106 cells per 60-mm plastic culture dish (Becton Dickinson, Lincoln, NJ) in DMEM containing 10% (vol/vol) DHS, 2% (vol/vol) FBS, 100 µg/ml streptomycin, 100 IU/ml penicillin, 5 µg/ml fungizone, 25 µg/ml gentamicin, 8 µg/ml trimethoprim, and 40 µg/ml sulfamethoxazole. The cells were cultured for 4 days in a humidified atmosphere of 5% CO2 in air at 37 C, after which time they formed confluent monolayers. Cells were rendered quiescent by the withdrawal of serum for 48 h before use. After stimulation for the times indicated in the individual figures, cells were washed with ice-cold PBS before lysing in the appropriate buffer.

Immunoblot analysis of MAPK and Raf-1 proteins
Following stimulation, cells were drained, scraped into 0.5 ml of Laemmli sample buffer (17), and sonicated for 5 sec. After boiling for 5 min, equal quantities of cell lysates were subjected to SDS-PAGE, and the separated proteins were transferred to PVDF membranes. For MAPK and raf-1 immunoblotting, the membranes were probed with mouse monoclonal anti-p42MAPK (1 µg/ml; UBI, Lake Placid, NY) or rabbit polyclonal anti-raf-1 antibody (0.5 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA) respectively. Immunoreactive bands were detected using horseradish peroxidase-conjugated secondary antibodies (Kirkegaard & Perry, Gaithersburg, MD) at a dilution of 1 in 3000 and enhanced chemiluminescence (LumiGlo, Kirkegaard & Perry).

MAPK activity assay
Following stimulation, cells were scraped into lysis buffer (137 mM NaCl, 25 mM NaF, 1 mM EGTA, 1.5 mM MgCl2, 1% (vol/vol) Triton X-100, 10% (vol/vol) glycerol, 20 mM Tris, pH 8.0) containing freshly added 100 µM Na3VO4, 200 µM phenylmethylsulfonylfluoride, aprotinin (1 µg/ml), leupeptin (1 µg/ml), and pepstatin (1 µg/ml) and frozen immediately on dry ice. After thawing, lysates were centifuged at 10,000 x g for 20 min at 4 C, and equal amounts of supernatant were immunoprecipitated with the anti-p42MAPK antibody. Immune complexes were collected on GammaBind Plus sepharose (Pharmacia, Uppsala, Sweden) and washed three times with lysis buffer lacking inhibitors. The sepharose beads were resuspended in 60 µl of reaction buffer (0.5 mM EGTA, 2.4 mM EDTA, 20 mM MgCl2, 50 µM NaF, 20 mM Tris, pH 7.5) in the presence of 2 µCi [{gamma}-32P]ATP, 50 µM unlabeled ATP, and 50 µg of the synthetic peptide, APRTPGGRR (synthesized by Dr. H. C. Chen), which contains amino acids 95–98 of bovine myelin basic protein, as substrate. After 30 min incubation at 30 C, the reaction was terminated by addition of ice-cold trichloroacetic acid (TCA) to a final concentration of 6% (wt/vol). Following removal of sepharose beads by centrifugation, the supernatant was applied to phosphocellulose filters (Whatman, Clifton, NJ), washed with 75 mM phosphoric acid, and radioactivity incorporated into the peptide substrate was measured by liquid scintillation spectrometry.

