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Ginsenosides may reverse the dexamethasone-induced down-regulation of glucocorticoid receptor
General and Comparative Endocrinology 140 (2005) 203–209 www.elsevier.com/locate/ygcen
Ginsenosides may reverse the dexamethasone-induced down-regulation of glucocorticoid receptor Changquan Linga,¤,1, Yong Lia,1, Xiaoyan Zhub, Chen Zhanga, Min Lic a
Department of Chinese Traditional Medicine, Changhai Hospital, Second Military Medical University, Shanghai 200433, China b Institute of Pathophysiology, Second Military Medical University, Shanghai 200433, China c Department of Navy Medicine, Second Military Medical University, Shanghai 200433, China Received 30 March 2004; revised 21 October 2004; accepted 9 November 2004
Abstract The eVects of glucocorticoid (GC) hormones are mediated via an intracellular receptor, the glucocorticoid receptor (GR). It has been established that glucocorticoid down-regulate GR. Ginsenosides (GSS) from extract of Panax ginseng have demonstrated glucocorticoid-like activities in homeostasis and regulation of immunity, etc. We hypothesize that ginsenosides might mediate some of their actions by binding to the GR. The present study is aimed to determine whether GSS can act like a GC analog in the activation of glucocorticoid response element-luciferase activity in HL7702 cells. We found that GSS alone had no eVect on the expression of reporter gene, but it enhanced dexamethasone (Dex)-induced transcription of reporter gene. To further explore the eVects of GSS, we examined the inXuence of GSS on the gene and protein expression as well as hormone binding activity of GR by semi-quantitative RT-PCR, Western blot, and radioligand-binding assay, respectively. GSS partially reversed the Dex-induced decrease in GR expression and hormone binding activity with an optimal dose of 25 g/ml, implicating a positive regulatory eVect of GSS on GR expression and binding activity. Therefore, our result suggests that GSS may reverse partially the dexamethasone-induced down-regulation of glucocorticoid receptor. 2004 Elsevier Inc. All rights reserved. Keywords: Ginsenosides; Glucocorticoid; Reporter gene; Glucocorticoid receptor; Up-regulation
1. Introduction Ginsengs have multiple pharmacological actions (Attele et al., 1999; Chen, 1996; Gillis, 1997; Kim et al., 1998) and have been used by traditional Chinese medicinal practitioners in the treatment of cardiovascular diseases and cancer as well as in improving homeostasis in stress. Recently, due to the increase in worldwide popularity of natural medicine in the prevention and treatment of diseases, the worldwide consumption of ginseng extract is on the rise. *
Corresponding author. Fax: +86 21 65562275. E-mail address: [email protected] (C. Ling). 1 Both authors contributed equally and should be considered as Wrst authors. 0016-6480/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2004.11.003
Ginsenosides (GSS), the major active components of ginseng, is one of the derivatives of triterpenoid dammarane consisting of 30 carbon atoms, steroid-like structure with sugar moieties attached, and about 30 diVerent forms have been isolated and identiWed from Panax ginseng and produce multiple pharmacological responses (Hakhoe, 1997). From laboratory studies, it exerts glucocorticoid-like activities and has been suggested that the pharmacological target sites for these compounds involve the hypothalamus–pituitary–adrenal axis due to the eVects upon serum levels of adrenocorticotropic hormone and corticosterone (Huang, 1999). However, the molecule responsible for the eVects of GSS is unknown, as is the cellular basis of the action of GSS. The overall eVects of GSS are quite complex due to their potential for multiple actions even in a single
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tissue (Attele et al., 1999). Previous reports demonstrated that GSS have anticarcinogenic eVects through a diVerent mechanism, either by direct cytotoxic eVects toward cancer cells or by induction of diVerentiation and inhibition of metastasis of cancer cells (Kim et al., 1998; Mochizuki et al., 1995; Ota et al., 1987; Wakabayashi et al., 1998). GSS can block many ionic channels, such as block subunits of brain Na+ channels reversibly, and it has been suggested that this eVect may contribute to its neuroprotective eVect during ischemia (Liu et al., 2001). Ginsenoside Rg1, one of the steroidal saponins, in particular, has estrogen-like activity by activating ERdependent gene transcription (Robble et al., 2002). It was also shown to have an immunomodulating eVect and may increase both humoral and cell-mediated immune responses (Kenarova et al., 1990; Kim et al., 1990). Glucocorticoid plays an important role during development and in many physiological and pathological processes. These include regulation of energy homeostasis, adaptation to stress, and modulation of central nervous system functions (Miller and Blake, 1995). It is generally believed that most, if not all, the eVects of GSS on cells are mediated via the glucocorticoid receptor (GR). Recently, we have reported that Shenfu decoction and Shemgami Powder, two Chinese Herbal mixtures both contain ginseng up-regulate the activity of GR in hepatic cytosol and strengthen organic resistance to heat stress and hemorrhagic shock in rats (Ling et al., 1999; Min et al., 2003). We suspected GSS was the one of the main molecular ingredients responsible for the actions of two mixtures. Moreover, the steroid backbone structure of GSS could make it a suitable candidate to interact and activate GR. Thus, we hypothesize that GSS might mediate some of their actions by binding to the glucocorticoid receptor, as they share many of the protective actions of glucocorticoid in various physiological systems. The present study was designed to study molecular mechanisms of the GC-like action of GSS. The ability to stimulate GRE dependent reporter gene expression was studied using human liver 7702 cells transfected with the glucocorticoid response element (GRE)-luciferase construct. In addition, the GR mRNA and protein expression and hormone binding activity of GR were tested by semi-quantitative RT-PCR, Western blot, and radioligand-binding assay, respectively.
