Research Article

Cellular & Molecular Immunology (2010) 7, 123–132; doi:10.1038/cmi.2010.1; published online 8 February 2010

Epigallocatechin-3-gallate (EGCG) attenuates inflammation in MRL/lpr mouse mesangial cells

Abigail Peairs1, Rujuan Dai2, Lu Gan3, Samuel Shimp4, M Nichole Rylander4, Liwu Li3 and Christopher M Reilly1,2

  1. 1Virginia College of Osteopathic Medicine, Blacksburg, VA, USA
  2. 2Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA
  3. 3Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA
  4. 4Department of Mechanical Engineering, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA

Correspondence: Dr A Peairs, Virginia College of Osteopathic Medicine, 1410 Prices Fork Rd, Blacksburg, VA 24060, USA. E-mail: apeairs@vcom.vt.edu

Received 21 September 2009; Revised 8 December 2009; Accepted 17 January 2010; Published online 8 February 2010.

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Abstract

Epigallocatechin-3-gallate (EGCG), a bioactive component of green tea, has been reported to exert anti-inflammatory effects on immune cells. EGCG is also shown to activate the metabolic regulator, adenosine 5'-monophosphate-activated protein kinase (AMPK). Reports have also indicated that EGCG inhibits the immune-stimulated phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway. The PI3K/Akt/mTOR pathway has been implicated in mesangial cell activation in lupus. Mesangial cells from MRL/lpr lupus-like mice are hyper-responsive to immune stimulation and overproduce nitric oxide (NO) and other inflammatory mediators when stimulated. In our current studies, we sought to determine the mechanism by which EGCG attenuates immune-induced expression of pro-inflammatory mediators. Cultured mesangial cells from MRL/lpr mice were pre-treated with various concentrations of EGCG and stimulated with lipopolysaccharide (LPS)/interferon (IFN)-γ. EGCG activated AMPK and blocked LPS/IFN-γ-induced inflammatory mediator production (iNOS expression, supernatant NO and interleukin-6). Interestingly, EGCG attenuated inflammation during AMPK inhibition indicating that the anti-inflammatory effect of EGCG may be partially independent of AMPK activation. Furthermore, we found that EGCG effectively inhibited the immune-stimulated PI3K/Akt/mTOR pathway independently of AMPK, by decreasing phosphorylation of Akt, suggesting an alternate mechanism for EGCG-mediated anti-inflammatory action in mesangial cells. Taken together, these studies show that EGCG attenuated inflammation in MRL/lpr mouse mesangial cells via the PI3K/Akt/mTOR pathway. Our findings suggest a potential therapeutic role for the use of EGCG to regulate inflammation and control autoimmune disease.

Keywords:

AMPK; inflammation; lupus; metabolism; MRL/lpr

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Introduction

The use of alternative medicine is widespread worldwide and on the rise in the United States. Patients with autoimmune disorders have increasingly sought alternative medicine as a complementary and primary means of mediating their disease. Epidemiological studies indicate that, in comparison to the United States, the incidence of lupus is considerably lower in China and Japan, the two leading green tea-consuming countries.1 The active compound in green tea, Epigallocatechin-3-gallate (EGCG), has been widely reported to exert anti-inflammatory effects in animals and humans.2 Thus, EGCG might be responsible, in part, for the geographical differences in the incidence of lupus. Green tea polyphenols consist mainly of catechins, including (−)-Epigallocatechin-3-gallate, the most abundant and strongest bioactive compound. EGCG has been shown to lower the incidence of cancers,3, 4 diabetes,5 arthritis,6 inflammatory mediator production and7, 8 oxidative stress,9, 10 and to reduce body weight and body fat.11 EGCG has also been shown to exert anti-inflammatory activity in immune cells.12, 13, 14 Hwang et al.15 have recently reported that one mechanism of EGCG anti-inflammatory action is through the activation of the adenosine 5'-monophosphate-activated protein kinase (AMPK). Activation of AMPK has been shown to inhibit the production of several proinflammatory mediators including tumor-necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, monocyte chemoattractant protein-1, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 with lipopolysaccharide (LPS) stimulation.16 Additionally, AMPK activation promotes macrophage polarization to an anti-inflammatory functional phenotype.17

