Epigallocatechin-3-Gallate (EGCG)-Inducible SMILE Inhibits STAT3-Mediated Hepcidin Gene Expression

Hepatic peptide hormone hepcidin, a key regulator of iron metabolism, is induced by inflammatory cytokine interleukin-6 (IL-6) in the pathogenesis of anemia of inflammation or microbial infections. Small heterodimer partner-interacting leucine zipper protein (SMILE)/CREBZF is a transcriptional corepressor of nuclear receptors that control hepatic glucose and lipid metabolism. Here, we examined the role of SMILE in regulating iron metabolism by inflammatory signals. Overexpression of SMILE significantly decreased activation of the Janus kinase 2-signal transducer and activator of transcription 3 (STAT3)-mediated hepcidin production and secretion that is triggered by the IL-6 signal in human and mouse hepatocytes. Moreover, SMILE co-localized and physically interacted with STAT3 in the nucleus in the presence of IL-6, which significantly suppressed binding of STAT3 to the hepcidin gene promoter. Interestingly, epigallocatechin-3-gallate (EGCG), a major component of green tea, induced SMILE expression through forkhead box protein O1 (FoxO1), as demonstrated in FoxO1 knockout primary hepatocytes. In addition, EGCG inhibited IL-6-induced hepcidin expression, which was reversed by SMILE knockdown. Finally, EGCG significantly suppressed lipopolysaccharide-induced hepcidin secretion and hypoferremia through induction of SMILE expression in mice. These results reveal a previously unrecognized role of EGCG-inducible SMILE in the IL-6-dependent transcriptional regulation of iron metabolism.


Introduction
Anemia of inflammation (AI), also called anemia of chronic disease, is the most common type of anemia, following iron deficiency anemia [1]. The major etiologies of AI are acute and chronic infections, autoimmune disorders, chronic kidney disease, malignancies and inflammation. The pathogenesis of AI is given through reduction in the lifespan of erythrocytes, impaired proliferation of erythroid progenitor cells and iron accumulation in the cells of the mononuclear phagocytic system by production of pro-inflammatory cytokines such as tumor necrosis factor α and interleukin-6 (IL-6) causing hypoferremia [1,2]. Iron is absorbed into the enterocytes, circulates through blood in a form of Antioxidants 2020, 9, 514 2 of 15 transferrin-bound iron and transports into another organs and tissues for utilization. In addition, iron recycling from macrophages for erythropoiesis is also required to maintain iron homeostasis [3,4]. Hepcidin, a hepatic peptide hormone, plays a key role in iron metabolism by binding to the cell surface the iron transporter ferroportin (FPN) and triggering its internalization and degradation [5,6]. It is reported that hypoxia-inducible factors decrease hepcidin expression during hypoxia and erythropoiesis [7,8], while hepcidin is mainly induced by activation of the IL-6/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in the pathogenesis of AI or microbial infections [4,9].
Epigallocatechin-3-gallate (EGCG), a type of polyphenol, is the richest and most potent catechin in green tea [10]. EGCG has many beneficial biological functions, such as anti-inflammatory, anti-carcinogenic and anti-oxidative, and neuroprotective functions and cholesterol-lowering effects, according to concentration, cell type and other factors. EGCG regulates numerous intracellular signaling pathways by interacting with membrane receptors, activating second messengers and modulating various transcription factors and metabolic enzymes and finally stimulating cellular protective systems [10,11]. In addition, it was reported that EGCG induces epithelial specific erythroblast transformation-specific transcription factor-1 expression, resulting in suppression of colorectal tumorigenesis [12]. In hepatoma cells, EGCG improves insulin sensitivity via reducing the levels of circulating free fatty acids, inflammation and lipotoxicity, mediated by glucose and palmitic acid [13]. However, whether there is a biological function of EGCG in iron metabolism is largely unknown.
Small heterodimer partner (SHP)-interacting leucine zipper protein (SMILE)/CREBZF belongs to the cAMP response element-binding and activating transcription factor (CREB/ATF) family of the basic region-leucine zipper (bZIP) family and could homodimerize, but it lacks the binding activity to DNA as a homodimer [14]. It is reported that curcumin, a major polyphenol found in turmeric, induces SMILE expression in hepatocytes and modulates expression of the endoplasmic reticulum stress-regulated target gene by suppressing the transcriptional activity of cAMP responsive element-binding protein H [15]. Interestingly, insulin-inducible SMILE suppresses hepatic gluconeogenesis by down-regulating the expression of gluconeogenic genes such as phosphoenolpyruvate carboxykinase and glucose-6-phosphatase and ameliorates hyperglycemia in diabetes [16]. In addition, ursodeoxychoic acid-inducible SMILE relieves hepatic lipid accumulation through inhibiting expression of liver X receptor α-mediated lipogenic genes [17]. Therefore, SMILE has been considered as a corepressor with a critical role in liver metabolism [18][19][20]. However, the role of SMILE in iron metabolism has not yet been elucidated. To this end, we examined the effect of SMILE on IL-6-dependent regulation of hepcidin expression in hepatocytes and further investigated the polyphenol EGCG-inducible SMILE effect on altering the iron metabolism that is triggered by an IL-6 signal.

