Abstract
We investigated whether endothelial nitrite oxide synthase (NOS) gene transfer inhibited cellular proliferation. Endothelial NOS and endothelin type A receptor genes were transferred into 293 cells, a human embryonic kidney cell line, by calcium-phosphate coprecipitation. The cytosolic free Ca2+ levels ([Ca2+]i) of transfected cells were estimated with fura-2 fluorescence. Thymidine incorporation was increased by endothelin-1 in type A receptor-transfected cells. The endothelial NOS gene transfer did not affect endothelin-1-induced increase in [Ca2+]i of type A receptor-transfected cells, but markedly inhibited mitogen-activated protein kinase andc-fos promoter activities. The endothelial NOS gene transfer also inhibited thymidine incorporation into type A receptor-transfected cells in response to endothelin-1, which was abolished in the presence of the NOS inhibitorNG-monomethyl-l-arginine acetate. The endothelin-1-induced increase in cell number was significantly suppressed by endothelial NOS gene transfer as well as by the mitogen-activated protein kinase inhibitor PD98059. These results indicate that endothelial NOS gene transfer inhibits cellular proliferation via inhibition of the mitogen-activated protein kinase cascade.
Nitric oxide (NO) plays various important roles, including regulation of vascular tone (Furchgott et al., 1980; Ignaro, 1989), neurotransmission (Bredt et al., 1989), and immunoregulation (Garthwaite et al., 1989). There are two classes of NO synthase (NOS), constitutive and inducible forms (Hibbs et al., 1988). The constitutive enzymes are calcium- and calmodulin dependent and were initially identified in brain (NOS I or nNOS) (Nathan et al., 1994) and endothelial cells (NOS III or eNOS) (Lamas et al., 1992; Marsden et al., 1993). They are regulated by reversible binding of calcium-calmodulin. The inducible NOS isoform (NOS II or iNOS) is typically expressed in cells only after exposure to cytokines and is calcium independent (Lowenstein et al., 1992; Ikeda et al., 1996; Studer et al., 1996). The inducible NOS contains a tightly bound calmodulin subunit and remains active even at resting levels of intracellular calcium. All three classes of NOS catalyze the five electron oxidation of l-arginine tol-citrulline and generate NO. This oxidation requires NADPH and transfers electrons through FAD and FMN to a heme group and tol-arginine. Tetrahydro-biopterin maintains the enzyme in its active homodimeric form, and is essential for NOS activity. In vascular smooth muscle cells and platelets, NO activates soluble guanylate cyclase, which increases intracellular guanosine 3′,5′-cyclic monophosphate, thereby inducing vasorelaxation and inhibition of platelet aggregation (Marin et al., 1990; deGraaf et al., 1992).
Previous studies have demonstrated that NO inhibits proliferation of various kinds of cells such as vascular smooth muscle cells (Garg et al., 1989a) and mesangial cells (Garg et al., 1989b). However, those studies relied on specific pharmacological tools such as NO donors at high pharmacological doses in the millimolar range rather than on authentic NO. NO donors contain several NO-related compounds, including peroxinitrate and oxidative products. Thus, the exact effects of authentic NO on cellular proliferation are still unclear. Recently, Sendai virus-liposome- or adenovirus-mediated endothelial NOS gene transfer into arteries has been reported to improve their vasomotor reactivities and inhibit neointimal formation in vivo (von der Lyden et al., 1995; Cable et al., 1997; Kullo et al., 1997a,b; Ooboshi et al., 1997). However, whether these effects are due to direct inhibition of smooth muscle cell proliferation or to inhibition of platelet aggregation or leukocyte adhesion by NO remains obscure.
Endothelin has been extensively investigated over the past years because of its important role in the pathogenesis of hypertension (Kohno et al., 1990), cardiac hypertrophy (Brown et al., 1995), and atherosclerosis (Lerman et al., 1991). Endothelin has three subtypes, endothelin-1, endothelin-2, and endothelin-3. Endothelin-1, the function of which is coupled to phospholipase C-mediated phosphoinositide hydrolysis (Takuwa et al., 1990), modulates vascular contractility and proliferation and is the most potent mammalian vasoconstrictor identified to date. In the present study, we transferred the endothelial NOS gene into 293 cells, a human embryonic kidney cell line, and investigated whether endogenously generated NO inhibited cellular proliferation in response to endothelin-1.
Materials and Methods
Cell Culture.
