Angiotensin II activation of cyclin D1-dependent kinase activity.

Angiotensin II (AII) binds to specific G protein-coupled receptors and is mitogenic in adrenal, liver epithelial, and vascular smooth muscle cells. Since the cyclin D1 gene encodes the regulatory subunit of the cyclin D1-dependent kinase (CD1K) required for phosphorylation of the retinoblastoma protein (pRB), an essential and rate-limiting step in G1 phase progression of the cell cycle, we examined the effect of AII on cyclin D1 expression and CD1K activity in the human adrenal cell line H295R. AII (10(-6) M) stimulated G1 phase progression within 12 h, with a maximal effect after 72 h. This action was antedated by the induction of cyclin D1 mRNA (3-fold), cyclin D1 nuclear protein abundance (4-fold), and CD1K activity (4-fold). AII induced cyclin D1 promoter activity 4-fold, via the AT1 receptor through an enhancer sequence at -954 base pairs. c-Fos and c-Jun bound the cyclin D1 -954 enhancer sequence, and the abundance of c-Fos within this complex was increased by AII treatment. AII induced extracellular signal-regulated kinase (ERK) activity 7-fold, and dominant-negative mutants of either p21(ras) or ERK reduced AII-stimulated cyclin D1 promoter activity. These findings suggest that AII may stimulate mitogenesis by increasing CD1K activity through a p21(ras)/ERK/activator protein 1 pathway.

The octapeptide angiotensin II (AII) 1 binds to specific high affinity receptors present in the adrenal cortex, liver epithelial cells, and in vascular smooth muscle cells, where it elicits a vast array of biological effects. AII increases DNA synthesis, cell proliferation, and steroidogenesis in cultured adrenal cortical cells, whether the cells are derived from the adrenal fasciculata or glomerulosa cell layer (1,2). AII also functions as a growth factor in cardiac fibroblasts, myocytes, and vascular smooth muscle cells (3)(4)(5). Many of the known biological actions of AII, including enhanced DNA synthesis, are mediated by stimulation of the AT 1 receptor (1,2).
The AT 1 receptor is a member of the G protein-coupled seven-transmembrane spanning receptor family (6,7). Binding of AII to the AT 1 receptor activates phospholipase C, which initiates a rapid release of inositol trisphosphate and diacylglycerol from phosphatidylinositol 4,5-bisphosphate, causing intracellular calcium release (8). The intracellular transmission of signaling by Ca 2ϩ and activation of cytosolic phospholipases involves, in part, sequential activation of p21 ras (9 -11) and thereby protein kinases, including the mitogen-activated protein kinases (MAPKs) (7,12). Previous studies showed that AII can stimulate phosphorylation of several intracellular signaling protein kinases at tyrosine residues including the extracellular signal-regulated kinases (ERKs) in vascular cells (13,14) and the related Stress Activated Protein Kinases (SAPKs or Jun N-terminal Kinases in hepatic cells (15)). In addition, AII stimulates tyrosine phosphorylation of p44/p56 SHC (13), the Jak family proteins Jak2 and Tyk2 (16), and focal adhesion kinase (FAK 125 ) in vascular smooth muscle cells (13).
Although these pathways of AII-mediated signal transduction have been studied extensively, the steps critical for the proliferative action of this hormone are not fully elucidated. Several transcription factors have been implicated in the proliferative signaling pathway induced by AII. The STAT proteins, STAT1 and STAT2, are transcriptionally activated upon AII-induced tyrosine phosphorylation (16). The STAT proteins contribute to the transcription factor complex known as SIF (sis inducing factor), and SIF binding is stimulated by AII in cardiac fibroblasts (17). SIF binds to the sis-inducible element sequences found in the promoter region of the c-fos and other immediate-early genes (18). In cultured adrenocortical and smooth muscle cells, AII increases the abundance of the mRNA encoding the early genes c-fos and c-jun (19 -21), which contribute to activity of the AP-1 transcription factor complex (22). It is likely that AII may induce these immediate-early genes through a STAT/SIF pathway.