Chemicals
Recombinant human IL-6 (R&D Systems, Inc., Minneapolis, MN, USA) and EGCG (Tocris Bioscience, Bristol, UK) were dissolved in deionized water for in vitro studies and in phosphate buffered saline (PBS) for in vivo studies. Lipopolysaccharide (O111:B4 LPS, L2630) was purchased from Sigma-Aldrich (St. Louis, MO, USA). We used 20 ng/mL of IL-6 or 100 µM of EGCG for in vitro studies and 100 mg/kg of EGCG or 1 mg/kg of LPS for in vivo studies, unless otherwise stated.

Animal Experiments
Female 8-week-old C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME, USA) were used for this study. For liver-specific FoxO1 knockout (FoxO1-LKO) studies, C57BL/6J mice containing floxed FoxO1 (FoxO1 f/f ) were obtained from Dr. Ronald A. DePinho (University of Texas MD Anderson Cancer Center). To produce the liver-specific FoxO1 knockout line (FoxO1-LKO), FoxO1 f/f animals were crossbred with C57BL/6J-Alb-Cre transgenic mice, which express Cre recombinase in hepatocytes under the control of the albumin promoter (Jackson Laboratory). All mice were acclimatized to a 12 h light/dark cycle at 22 ± 2 • C with free access to food and water in a specific pathogen-free facility.
To investigate the EGCG effect on LPS-dependent hepcidin expression, we carried out intraperitoneal injection of PBS (n = 5), EGCG (n = 5), LPS (n = 7) and EGCG plus LPS (n = 6). Mice were injected with LPS for 12 h after 2 h of the PBS and EGCG injection. All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Chonnam National University (CNU IACUC-H-2019-14).

Culture of Mouse Primary Hepatocytes
Mouse primary hepatocytes were isolated from wild-type (WT) and FoxO1-LKO mice (male or female, 20-25 g) by collagenase perfusion using a previously indicated method [25]. Primary hepatocytes were cultured in high-glucose DMEM supplemented with 10% FBS and 1% antibiotics. After attachment, the cells were treated with IL-6 and EGCG for 12 h.

Co-immunoprecipitation Analysis
Co-immunoprecipitation analysis was conducted using a FLAG immunoprecipitation kit (Sigma) according to the manufacturer's protocol. Briefly, HepG2 cells were transfected with vectors expressing HA-SMILE and FLAG-STAT3 and treated with IL-6 for 12 h. 200 µL of whole-cell lysates were prepared with IP lysis buffer (Thermo Fisher Scientific) and immunoprecipitated with anti-FLAG M2 affinity gel for 16 h. After the immunoprecipitation, proteins eluted with 3X FLAG peptide were analyzed with WB using anti-HA and anti-FLAG antibody (Santa Cruz Biotechnology).

Serum Iron and Hepcidin Measurement
All blood samples were taken using an intra-cardiac puncture of mice under anesthesia before killing. Serum iron was measured using a spectrophotometric method (TBA-200FR NEO). Serum hepcidin was measured using a Mouse Hepc (Hepcidin) ELISA kit (Elabscience, TX, USA), according to the manufacturer's protocols. For hepcidin measurement in cell culture media, Huh7 or AML12 cells were transfected with vector expressing SMILE and treated with IL-6 for 12 h after 2 h of serum starvation. Hepcidin levels were measured from the cell culture media using a Mouse Hepc (Hepcidin) ELISA kit (Elabscience) and a Human Hepcidin Quantikine ELISA kit (R&D Systems) according to the manufacturer's protocols.

