Non-genomic effects of ginsenoside-Re in endothelial cells via glucocorticoid receptor

FEBS Letters 581 (2007) 2423–2428

Non-genomic effects of ginsenoside-Re in endothelial cells via glucocorticoid receptor Kar Wah Leunga,*, Fung Ping Leungb, Yu Huangb, Nai Ki Maka, Ricky N.S. Wonga b

a Department of Biology, Hong Kong Baptist University, Hong Kong Department of Physiology, Chinese University of Hong Kong, Hong Kong

Received 13 January 2007; revised 13 April 2007; accepted 21 April 2007 Available online 30 April 2007 Edited by Robert Barouki

Abstract We demonstrated that ginsenoside-Re (Re), a pharmacological active component of ginseng, is a functional ligand of glucocorticoid receptor (GR) using competitive ligand-binding assay (IC50 = 156.6 nM; Kd = 49.7 nM) and reporter gene assay. Treatment with Re (1 lM) raises intracellular Ca2+ ([Ca2+]i) and nitric oxide (NO) levels in human umbilical vein endothelial cells as measured using fura-2 and 4-amino-5-methylamino-2 0 ,7 0 -difluorofluorescein diacetate, respectively. Western blot analysis shows that Re increased phosphorylation of endothelial nitric oxide synthase. These effects were abolished by GR antagonist RU486, siRNA targeting GR, non-selective cation channel blocker 2-aminoethyldiphenylborate, or in the absence of extracellular Ca2+, indicating Re is indeed an agonistic ligand for the GR and the activated GR induces rapid Ca2+ influx and NO production in endothelial cells.  2007 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. Keywords: Angiogenesis; Endothelial cell; Ginsenoside; Glucocorticoid receptor; Intracellular calcium concentration; Nitric oxide

1. Introduction Panax ginseng C.A. Meyer has been used as health tonics for centuries in Chinese medication [1]. The pharmacologically active components of ginseng are ginsenosides, which are broadly categorized into 20(S)-protopanaxatriol (PPT) and 20(S)-protopanaxadiol (PPD), based on the number and the position of the sugar moieties attached on the steroidal backbone. To date, more than 30 ginsenosides have been identified, in which ginsenoside Re, a PPT, makes up 0.1% (w/w) and 0.7% (w/w) of ginseng root and berry extracts, respectively [2]. Although, Re is a relative abundant ginsenoside, to date, its bioavailability in mammalian species has not been documented. Re was previously suggested to be a highly stable *

Corresponding author. Fax: +852 3411 5995. E-mail addresses: [email protected] (K.W. Leung), [email protected] (R.N.S. Wong). Abbreviations: 2-APB, 2-aminoethyldiphenylborate; DAF-FM, 4amino-5-methylamino-2 0 ,7 0 -difluorofluorescein; Dex, dexamethasone; eNOS, endothelial nitric oxide synthase; GR, glucocorticoid receptor; GRE, glucocorticoid responsive element; HUVEC, human umbilical vein endothelial cell; [Ca2+]i, intracellular calcium ion concentration; NO, nitric oxide

pro-angiogenic compound by upregulating in vitro proliferation, migration, chemoinvasion and tube formation of human umbilical vein endothelial cell (HUVEC), as well as ex vivo aortic sprouting and in vivo neovascularization [3,4]. Angiogenesis is the growth of new blood vessels from pre-existing vasculatures. It is a highly regulated process controlled by strict balance between the angiogenic promoters and inhibitors [5]. It is an important physiological process required for embryonic development, menstrual cycle and wound healing. Calcium is one of the major angiogenesis regulators and key mediator for endothelial cell proliferation [6,7], which is adopted by many reputed angiogenic factors including vascular endothelial growth factor (VEGF) [7–9]. Here, we demonstrated that Re stimulated the influx of calcium ion in HUVEC, at least partly, contributing to the angiogenic effects of Re. We had previously reported that another angiogenic PPT, ginsenoside-Rg1, induces rapid nitric oxide (NO) production and other angiogenic actions through activation of glucocorticoid receptor (GR) [10,11]. Since, Re shares a high structural correlation with Rg1 with only an additional rhamnose at C6 position (Fig. 1), we speculate that Re may also be a functional ligand of the GR. To elucidate whether Re has direct interaction with the GR, competitive ligand-binding assay and transfection reporter gene assay were employed. Our data established that Re binds to GR and induces a transient increase in intracellular calcium ion concentration ([Ca2+]i), via non-selective cation channel for phosphorylation and activation of endothelial nitric oxide synthase (eNOS) with an increase in NO production. Thus the angiogenic property of Re and its related ginsenosides can be exploited in angiotherapy.

