INTRODUCTION

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THE ras PROTOONCOGENE FAMILY is constituted by several 21-kDa guanine nucleotide-binding proteins that regulate growth and differentiation of mammalian cells (2). These guanosine triphosphatases (GTPases) couple the activation of cell surface receptor tyrosine kinases to multiple responses, including modulation of growth-related gene expression during mitogenesis (10, 36). p21^ras proteins bind GTP to achieve an active conformation that is rendered inactive on conversion to a GDP form by intrinsic GTPase activity (15). Reactivation is achieved by exchange of bound GDP for free cytosolic GTP (9). Two protein families have been identified that regulate Ras activity, guanine nucleotide-releasing factors, which catalyze the release of GDP, and GTPase-activating proteins, which accelerate GTP hydrolysis and Ras inactivation (20).

Overexpression of wild-type ras has been associated with oncogenic transformation in cultured NIH/3T3 cells (25) as well as induction of tumors in transgenic mice (19). ras mutants have been implicated in tumor formation in humans (12) as well as laboratory animals (18). More recently, ras has been implicated as a critical regulator of growth and differentiation in vascular smooth muscle cells (VSMC) (30). Single-point mutations in the ras gene affect intrinsic GTPase activity or guanine nucleotide exchange, resulting in persistent activation of Ras and a sustained mitogenic response (2). Mitogenic events involving Ras have been associated with modulation of protein kinase C (PKC) in several cell types. A Ras/PKC interaction is suggested by studies showing that transfection of a dominant negative kinase-inactive form of c-raf (17) or antisense c-raf blocked serum- and 12-O-tetradecanoyl phorbol13-acetate (TPA)-induced mitogenesis as well as transformation by Ras (29). Although these experiments suggested that both PKC and Ras function downstream of receptor tyrosine kinases and upstream of Raf-1 kinase, the critical point(s) of PKC involvement relative to Ras in mitogenic signaling remains debated. For example, microinjection of antibodies against phospholipase C inhibits DNA synthesis induced by Ras, suggesting that p21^ras is upstream of PKC (33), but a Ras-neutralizing monoclonal antibody can block TPA-induced mitogenesis in quiescent fibroblasts (36).

PKC is the designator for a group of serine/threonine protein kinases similar in size, structure, and function which serve as key regulators of mammalian growth and differentiation (21, 22). These kinases transduce signals from a wide variety of stimuli including growth factors, hormones, and neurotransmitters (21, 22). At least 12 PKC isoforms have been identified to date and classified into three structurally related groups (12): 1) conventional PKC (, I, II, ), 2) novel PKC (, , /L, , µ), and 3) atypical PKC (, , ). Conventional PKC are commonly referred to as calcium/phospholipid-dependent PKC and are responsive to cis-unsaturated fatty acids and lysophosphatidylcholine. Novel PKC differ from the classic PKC in that they lack a requirement for calcium in the activation process. Conventional and novel PKC are activated by TPA and undergo proteolytic degradation and downregulation on prolonged exposure to TPA (23). Atypical PKC appear to possess activation profiles independent of both calcium and diacylglycerol (DAG).

Transfection of naive VSMC with the c-Ha-ras^EJ oncogene induces a transformation process characterized by morphological changes, anchorage-independent growth, enhanced mitogenic responsiveness, and the acquisition of epidermal growth factor (EGF) responsiveness (31). Oncogene-transfected cells developed prominent processes, including the appearance of focal cellular arrangements giving rise to latticelike structures, and grow in soft agar. Enhanced mitogenic responsiveness directly correlated with a marked increase in ras expression relative to naive and vector controls at low serum concentrations. -Smooth muscle actin gene expression was reduced in c-Ha-ras^EJ VSMC suggesting that these cells have undergone dedifferentiation toward a more primitive state. In view of the proposed parallel between atherogenesis and carcinogenesis, the ability of oncogenic ras to influence growth-related signal transduction in VSMC suggests that ras may participate in the atherogenic process. In the present studies, we have examined the impact of c-Ha-ras^EJ overexpression on PKC-related signal transduction in VSMC. Our results suggest that c-Ha-ras^EJ circumvents a critical requirement for TPA-sensitive PKC isoforms during mitogenic stimulation of serum-starved VSMC.