RNA Interference
Small interfering RNAs against control (si-Con) and human SMILE (si-SMILE) were purchased from QIAGEN (Cat. No. 1027416). HepG2 cells were transfected with si-Con and si-SMILE using Lipofectamine RNAi MAX (Thermo Fisher Scientific) according to the manufacturer's instruction.

Cell Viability
HepG2 cells were treated with EGCG for 0 h, 6 h, 12 h and 24 h and incubated with MTT (final concentration: 0.5 mg/mL) for 2 h at 37 • C. The medium was removed, and the insoluble formazan product was dissolved in dimethyl sulfoxide. The absorbance was measured at 560 nm using a microplate reader (Biotek, Winooski, VT, USA). The absorbance of control cells was considered to represent 100% viability.

Statistical Analysis
Statistical analyses were performed using GraphPad Prism (GraphPad Software, CA, USA). The data are presented as the means ± SD or ± SEM. Statistical significance was estimated using a two-tailed Student's t-test. Differences were considered statistically significant at P < 0.05.

SMILE Inhibits IL-6-Mediated Hepcidin Production and Secretion
It is reported that IL-6 increases hepcidin expression through activation of the JAK2-STAT3 signaling pathway in response to inflammation [27]. To examine the effect of SMILE on IL-6-induced hepcidin expression in hepatocytes, we performed transient transfections with vectors expressing SMILE and hepcidin gene promoter in HepG2 cells, a human hepatoma cell line, and treated with IL-6. Interestingly, IL-6-induced promoter activity of human and mouse hepcidin gene was significantly decreased by SMILE (Figure 1a). In addition, SMILE inhibited hepcidin gene promoter activity by JAK2 and STAT3, respectively (Figure 1b,c). To further examine the inhibitory action of SMILE on IL-6-induced hepcidin gene transcription, we conducted adenoviral overexpression of GFP (Ad-GFP) or SMILE (Ad-SMILE) in HepG2 and AML12 cells, a non-transformed mouse liver cell line, treated with IL-6. Consistent with these results in transient transfection, overexpression of SMILE significantly suppressed induction of hepcidin mRNA expression by IL-6 stimulation in both cell lines (Figure 1d,e). Furthermore, we also found that SMILE inhibited IL-6-mediated hepcidin secretion in both human and mouse hepatoma cells (Figure 2a,d). Consequently, SMILE normalized decreased ferroportin expression by hepcidin (Figure 2b,c,e,f). These findings indicated that SMILE functions as a repressor of hepcidin transcriptional control by IL-6 signaling.

SMILE Represses Activity of The IL-6 Signaling Pathway via Inhibition of DNA-Binding of STAT3
To identify the molecular mechanism by which SMILE suppresses IL-6-mediated hepcidin expression, we first investigated subcellular localization of SMILE and STAT3 in HepG2 cells treated with IL-6. While STAT3 was mainly localized in cytoplasm in the absence of IL-6, SMILE was localized in the nucleus. However, they co-localized in the nucleus in the presence of IL-6 (Figure 3a,b). In addition, co-immunoprecipitation analysis demonstrated that IL-6 strongly increased the interaction between SMILE and STAT3 (Figure 3c). Moreover, chromatin immunoprecipitation (ChIP) assay showed that STAT3 binds to a STAT3-responsive element (STAT3-RE) on the hepcidin promoter in the presence of IL-6, which was almost entirely abrogated by overexpression of SMILE (Figure 3d). These results suggest that SMILE disrupts IL-6-mediated hepcidin expression by inhibiting STAT3 binding to the hepcidin promoter.
representation (c) in Huh7 cells. Hepcidin secretion (d), FPN expression (e) and graphical representation (f) in AML12 cells. All gels for western blot analysis in (b,e) were run under the same experimental conditions including equal amounts (100 μg) of protein. The independent experiments were repeated at least three times. The values are presented as means ± SD. ns; not significant. * P < 0.05, ** P < 0.01 using two-tailed Student's t-test.

