0013-7227/92/1313-1174$03.00/0 Endocrinology Copyright Q 1992 by The Endocrine
Vol. 131, No. 3 Printed in U.S.A.
Society
Mechanism of Angiotensin II-Induced Bovine Adrenocortical Cells* RAMA
NATARAJAN,
NOE GONZALES,
PETER J. HORNSBY,
Department of Diabetes, Endocrinology, and Metabolism, Duarte, California 91010; and the Department of Cellular (P.J.H.), Augusta, Georgia 30912
AND
Proliferation
JERRY
in
NADLER
City of Hope Medical Center, and Moleculur Biology, Medical
College of Georgia
ABSTRACT The peptide hormone angiotensin-II (AID is a potent vasoconstrictor and major regulator of aldosterone synthesis. In addition, AI1 also has growth-promoting effects. We have recently shown that the lipoxygenase (LO) pathway of arachidonic acid plays a major role in AII-induced aldosterone synthesis in adrenal glomerulosa cells. The LO pathway is also involved in the vasopressor and renin-inhibitory effects of AII. However, the role of LO products in AII-induced mitogenic effects have not yet been investigated. In the present studies we have evaluated the role of the LO pathway in AII-induced proliferative responses in a bovine adrenocortical cell clone termed AC1 cells. In addition, the potential receptor type and mechanism of AII-induced proliferation was studied by evaluating the effect of specific nonpeptide type 1 and type 2 AI1 receptor antagonists and the role of protein kinase-C (PKC).
AII-induced DNA synthesis was sisnificantlv attenuated bv two structurally dissimilar Li) inhibitors, b&alein and phenidone. In addition, the LO nroduct 12-hvdroxveicosatetraenoic acid (12-HETE) itself caused a &nificant in&ease”in DNA synthesis, suggesting that the 12LO pathway in part plays a role in AII-mediated mitogenesis. AIIinduced proliferative responses were blocked by the type 1 AI1 receptor antaaonist. Both AII- and 12-HETE-induced increases in DNA svnthe& were markedly inhibited by two PKC blockers, staurosporine and saneivamvcin. Further. both AI1 and 12-HETE could activate PKC by tranilocatmg it from the cytosol to the membrane fraction, as determined by Western immunoblotting. These results suggest that both 12-LO activation and protein kinase-C have an important role in AII-induced adrenal cell proliferation. (Endocrinology 13 1: 1174-1180, 1992)
A
activity, AI1 may have potent paracrine and autocrine actions that can lead to cardiovascular disease. One cell type in which A&induced proliferative responseshave been clearly documented are bovine adrenocortical cells (13). The present studies were conducted in a bovine adrenocortical clone termed AC1 cells. AI1 has been shown to increase DNA synthesis, cell proliferation, and steroidogenesisin thesecells (13, 14). The mechanismsof AII-induced mitogenic effects are not known. We have previously shown that the lipoxygenase (LO) pathway of arachidonic acid (AA) plays a key role in mediating AII-induced aldosterone synthesis in rat and human adrenal glomerulosa cells (15, 16). Further, the LO pathway has been implicated to play a role in the vasopressor and renin inhibitory effects of AI1 (17-19). LO products have also been shown to possessmitogenic properties. However, the role of the LO pathway in AII-induced proliferative responseshas not been studied. We evaluated the role of the LO pathway in AII-induced mitogenesisin AC1 cells in the present studies. In addition, one potential mechanismof AI1 and LO product-induced proliferation was studied by evaluating the role of protein kinase-C (PKC). Since two types of AI1 receptors have been identified (AT1 and AT1) (20-22), we also evaluated the effect of specific AT1 and AT2 receptor antagonists on AII-mediated proliferative effects.
NGIOTENSIN-II (AII) is a potent vasoconstrictor and also the major regulator of aldosterone secretion. In addition, AI1 also has potent growth-promoting effects. It is now increasingly apparent that these growth-modulating effects of AI1 may be key events in the development of cardiovascular disorders, such as hypertension and atherosclerosis.AI1 has both hypertrophic as well as hyperplastic effects on cells (1). Trophic effects of AI1 have been demonstrated in rat aortic vascular smooth muscle cells (VSMC) (2, 3) as well as in proximal tubular cells (4, 5). AI1 has been shown to have mitogenic effects when added with serum in human VSMC (6). However, there is still disagreement as to whether or not AI1 causesVSMC hyperplasia in vitro (2, 3). Support for the mitogenic and growthpromoting action of AI1 are the studies showing that AI1 can activate the protooncogenes c-fos, c-jun, and c-myc, and that AI1 can induce the expression of the potent mitogen plateletderived growth factor (7-10). Further, studiesshow an abundance of AI1 receptors in muscle and connective tissues of the fetus and that converting enzyme inhibitors prevent myointimal proliferative responsesafter vascular injury (11, 12). Therefore, along with its well known vasoconstrictive Received March 31, 1992. Address all correspondence and requests for reprints to: Rama Natarajan, Ph.D., Department of Diabetes, Endocrinology, and Metabolism, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, California 91010. * Presented in part at the National Meeting of the American Federation for Clinical Research, Seattle, WA, May 1991. This work was supported by grants from the American HeartAssociation, Los Angeles Affiliate (Grant-in-Aid 907-GI), and the NIH (ROl-DK-39721 and SCOR HL-44404).
Materials and Methods Materials AI1 (human, (Belmont, CA).
synthetic) was obtained from 12- and 15-hydroxyeicosatetraenoic
Peninsula acid
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Laboratories (12- and 15-
AII-INDUCED
PROLIFERATION
HETE) were obtained from Biomol Research Laboratories (Philadelphia, PA). They were added from lOOO-fold concentrates in dimethylsulfoxide (DMSO). The vehicle DMSO alone was added to the controls. The PKC inhibitor sangivamycin (23) was obtained as a gift from National Cancer Institute Chemotherapeutic Agents Repository, c/o ERC International (Rockville, MD). It was added as a solution in water. The PKC inhibitor staurosporine (24) was obtained from Boehrlnger Mannheim (Indianapolis, IN) and added from a lOOO-fold stock solution in DMSO. The LO inhibitor phenidone (2-phenylpyrazoline) was obtained from Sigma (St. Louis, MO), while baicalein (25) was purchased from Biomol Research Laboratories. Phenidone was dissolved in water, while baicalein was added in DMSO. The inhibitors are light sensitive and, therefore, kept protected from light. The cyclooxygenase (CO) blocker meclofenamate (from Sigma) was initially dissolved in ethanol to 10-r M, and further dilutions were made with distilled water. The nonpeptide AI1 receptor antagonists DuP753 (also known as losartan potassium) and PD123177 (21, 22) were gifts from DuPont (Wilmington, DE). All media for tissue culture were obtained from Sigma. Fetal calf serum was obtained from Irvine Scientific (Irvine, CA).
Culture of
celki
The AC1 cells were grown in Dulbecco’s Modified Eagle’s MediumHam’s F-12 (DME/FlZ) containing 10% fetal calf serum (FCS) and 4 rig/ml fibroblast growth factor (FGF; basic, bovine brain; R & D Systems, Minneauolis, MN) (13, 14). Thev zrew uniformlv for about 30-40 generations..Before an experiment; c&s were made-quiescent by serum starving for 48 h in DME/FlZ medium containing 0.2% BSA and 0.4% FCS. All experiments were conducted in DME/FlZ and 0.2% BSA.
Measurementof LO products, HETEs Confluent AC1 cells in lOO-mm dishes were serum starved and then placed in DME/FIZ and 0.2% fatty acid-free BSA. After preincubation for 10 min at 37 C, AI1 was added, and cells were incubated for a further 1 h. The reaction was terminated by cooling on ice. 12-HETE and 15HETE in the supernatants were extracted and quantitated by reverse phase HPLC and RIA, as described previously (15, 26).
rH]Thymidine incorporation studies Cells were plated on 12-well culture plates (3 x lO’/well). They were serum starved upon reaching near confluence and then placed in DME/ F12 and 0.2% BSA. Cells were preincubated for a period of 15 min with or without inhibitors, followed by the addition of AI1 or LO products. After 24 h, [3H]thymidine (1.5 @/well; New England Nuclear, Boston, MA) was added. Eighteen hours later, supernatants were aspirated, cells were washed twiccwith PBS, followed by twice with ice-cold 10% trichloroacetic acid solution. The cells were then washed with PBS and solubilized with 500 pi/well 1% sodium dodecyl sulfate (SDS) in 0.3 N NaOH. Radioactivity in these solutions was quantitated after the addition of liquid scintillation fluid.