The retinoblastoma protein (pRB) plays a critical role as an intermediary protein in proliferative responses (23,24). For instance, the vascular proliferation induced by intimal trauma was abrogated by the introduction of a dominant-negative mutant of the pRB protein, suggesting that inactivation of pRB was critical for normal proliferative signaling in vascular cells (25). Whether or not AII-mediated proliferative signaling involves inactivation of the pRB protein remains to be investigated. Inactivation of pRB is normally achieved by phosphorylation that is mediated by serine/threonine cyclin-dependent kinases (CDKs) (23, 24,26). A regulatory subunit of the G 1 phase CDKs, cyclin D1, forms a complex with the catalytic partners CDK4 and CDK6 to form an active holoenzyme that phosphorylates pRB (23, 24,26). Cyclin D1 is required for progression of the G 1 phase and is, therefore, a critical target for proliferative signals in G 1 (23,24). Immunoneutralization and antisense studies have demonstrated that cyclin D1 is capable of shortening the G 1 phase of the cell cycle, indicating that cyclin D1 is rate-limiting in fibroblast G 1 phase progression (27)(28)(29). Cyclin D1 expression is induced by several different growth factors including colony stimulating factor (CSF-1) and epidermal growth factor (30 -32). A role for cyclin D1 in AII signaling, to our knowledge, has not been examined. Several of the intermediary proteins implicated in AII signaling, including p21 ras and c-Jun, however, are capable of inducing either cyclin D1 mRNA levels or promoter activity in fibroblast cell lines (31,33,34). Accordingly, we reasoned that this hormone could stimulate cyclin D1 as well.
As AII induces cellular proliferation and DNA synthesis in adrenal cortical cells (1, 2), we used a human adrenal cell line to investigate a possible role of AII in regulating cyclin D1 expression. Because phosphorylation of the pRB protein appears to be necessary for its inactivation and this may be required for cell cycle progression, we examined the ability of AII to stimulate cyclin D1-dependent kinase activity using pRB as a substrate. Since we observed that AII induced cyclin D1 expression and promoter activity, we further examined the hypothesis that p21 ras and ERK may be involved in AII-mediated induction of the cyclin D1 promoter.

MATERIALS AND METHODS
Construction of Plasmid Vectors-The human cyclin D1 promoter was linked to the luciferase reporter gene using the vector pA 3 LUC (31). This vector includes the trimerized SV40 poly(A) termination site, which abolishes transcriptional readthrough (35) and does not contain the AP-1-responsive vector backbone sequences (36). A series of 5Ј promoter deletion constructions derived from this plasmid were described recently (31).
The human c-fos promoter from Ϫ361 to ϩ157 was cloned by PCR using oligodeoxyribonucleotide probes to the published sequence and human genomic DNA and was subcloned into the pA 3 LUC reporter. The 5Ј oligo primer sequence was 5Ј-GGT ACC GCC CGC GAG CAG TTC CCG-3Ј, and the 3Ј oligo sequence was 5Ј-AAG CTT CGT GGC GGT TAG GCA AAG-3Ј. The vector p 3 TP-LUX, which contains three collagenase AP-1 sites, was described previously (37,38). Restriction enzyme analysis and dideoxy DNA sequencing using an Applied Biosystems 373 automated sequencer confirmed the integrity of these constructs.
Cell culture, DNA transfection, and luciferase assays were performed as described previously (31,38), and the H295R cells (41) were described previously. Cells were maintained in a 1:1 mixture of Dulbecco's modified Eagle's and Ham's F-12 medium (DME/F12 containing pyridoxine HCl, L-glutamine and 15 mM HEPES; Life Technologies, Inc. Gaithersburg, MD) supplemented with insulin (6.25 mg/ml), transferrin (6.25 g/ml), selenium (6.25 g/ml), linoleic acid (5.35 g/ml), 1% ITS plus (Collaborative Research, Bedford, MA), 2% low protein serum replacement, (LPSR-1, Sigma, St. Louis, MO), and the antibiotics, 1% penicillin and 1% streptomycin. Cells were transfected by calcium phosphate precipitation, the medium was changed after 6 h, and luciferase activity was determined after an additional 24 h. At least three different plasmid preparations of each construct were used. In co-transfection experiments, a dose response was determined in each experiment with 300 and 600 ng of expression vector and the cyclin D1 promoter reporter plasmids (4.8 g). In co-transfection experiments, comparison was made between the effect of transfecting the expression vector with an equal amount of the parental empty expression vector to avoid spurious effects of the expression vector cassette. The fold effect was determined for 600 ng of expression vector. Luciferase assays were performed at room temperature using an Autolumat LB 953 (EG&G Berthold). Luciferase content was measured by calculating the light emitted during the initial 30 s of the reaction, and the values are expressed in arbitrary light units (38). The background activity from cell extracts was typically Ͻ150 arbitrary light units/30 s. Statistical analyses were performed using the Mann-Whitney U test. Significant differences were established as p Ͻ 0.05.