SMILE Represses Activity of The IL-6 Signaling Pathway via Inhibition of DNA-binding of STAT3
To identify the molecular mechanism by which SMILE suppresses IL-6-mediated hepcidin expression, we first investigated subcellular localization of SMILE and STAT3 in HepG2 cells treated with IL-6. While STAT3 was mainly localized in cytoplasm in the absence of IL-6, SMILE was localized in the nucleus. However, they co-localized in the nucleus in the presence of IL-6 ( Figure  3a,b). In addition, co-immunoprecipitation analysis demonstrated that IL-6 strongly increased the interaction between SMILE and STAT3 (Figure 3c). Moreover, chromatin immunoprecipitation (ChIP) assay showed that STAT3 binds to a STAT3-responsive element (STAT3-RE) on the hepcidin promoter in the presence of IL-6, which was almost entirely abrogated by overexpression of SMILE (Figure 3d). These results suggest that SMILE disrupts IL-6-mediated hepcidin expression by inhibiting STAT3 binding to the hepcidin promoter.  showing interaction between SMILE and STAT3. HepG2 cells were transfected with vectors encoding HA-SMILE and FLAG-STAT3 and treated with IL-6 for 12 h. Gels for western blot analysis were run under the same experimental conditions. (d) ChIP assay showing inhibitory effects of SMILE on STAT3 binding activity to hepcidin promoter. HepG2 cells were transfected with vectors encoding hepcidin promoter, HA-SMILE and FLAG-STAT3 and then treated with IL-6 for 12 h. Soluble chromatin was immunoprecipitated using anti-FLAG antibody. ChIP signals were measured using Q-PCR. STAT3-RE, STAT3-response element. The values are presented as means ± SD. ** P < 0.01, *** P < 0.001 using two-tailed Student's t-test.

EGCG Increases FoxO1 and SMILE Expression
The transcriptional repressive activity of SMILE is modulated by natural polyphenolic compounds such as curcumin, a major active component of turmeric, and resveratrol, a polyphenol found in red wine [15,20]. However, the effect of EGCG, a major polyphenol in green tea, on SMILE expression has not yet been elucidated. To this end, we examined SMILE gene promoter activity and mRNA expression in hepatocytes treated with EGCG for 12 h. As anticipated, we found that both human and mouse SMILE gene promoter activity were significantly induced by EGCG treatment (Figure 4a). In addition, mRNA expression of SMILE and forkhead box protein O1 (FoxO1), a downstream transcription factor of EGCG [28], was continuously increased with time in EGCG-treated AML12 cells (Figure 4b). Moreover, FoxO1 and SMILE mRNA levels were induced at high doses of EGCG (>100 µM) (Figure 4c). Indeed, FoxO1 and SMLE protein levels were induced in AML12 cells treated with 100 µM of EGCG for 12 h, while p-FoxO1 levels were unaltered at this condition (Figure 4d,e). Finally, to examine a cytotoxic effect of EGCG, we treated HepG2 cells with 100 µM of EGCG in a time-dependent manner and found that EGCG had no cytotoxic effect on HepG2 cells with 24 h of treatment (Figure 4f). transfected with vectors encoding hepcidin promoter, HA-SMILE and FLAG-STAT3 and then treated with IL-6 for 12 h. Soluble chromatin was immunoprecipitated using anti-FLAG antibody. ChIP signals were measured using Q-PCR. STAT3-RE, STAT3-response element. The values are presented as means ± SD. ** P < 0.01, *** P < 0.001 using two-tailed Student's t-test.

EGCG Increases FoxO1 and SMILE Expression
The transcriptional repressive activity of SMILE is modulated by natural polyphenolic compounds such as curcumin, a major active component of turmeric, and resveratrol, a polyphenol found in red wine [15,20]. However, the effect of EGCG, a major polyphenol in green tea, on SMILE expression has not yet been elucidated. To this end, we examined SMILE gene promoter activity and mRNA expression in hepatocytes treated with EGCG for 12 h. As anticipated, we found that both human and mouse SMILE gene promoter activity were significantly induced by EGCG treatment (Figure 4a). In addition, mRNA expression of SMILE and forkhead box protein O1 (FoxO1), a downstream transcription factor of EGCG [28], was continuously increased with time in EGCGtreated AML12 cells (Figure 4b). Moreover, FoxO1 and SMILE mRNA levels were induced at high doses of EGCG (>100 μM) (Figure 4c). Indeed, FoxO1 and SMLE protein levels were induced in AML12 cells treated with 100 μM of EGCG for 12 h, while p-FoxO1 levels were unaltered at this condition (Figure 4d,e). Finally, to examine a cytotoxic effect of EGCG, we treated HepG2 cells with 100 μM of EGCG in a time-dependent manner and found that EGCG had no cytotoxic effect on HepG2 cells with 24 h of treatment (Figure 4f).  The values are presented as means ± SD. * P < 0.05, *** P < 0.001 using two-tailed Student's t-test.