	MATERIALS AND METHODS

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Chemicals. Ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), Coomassie blue, sodium vanadate, and tris(hydroxymethyl)aminomethane (Tris) were purchased from Aldrich Chemical (Milwaukee, WI). Polydeoxyinosinic-deoxycytidylic acid [poly(dI-dC)] was from Boehringer Mannheim (Indianapolis, IN). TPA-responsive element (TRE) consensus sequence was obtained from Promega (Madison, WI). N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), benzamidine, and [methyl-³H]thymidine triphosphate (50 Ci/mmol) were obtained from ICN Biochemicals (Costa Mesa, CA). Acrylamide, sodium dodecyl sulfate (SDS), and pyronin Y were from Bio-Rad (Richmond, CA). Glycerol was obtained from J. T. Baker, and methanol was from Fisher Scientific (Fair Lawn, NJ). [-³²P]ATP (3,000 Ci/mmol) was from DuPont NEN (Boston, MA). All other chemicals were purchased from Sigma Chemical (St. Louis, MO).

Transfection and cell culture. The protocol for the stable c-Ha-ras^EJ or pSV2neo transfects used in the present studies has been described previously (31). Briefly, low-passage aortic SMC (P4) were seeded in 60-mm culture dishes at 50% confluence and incubated in serum-free Opti Eagle's minimum essential medium (MEM) for 24 h. pSV2neo with or without c-Ha-ras^EJ (40 µg/ml DNA) in 100 µl Tris-EDTA buffer was combined with 100 µl lipofectin reagent and allowed to incubate for 30 min at room temperature. Opti MEM (1.8 ml) was added, and the mixture was overlayered on cells and incubated for 24 h at 37°C. After transfection, cells were grown to confluence in medium containing 10% fetal bovine serum (FBS) and were split 1:3 for selection of transfected cells. Stable transfectants were selected by growing resistant colonies in the presence of 400 µg/ml Geneticin for 6 wk. Naive or transfected VSMC were maintained in medium 199 (GIBCO) supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 µg/ml amphotericin B in 5% CO₂-95% air at 37°C as described previously (27).

Homogenate preparation for measurements of PKC activity. VSMC were homogenized in buffer (volume adjusted to yield a final protein concentration 1 mg/ml) containing 0.32 M sucrose, 5 mM HEPES (pH 8.0), 5 mM benzamidine, 2 mM -mercaptoethanol, 3 mM EGTA, 5 µM magnesium sulfate, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 0.1 mg/ml leupeptin, 0.05 mg/ml pepstatin, 0.1 mg/ml aprotinin, and 1 mM sodium vanadate. Homogenates were centrifuged for 1 h at 100,000 g at 4°C to prepare cytosolic and particulate fractions. The supernatant from this centrifugation step was considered the cytosolic fraction. The remaining pellet, reconstituted in 150 µl of homogenization buffer supplemented with 0.1% (vol/vol) Triton X-100 and centrifuged at 3,000 g to remove Triton X-100-insoluble constituents, represented the particulate fraction. Protein concentration was determined by the method of Bradford (6) with bovine serum albumin as the standard.

PKC assay. Crude fractions containing 5 µg total protein were incubated in a reaction mixture containing 50 mM Tris · HCl (pH 7.6), 10 mM magnesium sulfate, 0.2 mM EDTA, 5 mM -mercaptoethanol, 10 µM ATP, and 15 µCi [-³²P]ATP as described (24). Histone IIIS was used as the phosphoacceptor substrate. PKC-dependent phosphorylation was measured in the presence of 0.1 µM TPA, 50 µg/ml phosphatidylserine, and 100 µg/ml adenosine 3',5'-cyclic monophosphate-dependent protein kinase inhibitor. PKC activity is defined as the difference between incorporated label in the presence of PKC activators or the PKC inhibitor PKC(19-36) (GIBCO). Calcium was not added to the reaction mixture to reduce interference by calcium-activated proteases and/or calcium-calmodulin-dependent kinases. Further, the use of phorbol esters for the activation of PKC renders low calcium concentrations adequate for activation (1, 8). The selective inhibitor PKC(19-36) was used to verify the specificity of the phosphorylation reactions by preventing TPA-stimulable kinase activity (not shown).