retains its activity after exposure with organic solvents such as acetonitrile and ethanol. The concentrations of four major ginsenosides were determined by reversedphase HPLC–MS/MS employing a quadrupole-ion trap mass spectrometer in samples (Rg1 D 20.88%, Re D 18.50%, Rb1 D 5.66%, and Rb2 D 2.38%). Dex and RU486 were purchased from Sigma–Aldrich Chemicals. The mouse monoclonal antibodies against -actin were purchased from Sigma–Aldrich Chemicals (St. Louis, MO). The polyclonal anti-human glucocorticoid receptor antibody was purchased from Santa Cruz (GR H300, USA). [1,2,4-3H]Dexamethasone (speciWc activity: 41.0 Ci/mmol) was purchased from Amersham–Pharmacia Biotech (Buckinghamshire, UK). Goat anti-rabbitHRP and goat anti-mouse-HRP conjugate was from Bio-Rad Laboratories (Hercules, CA). RPMI 1640 medium was obtained from Life Technologies (Grand Island, NY). GREtkLUC plasmid was kindly donated by S. Stoney Simons (NIH, Bethesda, MD), which contains two inverted repeats of the 23 bp glucocorticoid response element (GRE) in front of the thymidine kinase promoter driving the LUC gene (Sarlis et al., 1999). RLSV40 plasmid was purchased from Promega, as Renilla luciferase control reporter vector contains the SV40 early enhance/promoter region provides strong, constitutive expression of Renilla luciferase in variety of cell types. 2.2. Cell culturing HL7702 cells (human normal liver, from Cell Biological Research Institution of Chinese Academy of Sciences, Shanghai, China) were grown as monolayers in RPMI 1640 medium (Life Technologies) supplemented with 10% heat-inactivated calf serum, glutamine, and antibiotics, maintained in 5% CO2 atmosphere at 37 °C. GSS was dissolved in 70% ethanol at a concentration of 50 mg/ml and stored at ¡20 °C. Dex and RU486 were dissolved in anhydrous alcohol. All were diluted in the medium immediately before use (Wnal ethanol concentration <0.1%). In all experiments, control cultures were made up of medium, ethanol (0.1%), and the cell. Ethanol alone did not have any eVect on the parameters measured. 2.3. Cells transient co-transfections and luciferase activity
2. Materials and methods 2.1. Chemicals GSS, an extract from Panax ginseng stem and leaves, was obtained from Prof. Hanchen Zheng (Department of Natural Pharmacology, Second Military Medical University, Shanghai, China) with a purity greater than 95%. The extract is quite stable at room temperature and
Cells were seeded into 24 wells plate for 24 h (30–50% conXuence) prior to be co-transfected with pGRE-tkLUC and pRL-SV40-Renilla-LUC. Liposomes were used to deliver the plasmid DNA constructs to the HL7702 cells. For each well, 0.5 g of GREtkLUC plasmid DNA and 5 ng Renilla luciferase control vector pRL-SV40 were delivered to the cells in 600 l of solution containing Lipofectamine reagent diluted 50-fold in OPTI-MEM I (Gibco-BRL, Life Technologies, Rock-
C. Ling et al. / General and Comparative Endocrinology 140 (2005) 203–209
ville, MD, USA) according to the manufacturer’s protocol. The OPTI-MEM I medium was essentially serumfree. After 4 h of incubation at 37 °C in the transfection medium, cells were placed in medium for in phenol-red free MEM containing 10% charcoal-stripped calf serum for 12 h before being induced with indicated concentrations of dexamethasone, ginsenoside, or a mixture of both for 12–72 h. In those experiments, the cells were maintained in serum-free medium throughout the transfection procedure and during experimental treatment. The cells were lysed and assayed for reporter gene activity using the Dual-Luciferase Reporter Assay System according to the manufacturer’s instructions (Promega, USA). The activities of WreXy and Renilla luciferase are measured in a TD20/20 (Promega) luminometer sequentially from a single sample. Luciferase activities were normalized for transfection eYciency by dividing WreXy luciferase activity with that of Renilla luciferase and were expressed as fold induction compared with the group treated with vehicle. All transfections were done in triplicate and all experiments were repeated at least three times. 2.4. RNA isolation and RT-PCR analysis Total RNA was isolated with TRIzol Reagent (Gibco-BRL) according to the manufacturer’s instructions. BrieXy, Wrst-strand cDNA synthesis was performed with random hexanucleotide primer mixtures, total RNA (2.0 g/20 l reaction), and 15 U/20 l AMV reverse transcriptase (Promega) 45 min at 42 °C followed by a 5 min denaturation at 95 °C. PCR with thermostable polymerase (Taq, Promega) was carried out using human-speciWc GR primers. Primers for ampliWcation of GR gene were as follows: sense: 5⬘-CCT AAG GAC GGT CTG AAG AGC-3⬘, antisense: 5⬘-GCC AAG TCT TGG CCC TCT AT-3⬘ (484 bp) (Robert et al., 1996); primers for the control housekeeping gene GAPDH were: sense: 5⬘-TTC ATT GAC CTC AAC TAC ATG-3⬘, antisense: 5⬘-GTG GCA GTG ATG GCA TGG AC-3⬘ (443 bp). AmpliWcations of GR and GAPDH were for 200 ng template £ 28 cycles and 50 ng template £ 26 cycles, respectively; the ampliWcation process was the same: denaturation at 95 °C for 45 s, annealing at 54 °C for 60 s, and elongation at 72 °C for 60 s. The PCR products were electrophoresed on a 1.5% agarose gel in the presence of ethidium bromide, and absorbance were measured by densitometer. The ratio of GR to GAPDH was used to evaluate the signiWcance of the diVerence between various groups. 2.5. Western blot analysis Whole cell extracts were prepared in the lysis buVer M-PER. Lysates were then spun at 12,000 rpm for 15 min to remove insoluble substances. Twenty micrograms of total extract from each sample was separated
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by 12% SDS–polyacrylamide gel electrophoresis followed by electrotransfer onto a polyvinylidene diXuoride membrane (Immobilon-P, Millipore, USA). Membranes were blocked with 5% nonfat milk and incubated in a 1:1000 dilution of rabbit polyclonal anti-human GR antibody or in 1:5000 mouse monoclonal anti--actin (Sigma). The unbound primary antibody was washed away in Tris-buVered saline with 0.1% Tween 20 and the immune complexes were again probed with a secondary anti-mouse or anti-rabbit IgG antibody linked to peroxidase and detected by an enhanced chemiluminescence (ECL) Western blotting procedure (Amersham, UK). 2.6. Whole cell GR ligand binding assay HL7702 cells were cultured in RPMI 1640 medium supplemented with 10% charcoal-stripped calf serum in six-well plate to be treated. The cells were harvested, counted, and were washed three times with phosphatebuVered saline (PBS). Thereafter, medium was replaced with 0.5 ml Opti-MEM medium and then cultured with 2–20 nM [3H]Dex in the absence (total binding) and in the presence of 2000-fold molar excess of unlabeled Dex (nonspeciWc binding) for for 3 h at 21 °C. Incubation was terminated by the addition of 3 ml ice cold PBS, and free steroid was removed by centrifugation at 1500g for 5 min. After two successive washes again with ice cold PBS, the samples were added with 0.1 ml aminic acid, boiled for 5 min, and transferred into scintillation vials. Four milliliters of scintillation mixture was added, and the radioactivity was determined by scintillation counting. Cell-bound radioactivity in the presence of unlabeled Dex gave nonspeciWc binding. SpeciWc binding was calculated by subtracting this value from the total bound radioactivity in the absence of unlabeled Dex. Data from experiments were subjected to a Scatchard analysis and linear regression was performed to yield the equilibrium dissociation constant (Kd). Correlation coeYcients for these determinations were typically greater than 0.95. 2.7. Statistical analysis Data are expressed as means § SD, unless otherwise indicated. Student’s t test or ANOVA followed by ScheVés’s test was used for statistical analysis as appropriate. Statistical signiWcance was established at P < 0.05.