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sulfate-polyacrylamide gel electrophoresis (10% running gel, 4% stacking gel) according to the method of Laemmli (27). For Western blotting, gels were equilibrated in transfer buffer (35 mr.4 Tris base, 192 mu glycine, and 20% methanol, pH 8.3) and then transferred to nitrocellulose (Hybond, Amersham, Arlington Heights, IL), as described by Towbm et al. (ZB), in a semidry polyblot apparatus (American Bionetics, Inc, Emeryville, CA) for 40 min at 2.5 mamp/cm’ gel. The nonspecific sites were blocked with PBS containing 3% BSA at 4 C overnight. The membranes were then washed twice with PBST (PBS and 0.05% Tween20) and incubated for 2 h at room temperature with a 1:300 dilution of the PKC antibody in PBS containing 0.05% Tween-20, 1% BSA, and 20% FCS. The PKC antibody used was a generous gift from Dr. D. Cooper (University of South Florida, Tampa) (29). This antibody recognizes (Y-, @-, and y-isoforms of PKC. Washed membranes were then incubated for 1 h at room temperature with second antibody (goat antirabbit) conjugated with alkaline phosphatase (1:5000; Promega Corp., Madison, WI). Color development was carried out using substrate mixture (nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate) from Promega. Immunoblots were photographed and scanned with a computerized video densitometer (Applied Imaging Lynx DNA Vision, Santa Clara, CA). Absorbance of the major band at 80 kilodaltons (kDa) was taken as irnmunoreactive PKC. Values were expressed as arbitrary optical density units.
Data analysis Statistical analysis was performed on an IBM computer, using the PC!INFO Software. All results are expressed as the mean -C SE. Analysis of variance was used to compare control and experimental values. For multiple comparisons, Duncan’s test was also used. Changes in immunoreactive PKC levels are expressed in arbitrary optical density units obtained from a computerized video densitometer.
Results Effect of AII on immwwreactive 12-HETE and 15HETE levelsin AC1 cells Figure 1 depicts the effect of AI1 (lo-* M) on immunoreactive 12-HETE releasedby the AC1 cells. It is seenthat AI1 causesa significant increasein 12-HETE levels. Further, this increase was blocked by the LO inhibitor phenidone (basal, 77.4 +- 10.5 pg; IO-’ M AII, 120 f 15; P < 0.01 VS.basal; AI1 plus phenidone, 85.3 f 13 pg/ml; P C 0.04 VS.AII). These
Measurementof immunoreactivePKC Confluent cells in loo-mm dishes were serum starved and then placed in DME/F12 and 0.2% BSA. After preincubation at 37 C for 10 min, agents [AII, IP-HETE, or the phorbol ester tetradecanoyl phorbol acetate (TPA; Sigma)] and vehicle controls were added, and incubation was carried out for 15 min. Cells were then washed with ice-cold PBS, scraped, and centrifuged. Cell pellets were suspended in 1.2 ml lysis buffer containing 25 mu Tris-HCl buffer (pH 7.4), 250 mu sucrose, 0.5 mM EGTA, 0.5 mM EDTA, 1 mu phenylmethylsulfonylfluoride, 20 fig/ ml leupeptin, 2 pg/ml aprotinin, and 1 mu dithiothreitol and sonicated twice for 5 set each time at the maximum output of the microtip using a Branson sonicator (Branson Co., Danbury, CT). After saving an aliquot for protein estimation, the sonicate was ultracentrifuged (100,000 X g) for 1 h. The supernatants were saved as cytosol fractions, while the pellets were resuspended in lysis buffer containing 0.1% NP-40, sonicated briefly for 3 set, and saved as membrane fraction. Equal amounts of cytosol and membrane fractions were subjected to sodium dodecyl
i BASAL
FIG. 1. The effect of AI1 on immunoreactive 12-HETE levels in AC1 cells. Serum-starved cells in loo-mm dishes were treated for 1 h with AII. 12-HETE in the supernatants was extracted on Bond-Elut minicolumns and quantitated by RIA. Results are expressed as the mean + SE from four separate experiments, performed in triplicate. *, P < 0.01 vs. basal.
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1176
AII-INDUCED
PROLIFERATION
Endo. Vol131.
AC1 cells did not produce detectable immunoreactive 15HETE either in the basal state or after AI1 treatment (data not shown). Effects of LO and CO inhibitors
on AII-induced
1992 No 3
l-
DNA synthesis
Figure 2 shows the effect of LO blockers on AII-stimulated DNA synthesis,asevaluated by [3H]thymidine incorporation. We confirmed that AI1 causeda dose-dependent increase in DNA synthesis in these cells from 10-‘“-10-7 M [lo-” M AII, 129% of the control value (P < 0.02); low9M AII, 192 f 13% (P < 0.01); lo-’ M AII, 268 f 15% (P < 0.001); 1O-7M AII, 276 f 20% (P < 0.001 VS.basal)]. AI1 (lo-’ M) was used in most of the present studies. The nonselective LO inhibitor phenidone (10e5M) and the more selective 12-LO blocker, baicalein (10V5M) caused a significant attenuation of the AI1 effect on DNA synthesis (178 f 17% and 173 + 16% of the control value, respectively; P < 0.01 us. AI1 alone). Neither phenidone nor baicalein alone significantly altered basal [3H] thymidine incorporation. Further, we observed that the effect of these LO inhibitors was specific to AII, since they did not attenuate FGF-induced DNA synthesis (4 q/ml FGF, 634 + 77% of the control value; FGF plus 10m5M phenidone, 610 + 59%; FGF and 10m5M baicalein, 596 f 80%). In addition, our unpublished observations show that compared to the effects of a known PKC inhibitor, H-7, neither LO inhibitor baicalein nor phenidone blocked PKC activity in cell lysates, as measuredby the phosphorylation of a PKC-specific peptide using a PKC assay kit from Gibco-Bethesda Research Laboratories(catalog no. 3161 SA, Grand Island, NY; control, 68 pmol phosphate/min.mg protein; 50 PM H-7, 41 pmol; 10e5M phenidone, 72 pmol; 10e5M baicalein, 66 pmol). To examine whether the CO pathway of AA plays a role in AI1 action, the effect df the CO inhibitor meclofenamate (10m6M) on AII-induced DNA synthesis was studied. The results are shown in Fig. 3. Unlike the LO inhibitors, meclofenamate did not block the effects of AII. In fact, meclofenamate actually slightly potentiated the effects of AII.
Al!
All+. ~ meclcTen (10%
(10-w
3. The effect of a CO inhibitor, DNA synthesis. Results are expressed experiments, performed in triplicate. FIG.
1 Z-HETE (10-7M)
1 Z-HETE (ro-6M)
maclofsn
meclofenamate, as the mean
+
on AII-induced SE from three
1 J-HETE (10-7M)
15-HETE (lO.+M)
FIG. 4. The effect of the LO products 12- and 15HETE on DNA synthesis in AC1 cells. Results are expressed as the mean + SE from four experiments, performed in triplicate. *, P < 0.03; **, P < 0.01 (vs. basal).
Effect of HETEs
on DNA synthesis
We next examined the effect of direct addition of 12- and 15-HETE at dosesranging from 10-6-10-‘o M on DNA synthesis in AC1 cells. Figure 4 depicts the results from these studies. 12-HETE at 10m6and 10e7 M caused a significant increasein DNA synthesis. 12-HETE at lower concentrations (10-8-10-‘o M) did not alter DNA synthesis (data not shown). The effects of 12-HETE were relatively specific, since, in contrast to the stimulatory effect of 12-HETE, 15-HETE from 1Om6lo-” M did not significantly increase DNA synthesis.
All+
Baicalein (10-5M)
Phenidone
EL Baicalein
2. The effect of LO inhibitors on AII-induced DNA synthesis. Growth-arrested cells in 12-well dishes were preincubated for 15 min with freshly prepared LO inhibitors, followed by the addition of AII. [“H]Thymidine incorporation into the cells was then measured, as described in Materials and Methods. Results are expressed as the mean + SE from four to six experiments, performed in duplicate or triplicate. *, P < 0.001 US. basal; **, P < 0.01 vs. AII. FIG.
Effect of nonpeptide DNA synthesis
AII receptor antagonists
on AU-induced
Figure 5 shows the effect of the specific type 1 (ATI) AI1 receptor antagonist (DuP753, losartan potassium) and the specific type 2 (AT,) AI1 receptor antagonist (PD123177) on AII-induced DNA synthesis. We observed that DuP753 at 10e5and 10m6M attenuated the effects of AII. In contrast, the specific type 2 antagonist PD123177 was without any effect at the same doses. Neither DuP753 nor I’D123177 at these
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AII-INDUCED
-L-
-
.
