Mechanism of Catecholamine-Induced Proliferation of Vascular Smooth Muscle Cells
Background Catecholamines have been shown to aggravate atherosclerosis in animals and humans, and abnormal proliferation of vascular smooth muscle cells (VSMC) is a key event in the early stage of atherosclerosis. Catecholamines may be involved in such cell growth. Therefore, a series of experiments using cultured VSMC was performed to elucidate their possible mitogenic effect.
Methods and Results We examined the mitogenic effect of catecholamines using rat aortic smooth muscle cells (VSMC) by measuring [3H]thymidine incorporation, checking with flow cytometry, and counting the cell number directly. Furthermore, the catecholamine-activated signal transduction pathway was assessed by measurement of the formation of inositol 1,4,5-triphosphate, intracellular Ca2+ concentration, mitogen-activated protein kinase (MAPK) activity, and mitogenic gene expression. Norepinephrine (NE) and phenylephrine stimulated [3H]thymidine incorporation and cell growth. Clonidine and isoproterenol showed little of such effects. Prazosin was more effective than either yohimbine or propranolol in suppressing the mitogenic effect of NE, indicating that catecholamine-induced VSMC proliferation is mediated by α1-adrenoceptors. The α1-adrenoceptor activation was coupled to pertussis toxin–insensitive Gq-protein and triggered phosphoinositide hydrolysis with subsequent activation of protein kinase C and MAPK in VSMC. In response to NE, both 42- and 44-kD MAPK were activated and tyrosine was phosphorylated. α1-Adrenoceptor stimulation with NE also caused accumulation of c-fos, c-jun, and c-myc mRNA. Chloroethylclonidine completely blocked the α1-adrenoceptor–mediated mitogenesis.
Conclusions The effect of catecholamines appears to be mediated via the activation of the chloroethylclonidine-sensitive α1-adrenoceptors that triggers the phosphoinositide hydrolysis and activates the MAPK pathway, leading to DNA synthesis and cell proliferation.
Cardiovascular disease remains the chief cause of death in the world, including Taiwan, and atherosclerosis, the principal cause of myocardial and cerebral infarctions, accounts for the major cause of these deaths in the United States and Western Europe,1 probably in Taiwan as well. Hypertension, smoking, and psychological stress are among the main risk factors for atherosclerosis2 and are known to be associated with an increased level of catecholamines such as epinephrine and NE in plasma.3 In addition to regulating the blood vessel contraction, catecholamines have been shown to aggravate atherosclerosis in animals and humans,4 suggesting that they are likely to play a prominent role in the pathogenesis of atherosclerosis. In the development of advanced lesions of atherosclerosis, abnormal proliferation of VSMC is known to be a key event.1 Possibly, therefore, catecholamines also may show a long-term trophic effect on the growth of VSMC. In fact, catecholamines have been found to stimulate the proliferation of cultured VSMC5 and polyploidy formation of VSMC in vitro as well as in vivo.6 A recent in vivo study showed that infusion of PE, a synthetic specific α1-adrenoceptor agonist, induced cardiac hypertrophy and fibrosis.7 On the other hand, prazosin, a selective α1-adrenoceptor antagonist, reduces intimal hyperplasia in the rabbit abdominal aorta8 9 and the medial VSMC DNA synthesis stimulated by angiotensin II in the rat aorta.10
These observations suggest that α1-adrenoceptors are involved in NE-stimulated events. Two major subtypes of α1-adrenoceptor, α1A and α1B, have been identified in pharmacological studies.11 12 The α1A-adrenoceptor is insensitive to an alkylating agent, CEC, whereas the α1B receptor is irreversibly inactivated by CEC.13 The cDNA of an additional subtype, termed α1C-adrenoceptor, has been cloned from the bovine brain; the pharmacological property of this subtype is unknown, however.14 Recently, two laboratories have cloned an additional subtype cDNA from the brain, termed α1D, which has properties similar but not identical to those of the α1A subtype.15 16 These two cDNAs probably are coding for the same receptor subtype, which has recently been termed the α1A/D receptor.15 16 It is not known which subtype of the α1-adrenoceptor is involved in the growth of VSMC.