The 293 cells, a human embryonic kidney cell line, were maintained in Dulbecco's modified Eagle's medium/nutrient mixture F12 (DMEM/F12) (Life Technologies, Paisley, Scotland) supplemented with 10% fetal calf serum (CSL Limited, Melbourne, Australia), 1% penicillin/streptomycin, and 1% glutamate and incubated at 37°C under 5% CO2. The 293 cells were plated in 6-well culture dishes (Falcon Plastics, Cockeysville, MD) for Western blotting, luciferase assay, and NOS activity assay, or in 24-well dishes (Falcon Plasics) for thymidine incorporation assay.
Plasmids and Transfection.
The expression plasmids of human endothelin type A receptor (pCDM-ETAR) and endothelin type B receptor (pCDM-ETBR) were obtained from Riken Gene Bank (Ibaragi, Japan). The full-length bovine endothelial NOS cDNA, a kind gift from D. G. Harrison (Emory University School of Medicine, Atlanta, GA), was isolated from the Bluescript SK+ vector by restriction enzyme digestion with SalI (4096 base pairs). To generate pCMV-NOS, this SalI fragment was ligated into the SalI cloning site of the pCMV expression plasmid (4050 base pairs). The pCMV vectors were engineered by introducing the cytomegalovirus immediate early promoter, the human growth hormone first intron, and the simian virus 40 polyadenylation signal into pUC 18 vectors.
Twenty-four hours after plating, 293 cells were cotransfected with expression plasmids by the calcium-phosphate coprecipitation method. Briefly, 200 μl of 300 mmol/l CaCl2, 200 μl of 2× Hanks' balanced salt solution (280 mmol/l NaCl, 50 mmol/l HEPES, pH 7.1, 1.5 mmol/l Na2HPO4), and a total 4 μg of plasmid were mixed gently, and then added to the culture medium in 6-well dishes. For the 24-well dishes 1 μg of plasmid was used. After 6 h of incubation, the plates were rinsed twice with PBS, and further cultured in serum-free DMEM/F12 for 12 h. Transfection efficiency into 293 cells with calcium-phosphate coprecipitation was very high, and up to 50 to 80% of cells were successfully transfected when determined by X-gal staining after the transfection with pCMV-lacZ.
Mitogen-Activated Protein Kinase and c-fosPromoter Activity Assay.
Mitogen-activated protein kinase activity was measured with the PathDetect in vivo signal transduction pathway reporting system (Stratagene, La Jolla, CA) according to the manufacturer's instructions. Briefly, 293 cells were transfected withpFR-luc (GAL4 specific luciferase reporter gene),pFA-Elk (mitogen-activated kinase-specific transactivator GAL4 expression plasmid), pCDM-ETAR, and pCMV orpCMV-NOS with the calcium-phosphate coprecipitation method. After a 12-h incubation in serum-free medium, transfected cells were stimulated with 10−9 mol/l human endothelin-1 (Peptide Institute, Osaka, Japan) for 3 h, and then luciferase activities were measured. The c-fos promoter activation was measured by cotransfection of pfos/luc plasmids into 293 cells. After 12 h of incubation in serum-free medium, transfected cells were stimulated with 10−9 mol/l endothelin-1 for 6 h. Luciferase activity was measured with the luciferase assay system (Promega, Msdison, WI), and are expressed as relative light units.
Thymidine Incorporation Assay.
DNA synthesis in transfected 293 cells was measured by thymidine incorporation into the cells as reported previously (Ikeda et al., 1991). The cells were rinsed twice with PBS, cultured in serum-free DMEM/F12 for 12 h, and incubated with endothelin-1 for 12 h, followed by addition of [3H]thymidine (100 μCi/ml; Amersham, Arlington Heights, IL) for another 6 h. The cells were washed with PBS twice and then lysed with 1N NaOH and 0.1% SDS. Radioactivity was measured by liquid scintillation counting.
Immunoblot Analysis of Endothelial NOS Protein.