FoxO1 Increases SMILE Expression
Next, we tested if FoxO1 regulates SMILE gene transcription using wild-type (WT) and liver-specific FoxO1 knockout (FoxO1-LKO) hepatocytes. Overexpression of FoxO1 significantly induced SMILE gene promoter activity and mRNA expression (Figure 5a,b). In addition, EGCG treatment increased SMILE protein levels in WT hepatocytes but not in FoxO1-LKO hepatocytes (Figure 5c,d). Moreover, close investigation of the SMILE gene promoter revealed that a putative FoxO1 binding sequence was conserved in human and mouse (Figure 5e). Consistent with this investigation, FoxO1 was significantly recruited to the FoxO1-binding region of the SMILE gene promoter, as investigated using ChIP assay ( Figure 5f). These findings indicate that FoxO1 is a direct transcriptional regulator of SMILE gene in the presence of EGCG.
Next, we tested if FoxO1 regulates SMILE gene transcription using wild-type (WT) and liverspecific FoxO1 knockout (FoxO1-LKO) hepatocytes. Overexpression of FoxO1 significantly induced SMILE gene promoter activity and mRNA expression (Figure 5a,b). In addition, EGCG treatment increased SMILE protein levels in WT hepatocytes but not in FoxO1-LKO hepatocytes (Figure 5c,d). Moreover, close investigation of the SMILE gene promoter revealed that a putative FoxO1 binding sequence was conserved in human and mouse (Figure 5e). Consistent with this investigation, FoxO1 was significantly recruited to the FoxO1-binding region of the SMILE gene promoter, as investigated using ChIP assay (Figure 5f). These findings indicate that FoxO1 is a direct transcriptional regulator of SMILE gene in the presence of EGCG. ChIP signals were measured using Q-PCR. The independent experiments were repeated at least twice. The values are presented as means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 using two-tailed Student's t-test.

EGCG Attenuates IL-6-induced Hepcidin Expression through SMILE
In order to investigate the role of SMILE in EGCG action on regulation of hepcidin expression, we first examined the effect of EGCG on IL-6-mediated hepcidin expression in mouse primary hepatocytes. As expected, IL-6 significantly increased hepcidin mRNA expression, which was almost entirely reduced by EGCG (Figure 6a). Next, to demonstrate the role of SMILE in inhibiting hepcidin expression by EGCG, we treated HepG2 cells with IL-6 and EGCG after transfection of vector- showing FoxO1 binding to SMILE promoter. HepG2 cells were transfected with vectors encoding SMILE promoter and FLAG-FoxO1. Soluble chromatin was immunoprecipitated using anti-FLAG antibody. ChIP signals were measured using Q-PCR. The independent experiments were repeated at least twice. The values are presented as means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 using two-tailed Student's t-test.

EGCG Attenuates IL-6-Induced Hepcidin Expression through SMILE
In order to investigate the role of SMILE in EGCG action on regulation of hepcidin expression, we first examined the effect of EGCG on IL-6-mediated hepcidin expression in mouse primary hepatocytes. As expected, IL-6 significantly increased hepcidin mRNA expression, which was almost entirely reduced by EGCG (Figure 6a). Next, to demonstrate the role of SMILE in inhibiting hepcidin expression by EGCG, we treated HepG2 cells with IL-6 and EGCG after transfection of vector-carrying hepcidin gene promoter and SMILE knockdown using small interfering RNAs for SMILE (si-SMILE). SMILE mRNA and protein levels were significantly decreased by si-SMILE (Figure 6b,c). Consequently, the inhibitory effect of EGCG on IL-6-induced hepcidin promoter activity was significantly blocked by SMILE knockdown (Figure 6d). In addition, we observed that SMILE knockdown considerably reversed the effect of the EGCG treatment on IL-6-mediated induction of hepcidin mRNA expression (Figure 6e). These results demonstrated that SMILE is required for EGCG action on the IL-6-dependent transcriptional regulation of hepcidin gene expression.
Consequently, the inhibitory effect of EGCG on IL-6-induced hepcidin promoter activity was significantly blocked by SMILE knockdown (Figure 6d). In addition, we observed that SMILE knockdown considerably reversed the effect of the EGCG treatment on IL-6-mediated induction of hepcidin mRNA expression (Figure 6e). These results demonstrated that SMILE is required for EGCG action on the IL-6-dependent transcriptional regulation of hepcidin gene expression.

