Egg White Ovotransferrin Shows Osteogenic Activity in Osteoblast Cells

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Bioactive Constituents, Metabolites, and Functions

Egg White Ovotransferrin Shows Osteogenic Activity in Osteoblast Cells Nan Shang, and Jianping Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00069 • Publication Date (Web): 05 Mar 2018 Downloaded from http://pubs.acs.org on March 8, 2018

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Journal of Agricultural and Food Chemistry

Proliferation

Cell cycle

Alkaline Phosphatase

Ovotransferrin Bone formation Osteoblast cells

Type I collagen

Differentiation Bone resorption

Mineralization

ACS Paragon Plus Environment

Osteoprotegerin RANKL


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Egg White Ovotransferrin Shows Osteogenic Activity in Osteoblast Cells

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Nan Shang, Jianping Wu*

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Dept. of Agricultural, Food and Nutritional Science, University of Alberta,

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Edmonton, AB, Canada

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* Corresponding Author: Dr. Jianping Wu, 4-10 Ag/For Centre, Edmonton, AB,

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Canada T6G 2P5. Phone (780) 492-6885, Fax (780) 492-4265, e-mail:

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[email protected]

20 21 22 23 24 25


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Abstract

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Ovotransferrin, the major protein in egg white, is a member of transferrin family. The

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objective of this study was to study the effects of ovotransferrin on cell proliferation,

29

differentiation, and osteoclastogenesis of bone osteoblast cells. Effect of

30

ovotransferrin (concentrations ranging from 1 to 1000 µg/mL) on the proliferation,

31

differentiation and mineralization of mouse osteoblast cells MC3T3-E1 was

32

determined by 5-Bromo-2-deoxyuridine (BrdU) incorporation assay, Western blot,

33

immunofluorescence and Alizarin-S red staining, respectively. Our results showed

34

that ovotransferrin

35

differentiation (enhanced expression of alkaline phosphatase and type-I collagen), and

36

mineralization

37

Furthermore, ovotransferrin could increase the expression of osteoprotegerin (OPG)

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while decrease the expression of receptor activator of nuclear factor kappa-B ligand

39

(RANKL), suggesting its role on inhibition of bone resorption. This study

40

demonstrated for the first time that ovotransferrin might promote bone formation

41

while prevent bone resorption, which might open up a new application of egg white

42

protein ovotransferrin as a functional ingredient in bone health management.

stimulated proliferation (enhanced BrdU incorporation),

(increased

calcium

deposits)

in

a

dose-dependent

manner.

43 44

Keywords: ovotransferrin, osteoblast, proliferation, differentiation, mineralization,

45

RANKL/OPG


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Introduction

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Osteoporosis, defined as a skeletal disorder, is characterized by an impairment of

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bone mass, strength, and microarchitecture, and is caused by an imbalanced bone

49

formation and bone resorption, which is regulated respectively by osteoblast and

50

osteoclast.1 As a serious public health concern, osteoporosis affects over 200 million

51

people worldwide, leading to severe and localized pain, increased bone fractures, and

52

reduced height.2 Moreover, osteoporosis is also a major cause of morbidity and health

53

expenditure in ageing populations.3 Currently, antiresorptive medications such as

54

bisphosphonates, selective estrogen receptor modulators and hormones are the main

55

pharmacological treatment options for osteoporosis.4 Teriparatide, a recombinant

56

fragment of parathyroid hormone (PTH) has been used as an anabolic agent in

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osteoporosis treatment by stimulating the bone formation.5 Despite their great

58

therapeutic value, these agents are limited in their ability to restore bone mass, and are

59

associated with side effects such as nausea, irregular heartbeat, loose bowel

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movement, and even increased risk of cancer.6 Therefore, there is an increasing

61

interest in using natural components as the alternatives for the prevention and

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treatment of osteoporosis.7

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Ovotransferrin, a 78 kDa iron-binding glycoprotein found in avian egg white and in

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avian serum, is a member of transferrin family.8 Egg ovotransferrin displays several

65

bioactivities, including antimicrobial, antioxidant, and immunomodulating activities.8

66

Likewise, ovotransferrin is a component of innate immunity.9 In chicken

67

macrophages, ovotransferrin was reported to stimulate the production of IL-6, nitrite

68

and Matrix Metallo-proteinase, suggesting that ovotransferrin can modulate

69

macrophage and heterophil function.9 More recently, oral administration of egg

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ovotransferrin reduced the levels of inflammatory cytokines.10 Since inflammation is


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known to play an important role in bone metabolism,11 ovotransferrin may have

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beneficial effects on bone health. Previous study also suggested that the expression

73

levels of ovotransferrin and its receptor were increased during initial stages of bone

74

formation in developing chick embryo,12 further indicating its significant role on

75

cartilage and bone growth. Lactoferrin, another member of transferrin family, was

76

reported to increase osteoblast activity while decrease osteoclast activity in vitro.7

77

Injection of lactoferrin increases bone growth in adult male mice.3,7 Guo et al.13

78

further proved that orally administrated lactoferrin increases bone mineral density and

79

mechanical strength on ovariectomized-induced osteoporosis. In this research, we

80

investigated the effects of ovotransferrin on cell proliferation, differentiation, and

81

osteoclastogenesis of bone osteoblast cell MC3T3-E1, a widely used cell model in

82

osteoblastic activity study.

83 84

Materials and methods

85

Reagents

86

Ovotransferrin (Conalbumin from chicken egg white, at a purity of 98%) and

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Alizarin-S red stain were bought from Sigma Chemicals Co (St Louis, MO, USA). α-

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minimum essential medium (α-MEM) and Fetal Bovine Serum (FBS) were obtained

89

from Gibco/Invitrogen (Carlsbad, CA, USA). AlamarBlue cell viability reagent and

90

BrdU labeling reagent were bought from ThermoFisher Scientific Inc. (Waltham,

91

MA, USA). Propidium iodide flow cytometry kit was bought from Abcam plc.