All (10%)
,.
1
.*
All+
AlI+
DUP753
DUP753
PD123177
(lo-%
(10-Q)
(10%
PD123177 (lo-5M)
5. The effect of AI1 receptor antagonists on AII-induced DNA synthesis in AC1 cells. Serum-starved cells in 12-well plates were preincubated for 15 min with the receptor antagonists, followed by the addition of AII. [3H]Thymidine was added 24 h later, and the incorporation was measured as described in MaterMls and Methods. Results are expressed as the mean + SE from three experiments, performed in triplicate. *, P < 0.01 us. AII. FIG.
’ l
All (I o%
. .. 1 r-5
All+ SANG
All+ SANG
(10 -5.4)
(lo-i4)
SANG (10 -‘%)
1177
PROLIFERATION
control; AI1 plus 10e9 M staurosporine, 112 + 14%; P < 0.001). In addition, sangivamycin (10-l’ M) attenuated 12HETE-induced DNA synthesis ( 10m6M 12-HETE, 136 + 6% of the control; 12-HETE plus sangivamycin, 92 f 8%; P < 0.01). Figure 7 shows a representative Western immunoblot of the effects of AI1 (lo-’ M) and 12-HETE (lo-’ M) on the translocation of PKC from the cytosol to membrane fraction. AI1 as well as 12-HETE caused a decreasein cytosolic PKC and a marked increase in membrane PKC levels, showing that they can directly activate PKC. Figure 8 is a bar graph representation of Western blot data obtained by densitometric analysis. The changes in subcellular distribution of PKC (80 kDa) in response to various agents is seen. The positive control, TPA, which is a known activator of PKC, was also studied. 12-HETE (lo-’ M), TPA (10e7M) as well as AI1 (lo-’ M) causeda decreasein immunoreactive cytosolic PKC levels. Similarly, all three agents causeda marked increasein membrane PKC levels. Discussion
In the present studies we examined the role of the LO pathway in AII-induced proliferation in bovine AC1 cells
-
n SANG
(lo-gM)
FIG. 6. The effect of a specific PKC inhibitor, sangivamycin (SANG), on AII-induced mitogenesis. Cells were preincubated for 15 min with SANG before the addition of AII. Results are expressed as the mean + SE fromfour to six experiments, performedin triplicate.*, P < 0.01 us. basal;**, P < 0.04vs.AII; ***, P < 0.01 vs.AII.
80 kD
MC MC MC 12-HETE Control AII 10-T M lo-hl FIG. 7. Western immunoblot of the effects of AI1 and 12-HETE on the translocation of PKC (80 kDa) from the cytosol (C) to the membrane (M) fraction. Equal amounts of cytosolic and membrane fractions were electrophoresed, blotted onto nitrocellulose, and then probed with a 1:300 dilution of the PKC antibody. Similar results were obtained from two other experiments.
concentrations altered basal [3H]thymidine incorporation (10m5M DuP753, 94 + 18% of the control value; 10d5 M PD123177, 110 f 12%). Role of PKC in AII-
and HETE-induced
proliferation
The role of PKC was assessedboth by examining the effects of PKC inhibitors on AII- and 12-HETE-induced mitogenesisas well as by studying the ability of AI1 and 12HETE to translocate PKC from cytosol to the membrane. Figure 6 shows the effect of a specific PKC inhibitor, sangivamycin, on AII-induced DNA synthesis. Sangivamycin was found to be a potent inhibitor of AI1 action, being effective at dosesas low at 10-l’ and lo-’ M. Sangivamycin alone at these doseshad no effect on basal DNA synthesis. Higher dosesalso markedly blocked AI1 action, but were inhibitory to the basal effects. Staurosporine, another PKC inhibitor, decreased the effect of AI1 (lo-* M AII, 252 f 18% of the
CONTROL
All (lO@M)
12-HETE (lo-‘M)
TPA (lo-‘M)
FIG. 8. Bar graph representation of PKC Western blot data obtained by video densitometric analysis. The changes in subcellular distribution of PKC in response to the various agents are depicted. Similar results were obtained from two other experiments.
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1178
AII-INDUCED
and also studied whether PKC plays a role in the mitogenesis. These cellswere chosen for study becauseit had been shown previously that AI1 not only stimulates steroidogenesisin these cells, but also increasesDNA synthesis ([3H]thymidine incorporation) and proliferation (cell number) (13, 14). In these original studies the AII-mediated, but not FGF-mediated, proliferative response could be specifically blocked by a specific AI1 receptor antagonist Sar’-Ile5-Ile%AII. The potential mitogenic and growth-promoting effects of AI1 have elicited considerable attention due to the link with the development of cardiovascular disorders, especially as AI1 action has been shown to be enhanced in diseased states such as hypertension and diabetes mellitus (30, 31). Two distinct subtypes of AI1 receptors (AT, and AT2) have been revealed by their differential affinities for the nonpeptide drugs DuP753 and PD123177 (20-22) and differential sensitivity to dithiothreitol (32). AT1 receptors have high affinity for DuP753, and these receptors predominate in adrenal cortex (20), vascular smooth muscle (32), hepatocytes (33), and cardiocytes (34). The AT, receptor subtype is coupled to guanine nucleotide binding regulatory protein and phospholipase-C. This type of AI1 receptor has been shown to mediate AII-induced vasoconstriction and aldosterone secretion (21). Recently, it was observed that AII-induced hypertrophy in proximal tubular cells is also mediated by ATi receptors (5). In the present study we demonstrated for the first time that AR-induced proliferative effects in the adrenal are also mediated by the AT1 receptor. AT2 receptors have high affinity for PD123177, and these receptors predominate in adrenal medulla (20), brain (35), and ovarian granulosa cells (36). The function of the AT1 receptor is not yet fully known. We observed in the present studies that AI1 increased the 12-LO product 12-HETE formation in AC1 cells, and inhibition of the LO pathway with LO inhibitors, such as phenidone and baicalein, causeda significant attenuation of AIIinduced DNA synthesis in AC1 cells, thus suggesting that the LO pathway plays a role at least in part in the proliferative responsesof AII. We have shown (unpublished observation) that these LO inhibitors can clearly inhibit the LO pathway, but do not inhibit PKC activity, thus confirming that their inhibitory effect on AII-induced DNA synthesis is not secondary to their inhibitory effects on PKC. Further, both baicalein and phenidone specifically attenuated only AII-mediated, and not FGF-mediated, DNA synthesis. In contrast to the LO blockers, a CO blocker, meclofenamate, did not significantly alter AI1 action and, in fact, had a slight potentiating effect. Evidence suggeststhat vasodepressor prostaglandins, such as prostacyclin (PGIZ), PGE2, and PGD2, can dose-dependently decrease the growth of vascular smooth muscle cells (37). Hence, the potentiating effect of the CO blocker on AII-induced mitogenesismay be due to inhibition of the formation of antimitogenic PGs and/ or the channeling of more of the substrate arachidonic acid into the LO pathway. Support for this hypothesis comes from the observation that AI1 could stimulate the proliferation of endothelial cells in the presence of a CO blocker, indomethacin (38).
PROLIFERATION
Endo. Voll31.