Oligodeoxyribonucleotides and Electrophoretic Mobility Gel Shift Assays-The AP-1 site of the cyclin D1 promoter, the wild-type AP-1 (CD1AP-1wt) site, and a mutant AP-1 (CD1AP-1mt) site were synthesized as complementary oligodeoxyribonucleotide strands for electrophoretic mobility gel shift assays (EMSA). The antisense strands of these oligodeoxyribonucleotides were also used for PCR-directed amplification of the promoter. For the sequence of the cyclin D1 promoter AP-1 site oligodeoxyribonucleotides, CD1AP-1wt was TCC ATT CTG ACT CAT TTT TTT TAA, and CD1AP-1mt was TCC ATT CTG CCG CAT TTT TTT TAA. The sequences of the wild-type collagenase AP-1 oligodeoxyribonucleotides (42) used as competitor in EMSA was AP-1wt 5Ј-CGC TTG ATG AGT CAG CCG GAA. The sequence of the 3ЈCD1 amplimer used in PCR directed amplification of the promoter was 5Ј-TGG GGC TCT TCC TGG GCA.
EMSAs using nuclear extracts, in vitro translated proteins, or bacterially expressed c-Jun (Promega Corp., Madison, WI) were performed essentially as described previously (38). The cDNAs were transcribed in vitro and translated using the TNT-coupled reticulocyte lysate system according to the protocol of the suppliers (Promega). The programmed lysates (5 l) were incubated in a reaction mix (20 l) consisting of 20 mM HEPES, pH 7.8, 50 mM KCl, 1 mM EDTA, 10% glycerol, 1 mM dithiothreitol, and 50 g/ml poly(dI⅐dC) at room temperature for 15 min. ␥ 32 P-Labeled oligonucleotides (50 fmol, 50,000 cpm) were added to the reaction and incubated at room temperature for an additional 15 min. The protein-DNA complexes were analyzed by electrophoresis through a 5% polyacrylamide gel, with 0.5 ϫ Tris borate, EDTA buffer (TBE: 0.045 M Tris borate and 0.001 M EDTA) and 2.5% glycerol. For EMSA, 5-10 g of nuclear extracts were used in binding buffer containing 20 mM HEPES, pH 7.4, 40 mM KCl, 1 mM MgCl 2 , 0.1 mM EDTA, and 0.1% Nonidet P-40, to which 0.5 ng of ␥ 32 P-labeled probe and 2 g of sonicated salmon sperm DNA were added. Supershifts were performed using antibodies referred to as c-Jun (KM-1) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and c-Fos antibody (Santa Cruz Biotechnology, Inc.). The reaction products were separated on 5% polyacrylamide gel run in 0.5 ϫ Tris borate-EDTA at room temperature at 180 V for 2-4 h. The gels were dried and exposed to XAR5 (Kodak, Rochester, NY) radiographic film.
Reagents and Flow Cytometric Analyses-AII (Sigma) and the selective AT 1 receptor antagonist L-158,908 (DuPont Merck Pharmaceutical Co.) were reconstituted and stored as recommended by the manufacturer.
Flow cytometric analyses was carried out in a fluorescence-activated cell sorter (FACStar plus; Beckton Dickonson). DNA synthesis of the synchronized cells was determined by detection of 5-bromodeoxyuridine incorporation into DNA essentially as described (43). The cells were grown in six-well culture dishes, and 5-bromodeoxyuridine was added with serum. All nuclei were counterstained with Hoechst 33258 (Sigma).
Reverse Transcription-Polymerase Chain Reaction-Total RNA extracted from cells using Tri-reagent (Molecular Research, Inc., Cincinnati, OH), as described by the manufacturer, was subjected to reverse transcription-polymerase chain reaction with specific amplification of cyclin D1 mRNA. The cyclin D1 oligodeoxyribonucleotides were synthesized to anneal to sequences from exons 2 and 5 (44), spanning two introns, to give a 528-bp product. The 5Ј primer sequence is 5Ј-GTC TGC GAG GAA CAG AAG-3Ј and the 3Ј sequence is 5Ј-GCA GGC CCG GAG GCA GTC-3Ј. The primer sequences of the human ribosomal protein L19 (45) used as an internal standard for reverse transcriptionpolymerase chain reaction are 5Ј-CCA TGA GTA TGC TCA GGC TTC and 3Ј-TGC TCT TAG ACC TGC GG CCT and produced a 500-bp PCR product. Human ribosomal protein L19 primers were included in each PCR reaction, and relative cyclin D1 mRNA abundance was expressed as a ratio of specific transcript to internal standard. The PCR products were analyzed by electrophoresing a 20-l aliquot on a 5% polyacrylamide gel run in 0.5 ϫ Tris borate-EDTA at room temperature at 250 V for 3 h. Autoradiography was performed at Ϫ70°C with an intensifying screen using XAR5 film or a phosphorimager. Initial confirmation of the specificity of the 528-bp cyclin D1 PCR product was determined through subcloning and sequence analyses. No amplified products were detected in PCR reactions that lacked reverse transcriptase or mRNA. Quantitation was performed by densitometry using a Fuji Bio Imaging Analyzer BAS 2000.
