Protective effect of epigallocatechin-3-gallate on ischemia/reperfusion-induced injuries in the heart: STAT1 silencing flavonoid

Abstract

The beneficial effect of naturally occurring flavonoids in health is believed to be due to their strong antioxidant activity. However, recent laboratory evidence indicates the involvement of a more specific action. Here, we present evidence that, among a number of catechins present in green tea extract, only epigallocatechin-3-gallate (EGCG) exerts a strong inhibitory action on interferon-γ-elicited activation of signal transducer and activator of transcription 1 (STAT1). Protective action of EGCG in ischemia/reperfusion injury in the heart and the molecular mechanism of action, which has nothing to do with its anti-oxidant capacity are described.

Introduction

Green tea is one of the most ancient and widely consumed health drink in the world and has for long time been used as the popular remedy against common pathologies such as headaches, body aches and pains, bad digestion, mild depression and detoxification, and an elixir of long life [2]. Most of the beneficial effects of drinking green tea has been attributed to antioxidant properties of catechins present in it such as epigallocatechin-3-gallate (EGCG, a main polyphenol present in green tea leaves), epicatechin (EC), epigallocatechin (EGC) and epicatechin-3-gallate (ECG) (Fig. 1) [16]. An increasing body of literature evidence furthermore indicates that beneficial action of green tea drinking might be extended to a number of pathologies correlated to inflammation [4, 5, 11, 15, 17, 20, 22, 24, 26, 27, 35, 38]. Since pathogenesis of inflammatory diseases seems not to be exclusively correlated to redox state unbalance, a theory that the beneficial effect of green tea drinking might not simply be achieved by its strong antioxidant properties is emerging.

Fig. 1.

Structure of catechins present in green tea leaves

In 2001, we reported that EGCG but not other catechins present in green tea extract efficiently inhibits interferon-γ (IFN-γ)-elicited phosphorylation of tyrosine residue 701 and DNA-binding activity of signal transducer and activator of transcription 1 (STAT1) in different human carcinoma-derived cell lines [21]. STAT1 almost exclusively mediates the action of macrophage-derived IFN-γ, playing a pivotal role at the early phase of inflammation in regulating the expression of a number of inflammation-related genes [3].

Intracellular pathways activated upon recognition of IFN-γ by its specific receptors (IFNGR1/2) are well elucidated [1, 8]. Briefly, upon dimerisation of IFNGR1/2 followed by IFN-γ binding (Fig. 2b), tyrosine-autophosphorylation of tyrosine kinase 1 and 2 (JAK1/2) occurs. Successively, following SH2 domain-mediated recognition of activated JAK1/2 by IFNGR1/2 JAK1/2 tyrosine-phosphorylate IFNGR1/2 and resting STAT1 present in cytoplasm (Fig. 2b). Phosphorylated dimer of STAT1 enters in the nucleus and binds specifically to regulatory sequences to modulate the expression of target genes (Fig. 2d). Furthermore, up-regulation of JAK/STAT pathway is mediated at least by three different proteins: signal-transducing adapter molecules (STAMs) [19], STAT-interacting protein (StIP) and SH2B/Lnk/APS family (Fig. 2b). Negative regulator of JAK/STAT pathway includes suppressors of cytokine signaling (SOCS), protein inhibitors of activated STAT (PIAS) and protein tyrosine phosphatases (PTPs) such as SHP-1 (Fig. 2c) [6]. Recently discovered sumoylation (Fig. 2c) [10] seems not only to modify PIAS proteins [9[E9]] but also STATs themselves [29]. Despite some reports indicating the critical involvement of the change in redox state in modulating STAT1 activation [7, 12, 23, 25], results obtained by us showing that among all catechins present in green tea extract only EGCG is able to down-regulate STAT1 activation suggest that EGCG may not exert its activity by simply using its antioxidant capacity.

Fig. 2.

JAK1/2-STAT1 pathway. a Normal condition, b activation of IFN-γ-elicited STAT1 and it’s up-regulation, c down-regulation of STAT1, d induction of STAT1-dependent genes

A question arises: which is the molecular mechanism of action of EGCG in inhibiting STAT1 activation? Activated tyrosine-phosphorylated JAK1 and 2 are reported to deeply be involved in STAT1 and STAT3 signal transduction pathways [18]. Considering the fact that EGCG fails to inhibit intereukin-6-elicited activation of STAT3 [21], the possibility that EGCG targets JAK1/2 in inhibiting STAT1 activation is scarce. Involvement of tyrosine phosphatases in EGCG’s STAT1 inhibitory action also seems to be quite improbable, since they may regulate the tyrosine-phosphorylated state of a wide spectrum of proteins including STAT3. SOCS1/3 down-regulate STATs activation, therefore, they could be a target of EGCG. However, the notion that induction of SOCS expression is tightly regulated by STAT1 activation itself renders this possibility unlikely.

We hypothesize that the STAT1-inhibiting action of EGCG is correlated to its capacity to specifically interact with STAT1 protein. To evaluate this hypothesis we analyzed, by Surface Plasmon Resonance (SPR) method, the interaction between immobilized STAT1 and EGCG or ECG or quercetin, a strong antioxidant flavonoid without anti-STAT1 activity. Not only SPR analysis indicated that only EGCG directly interacts with STAT1 protein (Fig. 3) but also Kd value of EGCG toward STAT1 (640 nM) is far less than that of ECG (5.7 mM) and quercetin (43 μM), indicating that inhibitory action of EGCG on STAT1 DNA-binding activity is, at least partly, due to its capacity to strongly and directly interact with STAT1 protein, the notion in line with computer modeling analysis indicating that EGCG may strongly interact with STAT1 at the site near to SH2 domain, playing a critical role in phospho-tyrosine recognition (Fig. 4).

Fig. 3.

Sensograms indicating the dose-dependent interaction of EGCG with immobilized STAT1. Kd value was estimated by analyzing sensograms

Fig. 4.

Three-dimensional structure of STAT1. SH2 domain and putative EGCG binding region are indicated

To evaluate the possibility that only EGCG but not the other flavonoids without anti-STAT1 activity may act beneficially in animal model of diseases in which hyperactivation of STAT1 plays a deleterious role, we examined the effect of EGCG and quercetin in rat model of ischemia/reperfusion (I/R) injury in the heart. I/R-induced cardiomyocyte death by apoptosis is a pivotal event and STAT1 activation is deeply involved in it [32]. EGCG but not quercetin inhibits efficiently I/R-induced activation of STAT1 and protects heart from I/R-induced tissue and biochemical and functional damages [34]. Since EGCG and quercetin are equally strong antioxidant in this animal model, protective action of EGCG should be ascribed to its capacity to inhibit STAT1 activation. Furthermore, the aforementioned pathologies against which green tea/EGCG show protective action are also characterized by altered activation of STAT1 (Table 1), rendering it quite likely that anti-STAT1 capacity of green tea/EGCG is deeply involved in their beneficial action.

Table 1.

Some pathologies in which STAT1 plays a critical rule

Pathologies	References
Cardiovascular disease	[32]
UV-induced skin damage	[40]
Cerebral I/R injuries	[33]
Neurodegenerative disease	[13, 31]
Obesity	[30, 39]
Cancer	[14]
Diabetes	[28]
Atherosclerosis	[36]
Inflammation	[3]
Ageing	[37]

In conclusion, who knows if wisdom of ancient people preferring green tea over myriads of other plant-derived products (one might recall that tea, Camellia sinensis L., is the most drunk beverage only after water in the world) is bringing us to develop a new strategy to prevent and/or treat a number of inflammatory diseases!