1992 No 3
Further support for the role of the LO pathway was obtained by our observation that 12-HETE itself significantly increased DNA synthesis. The effect of 12-HETE was specific, since 15-HETE at the sameconcentrations did not have a significant effect. The dosesof 12-HETE required to elicit DNA synthesis (10-7-10-6 M) fall in the same range as the amount of 12-HETE released by these cells in responseto AII, i.e. 120 pg/ml, which is 0.4 PM. Further, HETEs are very rapidly incorporated into cell membrane phospholipids and are short lived in cell cultures (39, 40). Therefore, exogenously added LO products may not fully simulate endogenous product actions. Several LO products, including the HETEs, have been attributed mitogenic properties in other cells, such as endothelial cells and various tumor cells. In endothelial cells, the HETEs act via enhancement of diacylglycerol formation and presumably PKC activity (41), whereas in tumor cells, the HETEs induce new receptor formation and metastatic activity via direct activation of PKC (42). Other new evidence links LO products to proliferative effects, since they can increase the activity of ras by acting as inhibitors of GTPase-activating protein, which inhibits ras (43). In addition, epidermal growth factor-induced mitogenic activity has been linked to the formation of 15-LO metabolites of linoleic acid (44), and 12- and 15-LO products can induce c-fos expression (45). Thus, it seemslikely that the HETEs play a role not only in AII-induced steroidogenesis, but also at least in part in AII-mediated proliferative effects. The 12-LO pathway does not mediate all of AI1 effects in these AC1 cells, since LO inhibition could not completely block AII-induced mitogenesis. It is clear that AI1 action involves other signalling systems, including activation of polyinositolphosphate metabolism and Ca2+releasevia phospholipase-C, diacylglycerol synthesis via phospholipase-C and phospholipase-D, and tyrosine phosphorylation. Additional studies are now under way to evaluate the role of these additional signaling pathways in AII-mediated proliferative effects in ACI cells. In evaluating potential mechanisms of AI1 and HETEinduced proliferation, we examined the role of PKC, since PKC has been implicated as playing a key role in cell proliferation. Further, AI1 can activate PKC in adrenal cells (46, 47), and several fatty acids, including the HETEs, have been shown to activate PKC isolated from the brain (48). In the present studies we observed that PKC inhibitors, such as staurosporine and sangivamycin, could inhibit both AII- as well as HETE-induced DNA synthesis, suggestingthat PKC is one important signal for AII- and LO product-induced proliferative responses.Further, in immunoblot analysis using a PKC antibody, both AI1 and 12-HETE caused translocation of PKC from the cytosol to the membrane fraction, similar to the effects of the known PKC activator, TPA. The mechanism of how HETEs could activate PKC is not clear, since there is no direct evidence for a receptor for 12-HETE. However, it has been shown that exposure of cells to HETEs causes their rapid incorporation into membrane phospholipids (39, 40), folIowed by the release of diacylglycerol substituted with HETE in position 2, which could activate PKC (39). These results suggest that both 12-LO activation
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AII-INDUCED and PKC play an important proliferation.
role in AII-induced
PROLIFERATION
adrenal cell
17.
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18.
Acknowledgments The authors would like to thank assistance, and Ms. Linda Lanting assistance.
Nozawa
growth: 15
Ms. Jenny Yang for her secretarial and Hsin Yang for their technical
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(AII-1)
and
WF 1991 Pharrn
Sci
a nucleoside analog, Chem 263:1682-1692
is a
Tamaoki T, Nomoto H, Takahashi I, Kato Y, Morimoto M, Tomita F 1986 Staurosporine, a potent inhibitor of phospholipidiCa*+ dependent 402
25.
PBMWM, angiotensin
Loomis CR, Bell RM 1988 Sangivamvcin, potent
24.
165:196-203
II receptor subtype-specific ligands: DuI’753 (AII-2). J Pharmacol Exp Ther 255:584-592
Timmermans Nonpeptide 12:55-61
23.
Res Commun
Wong PC, Hart SD, Zaspel AM, Chiu AT, Ardecky RJ, Smith RD, Timmermans PBMWM 1990 Functional studies of nonpeptide angiotensin PD123177
II-stimulated protein svnthesis in cultured vascular smooth muscle cells. Hypertension 13~305-314 Geisterfer AAT. Peach Ml, Owens GK 1988 Aneiotensin II induces hvpertrouhv, not hvoer&ia, of cultured rat aohc smooth muscle c&. Ciri Res 62:7&756 Wolf G. Nielson EG 1990 Anziotensin II induces cellular hvuertrophy in. cultured murine proximal tubular cells. Am J ‘physiol 259:F768-F777 Wolf G, Nielson EG, Goldfarb S, Ziyadeh FN 1991 The influence of glucose concentration on angiotensin II-induced hypertrophy of proximal tubular cells in culture. Biochem Biophys Res Commun 176~902-909 Campbell-Boswell M, Robertson AL 1981 Effects of angiotensin II and vasopressin on human smooth muscle cells in vitro. Exp Mol Path01 351265-276
muscle: role of Ca2+ mobilization Biol Chem 264:526-530 9.
21.
V, Gordon HM, Tsuda T 1989 Angiotensin
through protein kinase C activation and calcium cultured vascular smooth muscle cells. Biochem mun 150:52-59 8
blockade.
K, Tuck ML, Golub M, Eggena P, Nadler JL, Stern N
Biochem
!&helling
by lipoxygenase
1990 Inhibition of lipoxygenase pathway reduces blood pressure in renovascular hypertensive rats. Am J Physiol 259:H1774-H1780 19. Antonipillai I, Horton R, Natarajan R, Nadler J 1989 A 12lipoxygenase product of arachidonate metabolism is involved in angiotensin action on renin release. Endocrinology 125:2028-2034 20. Chiu AT, Herblin WF, McCall DE, Ardee RJ, Carini DJ, Duncia
References 1.
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Biochem
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Res Commun
Sekiya K, Okuda H 1982 Selective
135:397-
inhibition of platelet lipoxvgenRes Commun~105:1096-ld 26. Nataraian R. Stern N. Nadler J 1988 Diacvlelvcerol urovides arachidor& acid for lipoxygenase pioducts thacme’diate angiotensin IIinduced aldosterone synthesis. Biochem Biophys Res Commun 156:717-724 27. Laemmli UK 1970 Cleavage of structural proteins during assembly of the head of bacteriophage Tr. Nature 227680-685 28. Towbin H, Staehelin T, Gordon J 1979 Electrophoretic transfer of proteins from polyacrylamide gel to nitrocellulose sheets. Proc Nat1 Acad Sci USA 76:4350-4354 29 Acevedo-Duncan M, Cooper DR, Standaert ML, Farese RV 1989 Immunological evidence that insulin activates protein kinase C in BCJH-1 myocytes. FEBS Lett 244:174-176 30. Scott-Burden T, Resink TJ, Hahn AWA, Buhler FR 1991 Angiotensin-induced growth related metabolism is activated in cultured smooth cells from spontaneously hypertensive rats and WistarKyoto rats. Am J Hypertension 4:183-188 31. Weidman P, Beretta-Piccoli C, Trost BN 1985 Pressor factors and responsiveness in hypertension accompanying diabetes mellitus. Hypertension 7:11-33-E-42 32. Whitebread S, Mele M, Kamber B, deGasporo M 1989 Preliminary biochemical characterization of two angiotensin II receptor subtypes. Biochem Biophys Res Commun 163:284-291 _ .33. Garcia-Sainz Al. Macias-Silva M 1990 Anziotensin II stimulates phosphoinositide turnover and phosphorylase through AI&l receptors in isolated rat hepatocytes. Biochem Biophys Res Con-u-nun 172:780-785 34. Baker KM, Singer HA, Aceto JF 1989 Angiotensin II receptormediated stimulation of cytosolic-free calcium and inositol phosphates in chick myocytes. J Pharmacol Exp Ther 251:578-585 35. Sumner8 C, Tang W, Zelezna 8, Raizada MK 1991 Angiotensin II receptor subtypes are coupled with distinct signal transduction mechanisms in neurons and astrocytes from rat brain. Proc Nat1 Acad Sci USA 88:7567-7571 36. Puce11 AG, Hodges JC, Sen I, Merlin Bumpers F, Husain A 1991 Biochemical properties of ovarian granulosa cell type 2-angiotensin II receptor. Endocrinology 128:1947-1959 37. Uehara Y, Ishimitsu T, Kimura H, Ishii M, Ikeda T, Sugimoto T 1988 Regulatory effects of eicosanoids on thymidine uptake by vascular smooth muscle cells of rats. Prostae;landins 36:847-857 38. Bagby SP, Holden W 1988 Angiotensin IIitimulates proliferation of aortic endothelial cells. Clin Res 36:259A (Abstract) 39. Legrand AB, Lawson JA, Meyrick BO, Blair IA, Oates JA 1991 Substitution of 15-hydroxyeicosatetraenoic acid in the phosphoiase by baicalein.