Western Blots-The abundance of cyclin D1 protein was determined by Western blotting as described previously (37) using a monoclonal cyclin D1 antibody (HD-11) (Santa Cruz Biotechnology, Inc.) and an antimouse second antibody. Reactive proteins were visualized by the enhanced chemiluminescence system (Amersham, Arlington Heights, IL). Quantitation was performed by densitometry using a Fuji Bio Imaging Analyzer BAS 2000.
p42 ERK , p44 ERK , and SAPK Immune Complex Assays-Assays were performed as described previously (31,37,46)  Cyclin D1 Immune Complex Assays-Immunoprecipitation kinase assays were performed essentially as described previously (47). Cells were suspended at 1 ϫ 10 6 to 5 ϫ 10 6 /ml in immunoprecipitation (RIPA) buffer at 4°C. Lysates were centrifuged at 10,000 ϫ g for 5 min, and the supernatants were precipitated for 4 h at 4°C with protein A-Sepharose beads precoated with saturating amounts of the cyclin D1 antibody (DCS-11; NeoMarker, Freeman, CA). Immunoprecipitated proteins on beads were washed twice with 1 ml of RIPA buffer and twice with kinase buffer (50 mM HEPES, pH 7.0, 10 mM MgCl 2 , 5 mM MnCl 2 , 1 mM dithiothreitol, and 10 M ATP). The beads were then suspended in 40 l of kinase buffer containing the protein substrate (2 g of soluble glutathione S-transferase-RB fusion protein, 2.5 mM EGTA, 10 mM ␤-glycerophosphate, 0.1 mM sodium orthovanadate, 0.1 mM NaF, 20 M ATP, and 10 Ci of [␥ 32 P]ATP (3000 Ci/mmol; 1 Ci ϭ 37 GBq). The samples were incubated for 30 min at 30°C with occasional mixing, and the samples were run after boiling in polyacrylamide gel sample buffer containing sodium dodecyl sulfate and separated by electrophoresis. Phosphorylated proteins were visualized by autoradiography of the dried gels, and quantitation was performed by densitometry using a Fuji Bio Imaging Analyzer BAS 2000.
The pRB substrate was prepared by transforming Escherichia coli with the vector pGEX-Rb (48) (a gift from Dr. E. Harlow) and growing the bacteria to an absorbance of 0.595 m. The fusion protein was induced by addition of 0.2 mM isopropylthioglycoside to the culture for 4 h. Cells were recovered by centrifugation (5000 ϫ g for 5 min) at 4°C and lysed on ice by sonication in a 1/10 volume of NETN buffer (50 mM Tris HCl, pH 7.5, 120 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40). The lysates were mixed with glutathione-Sepharose 4B (Pharmacia Biotech Inc.) and incubated for 2 h at 4°C. Beads were washed three times with kinase buffer, and the pRB fusion protein was released at 4°C by incubation in kinase buffer containing 2 mM reduced glutathione (Sigma).

RESULTS
AII Stimulates G 1 Phase Progression-To determine whether AII stimulates cell cycle progression in H295R cells, flow cytometric analysis was performed. Cells were treated with AII (10 Ϫ6 M) for 6 -72 h, and the proportion of cells in G 1 , S, and G 2 -M was determined at 12, 36, and 48 h (Table I). In six separate experiments (Table I), the relative proportion of cells in G 1 was reduced from 73 to 58% (p Ͻ 0.05) at 48 h, concomitant with an increase in cells in S phase (from 17 to 26%, p Ͻ 0.05) ( Table I). The proportion of cells in G 2 -M increased from 9.7 to 15% (p Ͻ 0.05). At 72 h, the proportion of cells in G 1 phase was further reduced to 51% (data not shown). Together, these studies indicate that AII decreases the proportion of cells in G 1 and increases the proportion of cells in S phase and G 2 -M, and that the effect persists after 48 h of treatment.
AII Stimulates Cyclin D1 Expression and Kinase Activity-To determine whether cyclin D1 expression is regulated by AII, the abundance of cyclin D1 mRNA was assessed using reverse transcription-polymerase chain reaction. Comparison of cyclin D1 mRNA abundance was made with the relative abundance of the human ribosomal protein L19 (45). Cyclin D1 mRNA was induced 4 -5-fold at 6 h of exposure to AII (Fig. 1A). At 12 h, the abundance of cyclin D1 mRNA remained elevated 2-3-fold, although it started to decline and had returned to baseline at 24 h (Fig. 1A).