Electrophoretic mobility shift assay. Electrophoretic mobility shift assays (EMSA) were carried out as described previously (5). VSMC were collected and lysed in an HEGD buffer [25 mM HEPES (pH 7.6), 1.5 mM EDTA, 10% glycerol, 1 mM dithiothreitol (DTT), and 0.1 mg/ml PMSF] using 20 strokes with a Dounce homogenizer. The homogenate was centrifuged at 12,000 g at 4°C for 5 min. The supernatant was discarded, and the pellet was extracted with 30 µl HEGDK buffer [25 mM HEPES (pH 7.6), 1.5 mM EDTA, 10% glycerol, 1 mM DTT, 0.1 mg/ml PMSF, and 0.5 M KCl] for 1 h on ice. Extracted pellets were centrifuged at 16,000 g for 5 min at 4°C, and the supernatant was designated as the nuclear extract. For EMSA, 10 µg of nuclear extract were incubated in a reaction mixture consisting of 18.8 mM HEPES, 40 mM KCl, 1.1 mM EDTA, 7.5% glycerol, 0.75 mM DTT, and 62.5 ng/µl poly(dI-dC) for 15 min at 20°C to reduce interference by nonspecific DNA-binding proteins. DNA (0.1 ng) labeled with [-³²P]ATP (3,000-5,000 Ci/mmol) was added for 15 min to determine TRE binding activity. Bound DNA was separated on a 5% polyacrylamide nondenaturing gel for 2.5 h at 120 V. Gels were dried and exposed to X-OMAT X-ray film for autoradiography.

DNA synthesis. VSMC were seeded at equal densities on tissue culture dishes and allowed to attach for 24 h. For TPA treatments, cells were synchronized in the G_o phase of the cell cycle by incubation in 0.1% FBS for 72 h as described previously (30), challenged for 24 h with 100 ng/ml TPA during the final 24 h of serum deprivation to downregulate PKC activity, and subsequently stimulated with 10% FBS in the presence of 1 µCi/ml [³H]thymidine for 24 h. Measurements of [³H]thymidine triphosphate incorporation into DNA were as described previously (26).

Western blot for extracellular signal-regulated kinase. Extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2) were electrophoretically separated from their phosphorylated forms and detected by Western blot to assess ERK activity in ras-transformed VSMC (35). VSMC were homogenized in buffer [20 mM Tris (pH 7.5), 0.5 mM EDTA, 0.5 mM EGTA, 100 mM -mercaptoethanol, 0.1 mM PMSF, 0.1 mg/ml leupeptin, 0.1 mg/ml aprotinin, 0.1 mg/ml antipain, 1 µM okadaic acid, 1 mM sodium vanadate, 1 mM sodium fluoride, 1 mM sodium pyrophosphate, and 0.5% Triton X-100] using 30 strokes in a Dounce homogenizer. VSMC proteins (20 µg) were separated on a 15-cm slab gel using a low cross-linker 15% SDS-polyacrylamide gel (final concentration in gel: 15% acrylamide and 0.42% bisacrylamide). Rainbow-colored protein molecular weight markers (Amersham, Arlington Heights, IL) were used to monitor maximum separation of proteins in the 30- to 46-kDa range during electrophoresis. The 20- to 60-kDa portion of the gel was excised and transferred onto a 0.2-µm nitrocellulose membrane using a Hoefer (San Francisco, CA) Mighty Small Transphor unit at 200 V for 1 h. Membranes were incubated with 5% nonfat dry milk in TBS-T [Tris-buffered saline containing Tween 20; 20 mM Tris (pH 7.6), 137 mM sodium chloride, and 0.1% Tween 20] for 1 h at room temperature to block nonspecific binding. Immunodetection was performed using the enhanced chemiluminescence system as described by the manufacturer (Amersham). For ERK1/ERK2 immunodetection, primary antibody (ERK1, no. sc-94; Santa Cruz Biotechnology, Santa Cruz, CA) was diluted 1:10,000 in TBS-T, biotinylated goat anti-rabbit immunoglobulin G (heavy and light chain) secondary antibody (Vector Laboratories, Burlingame, CA) was diluted 1:20,000 in TBS-T, and an avidin biotinylated hormone-related protein system was used for increased sensitivity (Vectastain Elite ABC kit; Vector Laboratories, Burlingame CA).

Statistics. Individual comparisons were made using Student's t-test or analysis of variance as appropriate. The P < 0.05 level was accepted as significant.