MRL/MPJ-Faslpr (MRL/lpr) mice develop SLE-like disease similar to humans and develop lymphadenopathy associated with proliferation of aberrant T cells, arthritis and autoimmune complex glomerulonephritis. The mice die at an average age of 22 weeks from renal disease. The composite genome distribution of autoantibodies produced by these mice is similar in spectrum to those seen in human SLE including antidouble stranded DNA antibodies and anti-Sm antibodies.18, 19 Mesangial cells are macrophage-like cells resident in the kidney possessing both immune and vascular functions. Mesangial cells, such as macrophages, produce nitric oxide (NO), superoxide, and other inflammatory mediators in response to LPS, IFN-γ and IL-1β.20, 21, 22 We and others23, 24, 25, 26 have reported iNOS expression in renal tissue of lupus patients and evidence of nitrated proteins in kidneys from MRL/lpr mice. In addition to NO, other inflammatory mediators released by mesangial cells have a pathogenic role in lupus (i.e. IFN-γ and TNF-α).26, 27 Given reports that EGCG administration decreases inflammatory mediator production, and lupus mouse mesangial cells exhibit a heightened state of inflammation, we sought to determine the mechanism by which EGCG attenuates immune-induced expression of proinflammatory mediators.

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Materials and methods

Animals

Eight-week-old female MRL/lpr mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Mice were maintained under specific pathogen-free conditions and used prior to the onset of disease. Mesangial cells were isolated from at least five mice by renal dissection, differential sieving and collagenase digestion, as we have previously described,21 and pooled for experimental procedures. Cultures were examined to ensure that they were homogeneous for expression of mesangial cell morphology and stained positively for α-smooth muscle actin. Cultures were maintained at 37 °C and 5% CO2 in a humidified atmosphere, in DMEM/F-12 media, supplemented with 10% fetal bovine serum (FBS) and antibiotics.

Reagents

IFN-γ was purchased from PharMingen (San Diego, CA, USA), while FBS and DMEM/F-12 from Gibco (Gaithersburg, MD, USA). The protein assay kits were purchased from Bio-Rad (Hercules, CA, USA). Anti-iNOS type II antibody was purchased from Transduction Laboratories (Lexington, KY, USA), β-actin from Ambion (Applied Biosystems, Foster City, CA, USA), and antibodies to AMPK, phospho-(Thr172)-AMPK, acetyl CoA carboxylase (ACC), phospho-ACC (p-ACC), Akt, phospho-Akt (Ser473), phospho-p70s6K (Thr389), nuclear factor (NF)-κB p65, and the inhibitors rapamycin and LY294002 were from Cell Signaling (Beverly, MA, USA). All other reagents, including EGCG, 5′-iodotubercidin, Adenosine-9-β-d-arabino-furanoside (Ara-A), Cell-lytic M lysis buffer, protease inhibitor cocktail, sodium orthovanadate, and LPS were purchased from Sigma (St Louis, MO, USA).

Experimental protocol

In each experiment, mesangial cells were plated on 6- or 12-well tissue culture plates, serum-starved (1% FBS) at confluence for 2 h, and pre-treated with indicated concentrations of EGCG for 1 h. After EGCG pre-treatment, cells were stimulated with LPS (1 µg/ml) and IFN-γ (300 U/ml) for the indicated treatment times. In some experiments, AMPK inhibitors (5′-iodotubercidin (0.1 µM) or Ara-A (500 µM)) were added to the cells for 30 min prior to EGCG treatment. In other experiments, the mTOR inhibitor rapamycin (10 nm) or phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (10 µM) was added to the cells for 1 h prior to EGCG treatment.

Nitrite production

Supernatants collected for 24 h after LPS/IFN-γ stimulation were analyzed for nitrite concentration (a stable reaction product of NO with oxygen) as described after conversion of nitrate to nitrite using nitrate reductase, G6P, NADPH, and G6PDH (Roche-Boehringer Mannheim, Indianapolis, IN, USA).28 Briefly, supernatants were analyzed by mixing an equal volume of sample with Griess reagents (1% sulfanilamide and 0.1% naphthylethylenediamene in 2.5% H3PO4) in a 96-well plate and the absorbance determined at 550 nm. The concentration of nitrite was calculated from a standard curve produced by the reaction of NaNO2 in the assay.

