行政院國家科學委員會補助專題研究計畫成果報告

行政院國家科學委員會補助專題研究計畫成果報告

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計畫類別：

þ

執行期間： 89 年 9 月 1 日至 90 年 7 月 31 日 計畫主持人：梁 有 志

共同主持人：

本成果報告包括以下應繳交之附件：

□赴國外出差或研習心得報告一份

□赴大陸地區出差或研習心得報告一份

□出席國際學術會議心得報告及發表之論文各一份

□國際合作研究計畫國外研究報告書一份

執行單位：台北醫學大學醫學系

中 華 民 國 90 年 8 月 30 日

行政院國家科學委員會專題研究計劃成果報告

篩選活化 PPAR

γ

Screening PPARγ ligands from natural products and study on the activation pathways

計劃編號: NSC 89－2320－B－038－075－

執行期限: 89 年 9 月 1 日 至 90 年 7 月 31 日

主持人: 梁 有 志 助教授 台北醫學大學醫學系

內容: 與本計畫有關已完成之研究成果(published results)

Suppression of inducible cyclooxygenase and nitric oxide synthase through activation of

peroxisome proliferator-activated receptor-γ by favonoids in mouse macrophages. Liang, Y.C., Tsai S.H., Tsai, D.C., Lin-Shiau, S.Y. and Lin, J.K. FEBS Letters, 496: 12-18, 2001.

中文摘要

PPARã (過氧化體增殖活化受體) 的活化與抗發炎作用有關。在本次計畫中，篩選 20 餘 種類黃素，發現三種類黃素 apigenin, chrysin 及 kaempferol 能有效地活化 PPARã。在吞噬細 胞中，過度表現 PPARã，可增強此三種類黃素之抑制 LPS 所活化的 COX-2 及 iNOS。然而在 體外的競爭結合分析，發現此三種類黃素只有微弱的 PPARã agonist 活性。有限度蛋白分解 試驗，顯示此三種類黃素會改變 PPARã 的構形，但不同於結合 BRL49653 之 PPARã。這些結 果顯示，此三種類黃素可作為 PPARã 之 allosteric effectors，結合在 PPARã 上，並活化之，但 結合的位置似乎不同於 BRL49653。

關鍵詞: 過氧化體增殖活化受體，類黃素，發炎反應，內環化脢，一氧化氮合成脢

Abstr act

PPARγ transcription factor has been implicated in anti-inflammatory response. Of the compounds tested, apigenin, chrysin, and kaempferol significantly stimulated PPARγ

transcriptional activity in a transient reporter assay. In addition, these three flavonoids strongly

enhanced the inhibition of inducible cyclooxygenase (COX-2) and nitric oxide synthase (iNOS)

promoter activities in LPS-activated macrophages which containing the PPARγ expression

plasmids. However, these three flavonoids exhibited weak PPARγ agonist activities in vitro

competitive binding assay. Limited protease digestion of PPARγ suggested these three flavonoids

produced a conformational change in PPARγ and the conformation differences in the receptor

bound to BRL49653 versus these three flavonoids. These results suggested that these three flavonoids might act as allosteric effectors and were able to bind to PPARγ and activate it, but it’s binding site might be different from the natural ligand BRL49653.

Key wor ds: Peroxisome proliferator-activated receptor-γ, Flavonoids, Inflammation,

Cyclooxygenase, Nitric oxide synthase.

Introduction

Peroxisome proliferator-activated receptors (PPARs) are transcription factors belonging to nuclear receptor gene family [1]. PPARs bind to specific response elements as heterodimers with the retinoid X receptor (RXR) and activate transcription in response to a variety of endogenous and exogenous ligands, including certain polyunsaturated fatty acids, arachidonic acid metabolites [2], and some antidiabetic drugs [3] and non-steroidal anti-inflammatory drugs (NSAIDs)[4]. Currently, PPARs subfamily has been defined as PPARα, PPARβ (also called PPARδ and NUC1) and PPARγ.