	RESULTS

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Transfection of c-Ha-ras^EJ into VSMC was associated with increased PKC activity in both the cytosolic and particulate compartments, relative to pSV2neo vector controls (Fig. 1). No difference in cytosolic or particulate PKC activities between vector and naive VSMC was observed (naive cytosol, 3,801 ± 158; naive particulate, 2,181 ± 613; vs. pSV2neo cytosol, 4,259 ± 204; pSV2neo particulate, 2,789 ± 416; n = 3). To further investigate the fidelity of the PKC signal-transduction cascade, VSMC were maintained in 0.1% FBS for 72 h and challenged with 100 ng/ml TPA for 3 h, and TRE-binding activity was evaluated in nuclear extracts from treated cells. Serum-restricted c-Ha-ras^EJ VSMC exhibited enhanced constitutive and TPA-inducible TRE-binding activities, relative to naive and vector controls (Fig. 2A). Specificity of the binding reaction was confirmed by addition of excess unlabeled TRE, which competitively eliminated the inducible bands, as well as by addition of unrelated DNA, which was without effect (Fig. 2B). Multiple TRE-binding complexes were observed in c-Ha-ras^EJ transfected VSMC but were absent in naive or vector controls. The multiple TRE-binding complexes observed in c-Ha-ras^EJ nuclear extracts were not present in nuclear extracts supplemented with PMSF, a broad spectrum protease inhibitor, indicating that these bands are degradation products (data not shown). These data demonstrate an intact PKC signal-transduction cascade in c-Ha-ras^EJ VSMC.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Protein kinase C (PKC) activity in cytosolic and particulate fractions of rat aortic smooth muscle cells (SMC) transfected with pSV2neo vector (open bars) or c-Ha-rasEJ oncogene (solid bars). SMC were processed for measurements of PKC activity as described in MATERIALS AND METHODS. Values represent means ± SE. * P < 0.05 vs. respective control. Similar results were observed in 3 separate experiments. cpm, Counts per minute.

View larger version (69K): [in this window] [in a new window]   View larger version (60K): [in this window] [in a new window]   Fig. 2.   A: induction of activator protein 1:12-O-tetradecanoyl phorbol-13-acetate (TPA)-responsive element (AP-1:TRE)-binding activity in nuclear extracts from naive, pSV2neo, or c-Ha-rasEJ SMC treated with TPA. Nuclear extracts from SMC treated with 100 ng/ml TPA for 2 or 3 h were incubated with a 32P-labeled TRE. Protein-DNA complexes were analyzed by gel electrophoresis and autoradiography. B: specificity for induction of AP-1:TRE-binding activity in nuclear extracts from naive, pSV2neo, or c-Ha-rasEJ SMC treated with TPA. Induced AP-1:TRE complexes are indicated and specificity of binding reaction is confirmed by addition of a 100-fold excess of unlabeled TRE, which competitively eliminated induced bands, as well as excess unlabeled nontarget DNA (DRE, dioxin responsive element), which had no effect on induced bands.