Two distinct intracellular signal pathways induce cell proliferation. One is activated by the receptor that contains the intrinsic protein tyrosine kinase. This type of receptor is activated by classic growth factors such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF).17 The other is activated by G-protein–coupled receptors such as thrombin and bombesin.17 Recent studies reveal that the cellular growth mediated by the intracellular signal pathway for the G-protein–coupled receptor involves a family of key kinases, MAPK, which belong to a family of serine/threonine protein kinases.18 19 20 MAPK are activated during proliferation and cell cycle transition triggered by a variety of stimuli,21 thereby indicating that MAPK are indeed important integrators of receptor-originated signals. Of particular relevance to VSMC hyperplasia in relation to MAPK activity is that certain transcription factors such as c-fos, c-jun, and c-myc have been identified as the substrates of MAPK.20 21 22 The α1-adrenoceptors are G-protein–coupled receptors,23 which mediate the vasoconstrictive effects of catecholamines. Although the mechanism for catecholamine-induced blood vessel contraction is well known, the cellular mechanism for catecholamine-induced VSMC proliferation, including whether or not the VSMC proliferation induced by α1-adrenergic stimulation involves the activation of MAPK, has not been elucidated. Here we report the results of a series of experiments in an attempt to elucidate the pathway catecholamines activate for VSMC proliferation. It becomes apparent that catecholamines exert their effect via the Gq-protein–coupled, CEC-sensitive α1-adrenoceptor pathway, including the activation of MAPK, which eventually leads to DNA synthesis and cell growth.
DMEM, FBS, antibiotics, and all other tissue culture reagents were obtained from GIBCO. Drugs were prepared in sterile saline and diluted to working concentration in DMEM. Unless otherwise noted, the concentrations of the drugs used were norepinephrine HCl (NE) 10 nmol/L, phenylephrine HCl (PE) 10 nmol/L, clonidine HCl 1 μmol/L, isoproterenol HCl 10 μmol/L, prazosin HCl 100 nmol/L, yohimbine HCl 10 μmol/L, propranolol HCl 10 μmol/L, staurosporine 30 nmol/L, PTX 50 ng/mL, W7 20 μmol/L, and PMA 500 nmol/L, which were all purchased from Sigma Chemical Co. Other chemicals, such as MBP, β-glycerophosphate, dithiothreitol, HEPES, and leupetin, were also obtained from Sigma. CEC (concentration used, 30 μmol/L) was obtained from Research Biochemicals International. Protein dye reagent, SDS, and bis-acrylamide were from Bio-Rad. [2-3H]Myoinositol (10 Ci/mmol), [methyl 1-1′,2′,-3H]thymidine (124 Ci/mmol), and [τ-32P]ATP (7000 Ci/mmol) were from Amersham Corp.
Aortic Smooth Muscle Cell Isolation and Culture
Medial explants were dissected from freshly harvested rat aortic strips and plated in 100-mm Petri dishes.24 Aortic smooth muscle cells (VSMC) from explants were grown in DMEM containing 10% FBS, 100 U/mL penicillin G, and 100 μg/mL streptomycin at 37°C in a humidified, 5% CO2 atmosphere. Explant-derived cells were initially treated with 0.1% trypsin–4 mmol/L EDTA for 3 minutes at 37°C, and thereafter the passage was made conventionally. Cells from passages 5 through 15 were used for all growth studies. The cells were characterized as smooth muscle cells by morphology and immunostaining with monoclonal antibody specific for smooth muscle α-actin (CGA-7). Cells were subcultured into 24-well plates in a medium containing 10% serum for 24 hours with an initial density of 5×104 cells per well. The cells were then placed in the medium without serum for 48 hours to render them quiescent.
