Enhancement by a-tocopheryl hemisuccinate of nitric oxide production induced by lypopolysaccharide and interferon-c through the upregulation of protein kinase C in rat vascular smooth muscle cells Kentaro Kogure, Motoki Morita, Susumu Hama, Sawa Nakashima, Akira Tokumura and Kenji Fukuzawa 1 Faculty of Pharmaceutical Sciences, University of Tokushima, Japan The effect of a-tocopheryl hemisuccinate (TS) on lipo- polysaccharide (LPS)/interferon-c (IFN)-induced nitric oxide production in rat vascular smooth muscle cells (VSMC) was examined. The LPS/IFN-induced NO pro- duction was enhanced by TS but not by the other a-tocopherol (a-T) derivatives a-tocopheryl acetate (TA) and a-tocopheryl nicotinate (TN), or a-T itself. a-T, TA and TN inhibited the enhancement by TS of LPS/IFN- induced NO production. The enhancing effect of TS was observed in the presence of LPS, but not IFN, suggesting that TS participates in the LPS-stimulated signal pathway leading to NO production. Protein kinase C (PKC) inhibitors, but not protein kinase A inhibitors, inhibited the enhancing effect of TS on LPS/IFN-induced NO produc- tion. Furthermore, TS enhanced the amount of PKCa in VSMC. From these results, we concluded that the enhan- cing effect of LPS/IFN-induced NO production was caused by upregulation of PKC in VSMC. Keywords: a-tocopheryl hemisuccunate; a-tocopherol; nitric oxide; vascular smooth muscle cells; protein kinase C. The nonantioxidant function of a-tocopherol (a-T) has been proposed [1–6]. It has been reported that a-T nonanti- oxidatively prevented the proliferation of smooth muscle cells through inhibition of protein kinase C (PKC), and the transcription of some genes such as CD36 and collagenase. We are interested in a-tocopheryl hemisuccinate (TS) as a key compound for clarification of the nonantioxidant function of a-T. TS is a naturally occurring amphiphilic compound initially isolated from a green barley extract that stimulates release of prolactin and growth hormone from pituitary cells in vitro [7]. Since then, various biological activities of TS such as inhibition of acetylcholine esterase activity [8] and induction of apoptosis in various cancer cells have been reported [9–16]. Furthermore, it has been reported that TS increased activities of c-Jun NH 2 -terminal kinases (JNK), extracellular signal-regulated kinases (ERK) and mitogen-activated protein kinase kinase (MEK1/2) [14,15]. In vascular smooth muscle cells (VSMC), NO is known to play a critical role in vasodilatory function and athero- sclerotic processes [17], and inducible NO production in the VSMC system has been well studied [18]. Recently, Kim et al. reported that TS itself induced nitric oxide (NO) production and inducible NO synthase (iNOS) expression in U937 human monoblasts through activation of nuclear factor-kappa B (NF-jB) [19]. However, because TS has been reported to inhibit NF-jB activation in various cell lines, such as human Jurkat T cells and human umbilical vein endothelial cells [20–23], the mechanism of the TS effect still remains unclear. Therefore, we investigated in detail the effect of TS on NO production in the VSMC system. The investigation would further give useful information about its nonantioxidant function and therapeutic possibilities for vascular diseases. In atherosclerotic plaques, cytokines such as tumor necrosis factor-a (TNF-a) and interleukin-1 secreted from macrophages and foam cells have been implicated in the pathogenetic events [24]. On the other hand, because these cytokines are responsible for iNOS expression through NF-jB factor activation, they are supposed to elicit the NO-dependent vasodilation to improve the decreased blood flow in the vascular lesions. To investigate the enhancing effect of TS on NO production under the atherosclerosis- like conditions, we used the system containing lipopolysac- charide (LPS) known as a stimulant of iNOS expression through a similar signaling cascade to these cytokines [24,25]. In this study, interferon-c (IFN), which was also reported to be secreted from T-cells in the lesions of Correspondence to K. Fukuzawa, Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi-1, Tokushima 770-8505, Japan. Fax: + 81 88 633 9572, E-mail: fukuzawa@ph.tokushima-u.ac.jp Abbreviations: AsA, ascorbic