Formononetin inhibits lipopolysaccharide-induced release of high mobility group box 1 by upregulating SIRT1 in a PPARδ-dependent manner

Background The release of high mobility group box 1 (HMGB1) induced by inflammatory signals acts as a cellular alarmin to trigger a chain of inflammatory responses. Although the inflammatory actions of HMGB1 are well studied, less is known about the therapeutic agents that can impede its release. This study investigated whether the isoflavonoid formononetin can modulate HMGB1 release in cellular inflammatory responses. Methods RAW264.7 murine macrophages were exposed to lipopolysaccharide (LPS) in the presence or absence of formononetin. The levels of HMGB1 release, sirtuin 1 (SIRT1) expression, and HMGB1 acetylation were analyzed by immunoblotting and real-time polymerase chain reaction. The effects of resveratrol and sirtinol, an activator and inhibitor of SIRT1, respectively, on LPS-induced HMGB1 release were also evaluated. Results Formononetin modulated cellular inflammatory responses by suppressing the release of HMGB1 by macrophages exposed to LPS. In RAW264.7 cells, formononetin significantly attenuated LPS-induced release of HMGB1 into the extracellular environment, which was accompanied by a reduction in its translocation from the nucleus to the cytoplasm. In addition, formononetin significantly induced mRNA and protein expression of SIRT1 in a peroxisome proliferator-activated receptor δ (PPARδ)-dependent manner. These effects of formononetin were dramatically attenuated in cells treated with small interfering RNA (siRNA) against PPARδ or with GSK0660, a specific inhibitor of PPARδ, indicating that PPARδ is involved in formononetin-mediated SIRT1 expression. In line with these effects, formononetin-mediated inhibition of HMGB1 release in LPS-treated cells was reversed by treatment with SIRT1-targeting siRNA or sirtinol, a SIRT1 inhibitor. By contrast, resveratrol, a SIRT1 activator, further potentiated the inhibitory effect of formononetin on LPS-induced HMGB1 release, revealing a possible mechanism by which formononetin regulates HMGB1 release through SIRT1. Furthermore, modulation of SIRT1 expression by transfection of SIRT1- or PPARδ-targeting siRNA significantly counteracted the inhibitory effects of formononetin on LPS-induced HMGB1 acetylation, which was responsible for HMGB1 release. Discussion This study shows for the first time that formononetin inhibits HMGB1 release by decreasing HMGB1 acetylation via upregulating SIRT1 in a PPARδ-dependent manner. Formononetin consequently exhibits anti-inflammatory activity. Identification of agents, such as formononetin, which can block HMGB1 release, may help to treat inflammation-related disorders.

chain reaction. The effects of resveratrol and sirtinol, an activator and inhibitor of SIRT1, respectively, on LPS-induced HMGB1 release were also evaluated.

Results.
Formononetin modulated cellular inflammatory responses by suppressing the release of HMGB1 by macrophages exposed to LPS. In RAW264.7 cells, formononetin significantly attenuated LPS-induced release of HMGB1 into the extracellular environment, which was accompanied by a reduction in its translocation from the nucleus to the cytoplasm. In addition, formononetin significantly induced mRNA and protein expression of SIRT1 in a peroxisome proliferator-activated receptor δ (PPARδ)-dependent manner. These effects of formononetin were dramatically attenuated in cells treated with small interfering RNA (siRNA) against PPARδ or with GSK0660, a specific inhibitor of PPARδ, indicating that PPARδ is involved in formononetin-mediated SIRT1 expression. In line with these effects, formononetin-mediated inhibition of HMGB1 release in LPS-treated cells was reversed by treatment with SIRT1targeting siRNA or sirtinol, a SIRT1 inhibitor. By contrast, resveratrol, a SIRT1 activator, further potentiated the inhibitory effect of formononetin on LPS-induced HMGB1 release, revealing a possible mechanism by which formononetin regulates HMGB1 release through SIRT1.