Raf-1 kinase assay
Following stimulation, cells were scraped into 0.5 ml of lysis buffer (150 mM NaCl, 2 mM EDTA, 1% (wt/vol) sodium deoxycholate, 1% (vol/vol) Nonidet P-40, 0.1% (wt/vol) SDS, 50 mM NaF, 10 mM sodium phosphate, pH 7.0) containing freshly added aprotinin (1 µg/ml), Na3VO4 (200 µM), ß-mercaptoethanol (14 mM) and phenylmethylsulfonylfluoride (1 mM). After centrifugation at 10,000 x g for 20 min, equal amounts of supernatant were immunoprecipitated with 2 µg of anti-raf-1 antibody. Immune complexes were collected on GammaBind Plus sepharose, washed three times in PAN buffer (10 mM piperazine-N,N'-bis[2-ethanesulfonic acid), pH 7.0, 20 µg/ml aprotinin, 100 mM NaCl), and resuspended in 100 µl total volume of PAN buffer containing 20 µCi [{gamma}-32P]ATP, 50 µM unlabeled ATP, and 2.5 µg of a catalytically inactive mutant GST-[K97A]-Mek-1 fusion protein (UBI, Lake Placid, NY) as substrate. After a 30-min incubation at 30 C, the reaction was terminated by the addition of ice-cold TCA to a final concentration of 6% (wt/vol). Precipitation of proteins was facilitated by the addition of 20 µg ovalbumin; the precipitates were collected by centrifugation and solubilized in Laemmli sample buffer (17). Samples were then boiled for 10 min, subjected to SDS-PAGE, and visualized in a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Ras activation assay
Serum-deprived cells were incubated for 16 h in phosphate-free DMEM containing 100 µCi/ml 32Pi. After washing, the cells were incubated in a buffered salt solution (123 mM NaCl, 2.4 mM KCl, 1.8 mM CaCl2, 10 mM glucose, 20 mM HEPES, pH 7.4) containing 0.1% (wt/vol) BSA for 10 min at 37 C. Following the addition Ang II (30 nM) for the required times, cells were washed with ice-cold PBS, drained, and scraped into lysis buffer (150 mM NaCl, 16 mM MgCl2, 1% (vol/vol) Nonidet P-40, 25 mM Tris, pH 7.5) containing 2 µg/ml Y13–259 rat monoclonal anti-v-Ha-ras antibody (Oncogene Science, Uniondale, NY). Insoluble material was removed by centrifugation and 20 µl of a 50% slurry of GammaBind Plus sepharose were added to the supernatants for 1 h at 4 C. The sepharose-bound immune complexes were collected, washed extensively with lysis buffer, and aspirated to dryness. Radiolabeled guanine nucleotides were extracted into 12 µl of elution buffer (0.2% (wt/vol) SDS, 2 mM DTT, 0.5 mM GTP, 0.5 mM GDP, 2 mM EDTA, pH 7.4) for 20 min at 68 C and resolved by ion-exchange TLC on PEI-cellulose plates containing a fluorescent indicator (EM Science, Gibbstown, NJ) with 0.75 M KH2PO4 (pH 3.4) as running buffer. Radioactivity migrating at the positions of GDP and GTP standards was visualized and quantitated by analysis with a PhosphorImager. The percentage of GTP was calculated using the formula: (GTP x 2/3)/(GDP + GTP x 2/3) x 100.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ang II activates MAPK in bovine adrenal glomerulosa cells
Because MAPK is activated by phosphorylation, we first examined whether Ang II is able to induce phosphorylation of the kinase in BAG cells. Using an anti-p42MAPK antibody, a single immunoreactive band of Mr 42,000 was detected in lysates prepared from untreated cells. Following stimulation with Ang II (100 nM), the MAPK band exhibited lower electrophoretic mobility (Fig. 1AGo), reflecting its phosphorylation on tyrosine and threonine residues (18, 19). Ang II-induced MAPK phosphorylation was evident after a lag time of 1 min, increased to a maximum between 5 and 10 min, and returned to the basal state by 60 min.



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  		Figure 1. Time-course of Ang II-stimulated MAPK activation. Serum-deprived glomerulosa cells were exposed to 100 nM Ang II for the indicated times. Cell lysates were prepared and subjected to (A) immunoblotting or (B) immunoprecipitation with an anti-p42MAPK antibody. In (A), the shift to a lower electrophoretic mobility correlates with phosphorylation of the kinase. A representative example is shown from three independent experiments. In (B), in vitro kinase activity was determined in MAPK immunoprecipitates incubated with [{gamma}-32P]ATP and a synthetic peptide substrate. Data represent the mean (± SEM) of radioactivity incorporated into the peptide from three independent experiments.

 
Consistent with these findings, Ang II also stimulated a transient, time-dependent increase in the enzymatic activity of p42MAPK immunoprecipitated from BAG cells. After a lag time of 1 min, kinase activity rapidly increased to reach a maximum (6.5-fold increase over control) at 5 min and returned to the basal level by 60 min (Fig. 1BGo). Ang II was a potent stimulus of MAPK activity in BAG cells, with a half-maximal effect at 3–10 pM and maximal activation by peptide concentrations as low as 300 pM (Fig. 2BGo). Similarly, maximal phosphorylation of MAPK was elicited by 500 pM Ang II (Fig. 2AGo).