92

(Toronto, ON, Canada). Collagen I alpha antibody was bought from Novus Biological

93

Canada ULC (Oakville, ON, Canada). Hoechst 33342 (Trihydrochloride), rabbit anti-

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mouse IgG (H+L) secondary antibody, and goat anti-rabbit IgG (H+L) secondary

95

antibody were bought from Molecular Probes (Waltham, MA, USA). BrdU mouse


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antibody was bought from Cell Signaling Technology Inc. (Danvers, MA, USA).

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Triton-X-100 was from VWR International (West Chester, PA, USA). Goat anti-

98

rabbit and donkey anti-mouse fluorochrome-conjugated secondary antibodies were

99

purchased from Licor Biosciences (Lincoln, NB, USA).

100

Cell Culture

101

The murine osteoblastic cell line MC3T3-E1 (subclone 4, ATCC CRL-2593) was

102

purchased from ATCC (Manassas, VA, USA) and cultured with ATCC suggested

103

protocol. Cells were cultured in α-MEM supplemented with 10% FBS and penicillin-

104

streptomycin in an incubator under 95% air and 5% CO2. Cells were subcultured

105

using 0.25% trypsin every 2 to 3 days. All experiments were performed on 80-90%

106

confluent cells grown in tissue culture grade plastic 96 or 48 well plates. To examine

107

the activity of egg white ovotransferrin, the cells were incubated with different

108

concentrations of ovotransferrin or 1000 µg/mL lactoferrin (the positive control) for

109

different time periods prior to BrdU incorporation, western blotting, and

110

immunofluorescence.

111

BrdU incorporation assay

112

The BrdU incorporation assay was performed similar to our previous studies.14 The

113

cells were seeded on 48 well tissue culture plates at a concentration of 1×104

114

cell/well, and incubated in α-MEM with 10% FBS. After 4 h of incubation, the

115

medium was changed and different concentrations of ovotransferrin were added.

116

Lactoferrin was also used as a positive control. Following 24 h of incubation, the cells

117

were washed with PBS and placed in fresh α-MEM with 1% FBS, containing 1%

118

bromodeoxyuridine (BrdU) for 1 h. The cells were then fixed in 70% ethanol for 20

119

min, treated with 1N hydrochloric acid (HCl) for 20 min to antigen exposure, then

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permeabilized with 0.1% Triton-X-100 in phosphate buffered saline for 5 min and


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blocked in 1% bovine serum albumin (BSA) in phosphate buffered saline for 60 min,

122

and finally incubated with mouse monoclonal antibody against BrdU (1:1000) at 4

123

°C. All the steps except addition of primary antibody were performed at room

124

temperature. Following overnight incubation with the primary antibody, the cells

125

were treated with anti-mouse secondary antibody for 30 min in the dark. Nuclei were

126

stained with the Hoechst33342 nuclear dye. Cells were visualized under an Olympus

127

IX81 fluorescent microscope. For each data point, 3 random fields were chosen. The

128

percentage of nuclei positive for BrdU staining was noted in each field and the mean

129

calculated.

130

Cell cycle analysis

131

Cell cycle distribution was analyzed by measuring DNA content using flow cytometer

132

(FACS Canto II, BD Bioscience, CA) according to the manufactory instruction of

133

propidium iodide flow cytometry kit. The cells were seeded on 6 well tissue culture

134

plates at a concentration of 1×104 cells/well, and incubated in α-MEM with 10% FBS

135

until confluence. After being treated with different concentrations of ovotransferrin or

136

1000 µg/mL lactoferrin for 24 h the adherent cells were washed once with PBS, then

137

trypsinized, and centrifuged at 500 g for 5 min. The cell pellets collected were gently

138

suspended in 400 µL PBS, slowly added 800 µL ice-cold 100% ethanol at 4 °C and

139

mixed for at least 2 h. After centrifugation at 500 g for 5 min, the cell pellets were

140

incubated with 200 µL staining solution (9.45 mL PBS containing 500 µL 20-fold

141

diluted propidium iodide stock solution and 50 µL 200×RNase) at 37 °C in the dark

142

for 30 min prior to the measurement.

143

Immunofluorescence

144

The immunofluorescence studies were performed similar to our previous studies.14

145

Briefly, cells were fixed in 4% formalin, permeabilized with 0.1% Triton-X-100 in


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PBS and immunostained overnight with a rabbit polyclonal antibody against type I

147

collagen. Cells were treated with Alexa Fluor546 (red) conjugated goat anti-rabbit

148

secondary antibody for 30 min in the dark. Nuclei were stained with the

149

Hoechst33342 nuclear dye (1:10000) for 10 min. After washing to remove unbound

150

antibody/dye, the immunostained cells were observed under an Olympus IX81

151

fluorescence microscope (Olympus, Tokyo, Japan). Images were obtained using

152

Metamorph imaging software (Molecular Devices, Sunnyvale, CA, USA). Mean

153

fluorescence intensity was calculated from the intensity of the red fluorescent signal

154

(determined by Adobe Photoshop Elements 2.0 software; Adobe Systems Inc., San

155

Jose, CA, USA) from 3 randomly selected fields per group.

