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Eicosapentaenoic Acid and Docosahexaenoic Acid Inhibit Vascular Smooth Muscle Cell Proliferation by Inhibiting Phosphorylation of Cdk2-cyclinE Complex
Biochemical and Biophysical Research Communications 254, 502–506 (1999) Article ID bbrc.1998.9976, available online at http://www.idealibrary.com on
Eicosapentaenoic Acid and Docosahexaenoic Acid Inhibit Vascular Smooth Muscle Cell Proliferation by Inhibiting Phosphorylation of Cdk2-cyclinE Complex Takashi Terano,* ,1 Tomoaki Tanaka,† Yasushi Tamura,‡ Masatoshi Kitagawa,§ Hideaki Higashi, ¶ Yasushi Saito,‡ Aizan Hirai\ *Department of Internal Medicine, Chiba Municipal Hospital, 827 Inohana Chuo-Ku, Chiba 260 Japan; †The Second Department of Internal Medicine, Chiba University School of Medicine, 1-8-1, Inohana, Chuo-ku, Chiba-city, Chiba 260 Japan; ‡Sasaki Research Institute, Kyoundou Hiratsuka Hospital, Hiratsuka, Kanagawa 254 Japan; §Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812 Japan; ¶ Banyu Tsukuba Research Institute, Merck Research Laboratories, Tsukuba Japan; and \Chiba Prefectural Togane Hospital, Togane, Chiba 283 Japan
Received December 8, 1998
Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the form of triacylglycerol (TG) were dose dependently incorporated into phospholipid fraction of vascular smooth muscle cells (VSMC) and suppressed the proliferation of VSMC. Flow cytometric analysis demonstrated both EPA and DHA inhibited G 1/S progression. EPA and DHA inhibited the phosphorylation of Cdk2 protein and Cdk2 kinase activity without altering the amount of cyclin E and p27 kip1 proteins and cyclin dependent kinase activating kinase activity by growth stimulation. This mechanisms remained to be clarified but this is the first report of a novel mechanisms of inhibition of DNA synthesis by EPA and DHA. © 1999 Academic Press Key Words: eicosapentaenoic acid; docosahexaenoic acid; vascular smooth muscle cells proliferation; Cdk2; p27 kip1; Cdk activating kinase.
Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), two major n-3 polyunsaturated fatty acids (PUFA) from marine lipids, has been reported to exert its anti-atherosclerotic effect through modulating various cell functions (1,2,3). Vascular smooth muscle cells (VSMC) are important in pathophysiology of atherosclerosis, because of their potential to proliferate and to accumulate lipid. We previously reported the antiproliferative effect of EPA through the modulation of growth factor signal transduction (4). EPA inhibited the binding of platelet derived growth factor (PDGF) to 1 To whom correspondence should be addressed. Fax: 81-43-2240719. E-mail: [email protected].
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
its receptor on VSMC (5) and suppressed PKC activation and c-fos mRNA expression at the level of transcription (6). But whether it affects other steps further down the pathway leading to DNA synthesis has not yet been clarified. Quiescent cells exit G 0 phase upon addition of growth factors and pass through G 1/S and G 2/M phase to complete cell cycle. Progression through the eukaryotic cell cycle is controlled by cyclins and their catalytic subunits, cyclin dependent kinases (cdks) (7,8). The active cyclin-cdk complexes are assumed to phosphorylate the retinoblastoma susceptibility gene products (pRB), thereby promoting cell cycle progression toward DNA replication (9). G 1 phase is the major control point for cell proliferation in mammalian cells and especially cdc2 family kinases (cdk2 and cdc2) seem to have crucial roles on cell cycle regulation (8). In this paper we reported the mechanisms through which EPA and DHA inhibited DNA synthesis with regards to G1 cyclins, Cdks, Cdk inhibitors (p27 kip1), and cyclin dependent kinase activating kinase (CAK) which played a central role in DNA synthesis (G 1/S progression). MATERIALS AND METHODS Preparation of fatty acids emulsions. Fatty acids emulsions of EPA (20:5 n-3), DHA (22:6 n-3), oleic acid (18:1 n-9), and linoleic acid (18:2 n-6) were prepared as previously reported (4). Each emulsion contained 20.6% of fatty acids triacylglycerol, 2.3% of glycerol, 1.2% of yolk egg PC and 0.004% tocopherol. Vascular smooth muscle cells (VSMC) culture. Medial smooth muscle cells were isolated from the thoracic aorta of 12 week old WKY rats and VSMC cultures were established using ex-plant method according to Ross et al. (10) as previously reported (11). VSMC were cultured in Dulbecco’s modified Eagles medium