3. Results 3.1. EVect of ginsenosides on glucocorticoid-inducible gene transcription To elucidate the GR-responsive transcriptional activation, we Wrst treated the HL7702 cells transfected transiently with pGREtkLUC and pRL-SV40 with dexa-
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methasone and RU 486, a glucocorticoid antagonist. Treatment of cells with 0.1 nM of dexamethasone for 24 h activated the reporter gene transcription approximately 5fold, and to peak induction value of 94-fold at 100 nM, suggesting that the dexamethasone-induced transactivation is dose-dependent (within 0.1–100 nM). As expected, the eVects were blocked by RU486 (Fig. 1A). In contrast, the reporter gene activity was not changed by the pretreatment with 6.25–100 g/ml of GSS (Fig. 1B), suggesting that GSS may not be interact with the GRE. To explore whether GSS changes the GC-inducible transactivation, cells were pretreated with 10 nM of Dex and with or without GSS. Surprisingly, GSS enhanced the Dex-induced transactivation when it was added simultaneously with Dex (Figs. 2A and B). The maximal induction obtained in the cells co-treated at 25 g/ml concentration of GSS for 36 h was 1.6-fold higher than that of Dex. This indicated that GSS enhances the GCinducible luciferase activity.
3.2. EVect of ginsenosides on the transcription and expression of GR To study the mechanisms of GSS enhancing the GCinduced GR transactivation, we examined the inXuence of GSS on the GR expression and GR down-regulation by Dex by semi-quantitative RT-PCR and Western blot analysis, respectively. Cells were pretreated with 25 g/ml concentration of GSS and simultaneously treated or untreated with 10 nM of Dex for 36 h. As shown in Figs. 3A and B, Dex alone treatment was found to induce down-regulation both of the expressed GR mRNAs and protein. Nevertheless, co-treatment with GSS reversed partially the down-regulation caused by Dex, the transcription and protein expression were turned out to be approximately 2-fold and 1.6-fold than that of Dex alone group. The gene and protein expression of GR in cells were not changed by the pretreatment with GSS.
Fig. 1. Induction of GRE-containing reporter gene by Dex and ginsenosides. (A) HL7702 cells were treated with increasing doses of Dex for 24 h before they were measured by luminometer. Values are expressed as fold induction § SD relative to the vehicle-treated from single experiment. The experiment was repeated an additional three times with similar results. Dex increase luciferase activity in a dose-dependent manner (vs control, *P < 0.05; n D 6). The maximal induction was 94-fold compared to control group. RU486 could speciWcally block the actions of Dex (P < 0.05). (B) The eVects of ginsenosides (GSS) on reporter gene induction. GSS could not induce the expression of reporter gene. Values are expressed as means § SD from three separate experiments.
Fig. 2. EVect of GSS on Dex-induced luciferase activity in HL7702 cells. The glucocorticoid responsive GREtkLUC reporter gene was introduced into the cells. The cells were incubated for 12 h, after which time they were simultaneously treated with GSS and Dex. (A) Cells were treated with 10 nM of Dex together with varying concentrations of GSS for 24 h. Luciferase activities are indicated as fold induction § SD relative to the Dex group. Values signiWcantly diVerent from Dex are marked *P < 0.05. (B) Transfectants were incubated with 10 nM of Dex and with or without 25 g/ ml of GSS for 12, 24, 36, 48, 60, or 72 h. Luciferase activities are indicated as fold induction § SD relative to the Dex-treated group at each time point. Values signiWcantly diVerent from Dex are marked *P < 0.05.
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Fig. 3. GSS blocked the inhibition of glucocorticoid on GR mRNA and protein expression. (A) RT-PCR analysis of RNA isolated from 7702 cells using hGR-speciWc primers. Values are expressed as relative abundance of GR mRNA relative to the Dex-treated from three independent experiments. *P < 0.05 compared with Dex alone group. (B) A portion of each whole cell extract was analyzed by Western blot using the anti-hGR antibody that recognizes an epitope common to the 94 kDa hGR and 34 Da -actin proteins. The experiment was repeated an additional three times with similar results. Results are means § SD of three independent experiments. *P < 0.05 vs Dex alone group.
Fig. 4. (A) Saturation binding analysis of GR on HL7702 cells. HL7702 cells (2 £ 106) were incubated 3 h with increasing concentration of [3H]Dex, with or without excess unlabeled Dex, without any treatment (I) or after being treated with 10 nM of Dex (䉱) or co-incubated with Dex and 25 g/ ml of GSS for 36 h (䊏). SpeciWc binding was determined. The concentration of free-labeled Dex still in solution after was calculated by subtracting the total bound radioactivity introduced in the incubation mixture. (B) Cell were treated for 36 h with various reagents at the concentration indicated. At the end of incubation, speciWc [3H]Dex binding was determined, and the number of binding sites per cell was calculated. The data represent means § SD of at least three experiments. *P < 0.01 vs Dex-treated group. (C) Scatchard transformation of the data. The Kd of the GR in 7702 cells treated with 10 nmol of Dex (䉱) or co-treated with Dex and GSS (䊏) is, respectively, 7.14 and 5.13 nM. The Kd of GR is 3.57 nM for control group.