Real-time RT-PCR

Cellular RNA was extracted by lysing cells with RNeasy kit reagents (Qiagen, Valencia, CA, USA) following the manufacturer’s protocol and mRNA was converted to cDNA using the high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Real-time RT-PCR was performed using the Taqman gene expression assay method (Applied Biosystems) on an iCycler IQ using specific primers targeting iNOS, IL-6, and expressed relative to the housekeeping gene hypoxanthine phosphoribosyltransferase (hprt1) (Applied Biosystems). The relative fold induction of corresponding target genes was calculated using the delta–delta-Ct method.

Western blotting

Whole cell lysates were prepared by lysing pelleted cells with Cell-lytic M lysis buffer supplemented with protease inhibitors and sodium orthovanadate. Protein concentrations were determined using the Bradford protein assay and equal amounts of protein were loaded onto a polyacrylamide gel and electrophoresed prior to transfer to a PVDF membrane. Membranes were blocked in 5% non-fat milk/Tris-buffered Saline with Tween (TBST) and then incubated overnight at 4 °C with the indicated primary antibodies and visualized after secondary antibody incubation using the ECL+ chemiluminescence kit (GE Healthcare, Piscataway, NJ, USA).

Preparation of nuclear extract

Nuclear extracts were prepared utilizing NE-PER nuclear extraction and cytoplasmic extraction reagents and protocol (Pierce Biotechnology, Rockford, IL, USA). Briefly, extracts were prepared by lysing pelleted cells in 100 µl of ice-cold hypotonic lysis solution (10 mM HEPES, pH 7.9, 10 mM KCl and 1.5 mM MgCl2) and vortexing vigorously for 15 s. Subsequently, 5.5 µl of Nonidet P-40 was added to the lysate, vortexed for 15 s and centrifuged at 16 000g for 5 min. The resulting pellet was washed, resuspended in 50 µl of hypertonic lysis solution (20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA and 25% glycerol), incubated for 5 min at 4 °C and centrifuged at 16 000g for 10 min. Supernatant containing the nuclear extract was removed, quantified with the Bradford protein assay kit, and stored at −80 °C.

Flow cytometry

Mesangial cells were serum-starved (1% FBS) for 2 h and pre-treated with EGCG for 1 h followed by stimulation with LPS/IFN-γ. Control cells were stimulated with LPS/IFN-γ only, or received no treatment. After 24 h, the cells were washed with phosphate-buffered saline, trypsinized and collected. Apoptosis/necrosis was evaluated using annexin V-FITC/propidium iodide staining29 as described previously.30 Cells were analyzed by flow cytometry, using a fluorescence activated cell sorter caliber (Becton Dickinson, Mountain View, CA, USA) and FlowJo software.

ELISA

IL-6 levels in supernatants were quantified by ELISA per the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA).

Statistics

Results shown represent means±SEM, with n=3 unless stated otherwise. Statistical analysis was performed by ANOVA with post hoc analyses, or Student’s t-test where appropriate, using GraphPad Prism version 4.0 for Windows (GraphPad Software, San Diego, CA, USA). Blots shown are representative of repeated experiments.

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Results

EGCG does not reduce cell viability

To ensure that the concentration of EGCG tested was not toxic to the mesangial cells, cell viability were evaluated utilizing annexin V staining coupled with flow cytometry. Mesangial cells were serum-starved for 2 h (1% FBS), and then EGCG (50 µM) was added for 1 h prior to LPS/IFN-γ for 24 h. The cells were collected and stained for annexin V and propidium iodide, and analyzed by flow cytometry (Figure 1). The results showed that there was no difference in apoptosis or necrosis between non-stimulated, LPS/IFN-γ stimulated or EGCG+ LPS/IFN-γ treated cells.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

EGCG administration and cell viability. Mesangial cells were serum-starved (1% FBS) at confluence for 2 h and pre-treated with EGCG for 1 h (or kept as controls), followed by LPS/IFN-γ stimulation for 24 h. Cells were collected by trypsinization, stained for annexin/propidium iodide and analyzed by flow cytometry. Data in each quadrant are the percentages of total cells, and shown as mean±SEM. Statistical analysis was performed by ANOVA. ANOVA: analysis of variance; EGCG: Epigallocatechin-3-gallate; FBS: fetal bovine serum; IFN: interferon; LPS: lipopolysaccharide.