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(DMEM) with 10% fetal calf serum (FCS) and cells of passage 5 to 10 were used. Cells at 70 – 80% confluence were made quiescent by withdrawal of FCS for 24 – 48 hours. Cells were then cultured for 13 hours in a growth medium (DMEM with 10% FCS) supplemented with 40 –160 mM of TG form of fatty acids (EPA, DHA, oleic acid and linoleic acid). Fatty acid composition and 3H-thymidine uptake. The fatty acid composition of the total phospholipid fraction of VSMC was measured by gas liquid chromatography as previously reported (12). After 13 hour incubation period, 1 mCi of 3H-thymidine was added to each well and incubated for a further 4 hours. DNA synthesis was measured by incorporation of 3H-thymidine into VSMC as previously reported (9). Flow cytometric analysis. Cells harvested by trypsinization were washed with PBS and stained with propidium iodide using CycleTEST PLUS DNA Reagent Kit (Becton Dickinson Immunocytometory System, San Jose, CA). The cell cycle profiles of samples were analyzed by FACScan (Becton Dickinson, San Jose, CA). Immunoprecipitation and immunoblotting. For immunoanalysis, harvested VSMC were washed with PBS and resuspended in 400 ml of ice cold buffer A (10 mM HEPES pH 7.9; 2 mM MgCl 2; 10 mM KCl; 0.1 mM EDTA; 0.1 mM EGTA; 1 mM DTT; 1 mM Na 3VO 4; 5 mM NaF; 0.5 mM p-aminophenyl methanesulfonyl fluoride hydrochloride (p-APMSF) and a cocktail of protease inhibitors (2.5 mg/ml of pepstatin A; 2.5 mg/ml of antipain; 2.5 mg/ml of chymostatin; 0.25 mg/ml leupeptin). The cells were lysed with lysis buffer(50 mM Hepes pH 7.0, 250 mM NaCl, 0.1% Nonidet P-40 (NP-40) and protease inhibitor cocktail. The homogenates were centrifuged for 30 sec in a microfuge. The nuclear pellet was resuspended in 50 ml buffer B (20 mM HEPES pH 7.9; 0.4 M NaCl; 1 mM EDTA; 1 mM EGTA; 1 mM DTT; 1 mM Na 3VO 4; 5 mM NaF; 0.5 mM p-APMSF) and the protease inhibitor cocktail. The tube was vigorously shaken for 15 min at 4°C. The nuclear extract was centrifuged for 5 min in a microfuge at 4°C and the supernatants were then mixed with SDS sample buffer (62.5 mM Tris-HCl buffer pH 6.8 containing 2% SDS, 5% 2-mercaptoethanol, 7% glycerol and 0.01% bromophenol blue) and boiled for 10 min and then 15 mg of each sample was subjected to SDS/PAGE on gels with an appropriate concentration of acrylamide. Proteis were then transferred electrophoretically to membrane. The blotted membranes were blocked,washed, then soaked in solutions of primary antibodies for 2 hours and visualized with enhanced chemiluminescence (ECL) western blotting kit (Amersham). Antibodies against cyclin E, cyclin H, Cdk2, Cdk7 and p27 kip1 were obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Anti-phospho-pRB antibody was raised against a phosphopeptide that encoded amino acids 801-815 of pRB (13). Assays of Cdk2 kinase activity and CAK activity. Whole cell lysate (300 mg/500 ml) was precleared by the incubation with 50 ml of proteinA-Separose CL-4B for 30min followed by centrifugation at 10000g for 15 min. Assay of Cdk2 kinase activity was performed by the phosphorylation of H1 histone. The clear supernatants were incubated with anti-cdk2 antibody and precipitated with protein A-Sepharose CL-4B. H1 histone kinase assay of the immunoprecipitates was performed at 30°C for 30 min in buffer containing 0.1 mg/ml of H1 histon, 50 mM ATP and 5 mCi 32P-ATP. Phosphorylated H1 histone were analyzed by SDS (12.5%) PAGE and auto radiography (14). Assay of CAK activity was performed by the phosphorylation of purified Cdk2-cyclinE complex using the immunoprecipitates prepared with anti-CAK antibody as previously reported (15).