2. Materials and methods 2.1. Experimental reagents Ginsenoside-Re (Re) is a reference compound (purity >98%) purchased form the Division of Chinese Materia Medica and Natural Products, National Institute for the Control of Pharmaceutical and Biological Products (NICPBP), Ministry of Public Health, China. Stock solution of Re (50 mM) was prepared in sterile DMSO. Culture medium M199, endothelial cell growth supplement (ECGS), GR antagonist RU486, 2-aminoethyldiphenylborate (2-APB) were purchased from Sigma (St. Louis, MO). eNOS inhibitor L-NMMA was purchased from Cayman Chemical (Ann Arbor, MI). Foetal bovine serum (FBS) was purchased from Gibco (Carlsbad, CA). Fura2-acetoxymethyl ester (AM), Pluronic F127 and NO sensitive fluorescent dye 4-amino-5-methylamino-2 0 ,7 0 -difluorofluorescein (DAF-FM) diacetate were obtained from Molecular Probes (Leiden, Netherlands).

0014-5793/$32.00  2007 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. doi:10.1016/j.febslet.2007.04.055

2424

K.W. Leung et al. / FEBS Letters 581 (2007) 2423–2428

Fig. 1. Illustrations depicted the molecular structures of ginsenosides Re and Rg1.

2.2. Cell culture and treatments HUVEC was obtained from Clonetics (San Diego, CA) and cultured in complete medium 199 supplemented with 20% FBS, 20 lg/ml ECGS, 90 U/ml heparin and 1% penicillin–streptomycin–neomycin (PSN) in a humidified incubator at 37 C with 5% CO2. HUVEC between passages 2–8 were used in these studies to ensure the genetic stability of the culture. For inhibitory assays, cells were pretreated, respectively with different antagonists or inhibitors, such as RU486 (10 lM), 2-APB (10 lM) and L-NMMA (10 lM) for 30 min in assay medium 199 containing only 90 U/ml heparin and 1% PSN. Cells transfected with the SmartPool siRNA for silencing GR (M-003424-02) (Dharmacon, Lafayette, CO) were incubated for 24 h prior to imaging. Knockdown efficacy of GR was previously demonstrated [10]. 2.3. Measurement of intracellular Ca2+ concentration ([Ca2+]i) [Ca2+]i was determined using the fluorescent Ca2+ indicator fura-2 AM. The circular coverslip containing the endothelial cells were pinned in a specially designed chamber. The chamber was placed on the stage of the Motic AE31 inverted microscope fitted with a ·20 objective. It was then illuminated with a computer-controlled shutter lambda 10-B filter wheel with mercury xenon light source (Intracellular Imaging Inc., University of Cincinnati, Cincinnati, OH, USA). Fura-2 loaded cells were excited with an alternating UV light at 340 and 380 nm. Images of the respective 510-nm emissions were collected in 2-s intervals using InCytIm2 ratio ion measurement programme version 5.27A (Intracellular Imaging Inc.). The emitted light was transmitted to the collecting device and then to a MICROPIXVGA camera. A calibration curve was constructed using a standard calibration kit (Molecular Probes). For calcium-free experiment, the assay medium was replaced by OCa2+-PSS (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM glucose, 2 mM EGTA, 5 mM HEPES, pH 7.4) during imaging. Three individual samples were analysis in each trial and each data point is the mean of three independent trials. 2.4. Measurement of NO NO production was monitored using fluorescent NO indicator DAF–FM diacetate. HUVEC was loaded with 1 lM DAF–FM for 30 min in assay medium. Detection was made by measuring fluorescence intensity excited at 495 nm and emitted at 515 nm with the same setup as mentioned in ‘‘Measurement of intracellular Ca2+ concentration ([Ca2+]i)’’.