Three PPAR isoforms differ in their tissue distribution and ligand specificity [5]. PPARα is predominantly expressed in tissues exhibiting high catabolic rate of fatty acids (heart, liver, and kidney), whereas PPARδ expression is ubiquitous, and its physiological role is unclear. PPARγ is expressed predominantly in adipose tissue, the adrenal gland, spleen, large colon and the immune system [6-9]. Several lines of evidence indicated that PPARγ plays an important role in regulating adipocyte differentiation and glucose homeostasis [10]. Both PPARα and PPARγ have been shown that also have anti-inflammatory actions through activating by arachidonic acid metabolites.

PPARα bind and be activated by leukotriene B4 [11], and the levels are induced at the transcriptional level by anti-inflammatory glucocorticoids [12]. PPARγ are activated by the

prostaglandin D

metabolite 15-deoxy-∆

prostaglandin J

(15d-PGJ

) and synthetic antidiabetic thiazolidinedione drugs (e.g. BRL49653 and ciglitizone) and resulted in negatively regulating the expression of pro-inflammatory genes, and suppressing tumor cell growth [13-16]. Furthermore, both PPARα and PPARγ are activated by a number of non-steroidal anti-inflammatory drugs, such as indomethacin [4]. Recently, the PPARγ agonists have been considered to inhibit production of monocyte inflammatory cytokines and the expression of iNOS [17, 18].

The flavonoids are a diverse family of chemicals commonly found in fruits and vegetables.

Flavonoids are plant polyphenolic compounds, which comprise several classes including flavonols, flavanones, flavanols and flavans. Epidemiological studies have shown that the consumption of vegetable, fruits and tea is associated with a decreased risk of cancer and cardio-vascular diseases, and flavonoids are believed to play an important role in preventing these diseases [19]. Numerous numbers of this family have anticarcinogenic [20], anti-inflammatory [21], cytostatic [22],

apoptotic [18], antioxidant [23], anti-angiogenic [24] and estrogenic [25] activities. Several reports

have also shown that flavonoids are potent modulators of both the expression, and activities of

specific cytochrome P450 genes/proteins [26]. These data indicate that certain flavonoids have

attracted attention as possible chemoprotective or chemotherapeutic agents.

NSAIDs such as aspirin, sodium salicylate, and indomethacin exert their anti-inflammatory effects in part by inhibition of IκB kinase-β (IKK-β), thereby preventing activation by NF-κB of genes involved in the inflammatory response [27]. However, indomethacin and several other NSAIDs (fenoprofen, ibuprofen, and flufenamic acid) are also PPARγ ligands and block production of inflammatory cytokines in human monocytes [17]. In addition, Ricote et al. [13] also

demonstrated that treatment of peritoneal macrophages with 15d-PGJ

or several synthetic PPARγ ligands reduce the expression of iNOS by interferon-γ and inhibited induction of gelatinase B and scavenger receptor A gene transcription in response to phorbol ester stimulation. Recently, we reported that apigenin and related flavonoids could suppress the transcriptional activity of COX-2 and iNOS in part through inhibition of IκB kinase activity [28]. The current study was designed to determine whether the anti-inflammatory effects of flavonoids were correlative with their activation of PPARγ.

2. Mater ials and Methods

2.1. Chemicals

LPS (Escherichia coli 0127:B8), flavone, 5-methoxyflavone, 7,8-dihydroxyflavone, apigenin, 3-hydroxyflavone, kaempferol, morin, quercetin, myricetin, rutin, genistein, indomethacin were purchased from Sigma Chemical Co. (St Louis, MO). Chrysin, luteolin, tangeretin, galangin, fisetin, pinocembrin, naringenin, isosakuranetin, eriodictyol, hesperetin, naringin, and biochanin A were purchased from Extrasynthese Inc. (Genay, France). Mouse interferon-γ was purchased from R & D systems Inc. (Minneapolis, MN). Two kinds of tea polyphenols, (-)-epigallocatechin-3-gallate and theaflavin-3,3’-digallate were purified as previously described [29].

2.2. Cell culture

The mouse macrophage cell lines RAW264.7 (ATCC, T1B71) were cultured as previously described [28]. Thioglycollate-elicited peritoneal macrophages were obtained from specific

pathogen-free female Balb/c mice as previously described [30]. For all assays except the luciferase assay, cells were plated in 60 mm dishes at 5×10

cells/dish and allowed to grow for 18-24h.

Treatment with vehicle (0.1% DMSO), test compounds and/or LPS or IFN-γ were carried out under serum-free conditions.