The PKC inhibitor calphostin C was used to evaluate the relative contributions of PKC to constitutive TRE-binding activity in serum-restricted VSMC. VSMC were serum deprived by incubation in 0.1% FBS for 72 h and were incubated with 100 ng/ml TPA during the final 24 h of serum starvation. This regimen is associated with downregulation of PKC activities in VSMC irrespective of their proliferative phenotype (3, 4). Calphostin C (100 nM; 5 h) reduced basal TRE-binding activity in c-Ha-ras^EJ VSMC by 30% (Fig. 3). Pretreatment of VSMC with TPA abolished the inhibitory actions of calphostin C on constitutive TRE-binding activity in c-Ha-ras^EJ VSMC. In addition, downregulation of PKC by TPA resulted in a modest increase of TRE-binding activity in both c-Ha-ras^EJ and pSV2neo VSMC. Collectively, these data suggest a minor contribution by PKC to the increase of constitutive TRE-binding activity in serum-starved c-Ha-ras^EJ VSMC. In contrast to TPA, treatment of serum-restricted c-Ha-ras^EJ and pSV2neo VSMC with 10% FBS for 3 h resulted in the induction of TRE-binding activity in nuclear extracts from pSV2neo, but not c-Ha-ras^EJ, VSMC (Fig. 4). Pretreatment of VSMC with 100 ng/ml TPA for 24 h to deplete PKC activity dramatically reduced FBS-mediated TRE-binding activity in vector controls.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effect of calphostin C on constitutive TRE-binding activity in nuclear extracts from pSV2neo or c-Ha-rasEJ SMC. SMC were serum deprived by incubation in 0.1% fetal bovine serum (FBS) for 72 h and exposed to DMSO or 100 ng/ml TPA during final 24 h of serum starvation to downregulate PKC activities. After this dosing regimen, cells were treated with 100 nM calphostin C for 5 h, and nuclear extracts were prepared as described in MATERIALS AND METHODS. Groups shown are DMSO control for calphostin C treatment (open bars) and calphostin C (solid bars). No differences between DMSO and naive groups were observed (data not shown). Nuclear extracts were incubated with a 32P-labeled TRE, and protein-DNA complexes were analyzed by gel electrophoresis and autoradiography.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   FBS-mediated TRE-binding activity in nuclear extracts from PKC-depleted pSV2neo or c-Ha-rasEJ SMC. Serum-starved SMC were treated with 100 ng/ml TPA for 24 h to downregulate PKC activity and were subsequently challenged with 10% FBS for 3 h. Nuclear extracts were prepared as described in MATERIALS AND METHODS. Groups shown are 0.1% FBS (open bars), 10% FBS (solid bars), DMSO + 10% FBS (hatched bars), and TPA + 10% FBS (crosshatched bars). Nuclear extracts were incubated with a 32P-labeled TRE, and protein-DNA complexes were analyzed by gel electrophoresis and autoradiography. Similar results were observed in 2 separate experiments.

To assess the impact of c-Ha-ras^EJ on PKC-dependent VSMC growth, we measured DNA synthesis in serum-starved PKC-depleted vector or c-Ha-ras^EJ VSMC. VSMC were serum deprived by incubation in 0.1% FBS for 72 h, incubated with 100 ng/ml TPA during the final 24 h of serum starvation, and subsequently treated with 10% FBS for 24 h in the presence of [³H]thymidine triphosphate. Depletion of TPA-sensitive PKC isoforms ablated serum-stimulated [³H]thymidine incorporation into DNA in naive and vector controls but was without effect in c-Ha-ras^EJ transfected VSMC (Fig. 5). After PKC downregulation, DNA synthetic rates in serum-deprived c-Ha-ras^EJ VSMC were increased ~50% relative to those of quiescent vector counterparts. DNA synthetic profiles in vector controls were comparable to naive VSMC after PKC downregulation (data not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Mitogen-induced DNA synthetic profiles in pSV2neo vector or c-Ha-rasEJ transfected rat aortic SMC after PKC depletion. SMC were incubated in 0.1% FBS for 72 h and treated with 100 ng/ml TPA during final 24 h of serum deprivation. PKC-depleted pSV2neo and c-Ha-rasEJ SMC were subsequently stimulated with 10% FBS for 24 h in presence of [3H]thymidine triphosphate. Groups represented are serum-deprived (open bars), 10% FBS-stimulated (solid bars), serum-deprived PKC-depleted (hatched bars), and 10% FBS-stimulated PKC-depleted (crosshatched bars) SMC. Values represent means ± SE. * P < 0.05 vs. serum-deprived SMC;  P < 0.05 vs. serum-deprived/PKC-depleted SMC;  P < 0.05 vs. pSV2neo serum-deprived SMC. Similar results were observed in 2 separate experiments.

Transfection of mammalian cells with oncogenic ras is associated with increased ERK activity. The active, phosphorylated form of ERK1 and ERK2 can be electrophoretically separated from the inactive form and detected by Western blot (35). This strategy was used to assess the impact of ras transfection and PKC downregulation on ERK activity in serum-restricted VSMC. VSMC were incubated in 0.1% FBS for 72 h, treated with 100 ng/ml TPA or DMSO during the final 24 h of serum starvation, and processed for measurements of ERK immunodetection as described in MATERIALS AND METHODS. Transfection of VSMC with c-Ha-ras^EJ was associated with increased expression of ERK2 (pSV2neo, 0.131 ± 0.014; c-Ha-ras^EJ, 0.220 ± 0.015 relative densitometric units), but not ERK1 (pSV2neo, 0.078 ± 0.007; c-Ha-ras^EJ, 0.081 ± 0.010 relative densitometric units), protein (Fig. 6). Under serum-starved conditions, the phosphorylated form of ERK2 was detected in both groups; however, the magnitude was dramatically increased in c-Ha-ras^EJ VSMC (band appearing above ERK2 in Fig. 6). The phosphorylated form of ERK1 was weakly detected and more difficult to resolve under these conditions. Downregulation of PKC activity by TPA did not decrease constitutive ERK activity in c-Ha-ras^EJ VSMC, as evidenced by the presence of the phosphorylated form of ERK2. Further, there appeared to be a modest increase in the intensity of the phosphorylated forms of ERK2 or ERK1 in pSV2neo and c-Ha-ras^EJ VSMC, respectively, after PKC depletion, consistent with a modest increase of constitutive TRE-binding activity in PKC-depleted cells (see Fig. 3).