Cells were rinsed with ice-cold PBS and resuspended into the lysis buffer (1% Nonidet P-40, 50 mmol/l Tris-HCl, pH 7.4, 150 mmol/l NaCl, 200 U/ml aprotinine, 1 mmol/l phenylmethylsulfonyl fluoride). After incubation on ice for 30 min, cell extracts were centrifuged to remove cell debris. Cell lysates (30 μg) were then separated through 7.5% polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membrane (Immunobin; Millipore, Bedford, MA). The membrane was incubated for 1 h at room temperature in Tris-buffered saline/Tween 20 (20 mmol/l Tris-HCl, pH 7.4, 150 mmol/l NaCl, 0.05% Tween 20) with 4% nonfat milk. The membrane was then incubated with anti-endothelial NOS antibody (Biomol, Plymouth Meeting, PA) (1:1000) overnight at 4°C in Tris-buffered/Tween 20. Specific binding of the antibody was visualized by the enhanced chemiluminescence detection system (Amersham) according to the manufacturer's instructions.
Measurement of NOS Activity.
To measure NOS enzyme activity,l-arginine to l-citrulline conversion was assayed in transfected 293 cells with the NOS detect assay kit (Stratagene) according to the manufacturer's instructions. Briefly, protein extracts prepared as described above were incubated for 60 min at 37°C in a solution of 10 mmol/ll-[3H]arginine (2183 MBq/mmol), 1 mmol/l NADPH, 1 μmol/l FAD, 1 μmol/l FMN, 100 nmol/l calmodulin, 600 μmol/l CaCl2, and 3 μmol/l tendothelin receptorahydrobiopterin in a final volume of 40 μl. The reaction was stopped by the addition of 400 μl of stop buffer (10 mmol/l EDTA, 50 mmol/l HEPES buffer, pH 5.5) to the reaction mixture. Then 100 μl of equilibrated resin was added to each mixture. Reaction samples were transferred to spin cups and centrifuged at 10,000g for 30 s. The radioactivity of the flowthrough was measured by liquid scintillation counting. Enzyme activity was expressed as citrulline production in picomoles per minute per milligram protein.
Measurement of [Ca2+]i.
The [Ca2+]i levels of transfected cells were estimated with fura-2 fluorescence. The cells were rinsed with physiological saline solution containing 140 mmol/l NaCl, 4.6 mmol/l KCl, 1 mmol/l MgCl2, 2 mmol/l CaCl2, 10 mmol/l glucose, and 10 mmol/l HEPES, pH 7.4. They were then loaded with 5 μmol/l fura-2 acetoxymethyl ester (fura-2/AM) for 60 min at 37°C. After aspiration of the fura-2/AM solution, the glass slides were rinsed and then placed in a quartz cuvette at 37°C in a fluorescence spectrometer (model CAF-100; Japan Spectrometer, Tokyo, Japan). The fluorescence was monitored at 500 nm with excitation wavelengths of 340 and 380 nm in the ratio mode. From the ratio of fluorescence at 340 and 380 nm, the [Ca2+]i was determined as described by Grynkiewicz et al. (1985), with the following expression: [Ca2+]i (nmol/l) =Kd × [(R −Rmin)/(Rmax− R)] × β, where R is the ratio of fluorescence of the sample at 340 and 380 nm, andRmax andRmin are determined by treating the cells with 50 μmol/l digitonin and 10 mmol/l MnCl2, respectively. The term β is the ratio of fluorescence of fura-2 at 380 nm at zero and saturating Ca2+. Kd is the dissociation constant of fura-2 for Ca2+, assumed to be 224 nm at 37°C.
Statistical Analysis.
Values are expressed as means ± S.E. of three or four samples, which represented at least three separate experiments. Differences of values were assessed by ANOVA with the least significant difference for multiple comparisons. Values ofP < .05 were considered statistically significant.
Results
It has been shown that endothelin-1 not only induces vasoconstriction but also promotes proliferation of vascular smooth muscle cells. Endothelin receptor is known to be functionally linked to G proteins. Endothelin-1 stimulation increases intracellular Ca2+ levels, activates protein kinase C and mitogen-activated protein kinase, and enhances transcriptional activation of the c-fos and c-myc proto-oncogenes through ETARs. First, we incubated 293 cells with endothelin-1 as a mitogenic stimulant. As shown in Fig. 1, the c-fos/luciferase activity did not change in response to 10−9 mol/l endothelin-1 in control cells, indicating that parental 293 cells do not express functional endothelin receptors. However, the c-fos/luciferase activity was increased in response to endothelin-1 in type A receptor-transfected cells.
We then investigated [3H]thymidine incorporation into 293 cells transfected with pCDM-ETAR or -ETBR. As shown in Fig. 2, [3H]thymidine incorporation into type A receptor-expressing cells was increased by endothelin-1 in a dose-dependent manner (10−10∼10−8 mol/l), whereas incorporation into type B receptor-expressing cells was not increased. These findings suggest that functional ETAR was induced and coupled with cellular proliferation.