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EGCG attenuates inflammation in mesangial cells stimulated with LPS/IFN-γ

We sought to determine if iNOS gene expression and NO production could be inhibited by EGCG treatment. Mesangial cells were serum-starved (1% FBS) for 2 h and treated with EGCG (50 µm) for 1 h prior to LPS/IFN-γ stimulation. After 24 h, iNOS protein and nitrate production were determined (Figure 2). We found that EGCG administration significantly inhibited both iNOS protein expression (Figure 2a) and supernatant NO levels (Figure 2b). Similar to the decrease in NO production, IL-6 production was significantly reduced when stimulated mesangial cells were pre-treated with EGCG (Figure 2c). Interestingly, we found that while the mRNA for IL-6 was reduced in stimulated cells pre-treated with EGCG as measured by real-time RT-PCR, the mRNA for iNOS was not significantly affected (Figure 2d–e).

Figure 2.
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EGCG inhibits LPS/IFN-γ-induced production of proinflammatory mediators in mesangial cells. Mesangial cells were serum-starved (1% FBS) for 2 h and pre-treated with EGCG for 1 h prior to LPS/IFN-γ stimulation. After 24 h LPS/IFN-γ stimulation, (a) iNOS protein, (b) NO and (c) IL-6 levels in the supernatant of MRL/lpr mouse mesangial cells were assessed by western blot, Griess reaction assay and ELISA respectively. (d) iNOS and (e) IL-6 mRNA levels after 16 h LPS/IFN-γ treatment were measured by real-time RT-PCR. Data are presented as mean±SEM and statistical analysis was performed by ANOVA with post hoc t-tests. *P<0.05 and **P<0.01 vs LPS/IFN-γ. ANOVA: analysis of variance; EGCG: Epigallocatechin-3-gallate; FBS: fetal bovine serum; IFN: interferon; iNOS: inducible nitric oxide synthase; LPS: lipopolysaccharide.

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EGCG increases phosphorylation of AMPK and ACC

AMPK activation has been shown to be induced by EGCG in a variety of cell types.15, 31, 32 Cultured mesangial cells were treated with various concentrations of EGCG for 1 h after which AMPK expression was measured. We found that EGCG increased AMPK phosphorylation in a concentration-dependent manner (Figure 3a). As sufficient AMPK activation was achieved with 50 µM EGCG, we used that concentration for subsequent experiments. In addition to AMPK activation, Figure 3b shows that EGCG administration causes phosphorylation and thus inactivation of the metabolic enzyme ACC.

Figure 3.
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EGCG activates AMPK. Mesangial cells from MRL/lpr mice were serum-starved (1% FBS) for 2 h and treated with EGCG for 1 h. (a) The effect of varying concentrations of EGCG on AMPK phosphorylation and (b) the effect of 50 µM EGCG pre-treatment on the phosphorylation of ACC and AMPK were assessed by western blot. Densitometric data of AMPK-p and ACC-p relative to β-actin are presented as mean±SEM. Statistical analysis was performed by ANOVA and t-test respectively. *P<0.05 vs control (0 µM EGCG). ACC: acetyl CoA carboxylase; AMPK: AMP-activated protein kinase; ANOVA: analysis of variance; EGCG: Epigallocatechin-3-gallate; FBS: fetal bovine serum.

