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Biochem Biophys Res Commun. 2008 September 5; 373(4): 584–588.
PMCID: PMC2832732

Epigallocatechin activates haem oxygenase-1 expression via protein kinase Cδ and Nrf2

Abstract

The Nrf2/anti-oxidant response element (ARE) pathway plays an important role in regulating cellular anti-oxidants, including haem oxygenase-1 (HO-1). Various kinases have been implicated in the pathways leading to Nrf2 activation. Here, we investigated the effect of epigallocatechin (EGC) on ARE-mediated gene expression in human monocytic cells. EGC time and dose dependently increased HO-1 mRNA and protein expression but had minimal effect on expression of other ARE-regulated genes, including NAD(P)H:quinone oxidoreductase 1, glutathione cysteine ligase and ferritin. siRNA knock down of Nrf2 significantly inhibited EGC-induced HO-1 expression. Furthermore, inhibition of PKC by Ro-31-8220 dose dependently decreased EGC-induced HO-1 mRNA expression, whereas MAP kinase and phosphatidylinositol-3-kinase pathway inhibitors had no significant effect. EGC stimulated phosphorylation of PKCαβ and δ in THP-1 cells. PKCδ inhibition significantly decreased EGC-induced HO-1 mRNA expression, whereas PKCα- and β-specific inhibitors had no significant effect. These results demonstrate for the first time that EGC-induced HO-1 expression occurs via PKCδ and Nrf2.

Keywords: Epigallocatechin, Haem oxygenase-1, Monocytic cells, Nrf2, Protein kinase C, Green tea polyphenols

Catechins, a family of plant polyphenols, exert anti-oxidant, anti-inflammatory and anti-proliferative effects in vitro and in vivo [1,2]. This may be in part due to their activation of Phase II enzymes and cellular anti-oxidants, including NAD(P)H:quinone oxidoreductase 1 (NQO1), catalase and enzymes involved in glutathione synthesis [3–5]. These and other cytoprotective molecules, including haem oxygenase-1 (HO-1, the rate-limiting enzyme in haem catabolism) and the iron-binding protein ferritin, contain anti-oxidant response elements (ARE) in the regulatory regions of their genes. The transcription factor NF-E2-related factor 2 (Nrf2) binds to this regulatory element and plays a key role in ARE-driven gene transcription [6]. Nrf2 knockout mice are deficient in cellular anti-oxidants and are susceptible to inflammation [7,8], demonstrating the importance of this pathway in cellular defence. Several kinase pathways have been implicated in activation of Nrf2 including the ERK and p38 MAP kinase pathways, phosphatidyl inositol 3 kinase and protein kinase C (PKC) [9–14].

Green tea is a rich source of catechins, comprising over 30% of its dry weight [15]. Green tea extract activates ARE-mediated reporter activity [16]. EGCG is the most abundant catechin in green tea and studies to date have focused on this compound. EGCG stimulates expression of many Nrf2-dependent genes in liver and intestine in mice and activates HO-1 expression in B lymphoblasts, epithelial and endothelial cells [13,14,17,18]. Epigallocatechin (EGC) is also a major catechin in green tea [19]. Although EGCG and EGC are structurally similar except for the addition of a 3-gallate group in EGCG, their biological effects differ extensively [3,20–26]. EGC is also more bioavailable than EGCG and other catechins and oral administration of EGC, but not EGCG, results in a significant increase in plasma anti-oxidant activity [27].

Monocytes play an essential role in the host response to oxidative stress and inflammation. In the present study, the effects of EGC on ARE-mediated gene expression were examined in THP-1 cells, a human monocytic cell line that we have previously used to examine ARE-mediated gene expression [9–11]. In addition, the role of Nrf2 and the various potential kinase pathways leading to Nrf2 activation were investigated.