Incorporation of [3H]thymidine into VSMC was measured with minor modifications as described previously.25 Quiescent cells were incubated with α-adrenoceptor agonists (NE, PE, or clonidine) or β-agonist (isoproterenol) in 1 mL DMEM/FBS-free medium for 20 hours, when 1 μCi/mL [3H]thymidine was added for pulse labeling. The cells were incubated for 4 more hours and then washed twice with 1 mL of PBS. The cells were treated with 10% trichloroacetic acid to precipitate the acid-insoluble material, from which the DNA was extracted with 0.1N NaOH. The DNA was collected on a Whatman GF/B filter and washed twice with 5 mL ice-cold PBS. The filter was then cut and shaken in 3.5 mL scintillation fluid for 24 hours before counting in a liquid scintillation counter.
VSMC were seeded at a density of 3×104 cells per 35-mm dish containing 3 mL serum-free DMEM supplemented with 10 μg/mL insulin, 1 mg/mL BSA, and specified amounts of agonists. The serum-free medium containing agonists was changed with a fresh one every day. On specified days, cells were washed with PBS, harvested with 0.5 mL trypsin/EDTA, centrifuged, and resuspended in 0.5 mL of DMEM containing 10% FBS. The number of cells was manually counted in a hemocytometer. Triplicate counts were taken for each plate, and quadruplicate plates were used for each determination.
To estimate the proportion of cells at various stages in different phases of the cell cycle, cellular DNA content was measured by flow cytometry 24 hours after stimulation of the cells. Cells were trypsinized, centrifuged at 1500 rpm for 3 minutes, washed with PBS, and then cleared of RNA with RNase A (0.1 mg/mL). The DNA was stained with propidium iodide (40 μg/mL) for 30 minutes at 37°C, and 2×107 cells were then analyzed for DNA with a FACstar cytofluorometer (Becton-Dickinson) by excitation at 488 nm and emission at 585 nm. Flow cytometric determination of DNA content was used as an index of cell proliferation.26
Assay of IP3 Content
VSMC were incubated with α1-agonists or α1-antagonists plus an α1-agonist at 37°C for 1 minute for IP3 formation. The reaction was terminated by the addition of 100 μL of ice-cold perchloric acid (20%) followed by 20-minute incubation in an ice bath. The mixture was centrifuged at 2000g for 15 minutes at 4°C, the supernatant was collected, and the pH was adjusted to 7.5 with 10N KOH solution. The KClO4 formed was allowed to precipitate for 30 minutes at 4°C and sedimented at 2000g for 15 minutes at 4°C. The amount of IP3 in the supernatant was determined by radioimmunoassay.
Measurement of Intracellular Ca2+
VSMC were cultured on a glass coverslip and incubated in a medium containing 5 μmol/L of the fura-2-acetoxymethyl ester (fura-2/AM), a Ca2+ indicator, for 45 minutes at 37°C. The loaded coverslip was then placed in a specially designed holder that positioned the coverslip diagonally in a polymethacrylate cuvette. Each cuvette contained 2.4 mL of Krebs-Henseleit solution that consisted of (in mmol/L) NaCl 117.5, KCl 5.6, MgSO4 1.18, NaH2PO4 1.2, NaHCO3 25.0, glucose 5.5, HEPES 25.0, and CaCl2 2.5. To this cuvette, 100 μL of drug solution containing the appropriate amount of drugs was added to make up to a total volume of 2.5 mL. The fluorescence was then measured at 37°C in a spectrophotometer (CAF-110, Jasco). The excitation wavelengths were 340 and 380 nm, and the emission wavelength was 510 nm. The ratio of the fluorescence excited at 340 nm (F340) to that at 380 nm (F380) was calculated from the illumination periods and referred to as R340/380. The ratio was used to estimate the intracellular Ca2+ concentration ([Ca2+]i).27