2.5. Western blot analysis Equal amount of proteins were separated by SDS–PAGE and transferred onto nitrocellulose membrane. The membrane was then incubated with antibody against phospho-eNOS (Ser1177) (Upstate Biotechnology Inc., Lake Placid, NY) for 3 h, followed by horseradish peroxidase-conjugated secondary antibodies and the banding were visualized by an ECL detection system. Then, the membranes were stripped and re-probed with antibody against and eNOS (Upstate Biotechnology Inc.). The bands were quantified by densitometric analysis using Metamorph software. 2.6. GR competitive ligand-binding assays Commercially available GR competitive binding assay (Invitrogen) was used according to the manufacturer’s instructions as previously described [10]. Briefly, serial 2-fold dilution of GR agonist dexamethasone (Dex) (0.9 nM to 1 lM) and Re (1.8 nM to 2 lM) were competed with a proprietary fluorescent glucocorticoid ligand (Fluormone) provided in the kit, for binding to the human GR. Fluorescence polarization was measured with a PTI Fluorescence Lifetime Scanning Spectrometer, and the 50% inhibitory concentration (IC50) and the equilibrium dissociation constant (Kd) were determined. 2.7. Reporter gene assay pGRE-TA-SEAP reporter plasmids (BD Biosciences, Palo Alto, CA) (0.5 lg/well of 24-well plate) or pTAL-SEAP (negative control) was transiently transfected into HUVEC using Lipofectamine (Invitrogen). pGRE-TA-SEAP contains tandem repeats of glucocorticoid responsive element (GRE) link to a downstream SEAP reporter gene. pTAL-SEAP is an identical plasmid but without the GREs. After transfection for 24 h, treatments were added and conditioned medium were harvested after another 24 h. SEAP activity was assayed according to the manufacturer’s protocol using the SEAP fluorescence detection kit (BD Biosciences, San Jose, CA). 2.8. Statistical analysis Numerical results were analyzed using one-way ANOVA with Duncan post hoc test. For [Ca2+]i and NO measurement, nonparametric analysis with Prism Software was employed. Values shown are means of at least N = 3 experiments with ±S.D. Differences were considered statistically significant at a value of P 6 0.05.

A

250 nM

250

+ Re

500 nM

200

Control + 2-APB -Ca2+

750 nM

150

1000 nM

eNOS

100 50 100

200

300

Time (sec) 3

* concentration

Relative Intracellular calcium

3

*

2 1 0 Control

+

2-APB

-Ca2+

+ Re 0

2

1

0

Control

+

2-APB + Re

- Ca2+

2-APB

- Ca2+ - Re

Fig. 2. Re induces elevation of [Ca2+]i in HUVEC. (A) Calcium indicator fura-2 loaded HUVEC was treated with Re at 250, 500, 750 and 1000 nM. The [Ca2+]i was monitored in 2-s intervals using InCytIm2 ratio ion measurement programme for 5 min. (B) Histogram showing the fold change in [Ca2+]i over control. Cells treated with Re (1 lM), or pretreatment with non-selective cation channel blocker 2APB (10 lM), or the use of Ca2+-free extracellular buffer, followed by treatment with Re (1 lM). Area under the curve representing total free calcium ion in 5 min was taken for analysis (N = 3; ±S.D.). *Means data significantly different from control at a value of P 6 0.05.

3. Results 3.1. Re stimulates [Ca2+]i via non-selective cation channels [Ca2+]i in HUVEC was monitored using fluorescence fura-2 AM dye. Re increased [Ca2+]i in a concentration-dependent manner with 50% effective concentration (EC50) at 316 nM and [Ca2+]i was peaked at 30 s after treatment (Fig. 2A). We also examined whether the Re-mediated [Ca2+]i elevation is a result of calcium influx from extracellular source or release of Ca2+ from internal reservoir. As shown in Fig. 2B, pretreatment with non-selective cation channel blocker 2-APB or depletion of extracellular source of Ca2+ suppressed the respective increase of [Ca2+]i, indicating the source of Ca2+ is external. 3.2. Re induces NO production Endothelial NOS is one of the direct targets of calcium signaling. As revealed by Western blot analysis, Re (1 lM) activated eNOS via the increase of eNOS phosphorylation (Ser1177) (Fig. 3A). Also, Re-enhanced production of NO as determined using DAF-FM diacetate fluorescence dye was in a dose-dependent manner (Fig. 3B). Fluorescence gradually accumulated in cells and reached plateau after peaking at 100 s (Fig. 3B). The EC50 for Re to induce NO production was 615 nM. Re-induced activation of eNOS and NO production were abrogated by pretreatment with 2-APB, absence of extracellular Ca2+ or eNOS inhibitor L-NMMA (Fig. 3A