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EGCG inhibits LPS/IFN-γ activation of the PI3K/Akt/mTOR pathway

Once we established that EGCG blocked immune-stimulated inflammatory mediator production in lupus mesangial cells, we focused our attention on delineating the mechanism by which EGCG blocks the inflammatory signaling cascade. We and others30, 33, 34, 35 have shown that LPS/IFN-γ activates the PI3K/Akt/mTOR pathway and induces iNOS. Furthermore, we have recently shown that EGCG decreases Akt phosphorylation.30 Here, we show that in mesangial cells stimulated with LPS/IFNγ, Akt-p increased reaching a peak after 30 min, at which point it started to decline. Pre-treatment with EGCG (50 µM for 1 h) significantly reduced Akt-p at all times tested without influencing total Akt levels (Figure 4a). As an additional control, we determined Akt levels relative to the housekeeping gene β-actin (Figure 4b). Furthermore, the reduction in Akt phosphorylation by EGCG pre-treatment was concentration-dependent (Figure 4c).

Figure 4.
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EGCG attenuates LPS/IFN-γ induction of Akt phosphorylation. Mesangial cells were serum-starved (1% FBS) for 2 h and pre-treated with EGCG (50 µM) for 1 h. (a) Akt-p levels relative to total Akt levels were measured for up to 2 h following LPS/IFN-γ stimulation and expressed as a fold change. (b) Akt-p levels relative to the housekeeping gene β-actin and (c) the effect of varying the concentration of EGCG on Akt phosphorylation are shown. All were assessed by western blot. Densitometry data are presented as mean±SEM and analyzed by ANOVA with post hoc t-tests. *P<0.01 vs control. Akt-p: Phosphorylated Akt; ANOVA: analysis of variance; EGCG: Epigallocatechin-3-gallate; FBS: fetal bovine serum; IFN: interferon; LPS: lipopolysaccharide.

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When activated, Akt is phosphorylated at serine 473 leading to downstream activation of mTOR and phosphorylation of p70s6k. Prior studies have shown that rapamycin decreases cytokine production by inhibiting mTOR in immune cells stimulated with LPS/IFN-γ showing the importance of the mTOR-p70s6k pathway as a key regulator of inflammation.36, 37 In our studies, we found that pre-treatment with EGCG blocked phosphorylation of p70s6K in cells stimulated with LPS/IFN-γ (Figure 5). When mesangial cells were pre-treated with either the PI3K inhibitor LY294002, or the mTOR inhibitor rapamycin, p70s6k phosphorylation was completely inhibited as expected (Figure 5a and b).

Figure 5.
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EGCG reduces phosphorylation of p70s6k. Mesangial cells were serum-starved (1% FBS) for 2 h and pre-treated with inhibitors for 1 h prior to EGCG treatment (50 µM) for 1 h and LPS/IFN-γ stimulation for 30 min. Levels of p70s6k phosphorylation were assessed for differences between control, EGCG and (a) the PI3K inhibitor LY294002 and (b) the mTOR inhibitor Rapamycin pre-treatments by western blot. Data are presented relative to β-actin as mean±SEM and statistical analysis was performed by ANOVA with post hoc t-tests. *P<0.05 EGCG treatment and **P<0.01 Inhibitor treatment vs control. ANOVA: analysis of variance; EGCG: Epigallocatechin-3-gallate; FBS: fetal bovine serum; IFN: interferon; LPS: lipopolysaccharide; mTOR: mammalian target of rapamycin; PI3K: phosphoinositide 3-kinase.

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Anti-inflammatory effect of EGCG is independent of AMPK

While it has recently been reported that EGCG can exert anti-inflammatory effects (reduction in cyclooxygenase-2) through the activation of AMPK in cancer cells,15, 38 we sought to determine if EGCG could block iNOS expression independently of AMPK. To inhibit AMPK activation, we pre-treated mesangial cells with a competitive AMPK inhibitor (Ara-A) and an inhibitor of adenosine kinase (5′-iodotubercidin) for 1 h prior to EGCG treatment and LPS/IFN-γ stimulation. Ara-A is the precursor of Ara-adenosine triphosphate (ATP), which acts as a competitive inhibitor for AMPK, while 5′-iodotubercidin indirectly inhibits AMPK activation by blocking intracellular AMP formation. As AMPK is quite sensitive to changes in the AMP/ATP ratio, this effectively reduces AMPK activation.39 In our studies, we found that pre-treatment with either Ara-A or 5′-iodotubercidin inhibited AMPK phosphorylation as determined by western blot analysis following EGCG treatment (Figure 6a). Although both inhibitors prevented AMPK phosphorylation, iNOS protein expression, supernatant NO levels and IL-6 production were still inhibited following EGCG treatment (Figure 6b–d). These results suggest that AMPK activation was not required for EGCG to attenuate immune-induced inflammation in mesangial cells.