Quiescent VSMC were stimulated with NE for the indicated time. After washing once with ice-cold PBS, the cells were lysed with ice-cold lysis buffer that consisted of (in mmol/L) HEPES 20, β-glycerophosphate 80, Na3VO4 0.2, dithiothreitol 2, EGTA 10, phenylmethylsulfonyl fluoride (PMSF) 0.1, and EDTA 2. The cells were then sonicated for 5 seconds and centrifuged at 14 000g at 4°C for 30 minutes (Beckman TL-100). The supernatant was used as the source of MAPK. Aliquots containing an equal amount of protein (2 to 3 mg) were used for immunoprecipitation. The kinase assay was performed using 32P phosphorylation of MBP as a measurement of MAPK activity as described by Morinelli et al.28 The reaction mixture (containing, in mmol/L, unless otherwise noted, β-glycerophosphate 5, HEPES 20, MgCl2 10, dithiothreitol 2, 0.02% Triton X-100, Na3VO4 0.1, ATP 0.05 as well as [τ-32P]ATP 5 μCi, and MBP 1 mg/mL) was incubated with 20 μL of the protein sample for 30 minutes at 25°C. Incorporation of 32P was measured as described by Erikson et al.29
Immunoprecipitation and Western Blot Analysis
Quiescent cells were treated with various agents for the indicated times. Cells were lysed in 1 mL of immunoprecipitation buffer (in mmol/L: HEPES 10, NaF 50, NaCl 50, EDTA 5, EGTA 5, Na3VO4 0.1, and PMSF 0.5; 0.01% Triton X-100 and 10 μg/mL leupetin). Cell lysates were centrifuged and the resultant supernatants incubated with anti-rat MAPK (erk-CT) at 4°C for 2 hours. All immune complexes were incubated with protein G–agarose at 4°C for 1 hour, and the immune complex bound to protein G–agarose was precipitated by centrifugation. After the immunoprecipitates were washed with 1 mL of the immunoprecipitation buffer, they were treated with 20 μL of sample buffer. Western blot analysis was then performed: proteins, separated by 10% SDS polyacrylamide gel electrophoresis, were treated first with 100 μL of antiphosphotyrosine antibody (Py20) followed by goat anti-mouse IgG conjugated to alkaline phosphatase (2000-fold dilution). The blots were developed by addition of alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (Promega Corp) in the presence of 0.1 mol/L Tris buffer, pH 9.0, and incubation for 30 minutes.
RNA Isolation and Northern Blot Analysis
Total RNA was extracted essentially as described by Chomczynski and Sacchi,30 fractionated with 1% agarose–3% formaldehyde gel electrophoresis, and transferred to a nylon filter by capillary action. The Northern blot procedure was as follows. The sample was UV cross-linked, prehybridized in hybridization buffer (50% formaldehyde, 10× SSPE buffer, 0.1% SDS, 5× Denhardt's solution, and 90 μg/mL salmon sperm DNA) at 42°C for 3 hours, and hybridized in the same buffer with 32P-labeled mouse c-fos, c-jun, and c-myc cDNA probes at 42°C for 16 hours. After hybridization, the blots were washed twice in 500 mL 2× SSC and 0.1% SDS at room temperature. The blots were partially dried and exposed to x-ray film (Kodak X-OMAT film) at −70°C with an intensifying screen.
All data are expressed as mean±SEM. One-way ANOVA was used for multiple group comparisons and the Student's t test for others. P<.05 was considered statistically significant.
Effect of Catecholamines on [3H]Thymidine Incorporation in VSMC
Cell proliferation must be preceded by DNA synthesis; therefore, the capability of catecholamines to stimulate the incorporation of [3H]thymidine into DNA was first determined by use of the procedure described in “Methods.” As seen in Fig 1⇓, both NE and the specific α1-agonist PE produced a concentration-dependent increase of [3H]thymidine incorporation into serum-deprived, quiescent VSMC. The concentrations of NE and PE that produced a 50% increase (EC50) of [3H]thymidine incorporation were 2.0±0.2 and 1.5±0.3 nmol/L, respectively. PE was more potent than the α2-agonist clonidine and the β-agonist isoproterenol in stimulating the incorporation (Fig 1⇓). We then measured the kinetics of DNA synthesis induced by NE in rat VSMC. As shown in Fig 2⇓, when the cells were incubated with NE for various time periods, no differences in DNA synthesis were observed between the control (untreated) and the NE-treated cells for up to 8 hours. However, after 8 hours, there was a significant increase in [3H]thymidine incorporation, which peaked in 20 hours.