156

Western blot

157

The cells were seeded on 48 well tissue culture plates at a concentration of 1×104

158

cells/well, and incubated in α-MEM with 10% FBS. The cells were treated with

159

different concentrations of ovotransferrin or 1000 µg/mL lactoferrin for 72 h. After

160

incubation, the culture medium was removed and the cells lysed in boiling hot

161

Laemmle’s buffer containing 50 µM dithiothreitol (DTT) and 0.2% Triton-X-100 to

162

prepare samples for western blot as described previously.14 These cell lysates were

163

run in SDS-PAGE, blotted to nitrocellulose membranes and immunoblotted with

164

antibodies against ALP, RANKL, OPG and the loading control α-tubulin. Anti-

165

tubulin was used at 0.4 µg/mL, while the others were used at 1 µg/mL. The protein

166

bands were detected by a Licor Odyssey BioImager and quantified by densitometry

167

using corresponding software (Licor Biosciences, Lincoln, NB, US). Each band of

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ALP, RANKL and OPG was normalized to its corresponding band of loading control.

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Cell lysates from untreated cells were loaded onto every gel. The results were

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expressed as percentage of the corresponding untreated control.


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Mineralization

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The mineralization studies were performed similar to previous studies.15 The degree

173

of mineralization was determined in the 12-well plates using Alizarin Red staining.

174

Cells were incubated with different concentrations of ovotransferrin for 10 days. The

175

medium was removed and cells were rinsed twice with PBS. The cells were fixed

176

with ice-cold 70% (v/v) ethanol for 1 h. The ethanol was removed by aspiration and

177

cells were washed twice with Milli-Q water. The cells were then stained with 1%

178

(w/v) Alizarin-S Red in Milli-Q (pH 4.2) for 10 min at room temperature. After

179

washing with Milli-Q water, the samples were observed under light and pictures were

180

photographed.

181

Statistics

182

All data are presented as mean±SEM (standard error of mean) of between 4 and 7

183

independent experiments. Data were analyzed using One Way analysis of variance

184

(ANOVA) with Dunnett’s post-hoc test for comparisons to control. The PRISM 6

185

statistical software (GraphPad Software, San Diego, CA) was used for the analyses.

186

P< 0.05 was considered significant.

187 188

Results

189

Ovotransferrin promotes cell proliferation of osteoblasts

190

Ovotransferrin did not show cytotoxicity to osteoblasts at concentrations up to 1000

191

µg/mL (data not shown). To investigate the effect of ovotransferrin on cell

192

proliferation, osteoblasts were incubated with thymidine analog BrdU, and the

193

incorporation of BrdU into newly synthesized DNA was detected by fluorescence

194

microscopy. As shown in Fig. 1, the percentage of BrdU positive cells significantly

195

increased after adding lactoferrin (positive control, 1000 µg/mL) or ovotransferrin at


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concentrations of 100 µg/mL and 1000 µg/mL. At the concentration of ovotransferrin

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(1000 µg/mL), the percentage of BrdU-incorporated cells increased to 18.3±0.7%,

198

which was twofold increment of the untreated group (10.4±1.2%). These results

199

indicated that ovotransferrin could promote osteoblast proliferation, suggesting the

200

potential role of ovotransferrin as an osteoinductive factor in bone formation.

201

Ovotransferrin inhibits the G0/G1 arrest but promotes S and G2/M arrest

202

To determine whether the promotion of cell proliferation can be attributed to cell

203

cycle arrest, cell nuclei were stained with propidium iodide and cell cycle was

204

analyzed by flow cytometry. As shown in Fig. 2, the percentage of cells in G0/G1

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phase decreased dose-dependently in the ovotransferrin groups, while increased in the

206

lactoferrin group. At the concentration of 1000 µg/mL ovotransferrin, the percentage

207

of cells in the G0/G1 phase decreased significantly to 74.3±0.6%, as compared to

208

86.4±0.4% in the untreated group and 87.6±3.0% in the lactoferrin group. On the

209

contrary to G0/G1 phase, the percentage of cells in S phase increased in the

210

ovotransferrin groups while decreased in lactoferrin group; the highest percentage

211

was shown at 100 µg/mL ovotransferrin treatment instead of 1000 µg/mL

212

ovotransferrin group. The percentage of cells in the S phase was increased

213

significantly to 15.9±0.3% in 100 µg/mL ovotransferrin treatment compared to

214

6.6±0.2% in the untreated group and 4.8±0.3% in the lactoferrin group.

215

Ovotransferrin increases expression of type I collagen in osteoblasts

216

Type I collagen expressed by osteoblasts play an important role as the structural

217

component in bone formation. A high level of type I collagen expression,

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biosynthesis, and secretion is a typical indicator of osteoblasts differentiation. Thus,

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the expression of type I collagen was also examined in this study to investigate the

220

effect of ovotransferrin on cell differentiation. As shown in Fig. 3, the expression of


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type I collagen increased after treatment with lactoferrin (positive control) and

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ovotransferrin; at a concentration of 100 µg/mL ovotransferrin, type I collagen

223

showed 1.5-fold increase compared with the untreated group. These results suggested

224

that ovotransferrin could stimulate the expression of bone matrix component type I

225

collagen, supporting further a beneficial role of ovotransferrin on promoting cell

226

differentiation.

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Ovotransferrin promotes expression of ALP in osteoblasts

228

Differentiation of osteoblast precursor to postmitotic matrix-producing osteoblasts is

229

one of the most important stages in bone formation. Determination of alkaline

230

phosphatase (ALP) expression is a widely used indicator for both osteoblast

231

differentiation and bone formation. As shown in Fig. 4, the expression of ALP was

232

significantly increased after treatment with lactoferrin (positive control) and

233

ovotransferrin (100 µg/mL and 1000 µg/mL) for 72 h. Interestingly, the maximal

234

stimulation was observed at 100 µg/mL, not at the concentration of 1000 µg/mL. The

235

expression level of ALP increased to 149.3±21.5% when treated with 100 µg/mL

236

ovotransferrin, compared to 138.3±13.7% for 1000 µg/mL treatment. Our results

237

suggested ovotransferrin could promote ALP expression when treated with

238

ovotransferrin, further supporting the ability of ovotransferrin on stimulating

239

osteoblast differentiation.