Biochem
Biophys
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1180
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nositide signaling pathway. J Biol Chem 266:7570-7577 40. Bandyopadhyay GK, Imagawa W, Wallace DR, Nandi S 1987 Proliferative effects of insulin and epidermal growth factor on mouse mammary epithelial cells in primary culture. J Biol Chem 263~7567-7573 41. Setty BNY, Graber JE, Stuart MJ 1987 The mitogenic effect of 15and 12-hydroxyeicosatetraenoic acid on endothelial cells may be mediated via diacylglycerol kinase inhibition. J Biol Chem 262:17613-17622 42. Grossi IM, Fitzgerald LA, Umbarger LA, Nelson KK, Diglio CA, Taylor JD, Honn KV 1989 Bidirectional control of membrane expression and/or activation of the tumor cell IRGpIIb/IIIa receptor and tumor cell adhesion by lipoxygenase products of arachidonic acid and linoleic acid. Cancer Res 49:1029-1037 43. Yu CL, Tsai M, Stacey DW 1990 Serum stimulation of NIH 3T3 cells induces the production of lipids able to inhibit GTPase-acti-
Endo. 1992 Voll31 *No 3
vating protein activity. Mol Cell Biol 10:6683-6689 44. Glasgow WC, Eling TE 1990 Epidermal growth factor stimulates linoleic acid metabolism in BALB/c 3T3 fibroblasts. Mol Pharmacol 38:503-510 45. Haliday EM, Ramesha CS, Ringold G 1991 TNF induces c-fos via a novel pathway requiring conversion of arachidonic acid to a lipoxygenase metabolite. EMBO J 10:109-115 46. Lang U, Valloton MB 1987 Angiotensin II but not potassium induces subcellular redistribution of protein kinase C in bovine adrenal glomerulosa cells. J Biol Chem 262:8047-8050 47. Nakano S, Carvallo P, Rocco S, Aguilera G 1990 Role of protein kinase C on the steroidogenic effect of angiotensin II in the rat adrenal glomerulosa cell. Endocrinology 126:125-133 48. Shearman MS, Naor Z, Sekiguchi K, Kishimoto A, Nishizuka Y 1989 Selective activation of the y-subspecies of protein kinase C from bovine cerebellum by arachidonic acid and its lipoxygenase metabolites. FEBS Lett 243:177-180
Physiologic Roles of the EGF System: EGF, TGF-a, and Their Receptor November 7-9,1992, Nashville, TN, Sponsored by NIDR/NIH Leading scientists will focus on advances in understanding of the potential roles of individual components of the EGF system in normal in vivo functions in intact tissues and organs in developing and mature animals. Possible physiological roles of the EGF system in several organ systems, including the oral cavity (salivary glands, teeth), the central nervous system, the kidney, and the reproductive systems will be featured. Additionally, the use of transgenic animals to study this system will be explored. A partial list of invited speakers includes: Stanley Cohen Ben Margolis James Rhodes Michael Hise Sudhansu Dey Robert Coffey
Graham Carpenter Kazuo Hosoi Mariann Blum Raymond Harris Richard DiAugustine Elaine Fuchs
Samuel Weiss Edward Gresik Robert Safirstein Jeff May Glen Hofmann
Poster presentations on all aspects of the EGF system are welcome. Those dealing with its possible normal physiological roles will be especially compatible with the general topic of the meeting. Deadline for submission of abstracts is September 28, 1992. Dr. Edward W. Gresik, Program Organizer, Department of Cell Biology and Anatomical Sciences, Sophie Davis School of Biomedical Education, City University of New York Medical School, 138th St. and Convent Ave., New York, NY 10031. Telephone: 212-650-6857 or 6858 or 6861. FAX: 212-650-6812.
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Vol. 131, No. 3 Printed in U.S.A.
Society
Mechanism of Angiotensin II-Induced Bovine Adrenocortical Cells* RAMA
NATARAJAN,
NOE GONZALES,
PETER J. HORNSBY,
Department of Diabetes, Endocrinology, and Metabolism, Duarte, California 91010; and the Department of Cellular (P.J.H.), Augusta, Georgia 30912
AND
Proliferation
JERRY
in
NADLER
City of Hope Medical Center, and Moleculur Biology, Medical
College of Georgia
ABSTRACT The peptide hormone angiotensin-II (AID is a potent vasoconstrictor and major regulator of aldosterone synthesis. In addition, AI1 also has growth-promoting effects. We have recently shown that the lipoxygenase (LO) pathway of arachidonic acid plays a major role in AII-induced aldosterone synthesis in adrenal glomerulosa cells. The LO pathway is also involved in the vasopressor and renin-inhibitory effects of AII. However, the role of LO products in AII-induced mitogenic effects have not yet been investigated. In the present studies we have evaluated the role of the LO pathway in AII-induced proliferative responses in a bovine adrenocortical cell clone termed AC1 cells. In addition, the potential receptor type and mechanism of AII-induced proliferation was studied by evaluating the effect of specific nonpeptide type 1 and type 2 AI1 receptor antagonists and the role of protein kinase-C (PKC).
AII-induced DNA synthesis was sisnificantlv attenuated bv two structurally dissimilar Li) inhibitors, b&alein and phenidone. In addition, the LO nroduct 12-hvdroxveicosatetraenoic acid (12-HETE) itself caused a &nificant in&ease”in DNA synthesis, suggesting that the 12LO pathway in part plays a role in AII-mediated mitogenesis. AIIinduced proliferative responses were blocked by the type 1 AI1 receptor antaaonist. Both AII- and 12-HETE-induced increases in DNA svnthe& were markedly inhibited by two PKC blockers, staurosporine and saneivamvcin. Further. both AI1 and 12-HETE could activate PKC by tranilocatmg it from the cytosol to the membrane fraction, as determined by Western immunoblotting. These results suggest that both 12-LO activation and protein kinase-C have an important role in AII-induced adrenal cell proliferation. (Endocrinology 13 1: 1174-1180, 1992)
A
activity, AI1 may have potent paracrine and autocrine actions that can lead to cardiovascular disease. One cell type in which A&induced proliferative responseshave been clearly documented are bovine adrenocortical cells (13). The present studies were conducted in a bovine adrenocortical clone termed AC1 cells. AI1 has been shown to increase DNA synthesis, cell proliferation, and steroidogenesisin thesecells (13, 14). The mechanismsof AII-induced mitogenic effects are not known. We have previously shown that the lipoxygenase (LO) pathway of arachidonic acid (AA) plays a key role in mediating AII-induced aldosterone synthesis in rat and human adrenal glomerulosa cells (15, 16). Further, the LO pathway has been implicated to play a role in the vasopressor and renin inhibitory effects of AI1 (17-19). LO products have also been shown to possessmitogenic properties. However, the role of the LO pathway in AII-induced proliferative responseshas not been studied. We evaluated the role of the LO pathway in AII-induced mitogenesisin AC1 cells in the present studies. In addition, one potential mechanismof AI1 and LO product-induced proliferation was studied by evaluating the role of protein kinase-C (PKC). Since two types of AI1 receptors have been identified (AT1 and AT1) (20-22), we also evaluated the effect of specific AT1 and AT2 receptor antagonists on AII-mediated proliferative effects.
NGIOTENSIN-II (AII) is a potent vasoconstrictor and also the major regulator of aldosterone secretion. In addition, AI1 also has potent growth-promoting effects. It is now increasingly apparent that these growth-modulating effects of AI1 may be key events in the development of cardiovascular disorders, such as hypertension and atherosclerosis.AI1 has both hypertrophic as well as hyperplastic effects on cells (1). Trophic effects of AI1 have been demonstrated in rat aortic vascular smooth muscle cells (VSMC) (2, 3) as well as in proximal tubular cells (4, 5). AI1 has been shown to have mitogenic effects when added with serum in human VSMC (6). However, there is still disagreement as to whether or not AI1 causesVSMC hyperplasia in vitro (2, 3). Support for the mitogenic and growthpromoting action of AI1 are the studies showing that AI1 can activate the protooncogenes c-fos, c-jun, and c-myc, and that AI1 can induce the expression of the potent mitogen plateletderived growth factor (7-10). Further, studiesshow an abundance of AI1 receptors in muscle and connective tissues of the fetus and that converting enzyme inhibitors prevent myointimal proliferative responsesafter vascular injury (11, 12). Therefore, along with its well known vasoconstrictive Received March 31, 1992. Address all correspondence and requests for reprints to: Rama Natarajan, Ph.D., Department of Diabetes, Endocrinology, and Metabolism, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, California 91010. * Presented in part at the National Meeting of the American Federation for Clinical Research, Seattle, WA, May 1991. This work was supported by grants from the American HeartAssociation, Los Angeles Affiliate (Grant-in-Aid 907-GI), and the NIH (ROl-DK-39721 and SCOR HL-44404).