Figure 6.
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EGCG attenuates LPS/IFN-γ-induced inflammation independently of AMPK activation. Mesangial cells were serum-starved (1% FBS) for 2 h and pre-treated with AMPK inhibitors (Ara-A and 5′-iodotubercidin) for 1 h prior to EGCG treatment (50 µM) for 1 h. (a) Following pretreatments, AMPK phosphorylation was assessed by western blot. Cells were also pre-treated and then stimulated with LPS/IFN-γ for 24 h and (b) iNOS protein expression, (c) supernatant NO and (d) IL-6 levels were assessed by western blot, Griess reaction assay and ELISA respectively. Data are presented as mean±SEM and statistical analysis was performed by ANOVA with post hoc t-tests. *P<0.01 vs LPS/IFN-γ; #P<0.05 vs non-stimulated control. AMPK: AMP-activated protein kinase; ANOVA: analysis of variance; Ara-A: adenosine-9-β-d-arabino-furanoside; EGCG: Epigallocatechin-3-gallate; FBS: fetal bovine serum; IFN: interferon; iNOS: inducible nitric oxide synthase LPS: lipopolysaccharide.

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EGCG reduces activation of Akt independently of AMPK

Although we and others30, 38, 40, 41 have reported that EGCG can exert its effects by activating AMPK, we sought to determine if EGCG may act independently of AMPK to reduce Akt activation. To determine if EGCG inhibits the PI3K/Akt/mTOR pathway independently of AMPK, mesangial cells were treated with AMPK inhibitors (Ara-A or 5′-iodotubercidin) and Akt phosphorylation was assessed following immune stimulation (Figure 7). We found that even during AMPK inhibition, EGCG significantly blocked LPS/IFN-γ-induced Akt phosphorylation. These findings indicate that EGCG reduces Akt activation independently of AMPK activation.

Figure 7.
Figure 7 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

EGCG attenuates LPS/IFN-γ-induced Akt phosphorylation independently of AMPK activation. Mesangial cells were serum-starved (1% FBS) for 2 h and pre-treated with the AMPK inhibitors Ara-A and 5′-iodotubercidin for 1 h prior to EGCG treatment for 1 h and LPS/IFN-γ stimulation for 30 min. Akt-p levels were assessed by western blot. Densitometric data of Akt-p levels relative to β-actin are presented as mean±SEM, and then compared to the non-stimulated control levels for a fold change. Statistical analysis was performed by ANOVA with post hoc t-tests. *P<0.01 vs LPS/IFN-γ. Akt-p: Phosphorylated Akt; AMPK: AMP-activated protein kinase; ANOVA: analysis of variance; EGCG: Epigallocatechin-3-gallate; FBS: fetal bovine serum; IFN: interferon; LPS: lipopolysaccharide.

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EGCG reduces activation of NF-κB

The NF-κB protein complex acts as a transcription factor and plays a key role in regulating the immune response.42, 43, 44 Consistent with this role, dysregulation of NF-κB has been linked to autoimmune diseases.45, 46, 47 In unstimulated cells, NF-κB is sequestered in the cytoplasm by inhibitor of κB which prevents the nuclear localization of NF-κB and sequesters them in the cytoplasm in an inactive state. Upon stimulation with LPS/IFN-γ, inhibitor of κB is phosphorylated, dissociates from the NF-κB complex, and is subsequently degraded. The liberated NF-κB rapidly translocates into the nucleus where it engages κB enhancer elements of many inflammation genes including IL-1α, IL-1β, TNF-α, leukocyte adhesion molecules (E-selectin, VCAM-1 and ICAM-1), and the iNOS promoter to induce NO production. Considering the critical role of NF-κB in regulating iNOS transcription and inflammatory protein production,48 we tested if EGCG treatment regulated NF-κB translocation in mesangial cells. Cells were serum-starved for 2 h, pre-treated with EGCG for 1 h and stimulated with LPS/IFN-γ for up to 30 min. EGCG significantly attenuated NF-κB translocation following LPS/IFN-γ stimulation (Figure 8).