240

Ovotransferrin promotes mineralization of osteoblasts

241

Mineralization is the last while the most important step of osteoblast differentiation

242

and is frequently used as a marker to characterize bone formation. To examine the

243

effect of ovotransferrin on mineralization in osteoblasts, MC3T3-E1 cells were

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incubated with ovotransferrin for 5 days, 10 days, 15 days and 20 days, and

245

mineralization assay was carried out using Alizarin red S staining. The magnitude of


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the absorbance for the developed color is proportional to the quantity of calcium

247

deposit, which is a direct indication of cell mineralization. As shown in Fig. 5, at

248

increasing incubation time, the calcium deposits were increased in MC3T3-E1 in all

249

groups. In the first 5 days, there was no significant difference between untreated

250

group and ovotransferrin treatment. At 10 days, a significantly increase of absorbance

251

was observed at 1000 µg/mL ovotransferrin group, indicating ovotransferrin

252

stimulated the calcium nodules and deposits in osteoblasts. At 20 days, the

253

absorbance was significantly increased at both 100 µg/mL and 1000 µg/mL

254

ovotransferrin groups as compared with the untreated group. These results indicated

255

that ovotransferrin could promote the calcium deposit and calcium nodules

256

production, suggesting that ovotransferrin could enhance mineralization.

257

Ovotransferrin inhibits expression of RANKL in osteoblasts

258

Besides the effect on bone formation, we also investigated the effect of ovotransferrin

259

on bone resorption. RANKL, produced by osteoblasts, acts as a chemoattractant to

260

initiate the osteoclastogenesis and contributes to osteoclast differentiation; therefore

261

expression of RANKL is critical in regulating osteoclasts formation and bone

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resorption.16 As shown in Fig. 6, expression of RANKL decreased after treated with

263

lactoferrin (positive control) and ovotransferrin in a dose-dependent manner. The

264

expression of RANKL was significantly decreased to 60.3±1.9% (100 µg/mL

265

ovotransferrin), or to 55.4±8.6% (1000 µg/mL ovotransferrin), or to 58.8±2.1%

266

(lactoferrin). These results indicated that ovotransferrin could inhibit the expression of

267

RANKL, suggesting its potential role on preventing osteoclastogenesis.

268

Ovotransferrin promotes expression of OPG in osteoblasts

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In addition to RANKL, osteoblasts also release OPG to counteract the function of

270

RANKL and to prevent osteoclast-mediated bone resorption.16 Thus, to confirm the


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effect of ovotransferrin on preventing bone resorption, expression of OPG was also

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determined by western blot. As shown in Fig. 7, the expression of OPG was

273

increasing in a dose-dependent manner, showing an 1.5-fold increase at 1000 µg/mL

274

ovotransferrin treatment, compared with the untreated group. These results suggested

275

that ovotransferrin could also promote the expression of OPG, which further

276

supported the protective role of ovotransferrin on preventing osteoclast-induced bone

277

resorption.

278 279

Discussion

280

Although a potential positive role of high-quality food protein intake on bone health

281

has been suggested,17 effect of egg proteins has not been studied. In addition to their

282

excellent essential amino acid profile and high digestibility, egg proteins are known to

283

impart a wide range of physiological benefits including antimicrobial, anticancer,

284

antioxidant and immunomodulation activities.18 In this study, we showed for the first

285

time that ovotransferrin could directly stimulate osteoblasts proliferation and

286

differentiation as an osteogenic agent. In addition, ovotransferrin also contributes to

287

inhibit osteoclastogenesis. The effects of ovotransferrin on both proliferation and

288

differentiation of osteoblasts are profound, being comparable to lactoferrin, a well-

289

accepted nutraceutical for bone formation. Lactoferrin, extracted from milk belonging

290

to transferrin family, is one of the most well studied bioactive proteins that acts as a

291

promoter of osteoblasts growth and increases calvarial formation in vivo.3 Some

292

reports claimed that the effects of lactoferrin on proliferation and survival of

293

osteoblasts even exceed other established anabolic factors such as transforming

294

growth factor-β, parathyroid hormone (PTH), amylin, and insulin.19 In this study, we

295

showed that ovotransferrin promoted osteoblast proliferation in a dose-depend


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manner, and reached significance at concentrations of 100 and 1000 µg/mL; in

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comparison, lactoferrin showed the same trend and the same magnitude.7 In general,

298

transferrin family is recognized to have stimulatory effects on proliferation. In

299

addition to the stimulation effects on osteoblasts, lactoferrin was also reported to

300

induce the growth and proliferation of human enterocytes,20 human endometrial

301

stromal cells 21 and human lymphoblastic T cells 22. Laskey et al. 23 gave the evidence

302

that transferrin supported cell proliferation by supplying iron for DNA synthesis. And

303

then transferrin has been studied in different cell lines and showed simulative effects

304

on lymphoblast cells,23 osteoblast cells,24 and oligodendrocytes progenitor cells25 with

305

different magnitude, while showed suppression effects on human hepatoma cells.26

306

Richie et al.27 reported that human milk-derived lactoferrin inhibited the mitogen and

307

alloantigen induced human lymphocytes proliferation. Zhang, Lima, & Rodrigues28

308

also described the potential anticancer activity of lactoferrin by inhibiting the growth

309

of breast cancer cells. Similarly, ovotransferrin has been successfully used in

310

inhibiting the proliferative response of mouse spleen lymphocytes stimulated by

311

lipopolysaccharide and phytohemagglutinin.29 Mizunoya et al.

