Ursolic Acid Isolated from the Leaves of Loquat (Eriobotrya japonica

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Bioactive Constituents, Metabolites, and Functions

Ursolic acid, isolated from the leaves of loquat (Eriobotrya japonica) inhibited osteoclast differentiation through targeting exportin 5 Hui Tan, Chong Zhao, Qinchang Zhu, Yoshinori Katakura, Hiroyuki Tanaka, Koichiro Ohnuki, and kuniyoshi shimizu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06954 • Publication Date (Web): 04 Mar 2019 Downloaded from http://pubs.acs.org on March 5, 2019

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Journal of Agricultural and Food Chemistry

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Ursolic acid, isolated from the leaves of loquat (Eriobotrya japonica) inhibited

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osteoclast differentiation through targeting exportin 5

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Hui Tan a, Chong Zhao b, Qinchang Zhu c, Yoshinori Katakura d, Hiroyuki Tanaka e, Koichiro

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Ohnuki f, Kuniyoshi Shimizu g, *

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a

Faculty of Health Science, Hokkaido University, Sapporo, 060-0812, Japan

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b

College of Food Science and Nutritional Engineering, China Agriculture University, Beijing, 100083,

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China

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c

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China

School of Pharmaceutical Sciences, Shenzhen University Health Science Center, Shenzhen, 518060,

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d

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812-8581, Japan

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e

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Fukuoka, 812-8582, Japan

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f

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Japan

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g

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819-0395, Japan

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*

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Shimizu)

Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu University, Fukuoka,

Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University,

Department of Biological and Environmental Chemistry, Kinki University, Fukuoka, 820-8555,

Department of Agro-environmental Sciences, Faculty of Agriculture, Kyushu University, Fukuoka,

Corresponding author. Tel./fax: +81 092 642 3002, email: [email protected] (Kuniyoshi

ACS Paragon Plus Environment


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Abstract

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One of the conventional strategies for treating osteoporosis is to eliminate the multinucleated

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osteoclast that are responsible for bone resorption. Our previous study revealed that ursolic acid,

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isolated from leaves of loquat that used as tasty tea in Japan, suppressed osteoclastogenesis. We

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confirmed that ursolic acid exhibited osteoclast differentiation inhibitory activity with IC50 value of

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5.4 ± 0. 96 M. To disclose its mechanism of action, this study first uses polymer-coated magnetic

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nanobeads to identify potential target proteins. As a result, we identified a nuclear exporter protein

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named exportin 5 (XPO5). Further studies demonstrated that knockdown of XPO5 significantly blocks

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osteoclast differentiation (P < 0.01). Expression profiling of mature microRNAs in the cells revealed

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that downregulation of XPO5 by small interfering RNA or by ursolic acid could downregulate the

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expression of mature microRNA let-7g-5p during osteoclast differentiation (P < 0.01). Collectively,

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our findings suggest that ursolic acid inhibits osteoclast differentiation through targeting XPO5, which

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provides further evidence for the healthy function of the tea. This study also provides new insights

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into the role of XPO5 and its mediated microRNAs in treatment for bone resorption diseases.

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Key words: osteoporosis, ursolic acid, exportin 5, microRNAs, let-7g-5p

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Introduction

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Osteoporosis is a metabolic bone disorder that occurs when bone resorption outpaces bone formation

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during bone remodeling. Bone resorption is the unique function of the osteoclast, and anti-osteoporosis

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therapies developed to date have targeted this cell 1. Osteoclasts are multinucleated giant cells, derived

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from hematopoietic stem cells and experiencing a series of differentiations and activations induced by

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the receptor activator of the nuclear factor- (RANK) and its ligand (RANKL) signaling pathway 2,

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3. Activation of RANK leads to rearrangements of cell shape that allows the tight binding of osteoclasts

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to bone. Subsequently the bone is eroded by the export of hydrogen ions generated via vacuolar H+-

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ATPase and degraded by the lytic enzymes tartrate-resistant acid phosphatase (TRAP) and lysosomal

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protease cathepsin K 4. The current treatment strategies for osteoporosis such as anti-resorptive agents

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bisphosphonate, which inhibit excessive bone resorption and are used for the treatment of bone

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diseases 5, 6. Odanacatib, a selective cathepsin K inhibitor, has been demonstrated to be a potent anti-

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resorptive drug in clinical trials 7. In addition, considerable efforts have been devoted to exploring or

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identifying develop new treatment targets and cost-effective dietary supplements from different natural

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resources. For example naturally occurring compound, reveromycin A is reported to be effective

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against bone resorption through inhibition of glyoxalase I 8, 9.