RESULTS Supplementation of EPA-TG from 40 mM to 160 mM dose dependently increased the content of EPA
in the total phospholipid fraction of VSMC without altering other fatty acids including arachidonic acid (AA) and DHA. EPA content increased from 0.6 to 2.2 mol% (an increase of more than 250%) by supplementation of 160 mM of EPA. DHA content increased dose dependently by the addition of DHA-TG (40-160 mM). DHA content increased from 2.1 to 3.6 mol% by supplementation of 160 mM of DHA-TG without altering other fatty acid contents. No significant change was observed in the fatty acid composition by the supplementation of oleic acid or linoleic acid-TG (40-160 mM). Detailed data of the change of fatty acid composition by PUFA supplementation were previously reported (16). As shown in Fig. 1, both EPA and DHA inhibited the incorporation of 3H-thymidine into VSMC in a dose dependent manner. The effect of EPA was more pronounced than that of DHA. No significant suppression of VSMC proliferation was observed by the addition of oleic acid or linoleic acid to the cell culture medium. Addition of EPA or DHA suppressed the transition of G 1 to S phase in VSMC, as determined d by flow cytometric analysis (Fig. 2). Percentages of G 1, S and G 2/M phase in serum-starved cells were 68, 13, and 19%, respectively. By stimulation with growth medium for 13 hours, percentages of G 1,S and G 2/M phase were changed to 17, 68, and 15%, respectively. By simultaneous addition of EPA or DHA to serum stimulated cells, the percentage of the cells in the G 1 and S phase were 55–57 and 23%, respectively. These data confirmed that EPA and DHA suppressed DNA synthesis by inhibiting G 1/S transition. Cdk2 protein increased by serum stimulation and EPA dose dependently inhibited phospholyrated form (active form, 33KDa) of Cdk2 protein during the stimulation of cellular prolifeation without affecting the amount of cyclin E, cyclin H, Cdk7 and p27 kip1 as shown in Fig. 3. DHA showed almost similar effect on the amount of Cdk2, cyclin E, p27 kip1, cyclin H and Cdk7 proteins (Fig. 3). Co-incubation of EPA and DHA (160 mM) almost completely suppressed Cdk2 kinase activity induced by serum (Fig. 4). CAK activity was not influenced by the coincubation with EPA or DHA (160 mM) (Fig. 4). DISCUSSION All proliferating mammalian cells pass through several checkpoits,mainly G 1 to S, and G 2 to M transitions. Cyclins have been demonstrated to promote cell cycle transitions by binding and activating specific cyclin dependent kinases (Cdks) (17). Progression from G 1 to S phase in mammalian cells involves activation of cyclinE/Cdk2 and cyclinD/Cdk4 (6). Cyclin E accumulates and activates cdk2 protein in G 1. However, cyclin accumulation and cdk binding do not constitute the
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FIG. 1. The effect of PUFA TG emulsions on VSMC proliferation, measured by 3H-thymidine uptake. Supplementation of EPA ( ) or ,,, 3 ,,, ,,,) inhibited H-thymidine incorporation into VSMC in a dose dependent manner. This inhibitory activity of EPA was more pronounced DHA (,,, ,, than that of DHA. Linoleic acid (,, ,,) or oleic acid (h) exerted no effect. Mean(SE). *p,0.01, **p,0.001 (compared with 0).
only levels of Cdk regulation. Additional models of regulation of Cdks include positive and negative phosphorylation events and accumulation or activation of inhibitory proteins. As one of inhibitory proteins for
FIG. 2. Cell cycle of VSMC was evaluated by flow cytometric analysis. (A) Serum-starved cells, (B) Cells stimulated with growth medium (FCS(1)) for 13 hours, (C) Cells stimulated with growth medium in the presence of EPA (160 mM), (D) Cells stimulated with growth medium in the presence of DHA (160 mM). This data is a representative of three separate experiments.
Cdk2, p27 kip1 has been identified in transforming growth factor b-arrested cells (18). Other mechanisms of Cdk2 activation in vivo are as follows; dephosphorylation of Thr-14 and Tyr-15 by cdc25 phosphatase and phosphorylation of Thr-160 by Cdk activating kinase (CAK) (19). Phosphorylation of Cdk2 influences its mobility on SDS-PAGE since the active Cdk2 molecule (33KDa) migrates further than the inactive one (34KDa) (Fig. 3) (20). EPA almost completely suppressed the increase in active phosphorylated Cdk2 (33KDa) by serum stimulation. DHA showed almost similar dose dependent effect on Cdk2 protein. Inhibition of phosphorylation of Cdk2 protein resulted in the inhibition of phosphorylated pRB protein accumulation, which was essential for G 1 /S progression. Oleic acid and linoleic acid did not show any effects on the amount of Cdk2, cyclin E, pRb and p27 kip1 proteins in VSMC (data not shown). Cdk2 kinase activity was completely inhibited by EPA and DHA at the concentration of 80-160 mM as shown in Fig. 4. EPA and DHA did not influence either the amount of cyclin H and Cdk7 proteins (Fig. 3) nor CAK activity (Fig. 4). Cdks are regulated by an intricate network of positive and negative regulators. Cdk kinase activity absolutely depends on binding to a cyclin subunit. Then the cyclin-Cdk complex is fully activated by phosphorylation on threonine residue in the Cdk catalytic domain.