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  		Figure 2. Concentration-dependent activation of MAPK by Ang II. Serum-deprived glomerulosa cells were exposed to the indicated concentrations of Ang II for 10 min, and MAPK phosphorylation (A) and activity (B) were determined as described in the legend to Fig. 1Go. In (A), a representative example is shown from three independent experiments. In (B), data represent the mean (± SEM) from three independent experiments.

 
We next determined the Ang II receptor subtype that mediates the MAPK phosphorylation in BAG cells. The AT1 angiotensin receptor is defined by its high sensitivity to DuP753, whereas the AT2 receptor is insensitive to DuP753 but has high affinity for PD123177 (20). Cells were therefore pretreated for 10 min with 10 µM DuP753 or 10 µM PD123177 and then exposed to 1 nM Ang II for an additional 10 min before immunoblotting for p42MAPK. Whereas Ang II-induced phosphorylation of MAPK was completely inhibited by the AT1-specific antagonist, DuP753, the AT2-specific antagonist, PD123177, had no effect (Fig. 3Go). Hence, Ang II induces phosphorylation (and, therefore, activation) of MAPK via the AT1 receptor subtype.



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  		Figure 3. MAPK phosphorylation is mediated via the AT1 receptor. Serum-deprived glomerulosa cells were pretreated with 10 µM DuP753 or 10 µM PD123177 for 10 min as indicated before exposure to 1 nM Ang II for a further 10 min. MAPK phosphorylation was then determined by immunoblotting. A representative example is shown from three independent experiments.

 
The role of Ca2+ in MAPK activation
In certain cell types, elevation of intracellular [Ca2+] can be sufficient to activate MAPK. For example, treatment with ionophores, such as ionomycin, and thapsigargin (an inhibitor of microsomal Ca2+-ATPase) stimulated MAPK activity in human foreskin fibroblasts (21). To investigate the role of Ca2+ in Ang II-induced MAPK activation in BAG cells, we incubated the cells with Ang II or thapsigargin in Ca2+ (1.2 mM)-containing, Ca2+-free, or Ca2+-free/EGTA (0.1 mM) medium. Treatment with thapsigargin (100 nM) for 10 min under each of the above conditions failed to stimulate MAPK phosphorylation or activity (Fig. 4Go), indicating that an increase in cytoplasmic [Ca2+] alone is insufficient to activate the kinase in BAG cells.



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  		Figure 4. Ca2+ is required for optimal MAPK activation. Serum-deprived glomerulosa cells were exposed to 100 nM Ang II or 100 nM thapsigargin for 10 min in Ca2+ (1.2 mM)-containing, Ca2+-free or Ca2+-free/0.1 mM EGTA medium as indicated. MAPK phosphorylation (A) and activity (B) were then determined as described in the legend to Fig. 1Go. In (A), a representative example is shown from three independent experiments. In (B), data represent the mean (± SEM) from three independent experiments.

 
The contribution of Ca2+ influx to the stimulatory effect of Ang II on MAPK activation was addressed by experiments in which extracellular Ca2+ was either omitted or Ca2+ influx was modified by inhibitors. As shown in Fig. 5Go. 100 µM La3+ (a nonspecific inhibitor of Ca2+ entry pathways) had no effect, and removal of external Ca2+ had only a minor effect on Ang II-induced MAPK activity (Fig. 4Go). Similarly, the dihydropyridine agonist, BayK (30 nM), and antagonist, nifedipine (2 µM), each had no effect on the Ang II-induced response (Fig. 5Go). However, removal of external Ca2+ combined with the addition of 0.1 mM EGTA significantly reduced the effect of Ang II on MAPK (Fig. 4Go). Taken together, these data suggest that elevation of intracellular [Ca2+], resulting either from Ca2+ influx or due to its release from intracellular stores, is not a major component of Ang II-induced MAPK activation. However, the impaired MAPK activation observed in the presence of Ca2+-free medium containing EGTA (which would deplete intracellular Ca2+) suggest a permissive role for Ca2+ in this response.



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  		Figure 5. Ca2+ influx does not activate MAPK. Serum-deprived glomerulosa cells were pretreated with vehicle, 100 µM LaCl3, 30 nM BayK or 2 µM nifedipine as indicated for 10 min before exposure to vehicle or 100 nM Ang II for a further 10 min. MAPK activity was then determined as described in the legend to Fig. 1Go. The data are representative of two independent experiments.