B DAF-FM Fluorescence (Arbitrary units)

0

B

p-eNOS

250 nM

290000

500 nM

280000

750 nM

270000

1000 nM

260000 250000 240000 230000 220000 210000 200000

0

100

200

300

400

500

600

Time (sec)

C DAF-FM Fluorescence (Arbitrary units)

Intracellular Calcium Concentration (nM)

A

2425 Ratio of p-eNOS/eNOS

K.W. Leung et al. / FEBS Letters 581 (2007) 2423–2428

1.5

*

1

0.5

0 Control

+

2-APB

-Ca2+

+ Re

L-NMMA 2-APB

-Ca2+

L-NMMA

- Re

Fig. 3. Re induces phosphorylation of eNOS and NO production. (A) Proteins (35 lg/lane) were separated on 10% SDS–PAGE and transferred onto nitrocellulose membrane. Protein loading was normalized with reference to total eNOS. Phosphorylated eNOS (Ser1177) was increased after Re (1 lM) treatment for 5 min. Total eNOS had no change in quantity in all treatment groups. (B) NO production as monitored in DAF-FM diacetate-fluorescent dye loaded HUVEC. Re increased NO production dose-dependently (250, 500, 750 and 1000 nM) in a time window of 10 min. (C) Histogram showing the fold changes in NO production over control. Cells treated with Re (1 lM), or pretreatment with non-selective cation channel blocker 2APB (10 lM), the use of Ca2+-free extracellular buffer, or NOS inhibitor L-NMMA (10 lM), followed by treatment with Re (1 lM). Area under the curve representing total NO production in 10 min was taken for analysis (N = 3; ±S.D.). *Means data significantly different from control at a value of P 6 0.05.

and C). These results provided evidence that Re-activated eNOS is downstream of Ca2+ elevation. 3.3. Re binds to the GR Owing to the structural similarity between Re and Rg1 (Fig. 1), Re was speculated to effect via the GR like Rg1. A competitive ligand-binding assay was performed to study the specific binding of Re to the GR using a proprietary fluorescent glucocorticoid ligand (Fluormone)-recombinant human

2426

A

350 300

Relative intracellular calcium concentration

Dex

250

Re

200 150 100

3

*

*

2.5 2 1.5 1 0.5 0 Control

+

RU486

50

-1

0

1

2

3

4

B

+ Re GR NS Control + RU486 siRNA siRNA

Log [Conc.] / nM p-eNOS

3

eNOS

*

*

NSsiRNA

RU486

1

0 Control

Re

Dex

RU486

Fig. 4. Competitive binding of Re to the GR and reporter gene assay. (A) The competitive binding was determined by a fluorescent polarization-based assay using recombinant human GR and a proprietary ligand (Fluormone). Dex served as a positive control. Fluorescent polarization was expressed in millipolarization (mP). Data represent the mean from three independent experiments. (B) HUVEC was transiently transfected with a reporter gene containing GRE. Dex (50 nM) and RU486 (10 lM) were used as positive and negative controls (N = 3; ±S.D.). *Means data significantly different from control at a value of P 6 0.05.

GR complex. Displacement of Fluormone from the complex by Re results in a shift in the fluorescent polarization of which the relative binding affinity of Re for the GR can be determined. The competitive ligand-binding assay indicated that Re is indeed a ligand of the GR. The Kd values for Dex and Re were 3.4 nM (IC50, 10.6 nM; R2 = 0.99) and 49.7 nM (IC50, 156.6 nM; R2 = 0.98), respectively (Fig. 4A). In the classical pathway of nuclear hormone receptor, the receptor-ligand complex undergoes conformation changes and translocates into the nucleus where it interacts with GRE located on the promoter regions of target genes [12]. In transfection reporter gene assay, Re was able to activate SEAP transcription of a promoter containing three copies of the GRE in HUVEC. Re (250 nM) showed a 2.4-fold SEAP induction over untreated control and this efficacy was comparable with that of Dex (Fig. 4B). Both Re and Dex had no effect on pTAL-SEAP, a vector identical to pERE-TA-SEAP but without the GRE (data not shown). RU486, a specific GR antagonist, was used as a negative control that showed