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In our previous study, the medical plant Eriobotrya japonica, which is used as the ingredient of healthy

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and tasty tea in Japan, was found to be effective in preventing the loss of ovariectomy-induced bone

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mineral density in vivo. Using a bioassay-guided approach, we first isolated ursolic acid as the

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strongest inhibitor of osteoclast differentiation 10, 11. Ursolic acid is a pentacyclic triterpenoid found in

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apple, rosemary and holy basil and possesses multiple functions, such as anti-bacterial, anti-allergy



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and anti-aging activities, so widely used as a natural nutraceutical agent

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mechanism by which ursolic acid inhibits osteoclast differentiation remains unclear. In this study, we

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performed an affinity-based proteomic screening to identify potential target proteins of ursolic acid to

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clarify its mechanism of action.


. However, the possible

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Materials and Methods

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Chemicals

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Ursolic acid (1) was purchased from Wako Pure Chemical Industries, Ltd., Osaka, Japan. Oleanolic

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acid (2) was purchased from Cayman Chemical Company, Ann Arbor, MI. RAW 264 murine

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macrophage cells were obtained from Riken Bio resource Center Cell Bank, Tsukuba, Japan. Minimal

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essential medium, alpha modification (α-MEM medium) was from Gibco BRL (Grand Island, NY,

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USA); fetal bovine serum (FBS) and antibiotics−antimycotics were obtained from Gibco and

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Invitrogen (Carlsbad, CA, USA), respectively. Receptor activator of NF-κB (RANKL) from

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Escherichia coli was purchased from PeproTech EC (London, UK), and tumor necrosis factor alpha

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(TNF-α) was obtained from Roche Molecular Biochemicals (Mannheim, Germany). Magnetic beads

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carrying an amino linker (TAS8848N1130) was obtained from Tamagawa Seiki Co., Kanagawa, Japan.

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XPO5 and negative control siRNAs were obtained from Ambion; Life Technologies, Gaithersburg,

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MD. Lipofectamine RNAiMAX and OptiMEM were obtained from Invitrogen, Paisley, UK. TRIzol

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reagent was bought from Life Technologies, Gaithersburg, MD.

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Preparation of ursolic acid and its analogues

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The purity of ursolic acid (1) (HPLC-purity of >95%) was confirmed by analytical HPLC (column,

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Inertsil ODS-3, 4.6 mm i.d.×150 mm; 90% methanol/10% water, 1 ml/min flow rate, Rt, 10 min,

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wavelength at 210 nm, Figure S1) and the molecular formula was determined on the basis of the ion

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peak at m/z 455.3665 [M-H]- by LCMS-IT-TOF spectra. 1H-NMR (400 MHz, DMSO-d6) ppm: 5.13

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(dd, J = 8; 4 Hz, H-12); 3.01 (dd, J = 10.4; 5.2 Hz, H-3); 2.12 (d, J = 12 Hz, H-18); 1.96 (dd, J = 8;



84

4 Hz, H-11); 0.92 (s, H-23); 0.67 (s, H-24); 0.75 (s, H-25); 0.71 (s, H-26); 1.04 (s, H-27); 0.80 (d, J =

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12 Hz, H-29); 0.89 (d, J = 12 Hz, H-30).

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Information Table S1).

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Ursolic acid methyl ester (2): The ursolic acid (70 mg) was dissolved in 2 ml of 20 %

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methanol/benzene and treated with 1 ml trimethylsilyldiazomethane (0.6 mol/l in 10% hexane) for 30

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min; the mixture was stirred at 50 °C for 1 h. After concentration, chromatography (EtOAc/n-

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hexane=1:3) afforded (2, 66.3 mg). The purity of (2) was checked by analytical HPLC (column,

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Inertsil ODS-3, 4.6 mm i.d.×150 mm; 90% methanol/10% water, 1 ml/min flow rate, Rt: 19 min) with

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more than 99%, wavelength at 210 nm. The chemical structures of (2) were confirmed by 13C NMR

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(Supplementary Information Table S1) 13.