The effect of AII on nuclear cyclin D1 protein levels was determined using Western blot analysis and the cyclin D1 antibody, HD-11. AII induced cyclin D1 protein levels 2-fold within 3 h, with a 5-fold increase at 6 h (Fig. 1B). Cyclin D1 protein abundance remained elevated at 12 and 24 h of treat-  1. AII induces cyclin D1 protein and kinase activity in H295R cells. A, reverse transcription-polymerase chain reaction was performed using mRNA derived from the AII (10 Ϫ6 M) treated H295R cells. Fold induction is shown with comparison to the human ribosomal protein L19 mRNA in the same samples. Data are shown for the mean of three separate experiments; bars, S.E. B, Western blot analysis of cyclin D1 protein levels, from H295R cells treated with AII (10 Ϫ6 M) for the time points indicated, was conducted using the monoclonal cyclin D1 antibody HD-11. The control lane contains in vitro translated cyclin D1. C, immune complex assays were conducted using extracts from H295R cells treated with AII for the time points indicated. Immunoprecipitation was conducted using the cyclin D1-specific antibody DCS-11, and phosphorylation of the GST-pRB substrate was performed as described in "Materials and Methods". The phosphorylated pRB band is indicated by the arrow (pRB). ment at a time when mRNA levels were declining (Fig. 1,  compare A and B).
Cyclin D1-dependent kinase activity was assessed by the immunoprecipitation assay, using the cyclin D1 antibody DCS-11 (49) with the GST-pRB protein as a substrate (47). In H295R cells treated with AII for 3-24 h, cyclin D1 kinase activity increased 2-fold after 12 h of treatment with AII and 6-fold after 24 h of treatment (Fig. 1C). Thus, the increase in activity was delayed by several hours compared to the increase in cyclin D1 mRNA (Fig. 1A) and protein levels (Fig. 1B), which were observed after only 6 h of exposure to AII.
AII Stimulates Cyclin D1 Promoter Activity in an AT 1 -dependent Manner-To determine whether AII was capable of inducing cyclin D1 promoter activity, the Ϫ1745 bp human cyclin D1 promoter fragment linked to the luciferase reporter gene (Ϫ1745 CD1LUC) was transiently transfected into H295R cells. Cells were then treated with AII to determine whether this hormone induces the cyclin D1 promoter and whether such an effect is concentration-dependent. AII stimulated cyclin D1 promoter activity in a dose-dependent manner, reaching a maximal increase (4-fold above basal) at 10 Ϫ7 and 10 Ϫ6 M (n ϭ 28; Fig. 2, A and B). Stimulation (2-fold) was observed at 10 Ϫ9 M AII. The 4-fold induction of the human cyclin D1 promoter by AII was completely inhibited by the AT 1 receptor antagonist L-158,809 (10 Ϫ6 M). (A representative example from six separate experiments is shown in Fig. 2A).
To examine further the specificity of the AII-dependent induction of the cyclin D1 promoter, cells were transfected with other native or synthetic promoters with identical plasmid backbone sequences. The pA 3 LUC vector alone was not induced by AII nor were the reporters TKLUC or RSVLUC, indicating that specific promoter sequences are required for AII induction. To examine the effect of AII on the steroidogenic P450 side chain cleavage (CYP11A1) gene, transient expression studies were conducted with the Ϫ2700 CYPLUC reporter (37,50). The Ϫ2700 CYPLUC promoter was induced 1.7-fold by AII (Fig. 2B).
Because AII treatment was previously shown to induce c-fos mRNA (21), we examined whether AII was capable of inducing the c-fos promoter in H295R cells. The c-fos LUC reporter, which contains human c-fos promoter sequences from Ϫ361 to ϩ157, was induced 5-fold by AII (10 Ϫ7 M). The AII stimulation of the c-fos promoter was dose-dependent, reaching a maximal 8-fold above basal at 10 Ϫ6 M. The induction of the c-fos LUC reporter by AII was also inhibited by the AT 1 receptor antagonist L-158,809 (10 Ϫ6 M) (Fig. 2C).