To determine the functional role of endogenous NO, we transfected the endothelial NOS gene into 293 cells by the calcium-phosphate coprecipitation method. As shown in Fig.3A, the 140-kD endothelial NOS protein was clearly present in transfected cells, whereas the cell lysate frompCMV (control plasmid)-transfected cells did not express the endothelial NOS protein. We also measured NOS activity inpCMV-NOS-transfected cells by the citrulline production. Citrulline production without Ca2+ indicates inducible NOS activity, whereas its production with Ca2+ represents Ca2+-dependent constitutive NOS activity. As shown in Fig. 3B, control cells showed neither inducible nor constitutive NOS activity, however, endothelial NOS-transfected cells demonstrated high constitutive but no inducible NOS activity. These findings indicate that endothelial NOS gene transfer into 293 cells was successful and functional. Therefore, we cotransfected ETAR and endothelial NOS genes into 293 cells in the following experiments.
ETAR mediates the mobilization of intracellular Ca2+ via activation of phospholipase C. As shown in Fig. 4, the addition of endothelin-1 (10−9 mol/l) did not increase [Ca2+]i of parental 293 cells, whereas endothelin-1 stimulation rapidly increased [Ca2+]i of type A receptor-transfected 293 cells. Preincubation of the cells with the type A receptor antagonist BQ-485 (10−8 mol/l; Calbiochem-Novabiochem Japan Ltd., Tokyo, Japan) for 10 min completely blocked the endothelin-1-induced increase in [Ca2+]i. However, endothelial NOS gene transfer did not influence the rise of [Ca2+]i in type A receptor-transfected cells induced by endothelin-1 (Table1).
We then investigated the effects of endothelial NOS gene transfer on cellular proliferation using c-fos and mitogen-activated protein kinase luciferase assays. The luciferase activity that represents c-fos promoter activity in control 293 cells was increased up to 3.1-fold by endothelin-1 (Fig.5). However, in endothelial NOS-transfected cells, the luciferase activity was significantly depressed under both endothelin-1-stimulated and unstimulated conditions. The luciferase activity that represents mitogen-activated protein kinase activity also was increased up to 12.8-fold with endothelin-1 stimulation (Fig. 6). However, in endothelial NOS-transfected cells, the luciferase activity was significantly depressed under both endothelin-1-stimulated and unstimulated conditions.
We further investigated DNA synthesis of endothelial NOS-transfected and untransfected 293 cells. As shown in Fig.7, endothelial NOS gene transfer significantly inhibited [3H]thymidine incorporation into 293 cells stimulated with endothelin-1 by 60.3%, and this inhibition was abolished in the presence of the NOS inhibitorNG-monomethyl-l-arginine acetate (1 mmol/l).
We also investigated the effects of endothelin-1 on cellular proliferation of 293 cells. As shown in Fig.8, the number of 293 cells was significantly increased in the presence of endothelin-1, which was significantly suppressed by endothelial NOS gene transfer as well as by the mitogen-activated protein kinase inhibitor PD98059 (10−5 mol/l; Biomol).
Discussion
Endothelin-1 activities are mediated by binding to specific cell-surface receptors. Two types of endothelin receptors, type A and type B receptors, have been identified, cloned, and sequenced (Arai et al., 1990; Sakurai et al., 1990). The contraction and proliferation of vascular smooth muscle cells in response to endothelin-1 are mediated via type A receptors (Yanagisawa et al., 1988; Eguchi et al., 1994). In the present study, we demonstrated that c-fos promoter activity and DNA synthesis of 293 cells were increased via type A receptor stimulation. Although parental 293 cells did not exhibit inducible or constitutive NOS activity, pCMV-NOS-transfected 293 cells induced functional constitutive NOS expression. Using these transfected cells, we demonstrated that endogenously generated NO inhibits cellular proliferation in response to endothelin-1.