acid; BHA, butylhydroxyl anisol; ERK, extracellular signal-regulated kinase; IFN, interferon-c;iNOS,indu- cible nitric oxide synthase; JNK, c-Jun NH 2 -terminal kinase; LPS, lipopolysaccharide; MEK, mitogen-activated protein kinase kinase; MyD88, myeloid differentiation protein; NF-jB, nuclear factor-kappa B; NO, nitric oxide; PKA, protein kinase A; PKC, protein kinase C; PP2A, protein phosphatase-2A; a-T, a-tocopherol; TA, a-tocopheryl acetate; TN, a-tocopheryl nicotinate; TRAF, tumor necrosis factor receptor; TS, a-tocopheryl hemisuccinate; VSMC, vascular smooth muscle cells. Enzymes: nitric oxide synthase (EC 1.14.13.39); protein kinase (EC 2.7.1.37). (Received 11 December 2001, revised 18 February 2002, accepted 21 March 2002) Eur. J. Biochem. 269, 2367–2372 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02894.x atherosclerosis, was further used together with LPS to strengthen LPS function. MATERIALS AND METHODS Materials RRR-a-Tocopheryl hemisuccinate (TS), RRR-a-tocopheryl acetate (TA) and (+/–)-a-tocopheryl nicotinate (TN) were purchased from Sigma Chemical Co. (St Louis, MO, USA) (Fig. 1). LPS was obtained from DIFCO Laboratories (Detroit, MI). Recombinant rat IFN was purchased from PEPRO TECH EC (London, UK). RRR-a-Tocopherol (a-T) waskindlyprovidedby Eisai Co.(Tokyo, Japan).Other reagents were of the highest grade commercially available. Treatment of VSMC with TS VSMC were isolated from rat thoracic aorta using the proteases elastase and collagenase as described previously [26]. The VSMC (1 · 10 6 cells) were seeded into 35-mm dishes, and were cultured for 24 h in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum in a CO 2 - incubator at 37 °CwithCO 2 in humidified air. Then, the medium containing serum was removed, and the cells were washed with phosphate buffered saline. Next, 2 mL of the medium containing TS without serum was added to the dishes. After treatment for 24 h with TS, LPS and IFN at final concentrations of 10 lgÆmL )1 and 100 UÆmL )1 were also added to the dishes for induction of NO production. Then, 48 h after the addition of LPS/IFN, the cells were subjected to various assays. Nitrite analysis The amount of NO was determined as production of nitrite, because NO generated by various stimuli was quickly oxidized to nitrite. Nitrite in the culture medium was measured colorimetrically using Griess reagent (1% sulfa- nilamide, 0.1% N-1-naphthyl-ethylenediamine dihydro- chloride) [27]. Nitrite diazotiates the aryl amine, and then the diazotiated product forms an azochromophore by coupling with naphthyl-ethylenediamine. Absorbance was measured at 550 nm in a Shimadzu UV-1600 spectropho- tometer, and nitrite concentration was determined using sodium nitrite as a standard. Western blotting of inducible NO synthase After removal of the culture medium for analysis of nitrite, cells were collected from the dish into a sample tube using a cell scraper. Buffer (2% SDS, 20% glycerol, 50 m M Tris/ HCl, pH 6.8) to dissolve cells was added to the cells, and then the cell suspension was sonicated in a bath-type sonicator for 5min. The amount of protein of the solubilized cells was determined with a bicinchoninic acid protein assay kit (PIERCE, Rockford, IL, USA) using bovine serum albumin as a standard. The solubilized cells were subjected to 10% SDS/polyacrylamide gel electrophoresis, and pro- teins were transferred electrophoretically to a poly(vinylidene difluoride) membrane. The membrane was treated with a rabbit anti-iNOS polyclonal antibody or rabbit anti-PKC polyclonal antibody at 1 : 1000 dilution and anti-(rabbit IgG) Ig horseradish peroxidase conjugated antibody at 1 : 5000 dilution as a primary antibody and a secondary antibody, respectively. The blots were detected with an enhanced chemiluminescence kit (Amersham International Plc, UK) and exposed to photographic films. The results of Western blot analysis were representative pictures of at least three independent experiments. Statistical analysis Data were expressed as the mean ± standard deviation of at least three independent experiments. Statistical significance was assessed by multiple-comparison test (Fisher’s protec- ted least significant difference method). A P value of < 0.01 was considered to be statistically significant. RESULTS Effect of TS on