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Acetyl-ursolic acid (3): 50 mg ursolic acid was dissolved in 50 mL chloroform, and then acetic

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anhydride/pyridine was added (10 ml, 1:1), and the mixture was heated at 100 °C for 20 min. After

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concentration, chromatography (EtOAc/n-hexane=1:5) gave (3, 50 mg). The purity of (3) was checked

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by analytical HPLC (column, Inertsil ODS-3, 4.6 mm i.d.×150 mm; 90% methanol/10% water, 1

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ml/min flow rate, Rt, 13.75 min, wavelength 210 nm). The molecular formula of (3) was determined

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with an ion peak at m/z 497.3682 [M-H]-) by LCMS-IT-TOF spectra. The chemical structures of (3)

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C NMR spectra were confirmed (Supplementary

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were confirmed by 13C NMR (Supplementary Information Table S1) 13.

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The purity of the oleanolic acid (4) (HPLC-purity of >99%) was confirmed the purity by analytical

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HPLC (column, Inertsil ODS-3, 4.6 mm i.d.×150 mm; 90% methanol/10% water, 1 ml/min flow rate,

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Rt, 12 min, wavelength 210 nm and molecular formula was confirmed with an ion peak at m/z


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455.3629 [M-H]-) by LCMS-IT-TOF spectra. 1H-NMR (400 MHz, DMSO-d6):  5.15 (t, 1, J=3.5 Hz,

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H-12), 3.01 (dd, 1, J = 12 and 4 Hz, H-3), 2.75 (d, 1, J = 8 Hz, H-18), 2.54 (dd, 2, J = 12 and 4 Hz,

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H-11), 0.97 (s, 3, H-23), 0.67 (s, 3, H-24), 0.75 (s, 3, H-25), 0.71 (s, 3, H-26), 1.09 (s, 3, H-27), 0.80

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(d, 3, J = 8.8 Hz, H-29), 0.89 (d, 3, J = 8.4 Hz, H-30),

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(Supplementary Information Table S1).

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Assay for osteoclastic TRAP activity and cell proliferation

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The assays for osteoclastic TRAP activity and cell proliferation were performed as described

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previously with slight modification 10. In brief, RAW 264 murine macrophage cells were cultured in

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a 96-well plate at a density of 1 × 105 cells/ml in -MEM medium with 10% FBS and 0.1% antibiotics-

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antimycotics. After 24 h, to induce osteoclast differentiation, the adherent cells were further co-

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cultured with 100 ng/ml RANKL and 50 ng/ml tumor necrosis factor alpha (TNF-α). Samples at 50,

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25, 20, 10, 5 and 1 g/ml were dissolved in DMSO (v/v, 0.1% in each well) and added into each well.

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Oleic acid was used as a positive control

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differentiation. The cells were stained for TRAP with a TRAP 387-A staining kit (Sigma-Aldrich (St.

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Louis, MO, USA) according to the manufacturer’s instructions. After 1 h of staining in the dark, the

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stained cells were washed with water and stained with hematoxylin solution for 2 min, then rinsed

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with 0.1 N NaOH. The number of TRAP-positive multinucleated cells with 3 and more nuclei in each

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well was counted. Cell proliferation was evaluated using a 3-(4,5-di-methylthiazol-2-yl)-2,5-

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diphenyltetrazolium bromide (MTT) assay with the indicated concentrations of samples for 48 h.

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Preparation of ligand-immobilized beads

14.

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C NMR spectra were also confirmed

The cells were then cultured for several days for



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500 l of 20 mM of ursolic acid (1), 50 l of 200 mM N-hydroxysuccinimide and 50 l of 200 mM

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1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in 400 l of N, N’-dimethylformamide were mixed

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at room temperature for two hours by microtube mixer. Magnetic beads carrying an amino linker were

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washed with N, N’-dimethylformamide three times. Subsequently, 2.5 mg beads were incubated with

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various concentrations of active ligand (0, 0.4, 2 and 10 mM), whose carboxyl group was modified by

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succinimide, in N, N’-dimethylformamide for 20h at room temperature using a microtube mixer. After

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the reaction, the supernatant was discarded and washed three times with N, N’-dimethylformamide.

