Epigallocatechin gallate activates miR-193a-3p and protects mice against glucocorticoid-induced osteoporosis by targeting NFATC1 expression

Purpose: To investigate the effect of epigallocatechin gallate (EGCG) on microRNAs in a mouse model of glucocorticoid-induced osteoporosis (GIOP), and the mechanism involved. Methods: Osteoclast-specific marker mRNA expressions, receptor activator of nuclear factor kappa-B ligand (RANKL), receptor activator of nuclear factor κ B (RANK), and miRNA expressions were determined using reverse transcription polymerase chain reaction (RT-qPCR) analysis. Western blotting was used to assay protein expressions, while miRNA and 3’UTR interaction studies were carried out with reporter assay. Results: Treatment with EGCG resulted in downregulation of glucocorticoid-induced expressions of RANKL, RANK and osteoclast-specific markers i.e. tumor necrosis factor receptor-associated factor 6, (TRAF6), nuclear factor of activated T cells 1 (NFATc1), cathepsin K, matrix metallopeptidase 9 (MMP9) and tartrate-resistant acid phosphatase (TRAP). Furthermore, EGCG treatment significantly reduced reactive oxygen species (ROS) levels and inflammatory cytokine expressions in GIOP mice. The expression of miRNA-targeting osteoclast marker mmu-mir-193-3p was significantly down-regulated in GIOP mice. However, EGCG treatment increased mmu-mir-193-3p expression and had specific interaction with NFATc1 3’UTR (3’-untranslated region). In vitro results showed that mmu-mir-193-3p mimics downregulated dexamethasone (DXM)-induced osteoclast-specific marker expressions. Conclusion: These results show that EGCG exerts a protective role against GIOP by upregulating miR193a-3p expressions.


INTRODUCTION
Glucocorticoid (GC) drugs are used to reduce immune responses during organ transplantation and inflammatory diseases [1]. Post-GC therapy patients develop various complications such as obesity, diabetes mellitus, cardiovascular disease and bone loss [2]. Clinical studies have revealed risk of asthma in 50% of patients who use GCs [3]. Glucocorticoids (GCs) induce osteoblast apoptosis, and target osteoclast bone resorptive activity, ultimately mediating osteoporosis. In the presence of receptor activator of RANKL and macrophage-colony stimulating factor (M-CSF), osteoclasts differentiate and fuse to form multinuclear osteoclasts which function in bone resorptive activity [4]. In this study, the role of miRNAs in GC-induced osteoporosis, and the protective effect of the antioxidant EGCG, were investigated. Epigallocatechin gallate (EGCG) possesses antioxidant property, scavenges free radicals and reduces oxidative stress [5]. The bioactivity of EGCG is due toits eight hydroxyl groups and gallate moiety [6]. It has been shown that EGCG treatment reduces high fat dietinduced weight gain [7], and regulates blood glucose and lipid levels [8].
Deregulated miRNA expressions are the prime causes of disease [9]. This study was aimed at investigating the miRNA expressions and the protective effect of EGCG against GIOPmediated miRNA expressions in mice.  [10].

EXPERIMENTAL Animals and treatment
The mice were allowed 1-week acclimatization to laboratory environment at a temperature of 22 ± 2 °C and relative humidity of 50 -52 %. The animals were divided into four groups: sham, EGCG, GIOP and GIOP+EGCG, with 6 mice per group. Glucocorticoid-induced osteoporosis was induced by treating the mice with dexamethasone (DXM) at a dose of 10 mg/kg intramuscularly for 10 weeks (3 times/per week). Mice in the sham group received vehicle during the treatment period, while mice in the EGCG group were given EGCG orally at a dose of 100 mg/kg daily for 10 weeks.

Measurement of reactive oxygen species
The tissue levels of ROS were determined using ROS detection kit (Abcam-ab113851), as per manufacturer's instructions.

Enzyme linked immunosorbent assay (ELISA) for serum cytokines
Serum cytokines (IL-6 and TNF-α) were assayed using ELISA kits, as per instructions in the kit manual.

Quantitative reverse transcription PCR (RT-qPCR)
Total tissue RNA was isolated using Trizol reagent. The RNA was converted into cDNA and the expressions of RANKL, RANK, TRAF6, NFATc1, Cathepsin K, MMP9 and TRAP were determined using RT-qPCR. Their expressions were calculated relative to that of β-actin which served as control.
Cell culture RAW 264.7 cells were stimulated with M-CSF and RANKL in the differentiation medium. To identify the effect of DXM on OC differentiation, 150nM DXM was used. Cells were transfected with 50nM miR-193a-3p mimics in DXM+miR-193a-3p group.

Reporter assay
Following overnight attachment, the cells were transfected with 50nM miR-193-3p mimics/Scramble and 1 μg of WT/MT NFATc1 3'UTR for 24 h. Reporter assay was carried out using Dual-Luciferase Reporter Assay System (Promega).

