Ursolic Acid Isolated from the Leaves of Loquat (Eriobotrya japonica

Mar 4, 2019 - One of the conventional strategies for treating osteoporosis is to eliminate the multinucleated osteoclasts that are responsible for bon...
1 downloads 0 Views 1013KB Size
Subscriber access provided by Washington University | Libraries

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

Journal of Agricultural and Food Chemistry

1

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

2

osteoclast differentiation through targeting exportin 5

3

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

4

Ohnuki f, Kuniyoshi Shimizu g, *

5

a

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

6

b

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

7

China

8

c

9

China

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

10

d

11

812-8581, Japan

12

e

13

Fukuoka, 812-8582, Japan

14

f

15

Japan

16

g

17

819-0395, Japan

18

*

19

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

Journal of Agricultural and Food Chemistry

20

Abstract

21

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

22

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

23

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

24

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

25

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

26

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

27

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

28

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

29

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

30

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

31

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

32

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

33

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

34

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

35

36

37

38

ACS Paragon Plus Environment

Page 2 of 32

Page 3 of 32

Journal of Agricultural and Food Chemistry

39

Introduction

40

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

41

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

42

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

43

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

44

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

45

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

46

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

47

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

48

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

49

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

50

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

51

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

52

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

53

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

54

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

55

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

56

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

57

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

58

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

59

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 4 of 32

12

60

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

61

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

62

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

63

clarify its mechanism of action.

ACS Paragon Plus Environment

. However, the possible

Page 5 of 32

Journal of Agricultural and Food Chemistry

64

Materials and Methods

65

Chemicals

66

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

67

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

68

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

69

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

70

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

71

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

72

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

73

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

74

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

75

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

76

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

77

reagent was bought from Life Technologies, Gaithersburg, MD.

78

Preparation of ursolic acid and its analogues

79

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

80

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

81

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

82

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

83

(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;

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

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 =

85

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

86

Information Table S1).

87

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

88

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

89

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

90

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

91

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

92

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

93

(Supplementary Information Table S1) 13.

94

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

95

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

96

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

97

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

98

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

99

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

13

C NMR spectra were confirmed (Supplementary

100

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

101

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

102

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

103

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

ACS Paragon Plus Environment

Page 6 of 32

Page 7 of 32

Journal of Agricultural and Food Chemistry

104

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

105

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,

106

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

107

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

108

(Supplementary Information Table S1).

109

Assay for osteoclastic TRAP activity and cell proliferation

110

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

111

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

112

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

113

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

114

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

115

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

116

Oleic acid was used as a positive control

117

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

118

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

119

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

120

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

121

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

122

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

123

Preparation of ligand-immobilized beads

14.

13

C NMR spectra were also confirmed

The cells were then cultured for several days for

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

124

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

125

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

126

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

127

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

128

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

129

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

130

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

131

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

132

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

133

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

134

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

135

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

136

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

137

Affinity purification of target protein with ligand-immobilized beads

138

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

139

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

140

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

141

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

142

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

143

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

144

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

ACS Paragon Plus Environment

Page 8 of 32

Page 9 of 32

Journal of Agricultural and Food Chemistry

145

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

146

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

147

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

148

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

149

Osaka, Japan).

150

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

151

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

152

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

153

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

154

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

155

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

156

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

157

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

158

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

159

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

160

using the Mascot search program and the NCBIInr database.

161

Western blotting

162

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

163

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

164

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 10 of 32

165

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

166

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

167

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

168

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

169

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

170

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

171

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

172

Healthcare Life Sciences).

173

SiRNA transfection and qRT-PCR

174

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

175

sequences:

176

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

177

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

178

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

179

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

180

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

181

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

182

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

183

study the effect of XPO5 on osteoclast differentiation.

184

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

185

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

XPO5

#1:

(sense)

5’-GAUUUGAUUUCGAUAGUGAtt-3’,

ACS Paragon Plus Environment

(antisense)

5’-

Page 11 of 32

Journal of Agricultural and Food Chemistry

186

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

187

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

188

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

189

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

190

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

191

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

192

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

193

CAATCTCATCTTCTCCACACAGG-3’;

194

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

195

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

196

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

197

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

198

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

199

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

200

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

201

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

202

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

203

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

204

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

205

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

206

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

GAPDH

ACS Paragon Plus Environment

forward

primer,

5’-

Journal of Agricultural and Food Chemistry

207

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

208

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

209

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

210

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

211

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

212

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

213

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

214

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

215

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

216

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

217

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.

218

The results were normalized against the U6 expression level.

219

Statistical analysis

220

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

221

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

222

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

223

Results

224

Molecular probe design

225

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

226

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

ACS Paragon Plus Environment

Page 12 of 32

Page 13 of 32

Journal of Agricultural and Food Chemistry

227

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

228

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

229

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

230

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

231

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

232

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

233

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

234

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

235

analogues in terms of their anti-osteoclastogenesis effect.

236

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

237

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

238

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

240

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

242

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

243

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

244

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

245

2B).

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

246

Identification of the possible target protein of ursolic acid

247

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,

249

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

250

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

251

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

252

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

265

protein was dependent on ursolic acid specifically.

ACS Paragon Plus Environment

Page 14 of 32

Page 15 of 32

Journal of Agricultural and Food Chemistry

266

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

267

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.

274

Exportin 5 is important for RANKL-induced osteoclastogenesis

275

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

276

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)

284

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 16 of 32

285

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

286

osteoclastogenesis

287

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

288

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

289

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

290

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

291

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

292

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

293

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

294

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

295

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

296

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

297

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

298

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

299

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

300

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

301

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

302

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

303

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

304

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

305

and three of them were significantly downregulated after XPO5 knockdown (