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Phosphorylaiton of this threonine residue is mediated by a Cdk-activating kinase (CAK). As a negative regulator for the kinase activity of Cdks, Cdk inhibitor p27 kip1 might be most important. In this experimental condition, positive and negative regulators of Cdk2, CAK activity and the protein amount of p27 kip1, respectively, were not influenced by EPA and DHA. Although the amounts of p27, p21 and p53 proteins were not changed in the present study (data of P21 and P53 were not shown), the possibility cannot be ruled out that undetectable change of the amount of these proteins in immunoblotting might be enough to modulate G 1/S transition. Soos et al. (21) and Poon et al. (22) reported that the amount of p27 was not changed during the cell cycle progression in MANCA hematopoietic B cells and in Swiss 3T3 mouse fibroblasts, respectively. Thus p27 may serve as a buffer for cyclin-Cdk complexes in G 1 phase. In summary, incubation of VSMC with EPA or DHA inhibited VSMC proliferation and inhibited the phosphorylation of cdk2 protein through the suppression of Ckd2 kinase activity without affecting the protein amounts of cyclinE and p27, and CAK activity. This is the first report of a novel mechanism of inhibition of DNA synthesis by EPA and DHA. This inhibition of
FIG. 4. Cdk2 kinase activity and CAK activity. EPA and DHA (160 mM) almost completely inhibited cdk2 kinase activity but did not influence CAK activity.
DNA synthesis was only observed in EPA and DHA treated cells among PUFA examined. The mechanisms of EPA and DHA on Cdk2 inhibition remained to be clarified. ACKNOWLEDGMENTS We thank Drs. I. Tatsuno, Y. Noguchi, and T. Shiina for their help and valuable advice and Ms. Y. Okuda, Y. Tsuchikawa, E. Miyagawa, and K. Tanuma for their excellent technical assistance.
REFERENCES
FIG. 3. Western blotting analysis of Cdk2, cyclin E, p27 kip1, Cdk7 and cyclin H. Cdk2 and cyclin E proteins increased by serum stimulation for 13 hours. Co-incubation of EPA or DHA almost completely suppressed the phosphorylated form of Cdk2 protein (33 KDa) without affecting those of cyclin E and p27 kip1. Cyclin H and Cdk7 were not altered by EPA and DHA.
1. Kromann, N., and Green, A. (1980) Acta Med. Scand. 208, 401– 406. 2. Dyerberg, J., Bang, H. O., Stoffersen, E., Moncada, S., and Vane, J. R. (1978) Lancet 2, 2117–2119. 3. Hirai, A., Terano, T., Tamura, Y., and Yoshida, S. (1989) J. Internal Med. 225 (Supp.), 69 –75. 4. Shiina, T., Terano, T., Saito, J., Tamura, Y., and Yoshida, S. (1993) Atherosclerosis 104, 95–103. 5. Terano, T., Shiina, T., Saito, J., Tamura, Y., and Yoshida, S. (1992) Jpn. J. Pharmacol. 58, 286. 6. Terano, T., Shiina, T., and Tamura, Y. (1996) Lipids 31, S301– S304. 7. Sherr, C. J. (1993) Cell 73, 1095–1065. 8. Fang, F., Orend G., Watanabe, N., Hunter, T., and Ruoslahti, E. (1996) Science 271, 499 –502. 9. Akiyama, T., Ohuchi, T., Sumida, S., Matsumoto, K., and Toyoshima, K. (1992) Proc. Natl. Acad. Sci. USA 89, 7900 –7904. 10. Ross, R., and Glomset, J. A. (1973) Science 180, 1332–1339. 11. Hasunuma, K., Terano, T., Tamura, Y., and Yoshida, S. (1991) Prostaglandins Leukot. Essent. Fatty Acids 42, 171–175. 12. Terano, T., Hirai, A., Hamazaki, T., Kobayashi, S., Fujita, T., Tamura, Y., and Kumagai, A. (1983) Atherosclerosis 46, 21–331. 13. Suzuki-Takahashi, I., Kitagawa, M., Saijo, M., Higashi, H., Ogino, H., Matsumoto, H., Taya, Y., Nishimura, S., and Okuyama, A. (1995) Oncogene 10, 1691–1698.