312

promoting activity of several egg white proteins in C2C12 myoblast cell line and

313

observed that ovalbumin and ovomucoid could stimulate the proliferation of

314

myoblasts while ovotransferrin decreased cell proliferation. Therefore, it seems that

315

the effect of ovotransferrin on cell proliferation as well as the sensitivity of the cells to

316

respond to ovotransferrin vary depending on the cell types.

317

Cell growth to high density often reflects loss of cell cycle control.31 Thus, cell cycle

318

analysis was performed to investigate the effect of ovotransferrin on cell cycle

319

control. Treatment of osteoblasts with ovotransferrin showed controlled cell cycle,

320

stimulated more cells at the S phase but less at G0/G1 phase, indicating active DNA


30

studied the growth

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321

synthesis and cell proliferation, which is consistent with BrdU incorporation results.

322

In addition to osteoblasts,32 controlled cell cycle also occurs in adipocytes,33, 34 as well

323

as mesenchymal cells.35 Cell cycle progression has been described as an important

324

mechanism contributing to bone cell activity. Glucocorticoids, a widely used

325

immunosuppressive and anti-inflammatory drug, has been reported to inhibit bone

326

formation by inhibiting cell cycle progression in osteoblasts and finally lead to

327

osteoporosis and increased fracture risk in chronic glucocorticoid-treated patients.31, 32

328

San Martin et al.36 proposed that deregulation of cell cycle may contribute to the

329

pathogenesis of osteosarcoma. Several osteogenic compounds have also been reported

330

to stimulate cell proliferation through affecting cell cycle. Cao et al.

331

icarii, a prenyl flavonoid from Epimedium considered as a strong candidate used in

332

bone tissue engineering, increased the percentage of S phase cell and decreased G1

333

phase in MC3T3-E1 cells. Polygonum amplexicaule var. sinense, a traditional herb,

334

also showed simulative effects on osteoblasts by affecting cell cycle progression.38

335

The osteoinductive activity of ovotransferrin is also showed by its capacity to

336

substantially promote osteoblast differentiation. The progress of bone formation was

337

classified into three stages: preosteoblast recruitment characterized by cell

338

proliferation and type I collagen secretion, followed by osteoblast precursor

339

differentiation into postmitotic matrix-producing osteoblasts accompanied with

340

downregulated replication, and osteoid mineralization by terminally differentiated

341

osteoblasts associated with increased extracellular matrix accumulation and ALP

342

expression and activity.39, 40 As the most abundant protein among all bone matrixes,

343

type I collagen is conductive to mineral deposition and also binds to non-collagenous

344

matrix proteins to initiate and regulate mineralization.41 Also, ALP is involved in

345

bone matrix formation before crystallization of the calcium and phosphate ions40 and


37

reported that

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346

may play a role in transport of phosphate.42 Therefore, type I collagen and ALP are

347

considered as the most important biomarkers to study osteoblastic activity.

348

Lactoferrin was reported to enhance osteoblast differentiation by increasing the

349

expression and activity of type I collagen and ALP in both mouse osteoblasts

350

MC3T3-E1 and human osteoblast-like cells SaOS-2.7, 43 Calcium phosphates such as

351

α-tricalcium phosphate (α-TCP) and tera-calcium phosphate (TetCP) have been

352

clinically applied as components of bone-substitutes materials. Ehara et al. 41 reported

353

that α-TCP and TetCP stimulated MC3T3-E1 differentiation through increasing ALP

354

activity and type I collagen expression. In this study, ovotransferrin promoted both

355

type I collagen and ALP expression, respectively, in the early and late phases,

356

suggesting that, in addition to promoting matrixes production and osteoblasts

357

differentiation in the early stage, it also affected late phase of calcification of bone

358

matrix.

359

Proteins from transferrin family are suggested to play an important role in cell

360

differentiation. Lactoferrin was reported to stimulate cell differentiation not only in

361

osteoblasts, but also other cell lines such as stromal cells ST2,7 mesenchymal cells,44

362

and human adipose stem cells.45 Macedo et al.

363

transferrin in T lymphocyte differentiation with hypotransferrinaemic mice and found

364

transferrin itself or a pathway triggered by the interaction of transferrin with its

365

receptor is crucial for normal early T-cell differentiation. Paez et al.

366

human transferrin in two oligodendroglial cell lines and found transferrin is required

367

for a more complete maturation of oligodendroglia.

368

As the most direct evidence of calcium deposit capability, mineralization was also

369

investigated to evaluate the effect of ovotransferrin on bone formation. Ovotransferrin

370

was found to increase bone-like tissue formation by MC3T3-E1 cells in a dose-

46

studied the possible involvement of


47

overexpressed

15

Page 17 of 35


371

dependent manner. This might be, in part, due to the increased cell activity and

372

increased production of extra-cellular matrix like type I collagen or other non-

373

collagenous proteins. The other possible reason for the enhanced formation of

374

calcified tissue is that ovotransferrin may function as suppliers of original materials

375

(e.g. amino acids) for bone formation. Ehara et al.

376

function of α-TCP and TetCP as calcium and phosphate materials for bone formation.

377

Above all, ovotransferrin acts to expand the pool of osteoblasts by exerting mitogenic

378

effects as well as driving differentiation of precursors to produce more mature

379

osteoblastic phenotypes capable of promoting bone matrix deposition and

380

mineralization. This anabolic potency suggests that ovotransferrin or its analogs

381

should be explored as therapies for osteoporosis that can restore skeletal strength

382

because most current interventions merely arrest further structural decline.