LPS/IFN-induced NO production in VSMC The addition of LPS induced small, but significant NO production, but additions of both LPS and IFN induced detectably high levels of NO in VSMC. As shown in Fig. 2A, approximately 20 l M of NO was produced by 10 lgÆmL )1 of LPS and 100 UÆmL )1 of IFN. Under these conditions, detectable induction of iNOS protein was observed by Western blotting (Fig. 2B). Thus, in subsequent experiments, we used LPS and IFN 10 lgÆmL )1 and 100 UÆmL )1 , respectively, to induce the NO production in VSMC. These concentrations are relatively high in com- parison with those used for induction of NO in macroph- ages but are similar to the amounts used in cells with low sensitivity to LPS and IFN [28]. NO production was not induced by 10 l M TS alone in the absence of LPS/IFN. However, treatment with TS 24 h before the addition of LPS/IFN enhanced LPS/IFN-induced NO production about twofold after a 48-h incubation Fig. 1. Structures of a-tocopherol (a-T) and T derivatives a-tocopheryl hemisuccinate (TS), a-tocopheryl acetate (TA) and a-tocopheryl nicoti- nate (TN). 2368 K. Kogure et al. (Eur. J. Biochem. 269) Ó FEBS 2002 (Fig. 2A). The enhancing effects of TS at concentrations higher than 10 l M were almost the same as that with 10 l M TS. Because TS has been reported to induce apoptosis in various cell lines [8–15,29], the inhibitory effect of TS on the cell growth at higher concentrations might prevent the increase in the enhancing effect of TS on LPS/IFN-induced NO production in VSMC. Therefore, we used a concentra- tion of 10 l M TS in this study, except in some cases. Furthermore, TS enhanced the amount of iNOS protein induced by LPS/IFN (Fig. 2B), as well as NO production. Accordingly, we concluded that the enhancing effect of TS on LPS/IFN-induced NO production is caused by enhance- ment of iNOS protein induction. Effects of a-T and its derivatives on the enhancement by TS of LPS/IFN-induced NO production We compared the effects of a-T and its derivatives TA and TN with that of TS on LPS/IFN-induced NO production. The additions of a-T, TA and TN did not induce NO production in the absence of LPS/IFN, like TS (data not shown). Furthermore, a-T, TA and TN, even at 50 l M ,did not enhance the LPS/IFN-induced NO production in VSMC in contrast to TS (Fig. 3). We further examined the effects of a-T, TA and TN on the enhancement by 10 l M TS of LPS/IFN-induced NO production. As shown in Fig. 3, 50 l M a-T significantly lowered the TS-enhanced NO production. In addition, a-T reduced the amount of TS-enhanced iNOS protein induced by LPS/IFN (data not shown). It is noteworthy that TA and TN also decreased the enhancing effect of TS on LPS/IFN- induced NO production. Because the antioxidant a-T prevented the enhancing effect of TS on LPS/IFN-induced NO production (Fig. 3), we examined the effect of antioxidants such as butylhyd- roxyl anisol (BHA) and ascorbic acid (AsA) on the enhancing effect of TS on LPS/IFN-induced NO produc- tion. Unexpectedly, neither BHA nor AsA affected the TS-enhanced NO production (data not shown). Effects of TS on NO productions induced by single additions of various concentrations of LPS and IFN NO production is reported to be stimulated by LPS and IFN through independent signal pathways; LPS and IFN stimulate cells by activating NF-jBandtheinterferon regulatory factor-1, respectively [25,29–31]. To determine which signal pathway of LPS or IFN was stimulated with TS, we examined the effects of TS on the NO production with LPS alone or IFN alone. As shown in Fig. 4, addition of LPS alone (10 mgÆmL )1 )orIFN alone (100 UÆmL )1 ) induced the NO productions but were very feeble as 1.1-fold and 1.4-fold of control, respectively. These results indicate that the detectable high NO production needs the presence of both LPS and IFN in our experimental conditions. TS even at a high concentration of 50 l M did not enhance IFN-induced NO production (Fig. 4A). On the other hand, LPS- dependent NO production was enhanced at 50 l M of TS (Fig. 4B). Addition of 10 l M TS also stimulated, but not significantly, the LPS-dependent NO production (data not shown). The TS-stimulated NO production increased with an increase in the concentration of LPS. From these results, TS was suggested to participate in the pathway