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14. Kitagawa, M., Okabe, T., Ogino, H., Matsumoto, H., SuzukiTakahashi, I., Kokubo, T., Higashi, H., Saitoh, S., Taya, Y., Yasuda, H., Ohba, Y., Nishimura, S., Tanaka, N., and Okuyama, A. (1993) Oncogene 8, 2425–2432. 15. Higashi, H., Suzuki-Takahashi, I., Saitoh, S., Segawa, K., Taya, Y., Okuyama, A., Nishimura, S., and Kitagawa, M. (1996) Eur. J. Biochem. 237, 460 – 467. 16. Saito, J., Terano, T., Hirai, A., Shiina, T., Tamura, Y., and Saito, Y. (1997) Atherosclerosis 131, 219 –228. 17. Nurse, P. (1990) Nature 344, 503–508.
18. Polyak, K., Kato, J., Solomon, M. J., Sherr, C. J., Massague, J., Roberts, J., and Koff, A. (1994) Genes Dev. 8, 9 –22. 19. Morgan, D. O. (1995) Nature 374, 131–134. 20. Dulic, V., Lees, E., and Reed, S. I. (1992) Science 257, 1958 – 1960. 21. Soos, T. J, Kiyokawa, H., Yan, J. S., Rubin, M. S., Giordano, A., DeBlasio, A., Bottega, S., Wong, B., Mendelsohn, J., and Koff, A. (1996) Cell Growth Diffrentiation 7, 135–146. 22. Poon, Y. C., Toyoshima, H., and Hunter, T. (1995) Mol. Biol. Cell 6, 1197–1213.
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Eicosapentaenoic Acid and Docosahexaenoic Acid Inhibit Vascular Smooth Muscle Cell Proliferation by Inhibiting Phosphorylation of Cdk2-cyclinE Complex Takashi Terano,* ,1 Tomoaki Tanaka,† Yasushi Tamura,‡ Masatoshi Kitagawa,§ Hideaki Higashi, ¶ Yasushi Saito,‡ Aizan Hirai\ *Department of Internal Medicine, Chiba Municipal Hospital, 827 Inohana Chuo-Ku, Chiba 260 Japan; †The Second Department of Internal Medicine, Chiba University School of Medicine, 1-8-1, Inohana, Chuo-ku, Chiba-city, Chiba 260 Japan; ‡Sasaki Research Institute, Kyoundou Hiratsuka Hospital, Hiratsuka, Kanagawa 254 Japan; §Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812 Japan; ¶ Banyu Tsukuba Research Institute, Merck Research Laboratories, Tsukuba Japan; and \Chiba Prefectural Togane Hospital, Togane, Chiba 283 Japan
Received December 8, 1998
Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the form of triacylglycerol (TG) were dose dependently incorporated into phospholipid fraction of vascular smooth muscle cells (VSMC) and suppressed the proliferation of VSMC. Flow cytometric analysis demonstrated both EPA and DHA inhibited G 1/S progression. EPA and DHA inhibited the phosphorylation of Cdk2 protein and Cdk2 kinase activity without altering the amount of cyclin E and p27 kip1 proteins and cyclin dependent kinase activating kinase activity by growth stimulation. This mechanisms remained to be clarified but this is the first report of a novel mechanisms of inhibition of DNA synthesis by EPA and DHA. © 1999 Academic Press Key Words: eicosapentaenoic acid; docosahexaenoic acid; vascular smooth muscle cells proliferation; Cdk2; p27 kip1; Cdk activating kinase.
Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), two major n-3 polyunsaturated fatty acids (PUFA) from marine lipids, has been reported to exert its anti-atherosclerotic effect through modulating various cell functions (1,2,3). Vascular smooth muscle cells (VSMC) are important in pathophysiology of atherosclerosis, because of their potential to proliferate and to accumulate lipid. We previously reported the antiproliferative effect of EPA through the modulation of growth factor signal transduction (4). EPA inhibited the binding of platelet derived growth factor (PDGF) to 1 To whom correspondence should be addressed. Fax: 81-43-2240719. E-mail: [email protected].