 
The role of PKC in MAPK activation
Because Ang II activates PKC (7), we investigated whether the kinase was able to activate MAPK in BAG cells. Treatment of the cells with the PKC-activating phorbol ester, phorbol 12-myristate, 13-acetate (PMA: 100 nM), induced phosphorylation and activation of MAPK in a time-dependent manner (Fig. 6Go). Maximal MAPK phosphorylation and activation occurred at 5–10 min, which correlated with the time required for maximal activation of MAPK in response to Ang II (Fig. 1BGo). However, in contrast to the transient effect of Ang II on MAPK activity, which returned to the basal level 60 min after addition of the ligand (Fig. 1BGo), the effect of PMA on MAPK activity was still evident (3.5-fold increase over control) at 60 min (Fig. 6BGo). Similarly, PMA-induced MAPK phosphorylation was still apparent at 60 min (Fig. 6AGo), at which time AngII-induced MAPK phosphorylation had returned to the basal level (Fig. 1AGo). Hence, stimulation of PKC alone is sufficient to activate MAPK in BAG cells.



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  		Figure 6. MAPK activation via PKC. Serum-deprived glomerulosa cells were exposed to 100 nM PMA for the indicated times. MAPK phosphorylation (A) and activity (B) were then determined as described in the legend to Fig. 1Go. In (A), a representative example is shown from three independent experiments. In (B), data represent the mean (± SEM) from three independent experiments.

 
To evaluate the dependence of Ang II-induced MAPK activation on PKC, BAG cells were preincubated with a high concentration (200 nM) of PMA for 24 h to down-regulate the kinase. The basal level of MAPK phosphorylation was slightly higher in PKC-depleted cells compared with PKC-replete cells (Fig. 7AGo), and the basal level of MAPK activity was increased by about 60% in PKC-depleted (compared with PKC-replete) cells (Fig. 7BGo). However, the magnitude of Ang II-induced MAPK activation in PKC-depleted cells was greatly reduced (110% increase over PKC-replete control; 40% increase over PKC-depleted control) compared with PKC-replete cells (300% increase over PKC-replete control). Consistent with these findings, PKC depletion partially attenuated Ang II-stimulated MAPK phosphorylation (Fig. 7AGo). Hence, PKC participates in a major (but not exclusive) pathway to MAPK activation in AngII-stimulated BAG cells.



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  		Figure 7. The role of PKC in Ang II-stimulated MAPK activation. Serum-deprived glomerulosa cells were pretreated for 24 h with vehicle or 200 nM PMA (to down-regulate PKC) before exposure to 100 nM Ang II or 100 nM PMA for 10 min. MAPK phosphorylation (A) and activity (B) were then determined as described in the legend to Fig. 1Go. In (A), a representative example is shown from three independent experiments. In (B), data represent the mean (± SEM) from three independent experiments.

 
MAPK activation is predominantly insensitive to pertussis toxin
In several cell types, MAPK activation via G protein-coupled receptors has been reported to be sensitive to pertussis toxin (PTX) (22, 23, 24). When the effects of the toxin on Ang II-induced MAPK activation were examined in BAG cells, PTX increased the basal level of MAPK activity by about 75%, although the magnitude of MAPK activity stimulated by Ang II was not significantly different in PTX-treated cells from that observed in control cells (Fig. 8Go). In terms of absolute magnitude, therefore, PTX had no effect on MAPK activation. However, if MAPK activation is evaluated as a fold increase over unstimulated (but PTX treated) cells, PTX inhibited the Ang II-stimulated increase in MAPK activation by 47% (from 3.4-fold to 1.8-fold). Hence, although a major component of the Ang II-induced MAPK activation is clearly insensitive to PTX, these data do not exclude a possible minor PTX-sensitive component in this response.



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  		Figure 8. Effect of PTX on MAPK activation. Serum-deprived glomerulosa cells were pretreated with vehicle or 200 ng/ml PTX as indicated for 16 h before exposure to vehicle or 100 nM Ang II for 10 min. MAPK activity was then determined. Data represent the mean (± SEM) of three independent experiments.