stimulated with LPS, and IFN is necessary for expansion of the TS effect. In addition, the TS (50 l M )-induced acceleration of NO production in the LPS alone system was inhibited by a-T, TA and TN (Fig. 4B, insert) as in the LPS/IFN combination system (Fig. 3). However, because the acceleration effect of TS on LPS-dependent NO production system was very small, the inhibiting Fig. 3. Effects of a-T and its derivatives (TA and TN) on LPS/IFN- induced NO production in VSMC treated without or with 10 l M TS. The concentrations of a-T, TA and TN were 50 l M .Otherexperi- mental conditions were as described in Fig. 2. Values are means ± SD (n ¼ 3). *P £ 0.01. Fig. 2. Enhancements by TS of LPS/IFN-induced NO production (A) and iNOS induction (B) in VSMC. The concentrations of LPS and IFN were 10 lgÆmL )1 and 100 UÆmL )1 , respectively. (A) NO was measured using the Griess reagent as nitrite. The amount of TS added alone was 10 l M . The doses of TS coadded with LPS/IFN are shown under the columns. Values are means ± SD (n ¼ 3). *P £ 0.01. (B) Induced iNOS in VSMC was detected by Western blotting using a rabbit anti- iNOS Ig. The concentration of TS was 10 l M . Ó FEBS 2002 Tocopheryl succinate-enhanced NO production (Eur. J. Biochem. 269) 2369 effects of a-T and its derivatives were not as clear as the effects observed in the system with LPS/IFN. Effects of protein kinase inhibitors on the enhancement by TS of LPS/IFN-induced NO production Protein kinases such as PKA and PKC, and various factors such as tumor necrosis factor receptor 6 (TRAF6) and myeloid differentiation protein (MyD88) are reported to participate in LPS-induced signal transduction [25,29–31]. BecauseTSwassuggestedtoaffectmainlythesignal pathway stimulated with LPS, we examined the effects of inhibitors of PKA and PKC on TS-enhanced NO produc- tion. As shown in Fig. 5A, the PKA inhibitors KT-5720 and H8 and the PKC inhibitors Ro31-2880 and GF109203X did not affect apparently LPS/IFN-induced NO production. In the combination system of 10 l M TS with LPS/IFN, the PKA inhibitors KT-5720 and H8 had no effect, but the PKC inhibitors Ro31-2880 and GF109203X significantly inhibited NO production. The degrees of inhibition of NO production with Ro31-2880 and GF109203X were approximately 70 and 30%, respectively. Effects of TS on the amount of PKCa We further examined the effects of TS on the amounts of PKCa and PKCb by Western blotting. As shown in Fig. 5B, PKCa was increased slightly by treatment with LPS/IFN. Addition of 10 l M TS to the LPS/IFN-system enhanced the amount of PKCa. However, we could not detect PKCb in control cells, and no change of PKCb involved with and without TS was observed in the LPS/IFN treated cells (data not shown). The amounts of other proteins related with the LPS-stimulated signal pathway, such as TRAF6 and MyD88 were not affected by the addition of TS in this study (data not shown). These results suggested that TS induced upregulation of PKCa. DISCUSSION In this study, to obtain information about the mode of actions of TS, we examined the effect of TS on LPS/IFN- induced NO production in VSMC. We found that TS activated LPS/IFN-induced NO production in VSMC through enhancement of iNOS protein synthesis, although TS itself did not induce NO production in the absence of LPS/IFN (Fig. 2). Previously, we found that TS was taken up immediately into the VSMC, but TS was not hydrolyzed to a-T and succinic acid [32]. Accordingly, the enhancement of LPS/IFN-induced NO production is attained by TS itself rather than a derivative. TS enhanced LPS-dependent but not IFN-dependent NO production, indicating that TS activated a LPS-stimulated signal pathway (Fig. 4). PKC is a key kinase in the LPS- stimulated signal pathway [30,31]. The findings that PKC inhibitors, not PKA inhibitors, inhibited TS-enhanced NO production, and the level of NO production inhibited with PKC inhibitor Ro31-2880 was almost the same as that of LPS/IFN-induced NO production without TS (Fig. 5A), suggesting that the enhancement of NO production with TS was strongly dependent on PKC activity. Furthermore, an increase in the amount of PKCa by TS treatment (Fig. 5B) suggested that TS enhanced LPS-dependent NO production through upregulation of PKCa. Fig. 5. Effects of PKA inhibitors (KT-5720 and