EGCG Inhibits LPS-induced Hepcidin Expression and Hypoferremia in Mice
Based on these findings in cultured cells, we next tested if EGCG suppresses hepatic hepcidin production and secretion by lipopolysaccharide (LPS), a key factor of IL-6 production [29]. As expected, intraperitoneal injection of LPS into mice caused a significant induction of IL-6 and hepcidin levels in liver and serum and a reduction of serum iron levels (Figure 7a-d). However, EGCG treatment significantly increased hepatic FoxO1 and SMILE expression (Figure 7e-h) and reversed the LPS effect on hepcidin and serum iron levels without significant changes in the IL-6 expression (Figure 7a-c). Taken together, these results suggest that EGCG-inducible SMILE acts as an important negative regulator for the IL-6-depedent induction of hepcidin expression (Figure 8).

EGCG Inhibits LPS-Induced Hepcidin Expression and Hypoferremia in Mice
Based on these findings in cultured cells, we next tested if EGCG suppresses hepatic hepcidin production and secretion by lipopolysaccharide (LPS), a key factor of IL-6 production [29]. As expected, intraperitoneal injection of LPS into mice caused a significant induction of IL-6 and hepcidin levels in liver and serum and a reduction of serum iron levels (Figure 7a-d). However, EGCG treatment significantly increased hepatic FoxO1 and SMILE expression (Figure 7e-h) and reversed the LPS effect on hepcidin and serum iron levels without significant changes in the IL-6 expression (Figure 7a-c). Taken together, these results suggest that EGCG-inducible SMILE acts as an important negative regulator for the IL-6-depedent induction of hepcidin expression (Figure 8). Serum hepcidin levels (c). IL-6, FoxO1 and SMILE mRNA levels in liver (d-f). Western blot analysis showing FoxO1 and SMILE expression (g). Graphical representation of FoxO1 (right) and SMILE (left) (h). mRNA levels were measured using Q-PCR. Gels for western blot analysis were run under the same experimental conditions. The values are presented as means ± SEM in a-f and ± SD in h. ns; not significant (Con vs. EGCG). * P < 0.05, ** P < 0.01, *** P < 0.001 using two-tailed Student's t-test. . mRNA levels were measured using Q-PCR. Gels for western blot analysis were run under the same experimental conditions. The values are presented as means ± SEM in (a-f) and ±SD in h. ns; not significant (Con vs. EGCG). * P < 0.05, ** P < 0.01, *** P < 0.001 using two-tailed Student's t-test. Serum hepcidin levels (c). IL-6, FoxO1 and SMILE mRNA levels in liver (d-f). Western blot analysis showing FoxO1 and SMILE expression (g). Graphical representation of FoxO1 (right) and SMILE (left) (h). mRNA levels were measured using Q-PCR. Gels for western blot analysis were run under the same experimental conditions. The values are presented as means ± SEM in a-f and ± SD in h. ns; not significant (Con vs. EGCG). * P < 0.05, ** P < 0.01, *** P < 0.001 using two-tailed Student's t-test.