Materials and methods

Materials. LY333531 was purchased from Alexis Biotechnologies (Nottingham, UK). SB203580, Ro-31-8220, rottlerin, LY294002, Go6976 and PD98059 were obtained from Calbiochem (Nottingham, UK). (−)Epigallocatechin and all other chemicals were purchased from Sigma (Poole, UK).

Cell culture. THP-1, a human monocytic leukaemia cell line [28], was purchased from ECACC (Porton Down, UK) and cultured in RPMI 1640 medium supplemented with 10% foetal calf serum, 2 mM l-glutamine (Biowhittaker, Wokingham, UK) and 2-mercaptoethanol. Cells were maintained in a humidified atmosphere at 37 °C and 5% CO2.

Western immunoblotting. Cells (1 × 106) were unstimulated or stimulated with EGC and whole cell lysates prepared, proteins separated and immunoblotting carried out as previously described [11]. Antibodies were purchased from the following: mouse anti-human HO-1 antibody (Stressgen Biotechnologies Corporation, Victoria, Canada); rabbit anti-human phosphorylated PKCαβ and PKCδ antibodies (Cell Signalling Technology, Beverley, USA); mouse anti-human endogenous PKCδ antibody (BD Biosciences, CA, USA); goat anti-mouse and goat anti-rabbit secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, USA); mouse anti-human β-actin antibody (Sigma).

Real-time PCR. Cells (5 × 105) were unstimulated or stimulated with EGC for various times at 37 °C. In some experiments, cells were pre-treated with kinase inhibitors for 30 min prior to EGC stimulation. RNA extraction, reverse transcription, and real-time PCR were carried out as previously described [10]. Relative quantitative mRNA expression of HO-1, NQO1, GCLM or ferritin was normalized to 18s ribosomal unit mRNA expression.

Nrf2 siRNA transfection. Nrf2 siRNA sense sequences 5′-GAGUAUGAGCUGGAAAAACtt-3′ (siNrf2 A) [29], 5′-CCUUAUAUCUCGAAGUUUUtt-3′ (siNrf2 B), their complementary antisense sequences and negative controls were obtained from Ambion as purified annealed duplexes. THP-1 cells (5 × 104/well) were transfected in serum-free media with control siRNA or Nrf2-targeted siRNA (30 nM final concentration), using Oligofectamine transfection reagent according to the manufacturer’s instructions (Invitrogen). Transfected cells were incubated for 48 h, with addition of 10% FCS at 4 h. Following this, cells were stimulated with EGC for 4 h before RNA extraction and real-time PCR analysis.

PKCδ antisense oligodeoxyribonucleotide (ODN) transfection. THP-1 cells (5 × 104) were transfected in serum-free media with sense or antisense ODN to PKCδ, using Oligofectamine transfection reagent (Invitrogen), as previously described [10]. Following transfection, cells were unstimulated or stimulated with EGC for 4 h, total RNA extracted and real-time PCR performed.

Statistical analyses. Where indicated, statistical analyses were performed using paired Student’s t test. Results are means ± SD of three independent experiments. Results with p < 0.05 were considered statistically significant.

Results

EGC increases HO-1 expression in THP-1 cells

EGC increased HO-1 mRNA expression in THP-1 cells, peaking at 4 h (p < 0.01), remaining elevated at 8 h (p < 0.01) and returning to baseline by 24 h (Fig. 1A). This correlated with an elevation in HO-1 protein expression by 4 h, which further increased by 8 h and decreased, but was still evident, at 24 h (Fig. 1B). EGC also time and dose dependently increased GCLM mRNA expression in THP-1 cells with maximal induction at 4 h. However, the induction was much weaker than that seen with HO-1 (GCLM at 4 h, 50 μM EGC, 2.5 ± 0.5, p < 0.01; 100 μM EGC, 3.0 ± 1.30, p < 0.01, mean fold increase above control ± SD, n = 7). EGC (12.5–100 μM) had no significant effect on either NQO1 or ferritin gene expression up to 24 h (data not shown). To ensure that EGC-induced HO-1 expression was not the result of toxicity, THP-1 cells pre-incubated with EGC for 24 h were analysed by MTT assay. At concentrations up to 125 μM THP-1 cells remained 96% (±2.3) viable compared to control cells, suggesting that the EGC concentrations used in this study did not exert cytotoxic effects.