H8) and PKC inhibitors (Ro31-8220 and GF109203X) on the enhancement by TS of LPS/IFN- induced NO production in VSMC (A), and Western blot analysis of PKCa in control VSMC, VSMC treated with LPS/IFN and VSMC treated with TS and LPS/IFN (B). KT: KT-5720, Ro: Ro31-8220, GF: GF109203X. The concentrations of KT, H8, Ro, GF and TS were 1, 20,5,10and10l M , respectively. Values are means ± SD (n ¼ 3). *P £ 0.01. Other experimental conditions were as described in Fig. 2. Fig. 4. Effects of TS on NO production induced with various amount of IFN (A) or LPS (B) in VSMC. VSMC was treated without (open column) or with 50 l M TS (closed column). The insert graph of (B) shows the inhibition effects of a-T, TA and TN on the enhancement by 50 l M TS of LPS-induced NO production. Values are means ± SD (n ¼ 3). *P £ 0.01. 2370 K. Kogure et al. (Eur. J. Biochem. 269) Ó FEBS 2002 TS enhanced the LPS/IFN-induced NO production but TA and TN did not (Fig. 3), although their antioxidative OH-moiety is masked. We hypothesized that an am- phiphilic characteristic of TS is important for its enhancing effect, because among the a-T derivatives examined only TS has an amphiphilic structure of the polar carboxyl moiety and the hydrophobic isoprene moiety. The enhancing effect of TS on LPS/IFN-induced NO production was inhibited by the coexistence of a-T, TA and TN (Figs 3 and 4), but not of the antioxidants BHA and AsA. These results suggested that active oxygens and free radicals did not participate in the TS effect, and that the inhibitory effect of a-T was mediated by a nonantioxidative reaction. a-T was reported to decrease PKCa activity due to activation of protein phosphatase 2A (PP2A) in smooth muscle cells [2– 6]. These studies suggest that the inhibitory effect of a-T was due to reduction of accelerated PKC activity with TS in VSMC. TA and TN also showed the inhibition effect like a- T on TS-activated NO production, suggesting that the action target of both TA and TN is the same as that of a-T. It is very interesting that a-T and TS showed opposite effects on PKC in this study, although the structures of both are very similar. Perhaps, the negatively charged carboxyl moiety of TS is important for upregulation of PKC. Recently, Neuzil et al. reported the opposite findings to ours. They proposed that TS-induced apoptosis in hepatopoietic and cancer cell lines is caused by the prevention of PKC activity due to activation of PP2A, similar to a-T activation of PP2A [13]. The reason for the inconsistency between our results and those of Neuzil et al. is unclear; it may be caused by differences in the response, delivery and distribution of TS in each cell line. Recently, we found that TS-induced apoptosis of VSMC was caused by the stimulation of superoxide production due to the activation of NADPH oxidase [32]. As the activation of NADPH oxidase is reported to be associated with the activation of PKC [33,34], the PKC-dependent mechanism of TS-induced enhancement of NO production observed in this study are consistent with the mechanism of TS-induced apoptosis. Yu et al. reported recently that activation of ERK is required for TS-induced apoptosis of human breast cancer cells [15]. Kim et al. reported that the addition of TS alone induced NO production in human U937 monoblasts possibly by the activation of NF-jB [19]. Further study is necessary to clarify the relationship between the activation of these factors participating in cell survival signaling pathwaysandTSeffects. In this study, we found for the first time that TS enhanced LPS/IFN-induced NO production in VSMC, and that the TS effect would be induced through upreg- ulation of PKCa. 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(1999) Stimulation of respiratory burst by cyclocommunin in rat neutrophils is associated with the increase in cellular Ca 2+ and protein kinase C activity. Free Rad. Biol. Med. 26, 580–588. 2372 K. Kogure et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . Enhancement by a-tocopheryl hemisuccinate of nitric oxide production induced by lypopolysaccharide and interferon -c through the upregulation of protein. LPS/IFN. Effects of protein kinase inhibitors on the enhancement by TS of LPS/IFN -induced NO production Protein kinases such as PKA and PKC, and various factors such