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Unreacted residues were masked using 50 l triethylamine and 0.2 mmol of acetic anhydride in 430

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l N, N’-dimethylformamide for 2h at room temperature by a mixer. For the ligands with hydroxyl

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groups, we applied a deacetylation reaction by resuspending the beads in 500 ml of 0.1 M sodium

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hydroxide and mixing for 30 min at room temperature. After being washed with ultrapure water three

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times, the ursolic acid-immobilized beads were stored in 50% methanol at 4 °C. Acetylate-ursolic acid

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(3) or oleanolic acid, (4) used as a negative control, was prepared by the same method.

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Affinity purification of target protein with ligand-immobilized beads

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The extraction of differential proteins from RAW 264 cells were separated by the ProteoExtract

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subcellular proteome extraction kit (Merck Millipore Co., Tokyo) according to subcellular localization.

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Ligand-immobilized beads (0.5 mg) were washed three times with 100 mM KCL buffer solution (200

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l) and then co-cultured with each cell fraction by performing a binding reaction for 4h at 4 °C. In a

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competition binding assay, the free ligand was preincubated with protein lysis for two hours (DMSO

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5%, ligand conc. 1 mM). After incubation, the beads were separated magnetically, the supernatant was

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discarded. They were then washed extensively with 100 mM KCL buffer solution (200 l) 4~5 times.


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The non-covalence binding proteins were eluted with 1 M KCL buffer solution (30 l) and separated

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magnetically. The covalence binding proteins were obtained by heating for 5 min at 98 °C from the

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remaining beads. After then eluted sample underwent SDS-PAGE on 5~20% gel. Finally, silver

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staining of the gel was performed using a silver stain MS kit (Wako Pure Chemical Industry, Ltd.,

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Osaka, Japan).

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Identification of the target protein by nano-LC-MS/MS

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The 120 kDa band was cut and treated with trypsin. The peptide fragments were analyzed using a

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nanoLC-MS/MS Bio Nano LC system (KYA TECH) coupled a with QSTAR XL quadrupole time-of-

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flight mass spectrometer (Applied Biosystems, Foster, City, CA). Tryptic peptides were fractionated

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with a HiQ sil C18W-3 column (0.1 mmφ×50 mm; KYA TECH, Tokyo). A mobile phase was

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composed of solvent A with 2% acetonitrile (0.1% formic acid) and solvent B with 80% acetonitrile

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(0.1% formic acid). The mobile phase was consecutively programmed as follows: B conc. 0-10 min

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0%, 40 min 50%, 50 min 100%, 70 min 100%, and 80 min 0%. The flow rate was 200 nL/min. An

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electrospray voltage of 1.8 kV was applied. Spectra were collected in positive ion mode and in cycles

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of one full MS scan (m/z: 400-1800). The protein was identified from peptide mass fingerprinting

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using the Mascot search program and the NCBIInr database.

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Western blotting

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Affinity purified proteins were resolved by SDS-PAGE and detected by western blotting with anti-

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XPO5 antibody. The indicated concentrations (0, 0.4, 2 and 10 mM) of purified proteins were applied

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in equal amounts to each pane. After SDS-PAGE, proteins were electroblotted onto an immobilon



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polyvinylidene difluoride transfer membrane (Merck Millipore Co.). The absorbent paper (CB-09A,

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85×90 mm) and EzFastBlot (blot buffer) were obtained from ATTO Co. (Tokyo). Precision Plus

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Protein All Blue standards were purchased from Bio-Rad, Tokyo. The blots were blocked with TBST

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three times for 5 min each and incubated with anti-XPO5 rabbit monoclonal antibody (ERP8452;

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Abcam, Tokyo) for 1h, then washed, incubated with peroxidase-conjugated secondary antibody (anti-

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rabbit IgG, MP biomedicals, Japan), and finally detected by Clarity Western ECL chemiluminescent

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substrate (Bio-Rad). The bands were analyzed by an ImageQuant LAS-4000 imaging system (GE

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Healthcare Life Sciences).