Figure 8.
Figure 8 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

EGCG attenuates NF-κB translocation. Mesangial cells were serum-starved (1% FBS) for 2 h and pre-treated with EGCG (50 µM) for 1 h prior to LPS/IFN-γ stimulation for up to 30 min. Nuclear levels of NF-κB were assessed by western blot. Data of nuclear NF-κB levels compared to the non-stimulated control levels for a fold change are presented as mean±SEM. Statistical analysis was performed by ANOVA with post hoc t-tests. P<0.05 between EGCG pre-treated and control groups. ANOVA: analysis of variance; EGCG: Epigallocatechin-3-gallate; FBS: fetal bovine serum; IFN: interferon; LPS: lipopolysaccharide; NF: nuclear factor.

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Discussion

We and others30, 49, 50 have previously shown that the AMPK activator AICAR inhibits immune stimulated inflammatory mediator production. In our current studies, we sought to determine if the polyphenol EGCG would exert similar effects on AMPK activation and inhibition of inflammation. Furthermore, we sought to define the mechanism by which EGCG modulates the immune stimulated PI3K/Akt/mTOR signaling cascade. Our results showed that EGCG reduced Akt and p70s6k phosphorylation while increasing AMPK activation. Interestingly, when AMPK was inhibited with either Ara-A or 5′-iodotubercidin, EGCG was still able to block the PI3K/Akt/mTOR signaling cascade. This suggests that EGCG has the ability to exert anti-inflammatory effects independent of AMPK activation.

In our previous studies, we reported that EGCG activated AMPK and inhibited Akt phosphorylation.30 In our current studies, we show that AMPK activation is not essential for the reduction in Akt activation to occur by EGCG administration. The effect of EGCG on Akt is important in the context of SLE as Akt activation has been shown to enhance mesangial cell proliferation, hypertrophy, migration, and extracellular matrix protein production which can all contribute to glomerular injury.51, 52 Akt is a serine/threonine kinase (also known as protein kinase B) that plays a key role in cellular metabolism, growth and survival.53 Although Akt is activated by phosphorylation of both Thr308 and Ser473 residues, the Ser473 phosphorylation site is considered key as it stabilizes the active conformational state of Akt.54 Akt is also considered an important regulator of several signaling pathways that can lead to autoimmunity.55 There have been mixed reports regarding the effects of EGCG on Akt depending on the cell type studied, as EGCG is shown to reduce Akt phosphorylation in endothelial cells, tumor associated endothelial cells and caco-2 cells,56, 57, 58, 59 but activate Akt in lung carcinoma cells and osteoblasts.60, 61 Here, we show that EGCG reduces Akt activation and subsequently inflammation in immune stimulated mesangial cells.

The role of Akt in modulating inflammation specifically in immune cells has been examined in several cell types, with discrepant results. While some reports have shown that activation of the PI3K pathway serves as a negative regulator of inflammation,62, 63 others have reported that activation of Akt contributes to the inflammatory phenotype in immune cells.37 Furthermore, as constitutive activation of PI3K results in SLE-like disease,64, 65 it has been suggested that targeting Akt activation in the setting of SLE may have therapeutic effects.55 Here, we have shown that activation of Akt subsequent to LPS/IFN-γ stimulation corresponds to increased iNOS protein expression and activity. EGCG prevented Akt phosphorylation and subsequent iNOS, NO and IL-6 production in stimulated mesangial cells. This can have significant effects on SLE, as mesangial cells from lupus mice are hypersensitive to immune stimulation and may be predisposed to increased activation of the Akt pathway.66 Importantly, it has been reported that inhibition of the PI3K pathway reduces glomerulonephritis and increases life span in SLE mice.67 Furthermore, lymphocytes in the B6.Sle1.Sle3 lupus mouse model exhibited enhanced phosphorylation of Akt and p70s6k,68 indicating that downregulating this pathway may indeed be therapeutic in reducing inflammation in SLE.