INTRODUCTION
High mobility group box 1 (HMGB1), a non-histone DNA-binding protein, is a well-conserved nuclear protein that has multiple functions depending on its cellular location. In the nucleus, HMGB1 plays roles in DNA replication, transcription, recombination, and maintenance of chromosome stability (Stros, 2010). However, when released by stressed cells, HMGB1 plays a critical role in the inflammatory response and is a late proinflammatory marker in many diseases including sepsis (Andersson & Harris, 2010;Abdulahad et al., 2010;Sims et al., 2010;Stros, 2010;Zhang et al., 2009). Recent reports show that post-translational modifications of HMGB1, such as acetylation and phosphorylation, are associated with its translocation and release in inflammatory cells exposed to pathogen-related molecules including lipopolysaccharide (LPS) (Bonaldi et al., 2003;Ito et al., 2007;Youn & Shin, 2006). The importance of extracellular HMGB1 in the inflammatory response has been demonstrated in inflammatory conditions; a neutralizing anti-HMGB1 antibody and HMGB1 antagonists attenuate cellular damage induced by inflammation (Wang et al., 1999;Davé et al., 2009). These reports indicate the importance of pathways or molecules that regulate HMGB1 release from activated inflammatory cells.
Furthermore, we showed that activation of PPARδ and PPARγ by specific ligands induces SIRT1 expression in human coronary artery endothelial cells (Kim et al., 2012) and RAW264.7 cells (Hwang et al., 2014). Thus, we hypothesized that formononetin may modulate cellular inflammatory responses by inhibiting HMGB1 release via upregulation of SIRT1. Here, we show that formononetin reduces LPS-induced HMGB1 acetylation by upregulating SIRT1 in a PPARδdependent manner, thereby blocking HMGB1 release into the extracellular environment. determined by a hemocytometer. For MTT assay, the cells were incubated for final 2h in medium containing MTT solution (0.1 mg/ml). Following removing the medium, the absorbance at 570 nm was measured using formazan crystals solution dissolved in acidified isopropanol.

Western blot analysis
Protein levels were analyzed by immunoblot as described previously (Hwang et al., 2015).
Briefly, RAW264.7 cells washed with ice-cold PBS were lysed and aliquots of the resulting whole-cell lysates or conditioned media were analyzed by immunoblot with indicated antibodies.

Measurement of extracellular HMGB1
Levels of HMGB1 released into culture media were determined using a previously described method (Hwang et al., 2012). Briefly, the relative amounts of HMGB1 were determined in the conditioned media of RAW264.7 cells treated with the indicated reagents for the indicated durations. The 80% ice-cold acetone was used to precipitate the proteins in the conditioned media. After centrifugation, the pellets were obtained and washed with 80% ice-cold acetone.
Following resuspension in SDS-PAGE sample buffer, the levels of HMGB1 released into culture media were analyzed by immunoblot.

Fractionation of nuclear and cytoplasmic proteins
Cellular fractions were prepared using a previously described method (Hwang et al., 2015). containing cytosolic fraction was obtained and the resulting pellet was lysed by a PRO-PREP Protein Extraction Solution. Following standing for 20 min on ice, the nuclear fraction (supernatant) was obtained by centrifugation.

Reporter gene assay
The luciferase construct containing mouse SIRT1 promoter was a gift from Dr. Toren Finkel (NIH, MD, USA). The promoter activity of SIRT1 was measured as described previously (Hwang et al., 2014). Briefly, 1 μg of the SIRT1 luciferase reporter plasmid and 0.5 μg of pSV β-Gal (SV40 β-galactosidase expression vector) were introduced into RAW264.7 cells by SuperFect reagent (Qiagen, Valencia, CA, USA). After 38 h, the cells were treated with GSK0660 for 30 min prior to stimulation with formononetin for 24 h. Then, the cells were lysed by adding the luciferase reporter lysis buffer (Promega) and then aliquots of the lysates were used to determine luciferase activity.

Co-immunoprecipitation
Immunoprecipitation was performed using a previously described method (Hwang et al., 2015).
Briefly, the protein G Sepharose was added to whole-cell lysates to pre-clear and then the precleared lysates were mixed with 1 μg of an anti-HMGB1 antibody. After incubation overnight at 4°C, the mixture was reacted with protein G Sepharose for 4 h. Mixtures were extensively washed with PBS and then boiled in gel-loading buffer. The immunoblot analysis was performed using an anti-acetyl-lysine antibody (Santa Cruz Biotechnology).