A Distal Enhancer Sequence Is Required for AII-dependent Induction of the Cyclin D1 Promoter-The DNA sequences required for AII-dependent induction of the cyclin D1 gene were investigated further using a series of 5Ј promoter deletions of the cyclin D1 promoter (Fig. 3A). Deletion of sequences from 1093 to Ϫ964 did not affect induction by AII, whereas deletion from Ϫ964 to Ϫ420 reduced induction from 3-to 1.3-fold (Fig.  3B). The region of the cyclin D1 promoter required for activation by AII was, therefore, located between Ϫ964 and Ϫ420. Within this region, sequences resembling an AP-1 site were identified. The effect of point mutations of these AP-1 like sequences in the context of the Ϫ964-bp promoter fragment was determined. Mutation of the AP-1 site at Ϫ954 reduced basal reporter activity by 60% and virtually abolished induction by AII (1.4-fold, Fig. 3C). p21 ras Is Required for AII Induction of the Cyclin D1 Promoter-Because of the evidence for p21 ras in AP-1-mediated signal transduction, the role of p21 ras in basal and AII-regulated cyclin D1 promoter activity was examined using transient expression studies and an expression vector for the dominantnegative p21 ras mutant (Ras N17). Comparison was made with the effect of the empty expression vector cassette. Ras N17 reduced basal level activity of the Ϫ1745 CD1LUC reporter by 10% (p Ͻ 0.01), whereas AII (10 Ϫ6 M) increased it 4-fold (Fig.  4A). Overexpression of p21 ras N17 reduced AII-induced cyclin D1 transcription by 60% (Fig. 4A).
The effect of an activating p21 ras mutant on cyclin D1 promoter activity was also examined. Ras Leu 61 induced cyclin D1 reporter activity 6-fold compared with basal activity, and the addition of AII induced it even further (13-fold, Fig. 4B). By contrast, the expression vector encoding the double mutant, Ras Leu 61/Ser 186, which is incapable of insertion in the plasma membrane, did not induce cyclin D1 promoter activity (1-fold, n ϭ 9; data not shown).
As a form of positive control, the effect of p21 ras N17 on the AII induction of the c-fos promoter was determined (Fig. 4C). Basal c-fos promoter activity was reduced to 50% of wild type by overexpression of the N17Ras vector. The induction of this promoter by AII was reduced 60% by N17Ras (Fig. 4C). Ras Leu 61 induced c-fos LUC reporter activity 9-fold (Fig. 4D).
ERK Activity Is Both Induced by AII and Required for AII Induction of the Cyclin D1 Promoter-Several MAPK pathways have been implicated in p21 ras -mediated signal transduction.
To determine whether AII induces ERK activity in H295R cells, immune complex assays were performed on extracts derived from cells treated with AII (10 Ϫ6 M) for 5 min to 24 h. AII increased ERK activity (7-fold) within 5 min; after 1 h of exposure to AII, ERK activity started to decline, but it remained elevated at 6 h and returned to basal within 24 h (Fig. 5A). The kinetics of ERK induction by AII were similar whether immunoprecipitation was performed with the p44 ERK or the p42 ERK antibodies (data not shown). Immunoprecipitation kinase assays performed on AII-treated extracts using antibody directed toward the SAPKs (46), and GST c-Jun as substrate, demonstrated that SAPK activity was increased 3-5-fold (Fig. 5B). A maximal 5-fold increase was observed at 30 min, followed by a return toward baseline after 1 h (Fig. 5B).
Because previous studies in fibroblast cell lines had demonstrated that the c-fos promoter was induced by ERK, the effect of activating or dominant-negative ERK expression plasmids on basal and AII-induced c-fos promoter activity was assessed. Dominant-negative ERK expression vectors from human (ERK Ϫ h) (39) or Xenopus (ERK Ϫ x) (40) reduced AII-induced c-fos promoter activity by 60% (data not shown).
AII Stimulates AP-1 Binding to the Cyclin D1 Ϫ954 Region-Because AII induction of the cyclin D1 promoter required sequences within the Ϫ954 region AP-1 site, EMSA was performed with nuclear extracts from H295R cells to characterize the nature of the complex binding this site (Fig. 6A). Comparison was made with the binding of nuclear protein to the collagenase AP-1 site probe. The cyclin D1 AP-1 site bound a complex with similar electrophoretic mobility to the complex binding the wild-type collagenase AP-1 site probe (Fig. 6A,  lanes 1 versus 8). Binding to the cyclin D1 AP-1 site was competed by 100-fold molar excess of cyclin D1 AP-1 site probe or 100-fold molar excess of the collagenase AP-1 site (Fig. 6A,  lanes 2 and 4). Mutant AP-1 sequences did not inhibit binding of the nuclear complex to the cyclin D1 Ϫ954 region (Fig. 6A,  lane 3). The c-Jun antibody supershifted nuclear protein complex binding to the cyclin D1 Ϫ954 region (Fig. 6A, lane 5), whereas addition of the c-Fos antibody supershifted most of the complex binding to the cyclin D1 Ϫ954 region (Fig. 6A, lane 6). Equal amounts of control serum neither inhibited nuclear protein binding to the probe nor induced a supershift, indicating the specificity of the supershift observed (Fig. 6A, lane 7).