 
Ang II activates raf-1 kinase
To identify further elements involved in MAPK activation, the effect of Ang II on raf-1 kinase was investigated in BAG cells. Immunoblotting with an anti-raf-1 antibody revealed that Ang II induced a time-dependent shift of raf-1 to a lower electrophoretic mobility (Fig. 9AGo), an effect that correlates with hyperphosphorylation of the kinase (25). However, this mobility shift was considerably delayed in comparison to the effect of Ang II on MAPK phosphorylation in the same experiment. Whereas MAPK phosphorylation was detected as early as 1 min after addition of the peptide, no mobility shift of raf-1 was apparent before 5 min. Furthermore, whereas MAPK phosphorylation decreased from its peak at 5–10 min to the basal level by 60 min, the mobility shift of raf-1 was sustained and was still maximal at 60 min (Fig. 9AGo).



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  		Figure 9. Ang II activates and hyperphosphorylates Raf-1 kinase. Serum-deprived glomerulosa cells were exposed to 100 nM Ang II for the indicated times. Cell lysates were prepared and subjected to (A) immunoblotting, or (B) immunoprecipitation with an anti-raf-1 antibody. At each time point in (A), the shift of raf-1 to a lower electrophoretic mobility (which correlates with hyperphosphorylation of the kinase) was compared with the Ang II-induced mobility shift of MAPK. A representative example is shown from three independent experiments. In (B), in vitro kinase activity was determined in raf-1 immunoprecipitates incubated with a catalytically inactive mutant GST-[K97A]Mek-1 fusion protein as substrate. The data are representative of three independent experiments.

 
In further experiments, the time-course of raf-1 phosphorylation was compared with the enzymatic activity of p74raf-1 immunoprecipitated from BAG cells. No raf-1 activity was detected in control cells, but Ang II (100 nM) stimulated a transient increase in raf-1 activity that reached a maximum at 5 min (when raf-1 phosphorylation was just detectable) and returned toward the basal level by 60 min (when raf-1 phosphorylation was maximal) (Fig. 9BGo). Thus, the time-course of Ang II-induced raf-1 phosphorylation and the consequent shift in its electrophoretic mobility do not correlate with activation, but rather with inactivation, of the kinase.

We next investigated the role of PKC in raf-1 phosphorylation. Short-term (5 min) treatment of BAG cells with PMA caused a shift in raf-1 mobility (Fig. 10AGo), indicating that activation of PKC alone is sufficient to phosphorylate raf-1. Furthermore, depletion of PKC by prolonged PMA treatment completely abrogated the raf-1 mobility shift induced by Ang II (Fig. 10AGo). These results indicate that the raf-1 phosphorylation (which causes a shift in its electrophoretic mobility) induced by Ang II is mediated by PKC.



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  		Figure 10. The role of PKC in raf-1 activation and hyperphosphorylation. Serum-deprived glomerulosa cells were pretreated for 24 h with vehicle or 200 nM PMA (to down-regulate PKC) before exposure to 100 nM Ang II or 100 nM PMA for 5 min as indicated. Raf-1 hyperphosphorylation (A) and activity (B) were then determined as described in the legend to Fig. 9Go. Representative examples are shown from three independent experiments.

 
We also assessed the role of PKC in Ang II-induced raf-1 activation. Short-term (5 min) treatment of the cells with PMA increased raf-1 activity. However, while PKC depletion by prolonged exposure to high dose PMA abolished the acute effect of PMA on raf-1, the ability of Ang II to stimulate raf-1 was unaffected (Fig. 10BGo). These data indicate that, although PKC activation is sufficient to activate raf-1 kinase, the pathway by which Ang II activates raf-1 does not require PKC.

Ang II activates ras
Because ras acts as an upstream regulator of raf-1 kinase in growth factor-stimulated cells, we investigated the effect of Ang II on ras activation in BAG cells. As ras is activated by the exchange of bound GDP for GTP, the percentage of radiolabeled GTP (as a proportion of GTP + GDP) bound to p21ras immunoprecipitated from 32Pi-labeled BAG cells was measured. The basal %GTP bound to ras varied from 5.3% to 9.3% (n = 3). Ang II (30 nM) stimulated a rapid but transient exchange of ras-bound GDP for GTP that reached a maximum (mean 70% increase over control, n = 3) at 5–10 min and declined thereafter to near the prestimulation level by 30 min (Fig. 11Go). The time-course of Ang II-induced ras activation therefore correlates with both raf-1 and MAPK activation in BAG cells.