Antiproliferative effects of NO have been extensively investigated. However, most previous studies relied on specific pharmacological tools such as NO donors at high pharmacological doses. Recently, Kullo et al. (1997c) and Sharma et al. (1999) reported that gene transfer of endothelial NOS to vascular smooth muscle cells resulted in the expression of a functional enzyme and inhibition of cellular proliferation in response to fetal calf serum or platelet-derived growth factor. The gene transfer method is thought to be one of promising new tools for investigating the effects of certain molecules and their signaling pathways. In this study, we investigated whether NO generated from overexpression of endothelial NOS by 293 cells could inhibit cellular proliferative activities and which pathways were involved. These 293 cells are easily transduced by the calcium-phosphate coprecipitation method, and because parental 293 cells have no functional endothelin receptors, we reconstructed an endothelin-sensitive cell system.
ETAR is known to be functionally linked to G proteins. Activation of G proteins results in activation of the phospholipase C cascade and increases diasylglycerol and [Ca2+]i, which activates protein kinase C. In the present study, [Ca2+]i was increased in response to endothelin-1 in type A receptor-transfected cells. However, the [Ca2+]i response to endothelin-1 in endothelial NOS-transfected cells was not different from that in the untransfected control cells. These findings suggest that endogenous NO does not modulate the phospholipase C-calcium system. However, exogenous NO has been reported to inhibit protein kinase C gene expression and its activity in a variety of cells. Jun et al. (1994) reported that lipopolysaccharide- or phorbol 12-myristate 13-acetate-induced NO or the NO donor sodium nitroprusside inhibited the expression of the protein kinase C delta gene in murine peritoneal macrophages. Studer et al. (1996) reported that the NO donorS-nitroso-N-acetylpenicillamine inhibited basal protein kinase C activity in mesangial cells. The modulation of protein kinase C activity is important for cellular proliferation and protein synthesis. We investigated whether the expression of the intermediary signaling element mitogen-activated protein kinase, which is downstream of protein kinase C, and the proto-oncogene c-fos were modulated by endothelial NOS gene transfer. Various growth factors, including endothelin-1, are known to activate mitogen-activated protein kinase and c-fos (Komuro et al., 1988; Jones et al., 1995). We observed that mitogen-activated protein kinase and c-fospromoter activities were increased in ETAR-reconstituted cells with endothelin-1 stimulation. However, their activities as well as [3H]thymidine incorporation and cellular proliferation in response to endothelin-1 were significantly suppressed in endothelial NOS-transfected cells. In addition, the mitogen-activated protein kinase inhibitor PD98059 significantly inhibited proliferation of 293 cells induced by endothelin-1. Thus, the antiproliferative effect of authentic NO appears to occur after the phospholipase C-calcium system but before the mitogen-activated protein kinase pathway. However, there are multiple pathways induced by endothelin-1 that could ultimately lead to cellular proliferation, and precise mechanisms of antiproliferative effects of authentic NO remain unclear.
Endothelin-1, a potent vasoconstrictor peptide secreted from endothelial cells, has been implicated in a number of human diseases including hypertension, atherosclerosis, and restenosis after angioplasty. These pathological processes are thought to mediate the cell-proliferative effects of endothelin-1. Azuma et al. (1994)reported that endothelin-1 and endothelin receptor gene expression was enhanced in the neointima of rat carotid artery after balloon injury. There are also several reports that endothelin-1 infusion accelerates and endothelin receptor antagonist administration reduces neointimal formation after balloon injury (Douglas et al., 1993, 1994;Trachtenberg et al., 1993). Therefore, endothelin-1-induced cellular proliferation may contribute to the development of vascular remodeling in response to the vascular injury.
Our results revealed that endogenously generated NO inhibits cellular proliferation in response to endothelin-1 via suppression of the mitogen-activated protein kinase cascade, suggesting that endothelial NOS gene transfer can improve the clinical course of endothelin-1-related proliferative vascular disorders.
Footnotes
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Send reprint requests to: Uichi Ikeda, M.D., Ph.D., Department of Cardiology, Jichi Medical School, Minamikawachi-machi, Tochigi 329-0498, Japan. E-mail:uikeda{at}jichi.ac.jp
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↵1 This work was supported by grants from the Ministry of Health and Welfare (no. 10C-1) and the Ministry of Education, Science, Sports and Culture (no. 10670675).
- Abbreviations:
- NO
- nitric oxide
- NOS
- nitric-oxide synthase
- DMEM/F12
- Dulbecco's modified Eagle's medium/nutrient mixture F12
- fura-2/AM
- fura-2 acetoxymethyl ester
- ETAR
- endothelin type A receptor
- Received April 7, 1999.
- Accepted October 1, 1999.
- The American Society for Pharmacology and Experimental Therapeutics