2.3. Determination of PGE

and nitrite

The cultured medium of control and treated cells were collected, centrifuged, and stored at -70°C, until tested. The level of PGE

released into culture media was quantified using a specific enzyme immunoassay (EIA) according to the manufacturer’s instructions (Amersham). The nitrite

concentration in the cultured medium was measured as an indicator of NO production according to the Griess reaction [28].

2.4. Plasmids

The PPARγ expression plasmid and AOx-TK reporter plasmid were generously provided by Professor Christopher K. Glass (California University)[13].

The mouse iNOS promoter plasmid was generously provided by Professor Charles J. Lowenstein (Johns Hopkins University)[31]. The mouse COX-2 promoter plasmid containing a 1035 bp fragment, -966 to＋70 relative to the transcription start, and constructed as previously described [28]. To generate the pGEX-2T-PPARγ LBD chimeric receptor expression plasmid, cDNA encoding the ligand binding domains (LBD) of the mouse PPARγ1(amino acids 174-475) were amplified by polymerase chain reaction and subcloned into the pGEX-2T expression plasmid.

Transient cotransfection and luciferase activity assay using these plasmids were performed as described previously [28].

2.5. Ligand binding assay

The Gst-PPARγ LBD was expressed in JM109 Escherichia coli [32] and the fusion proteins were bound to the Glutathione Sepharose-4B beads according to the manufacturer’s instructions

(Pharmacia Biotech). For competition binding assay, 10 µl of Glutathione Sepharose-4B beads containing 0.1 µg of Gst-PPARγ LBD chimeric protein were incubated with or without unlabeled flavonoids at 4℃ for 12h in buffer containing 10 mM Tris (pH 7.4), 50 mM KCl, 10 mM

dithiothreitol and proteinase inhibitors, then added [

H]BRL49653 (specific activity, 60 Ci/mmol) for additional 8 h. Bound [

H]BRL49653 was precipitated from free radioactivity by centrifugation, and washed three times with PBS. The beads containing [

H]BRL49653 were collected and

quantitated by liquid scintillation counting.

2.6. Limited protease digestion assay

The protease digestion assays were performed by the method of Allen et al. [33], with some modification. The PPARγ expression plasmid [13] was used to synthesize [

S]-radiolabeled PPARγ in a coupled transcription/translation system according to the protocol of the manufacturer

(Promega). Approximately 5 µl of the transcription/translation reactions was preincubated with 1 µl of tested compounds for 20 min at 25 ℃. Trypsin was added and allowed to proceed for 10 min at 25 ℃, then terminated by the addition of SDS sample loading buffer and boiling for 8 min. The products of the digestion were separated by electrophoresis through a 12% SDS-polyacrylamide gel.

Labeled PPARγ was visualized by autoradiography.

3. Results

3.1. Apigenin, chrysin, and kaempferol activated PPARγ in macrophages

A series of flavonoids including flavone, 5-methoxyflavone, 7,8-dihydroxyflavone, apigenin,

3-hydroxyflavone, kaempferol, morin, quercetin, myricetin, rutin, genistein, chrysin, luteolin,

tangeretin, galangin, fisetin, pinocembrin, naringenin, isosakuranetin, eriodictyol, hesperetin,

naringin, and biochanin A, tea polyphenols (Table 1), and indomethacin were first tested with

regard to their activation effects on PPARγ in RAW264.7 cells. As RAW264.7 cells express very low levels of PPARγ [13] and required transfection of a PPARγ expression plasmid. The cells therefore allowed a direct assessment of the role of PPARγ in mediating the effects of these

flavonoids on macrophage gene expression. The PPARγ expression plasmid was cotransfected into RAW264.7 cells with a reporter construct containing three copies of the acyl CoA oxidase PPAR responsive element (PPRE) upstream of the thymidine kinase (TK) promoter driving luciferase gene expression. In the absence of a cotransfected PPARγ expression plasmid, treatment of RAW264.7 macrophages with the tested flavonoids at 10 µM had little effect on activation of PPARγ. However, when a PPARγ expression plasmid was cotransfected into the cells, apigenin, chrysin, and kaempferol significantly induced the PPARγ 8.13, 5.60 and 7.66-fold, respectively (p<

0.05) (Fig. 1A). The positive control of indomethacin (100 µM) strongly induced the PPARγ activity 13.34-fold which was compared with the mock experiment. Apigenin, chrysin, and kaempferol increased the PPARγ activity in a dose-dependent manner, with the EC

of

approximately 5 µM, 10 µM, and 10 µM, respectively (Fig. 1B). However, there was a cytotoxic effect in RAW264.7 cells with 20 µM of apigenin and a decrease in the activation of PPARγ.