The effect of AII treatment on the nature of the complex binding to the cyclin D1 AP-1 site was examined using nuclear extracts from H295R cells treated with AII (10 Ϫ6 M) for 1 to 24 h (Fig. 6B). When equal amounts of total nuclear protein were incubated with either the cyclin D1 AP-1 site (Fig. 6B) or the collagenase AP-1 site (data not shown), the amount of protein binding increased with time of exposure to AII. The increase in binding was observed within 1 h and continued to increase at 24 h (Fig. 6B). Supershift assays were also performed using the c-Jun or c-Fos antibodies. The amount of c-Fos supershifted from the nuclear complex binding the cyclin D1 AP-1 site also increased with AII treatment (Fig. 6B). The amount of c-Fos supershifted from complexes bound to the collagenase AP-1 site also increased in nuclear extracts from cells treated with AII (data not shown). The c-Jun antibody efficiently shifted most of the complex binding the cyclin D1 AP-1 site at each of the time points studied, suggesting that c-Jun remained an important component of the complex binding this region in AII-treated cells.

DISCUSSION
Cyclin D1, the regulatory subunit of several CDKs, is required for and is capable of shortening the G 1 phase progression of the cell cycle (27)(28)(29). The induction of cyclin D1 serine/ threonine kinase activity promotes cell cycle progression and cellular proliferation by phosphorylating and inactivating the substrate pRB (23, 24). Inactivation of pRB is an essential prerequisite for passage of the cell through the restriction point, after which time the cell is irrevocably committed to cellular division (24). We now show that AII promotes cell cycle progression, cyclin D1 promoter activity, cyclin D1 mRNA levels, and cyclin D1 protein abundance in H295R cells. Furthermore, AII induced the activity of the cyclin D1-dependent kinase (CD 1 K) capable of phosphorylating and inactivating the pRB protein. Therefore, our finding that AII induced CD 1 K activity, assessed using pRB as a substrate, links G protein-coupled receptor signaling to the cell cycle regulatory machinery.
The induction of cyclin D1 promoter activity, mRNA levels, protein abundance, and then CD 1 K activity by AII occurred sequentially. We found a rapid induction of cyclin D1 mRNA by AII consistent with regulation, at least initially, at the transcriptional level, as shown previously for other growth factors (30 -32). Furthermore, our findings are also consistent with a model proposed previously in which the induction of CD 1 K activity occurs through temporally sequential induction and complex assembly. The induction of CDK activity followed an increase in protein abundance (26,47,51). The mechanisms responsible for the brief delay in AII-enhanced CD 1 K activity after cyclin D1 protein levels had increased substantially (5fold at 6 h) are unknown. The induction of CD 1 K activity may require a greater amount of cyclin D1 protein or may require the association of CD 1 K with other cofactors, such as the cyclinassociated kinase (26,47,51).
AII was further shown to stimulate the cyclin D1 promoter, and the dominant-negative p21 ras N17 mutant antagonized a component of AII-induced transcriptional induction of the cyclin D1 promoter. Because AII is involved in the normal proliferative response to intimal trauma (52)(53)(54) and dominantnegative p21 ras mutants inhibited part of the vascular proliferative response induced by vascular injury in vivo (55), it is likely that p21 ras conveys an important component of AII signaling in vivo. Both p21 ras and pp60 c-src have complementary roles in several different signal transduction pathways. A component of phospholipase C␥-1 activation by AII requires the pp60 c-src in rat aortic smooth muscle cells (56). Thus, both p21 ras and pp60 c-src have been implicated in AII signaling. Interestingly, overexpression of either p21 ras or pp60 c-src was capable of inducing cyclin D1 expression and G 1 phase progression in fibroblast cell lines (34). In our studies, constitutively active p21 ras mutants induced cyclin D1 promoter activity. In addition, pp60 v-src is also capable of activating the cyclin D1 promoter in a robust manner. 2 Whether the residual p21 rasindependent component of AII-mediated induction of the cyclin D1 promoter involves pp60 c-src remains to be determined.
Previous studies investigating the signal transduction pathway conveying the mitogenic action of AII demonstrated the rapid phosphorylation of intermediary kinases at tyrosine residues (13,14,16). The induction of tyrosine phosphorylation is a common feature of many other G protein-coupled receptors. AII activates phospholipase C and Ca ϩ2 pathways in cultured glomerulosa cells (8,57), and induction of these secondary messengers have been shown to induce ERK (9 -11). AII was also shown previously to stimulate tyrosine phosphorylation of ERK (13,14). Our study demonstrates a requirement for ERK in the induction of the cyclin D1 promoter by AII as the dominant-negative ERK mutants reduced AII-mediated cyclin D1 promoter activity. This finding is consistent with studies performed in other cell types in which the induction of ERK activity by AII was associated with cellular proliferation. Our studies provide a mechanism by which the induction of ERK activity by AII may be linked directly to proliferative signaling through inducing cyclin D1 expression and pRB phosphorylation.