GR

NS-

siRNA

siRNA

- Re

3 2.5 2 1.5 1 0.5 0

*

Control

+

*

RU486

GR NSsiRNA siRNA

- Re

C

2

DAF-FM Fluorescence (Arbitrary units)

Relative Fluorescence intensity

B

GR siRNA

+ Re

Ratio of p-eNOS/eNOS

Fluorescence polarization (mP)

A

K.W. Leung et al. / FEBS Letters 581 (2007) 2423–2428

1.5

*

*

1

0.5

0 Control

+

RU486

GR

NS-

siRNA

siRNA

+ Re

RU486

GR

NS-

siRNA

siRNA

- Re

Fig. 5. Effects of Re are GR dependent. Cells treated with Re (1 lM), or pretreatment with GR antagonist RU486 (10 lM), or transfected with siRNA targeting GR or non-specific (NS)-siRNA, followed by treatment with Re (1 lM). (A) Histogram showing the fold change in [Ca2+]i over control. Area under the curve representing total free calcium ion in 5 min was taken for analysis. (N = 3; ±S.D.). (B) Western blot analysis probing with antibody against phosphorylated eNOS. Protein loading was normalized with reference to total eNOS. (C) Histogram showing the fold changes in NO production over control. Area under the curve representing total NO production in 10 min was taken for analysis (N = 3; ±S.D.). *Means data significantly different from control at a value of P 6 0.05.

no transactivation of the reporter gene (Fig. 4B). Taken together, these results demonstrate that Re is indeed a functional agonist for GR. 3.4. Effects of Re are GR-dependent Next, we examine whether Re effects via GR. In the presence of specific GR antagonist RU486 (10 lM) or using siRNA to knockdown GR, Re-induced elevation of [Ca2+]i, eNOS phosphorylation and NO production were significantly reduced (Fig. 5A–C), while pretreatment with specific estrogen receptor (ER) antagonist ICI 182 780 (data not shown), or transfection with non-specific siRNA had no inhibitory effects, suggesting the involvement of GR, but not ER, in the rapid responses initiated by Re.