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  		Figure 11. Ang II activates Ras. Serum-deprived glomerulosa cells were labeled for 16 h with 32Pi before exposure to 30 nM Ang II for the indicated times. Radiolabeled guanine nucleotides bound to immunoprecipitated ras were resolved by TLC as described in the text. After quantitation in a PhosphorImager, GTP was expressed as the percentage of (GTP + GDP) bound to ras. Each point represents the mean percentage (± SEM) of the maximum increase in GTP bound to ras from three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Hypertrophic and hyperplastic effects of Ang II have been observed in a variety of target cells. Because of its potential involvement in the mechanisms of hypertension, the growth effects of Ang II have been extensively studied in vascular smooth muscle cells. For example, Ang II stimulates protein synthesis and hypertrophy in aortic smooth muscle cells (26, 27) and causes proliferation of renal arteriolar smooth muscle cells (28). Mitogenic responses to Ang II have also been reported for other cardiovascular cell types such as cardiac fibroblasts (29), as well as noncardiovascular cells such as rat intestinal epithelial cells (30). However, a major trophic target of Ang II is the adrenal cortex, where mitogenic effects of the peptide have long been recognized. This action of Ang II has been analyzed largely in cells derived from the fasciculata/reticularis zones of the bovine adrenal cortex (31, 32, 33, 34). Because the glomerulosa zone is the region from which the adrenal cortex grows and regenerates (35), we have studied growth responses in cells derived from this tissue. Indeed, we have previously reported that Ang II, acting via the G protein-coupled AT1 receptor, is mitogenic in primary cultures of BAG cells (6).

We found that Ang II activates the major mitogenic signaling intermediate, MAPK, in BAG cells, thus providing a probable mechanistic explanation for the mitogenic effects of the peptide in these cells. Although the pathway from receptor tyrosine kinases to MAPK activation is well characterized, the mechanisms whereby G protein-coupled receptors (which lack intrinsic kinase activity) activate MAPK are not well defined. In contrast to the ras-dependent pathway activated by receptor tyrosine kinases, G protein-coupled receptors appear to activate multiple ras-dependent and -independent pathways to MAPK (12, 13, 14). Receptors that couple via Gq to phosphoinositide hydrolysis (such as the AT1 receptor) stimulate the formation of diacylglycerol and elevate intracellular [Ca2+] (7). In several cell types, the resultant activation of PKC plays an important role in MAPK activation (12, 13, 14), although elevated intracellular [Ca2+] also appears to be sufficient to activate MAPK in some cell types (21).

Ang II increases intracellular [Ca2+] in BAG cells, both via the release of Ca2+ from intracellular stores and by the activation of Ca2+ influx (7). However, neither blockade nor activation of Ca2+ channels had any effect on Ang II-stimulated MAPK activation. Also, the release of Ca2+ from intracellular stores by thapsigargin failed to activate MAPK. Hence, elevation of intracellular [Ca2+] does not appear to contribute significantly to Ang II-induced MAPK activation. However, Ca2+ does appear to play a permissive role in this response because incubation of cells in Ca2+-free medium containing EGTA (which depletes intracellular Ca2+) significantly reduced Ang II-stimulated MAPK activation.

We examined the role of PKC in Ang II-induced MAPK activation by down-regulating the kinase with a high dose of PMA. Because PKC depletion attenuated, but did not abolish, Ang II-induced MAPK activation, the peptide appears to activate at least two pathways to MAPK: a major, PKC-dependent pathway and a minor, PKC-independent pathway. In contrast to its partial inhibitory effect on MAPK activation, PKC depletion had no effect on Ang II-induced raf-1 activation. Because raf-1 kinase lies immediately downstream of ras, it is reasonable to assume, therefore, that the ras/raf-1 activation pathway represents the PKC-independent limb of the Ang II-induced MAPK response in BAG cells.

Previous studies of the role of PKC in ras activation are conflicting. For example, the expression of a dominant-negative ras mutant failed to block PMA-stimulated MAPK phosphorylation in fibroblasts (36), whereas phorbol ester-induced MAPK activation was dependent on ras in T cells (37). Because ras/raf-1 activation is independent of PKC in BAG cells, the site(s) of PKC action in these cells probably lies downstream of raf-1 at the level of Mek and/or MAPK itself. However, because PKC appears unable to directly activate either of these kinases in some cells (14), it is possible that it may act on alternative (non-raf) Mek activators such as Mek kinase or mos (12, 13, 14).