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
its receptor on VSMC (5) and suppressed PKC activation and c-fos mRNA expression at the level of transcription (6). But whether it affects other steps further down the pathway leading to DNA synthesis has not yet been clarified. Quiescent cells exit G 0 phase upon addition of growth factors and pass through G 1/S and G 2/M phase to complete cell cycle. Progression through the eukaryotic cell cycle is controlled by cyclins and their catalytic subunits, cyclin dependent kinases (cdks) (7,8). The active cyclin-cdk complexes are assumed to phosphorylate the retinoblastoma susceptibility gene products (pRB), thereby promoting cell cycle progression toward DNA replication (9). G 1 phase is the major control point for cell proliferation in mammalian cells and especially cdc2 family kinases (cdk2 and cdc2) seem to have crucial roles on cell cycle regulation (8). In this paper we reported the mechanisms through which EPA and DHA inhibited DNA synthesis with regards to G1 cyclins, Cdks, Cdk inhibitors (p27 kip1), and cyclin dependent kinase activating kinase (CAK) which played a central role in DNA synthesis (G 1/S progression). MATERIALS AND METHODS Preparation of fatty acids emulsions. Fatty acids emulsions of EPA (20:5 n-3), DHA (22:6 n-3), oleic acid (18:1 n-9), and linoleic acid (18:2 n-6) were prepared as previously reported (4). Each emulsion contained 20.6% of fatty acids triacylglycerol, 2.3% of glycerol, 1.2% of yolk egg PC and 0.004% tocopherol. Vascular smooth muscle cells (VSMC) culture. Medial smooth muscle cells were isolated from the thoracic aorta of 12 week old WKY rats and VSMC cultures were established using ex-plant method according to Ross et al. (10) as previously reported (11). VSMC were cultured in Dulbecco’s modified Eagles medium
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(DMEM) with 10% fetal calf serum (FCS) and cells of passage 5 to 10 were used. Cells at 70 – 80% confluence were made quiescent by withdrawal of FCS for 24 – 48 hours. Cells were then cultured for 13 hours in a growth medium (DMEM with 10% FCS) supplemented with 40 –160 mM of TG form of fatty acids (EPA, DHA, oleic acid and linoleic acid). Fatty acid composition and 3H-thymidine uptake. The fatty acid composition of the total phospholipid fraction of VSMC was measured by gas liquid chromatography as previously reported (12). After 13 hour incubation period, 1 mCi of 3H-thymidine was added to each well and incubated for a further 4 hours. DNA synthesis was measured by incorporation of 3H-thymidine into VSMC as previously reported (9). Flow cytometric analysis. Cells harvested by trypsinization were washed with PBS and stained with propidium iodide using CycleTEST PLUS DNA Reagent Kit (Becton Dickinson Immunocytometory System, San Jose, CA). The cell cycle profiles of samples were analyzed by FACScan (Becton Dickinson, San Jose, CA). Immunoprecipitation and immunoblotting. For immunoanalysis, harvested VSMC were washed with PBS and resuspended in 400 ml of ice cold buffer A (10 mM HEPES pH 7.9; 2 mM MgCl 2; 10 mM KCl; 0.1 mM EDTA; 0.1 mM EGTA; 1 mM DTT; 1 mM Na 3VO 4; 5 mM NaF; 0.5 mM p-aminophenyl methanesulfonyl fluoride hydrochloride (p-APMSF) and a cocktail of protease inhibitors (2.5 mg/ml of pepstatin A; 2.5 mg/ml of antipain; 2.5 mg/ml of chymostatin; 0.25 mg/ml leupeptin). The cells were lysed with lysis buffer(50 mM Hepes pH 7.0, 250 mM NaCl, 0.1% Nonidet P-40 (NP-40) and protease inhibitor cocktail. The homogenates were centrifuged for 30 sec in a microfuge. The nuclear pellet was resuspended in 50 ml buffer B (20 mM HEPES pH 7.9; 0.4 M NaCl; 1 mM EDTA; 1 mM EGTA; 1 mM DTT; 1 mM Na 3VO 4; 5 mM NaF; 0.5 mM p-APMSF) and the protease inhibitor cocktail. The tube was vigorously shaken for 15 min at 4°C. The nuclear extract was centrifuged for 5 min in a microfuge at 4°C and the supernatants were then mixed with SDS sample buffer (62.5 mM Tris-HCl buffer pH 6.8 containing 2% SDS, 5% 2-mercaptoethanol, 7% glycerol and 0.01% bromophenol blue) and boiled for 10 min and then 15 mg of each sample was subjected to SDS/PAGE on gels with an appropriate concentration of acrylamide. Proteis were then transferred electrophoretically to membrane. The blotted membranes were blocked,washed, then soaked in solutions of primary antibodies for 2 hours and