Statistical analysis
Data are expressed as means ± standard error (SE). The significance in statistical analysis was evaluated by a one-way ANOVA, followed by Tukey-Kramer test. A value of p<0.05 was considered statistically significant.

Formononetin inhibits LPS-induced release of HMGB1 in both murine and human macrophages
To determine the optimal concentration of formononetin, we determined the viability of The level of HMGB1 released into culture media was increased in RAW264.7 cells exposed to LPS, and this increase was markedly reduced in the presence of herbal compounds. In particular, the effects of formononetin and curcumin were superior to those of genistein ( Figure 1C). By contrast, neither LPS nor formononetin affected the expression level of endogenous HMGB1 ( Figure 1D). Similar results were obtained from the human primary macrophages, indicating that formononetin affects LPS-induced HMGB1 release, but not HMGB1 expression, in both murine and human macrophages ( Figure 1E).
HMGB1 is reported to translocate from the nucleus into the cytoplasm in response to inflammatory signals such as LPS (Bonaldi et al., 2003;Youn & Shin, 2006). Therefore, we exposed to LPS, this was significantly suppressed by formononetin ( Figure 1F). However, the expression of HMGB1 mRNA was not affected by formononetin in cells treated with or without LPS ( Figure 1G). These results suggest that formononetin decreases the release of HMGB1 by inhibiting its translocation rather than expression in LPS-primed RAW264.7 cells. µM formononetin ( Figure 2B). Similarly, the mRNA level of SIRT1 was time-dependently upregulated by formononetin ( Figure 2C). In addition, the inhibitory effect of formononetin on the LPS-stimulated release of HMGB1 was significant at 6 h pretreatment and the maximal inhibitory effect of formononetin was observed with a pre-treatment of 24 h which corresponds to the time of maximal induction of SIRT1 expression upon formononetin treatment ( Figure 2D).
To elucidate the mechanisms by which formononetin induces SIRT1 expression, we determined the effects of Act D (a RNA synthesis inhibitor) and CHX (a protein synthesis inhibitor). While formononetin significantly increased mRNA expression of SIRT1, this was significantly reduced in the presence of Act D or CHX ( Figure 2E). These results indicate that de novo synthesis of mRNA as well as of proteins that act on the SIRT1 gene promoter is indispensable for the induction of SIRT1 mRNA by formononetin in RAW264.7 cells.

Formononetin induces SIRT1 expression via PPARδ in RAW264.7 cells
To further examine the mechanisms by which formononetin upregulates SIRT1 expression, we evaluated the role of PPARδ, a nuclear receptor that regulates the transcription of a variety of target genes (Kidani & Bensinger, 2012;Mangelsdorf et al., 1995), by transfecting RAW264.7 cells with siRNA against PPARδ. The protein level of PPARδ was reduced in cells transfected with PPARδ-targeting siRNA, but not in cells transfected with control siRNA composed of a pool of nonspecific sequences ( Figure 2F). Transfection of PPARδ-targeting siRNA attenuated the induction of SIRT1 expression by formononetin, whereas transfection of control siRNA did not ( Figure 2G). In line with these findings, GSK0660, a specific inhibitor of PPARδ, significantly attenuated the formononetin-induced increase in SIRT1 promoter activity ( Figure 2H). These results suggest that formononetin upregulates SIRT1 expression via PPARδ at the transcriptional level.