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SiRNA transfection and qRT-PCR

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XPO5 siRNA duplexes were designed from the mouse XPO5 cDNA sequences with the following

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sequences:

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UCACUAUCGAAAUCAAAUCgg-3’; XPO5 #2: (sense) 5’-CUAACAUACAAACACCUAUtt-3’,

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(antisense) 5’-AUAGGUGUUUGUAUGUUAGca-3’. An siRNA with sequences that do not target

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any gene product was used as a negative control. Cy3-labeled siRNA was used for analyzing the

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transfection stability and efficiency. RAW 264 cells at 0.5×104 cells/well in 24-well plates were

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transfected with siRNAs using Lipofectamine RNAiMAX according to the manufacturer’s

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instructions. Transfection reactions were performed in serum-free OptiMEM. After 24h, the

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transfected cells were cultured in the presence of 100 ng/ml RANKL and 50 ng/ml TNF-α for 60h to

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study the effect of XPO5 on osteoclast differentiation.

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XPO5 knockdowns after 24h siRNA treatment with RAW 264 cells were measured by RT-PCR. Total

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RNA prepared from cells was extracted with the High Pure RNA Isolation Kit (Roche Diagnostics

XPO5

#1:

(sense)

5’-GAUUUGAUUUCGAUAGUGAtt-3’,


(antisense)

5’-

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GmbH, Mannheim, Germany), cDNA was prepared using the ReverTra Ace qPCR RT kit (Toyobo,

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Tokyo) according to the manufacturer’s instructions. Single amplicon amplification was confirmed

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using melting curve analysis, and absence of primer dimers and genomic DNA amplification by

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agarose gel electrophoresis, followed by a direct sequencing. qRT-PCR was performed using the

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KAPA SYBR Fast qPCR kit (KAPA Biosystems, Woburn, MA) and the Thermal Cycler Dice Real

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Time System TP-800 instrument (TaKaRa, Shiga, Japan). The PCR primer sequences used were as

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follows: XPO5 forward primer, 5’-CCTCTCTTCACCTACCTCCACA-3’ and reverse primer, 5’-

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CAATCTCATCTTCTCCACACAGG-3’;

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AAATGGTGAAGGTCGGTGTG-3’ and reverse primer 5’-TGAAGGGGTCGTTGATGG-3’. The

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samples were analyzed in triplicate. Results were normalized against the GAPDH expression level.

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Microarray analysis and mature miRNAs let-7g-5p measurement

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RAW 264 cells at 3×105 cells/well in 6-well plate. After 24h, cells were treated with XPO5 siRNA and

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5 g/ml ursolic acid followed by adding 100 ng/ml RANKL and 50 ng/ml TNF-α for cell

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differentiation. After 24h, total RNA was isolated from RAW 264 cells using TRIzol Reagent and

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purified using the SV Total RNA Isolation System (Promega, Madison, WI). Expression levels of

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mature miRNA were analyzed according to the manufacturer’s instructions, the cRNA was amplified

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and labeled using a Low input quick amp labeling kit (Agilent Technologies, Santa Clara, CA), and

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hybridized using a SurePrint G3 Human Gene Expression Microarray (8 × 60K v2; Agilent). All

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hybridized microarray slides were scanned using an Agilent scanner. Relative hybridization intensities

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and background hybridization values were calculated using Agilent Feature Extraction Software 15.

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Cells were treated same as above mentioned. A small RNA enrichment procedure was performed with

GAPDH


forward

primer,

5’-


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the mirVana miRNA Isolation Kit (Ambion). Extraction was carried out according to the

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manufacturer’s instruction. TaqMan advanced miRNA assay (Applied Biosystems) were used to

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quantify the level of mature miRNAs let-7g-5p (Inventoried, #4427975). Each reverse transcriptase

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(RT) reaction contained 10 ng of purified small RNAs, 3 l of 5×RT primer mixture with 7 l of

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master mix including 100mM dNTPs, MultiScribe Reverse Transcriptase 50 U/l, 10×Reverse

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Transcription buffer, and 20 U/l RNase inhibitor. The reaction mixture was incubated at 16 °C for 30

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min, 42 °C for 30 min and 85 °C for 5 min. The concentration and purity of RNA was checked by

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nanodrop. qPCR reactions were done in triplicate, and each reaction mixture included 1 l of

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20×TaqMan small assay, 1.33 l of the product of the RT reaction and 10 l of 2×TaqMan Universal

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PCR Master Mix II. The reaction mixtures were incubated in a Thermal Cycler Dice Real Time System

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TP-800 instrument at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min.