visualized with enhanced chemiluminescence (ECL) western blotting kit (Amersham). Antibodies against cyclin E, cyclin H, Cdk2, Cdk7 and p27 kip1 were obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Anti-phospho-pRB antibody was raised against a phosphopeptide that encoded amino acids 801-815 of pRB (13). Assays of Cdk2 kinase activity and CAK activity. Whole cell lysate (300 mg/500 ml) was precleared by the incubation with 50 ml of proteinA-Separose CL-4B for 30min followed by centrifugation at 10000g for 15 min. Assay of Cdk2 kinase activity was performed by the phosphorylation of H1 histone. The clear supernatants were incubated with anti-cdk2 antibody and precipitated with protein A-Sepharose CL-4B. H1 histone kinase assay of the immunoprecipitates was performed at 30°C for 30 min in buffer containing 0.1 mg/ml of H1 histon, 50 mM ATP and 5 mCi 32P-ATP. Phosphorylated H1 histone were analyzed by SDS (12.5%) PAGE and auto radiography (14). Assay of CAK activity was performed by the phosphorylation of purified Cdk2-cyclinE complex using the immunoprecipitates prepared with anti-CAK antibody as previously reported (15).
RESULTS Supplementation of EPA-TG from 40 mM to 160 mM dose dependently increased the content of EPA
in the total phospholipid fraction of VSMC without altering other fatty acids including arachidonic acid (AA) and DHA. EPA content increased from 0.6 to 2.2 mol% (an increase of more than 250%) by supplementation of 160 mM of EPA. DHA content increased dose dependently by the addition of DHA-TG (40-160 mM). DHA content increased from 2.1 to 3.6 mol% by supplementation of 160 mM of DHA-TG without altering other fatty acid contents. No significant change was observed in the fatty acid composition by the supplementation of oleic acid or linoleic acid-TG (40-160 mM). Detailed data of the change of fatty acid composition by PUFA supplementation were previously reported (16). As shown in Fig. 1, both EPA and DHA inhibited the incorporation of 3H-thymidine into VSMC in a dose dependent manner. The effect of EPA was more pronounced than that of DHA. No significant suppression of VSMC proliferation was observed by the addition of oleic acid or linoleic acid to the cell culture medium. Addition of EPA or DHA suppressed the transition of G 1 to S phase in VSMC, as determined d by flow cytometric analysis (Fig. 2). Percentages of G 1, S and G 2/M phase in serum-starved cells were 68, 13, and 19%, respectively. By stimulation with growth medium for 13 hours, percentages of G 1,S and G 2/M phase were changed to 17, 68, and 15%, respectively. By simultaneous addition of EPA or DHA to serum stimulated cells, the percentage of the cells in the G 1 and S phase were 55–57 and 23%, respectively. These data confirmed that EPA and DHA suppressed DNA synthesis by inhibiting G 1/S transition. Cdk2 protein increased by serum stimulation and EPA dose dependently inhibited phospholyrated form (active form, 33KDa) of Cdk2 protein during the stimulation of cellular prolifeation without affecting the amount of cyclin E, cyclin H, Cdk7 and p27 kip1 as shown in Fig. 3. DHA showed almost similar effect on the amount of Cdk2, cyclin E, p27 kip1, cyclin H and Cdk7 proteins (Fig. 3). Co-incubation of EPA and DHA (160 mM) almost completely suppressed Cdk2 kinase activity induced by serum (Fig. 4). CAK activity was not influenced by the coincubation with EPA or DHA (160 mM) (Fig. 4). DISCUSSION All proliferating mammalian cells pass through several checkpoits,mainly G 1 to S, and G 2 to M transitions. Cyclins have been demonstrated to promote cell cycle transitions by binding and activating specific cyclin dependent kinases (Cdks) (17). Progression from G 1 to S phase in mammalian cells involves activation of cyclinE/Cdk2 and cyclinD/Cdk4 (6). Cyclin E accumulates and activates cdk2 protein in G 1. However, cyclin accumulation and cdk binding do not constitute the
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FIG. 1. The effect of PUFA TG emulsions on VSMC proliferation, measured by 3H-thymidine uptake. Supplementation of EPA ( ) or ,,, 3 ,,, ,,,) inhibited H-thymidine incorporation into VSMC in a dose dependent manner. This inhibitory activity of EPA was more pronounced DHA (,,, ,, than that of DHA. Linoleic acid (,, ,,) or oleic acid (h) exerted no effect. Mean(SE). *p,0.01, **p,0.001 (compared with 0).