383

Bone mass is regulated by continuous remodeling, which is based on a delicate

384

balance between osteoblastic bone formation and osteoclastic bone resorption. The

385

maturation and activation of osteoclasts is dependent on the RANK/RANKL/OPG

386

system. Osteoblasts are the primary source of receptor activator of nuclear factor κB

387

ligand (RANKL), which stimulates osteoclast differentiation and activates bone

388

resorption after binding to RANK on osteoprogenitor cells.16 Thus, the osteoblastic

389

bone resorption depends on a delicate balance between the expression of RANKL and

390

OPG. Ovotransferrin inhibited RANKL expression while promoted OPG expression

391

in osteoblasts, indicating its inhibitory effect on bone resorption. Bone resorption

392

prevention was also reported for milk protein,48 soybean protein and soybean

393

isoflavone.49 Lactoferrin reduced bone-resorbing activity was due to inhibited

394

RANKL expression while promoted OPG expression.48 Some drugs, like denosumab,

395

showed prevention of bone resorption by inhibiting RANKL expression as well.50

41


also implied the possible

16


Page 18 of 35

396

Besides the regulation of RANK/RANKL/OPG system, ovotransferrin was shown

397

anti-inflammatory activity.9 Given the inflammatory nature of osteoporotic condition,

398

ovotransferrin may play a role in counterbalancing the inflammation-induced bone

399

resorption. The anti-inflammatory property of ovotransferrin may complement with

400

its osteogenic effect on bone cells.

401

In conclusion, this study demonstrated for the first time that the egg white protein

402

ovotransferrin could function as an osteogenic agent by stimulated proliferation and

403

differentiation of osteoblasts as well as inhibited osteoclastogenesis. These findings

404

suggested an important role of ovotransferrin in mediating bone remodeling and

405

rebuilding the balance between bone formation and resorption. Although further

406

research is warranted to test its in vivo efficacy, ovotransferrin shows the potential as

407

a complementary therapeutic target for alleviation of bone disorders such as

408

osteoporosis as well as an anabolic agent for promotion of bone repair.

409 410

Abbreviations Used

411

BrdU, 5-Bromo-2-deoxyuridine; ALP, alkaline phosphatase; OPG, osteoprotegerin;

412

RANKL, receptor activator of nuclear factor kappa-B ligand; α-MEM, α-Minimum

413

Essential Medium; DTT, dithiothreitol; FBS, fetal bovine serum; PBS, phosphate

414

buffered saline; PI, propidium iodide;

415 416

Acknowledgements

417

This research was funded by grants from Egg Farmers of Canada and Natural

418

Sciences and Engineering Research Council of Canada (NSERC). The funders had no

419

role in the study design, data collection and analysis, decision to publish or

420

preparation of the manuscript.


17

Page 19 of 35


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Reference

422

1. Eastell, R. Prevention and management of osteoporosis. Medicine. 2017, 45(9),

423

565-569.

424

2. International Osteoporosis Foundation. URL: https://www.iofbonehealth.org.

425

Access Date: Nov. 13. 2017

426

3. Naot, D., Grey, A., Reid, I. R., Cornish, J. Lactoferrin - a novel bone growth factor.

427

Clin Med Res. 2005, 3(2), 93-101.

428

4. Chen, J. S., Sambrook, P. N. Antiresorptive therapies for osteoporosis: a clinical

429

overview. Nat Rev Endocrinol. 2012, 8(2), 81-91.

430

5. Rosen, C. J. What’s new with PTH in osteoporosis: where are we and where are we

431

headed? TEM. 2004, 15(5), 229-233.

432

6. Salari, S. P., Abdollahi, M., Larijani, B. Current, new and future treatments of

433

osteoporosis. Rheumato Int. 2011, 31(3): 289-300.

434

7. Cornish, J., Callon, K. E., Naot, D., Palmano, K. P., Banovic, T., Bava, U., Reid, I.

435

R. Lactoferrin is a potent regulator of bone cell activity and increases bone formation

436

in vivo. Endocrinology. 2004, 145(9), 4366-4374.

437

8. Giansanti, F., Leboffe, L., Pitari, G., Ippoliti, R., Antonini, G. Physiological roles

438

of ovotransferrin. Biochim Biophys Acta -General Subjects. 2012, 1820(3): 218-225.

439

9. Xie, H., Huff, G. R., Huff, W. E., Balog, J. M., Rath, N. C. Effects of

440

ovotransferrin on chicken macrophages and heterophil-granulocytes. Dev Comp

441

Immunol. 2002, 26, 805-815.

442

10. Andersen, C. J. Bioactive egg components and inflammation. Nutrients. 2015,

443

7(9), 7889-7913.

444

11. Redlich, K., Smolen, J. S. Inflammatory bone loss: pathogenesis and therapeutic

445

intervention. Nat Rev Drug Discov. 2012, 11(3): 234-250.


18


Page 20 of 35

446

12. Gentili, C., Doliana, R., Bet, P., Campanile, G., Colombatti, A., Cancedda, F. D.,

447

Cancedda, R. Ovotransferrin and ovotransferrin receptor expression during

448

chondrogenesis and endochondral bone formation in developing chick embryo. J Cell

449

Biol. 1994, 124(4), 579-588.

450

13. Guo, H. Y., Jiang, L., Ibrahim, S. A., Zhang, L., Zhang, H., Zhang, M., Ren, F. Z.

451

Orally administered lactoferrin preserves bone mass and microarchitecture in

452

ovariectomized rats. J Nutr. 2009, 139(5): 958-964.