Effect of Catecholamines on Cell Number
To see whether or not the [3H]thymidine incorporation correlated cell proliferation, we checked to see whether or not NE and other drugs induced an increase in cell number. The VSMC were grown in serum-free medium in the absence (control) or presence of NE, PE, clonidine, or isoproterenol, and the number of cells was counted. As shown in Fig 3⇓, both NE and PE indeed increased the number of cells. In contrast, treatment with clonidine or isoproterenol did not significantly increase the cell number. The magnitude of the mitogenic effect of NE was comparable to that of PDGF and endothelin (data not shown). The mitogenic effect was also assessed by flow cytometry. As shown in Fig 4A⇓, in unstimulated quiescent VSMC, 95% of the cells were in the growth-arrested (G0/G1) phase of the cell cycle, whereas after 24-hour stimulation with NE, 60% of the VSMC entered the S phase of the cell cycle (Fig 4B⇓). Prazosin, an α1-antagonist, nearly completely abolished this response by NE (Fig 4C⇓). These observations indicated that the increase in [3H]thymidine incorporation and the mitogenic effect were the manifestation of the α1-adrenergic stimulation evoked by the drugs.
Mechanism of Mitogenic Effect of NE on VSMC
The determination of the mechanism responsible for the mitogenic effect of NE on VSMC was attempted by performing the following experiments. Cells were preincubated for 30 minutes with CEC or prazosin and then added with NE, and the [3H]thymidine incorporation was measured. As shown in Fig 5⇓, the two α1-antagonists greatly inhibited the [3H]thymidine incorporation, whereas propranolol, a β-antagonist, or yohimbine, an α2-antagonist, did not. The [3H]thymidine incorporation also was blocked by staurosporine at the concentration (30 nmol/L) that inhibited PKC but not by PTX at the concentration (50 ng/mL, 24-hour pretreatment) that inactivated Giα (Fig 5⇓). It should be noted that none of these antagonists were cytotoxic to the cells at the concentrations used (data not shown). NE also did not affect cAMP formation in rat VSMC (data not shown). Taken together, these data indicate that PTX-insensitive G-protein (Gq) and PKC were involved in the NE mitogenic signal system, which was effected via the activation of the CEC-sensitive α1-adrenoceptor(s).
Transductional Pathway Linked toα1-Adrenoceptor–Mediated Stimulation of Cell Growth
It appears that the α1-adrenoceptor in the signal pathway was involved in the [3H]thymidine incorporation. This was next examined. The hydrolysis of phosphoinositide appeared to be the major signal transduction pathway in the control of cell proliferation.17 In VSMC, NE rapidly (within 1 minute) increased IP3 formation to 2.5±0.3-fold after stimulation (Table 1⇓). NE also caused a rapid rise in [Ca2+]i (Fig 6A⇓). When cells were preincubated for 30 minutes with CEC, NE failed to trigger IP3 formation as well as the intracellular Ca2+ mobilization, whereas preincubation with PTX did not show such blocking effect (Table 1⇓ and Fig 6⇓, B and C).
MAPK, another group of components in the pathway, have been shown to be activated during the transition of entering the S phase from the G0/G1 phase of the cell cycle.31 NE appeared to facilitate the transition of the cell cycle from the G0/G1 phase to the S phase. Therefore, whether or not NE activated MAPK was checked. As shown in Fig 7⇓, NE rapidly activated MAPK in VSMC: MAPK activity was measurable 5 minutes after stimulation, peaked at 10 minutes, and then decreased thereafter. NE activated MAPK in a concentration-dependent manner with an EC50 of ≈1.1±0.1 nmol/L. This was similar to the case of [3H]thymidine incorporation.