only levels of Cdk regulation. Additional models of regulation of Cdks include positive and negative phosphorylation events and accumulation or activation of inhibitory proteins. As one of inhibitory proteins for
FIG. 2. Cell cycle of VSMC was evaluated by flow cytometric analysis. (A) Serum-starved cells, (B) Cells stimulated with growth medium (FCS(1)) for 13 hours, (C) Cells stimulated with growth medium in the presence of EPA (160 mM), (D) Cells stimulated with growth medium in the presence of DHA (160 mM). This data is a representative of three separate experiments.
Cdk2, p27 kip1 has been identified in transforming growth factor b-arrested cells (18). Other mechanisms of Cdk2 activation in vivo are as follows; dephosphorylation of Thr-14 and Tyr-15 by cdc25 phosphatase and phosphorylation of Thr-160 by Cdk activating kinase (CAK) (19). Phosphorylation of Cdk2 influences its mobility on SDS-PAGE since the active Cdk2 molecule (33KDa) migrates further than the inactive one (34KDa) (Fig. 3) (20). EPA almost completely suppressed the increase in active phosphorylated Cdk2 (33KDa) by serum stimulation. DHA showed almost similar dose dependent effect on Cdk2 protein. Inhibition of phosphorylation of Cdk2 protein resulted in the inhibition of phosphorylated pRB protein accumulation, which was essential for G 1 /S progression. Oleic acid and linoleic acid did not show any effects on the amount of Cdk2, cyclin E, pRb and p27 kip1 proteins in VSMC (data not shown). Cdk2 kinase activity was completely inhibited by EPA and DHA at the concentration of 80-160 mM as shown in Fig. 4. EPA and DHA did not influence either the amount of cyclin H and Cdk7 proteins (Fig. 3) nor CAK activity (Fig. 4). Cdks are regulated by an intricate network of positive and negative regulators. Cdk kinase activity absolutely depends on binding to a cyclin subunit. Then the cyclin-Cdk complex is fully activated by phosphorylation on threonine residue in the Cdk catalytic domain.
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Phosphorylaiton of this threonine residue is mediated by a Cdk-activating kinase (CAK). As a negative regulator for the kinase activity of Cdks, Cdk inhibitor p27 kip1 might be most important. In this experimental condition, positive and negative regulators of Cdk2, CAK activity and the protein amount of p27 kip1, respectively, were not influenced by EPA and DHA. Although the amounts of p27, p21 and p53 proteins were not changed in the present study (data of P21 and P53 were not shown), the possibility cannot be ruled out that undetectable change of the amount of these proteins in immunoblotting might be enough to modulate G 1/S transition. Soos et al. (21) and Poon et al. (22) reported that the amount of p27 was not changed during the cell cycle progression in MANCA hematopoietic B cells and in Swiss 3T3 mouse fibroblasts, respectively. Thus p27 may serve as a buffer for cyclin-Cdk complexes in G 1 phase. In summary, incubation of VSMC with EPA or DHA inhibited VSMC proliferation and inhibited the phosphorylation of cdk2 protein through the suppression of Ckd2 kinase activity without affecting the protein amounts of cyclinE and p27, and CAK activity. This is the first report of a novel mechanism of inhibition of DNA synthesis by EPA and DHA. This inhibition of
FIG. 4. Cdk2 kinase activity and CAK activity. EPA and DHA (160 mM) almost completely inhibited cdk2 kinase activity but did not influence CAK activity.
DNA synthesis was only observed in EPA and DHA treated cells among PUFA examined. The mechanisms of EPA and DHA on Cdk2 inhibition remained to be clarified. ACKNOWLEDGMENTS We thank Drs. I. Tatsuno, Y. Noguchi, and T. Shiina for their help and valuable advice and Ms. Y. Okuda, Y. Tsuchikawa, E. Miyagawa, and K. Tanuma for their excellent technical assistance.
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FIG. 3. Western blotting analysis of Cdk2, cyclin E, p27 kip1, Cdk7 and cyclin H. Cdk2 and cyclin E proteins increased by serum stimulation for 13 hours. Co-incubation of EPA or DHA almost completely suppressed the phosphorylated form of Cdk2 protein (33 KDa) without affecting those of cyclin E and p27 kip1. Cyclin H and Cdk7 were not altered by EPA and DHA.
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