3.3. EVect of ginsenosides on GR speciWc binding aYnity To elucidate the eVect of GSS on the Dex-induced changes of GR-binding aYnity and the number of the binding sites, we performed Scatchard analysis of the binding value obtained by progressive saturation of the receptor on HL7702 cells. The results are presented in Fig. 4. The data show that HL7702 cells express constitutively an average number of 49,700 sites per cell and that this number decreased to 21,700 receptors per cell after treatment with 10 nM Dex for 36 h. However, the number increased to 34,700 receptors per cell in the cells co-treated with GSS (Fig. 4C). As shown in Figs. 4A and B, 10 nM Dex resulted in increase in the GR Kd from 3.57 § 0.53 to 7.14 § 0.96 nM compared with vehicle alone. The calculated Kd value of the Dex binding sites in pretreated cells with GSS (5.13 § 0.42 nM) is not signiWcantly diVerent from that of Dex treatment. These results indicated that GSS induced an increase in the number of
binding sites without altering aYnity of GR down-regulation by Dex.
4. Discussion In the present study, we examined the eVect of GSS on the transactivation induced by glucocorticoid as well as expression and binding activity of GR in HL7702 cells. Our results indicated that GSS do not interact directly with GR, as demonstrated by its inability to induce transcription of GRE-dependent luciferase reporter gene. However, Dex-induced transcription was synergistically enhanced by the treatment of GSS, and a reversal eVect of GSS on Dex-induced down-regulation of expression and hormone binding activity of GR was observed. Scatchard analysis provided evidence that the increased hormone binding activity. Western blot and RT-PCR analyses showed that both Dex-induced down-
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regulation of protein and mRNA levels of GR were increased after treated with GSS. GSS can mediate these actions at an optimal dose of 25 g/ml. Thus, our results suggests that glucocorticoid-like eVects of GSS may be a consequence of reversing partially the dexamethasoneinduced down-regulation of glucocorticoid receptor. Glucocorticoid therapy is one of the most eVective medications in the acute and chronic management of inXammatory diseases such as allergic diseases and autoimmune diseases. GC exerts the functions that are crucial for life, and therefore complete GC resistance is uncommon. However, there is large variation in responsiveness to GC therapy between individuals. The actions of GC are mediated by an intracellular receptor, the glucocorticoid receptor, a member of the nuclear receptor family of ligand-dependent transcription factors. Upon activation by their ligand, these receptors can act as transcription factors. In addition, the presence of GR is essential for survival at birth, and its function is important for proper regulation of many physiological processes. This is known from human pathology (Miller and Blake, 1995), in vitro and in vivo experiments, and the analysis of genetically manipulated mice (Cole et al., 1995; Reichardt et al., 1998; Tronche et al., 1999). Nevertheless, previous studies proved that glucocorticoid receptor agonists may result in a signiWcant down-regulation of the expression of the GR (Dong et al., 1988; Marcel and Schaaf, 2003; Rosewicz et al., 1988; Svec and Rudis, 1981). The mechanism of homologous down-regulation is poorly understood. However, it has been shown that down-regulation takes place at both the mRNA and the protein level and that multiple mechanisms play a role (Burnstein et al., 1994). When GC acts as protective factor for adaptation to stress, it also down-regulates the expression of GR. The down-regulation of GC on GR is one of reasons result in GC resistance and could therefore seriously decrease the eYcacy of GC used clinically. Serious side eVects will also present in those patients who take great a dosage of GC for long time. Therefore, it is critical to the development of newer more eVective therapies for these diYcult to manage disease conditions. GSS, an extract from ginseng stem and leaves, is a diverse group of steroidal saponins that produce multiple pharmacological responses and have been used in the treatment of cardiovascular diseases and cancer as well as in improving overall stress tolerance. Although many studies have been designed to study the pharmacological action of GSS, the detailed mechanism of the action of GSS has yet to be determined. GSS is one of the derivatives of triterpenoid dammarane consisting of 30 carbon atoms, steroid-like structure with sugar moieties attached. The steroid backbone structure of GSS could make it a suitable candidate to interact and activate GR. A previous report also indicated that ginsenoside-Rg1, a monosomic saponin from GSS, did bind to the glucocor-
ticoid receptor to induce the expression of reporter genes driven by GREs. At the same time, ginsenoside-Rg1 treatment led to the down-regulation of intracellular GR content, which was similar to the eVect of Dex (Lee et al., 1997). We therefore examined the inductive eVect of GSS on GRE-tk-luc gene. Unexpectedly, GSS alone could not induce the reporter gene expression. It seems that there are some discrepancy between our results and the report above. The reason(s) for the discrepancy are unclear but may be explained by the fact that diVerence in medical component. It should be noted that GSS contains many other monosomic ginsenosides besides Rg1 that might contribute to either a synergistic or antagonistic eVect of Rg1 on its target receptor. Whereas the actions ginsenoside-Rg1 exerts, also provides further support for the notion that GSS could have glucocorticoid-like activity. Future studies will be needed to clarify the complex interactions among the diVerent GSS. In the present study, we attempt to studied the mechanism of GSS enhancing the GC-inducible luciferase activity. The data demonstrate that despite the fact that GSS produces biochemical eVects of elevating GC eYcacy, GSS does not directly activate the glucocorticoid receptor. Glucocorticoid-like eVects may be a consequence of reversing partially the GC-induced downregulation of GR. To sum up, the result provides support for the notion that GSS contains glucocorticoidlike activity. The mechanisms of GSS regulate the expression and binding activity of GR down-regulation by Dex noticed in the present study is not fully understood. Nevertheless, our results strongly support the concept that GSS may oVer a new and exciting possibility for enhancing GC eYciency. The discovery also helps in understanding the miracle action of ginseng in the prevention and treatment of many diseases. The potential application of GSS in the treatment of glucocorticoid-resistant diseases warrants further detailed study.