Materials and Methods Materials AI1 (human, (Belmont, CA).
synthetic) was obtained from 12- and 15-hydroxyeicosatetraenoic
Peninsula acid
1174
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Laboratories (12- and 15-
AII-INDUCED
PROLIFERATION
HETE) were obtained from Biomol Research Laboratories (Philadelphia, PA). They were added from lOOO-fold concentrates in dimethylsulfoxide (DMSO). The vehicle DMSO alone was added to the controls. The PKC inhibitor sangivamycin (23) was obtained as a gift from National Cancer Institute Chemotherapeutic Agents Repository, c/o ERC International (Rockville, MD). It was added as a solution in water. The PKC inhibitor staurosporine (24) was obtained from Boehrlnger Mannheim (Indianapolis, IN) and added from a lOOO-fold stock solution in DMSO. The LO inhibitor phenidone (2-phenylpyrazoline) was obtained from Sigma (St. Louis, MO), while baicalein (25) was purchased from Biomol Research Laboratories. Phenidone was dissolved in water, while baicalein was added in DMSO. The inhibitors are light sensitive and, therefore, kept protected from light. The cyclooxygenase (CO) blocker meclofenamate (from Sigma) was initially dissolved in ethanol to 10-r M, and further dilutions were made with distilled water. The nonpeptide AI1 receptor antagonists DuP753 (also known as losartan potassium) and PD123177 (21, 22) were gifts from DuPont (Wilmington, DE). All media for tissue culture were obtained from Sigma. Fetal calf serum was obtained from Irvine Scientific (Irvine, CA).
Culture of
celki
The AC1 cells were grown in Dulbecco’s Modified Eagle’s MediumHam’s F-12 (DME/FlZ) containing 10% fetal calf serum (FCS) and 4 rig/ml fibroblast growth factor (FGF; basic, bovine brain; R & D Systems, Minneauolis, MN) (13, 14). Thev zrew uniformlv for about 30-40 generations..Before an experiment; c&s were made-quiescent by serum starving for 48 h in DME/FlZ medium containing 0.2% BSA and 0.4% FCS. All experiments were conducted in DME/FlZ and 0.2% BSA.
Measurementof LO products, HETEs Confluent AC1 cells in lOO-mm dishes were serum starved and then placed in DME/FIZ and 0.2% fatty acid-free BSA. After preincubation for 10 min at 37 C, AI1 was added, and cells were incubated for a further 1 h. The reaction was terminated by cooling on ice. 12-HETE and 15HETE in the supernatants were extracted and quantitated by reverse phase HPLC and RIA, as described previously (15, 26).
rH]Thymidine incorporation studies Cells were plated on 12-well culture plates (3 x lO’/well). They were serum starved upon reaching near confluence and then placed in DME/ F12 and 0.2% BSA. Cells were preincubated for a period of 15 min with or without inhibitors, followed by the addition of AI1 or LO products. After 24 h, [3H]thymidine (1.5 @/well; New England Nuclear, Boston, MA) was added. Eighteen hours later, supernatants were aspirated, cells were washed twiccwith PBS, followed by twice with ice-cold 10% trichloroacetic acid solution. The cells were then washed with PBS and solubilized with 500 pi/well 1% sodium dodecyl sulfate (SDS) in 0.3 N NaOH. Radioactivity in these solutions was quantitated after the addition of liquid scintillation fluid.
1175
sulfate-polyacrylamide gel electrophoresis (10% running gel, 4% stacking gel) according to the method of Laemmli (27). For Western blotting, gels were equilibrated in transfer buffer (35 mr.4 Tris base, 192 mu glycine, and 20% methanol, pH 8.3) and then transferred to nitrocellulose (Hybond, Amersham, Arlington Heights, IL), as described by Towbm et al. (ZB), in a semidry polyblot apparatus (American Bionetics, Inc, Emeryville, CA) for 40 min at 2.5 mamp/cm’ gel. The nonspecific sites were blocked with PBS containing 3% BSA at 4 C overnight. The membranes were then washed twice with PBST (PBS and 0.05% Tween20) and incubated for 2 h at room temperature with a 1:300 dilution of the PKC antibody in PBS containing 0.05% Tween-20, 1% BSA, and 20% FCS. The PKC antibody used was a generous gift from Dr. D. Cooper (University of South Florida, Tampa) (29). This antibody recognizes (Y-, @-, and y-isoforms of PKC. Washed membranes were then incubated for 1 h at room temperature with second antibody (goat antirabbit) conjugated with alkaline phosphatase (1:5000; Promega Corp., Madison, WI). Color development was carried out using substrate mixture (nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate) from Promega. Immunoblots were photographed and scanned with a computerized video densitometer (Applied Imaging Lynx DNA Vision, Santa Clara, CA). Absorbance of the major band at 80 kilodaltons (kDa) was taken as irnmunoreactive PKC. Values were expressed as arbitrary optical density units.
Data analysis Statistical analysis was performed on an IBM computer, using the PC!INFO Software. All results are expressed as the mean -C SE. Analysis of variance was used to compare control and experimental values. For multiple comparisons, Duncan’s test was also used. Changes in immunoreactive PKC levels are expressed in arbitrary optical density units obtained from a computerized video densitometer.
Results Effect of AII on immwwreactive 12-HETE and 15HETE levelsin AC1 cells Figure 1 depicts the effect of AI1 (lo-* M) on immunoreactive 12-HETE releasedby the AC1 cells. It is seenthat AI1 causesa significant increasein 12-HETE levels. Further, this increase was blocked by the LO inhibitor phenidone (basal, 77.4 +- 10.5 pg; IO-’ M AII, 120 f 15; P < 0.01 VS.basal; AI1 plus phenidone, 85.3 f 13 pg/ml; P C 0.04 VS.AII). These
Measurementof immunoreactivePKC Confluent cells in loo-mm dishes were serum starved and then placed in DME/F12 and 0.2% BSA. After preincubation at 37 C for 10 min, agents [AII, IP-HETE, or the phorbol ester tetradecanoyl phorbol acetate (TPA; Sigma)] and vehicle controls were added, and incubation was carried out for 15 min. Cells were then washed with ice-cold PBS, scraped, and centrifuged. Cell pellets were suspended in 1.2 ml lysis buffer containing 25 mu Tris-HCl buffer (pH 7.4), 250 mu sucrose, 0.5 mM EGTA, 0.5 mM EDTA, 1 mu phenylmethylsulfonylfluoride, 20 fig/ ml leupeptin, 2 pg/ml aprotinin, and 1 mu dithiothreitol and sonicated twice for 5 set each time at the maximum output of the microtip using a Branson sonicator (Branson Co., Danbury, CT). After saving an aliquot for protein estimation, the sonicate was ultracentrifuged (100,000 X g) for 1 h. The supernatants were saved as cytosol fractions, while the pellets were resuspended in lysis buffer containing 0.1% NP-40, sonicated briefly for 3 set, and saved as membrane fraction. Equal amounts of cytosol and membrane fractions were subjected to sodium dodecyl
i BASAL
FIG. 1. The effect of AI1 on immunoreactive 12-HETE levels in AC1 cells. Serum-starved cells in loo-mm dishes were treated for 1 h with AII. 12-HETE in the supernatants was extracted on Bond-Elut minicolumns and quantitated by RIA. Results are expressed as the mean + SE from four separate experiments, performed in triplicate. *, P < 0.01 vs. basal.
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1176
AII-INDUCED
PROLIFERATION
Endo. Vol131.
AC1 cells did not produce detectable immunoreactive 15HETE either in the basal state or after AI1 treatment (data not shown). Effects of LO and CO inhibitors
on AII-induced
1992 No 3
l-
DNA synthesis
Figure 2 shows the effect of LO blockers on AII-stimulated DNA synthesis,asevaluated by [3H]thymidine incorporation. We confirmed that AI1 causeda dose-dependent increase in DNA synthesis in these cells from 10-‘“-10-7 M [lo-” M AII, 129% of the control value (P < 0.02); low9M AII, 192 f 13% (P < 0.01); lo-’ M AII, 268 f 15% (P < 0.001); 1O-7M AII, 276 f 20% (P < 0.001 VS.basal)]. AI1 (lo-’ M) was used in most of the present studies. The nonselective LO inhibitor phenidone (10e5M) and the more selective 12-LO blocker, baicalein (10V5M) caused a significant attenuation of the AI1 effect on DNA synthesis (178 f 17% and 173 + 16% of the control value, respectively; P < 0.01 us. AI1 alone). Neither phenidone nor baicalein alone significantly altered basal [3H] thymidine incorporation. Further, we observed that the effect of these LO inhibitors was specific to AII, since they did not attenuate FGF-induced DNA synthesis (4 q/ml FGF, 634 + 77% of the control value; FGF plus 10m5M phenidone, 610 + 59%; FGF and 10m5M baicalein, 596 f 80%). In addition, our unpublished observations show that compared to the effects of a known PKC inhibitor, H-7, neither LO inhibitor baicalein nor phenidone blocked PKC activity in cell lysates, as measuredby the phosphorylation of a PKC-specific peptide using a PKC assay kit from Gibco-Bethesda Research Laboratories(catalog no. 3161 SA, Grand Island, NY; control, 68 pmol phosphate/min.mg protein; 50 PM H-7, 41 pmol; 10e5M phenidone, 72 pmol; 10e5M baicalein, 66 pmol). To examine whether the CO pathway of AA plays a role in AI1 action, the effect df the CO inhibitor meclofenamate (10m6M) on AII-induced DNA synthesis was studied. The results are shown in Fig. 3. Unlike the LO inhibitors, meclofenamate did not block the effects of AII. In fact, meclofenamate actually slightly potentiated the effects of AII.