Fig. 1
EGC increases HO-1 expression. (A) THP-1 cells were treated with 0–100 μM EGC for 4, 8 or 24 h. RNA was extracted, reverse transcribed and real-time PCR performed for HO-1 mRNA expression. Mean ± SD, ...

Role of Nrf2 in EGC-induced HO-1 expression

Nrf2 plays a key role in HO-1 regulation. The role of Nrf2 in EGC-induced HO-1 expression was confirmed by use of siRNA. THP-1 cells were transiently transfected with control or two different Nrf2 siRNAs and Nrf2 mRNA expression measured by real-time PCR. Fig. 2A demonstrates that both Nrf2 siRNAs significantly inhibited Nrf2 mRNA expression to a similar extent when compared with the negative control (black bars, p = 0.004, p = 0.0002, respectively). These siRNAs also inhibited Nrf2 protein expression (data not shown). In addition, they significantly inhibited EGC-induced HO-1 mRNA expression (grey bars, A, 60.7 ± 8.9, p = 0.01; B, 66.1 ± 2.6, p = 0.0007; mean ± SD% inhibition, n = 3), confirming that Nrf2 plays a key role in this pathway in THP-1 cells.

Fig. 2
Nrf2 regulates EGC-induced HO-1 mRNA expression. THP-1 cells were transfected with control or Nrf2-targeted siRNA in serum-free media for 4 h followed by addition of 10% FCS for 44 h. Following this, cells were stimulated with EGC for ...

Investigation of kinase pathways regulating EGC-induced HO-1 expression

THP-1 cells were pre-treated with LY294002 (a PI3K inhibitor), SB203580 (a p38 MAPK inhibitor), PD98059 (an ERK MAPK pathway inhibitor) or Ro-31-8220 (a pan-PKC inhibitor) prior to stimulation with EGC. LY294002, SB203580 and PD98059 had no significant effect on EGC-induced HO-1 mRNA expression. However, pre-treatment with Ro-31-8220 abolished EGC-induced HO-1 mRNA expression (p < 0.05) (Fig. 3A), suggesting that this response is regulated by PKC. The effect of Ro-31-8220 upon EGC-induced HO-1 mRNA expression was concentration-dependent, confirming the specificity of this effect (Fig. 3B). Ro-31-8220 (5 μM) also completely abolished EGC-induced GCLM mRNA expression in these cells (data not shown). Furthermore, EGC induced phosphorylation of PKC within 5 min, peaked at 20 min (Fig. 3C) and remained phosphorylated at 4 and 8 h (data not shown). These results suggest that PKC plays a key role in EGC-induced HO-1 (and GCLM) expression in THP-1 cells.

Fig. 3
EGC-induced HO-1 mRNA expression is PKC-dependent. THP-1 cells were pre-treated with (A) 25 μM LY294002 (LY), 25 μM PD98059 (PD), 10 μM SB203580 (SB) or 5 μM Ro-31-8220 (Ro) or (B) varying ...