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (39870920, 30472271, and 30025045). We are very grateful to Prof. Renbao Xu for critical reading of the manuscript. The author also thanks Hanchen Zheng for the supply of ginsenosides and Yidong Li for GREtkLUC plasmid.
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Ginsenosides may reverse the dexamethasone-induced down-regulation of glucocorticoid receptor Changquan Linga,¤,1, Yong Lia,1, Xiaoyan Zhub, Chen Zhanga, Min Lic a
Department of Chinese Traditional Medicine, Changhai Hospital, Second Military Medical University, Shanghai 200433, China b Institute of Pathophysiology, Second Military Medical University, Shanghai 200433, China c Department of Navy Medicine, Second Military Medical University, Shanghai 200433, China Received 30 March 2004; revised 21 October 2004; accepted 9 November 2004
Abstract The eVects of glucocorticoid (GC) hormones are mediated via an intracellular receptor, the glucocorticoid receptor (GR). It has been established that glucocorticoid down-regulate GR. Ginsenosides (GSS) from extract of Panax ginseng have demonstrated glucocorticoid-like activities in homeostasis and regulation of immunity, etc. We hypothesize that ginsenosides might mediate some of their actions by binding to the GR. The present study is aimed to determine whether GSS can act like a GC analog in the activation of glucocorticoid response element-luciferase activity in HL7702 cells. We found that GSS alone had no eVect on the expression of reporter gene, but it enhanced dexamethasone (Dex)-induced transcription of reporter gene. To further explore the eVects of GSS, we examined the inXuence of GSS on the gene and protein expression as well as hormone binding activity of GR by semi-quantitative RT-PCR, Western blot, and radioligand-binding assay, respectively. GSS partially reversed the Dex-induced decrease in GR expression and hormone binding activity with an optimal dose of 25 g/ml, implicating a positive regulatory eVect of GSS on GR expression and binding activity. Therefore, our result suggests that GSS may reverse partially the dexamethasone-induced down-regulation of glucocorticoid receptor. 2004 Elsevier Inc. All rights reserved. Keywords: Ginsenosides; Glucocorticoid; Reporter gene; Glucocorticoid receptor; Up-regulation
1. Introduction Ginsengs have multiple pharmacological actions (Attele et al., 1999; Chen, 1996; Gillis, 1997; Kim et al., 1998) and have been used by traditional Chinese medicinal practitioners in the treatment of cardiovascular diseases and cancer as well as in improving homeostasis in stress. Recently, due to the increase in worldwide popularity of natural medicine in the prevention and treatment of diseases, the worldwide consumption of ginseng extract is on the rise. *
Corresponding author. Fax: +86 21 65562275. E-mail address: [email protected] (C. Ling). 1 Both authors contributed equally and should be considered as Wrst authors. 0016-6480/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2004.11.003
Ginsenosides (GSS), the major active components of ginseng, is one of the derivatives of triterpenoid dammarane consisting of 30 carbon atoms, steroid-like structure with sugar moieties attached, and about 30 diVerent forms have been isolated and identiWed from Panax ginseng and produce multiple pharmacological responses (Hakhoe, 1997). From laboratory studies, it exerts glucocorticoid-like activities and has been suggested that the pharmacological target sites for these compounds involve the hypothalamus–pituitary–adrenal axis due to the eVects upon serum levels of adrenocorticotropic hormone and corticosterone (Huang, 1999). However, the molecule responsible for the eVects of GSS is unknown, as is the cellular basis of the action of GSS. The overall eVects of GSS are quite complex due to their potential for multiple actions even in a single
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tissue (Attele et al., 1999). Previous reports demonstrated that GSS have anticarcinogenic eVects through a diVerent mechanism, either by direct cytotoxic eVects toward cancer cells or by induction of diVerentiation and inhibition of metastasis of cancer cells (Kim et al., 1998; Mochizuki et al., 1995; Ota et al., 1987; Wakabayashi et al., 1998). GSS can block many ionic channels, such as block subunits of brain Na+ channels reversibly, and it has been suggested that this eVect may contribute to its neuroprotective eVect during ischemia (Liu et al., 2001). Ginsenoside Rg1, one of the steroidal saponins, in particular, has estrogen-like activity by activating ERdependent gene transcription (Robble et al., 2002). It was also shown to have an immunomodulating eVect and may increase both humoral and cell-mediated immune responses (Kenarova et al., 1990; Kim et al., 1990). Glucocorticoid plays an important role during development and in many physiological and pathological processes. These include regulation of energy homeostasis, adaptation to stress, and modulation of central nervous system functions (Miller and Blake, 1995). It is generally believed that most, if not all, the eVects of GSS on cells are mediated via the glucocorticoid receptor (GR). Recently, we have reported that Shenfu decoction and Shemgami Powder, two Chinese Herbal mixtures both contain ginseng up-regulate the activity of GR in hepatic cytosol and strengthen organic resistance to heat stress and hemorrhagic shock in rats (Ling et al., 1999; Min et al., 2003). We suspected GSS was the one of the main molecular ingredients responsible for the actions of two mixtures. Moreover, the steroid backbone structure of GSS could make it a suitable candidate to interact and activate GR. Thus, we hypothesize that GSS might mediate some of their actions by binding to the glucocorticoid receptor, as they share many of the protective actions of glucocorticoid in various physiological systems. The present study was designed to study molecular mechanisms of the GC-like action of GSS. The ability to stimulate GRE dependent reporter gene expression was studied using human liver 7702 cells transfected with the glucocorticoid response element (GRE)-luciferase construct. In addition, the GR mRNA and protein expression and hormone binding activity of GR were tested by semi-quantitative RT-PCR, Western blot, and radioligand-binding assay, respectively.