Al!
All+. ~ meclcTen (10%
(10-w
3. The effect of a CO inhibitor, DNA synthesis. Results are expressed experiments, performed in triplicate. FIG.
1 Z-HETE (10-7M)
1 Z-HETE (ro-6M)
maclofsn
meclofenamate, as the mean
+
on AII-induced SE from three
1 J-HETE (10-7M)
15-HETE (lO.+M)
FIG. 4. The effect of the LO products 12- and 15HETE on DNA synthesis in AC1 cells. Results are expressed as the mean + SE from four experiments, performed in triplicate. *, P < 0.03; **, P < 0.01 (vs. basal).
Effect of HETEs
on DNA synthesis
We next examined the effect of direct addition of 12- and 15-HETE at dosesranging from 10-6-10-‘o M on DNA synthesis in AC1 cells. Figure 4 depicts the results from these studies. 12-HETE at 10m6and 10e7 M caused a significant increasein DNA synthesis. 12-HETE at lower concentrations (10-8-10-‘o M) did not alter DNA synthesis (data not shown). The effects of 12-HETE were relatively specific, since, in contrast to the stimulatory effect of 12-HETE, 15-HETE from 1Om6lo-” M did not significantly increase DNA synthesis.
All+
Baicalein (10-5M)
Phenidone
EL Baicalein
2. The effect of LO inhibitors on AII-induced DNA synthesis. Growth-arrested cells in 12-well dishes were preincubated for 15 min with freshly prepared LO inhibitors, followed by the addition of AII. [“H]Thymidine incorporation into the cells was then measured, as described in Materials and Methods. Results are expressed as the mean + SE from four to six experiments, performed in duplicate or triplicate. *, P < 0.001 US. basal; **, P < 0.01 vs. AII. FIG.
Effect of nonpeptide DNA synthesis
AII receptor antagonists
on AU-induced
Figure 5 shows the effect of the specific type 1 (ATI) AI1 receptor antagonist (DuP753, losartan potassium) and the specific type 2 (AT,) AI1 receptor antagonist (PD123177) on AII-induced DNA synthesis. We observed that DuP753 at 10e5and 10m6M attenuated the effects of AII. In contrast, the specific type 2 antagonist PD123177 was without any effect at the same doses. Neither DuP753 nor I’D123177 at these
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AII-INDUCED
-L-
-
.
All (10%)
,.
1
.*
All+
AlI+
DUP753
DUP753
PD123177
(lo-%
(10-Q)
(10%
PD123177 (lo-5M)
5. The effect of AI1 receptor antagonists on AII-induced DNA synthesis in AC1 cells. Serum-starved cells in 12-well plates were preincubated for 15 min with the receptor antagonists, followed by the addition of AII. [3H]Thymidine was added 24 h later, and the incorporation was measured as described in MaterMls and Methods. Results are expressed as the mean + SE from three experiments, performed in triplicate. *, P < 0.01 us. AII. FIG.
’ l
All (I o%
. .. 1 r-5
All+ SANG
All+ SANG
(10 -5.4)
(lo-i4)
SANG (10 -‘%)
1177
PROLIFERATION
control; AI1 plus 10e9 M staurosporine, 112 + 14%; P < 0.001). In addition, sangivamycin (10-l’ M) attenuated 12HETE-induced DNA synthesis ( 10m6M 12-HETE, 136 + 6% of the control; 12-HETE plus sangivamycin, 92 f 8%; P < 0.01). Figure 7 shows a representative Western immunoblot of the effects of AI1 (lo-’ M) and 12-HETE (lo-’ M) on the translocation of PKC from the cytosol to membrane fraction. AI1 as well as 12-HETE caused a decreasein cytosolic PKC and a marked increase in membrane PKC levels, showing that they can directly activate PKC. Figure 8 is a bar graph representation of Western blot data obtained by densitometric analysis. The changes in subcellular distribution of PKC (80 kDa) in response to various agents is seen. The positive control, TPA, which is a known activator of PKC, was also studied. 12-HETE (lo-’ M), TPA (10e7M) as well as AI1 (lo-’ M) causeda decreasein immunoreactive cytosolic PKC levels. Similarly, all three agents causeda marked increasein membrane PKC levels. Discussion
In the present studies we examined the role of the LO pathway in AII-induced proliferation in bovine AC1 cells
-
n SANG
(lo-gM)
FIG. 6. The effect of a specific PKC inhibitor, sangivamycin (SANG), on AII-induced mitogenesis. Cells were preincubated for 15 min with SANG before the addition of AII. Results are expressed as the mean + SE fromfour to six experiments, performedin triplicate.*, P < 0.01 us. basal;**, P < 0.04vs.AII; ***, P < 0.01 vs.AII.
80 kD
MC MC MC 12-HETE Control AII 10-T M lo-hl FIG. 7. Western immunoblot of the effects of AI1 and 12-HETE on the translocation of PKC (80 kDa) from the cytosol (C) to the membrane (M) fraction. Equal amounts of cytosolic and membrane fractions were electrophoresed, blotted onto nitrocellulose, and then probed with a 1:300 dilution of the PKC antibody. Similar results were obtained from two other experiments.
concentrations altered basal [3H]thymidine incorporation (10m5M DuP753, 94 + 18% of the control value; 10d5 M PD123177, 110 f 12%). Role of PKC in AII-
and HETE-induced
proliferation
The role of PKC was assessedboth by examining the effects of PKC inhibitors on AII- and 12-HETE-induced mitogenesisas well as by studying the ability of AI1 and 12HETE to translocate PKC from cytosol to the membrane. Figure 6 shows the effect of a specific PKC inhibitor, sangivamycin, on AII-induced DNA synthesis. Sangivamycin was found to be a potent inhibitor of AI1 action, being effective at dosesas low at 10-l’ and lo-’ M. Sangivamycin alone at these doseshad no effect on basal DNA synthesis. Higher dosesalso markedly blocked AI1 action, but were inhibitory to the basal effects. Staurosporine, another PKC inhibitor, decreased the effect of AI1 (lo-* M AII, 252 f 18% of the
CONTROL
All (lO@M)
12-HETE (lo-‘M)
TPA (lo-‘M)
FIG. 8. Bar graph representation of PKC Western blot data obtained by video densitometric analysis. The changes in subcellular distribution of PKC in response to the various agents are depicted. Similar results were obtained from two other experiments.
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1178
AII-INDUCED
and also studied whether PKC plays a role in the mitogenesis. These cellswere chosen for study becauseit had been shown previously that AI1 not only stimulates steroidogenesisin these cells, but also increasesDNA synthesis ([3H]thymidine incorporation) and proliferation (cell number) (13, 14). In these original studies the AII-mediated, but not FGF-mediated, proliferative response could be specifically blocked by a specific AI1 receptor antagonist Sar’-Ile5-Ile%AII. The potential mitogenic and growth-promoting effects of AI1 have elicited considerable attention due to the link with the development of cardiovascular disorders, especially as AI1 action has been shown to be enhanced in diseased states such as hypertension and diabetes mellitus (30, 31). Two distinct subtypes of AI1 receptors (AT, and AT2) have been revealed by their differential affinities for the nonpeptide drugs DuP753 and PD123177 (20-22) and differential sensitivity to dithiothreitol (32). AT1 receptors have high affinity for DuP753, and these receptors predominate in adrenal cortex (20), vascular smooth muscle (32), hepatocytes (33), and cardiocytes (34). The AT, receptor subtype is coupled to guanine nucleotide binding regulatory protein and phospholipase-C. This type of AI1 receptor has been shown to mediate AII-induced vasoconstriction and aldosterone secretion (21). Recently, it was observed that AII-induced hypertrophy in proximal tubular cells is also mediated by ATi receptors (5). In the present study we demonstrated for the first time that AR-induced proliferative effects in the adrenal are also mediated by the AT1 receptor. AT2 receptors have high affinity for PD123177, and these receptors predominate in adrenal medulla (20), brain (35), and ovarian granulosa cells (36). The function of the AT1 receptor is not yet fully known. We observed in the present studies that AI1 increased the 12-LO product 12-HETE formation in AC1 cells, and inhibition of the LO pathway with LO inhibitors, such as phenidone and baicalein, causeda significant attenuation of AIIinduced DNA synthesis in AC1 cells, thus suggesting that the LO pathway plays a role at least in part in the proliferative responsesof AII. We have shown (unpublished observation) that these LO inhibitors can clearly inhibit the LO pathway, but do not inhibit PKC activity, thus confirming that their inhibitory effect on AII-induced DNA synthesis is not secondary to their inhibitory effects on PKC. Further, both baicalein and phenidone specifically attenuated only AII-mediated, and not FGF-mediated, DNA synthesis. In contrast to the LO blockers, a CO blocker, meclofenamate, did not significantly alter AI1 action and, in fact, had a slight potentiating effect. Evidence suggeststhat vasodepressor prostaglandins, such as prostacyclin (PGIZ), PGE2, and PGD2, can dose-dependently decrease the growth of vascular smooth muscle cells (37). Hence, the potentiating effect of the CO blocker on AII-induced mitogenesismay be due to inhibition of the formation of antimitogenic PGs and/ or the channeling of more of the substrate arachidonic acid into the LO pathway. Support for this hypothesis comes from the observation that AI1 could stimulate the proliferation of endothelial cells in the presence of a CO blocker, indomethacin (38).