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The results were normalized against the U6 expression level.

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Statistical analysis

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All values are expressed as the means ± S.D. Student’s t-test and one-way analysis of variance

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(ANOVA) with the Tukey–Kramer test were done for multiple comparisons. Differences between

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means at the 5% confidence level were considered statistically significant.

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Results

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Molecular probe design

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To profile and identify the targets of ursolic acid, we first evaluated the structure-activity relationship

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of ursolic acid (1) and its three typical analogues (2, 3 and 4; Figure 1) against RANKL-induced


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osteoclast differentiation evaluated by using TRAP as a specific marker. We calculated the IC50

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values against the RANKL-induced osteoclast differentiation and the CC 50 values for the cell

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viability of ursolic acid and its analogues (for individual concentrations see Supplemental

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Information Tables S2 and S3). The relative effectiveness of the compounds for inducing

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cell death compared to inhibiting osteoclastogenesis was defined as the selectivity index (S.I.,

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calculated as the ratio of the CC50 value to the IC 50 value). It is desirable to have a high S.I.

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(S.I. >1), which provides information on the potential for selective osteoclast inhibition. As

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shown in Figure 1, ursolic acid (1) and its methyl ester (2) were more potent than the other

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analogues in terms of their anti-osteoclastogenesis effect.

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The results clearly indicated that the hydroxyl group at the R1 position is essential for the activity,

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because (3) caused a dramatic loss of activity. Similarly, the total difference between (1) and (4)

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demonstrated the importance of the position of the methyl groups at C-29 and C-30 on the E-ring to

239

the activities of these compounds, since (4) showed limited activity. However, modification of the R2

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position of ursolic acid (1) to (2) seemed not to cause a significant loss of activity. Based on the above

241

results, we decided to keep the essential C-3 hydroxyl group and two methyl groups free, and ursolic

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acid was covalently conjugated with the amino-functionalized magnetic beads through amidation of

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the carboxylic group in the side chain of the ursolic acid. On the other hand, inactive analogues (3)

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and (4) were synthesized with magnetic beads with the same reaction as negative controls (Figure

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2B).



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Identification of the possible target protein of ursolic acid

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To identify the cellular target of ursolic acid, we performed affinity purification using ursolic acid-

248

immobilized beads. The proteins interacted with ligand-conjugated beads at various concentrations,

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and only the beads were investigated. After extensively washing, binding proteins were eluted and

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subjected to SDS-PAGE for silver staining. Some specific bands were cut and treated with trypsin.

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Then proteins sequencing using mass spectrometry was performed (Figure 2A). As a result, three

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bands were specifically eluted in the region of 100 - 150 kDa (Figure 2C). Since highly abundant

253

proteins of cell lysates might non-specifically bind either to the linker or the ligand itself, to distinguish

254

the target proteins from non-specific binding proteins, we performed a competitive inhibition of

255

binding by pre-incubation with an excess amount of free ligand. Proteins lysates were premixed with

256

excess amounts of free ursolic acid (1 mM) in advance. After 2 h of incubation with ursolic acid-

257

immobilized magnetic beads, we found that three candidates for specific target proteins with a lower

258

recovery rate after being premixed with free ursolic acid. These results suggest that the three target

259

proteins may specifically bind with ursolic acid (Figure 2D).

260

For further confirmation, we used two inactive analogs of ursolic acid (NC-3 or NC-4) to distinguish

261

specific target proteins from non-specific binding proteins by the same elution method. As Figure 2E

262

shows, the target protein (solid arrow) at ~120 kDa was the only band with ursolic acid-immobilized

263

magnetic beads. Slight bands for another two targets (dashed arrows) appeared at around ~100 kD in

264

negative controls (3) and (4) immobilized magnetic beads. These results showed that the ~120 kDa

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protein was dependent on ursolic acid specifically.