retains its activity after exposure with organic solvents such as acetonitrile and ethanol. The concentrations of four major ginsenosides were determined by reversedphase HPLC–MS/MS employing a quadrupole-ion trap mass spectrometer in samples (Rg1 D 20.88%, Re D 18.50%, Rb1 D 5.66%, and Rb2 D 2.38%). Dex and RU486 were purchased from Sigma–Aldrich Chemicals. The mouse monoclonal antibodies against -actin were purchased from Sigma–Aldrich Chemicals (St. Louis, MO). The polyclonal anti-human glucocorticoid receptor antibody was purchased from Santa Cruz (GR H300, USA). [1,2,4-3H]Dexamethasone (speciWc activity: 41.0 Ci/mmol) was purchased from Amersham–Pharmacia Biotech (Buckinghamshire, UK). Goat anti-rabbitHRP and goat anti-mouse-HRP conjugate was from Bio-Rad Laboratories (Hercules, CA). RPMI 1640 medium was obtained from Life Technologies (Grand Island, NY). GREtkLUC plasmid was kindly donated by S. Stoney Simons (NIH, Bethesda, MD), which contains two inverted repeats of the 23 bp glucocorticoid response element (GRE) in front of the thymidine kinase promoter driving the LUC gene (Sarlis et al., 1999). RLSV40 plasmid was purchased from Promega, as Renilla luciferase control reporter vector contains the SV40 early enhance/promoter region provides strong, constitutive expression of Renilla luciferase in variety of cell types. 2.2. Cell culturing HL7702 cells (human normal liver, from Cell Biological Research Institution of Chinese Academy of Sciences, Shanghai, China) were grown as monolayers in RPMI 1640 medium (Life Technologies) supplemented with 10% heat-inactivated calf serum, glutamine, and antibiotics, maintained in 5% CO2 atmosphere at 37 °C. GSS was dissolved in 70% ethanol at a concentration of 50 mg/ml and stored at ¡20 °C. Dex and RU486 were dissolved in anhydrous alcohol. All were diluted in the medium immediately before use (Wnal ethanol concentration <0.1%). In all experiments, control cultures were made up of medium, ethanol (0.1%), and the cell. Ethanol alone did not have any eVect on the parameters measured. 2.3. Cells transient co-transfections and luciferase activity
2. Materials and methods 2.1. Chemicals GSS, an extract from Panax ginseng stem and leaves, was obtained from Prof. Hanchen Zheng (Department of Natural Pharmacology, Second Military Medical University, Shanghai, China) with a purity greater than 95%. The extract is quite stable at room temperature and
Cells were seeded into 24 wells plate for 24 h (30–50% conXuence) prior to be co-transfected with pGRE-tkLUC and pRL-SV40-Renilla-LUC. Liposomes were used to deliver the plasmid DNA constructs to the HL7702 cells. For each well, 0.5 g of GREtkLUC plasmid DNA and 5 ng Renilla luciferase control vector pRL-SV40 were delivered to the cells in 600 l of solution containing Lipofectamine reagent diluted 50-fold in OPTI-MEM I (Gibco-BRL, Life Technologies, Rock-
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ville, MD, USA) according to the manufacturer’s protocol. The OPTI-MEM I medium was essentially serumfree. After 4 h of incubation at 37 °C in the transfection medium, cells were placed in medium for in phenol-red free MEM containing 10% charcoal-stripped calf serum for 12 h before being induced with indicated concentrations of dexamethasone, ginsenoside, or a mixture of both for 12–72 h. In those experiments, the cells were maintained in serum-free medium throughout the transfection procedure and during experimental treatment. The cells were lysed and assayed for reporter gene activity using the Dual-Luciferase Reporter Assay System according to the manufacturer’s instructions (Promega, USA). The activities of WreXy and Renilla luciferase are measured in a TD20/20 (Promega) luminometer sequentially from a single sample. Luciferase activities were normalized for transfection eYciency by dividing WreXy luciferase activity with that of Renilla luciferase and were expressed as fold induction compared with the group treated with vehicle. All transfections were done in triplicate and all experiments were repeated at least three times. 2.4. RNA isolation and RT-PCR analysis Total RNA was isolated with TRIzol Reagent (Gibco-BRL) according to the manufacturer’s instructions. BrieXy, Wrst-strand cDNA synthesis was performed with random hexanucleotide primer mixtures, total RNA (2.0 g/20 l reaction), and 15 U/20 l AMV reverse transcriptase (Promega) 45 min at 42 °C followed by a 5 min denaturation at 95 °C. PCR with thermostable polymerase (Taq, Promega) was carried out using human-speciWc GR primers. Primers for ampliWcation of GR gene were as follows: sense: 5⬘-CCT AAG GAC GGT CTG AAG AGC-3⬘, antisense: 5⬘-GCC AAG TCT TGG CCC TCT AT-3⬘ (484 bp) (Robert et al., 1996); primers for the control housekeeping gene GAPDH were: sense: 5⬘-TTC ATT GAC CTC AAC TAC ATG-3⬘, antisense: 5⬘-GTG GCA GTG ATG GCA TGG AC-3⬘ (443 bp). AmpliWcations of GR and GAPDH were for 200 ng template £ 28 cycles and 50 ng template £ 26 cycles, respectively; the ampliWcation process was the same: denaturation at 95 °C for 45 s, annealing at 54 °C for 60 s, and elongation at 72 °C for 60 s. The PCR products were electrophoresed on a 1.5% agarose gel in the presence of ethidium bromide, and absorbance were measured by densitometer. The ratio of GR to GAPDH was used to evaluate the signiWcance of the diVerence between various groups. 2.5. Western blot analysis Whole cell extracts were prepared in the lysis buVer M-PER. Lysates were then spun at 12,000 rpm for 15 min to remove insoluble substances. Twenty micrograms of total extract from each sample was separated
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by 12% SDS–polyacrylamide gel electrophoresis followed by electrotransfer onto a polyvinylidene diXuoride membrane (Immobilon-P, Millipore, USA). Membranes were blocked with 5% nonfat milk and incubated in a 1:1000 dilution of rabbit polyclonal anti-human GR antibody or in 1:5000 mouse monoclonal anti--actin (Sigma). The unbound primary antibody was washed away in Tris-buVered saline with 0.1% Tween 20 and the immune complexes were again probed with a secondary anti-mouse or anti-rabbit IgG antibody linked to peroxidase and detected by an enhanced chemiluminescence (ECL) Western blotting procedure (Amersham, UK). 2.6. Whole cell GR ligand binding assay HL7702 cells were cultured in RPMI 1640 medium supplemented with 10% charcoal-stripped calf serum in six-well plate to be treated. The cells were harvested, counted, and were washed three times with phosphatebuVered saline (PBS). Thereafter, medium was replaced with 0.5 ml Opti-MEM medium and then cultured with 2–20 nM [3H]Dex in the absence (total binding) and in the presence of 2000-fold molar excess of unlabeled Dex (nonspeciWc binding) for for 3 h at 21 °C. Incubation was terminated by the addition of 3 ml ice cold PBS, and free steroid was removed by centrifugation at 1500g for 5 min. After two successive washes again with ice cold PBS, the samples were added with 0.1 ml aminic acid, boiled for 5 min, and transferred into scintillation vials. Four milliliters of scintillation mixture was added, and the radioactivity was determined by scintillation counting. Cell-bound radioactivity in the presence of unlabeled Dex gave nonspeciWc binding. SpeciWc binding was calculated by subtracting this value from the total bound radioactivity in the absence of unlabeled Dex. Data from experiments were subjected to a Scatchard analysis and linear regression was performed to yield the equilibrium dissociation constant (Kd). Correlation coeYcients for these determinations were typically greater than 0.95. 2.7. Statistical analysis Data are expressed as means § SD, unless otherwise indicated. Student’s t test or ANOVA followed by ScheVés’s test was used for statistical analysis as appropriate. Statistical signiWcance was established at P < 0.05.