Western blot
The cells were lysed using RIPA lysis buffer. Then, 50µl of protein was subjected to gradient SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane. Then, the membrane was incubated overnight with primary antibodies for TRAF6, NFATc1, cathepsin K, MMP9 and TRAP at 4 °C. The membrane was washed thrice with TBST, and incubated with horse radish-conjugated secondary antibody (1:1000) for 1 h at room temperature. The blots were visualized using enhanced chemiluminescence (ECL), and the images were analyzed.

Statistical analysis
The results are expressed as mean ± standard deviation. Each experiment was conducted in triplicate. Statistical differences were analyzed with analysis of variance, using Prism6 (GraphPad Software Inc). Values of p < 0.001 were taken as indicative of statistical significance. Figure 1 A shows significant upregulation of the expressions of RANK and RANKL in GIOP mice model, when compared to sham mice. However, EGCG treatment downregulated the expressions of RANKL and RANK, when compared to GIOP mice. Furthermore, the upregulated osteoclastspecific mRNA and protein expressions (TRAF6, NFATc1, MMP9, Cathepsin K and TRAP) in GIOP mice group were significantly downregulated in EGCG-treated GIOP mice group. These results show that EGCG exerted protective effect against glucocorticoid-induced osteoporosis.

DISCUSSION
In this study, EGCG-mediated increases in miR-193-3p expression had protective effect against GIOP-induced oxidative stress and osteoclast markers.
Glucocorticoids mediate osteoclast differentiation through coordinated mechanisms involving osteoclasts and osteoblasts [11]. Osteoblasts secrete RANKL which interact with RANK (RANK-RANKL) to initiate osteoclast differentiation. In the present study, the glucocorticoid upregulated the expressions of RANK and RANKL. Moreover, there was significant upregulations of osteoclast-specific marker mRNA and protein expressions in GIOP mice. However, EGCG treatment in GIOP mice resulted in protective effect against osteoclast differentiation through downregulation of RANKL, RANK and osteoclast markers. Previous studies have shown that GIOP increased CTSK and Runx2 expressions in a rat model [12]. Glucocorticoids upregulated RANKL expression in the osteoblasts and promoted osteoclast differentiation in human PBMCs (monocytes) [13]. Moreover, it has been reported that glucocorticoids stimulated osteoclast differentiation in mouse calvarial bones [14].
Oxidative stress and inflammatory cytokines are implicated in bone destruction [14][15][16]. Chronic inflammation is associated with bone resorption. The roles TNF-α, IL-1β and IL-6 [17][18][19][20] and oxidative stress [21] in osteoclastogenesis have been reported. In this study, the increases in levels of ROS and inflammatory cytokines due to GIOP were significantly downregulated by EGCG treatment. The reno-protective role of EGCG has been demonstrated in unilateral ureteral obstruction (UUO) mice model through upregulation of Nrf2/HO-1 signaling pathway [22]. Moreover, a study has shown that EGCG attenuated uric acid-mediated inflammatory responses through downregulation of the NOTCH signaling pathway [23].
The miRNA-mediated protein regulation plays a critical role in disease control. Among the various shortlisted miRNAs, mmu-miR-193-3p was significantly downregulated in GIOP, with specific target for NFATc1. This particular miRNA was upregulated by EGCG treatment in GIOP mice. Nuclear factor-activated T cell c1 (NFATc1) is a transcription factor that increases the expressions of the target proteins TRAP and cathepsin K [24,25]. Evidence has shown that NFATc1 alone is sufficient for osteoclastogenesis process [24]. Deficiency of NFATc1 prevents osteoclast formation, while overexpression of NFATc1 enhances osteoclastogenesis [24]. From the in vitro studies, miR-193-3p mimics in the presence of DEX treatment significantly downregulated the expressions of NFATc1 and osteoclast markers (TRAF6, cathepsin K, MMP9 and TRAP) at both mRNA and protein levels.

CONCLUSION
These findings show the protective role of EGCG against GIOP-induced osteoclast marker expressions via miR-198-3p/NFATc1 targeting. Thus, the antioxidant EGCG has promising potential as a therapeutic agent for glucocorticoids-induced osteoporosis.

Conflict of interest
No conflict of interest is associated with this work.

Contribution of authors
We declare that this work was done by the author(s) named in this article and all liabilities pertaining to claims relating to the content of this article will be borne by the authors. Ben Dou, Xiaolan Liu-conceived and designed the study; Xiaohui Wu, Yisong Xie-collected and analyzed the data; Ben Dou, Hongliang Ruan -wrote the manuscript. Xiaolan Liu-Approved final version of the manuscript. All authors read and approved the manuscript for publication.

Open Access
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