EGC-induced HO-1 expression is regulated by PKCδ

The PKC family of proteins comprises at least 10 serine/threonine kinases [30]. We have shown that LPS and curcumin-induced HO-1 expression in THP-1 cells are mediated via a classical PKC isoform and PKCδ, respectively [10,11]. The potential role of these PKC isoforms in regulating EGC-induced HO-1 expression in THP-1 cells was examined. EGC induced phosphorylation of classical PKCs(αβ) and PKCδ by 5 min, which was sustained at 30 min (Fig. 4A). This was not the result of a change in expression of unphosphorylated PKC isoforms. Cells were also pre-treated with LY333531 (a PKC β12 inhibitor), Go6976 (a PKCα/β1 inhibitor) or rottlerin (which inhibits PKCδ and θ at the concentrations used) prior to stimulation with EGC and real-time PCR performed. LY333531 and Go6976 exerted no significant effect on EGC-induced HO-1 mRNA expression, suggesting that classical PKC isoforms are not important for this pathway. However, 15 μM rottlerin significantly inhibited EGC-induced HO-1 mRNA expression (p < 0.05) (Fig. 4B), suggesting that, similarly to curcumin, PKCδ may be important in HO-1 induction by EGC. Rottlerin, but not Go6976 or LY333531, also inhibited EGC-induced GCLM mRNA expression in THP-1 cells by 61% (data not shown). As rottlerin may also inhibit PKCθ and other kinase pathways, PKCδ antisense ODN were employed to confirm specificity. PKCδ antisense ODN inhibited PKCδ protein expression in THP-1 cells [10]. Transfection of PKCδ sense ODN did not affect EGC-induced HO-1 mRNA expression (Fig. 4C). However, transfection of antisense ODN prior to EGC stimulation resulted in a 78% reduction (p < 0.005) in EGC-induced HO-1 mRNA expression, confirming a role for PKCδ in the regulation of EGC-induced HO-1 expression.

Fig. 4
EGC-induced HO-1 expression is PKCδ-dependent. (A) THP-1 cells were treated with 100 μM EGC for indicated times, whole cell extracts prepared and Western blotting performed. Representative of three independent experiments. (B) ...

Discussion

Green tea extract activates ARE-dependent gene expression and studies to date have focused on EGCG. In the present study, EGC, a more bioavailable catechin found in green tea, induced HO-1 expression and GCLM expression in human monocytic THP-1 cells. Furthermore, EGC-induced HO-1 expression was regulated by Nrf2 and PKCδ.

We have previously reported the activation of Nrf2/ARE-mediated gene expression by dietary anti-oxidants in THP-1 cells. Alpha lipoic acid activates Nrf2-mediated HO-1 expression [9] and curcumin activates expression of Nrf2-regulated HO-1, NQO1, glutathione cysteine ligase and ferritin [10]. However, other dietary anti-oxidants including ascorbic acid, alpha tocopherol, gamma tocopherol and resveratrol do not activate these genes in THP-1 cells (unpublished). In the present study, EGC activated HO-1 and GCLM, but not NQO1 or ferritin expression in THP-1 cells. EGCG minimally increases HO-1 mRNA expression in THP-1 cells (unpublished). EGCG activates HO-1 mRNA and protein expression in epithelial and endothelial cells at similar concentrations used in our study [14,18]. However, in contrast to our results, 100 μM EGC had no effect on HO-1 protein expression in endothelial cells, which may be due to differences in cell type. Nrf2 regulates EGCG-induced HO-1 in B lymphoblasts and epithelial cells [13,18]. Here, EGC also activated Nrf2 in THP-1 cells, and Nrf2 silencing significantly suppressed EGC-induced HO-1 expression, suggesting a key role for this transcription factor in this pathway. However, EGC did not activate ARE-driven reporter activity in HepG2 cells transfected with an ARE reporter [3].

Small differences in catechin structure result in wide-ranging biological effects. The presence of a 3-gallate group in EGCG and epicatechin gallate (ECG) results in pharmacokinetic differences. EGC is more bioavailable than either EGCG or ECG and oral administration of EGC results in a higher plasma anti-oxidant activity than that seen with gallated catechins [27], suggesting that it may be more important in vivo. In addition, cellular effects differ between gallated and non-gallated catechins. EGCG and ECG, but not EGC, bind to oestrogen receptors in MCF7 cells [21]. However, in the same study, EGCG, but not EGC or the gallated ECG, activated oestrogen receptor-mediated gene expression. Catechins also exhibit varying cytotoxic effects in different cells [3,22]. In addition, EGCG, but not EGC, (i) binds to the metastasis-associated laminin receptor in tumour cells, (ii) disrupts liposome membrane structure, (iii) inhibits CYP450 isoforms, (iv) inhibits proteasome activity and (v) suppresses Type I collagen and matrix metalloproteinase 1 production [20,23–26].