3. Results 3.1. EVect of ginsenosides on glucocorticoid-inducible gene transcription To elucidate the GR-responsive transcriptional activation, we Wrst treated the HL7702 cells transfected transiently with pGREtkLUC and pRL-SV40 with dexa-
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methasone and RU 486, a glucocorticoid antagonist. Treatment of cells with 0.1 nM of dexamethasone for 24 h activated the reporter gene transcription approximately 5fold, and to peak induction value of 94-fold at 100 nM, suggesting that the dexamethasone-induced transactivation is dose-dependent (within 0.1–100 nM). As expected, the eVects were blocked by RU486 (Fig. 1A). In contrast, the reporter gene activity was not changed by the pretreatment with 6.25–100 g/ml of GSS (Fig. 1B), suggesting that GSS may not be interact with the GRE. To explore whether GSS changes the GC-inducible transactivation, cells were pretreated with 10 nM of Dex and with or without GSS. Surprisingly, GSS enhanced the Dex-induced transactivation when it was added simultaneously with Dex (Figs. 2A and B). The maximal induction obtained in the cells co-treated at 25 g/ml concentration of GSS for 36 h was 1.6-fold higher than that of Dex. This indicated that GSS enhances the GCinducible luciferase activity.
3.2. EVect of ginsenosides on the transcription and expression of GR To study the mechanisms of GSS enhancing the GCinduced GR transactivation, we examined the inXuence of GSS on the GR expression and GR down-regulation by Dex by semi-quantitative RT-PCR and Western blot analysis, respectively. Cells were pretreated with 25 g/ml concentration of GSS and simultaneously treated or untreated with 10 nM of Dex for 36 h. As shown in Figs. 3A and B, Dex alone treatment was found to induce down-regulation both of the expressed GR mRNAs and protein. Nevertheless, co-treatment with GSS reversed partially the down-regulation caused by Dex, the transcription and protein expression were turned out to be approximately 2-fold and 1.6-fold than that of Dex alone group. The gene and protein expression of GR in cells were not changed by the pretreatment with GSS.
Fig. 1. Induction of GRE-containing reporter gene by Dex and ginsenosides. (A) HL7702 cells were treated with increasing doses of Dex for 24 h before they were measured by luminometer. Values are expressed as fold induction § SD relative to the vehicle-treated from single experiment. The experiment was repeated an additional three times with similar results. Dex increase luciferase activity in a dose-dependent manner (vs control, *P < 0.05; n D 6). The maximal induction was 94-fold compared to control group. RU486 could speciWcally block the actions of Dex (P < 0.05). (B) The eVects of ginsenosides (GSS) on reporter gene induction. GSS could not induce the expression of reporter gene. Values are expressed as means § SD from three separate experiments.
Fig. 2. EVect of GSS on Dex-induced luciferase activity in HL7702 cells. The glucocorticoid responsive GREtkLUC reporter gene was introduced into the cells. The cells were incubated for 12 h, after which time they were simultaneously treated with GSS and Dex. (A) Cells were treated with 10 nM of Dex together with varying concentrations of GSS for 24 h. Luciferase activities are indicated as fold induction § SD relative to the Dex group. Values signiWcantly diVerent from Dex are marked *P < 0.05. (B) Transfectants were incubated with 10 nM of Dex and with or without 25 g/ ml of GSS for 12, 24, 36, 48, 60, or 72 h. Luciferase activities are indicated as fold induction § SD relative to the Dex-treated group at each time point. Values signiWcantly diVerent from Dex are marked *P < 0.05.
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Fig. 3. GSS blocked the inhibition of glucocorticoid on GR mRNA and protein expression. (A) RT-PCR analysis of RNA isolated from 7702 cells using hGR-speciWc primers. Values are expressed as relative abundance of GR mRNA relative to the Dex-treated from three independent experiments. *P < 0.05 compared with Dex alone group. (B) A portion of each whole cell extract was analyzed by Western blot using the anti-hGR antibody that recognizes an epitope common to the 94 kDa hGR and 34 Da -actin proteins. The experiment was repeated an additional three times with similar results. Results are means § SD of three independent experiments. *P < 0.05 vs Dex alone group.
Fig. 4. (A) Saturation binding analysis of GR on HL7702 cells. HL7702 cells (2 £ 106) were incubated 3 h with increasing concentration of [3H]Dex, with or without excess unlabeled Dex, without any treatment (I) or after being treated with 10 nM of Dex (䉱) or co-incubated with Dex and 25 g/ ml of GSS for 36 h (䊏). SpeciWc binding was determined. The concentration of free-labeled Dex still in solution after was calculated by subtracting the total bound radioactivity introduced in the incubation mixture. (B) Cell were treated for 36 h with various reagents at the concentration indicated. At the end of incubation, speciWc [3H]Dex binding was determined, and the number of binding sites per cell was calculated. The data represent means § SD of at least three experiments. *P < 0.01 vs Dex-treated group. (C) Scatchard transformation of the data. The Kd of the GR in 7702 cells treated with 10 nmol of Dex (䉱) or co-treated with Dex and GSS (䊏) is, respectively, 7.14 and 5.13 nM. The Kd of GR is 3.57 nM for control group.