Discussion
Iron homeostasis is maintained by the tight regulation of dietary iron absorption from enterocytes and release of iron from macrophages and hepatocytes. Hepcidin, an iron homeostatic hormone, is regulated through two major signaling pathways: inflammatory cytokine IL-6-dependent activation of JAK2-STAT3 signaling and excessive iron-mediated activation of bone morphogenetic protein-6 (BMP-6)-SMADs signaling [30]. Recently, we reported that SHP is a transcriptional corepressor of BMP-6-SMAD1/5/8-activated hepcidin expression triggered by an iron signal [31]. However, transcriptional corepressors of STAT3 in IL-6-mediated hepcidin induction have not yet been elucidated. In this study, we identified SMILE as a transcriptional corepressor of STAT3 suppressing hepcidin production and eventual hypoferremia. Indeed, the ChIP assay revealed that SMILE directly interacted with STAT3 and almost entirely blocked IL-6-induced STAT3 binding to hepcidin promoter. Previously, we also demonstrated that in response to Salmonella typhimurium, nuclear receptor estrogen-related receptor γ (ERRγ), known as a SMILE-interacting protein [20], mediates IL-6-induced hepcidin expression in mouse livers [22]. These results suggest that SMILE plays a critical role as transcriptional corepressor of the IL-6 signal in hepatocytes by inhibiting transcriptional activity of both STAT3 and ERRγ on hepcidin expression. In addition, it is reported that SMILE controls liver regeneration by inhibiting STAT3 activation in carbon tetrachloride-treated mice [32], indicating that the mechanism by which SMILE suppresses STAT3 activation could be applied for other cell physiologies.
EGCG, a major polyphenolic component of green tea, is known to have antioxidant effects by inducing expression of antioxidant enzymes including superoxide dismutase (SOD) and glutathione peroxidase. Interestingly, however, EGCG acts as a pro-oxidant by producing reactive oxygen species (ROS), such as hydrogen peroxide and hydroxyl radicals, at high concentrations (>50 µM) [10]. For example, EGCG activated AMP-dependent kinase (AMPK) by inducing ROS-dependent CaMKKβ and liver kinase B1 (LKB1) activity in hepatocytes, adipocytes and endothelial cells as well as cancer cells [33,34]. In this study, we found that EGCG increases SMILE gene expression at high concentration in hepatocytes, suggesting that the pro-oxidant effect of EGCG contributes to inducing SMILE expression. These findings are further supported by a previous report showing that curcumin, a polyphenol, significantly induces SMILE expression by activating the LKB1-AMPK pathway [15]. In addition, we showed that EGCG increases FoxO1 expression, resulting in induction of SMILE in hepatocytes. These findings are further supported by the results showing that EGCG-induced SMILE expression was almost entirely blocked in FoxO1-LKO hepatocytes. Recently, it is also reported that EGCG significantly increased FoxO1 expression in liver [28]. These results suggest that FoxO1 plays a critical role in EGCG-mediated SMILE induction. However, the antioxidant function of EGCG is reported to inhibit FoxO1 transcriptional activity by activating Akt, which phosphorylates FoxO1 in adipocytes [35]. In addition, it is reported that EGCG downregulates FoxO1 without affecting insulin signaling in hepatocytes [36]. These results indicate that FoxO1 activity and cellular redox state are intrinsically linked to each other because ROS modulates FoxO1 activity at the transcriptional and posttranslational levels, which in turn regulates expression of antioxidant proteins, such as SOD, peroxiredoxins and selenoprotein P, which contribute to cellular antioxidant defense [37]. Therefore, these findings suggest that FoxO1 might act as a cellular redox sensor linking between anti-and pro-oxidant functions of EGCG. However, the detailed mechanisms by which EGCG regulates FoxO1 in hepatocytes needs to be further characterized.
AI is a form of anemia found in several disease states such as chronic infections, chronic immune activation and malignancies. These conditions produce massive induction of IL-6, which elevates hepcidin production and secretion from the liver, which in turn promotes degradation of FPN and eventual hypoferremia due to reduced access of iron to the circulation [6]. Therefore, pharmacological control of IL-6-dependent hepcidin expression can provide a therapeutic approach to ameliorating AI. In the current study, we elucidated that SMILE acts as a transcriptional corepressor of IL-6-dependent hepcidin expression by inhibiting STAT3 binding to hepcidin promoter, leading to reduced hepcidin secretion from hepatocytes ( Figure 8). In addition, EGCG increased SMILE expression through FoxO1 induction, which reversed the IL-6 effect on hepcidin production and eventual hypoferremia in mice. Taken together, these findings suggest that EGCG that induces SMILE expression would have potential for treating AI.