453

14. Wang, L., Chakrabarti, S., Davidge, S. T., Wu, J. Modulatory effects of egg white

454

ovotransferrin-derived tripeptide IRW (Ile-Arg-Trp) on vascular smooth muscle cells

455

against angiotensin II stimulation. J Agric Food Chem. 2016, 64(39), 7342-7347.

456

15. Reinholz, G. G., Getz, B., Pederson, L., Sanders, E. S., Subramaniam, M., Ingle,

457

J. N., Spelsberg, T. C. Bisphosphonates directly regulate cell proliferation,

458

differentiation, and gene expression in human osteoblasts. Cancer Res. 2000, 60(21),

459

6001-6007.

460

16. Rucci, N. Molecular biology of bone remodelling. Clin Cases Miner Bone Metab.

461

2008, 5(1), 49-56.

462

17. Shams-White, M. M., Chung, M., Du, M., Fu, Z., Insogna, K. L., Karlsen, M. C.,

463

LeBoff, M. S., Shapses, S. A., Sackey, J., Wallace, T. C., Weaver, C. M. Dietary

464

protein and bone health: a systematic review and meta-analysis from the National

465

Osteoporosis Foundation. Am J Clin Nutr. 2017, 105(6): 1528-1543.

466

18. Wu, J., Majumder, K., Gibbons, K. Bioactive proteins and peptides from egg

467

proteins. Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals;

468

Mine, Y., Li-Chan, E., Jiang, B.; Blackwell Publishing Ltd. and Institute of Food

469

Technologists. 2011, 247-264.


19

Page 21 of 35


470

19. Cornish, J., Callon, K. E., Lin, C. Q., Xiao, C. L., Gamble, G. D., Cooper, G. J. S.,

471

Reid, I. R. Comparison of the Effects of Calcitonin Gene-Related Peptide and Amylin

472

on Osteoblasts. J Bone Miner Res. 1999, 14(8), 1302-1309.

473

20. Buccigrossi, V., De Marco, G., Bruzzese, E., Ombrato, L., Bracale, I., Polito, G.,

474

Guarino, A. Lactoferrin induces concentration-dependent functional modulation of

475

intestinal proliferation and differentiation. Pediatr Res. 2007, 61(4), 410-414.

476

21. Yanaihara, A., Toma, Y., Saito, H., Yanaihara, T. Cell proliferation effect of

477

lactoferrin in human endometrial stroma cells. Mol Hum Reprod. 2000, 6(5): 469-473

478

22. Bi, B. Y., Lefebvre, A. M., Duś, D., Spik, G., Mazurier, J. Effect of lactoferrin on

479

proliferation and differentiation of the Jurkat human lymphoblastic T cell line. Arch

480

Immunol Ther Exp. 1997, 45(4), 315-320

481

23. Laskey, J., Webb, I., Schulman, H. M., Ponka, P. Evidence that transferrin

482

supports cell proliferation by supplying iron for DNA synthesis. Exp Cell Res. 1988,

483

176(1), 87–95.

484

24. Tsunoi, M., Hakeda, Y., Kurihara, N., Maeda, N., Utsum, N., Kumegawa, M.

485

Effect of transferrin on alkaline phosphatase activity and collagen synthesis in

486

osteoblastic cell derived from newborn mouse calvaria. Exp Cell Res. 1984, 153, 240–

487

244.

488

25. Silvestroff, L., Franco, P. G., Pasquini, J. M. Neural and oligodendrocyte

489

progenitor cells: transferrin effects on cell proliferation. ASN Neuro. 2013, 5(1),

490

e00107.

491

26. Lee, A. W. M., Oates, P. S., Trinder, D. Effects of cell proliferation on the uptake

492

of transferrin-bound iron by human hepatoma cells. Hepatology. 2003, 38(4), 967–

493

977.


20


Page 22 of 35

494

27. Richie, E. R., Hilliard, J. K., Gilmore, R., Gillespie, D. J. Human milk-derived

495

lactoferrin inhibits mitogen and alloantigen induced human lymphocyte proliferation.

496

J Reprod Immunol. 1987, 12(2), 137–148.

497

28. Zhang, Y., Lima, C. F., Rodrigues, L. R. In vitro evaluation of bovine lactoferrin

498

potential as an anticancer agent. Int Dairy J. 2015, 40, 6–15.

499

29. Otani, H., Odashima, M. Inhibition of proliferative responses of mouse spleen

500

lymphocytes by lacto- and ovotransferrins. Food Agric Immunol. 1997, 9(3), 193-201

501

30. Mizunoya, W., Tashima, A., Sato, Y., Tatsumi, R., Ikeuchi, Y. The growth-

502

promoting activity of egg white proteins in the C2C12 myoblast cell line. Anim Sci J.

503

2015, 86(2), 194-199.

504

31. Smith, E., Coetzee, G. A., Frenkel, B. Glucocorticoids inhibit cell cycle

505

progression in differentiating osteoblasts via glycogen synthase kinase-3β. J Biol

506

Chem. 2002, 277(20), 18191–18197.

507

32. Smith, E., Redman, R. A., Logg, C. R., Coetzee, G. A., Kasahara, N., Frenkel, B.

508

Glucocorticoids

509

Dissociation of cyclin A-cyclin-dependent kinase 2 from E2F4-p130 complexes. J

510

Biol Chem. 2000, 275(26), 19992–20001.

511

33. Richon, V. M., Lyle, R. E., McGehee, R. E. Regulation and expression of

512

retinoblastoma proteins p107 and p130 during 3T3-L1 adipocyte differentiation. J

513

Biol Chem. 1997, 272(15), 10117–10124.