Akt lies upstream of mTOR and its effector molecule p70s6k. In our studies, we show that p70s6k phosphorylation was increased in immune-stimulated mesangial cells, and the addition of EGCG reduced p70s6k activation. Inhibition of the Akt/mTOR pathway has been shown to have therapeutic efficacy in autoimmune and inflammatory diseases.69 Rapamycin is an inhibitor of mTOR and approved for the prevention of allograft rejection. Rapamycin has been used as an immunosuppressive agent and several reports have shown that rapamycin decreased the development of glomerulonephritis in NZB/W and MRL/lpr mice.70, 71, 72, 73 Although inhibition of mTOR has been shown to exert beneficial effects in lupus mice, other reports have suggested a negative outcome with the use of rapamycin in regulating anti-GBM glomerulonephritis, depending on timing of administration.74 mTOR blockage by rapamycin has also been shown to induce proteinuria and renal deterioration and induce focal segmental glomerulonephritis.75, 76 Conversely, rapamycin efficiently inhibited monocyte chemoattractant protein-1, a chemokine produced by immune cells associated with inflammatory lupus.77 Therefore, given the disparate reports showing the effect of mTOR regulation on inflammation, caution should be used in interpretation of the data.

Although Huang et al.40 have recently reported that EGCG inhibited the mTOR pathway through AMPK activation in cancer cells, we did not find this to be the case in mesangial cells. It is possible that EGCG exerts differential effects depending on the cell type tested. While those authors showed that EGCG significantly reduced the viability in cancer cells at the concentration used to activate AMPK and reduce mTOR activation (80 µM), we did not observe a decrease in viability in mesangial cells at the 50 µM concentration sufficient for reducing Akt/mTOR activation. Those authors also used a different AMPK inhibitor (compound c), whereas we used 5′-iodotubercidin and Ara-A which may account for the differences in AMPK modulation. Finally, it cannot be ruled out that EGCG may act through multiple mechanisms including both AMPK-dependent and -independent pathways which may depend on the cell type tested. Although our initial hypothesis that EGCG would act solely through AMPK to decrease inflammation was not supported, multiple alternate pathways were modulated by EGCG to result in reduced inflammation.

NF-κB is a nuclear transcription factor that regulates genes involved in the inflammatory response. The reduction in NF-κB translocation with EGCG treatment correlated with the observed reduction in IL-6 and NO production indicating that EGCG exerts inhibitory effects on NF-κB activation. Interestingly, we found that iNOS mRNA was not significantly reduced, indicating that there are other inflammatory signal transduction cascades that were not mediated by EGCG administration. This could account for the difference observed in mRNA expression levels. The decrease in nitrite and iNOS protein levels suggests translational inhibition of the iNOS message. One explanation could include that EGCG is known to interfere with the activity of the chaperone protein heat shock protein-90, which is known to help stabilize the iNOS protein and prevent its degradation.78 We are currently exploring the role of EGCG in heat shock protein-90 inhibition and iNOS protein expression.

In summary, we have identified and characterized a critical function of EGCG to reduce activation of mTOR independently of AMPK in mesangial cells. EGCG inhibits Akt and p70s6k, both of which are involved in the mTOR pathway. The reduction of Akt-p by EGCG led to a decrease in inflammatory mediators in immune-stimulated mesangial cells. These findings suggest that EGCG may act as a therapeutic to specifically regulate the Akt/mTOR pathway to decrease inflammation associated with autoimmunity.

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Acknowledgements

This project was funded by grants from the Harvey Peters Foundation (Peairs), Arthritis Foundation (Reilly) and the NIH/NIAD grant 1R15A1072756 (Reilly). The authors would also like to acknowledge the laboratory assistance of Krystina Cocco, Sarah Muse, Nicole Regna and David Fulbrook, in addition to Melissa Makris for flow cytometry.