Flavone, 7,8-dihydroxyflavone, 3-hydroxyflavone, luteolin, galangin, genistein, biochanin A also increased the PPARγ activities when RAW264.7 cells were transfected with the expression plasmid of PPARγ. However, the induction folds of these flavonoids showed no significant difference in activation of PPARγ compared with control RAW264.7 cells that were transfected with PPARγ expression plasmid (Fig. 1A, land 2, 2.74-fold). The other tested flavonoids were unable to activated the PPARγ, and the data were not shown in Fig. 1A.

3.2. Apigenin, chrysin and kaempferol enhanced the inhibition of COX-2 and iNOS promoters’

activities in a PPARγ-dependent manner

RAW264.7 cells was transiently transfected with the reporter plasmids of COX-2 or iNOS and both promoters’ activities were markedly increased when RAW264.7 cells were treated with LPS (Fig. 2). Both promoters’ activities were inhibited by concurrent treatment of the cells with apigenin, chrysin and kaempferol. Moreover, transfection of PPARγ expression plasmid enhanced the inhibitory effects of these three flavonoids (Fig. 2A&B). The results suggested that apigenin, chrysin and kaempferol inhibited the promoter’ activities of COX-2 and iNOS genes partially through PPARγ pathways.

3.3. Apigenin, chrysin, and kaempferol bound with PPARγ and induced conformational change in PPARγ

We next sought to determine whether these three flavonoids activated PPARγ through direct

interaction with the PPARγ receptor. The abilities of these flavonoids to bind to PPARγ were

assessed in a competition binding assay using [

H] BRL49653 and the Glutathione Sepharose beads

containing Gst-PPARγ LBD fusion protein. As shown in Fig. 3, [

H] BRL49653 bound specifically

and saturably to Gst-PPARγ LBD beads with a Kd of 8 nM (Fig. 3A & B). No binding was detected in control Gst-PPARγ LBD beads (Data not shown). Apigenin, chrysin, and kaempferol competed with [

H] BRL49653 for binding to the PPARγ LBD in a dose-dependent manner, with an IC

of approximately 50 µM. Proteolytic analysis have been used for several experiments to demonstrate that ligands of nuclear receptor can, upon binding, specifically alter the conformation of the receptor [33, 34]. This conformational change was reflected by the increased resistance of the receptor to partial digestion by proteases. To determine if there were conformational differences in PPARγ bound to these three flavonoids, a limited trypsin digestion on a [

S] methionine-labeled PPARγ was performed. As shown in Fig. 4, incubation of PPARγ with increasing concentrations of trypsin in the absence of ligand led to the complete digestion of PPARγ. In contrast, BRL49653 induced a stronger protection of the 22-, 29- and 30-kDa fragments. These three flavonoids binding yielded 29- and 30-kDa protected fragments, especially 30-kDa band. These results indicated that these three flavonoids were able to bind to PPARγ and flavonoids-bound PPARγ had a distinct trypsin digestion pattern compared with the BRL49653-bound receptor.

4. Discussion

Flavonoids are naturally occurring plant polyphenols found in abundance in the diets rich in fruit, vegetables and plant-derived beverages such as tea. The PPARγ ligands share certain

structural characteristics including a lipophilic backbone and an acid moiety, usually a carboxylate.