Two isoforms of MAPK have been identified: one with a molecular weight of 42 kD and the other of 44 kD. In addition, the activation of MAPK requires phosphorylation at both tyrosine and threonine residues.19 Therefore, we next asked whether or not the NE-induced activation of MAPK was accompanied by tyrosine phosphorylation. Cell lysates were immunoprecipitated with anti-mouse MAPK 1 (erk 1) and anti-MAPK 2 (erk 2) antibodies, and Western blotting with antiphosphotyrosine antibody (Py20) was carried out. As shown in Fig 8A⇓, tyrosine phosphorylation in both 42- and 44-kD MAPK was observed at 5 minutes after stimulation with NE, reaching a maximum at 10 minutes, and then markedly decreased at 60 minutes, consistent with the above result. Since NE has been shown to increase IP3 and evoke diacylglycerol generation from the hydrolysis of phosphatidylinositol 4,5-bis-phosphate, resulting in intracellular Ca2+ mobilization and PKC activation, the role of PKC in the NE-induced MAPK activation was examined. The activation of MAPK by NE was almost completely inhibited by staurosporine, a PKC inhibitor, but not by W7, a Ca2+/calmodulin kinase inhibitor (Table 2⇓). When cells were pretreated with PMA for 24 hours (which activates and then downregulates classic PKC) and subsequently challenged with NE, 42- and 44-kD MAPK tyrosine phosphorylation failed to be induced (Fig 8B⇓). These results demonstrate that NE-induced MAPK activation involves the activation of PKC.
Effect of NE on Production of mRNAs of Transcription Factors
As mentioned earlier, certain transcription factors (such as c-fos, c-jun, and c-myc) have been identified as substrates for MAPK.20 21 22 Therefore, the production of the mRNAs of these factors was checked. As shown in Fig 9⇓, NE caused a rapid and transient increase of c-fos, c-jun, and c-myc mRNA. Both prazosin and CEC completely blocked this increase by NE (Fig 9⇓), whereas propranolol did not.
Catecholamines (NE and PE) stimulate [3H]thymidine incorporation and growth of rat aortic smooth muscle cells, consistent with the observations of others.5 6 7 Prazosin, as observed by other investigators,8 9 10 and CEC, both α1-adrenoceptor antagonists, significantly block the mitogenic effect of NE. In contrast, α2-agonist (clonidine) and β-agonist (isoproterenol) fail to show the mitogenic effect. Furthermore, propranolol, a β-antagonist, and yohimbine, an α2-antagonist, also did not show the inhibitory effect. Thus, catecholamines appear to induce VSMC proliferation via the activation of α1-adrenoceptors.
The effect of catecholamines facilitates the transition of the cells from the G0/G1 phase to the S phase of the cell cycle as shown in the result of flow cytometry. The NE concentrations that induce the mitogenic effect range from 0.03 to 100 nmol/L. The plasma concentration of catecholamines reaches 5 nmol/L even when a human is in a mild stress state such as in public speaking,3 and the increase in plasma catecholamines accelerates and aggravates the degree of atherosclerosis.4 32 Therefore, the mitogenic effect of catecholamines in the ranges below 5 nmol/L as shown in this study could be physiologically and pathologically meaningful in vivo.
NE appears to show its effect by activating various α1-adrenoceptor subtypes. There are at least four subtypes: α1A, α1B, α1C, and α1A/D.14 15 16 As shown above, CEC, which irreversibly inactivates the α1B-receptor,13 completely blocked the mitogenic effect of NE and its signal transduction system (ie, the IP3 formation, [Ca2+]i increase, MAPK activation, and accumulation of the mRNA of transcription factors). Others15 16 have shown that CEC also partially blocked the activity of the cloned α1A/D-receptor. Thus, the mitogenic effect of NE on rat VSMC appears to be mediated by the α1B-adrenoceptor and possibly also by the α1A/D-receptor but not α1A, which is insensitive to CEC. These results are consistent with the finding of Okazaki et al,33 who, using RNase protection assay, show that both α1B and α1A/D mRNAs are expressed in rat aorta.