K.W. Leung et al. / FEBS Letters 581 (2007) 2423–2428

4. Discussion Panax ginseng has been used extensively in countries, including the United States and many parts of Europe. The pharmacologically active ginsenosides were demonstrated to exert beneficial effects to the cardiovascular system, in which ginsenoside-Re was reported to stimulate vasodilation and angiogenesis in vivo [3,13]. Angiogenesis is a fundamental process in both physiological and pathological conditions. Therapeutical angiogenesis is now drawing more attention as a treatment of chronic wound or gastric ulcer [14,15]. In this study, we demonstrated that Re stimulated Ca2+ influx via non-selective cation channel, the onset of calcium influx was rapid following by a quick decline (Fig. 2). The necessity in limiting the duration of [Ca2+]i elevation may due to the fact that prolonged elevation of calcium can be toxic and may trigger apoptosis [16,17]. Calcium signaling regulates a diversity of cellular responses, including cell cycle progression and NO production [18]. Yet, basal [Ca2+]i in the endothelial cells is insufficient in regulating the minimal activity of eNOS and briefly elevation of [Ca2+]i is thus required to facilitate the activation of eNOS [19,20]. Our results shown that Re induces peak NO production (100 s) follows elevation in [Ca2+]i at around 60 s. Previous reports on Rg1, another angiogenic ginsenoside of the same structural category as Re, and Dex, a specific GR agonist, showed a peak eNOS phosphorylation and NO production were observed at 30–60 min via PI3K/Akt pathway [10,21]. The time-point discrepancy between Re and Rg1/Dex can be explained by the fact that regulation of eNOS activation is bimodal. Calcium-calmodulin activation of eNOS leads to immediate NO synthesis until the binding of the heat shock protein (hsp)-90 to the eNOS, and then Akt phosphorylation activation of eNOS takes over and accounts for the second phase [22]. Re was recently demonstrated to be able to stimulate NO release via the PI3K/Akt pathway in cardiac myocytes [23], we believe it would exhibit the same actions in endothelial cells like Rg1 [10]. Calcium-calmodulin-dependent activation of eNOS and NO production is a major determinant of the vascular tone [24–26], indicating Re can potentially be used as a new modality in treating hypertension. Moreover, there are more evident that NO is more than just a vasodilator, it was also shown to play a key role in postnatal angiogenesis, though the precise mechanisms of how NO regulates angiogenesis is still unclear [27–30]. From the inhibitory assays, both GR antagonist RU486 and GR siRNA significantly reduced the Re-mediated Ca2+ influx and NO production from the endothelial cells (Fig. 5). Estrogen receptor (ER), the second largest population of nuclear receptors in the endothelial after GR, was also investigated for the possible role in the Re-induced non-genomic responses in endothelial cells. Pretreatment with specific ER antagonist ICI 182,780 have no inhibitory effects (data not shown), indicating the effects of Re is GR-mediated, but not ER. Further, we established a direct correlation between Re and the GR using competitive ligand-binding and reporter gene assays. We demonstrated for the first time that Re is a functional ligand of the GR and the Kd and IC50 were 49.7 nM and 156.6 nM, respectively (Fig. 4A). Our previous study had reported Rg1 binds to GR with a slightly stronger affinity than that of Re as demonstrated using the same competitive binding assay [10]. The lower affinity of Re, in comparison with Rg1, might due to presence of the additional rhamnose at the C-6

2427

position that causing steric bump during Re–GR interaction, complete understanding warrants detailed structural analysis. GR, a member of the nuclear receptor superfamily, exists abundantly in almost all cell types. Besides the classical transcriptional activation of GR, there is accumulating evidence showing the physiological importance of the GR-mediated non-transcriptional ‘‘rapid’’ pathways [21,31,32]. The ability for Re to stimulate rapid calcium influx and NO production, as well as to transactivate genes regulated by GRE, suggesting Re is both non-transcriptional and transcriptional agonist to GR. Thus, administration of Re could modulate a broader spectrum of biological processes, including secretion, metabolism, gene transcription and even apoptosis, via GR-mediated calcium signaling [33–36]. Collectively, we have clearly demonstrated that the ginsenoside-Re can act as an agonist for GR, eliciting Ca2+ influx and NO production. Re, as an angiogenic modulator, can be exploited to be a new modality in angiotherapy to treat pathological conditions involving insufficient angiogenesis, for examples, chronic wounds and ischemic diseases. Acknowledgements: The present work was supported by Earmarked Research Grants (HKBU 2171/03M, HKBU2476/06M to Ricky N.S. Wong and CUHK 4362/04M to Yu Huang) of the Research Grant Council, Hong Kong SAR Government. F.P.L. is a post-doctoral fellow supported by CUHK 4362/04M.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2007. 04.055. References [1] Attele, A.S., Wu, J.A. and Yuan, C.-S. (1999) Ginseng pharmacology. Multiple constituents and multiple actions. Biochem. Pharmacol. 58, 1685–1693. [2] Attele, A.S., Zhou, Y.P., Xie, J.T., Wu, J.A., Zhang, L., Dey, L., Pugh, W., Rue, P.A., Polonsky, K.S. and Yuan, C.S. (2002) Antidiabetic effects of Panax ginseng berry extract and the identification of an effective component. Diabetes 51, 1851– 1858. [3] Huang, Y.C., Chen, C.T., Chen, S.C., Lai, P.H., Liang, H.C., Chang, Y., Yu, L.C. and Sung, H.W. (2005) A natural compound (ginsenoside Re) isolated from Panax ginseng as a novel angiogenic agent for tissue regeneration. Pharm. Res. 22, 636–646. [4] Yu, L.C., Chen, S.C., Chang, W.C., Huang, Y.C., Lin, K.M., Lai, P.H. and Sung, H.W. (2007) Stability of angiogenic agents, ginsenoside Rg1 and Re, isolated from Panax ginseng: in vitro and in vivo studies. Int. J. Pharm. 10 (328), 168–176. [5] Folkman, J. (2003) Fundamental concepts of the angiogenic process. Curr. Mol. Med. 3, 643–651. [6] Patton, A.M., Kassis, J., Doong, H. and Kohn, E.C. (2003) Calcium as a molecular target in angiogenesis. Curr. Pharm. Des. 9, 543–551. [7] Antoniotti, S., Fiorio Pla, A., Pregnolato, S., Mottola, A., Lovisolo, D. and Munaron, L. (2003) Control of endothelial cell proliferation by calcium influx and arachidonic acid metabolism: a pharmacological approach. J. Cell Physiol. 197, 370–378. [8] Cheng, H.W., James, A.F., Foster, R.R., Hancox, J.C. and Bates, D.O. (2006) VEGF activates receptor-operated cation channels in human microvascular endothelial cells. Arterioscler. Thromb. Vasc. Biol. 26, 1768–1776. [9] Li, Z., Chen, X., Niwa, Y., Sakamoto, S. and Nakaya, Y. (2001) Involvement of Ca2+-activated K+ channels in ginsenosides-