Although flavonoids only have a similar lipophilic backbone, several flavonoids also bind to PPARγ in vitro (Fig. 4). Several reports have shown that treatment of various fibroblast and

mesenchymal stem cells lines with PPARγ ligands, including 15d-PGJ

, the anti-diabetic drugs, and several NSAIDs, promotes their efficient conversion to adipocytes [3, 4, 35-37]. However,

treatment of C3H10T1/2 stem cells with 10 µM of apigenin, chrysin, and kaempferol failed to promote adipocytes differentiation as indicated by Oil Red O staining (Data not shown). Northern blot analysis indicated that PPARγ expression levels were not induced in response to treatment with these three flavonoids in fibroblast cells (Data not shown). As we known, adipocyte differentiation required forced expression of PPARγ and was significantly enhanced in the presence of PPARγ activators [38]. For example, treatment of C3H10T1/2 cells with BRL49653 increased PPARγ expression levels approximately 3-fold, and resulted in efficient adipocyte differentiation [3]. In addition, while flavonoids were potent inhibitors of several kinases involved in signal transduction, mainly protein kinase C [39] and tyrosine kinase [40, 41]. However, adipocyte differentiation is characterized by a coordinate increase in adipocyte-specific gene expression through activating of gene transcription. Therefore, they were insufficient to initiate the adipogenic signaling cascade in a mesenchymal stem cell line. Devchand et al. [11] proposed that leukotriene B4, an agonist of the related receptor PPARα, has anti-inflammatory activity. The flavonoids might also activate the PPARα and have anti-inflammatory effects, but PPARα is not expressed in activated macrophages.

Of the tested flavonoids (Table 1), the groups of flavanones and flavan-3-ol were inefficient on

activation of PPARγ. This result suggested that the C2-C3 double bond of C ring was essential for their activation of PPARγ. Some flavonoids of flavones, flavonols and isoflavones groups were able to activate PPARγ. The activation of PPARγ seems to be dependent on the number and the position of hydroxyl residues. The hydroxyl residues of the 5 and 7 positions of A ring and 4’ position of B ring were important factors for activation of PPARγ, such as apigenin, chrysin and kaempferol.

However, additional hydroxyl residue of 3’ position of B ring resulted in decrease the activation of PPARγ, such as luteolin and qucrcetin.

These three flavonoids were able to activate PPARγ in a transient reporter assay, with an EC

of approximately 5~10 µM. However, they needed a higher concentration to bind to Gst-PPARγ in vitro competitive binding assay (Fig. 3). The high concentration of IC

(50 µM) suggested that these three flavonoids might not directly bind to PPARγ or bind to PPARγ in the other sites. In limited protease digestion assay (Fig. 4) indicated that PPARγ has a conformational difference in the receptor bound to the three flavonoids versus natural ligand BRL49653. These results suggested that these three flavonoids might act as allosteric effectors, and were able to bind to PPAR γ and activate it, but it’s binding site might be different from the natural ligand BRL49653. Based on the different binding kinetics, it may interpret the fact that these three flavonoids did not promote differentiation of C3H10T1/2 stem cells to adipocyte. Our previous studies have shown that apigenin was able to inhibit IκB kinase activity and prevent the activation of NF-κB, and then suppress the promoter activities of COX-2 and iNOS [28]. Therefore, apigenin was a more stronger inhibitor of COX-2 and iNOS promoters activities than BRL49653 in the absence and presence of transfected PPARγ expression plasmid (Fig. 2). However apigenin was more effective in the presence of PPARγ than absence of PPARγ in inhibition of COX-2 and iNOS promoters. These results indicated that these three flavonoids inhibited the expression of COX-2 and iNOS partially through activating PPARγ. In RAW264.7 cells, the base level of PPARγ protein was very low, so BRL49653 could not activate it and then to suppress the activities of COX-2 and iNOS promoters in the absence of transfected PPARγ expression plasmid.

Our results suggested that certain flavonoids could activate PPARγ, then to inhibit the protein expression of COX-2. Indomethacin was able to inhibit COX activity without affecting the protein levels of PPARs at lower concentration [42]. At higher concentration, we thought that

anti-inflammatory activity of indomethacin might be also mediated through activation of PPARγ then inhibition of COX expression. Since indomethacin also acts as a PPARγ agonist, promoting adipocyte differentiation.

Our previous studies demonstrated that the anti-inflammatory properties of apigenin might be mediated through inhibition of IκB kinase activity. In this study, we showed that apigenin was also an efficacious activator of PPARγ which regulated inflammatory responses. These results suggested that apigenin, chrysin, and kaempferol were possible activators of PPARγ, and might have

therapeutic applications in inflammatory diseases, such as atherosclerosis and rheumatoid arthritis.

These findings also provide a significant molecular basis for explaining how dietary flavonoids are

active in preventing cancer and inflammation.

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