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Western blot analysis of extracellular signal-regulating kinase 1 and 2 (ERK1 and ERK2) expression in serum-starved PKC depleted SMC. SMC were incubated in 0.1% FBS for 72 h and treated with 100 ng/ml TPA during final 24 h of serum deprivation. Cell proteins (20 µg) from pSV2neo and c-Ha-rasEJ SMC were separated on a 15% SDS-polyacrylamide gel and blotted to nitrocellulose, and ERK1/ERK2 expression was detected by enhanced chemiluminescence as described in MATERIALS AND METHODS. Similar results were obtained in 2 separate experiments.

	DISCUSSION

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ras Protooncogenes have been firmly established as central regulators of mammalian cell growth and differentiation (2). Overexpression of wild-type or mutant Ras proteins is associated with transformation of somatic cells, including VSMC (31). In the present studies, we have examined the impact of c-Ha-ras^EJ on PKC-related signaling in serum-starved VSMC and report that modulation of VSMC growth and differentiation by c-Ha-ras^EJ involves deregulation of this pathway. This conclusion is consistent with the observation that serum induces PKC-dependent TRE-binding activity in pSV2neo, but not c-Ha-ras^EJ, VSMC (Fig. 4) and that PKC depletion by TPA abrogates the DNA synthetic response to serum in vector controls but does not inhibit mitogenic responsiveness in c-Ha-ras^EJ VSMC (Fig. 5). Because long-term treatment with TPA downregulates PKC activity in proliferative VSMC overexpressing ras (3, 4), our findings suggest that Ras circumvents a requirement for TPA-sensitive PKC activities in mitogen-induced growth. In addition, transformation of mammalian cells with the ras oncogene has been associated with increased TPA-inducible protease activity (37). Consistent with this observation, multiple TRE-binding complexes are observed in TPA-treated VSMC (Fig. 2) but not in nuclear extracts supplemented with the protease inhibitor PMSF (data not shown).

VSMC transfected with the ras oncogene exhibited a marked proliferative advantage over controls under restrictive and growth-permissive serum conditions (Fig. 5). Western blot analysis of homogenates from serum-starved cells demonstrated increased expression of ERK2 and the phosphorylated form of ERK2 in c-Ha-ras^EJ VSMC, relative to vector controls (Fig. 6), consistent with the constitutive activation of this pathway by oncogenic ras (35). Thus, under serum-restrictive conditions, the Ras:ERK pathway appears to contribute to the enhanced proliferative advantage of c-Ha-ras^EJ VSMC. Downregulation of PKC by TPA did not modulate the constitutive activation of ERK2 (Fig. 6) or mitogen-induced DNA synthesis (Fig. 5) in c-Ha-ras^EJ VSMC, suggesting that TPA-sensitive PKC isoforms and oncogenic Ras are not positioned linearly on a single signal-transduction pathway in VSMC. In contrast, PKC depletion modestly increased the appearance of phospho-ERK2 in pSV2neo VSMC and phospho-ERK1 in c-Ha-ras^EJ VSMC (Fig. 6), and this response directly correlated with a modest increase of TRE-binding activity (Figs. 3 and 4), suggesting that TPA-sensitive PKC isoforms may negatively regulate the ERK pathway.