514

34. Yeh, W. C., Bierert, B. E., Mcknightt, S. L. Rapamycin inhibits clonal expansion

515

and adipogenic differentiation of 3T3-L1 cells. Proc Natl Acad Sci. 1995,

516

92(November), 11086–11090.

517

35. Hall, B. K., Miyake, T. All for one and one for all: Condensations and the

518

initiation of skeletal development. Bioessays. 2000, 22(2), 138–147.

inhibit

developmental

stage-specific


osteoblast

cell

cycle:

21

Page 23 of 35


519

36. San Martin, I. A., Varela, N., Gaete, M., Villegas, K., Osorio, M., Tapia, J. C.,

520

Galindo, M. Impaired cell cycle regulation of the osteoblast-related heterodimeric

521

transcription factor Runx2-Cbfβ in osteosarcoma cells. J Cell Physiol. 2009, 221(3),

522

560-571.

523

37. Cao, H., Ke, Y., Zhang, Y., Zhang, C. J., Qian, W., Zhang, G. L. Icariin stimulates

524

MC3T3-E1 cell proliferation and differentiation through up-regulation of bone

525

morphogenetic protein-2. Int J Mol Med. 2012, 29(3), 435-439.

526

38. Xiang, M. X., Su, H. W., Hu, J., Yan, Y. J. Stimulative effects of Polygonum

527

amplexicaule var. sinense on osteoblastic MC3T3-E1 cells. Pharm Biol. 2011, 49(10),

528

1091-1096.

529

39. Owen, T. A., Aronow, M., Shalhoub, V., Barone, L. M., Wilming, L., Tassinari,

530

M. S., Stein, G. S. Progressive development of the rat osteoblast phenotype In vitro -

531

reciprocal relationships in expression of genes associated with osteoblast proliferation

532

and differentiation during formation of the bone extracellular-matrix. J Cell Physiol.

533

1990, 143(3), 420–430.

534

40. Lee, Y. K., Song, J., Lee, S. B., Kim, K. M., Choi, S. H., Kim, C. K., Kim, K. N.

535

Proliferation, differentiation, and calcification of preosteoblast-like MC3T3-E1 cells

536

cultured onto noncryrstalline calcium phosphate glass. J Biomed Mater Res A. 2004,

537

69(1), 188-195.

538

41. Ehara, A., Ogata, K., Imazato, S., Ebisu, S., Nakano, T., Umakoshi, Y. Effects of

539

α-TCP and TetCP on MC3T3-E1 proliferation, differentiation and mineralization.

540

Biomaterials. 2003, 24(5), 831-836.

541

42. Millán, J. L., Whyte, M. P. Alkaline phosphatase and hypophosphatasia. Calcif

542

Tissue Int. 2016, 98, 398-416.


22


Page 24 of 35

543

43. Fritsche, K. L., Setchell, K. D. R., Conneely, O. M., Beck, M. A., Prasad, K. N.,

544

Keen, C. L., Klurfeld, D. M. Anti-inflammatory activities of lactoferrin. J Am Coll

545

Nutr. 2001, 20(5 SUPPL.), 396S-397S.

546

44. Yagi, M., Suzuki, N., Takayama, T., Arisue, M., Kodama, T., Yoda, Y., Ito, K.

547

Effects of lactoferrin on the differentiation of pluripotent mesenchymal cells. Cell

548

Biol Int. 2009, 33(3), 283–289.

549

45. Ying, X., Cheng, S., Wang, W., Lin, Z., Chen, Q., Zhang, W., Zhu Lu, C. Effect

550

of lactoferrin on osteogenic differentiation of human adipose stem cells. Int Orthop.

551

2012, 36(3), 647–653.

552

46. Macedo, M. F., De Sousa, M., Ned, R. M., Mascarenhas, C., Andrews, N. C.,

553

Correia-Neves, M. Transferrin is required for early T-cell differentiation.

554

Immunology. 2004, 112(4), 543–549.

555

47. Paez, P. M., García, C. I., Campagnoni, A. T., Soto, E. F., Pasquini, J. M.

556

Overexpression of human transferrin in two oligodendroglial cell lines enhances their

557

differentiation. Glia. 2005, 52(1), 1–15.

558

48. Lorget, F., Clough, J., Oliveira, M., Daury, M. C., Sabokbar, A., Offord, E.

559

Lactoferrin reduces in vitro osteoclast differentiation and resorbing activity. Biochem

560

Biophys Res Commun. 2002, 296(2), 261–266.

561

49. Messina, M., Messina, V. Soyfoods, soybean isoflavones, and bone health: a brief

562

overview. J Ren Nutr. 2000, 10(2), 63–68.

563

50. Hamdy, N. A. T. Denosumab: RANKL inhibition in the management of bone loss.

564

Drugs Today. 2008, 44(1), 7.

565 566 567


23

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568

Figure captions

569

Fig. 1 Effects of egg white ovotransferrin on cell proliferation in osteoblast cell

570

MC3T3-E1

571

MC3T3-E1 cells were seeded into 48-well plate for 4 h and treated with 1000 µg/mL

572

lactoferrin or different concentrations (1000, 100, 10, and 1 µg/mL) of ovotransferrin

573

for 24 h prior to adding BrdU reagent, fixed, permeabilized and immunostained. Cell

574

nuclei were counter-stained with Hoechst33342 dye. Percentage of BrdU(+) nuclei

575

were counted in 3 random fields per group and their mean value determined. Data are

576

mean±SEM from 4-6 independent experiments. *, ** and *** indicate p

Egg White Ovotransferrin Shows Osteogenic Activity in Osteoblast Cells

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