The activation of the α1B-receptor is linked to the stimulation of phosphoinositide hydrolysis, which produces two second messengers, diacylglycerol and IP3.34 The former is known to activate PKC and the latter to release Ca2+ from intracellular storage. The activation of PKC and increase of [Ca2+]i appear to account for most of the early proliferative events.17 Such a pathway is apparently activated by the G-protein–coupled, growth-promoting agents such as angiotensin II35 and endothelin.36 We showed that 10 nmol/L NE caused the increase in IP3 formation and [Ca2+]i. Thus, the phosphoinositide hydrolysis is presumably stimulated by NE and may be essential for the mitogenic effect on VSMC. Generally, α1-adrenoceptors are found to be coupled to G-proteins in most cell types. NE, however, did not affect the cAMP formation, thus excluding the possibility that the α1-adrenoceptor is linked to Gi-protein. This is reinforced by the observation that the NE-induced VSMC proliferation was unaffected by the inhibition of Gi-protein with PTX pretreatment. However, it was abolished by staurosporine at concentrations that inhibit PKC. Taken together, it appears that PTX-insensitive G-protein (Gq instead of Gi) and PKC are involved in the NE-stimulated mitogenic signal via the activation of CEC-sensitive α1-adrenoceptors.
The proliferation of VSMC requires incubation with NE for at least 8 hours. This suggests that the activation of specific components of the intracellular signal pathway is required before the induction of cell proliferation. Recently, a novel group of serine/threonine kinases, MAPK, which are activated by various growth factors (eg, PDGF,37 EGF,17 thrombin,17 angiotensin II,35 and vasopressin38 ), has been described. We have shown above the activation of the 42- and 44-kD MAPK by NE as a function of time and concentration. Furthermore, this activation is accompanied by tyrosine phosphorylation, which is a part of the established mechanism for the activation of MAPK.39 As far as we know, this is the first demonstration that NE induces tyrosine phosphorylation that signifies the activation of MAPK in rat VSMC. The α1-adrenoceptor is associated with GTP-binding protein,23 and on the basis of its predicted sequence, it lacks an intracellular tyrosine kinase domain.40 Thus, the stimulation of the MAPK activity by NE is probably indirect. Vasopressin, angiotensin II, thrombin, and phorbol ester stimulate PKC in VSMC41 and induce tyrosine phosphorylation and MAPK activity38 in these cells. Thus, we postulate that the activation of MAPK is downstream from PKC in the signal transduction pathway responsive to NE in VSMC. This is supported by the observation that the activation of MAPK was sensitive to staurosporine, a PKC inhibitor, but not to W7, a Ca2+/calmodulin kinase inhibitor. Furthermore, the downregulation of classic PKC with a pretreatment of the cells with PMA suppressed the activation of MAPK by NE.
Recent studies indicate that extracellular signals cause cell proliferation by modulating transcription factor activity in the nucleus via protein phosphorylation cascades,42 and certain transcription factors have been shown to be substrates for MAPK.20 21 22 In the present study, we show that NE activates MAPK and produces c-fos, c-jun, and c-myc mRNA in VSMC. Both prazosin and CEC completely blocked such increase. Therefore, it appears that the activation of MAPK by the signal from the α1-adrenoceptor on the cell surface leads to phosphorylation of the transcription factors that in turn leads to the expression of genes for DNA synthesis and cell proliferation.
The mechanism of VSMC proliferation induced by NE may be as follows: NE binds to a CEC-sensitive, α1-adrenoceptor–Gq complex and stimulates the hydrolysis of phosphoinositide, producing IP3, which causes the rise of [Ca2+]i and diacylglycerol, which activates PKC. The activation of PKC in turn activates MAPK, causing phosphorylation of transcription factors, which then induces gene expression, leading to DNA synthesis that results in growth of the cell. Thus, circulating catecholamines may contribute to the pathogenesis of atherosclerosis by activating CEC-sensitive α1-adrenoceptors. Future studies will be directed toward analyzing whether or not other cytoplasmic kinases are activated through the stimulation of α1-adrenoceptors.
Selected Abbreviations and Acronyms
|MAPK||=||mitogen-activated protein kinase(s)|
|MBP||=||myelin basic protein|
|PKC||=||protein kinase C|
|PMA||=||phorbol 12-myristate 13-acetate|
|VSMC||=||vascular smooth muscle cell(s)|
This work was supported by research grants CMRP 576 from Chang Gung Medical Research Foundation and NSC 84-2331-B182-095 from the National Science Council, the Republic of China.
- Received October 19, 1995.
- Revision received January 16, 1996.
- Accepted January 22, 1996.
- Copyright © 1996 by American Heart Association
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