2428

[10]

[11]

[12]

[13]

[14] [15] [16] [17] [18] [19] [20]

[21]

[22]

K.W. Leung et al. / FEBS Letters 581 (2007) 2423–2428 induced aortic relaxation in rats. J. Cardiovasc. Pharmacol. 37, 41–47. Leung, K.W., Cheng, Y.K., Mak, N.K., Chan, K.K., Fan, T.P. and Wong, R.N. (2006) Signaling pathway of ginsenoside-Rg1 leading to nitric oxide production in endothelial cells. FEBS Lett. 580, 3211–3216. Leung, K.W., Pon, Y.L., Wong, R.N. and Wong, A.S. (2006) Ginsenoside-Rg1 induces vascular endothelial growth factor expression through the glucocorticoid receptor-related phosphatidylinositol 3-kinase/Akt and beta-catenin/T-cell factor-dependent pathway in human endothelial cells. J. Biol. Chem. 281, 36280–36288. Dahlman-Wright, K., Wright, A., Carlstedt-Duke, J. and Gustafsson, J.A. (1992) DNA-binding by the glucocorticoid receptor: a structural and functional analysis. J. Steroid Biochem. Mol. Biol. 41, 249–272. Scott, G.I., Colligan, P.B., Ren, B.H. and Ren, J. (2001) Ginsenosides Rb1 and Re decrease cardiac contraction in adult rat ventricular myocytes: role of nitric oxide. Br. J. Pharmacol. 134, 1159–1165. Fan, T.P., Yeh, J.C., Leung, K.W., Yue, P.Y. and Wong, R.N. (2006) Angiogenesis: from plants to blood vessels. Trends Pharmacol. Sci. 27, 297–309. Zhang, S.C. (1985) Experimental studies on action of ginseng flower saponins and ginsenoside Re in gastric ulcers in rats. Zhong Yao Tong Bao 10, 43–44. Berridge, M.J. (1994) The biology and medicine of calcium signaling. Mol. Cell Endocrinol. 98, 119–124. Choi, J. and Husain, M. (2006) Calmodulin-mediated cell cycle regulation: new mechanisms for old observations. Cell Cycle 5, 2183–2186. Wang, H.G., Pathan, N., Ethell, I.M., Krajewski, S., Yamaguchi, Y. and Shibasaki, F. (1999) Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD. Science 284, 339–343. Presta, A., Liu, J., Sessa, W.C. and Stuehr, D.J. (1997) Substrate binding and calmodulin binding to endothelial nitric oxide synthase coregulate its enzymatic activity. Nitric Oxide 1, 74–87. Forstermann, U., Pollock, J.S., Schmidt, H.H., Heller, M. and Murad, F. (1991) Calmodulin-dependent endothelium-derived relaxing factor/nitric oxide synthase activity is present in the particulate and cytosolic fractions of bovine aortic endothelial cells. Proc. Natl. Acad. Sci. USA 88, 1788–1792. Moghadam, A.H., Simoncini, T., Yang, Z., Limbourg, F.P., Plumier, J.C., Rebsamen, M.C., Hsieh, C.M., Chui, D.S., Thomas, K.L., Prorock, A., Laubach, V.E., Moskowitz, M.A., French, B.A., Ley, K. and Liao, J.K. (2002) Acute cardiovascular protective effects of corticosteroids are mediated by non-transcriptional activation of endothelial nitric oxide synthase. Nat. Med. 8, 473–479. Brouet, A., Sonveaux, P., Dessy, C., Moniotte, S., Balligand, J.L. and Feron, O. (2001) Hsp90 ensures the transition from the early Ca2+-dependent to the late phosphorylation-dependent activation of the endothelial nitric-oxide synthase in vascular endothelial