SIRT1 is essential for inhibition of LPS-induced HMGB1 release by formononetin
To investigate the direct effect of SIRT1 on LPS-induced HMGB1 release, we examined the levels of SIRT1 protein and released HMGB1 in RAW264.7 cells exposed to LPS in the presence or absence of formononetin. A high level of HMGB1 was released upon LPS treatment, whereas this was reduced in the presence of formononetin. On the other hand, the level of SIRT1 protein was significantly suppressed in LPS-treated RAW264.7 cells. However, this LPS-mediated repression of SIRT1 was recovered in the presence of formononetin, indicating that SIRT1 is critical for modulation of LPS-induced HMGB1 release by formononetin ( Figure 3A).
To further clarify the functional significance of formononetin-mediated upregulation of SIRT1 in RAW264.7 cells, we manipulated the expression and activity of SIRT1 using siRNA or chemicals. The levels of SIRT1 protein were diminished in cells transfected with SIRT1 siRNA, however control siRNAs had no effect on the levels of either protein ( Figure 3B). Transfection of SIRT1-targeting siRNA significantly attenuated the inhibitory effect of formononetin on LPSinduced HMGB1 release ( Figure 3C). Consistently, inhibition of SIRT1 activity by sirtinol also Manuscript to be reviewed prevented inhibition of HMGB1 release by formononetin ( Figure 3D). By contrast, activation of SIRT1 by resveratrol inhibited LPS-induced HMGB1 release. Furthermore, resveratrol treatment potentiated the inhibitory effects of formononetin, suggesting that SIRT1 plays a role in the suppression of HMGB1 release by formononetin ( Figure 3E). These results indicate that formononetin inhibits LPS-induced HMGB1 release by regulating SIRT1 expression.

SIRT1-mediated deacetylation of HMGB1 underlies the inhibition of its release by formononetin
Inflammatory signal-mediated acetylation of HMGB1 is critical for its release into the extracellular compartment and acetylated HMGB1 is a substrate of SIRT1 (Bonaldi et al., 2003;Hwang et al., 2014;Rickenbacher et al., 2014); therefore, we evaluated whether formononetin affects LPS-induced acetylation of HMGB1. When RAW264.7 cells were stimulated with LPS for 6 h, the level of acetylated HMGB1 in an immunoprecipitate obtained using an anti-HMGB1 antibody was significantly enhanced. However, formononetin reduced this increase in acetylated HMGB1 in a concentration-dependent manner, indicating that formononetin is involved in the deacetylation of HMGB1 primed by LPS ( Figure 4A).
To evaluate whether this inhibition of LPS-induced HMGB1 acetylation by formononetin correlates with the level of SIRT1 in RAW264.7 cells, we knocked down SIRT1. Transfection of SIRT1-targeting siRNA significantly prevented the decrease in acetylated HMGB1 by formononetin in LPS-exposed RAW264.7 cells ( Figure 4B). Transfection of PPARδ-targeting siRNA elicited the same effect ( Figure 4C). These results indicate that formononetin reduces HMGB1 acetylation via PPARδ-mediated upregulation of SIRT1, thereby inhibiting the release of HMGB1 into the extracellular milieu. Manuscript to be reviewed translational modifications such as acetylation and phosphorylation (Bonaldi et al., 2003;Youn & Shin, 2006). Consistent with previous studies, formononetin inhibited LPS-induced acetylation of HMGB1, leading to suppression of its release. This effect of formononetin on HMGB1 release was intimately correlated with the level of SIRT1 expression, indicating that SIRT1 deacetylates HMGB1 and thereby inhibits its release. This result is in line with previous reports indicating that SIRT1 deacetylates inflammation-related transcription factors such as AP-1 and NF-κB, and thereby modulates the progression of inflammation by suppressing the transcription of diverse inflammation-related genes (Yang et al., 2007;Yeung et al., 2004;. These results provide a rationale for the use of SIRT1 activators as therapeutic agents in inflammatory diseases as shown previous studies using resveratrol to activate the SIRT1 (Xu et al., 2014;Dong et al., 2015). In fact, a recent study demonstrated that inflammatory diseases are closely associated with a reduced SIRT1 protein level (Xie et al., 2013). Because release of HMGB1 is intimately correlated with its post-translational modifications along with decreased SIRT1 expression, it may be possible to suppress inflammatory reactions by inducing SIRT1 expression using formononetin.
PPARδ was initially shown to promote SIRT1 expression in human hepatocytes via an unconventional mechanism in which specificity protein 1 plays a central role, rather than the PPAR-response element (Okazaki et al., 2010). PPARδ activation also induces SIRT1 expression in vascular endothelial cells (Kim et al., 2012 Manuscript to be reviewed  reporter plasmid and 0.5 μg of pSV b-Gal for 38 h were pretreated with GSK0660 for 30 min and then exposed to formononetin for 24 h. Luciferase activity was normalized to βgalactosidase activity. The results are expressed as the means ± SE (n=3). * p<0.05, ** p<0.01