Overexpression of ERK is capable of inducing cellular proliferation (58,59), and suppression of ERK activity antagonizes cellular proliferative responses (60). ERK induction may impart genotypic cues that may vary with the cell type (60). The sustained induction of ERK activity is associated with a proliferative response in fibroblasts, whereas PC12 cells undergo differentiation (58 -60). In the studies described herein, the induction of cell cycle progression by AII in H295R cells was associated with a rapid and sustained activation of ERK. The mechanisms by which activation of ERK triggers cellular proliferation are unknown: however, genes capable of modulating cell cycle progression, such as cyclin D1, represent likely targets.
The induction of AP-1 binding to the cyclin D1 AP-1 site by AII is likely mediated through a mechanism that involves the induc-2 G. Watanabe, R. J. Lee, and R. G. Pestell, unpublished data.
FIG. 5. ERK is required for AII-induced cyclin D1 and c-fos promoter activity. H295R cells were treated with AII (10 Ϫ6 M) for the time points as indicated and ERK (A) and SAPK (B) activity was determined. Extracts were immunoprecipitated using the anti-ERK antibody ERK2, p42 MAPK , or K-23 or the SAPK antibody (46). Relative fold induction was determined by comparison with untreated cells using densitometry. The data of a representative experiment are shown. (Similar results were obtained using the anti-ERK antibody ERK1 (C16). C, the Ϫ1745 CD1LUC reporter was co-transfected with the dominant-negative ERK expression vectors either alone or in the presence of AII. The dominant-negative ERK expression vector ERK Ϫ h is the human ERK dominant negative (39) and ERK Ϫ x is the Xenopus form (40). The mean data (bars, S.E.) of 8 -13 separate transfections are shown.
tion of c-Fos. AII treatment increased c-Fos abundance within the AP-1 complex, and the induction of c-Fos enhanced the affinity of AP-1 proteins for AP-1 sites (61). Furthermore, the c-fos promoter was induced by AII in a p21 ras and ERK-dependent manner (Fig. 2). Both ERK and SIF, which can be induced by AII (17), have been shown to induce the c-fos promoter (18,62); ERK2 phosphorylates and potentiates the activation of TCF/Elk1 and thereby induces c-Fos expression (63,64). At least a significant component of the TCF/Elk1-mediated activation is p21 rasdependent (62). Induction of ERK activity phosphorylates inhibitory DNA binding domains of c-Jun, thereby enhancing c-Jun binding to AP-1 sequences (61,65).
AII is well known to activate through a G protein-coupled receptor signaling pathway (6,7). The induction of SAPK activity by AII demonstrated in this study is consistent with a recent study in which overexpression of activating mutants of G␣ 12 and G␣ 13 lead to the induction of SAPK activity (66). In a very recent study by Zohn et al. (15), AII also stimulated SAPK (Jun kinase) activity in the rat liver epithelial cell line GN4 (15), and this effect was reduced by calcium chelating agents, consistent with an intermediary role for Ca 2ϩ (15). The induction of SAPK activity and cyclin D1 promoter and kinase activity by AII are consistent with an intermediary role for the targets of SAPK, c-Jun (46), or ATF-2 (67) in regulation of CD 1 K activity. AII induces c-jun mRNA in cultured human and bovine adrenal cells (21,68). Previous antisense and immunodepletion studies were consistent with a role for c-Jun and AP-1 activity in promoting G 1 phase progression and DNA synthesis (69,70). Although the intermediary targets genes conveying AP-1-dependent G 1 phase progression were unclear, c-Jun is capable of activating the cyclin D1 promoter through the Ϫ954 region (31). The induction of SAPK activity by AII may contribute to enhanced transactivation by c-Jun (46,71) and thereby induce cyclin D1 promoter activity.
AII is involved in the excessive proliferation of vascular smooth muscle cells following angioplasty, which plays an important role in restenosis (52,53). Because AII induces c-Fos and c-Jun in vascular smooth muscle cells (19,20), it will be of interest to determine whether AII induces AP-1 activity, cyclin D1 expression, and thereby mitogenesis in these cells. Furthermore, constitutive activation of G protein-coupled receptors is associated in some circumstances with tumor formation, although the molecular targets involved remain to be elucidated (72). Since overexpression of cyclin D1 is associated with a variety of tumors and cyclin D1 overexpression in the breast of transgenic animals has been shown to induce breast tumor formation (73), it will be of interest to determine the role of cyclin D1 in activating G protein-coupled receptor tumor formation.