Transfection of C₃H/10T1/2 cells with the ras oncogene results in the downregulation of PKC (14). In contrast, the PKC signal-transduction cascade remains intact and is upregulated in c-Ha-ras^EJ VSMC. This statement is supported by the observation that PKC activity (Fig. 1) and TRE-binding activity (Fig. 2) are increased in c-Ha-ras^EJ VSMC, relative to vector control. Constitutive TRE-binding activity in serum-starved c-Ha-ras^EJ VSMC can be partially inhibited by calphostin C, a specific PKC inhibitor, and the inhibitory properties of calphostin C on TRE-binding activity are lost in PKC-depleted VSMC (Fig. 3). Collectively, these data suggest that PKC activity contributes to constitutive TRE-binding activity in serum-starved c-Ha-ras^EJ VSMC. The impact of ras transfection on PKC-related signaling may be due to increased DAG production, as shown previously in VSMC overexpressing c-Ha-ras (28). DAG is an endogenous activator of PKC, which could account for increased PKC activities (Fig. 1) and constitutive activator protein 1 (AP-1):TRE-binding activity (Fig. 2) in the absence of mitogens in c-Ha-ras^EJ VSMC (22). However, PKC depletion modestly increases TRE-binding activity (Figs. 3 and 4), apparently through an ERK-dependent mechanism (Fig. 6), but does not modulate the growth behavior of serum-starved VSMC (Fig. 5). Thus the role of PKC in ras-regulated growth programming remains unclear.

The mechanism for ras-mediated deregulation of PKC-dependent VSMC growth is not known but may be related to the activation of dormant mitogenic pathways that do not require PKC. For example, c-Ha-ras^EJ VSMC have acquired EGF responsiveness (31), and the EGF receptor couples directly to Ras via adapter proteins (e.g., Grb2) and guanine nucleotide-releasing factors for ras (e.g., Sos1) (32). Ras, in turn, is associated with the activation of ERK, which regulate several distinct nuclear transcription factors associated with mitogen-induced growth (7). The Ras:ERK pathway is predominantly associated with the induction of AP-1:TRE-binding activity (16); however, transcriptional activities such as the serum-response element binding complex (p62^TCF/Elk-1) are also positively regulated by this pathway (7). Within this context, the mitogenic response of c-Ha-ras^EJ VSMC to serum was not associated with the induction of TRE-binding activity (Fig. 4). The observation that TPA treatment dramatically increases TRE-binding activity in serum-starved c-Ha-ras^EJ VSMC (Fig. 2) suggests that this observation cannot be explained by Ras-mediated downregulation of AP-1. Collectively, these data indicate that mitogen-induced growth in VSMC transformed by oncogenic ras does not proceed through the Ras:ERK:AP-1 pathway. It is important to note that, whereas c-Ha-ras^EJ VSMC appeared capable of proliferation in the absence of serum, the proliferative advantage afforded these cells appeared to be sensitive to the presence of mitogens as indicated by the dramatic increase in DNA synthesis after challenge of serum-starved c-Ha-ras^EJ VSMC with FBS (Fig. 5).

Although elucidation of the mitogenic pathway in c-Ha-ras^EJ VSMC requires further investigation, several candidate pathways can be identified. In addition to the serum-response element binding complex (7), Ras is associated with activation of PKC, an isoform insensitive to downregulation by phorbol esters (13) and present in VSMC (34). Thus mitogenic responsiveness via this pathway would likely be insensitive to PKC depletion. Alternatively, it is well known that oncogenic transformation of mammalian cells is associated with abnormal progression into the early phases of the cell cycle (11). Under serum-restrictive growth conditions, c-Ha-ras^EJ VSMC exhibit a significant proliferative advantage (Fig. 5), suggesting that they do not arrest in G_o. Thus serum deprivation may result in the accumulation of transformed cells at a stage of the cell cycle distal to a requirement for PKC-related signaling for subsequent cell-cycle progression. Either of these hypotheses is consistent with the observation that FBS-mediated TRE-binding activity is observed in serum-starved pSV2neo, but not c-Ha-ras^EJ, VSMC (Fig. 4).

In conclusion, the overall deregulation of VSMC growth and differentiation programming by oncogenic ras involves deregulation of PKC-related signal transduction. c-Ha-ras^EJ VSMC exhibit upregulated PKC-related signaling; however, this pathway does not appear to contribute to the proliferative phenotype under serum-restrictive conditions. In contrast to naive and vector controls, phorbol ester-sensitive PKC isoforms are not required for mitogenic responsiveness in serum-starved c-Ha-ras^EJ VSMC.