3.3. EVect of ginsenosides on GR speciWc binding aYnity To elucidate the eVect of GSS on the Dex-induced changes of GR-binding aYnity and the number of the binding sites, we performed Scatchard analysis of the binding value obtained by progressive saturation of the receptor on HL7702 cells. The results are presented in Fig. 4. The data show that HL7702 cells express constitutively an average number of 49,700 sites per cell and that this number decreased to 21,700 receptors per cell after treatment with 10 nM Dex for 36 h. However, the number increased to 34,700 receptors per cell in the cells co-treated with GSS (Fig. 4C). As shown in Figs. 4A and B, 10 nM Dex resulted in increase in the GR Kd from 3.57 § 0.53 to 7.14 § 0.96 nM compared with vehicle alone. The calculated Kd value of the Dex binding sites in pretreated cells with GSS (5.13 § 0.42 nM) is not signiWcantly diVerent from that of Dex treatment. These results indicated that GSS induced an increase in the number of
binding sites without altering aYnity of GR down-regulation by Dex.
4. Discussion In the present study, we examined the eVect of GSS on the transactivation induced by glucocorticoid as well as expression and binding activity of GR in HL7702 cells. Our results indicated that GSS do not interact directly with GR, as demonstrated by its inability to induce transcription of GRE-dependent luciferase reporter gene. However, Dex-induced transcription was synergistically enhanced by the treatment of GSS, and a reversal eVect of GSS on Dex-induced down-regulation of expression and hormone binding activity of GR was observed. Scatchard analysis provided evidence that the increased hormone binding activity. Western blot and RT-PCR analyses showed that both Dex-induced down-
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regulation of protein and mRNA levels of GR were increased after treated with GSS. GSS can mediate these actions at an optimal dose of 25 g/ml. Thus, our results suggests that glucocorticoid-like eVects of GSS may be a consequence of reversing partially the dexamethasoneinduced down-regulation of glucocorticoid receptor. Glucocorticoid therapy is one of the most eVective medications in the acute and chronic management of inXammatory diseases such as allergic diseases and autoimmune diseases. GC exerts the functions that are crucial for life, and therefore complete GC resistance is uncommon. However, there is large variation in responsiveness to GC therapy between individuals. The actions of GC are mediated by an intracellular receptor, the glucocorticoid receptor, a member of the nuclear receptor family of ligand-dependent transcription factors. Upon activation by their ligand, these receptors can act as transcription factors. In addition, the presence of GR is essential for survival at birth, and its function is important for proper regulation of many physiological processes. This is known from human pathology (Miller and Blake, 1995), in vitro and in vivo experiments, and the analysis of genetically manipulated mice (Cole et al., 1995; Reichardt et al., 1998; Tronche et al., 1999). Nevertheless, previous studies proved that glucocorticoid receptor agonists may result in a signiWcant down-regulation of the expression of the GR (Dong et al., 1988; Marcel and Schaaf, 2003; Rosewicz et al., 1988; Svec and Rudis, 1981). The mechanism of homologous down-regulation is poorly understood. However, it has been shown that down-regulation takes place at both the mRNA and the protein level and that multiple mechanisms play a role (Burnstein et al., 1994). When GC acts as protective factor for adaptation to stress, it also down-regulates the expression of GR. The down-regulation of GC on GR is one of reasons result in GC resistance and could therefore seriously decrease the eYcacy of GC used clinically. Serious side eVects will also present in those patients who take great a dosage of GC for long time. Therefore, it is critical to the development of newer more eVective therapies for these diYcult to manage disease conditions. GSS, an extract from ginseng stem and leaves, is a diverse group of steroidal saponins that produce multiple pharmacological responses and have been used in the treatment of cardiovascular diseases and cancer as well as in improving overall stress tolerance. Although many studies have been designed to study the pharmacological action of GSS, the detailed mechanism of the action of GSS has yet to be determined. GSS is one of the derivatives of triterpenoid dammarane consisting of 30 carbon atoms, steroid-like structure with sugar moieties attached. The steroid backbone structure of GSS could make it a suitable candidate to interact and activate GR. A previous report also indicated that ginsenoside-Rg1, a monosomic saponin from GSS, did bind to the glucocor-
ticoid receptor to induce the expression of reporter genes driven by GREs. At the same time, ginsenoside-Rg1 treatment led to the down-regulation of intracellular GR content, which was similar to the eVect of Dex (Lee et al., 1997). We therefore examined the inductive eVect of GSS on GRE-tk-luc gene. Unexpectedly, GSS alone could not induce the reporter gene expression. It seems that there are some discrepancy between our results and the report above. The reason(s) for the discrepancy are unclear but may be explained by the fact that diVerence in medical component. It should be noted that GSS contains many other monosomic ginsenosides besides Rg1 that might contribute to either a synergistic or antagonistic eVect of Rg1 on its target receptor. Whereas the actions ginsenoside-Rg1 exerts, also provides further support for the notion that GSS could have glucocorticoid-like activity. Future studies will be needed to clarify the complex interactions among the diVerent GSS. In the present study, we attempt to studied the mechanism of GSS enhancing the GC-inducible luciferase activity. The data demonstrate that despite the fact that GSS produces biochemical eVects of elevating GC eYcacy, GSS does not directly activate the glucocorticoid receptor. Glucocorticoid-like eVects may be a consequence of reversing partially the GC-induced downregulation of GR. To sum up, the result provides support for the notion that GSS contains glucocorticoidlike activity. The mechanisms of GSS regulate the expression and binding activity of GR down-regulation by Dex noticed in the present study is not fully understood. Nevertheless, our results strongly support the concept that GSS may oVer a new and exciting possibility for enhancing GC eYciency. The discovery also helps in understanding the miracle action of ginseng in the prevention and treatment of many diseases. The potential application of GSS in the treatment of glucocorticoid-resistant diseases warrants further detailed study.
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (39870920, 30472271, and 30025045). We are very grateful to Prof. Renbao Xu for critical reading of the manuscript. The author also thanks Hanchen Zheng for the supply of ginsenosides and Yidong Li for GREtkLUC plasmid.
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