PROLIFERATION
Endo. Voll31.
1992 No 3
Further support for the role of the LO pathway was obtained by our observation that 12-HETE itself significantly increased DNA synthesis. The effect of 12-HETE was specific, since 15-HETE at the sameconcentrations did not have a significant effect. The dosesof 12-HETE required to elicit DNA synthesis (10-7-10-6 M) fall in the same range as the amount of 12-HETE released by these cells in responseto AII, i.e. 120 pg/ml, which is 0.4 PM. Further, HETEs are very rapidly incorporated into cell membrane phospholipids and are short lived in cell cultures (39, 40). Therefore, exogenously added LO products may not fully simulate endogenous product actions. Several LO products, including the HETEs, have been attributed mitogenic properties in other cells, such as endothelial cells and various tumor cells. In endothelial cells, the HETEs act via enhancement of diacylglycerol formation and presumably PKC activity (41), whereas in tumor cells, the HETEs induce new receptor formation and metastatic activity via direct activation of PKC (42). Other new evidence links LO products to proliferative effects, since they can increase the activity of ras by acting as inhibitors of GTPase-activating protein, which inhibits ras (43). In addition, epidermal growth factor-induced mitogenic activity has been linked to the formation of 15-LO metabolites of linoleic acid (44), and 12- and 15-LO products can induce c-fos expression (45). Thus, it seemslikely that the HETEs play a role not only in AII-induced steroidogenesis, but also at least in part in AII-mediated proliferative effects. The 12-LO pathway does not mediate all of AI1 effects in these AC1 cells, since LO inhibition could not completely block AII-induced mitogenesis. It is clear that AI1 action involves other signalling systems, including activation of polyinositolphosphate metabolism and Ca2+releasevia phospholipase-C, diacylglycerol synthesis via phospholipase-C and phospholipase-D, and tyrosine phosphorylation. Additional studies are now under way to evaluate the role of these additional signaling pathways in AII-mediated proliferative effects in ACI cells. In evaluating potential mechanisms of AI1 and HETEinduced proliferation, we examined the role of PKC, since PKC has been implicated as playing a key role in cell proliferation. Further, AI1 can activate PKC in adrenal cells (46, 47), and several fatty acids, including the HETEs, have been shown to activate PKC isolated from the brain (48). In the present studies we observed that PKC inhibitors, such as staurosporine and sangivamycin, could inhibit both AII- as well as HETE-induced DNA synthesis, suggestingthat PKC is one important signal for AII- and LO product-induced proliferative responses.Further, in immunoblot analysis using a PKC antibody, both AI1 and 12-HETE caused translocation of PKC from the cytosol to the membrane fraction, similar to the effects of the known PKC activator, TPA. The mechanism of how HETEs could activate PKC is not clear, since there is no direct evidence for a receptor for 12-HETE. However, it has been shown that exposure of cells to HETEs causes their rapid incorporation into membrane phospholipids (39, 40), folIowed by the release of diacylglycerol substituted with HETE in position 2, which could activate PKC (39). These results suggest that both 12-LO activation
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AII-INDUCED and PKC play an important proliferation.
role in AII-induced
PROLIFERATION
adrenal cell
17.
Stern N, Golub M, Nozawa K, Berger M, Knoll E, Yanagawa N, Nataraian R, Nadler JL, Tuck M 1989 Selective inhibition of angiothnsin II-mediated~vasoconstriction Am J Physiol257:H434-H443
18.
Acknowledgments The authors would like to thank assistance, and Ms. Linda Lanting assistance.
Nozawa
growth: 15
Ms. Jenny Yang for her secretarial and Hsin Yang for their technical
JV, Pease LJ, Wong PC, Wexler RR, Johnson AL, Timmermans PBMWM 1989 Identification of angiotensin II receptor subtypes.
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H, Furkumoto
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of the c-fos gene ion mobilization in Biophys Res Com-
Taubman MB, Berk SC, Izumo S, Tsuda T, Alexander RW, NadalGinard B 1989 Anglotensin II induces c-fos mRNA in aortic smooth and protein
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of atria1 natriuretic polypeptide and angiotensin II on protooncogene expression and vascular cell growth. Biochem Biophys Res Commun 176:16011609 10. Naftilan AJ, Pratt RE, Dzau VJ 1989 Induction of platelet-derived growth factor A-chain and c-myc gene expressions by angiotensin ~;;4ultured rat vascular smooth muscle cells. J Clin Invest 83:141911.
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22.
Wong PC, Chiu AT, Herblin II receptor
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II-stimulated protein svnthesis in cultured vascular smooth muscle cells. Hypertension 13~305-314 Geisterfer AAT. Peach Ml, Owens GK 1988 Aneiotensin II induces hvpertrouhv, not hvoer&ia, of cultured rat aohc smooth muscle c&. Ciri Res 62:7&756 Wolf G. Nielson EG 1990 Anziotensin II induces cellular hvuertrophy in. cultured murine proximal tubular cells. Am J ‘physiol 259:F768-F777 Wolf G, Nielson EG, Goldfarb S, Ziyadeh FN 1991 The influence of glucose concentration on angiotensin II-induced hypertrophy of proximal tubular cells in culture. Biochem Biophys Res Commun 176~902-909 Campbell-Boswell M, Robertson AL 1981 Effects of angiotensin II and vasopressin on human smooth muscle cells in vitro. Exp Mol Path01 351265-276
muscle: role of Ca2+ mobilization Biol Chem 264:526-530 9.
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K, Tuck ML, Golub M, Eggena P, Nadler JL, Stern N
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1990 Inhibition of lipoxygenase pathway reduces blood pressure in renovascular hypertensive rats. Am J Physiol 259:H1774-H1780 19. Antonipillai I, Horton R, Natarajan R, Nadler J 1989 A 12lipoxygenase product of arachidonate metabolism is involved in angiotensin action on renin release. Endocrinology 125:2028-2034 20. Chiu AT, Herblin WF, McCall DE, Ardee RJ, Carini DJ, Duncia
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Physiologic Roles of the EGF System: EGF, TGF-a, and Their Receptor November 7-9,1992, Nashville, TN, Sponsored by NIDR/NIH Leading scientists will focus on advances in understanding of the potential roles of individual components of the EGF system in normal in vivo functions in intact tissues and organs in developing and mature animals. Possible physiological roles of the EGF system in several organ systems, including the oral cavity (salivary glands, teeth), the central nervous system, the kidney, and the reproductive systems will be featured. Additionally, the use of transgenic animals to study this system will be explored. A partial list of invited speakers includes: Stanley Cohen Ben Margolis James Rhodes Michael Hise Sudhansu Dey Robert Coffey
Graham Carpenter Kazuo Hosoi Mariann Blum Raymond Harris Richard DiAugustine Elaine Fuchs
Samuel Weiss Edward Gresik Robert Safirstein Jeff May Glen Hofmann
Poster presentations on all aspects of the EGF system are welcome. Those dealing with its possible normal physiological roles will be especially compatible with the general topic of the meeting. Deadline for submission of abstracts is September 28, 1992. Dr. Edward W. Gresik, Program Organizer, Department of Cell Biology and Anatomical Sciences, Sophie Davis School of Biomedical Education, City University of New York Medical School, 138th St. and Convent Ave., New York, NY 10031. Telephone: 212-650-6857 or 6858 or 6861. FAX: 212-650-6812.
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