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The band of protein at ~120 kDa was excised carefully from the SDS-PAGE gel, identified by nano

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LC-MS/MS and analyzed by Mascot (Supplementary Information Table S4). Through the LC-

268

MS/MS analysis result, we identified XPO5 as the likely target of ursolic acid (1). XPO5 has a

269

predicted molecular size of 136.3 kDa, which matches the expected band observed in SDS-PAGE.

270

Based on the hint from LC-MS/MS, the existence of XPO5 was re-confirmed in a pull-down assay

271

using an anti-XPO5 antibody through western blotting, further confirming XPO5 as the target of

272

ursolic acid (1) (Supplementary Information Figure S5). Thus, multiple lines of evidence converged

273

to support XPO5 as the likely target of ursolic acid in RAW 264 cells.

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Exportin 5 is important for RANKL-induced osteoclastogenesis

275

To investigate whether the loss of XPO5 activity could suppress osteoclastogenesis, we performed an

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siRNA knockdown experiment in RANKL-induced osteoclastogenesis. We selected two specific

277

siRNAs for XPO5 (XPO5#1 and XPO5#2) and one negative control siRNA in our experiment. qRT-

278

PCR results showed that mRNA levels were successfully reduced by around 33% and 56% in XPO5#1

279

treated cells at 10 pmol and 100 pmol around 28% and 52% in XPO5#2 treated cells at 10 pmol and

280

100 pmol, respectively (Figure 3A). We used a 100 pmol concentration of siRNA for knockdown

281

since higher concentrations of siRNA showed higher transfection efficiencies. After a 60h co-culture

282

with RANKL and TNF-α, osteoclast differentiation in XPO5-silenced cells was significantly inhibited

283

in comparison with non-functioning siRNA-treated cells (negative control) and untreated cells (control)

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(Figure 3B). These results suggested that XPO5 activity was necessary for osteoclastogenesis.



Page 16 of 32

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XPO5 deficiency influenced mature miRNA let-7g-5p expression during

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osteoclastogenesis

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Here we show evidence that ursolic acid interacted directly with XPO5 and that the loss function of

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XPO5 directly impaired osteoclastogenesis. It is widely known that XPO5 is mainly responsible for

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exporting precursor miRNAs (pre-miRNAs) through the nuclear membrane to the cytoplasm into

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mature miRNAs 16. In mammalian miRNA biogenesis, the primary transcripts of miRNA genes (pri-

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miRNAs) are cleaved into hairpin intermediates (pre-miRNAs) by nuclear Drosha and further

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processed to mature miRNAs by cytosolic Dicer 17. The highly specific interactions between XPO5

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and pre-miRNAs implicate XPO5 as an essential factor in miRNA biogenesis (Figure 5)

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hypothesized that the loss function of XPO5 may trap pri-miRNA or pre-miRNAs in the nucleus,

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reduce miRNA processing and diminish mature miRNA production. Mature miRNAs contribute to

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the regulation of endogenous genes. Consequently, the phenotype observed in XPO5 knockdown

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osteoclast cells could be caused by impaired mature miRNA expressions during osteoclastogenesis.

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To identify the mature miRNAs expression signature in RANKL-induced osteoclastogenesis with or

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without XPO5 knockdown for 24 hours, we compared a total of 1248 mature miRNA expression

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profiles. Using XPO5-silenced cells, we first revealed the involvement of XPO5 in miRNA maturation

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in RAW 264 cells. The data showed that relatively high expression levels of more than 185 miRNAs

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were observed after XPO5 knockdown, 14.82% of the total number of reads in the library. This result

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is consistent with the findings of Kim et al. 19, who showed a modest reduction of miRNA levels in

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XPO5-knockout cells. Among these 185 mature miRNAs, 96 (7.7% of the total tested) were reduced

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and three of them were significantly downregulated after XPO5 knockdown (

Ursolic Acid Isolated from the Leaves of Loquat (Eriobotrya japonica

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