Several kinase pathways may regulate EGC-induced HO-1 and GCLM expression in THP-1 cells. ALA and curcumin induce HO-1 expression through p38 MAPK in THP-1 cells [9,10]. EGCG also activates Nrf2 expression in B lymphoblasts via p38 MAP kinase [13]. Here, the p38 MAPK inhibitor SB203580 did not significantly inhibit EGC-induced HO-1 mRNA expression, suggesting this pathway is not involved. EGC activates ERK MAP kinases in Ehrlich ascites tumour cells and HepG2 cells [3,22] and EGCG-induced HO-1 expression in epithelial and endothelial cells is regulated by ERK MAP kinase and PI3K [14,18]. However, we found that inhibitors of these pathways had no effect on EGC-induced HO-1 expression in THP-1 cells.

The present study implicates a role for PKCδ in Nrf2-regulated EGC-induced HO-1 expression. PKC regulates ARE-mediated gene expression in various cell types [10–12]. PKC phosphorylates Nrf2 on Ser40, enabling dissociation from its inhibitor Keap 1 [31]. EGCG activates PKC in neuronal, astroglioma and phaeochromacytoma cells [32–34]. However, PKC inhibitors did not affect EGCG-induced HO-1 expression in endothelial cells [14]. We have previously shown that a classical PKC regulates LPS-induced HO-1 expression in monocytes [11]. However, inhibitors of classical PKC had no significant effect in this study. However, PKCδ inhibitors suppressed EGC-induced HO-1 expression. In addition, Ro-31-8220 and rottlerin, but not Go6976 or LY333531, significantly inhibited EGC-induced GCLM expression, suggesting that PKCδ also regulates EGC-induced GCLM mRNA expression in THP-1 cells. Curcumin-induced HO-1 and GCLM expression are mediated via PKCδδ in human monocytes and THP-1 cells, suggesting a common pathway of activation for these polyphenols. However, p38 also regulates curcumin-induced HO-1 expression in THP-1 cells [10], suggesting some divergence in the two pathways. Based on our studies and other recent reports [10,12,35], PKCδ is emerging as an important member of the signalling pathways leading to Nrf2/ARE-mediated gene expression and its exact role warrants further investigation.

GCLM is the regulatory unit of the rate-limiting enzyme of glutathione synthesis and GCLM induction leads to an increase in glutathione in cells [36]. In the present study, EGC weakly increased GCLM mRNA expression in THP-1 cells. In contrast, EGC decreased glutathione concentrations in Ehrlich ascites tumour cells but this correlated with cell viability [22]. EGC was not cytotoxic at the concentrations used here, suggesting this may be a reason for the differences between the two studies. Interestingly, although NQO1 and ferritin are also regulated by the ARE and green tea extract activates NQO1 in vitro and in vivo [3,4], EGC did not activate these genes in THP-1 cells, suggesting that an alternative catechin may be responsible for the effect of green tea extracts on NQO1 expression or that this effect is cell type-specific.

In conclusion, EGC, in addition to EGCG, contributes to the activation of cellular anti-oxidants by catechins, including HO-1 and this, in turn, may partially be responsible for the beneficial effects of green tea extracts in vitro and in vivo. HO-1 plays a protective role in models of vascular diseases [37,38], suggesting that activation of HO-1 by EGC warrants further investigation. In addition, PKCδ regulates EGC-induced HO-1 expression, confirming that this kinase is emerging as an important member of the signalling pathways leading to HO-1.

Acknowledgment

The MRC is acknowledged for funding this study.

References

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