The reported effects of PKC on raf-1 phosphorylation and its relationship to raf-1 activity are contradictory. In fibroblasts, PKC phosphorylated and directly activated raf-1 (38, 39) and the expression of a dominant-negative raf-1 mutant blocked PMA-induced gene transcription (40). Conversely, mutation of the raf-1 site phosphorylated by PKC was found to have no effect on raf-1 activation in T cells (41). In BAG cells, Ang II stimulated a raf-1 phosphorylation event that was detectable as a shift in the electrophoretic mobility of the kinase, but this mobility shift did not correlate temporally with raf-1 activation. Furthermore, whereas raf-1 activation was independent of PKC, its phosphorylation was PKC dependent. It is apparent, therefore, that the raf-1 phosphorylation event induced by Ang II that causes a mobility shift cannot be responsible for activating the kinase but correlates at least temporally with its deactivation. Nevertheless, it remains to be determined whether delayed PKC-mediated raf-1 phosphorylation is causally related to deactivation of the kinase.

One group of G protein-coupled receptors capable of activating MAPK operate independently of PKC via PTX-sensitive G proteins (22, 23, 24). The resultant liberation of Gß{gamma} complexes is believed to mediate a sequence of events similar to that activated by receptor tyrosine kinases (15, 16). These include the tyrosine phosphorylation of shc and its association with Grb2, and also involve the participation of a hitherto unidentified component(s) presumed to contain pleckstrin homology domains (which are capable of interaction with liberated Gß{gamma} complexes) (42). In BAG cells, PTX had no major effect on Ang II-stimulated MAPK activation, and both the mitogenic and early gene responses of BAG cells to Ang II appear to be mediated by PTX-insensitive pathways (6). However, because we cannot rule out a minor PTX-sensitive component to MAPK activation, it will be interesting to determine the sensitivity to PTX of signaling intermediates upstream of MAPK activated by Ang II in BAG cells.

Activation of MAPK by Ang II has previously been demonstrated in several cell types including rat vascular smooth muscle cells (43, 44, 45), cardiac myocytes (46), bovine adrenal fasciculata/reticularis cells (34), CHO cells stably transfected with the human AT1 receptor (47), and H295R human adrenal cells (48). However, reports of the role of PKC in MAPK activation are conflicting. Unlike BAG cells, PKC depletion had no effect on MAPK activation in cardiac myocytes (46) or vascular smooth muscle cells (44), whereas (like BAG cells) PKC depletion significantly reduced MAPK activation in AT1-expressing CHO cells (47) and abolished it in bovine adrenal fasciculata/reticularis cells (34). Ang II activated ras in vascular smooth muscle cells (43, 44, 45, 49), ras (50), and raf-1 (46) in cardiac myocytes, and raf-1 in AT1-expressing CHO cells (47). However, in contrast to the PKC-independence of raf-1 activation in BAG cells, raf-1 activation was dependent on PKC in cardiac myocytes (46) and AT1-expressing CHO cells (47). In vascular smooth muscle cells, ras activation was independent of PKC (44). Where tested, PTX had no effect on MAPK activation by Ang II in any of these cells, although both PTX-sensitive (43) and -insensitive (44) ras activation by Ang II have been reported in vascular smoooth muscle cells. The causal relationship between ras/raf-1 activation and MAPK activation in some Ang II-stimulated cells has been addressed by expressing dominant-negative mutants of each factor, with conflicting results: whereas ras was not required for MAPK activation in vascular smooth muscle cells (43, 45) and AT1-expressing CHO cells (47), dominant-negative ras reduced c-fos and cyclin D1 transcription in H295R adrenal cells (48). Similarly, dominant-negative raf-1 abolished, and manumycin (an inhibitor of ras farnesyl transferase) reduced MAPK activation in cardiac myocytes (46). Taking all these findings together, it is apparent that the pathways leading to MAPK activation from the AT1 receptor, and the role played by PKC in these pathways, vary considerably between different cell types, consistent with multiple mechanisms converging on MAPK.

In summary, we have demonstrated that Ang II activates multiple pathways to MAPK in BAG cells: a major, PKC-dependent pathway and a minor PKC-independent pathway which appears to operate via ras and raf-1 kinase. Further analysis of the upstream signaling pathways to MAPK should facilitate our understanding of the mechanisms underlying the mitogenic effects of Ang II in BAG cells.


   Acknowledgments
 
We thank Xue Zhao and Annamaria Zolyomi for preparing bovine adrenal glomerulosa cells.


   Footnotes
 
1 Recipient of an International Fellowship (FS/95018) from the British Heart Foundation. Back

Received May 27, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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