[23]

[24] [25] [26] [27]

[28] [29]

[30]

[31] [32]

[33]

[34]

[35] [36]

growth factor-exposed endothelial cells. J. Biol. Chem. 276, 32663–32669. Furukawa, T., Bai, C.X., Kaihara, A., Ozaki, E., Kawano, T., Nakaya, Y., Awais, M., Sato, M., Umezawa, Y. and Kurokawa, J. (2006) Ginsenoside Re, a main phytosterol of Panax ginseng, activates cardiac potassium channels via a nongenomic pathway of sex hormones. Mol. Pharmacol. 70, 1916–1924. Wyatt, A.W., Steinert, J.R. and Mann, G.E. (2004) Modulation of the L -arginine/nitric oxide signalling pathway in vascular endothelial cells. Biochem. Soc. Symp. 71, 143–156. Berridge, M.J. (1997) Elementary and global aspects of calcium signalling. J. Physiol. 499, 291–306. Himpens, B., Missiaen, L. and Casteels, R. (1995) Ca2+ homeostasis in vascular smooth muscle. J. Vasc. Res. 32, 207–219. Ziche, M., Morbidelli, L., Choudhuri, R., Zhang, H.T., Donnini, S., Granger, H.J. and Bicknell, R. (1997) Nitric oxide synthase lies downstream from vascular endothelial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis. J. Clin. Invest. 99, 2625–2634. Ferrara, N., Houck, K., Jakeman, L. and Leung, D.W. (1992) Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocr. Rev. 13, 18–32. Morbidelli, L., Chang, C.H., Douglas, J.G., Granger, H.J., Ledda, F. and Ziche, M. (1996) Nitric oxide mediates mitogenic effect of VEGF on coronary venular endothelium. Am. J. Physiol. 270, H411–H415. Papapetropoulos, A., Garcia-Cardena, G., Madri, J.A. and Sessa, W.C. (1997) Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. J. Clin. Invest. 100, 3131–3139. Limbourg, F.P. and Liao, J.K. (2003) Nontranscriptional actions of the glucocorticoid receptor. J. Mol. Med. 81, 168–174. Limbourg, F.P., Huang, Z., Plumier, J.C., Simoncini, T., Fujioka, M., Tuckermann, J., Schutz, G., Moskowitz, M.A. and Liao, J.K. (2002) Rapid non-transcriptional activation of endothelial nitric oxide synthase mediates increased cerebral blood flow and stroke protection by corticosteroids. J. Clin. Invest. 110, 1729–1738. Lee, G.S., Choi, K.C. and Jeung, E.B. (2006) Glucocorticoids differentially regulate expression of duodenal and renal calbindinD9k through glucocorticoid receptor-mediated pathway in mouse model. Am. J. Physiol. Endocrinol. Metab. 290, E299–E307. Nambiar, M.P., Enyedy, E.J., Fisher, C.U., Warke, V.G. and Tsokos, G.C. (2001) High dose of dexamethasone upregulates TCR/CD3-induced calcium response independent of TCR zeta chain expression in human T lymphocytes. J. Cell Biochem. 83, 401–413. Lam, M., Dubyak, G. and Distelhorst, C.W. (1993) Effect of glucocorticosteroid treatment on intracellular calcium homeostasis in mouse lymphoma cells. Mol. Endocrinol. 7, 686–693. Kerr, D.S., Campbell, L.W., Thibault, O. and Landfield, P.W. (1992) Hippocampal glucocorticoid receptor activation enhances voltage-dependent Ca2+ conductances: relevance to brain aging. Proc. Natl. Acad. Sci. USA 89, 8527–8531.