Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : March 31, 2020
Accepted : April 20, 2020
Published online : June 30, 2020
doi:10.24659/gsr.7. 2_142

Original article

Effect of glycated human serum albumin on BMP2-induced osteoblastgenesis
in C2C12 cells
Rina Yamamoto 1), Wakako Takabe 1, 2), Masayuki Yagi 2), Yoshikazu Yonei 1)
1) Anti-Aging Medical Research Center and Glycation Stress Research Center, Graduate School of Life and Medical Sciences,
Doshisha University, Kyoto, Japan
2) Department of Materials and Life Science​, Faculty of Science and Technology​, Shizuoka Institute of Science and Technology​,
Shizuoka, Japan

Abstract

Aim: Bone remodeling by which mature bone tissue is removed by osteoclast and new bone tissue is formed by osteoblast, is
important to maintain bone mass and quality. Imbalanced bone homeostasis increases fracture risks and lead to osteoporosis.
Accumulation of advanced glycation end products (AGEs) plays a role in diabetic complications and patients with diabetes
mellitus have a higher incidence rate of osteoporosis than healthy subjects. In this research, we investigated the effect of AGEs
on osteoblast differentiation (osteoblastgenesis).
Methods: For osteoblstgenesis, mouse myoblast C2C12 cells were stimulated with bone morphogenetic protein 2 (BMP2). To
determine the effect of AGEs on osteoblastgenesis, cells were treated with BMP2 with or without AGEs, which were formedby glyceraldehyde and human serum albumin (HSA-glycer), and we performed quantitative real-time PCR analysis against
alkarine phosphatase (ALP) and osteocalcin (OC), marker of osteoblastgenesis. We further investigated the effect of AGEs on
the expression of runt related transcription factor 2 (Runx2) and osterix, major transcription factors of osteoblastgenesis. To
see the effect of AGEs on Smad pathway, which partially regulates the BMP2 signaling pathway, western blot analyses against
phosphorylation of Smad 1/5/9 were examined. Finally, to evaluate the effect of AGEs on bone turnover, we investigated theratio of osteoprotegerin (OPG) and receptor activator of NF- кB ligand (RANKL), a major indicator of bone turnover.
Results: HSA-glycer inhibited BMP2-induced expression of ALP and OC. The expression of transcription factors andphosphorylation of Smad 1/5/9 were partially suppressed by HSA-glycer. Decreasing RANKL/OPG ratios by HSA-glycer
suggests that AGEs are likely to delay bone turnover.
Conclusion: Our findings indicate that AGEs may inhibit BMP2-induced osteoblastgenesis via alteration of the Smadpathway.

KEY WORDS: glycative stress, AGEs, bone morphogenetic protein 2, osteoblastgenesis, osteoporosis, bone turnover

Introduction
Osteoporosis is a disease which is characterized by low
bone mass and deterioration of the quality of bone tissue 1). In
Japan, over twelve million people suffered from osteoporosis
in 2015 2) and patients faced fracture risks, leading to the lossof their quality of life. While Osteoporosis can affect people
at almost any age, it is most common among those 50 yearsof age or older, especially postmenopausal women 3, 4). Other
than the age, Schwartz et al. demonstrated that older womenwith type 2 diabetes have higher fracture rates than non-diabetic women 5, 6). Both bone mass (or bone mineral density,
BMD) and bone quality are important for bone strength,
however, meta analyses found that type 2 diabetic patients
have normal or high BMD compared to healthy subjects 7).
Thus, plenty of studies have been conducted to clarify thecontribution of diabetes toward the loss of bone quality.
Diabetes mellitus is a chronic symptom in which thelevels of glucose in the blood are too high. Excess blood sugar
reacts with proteins non-enzymatically and forms advanced
glycation end products (AGEs). Several lines of evidence haveshown that the accumulation of AGEs is implicated in multiple

Corresponding author: Wakako Takabe, PhD
Department of Materials and Life Science​, Faculty of Science and Technology​,
Shizuoka Institute of Science and Technology​
2200 -2 Toyosawa, Fukuroi, Shizuoka 4 37-8555, Japan
TEL: +81-538-45- 0164 e-mail: takabe.wakako@sist.ac.jp

Co-authors: Yamamoto R, rina12210210@gmail.com;
Yagi M, myagi@mail.doshisha.ac.jp; Yonei Y, yyonei@mail.doshisha.ac.jp_ 14 2 _Glycative Stress Research 2020; 7 ( 2) : 142-151(c) Society for Glycative Stress ResearchGlycative Stress Researchdiseases 8 -13), including osteoporosis 14). Recently, Takeuchi
et al. demonstrated that glyceraldehyde, which is synthesized
in fructolysis, formed more highly toxic AGEs than glucose 15).
To maintain the mechanical properties of bones, type I collagen
which is a major protein in bone tissue form the enzymaticcross-link as a post- transcriptional modification 16). While,patients with osteoporosis and diabetes mellitus, AGEs playa role for forming dispensable cross-link in bone collagen,leading to the loss of bone resilience 17). Moreover, Shiraki M.
et al. reported that pentosidine, which is a fluorescent crosslinking type of AGEs, is an independent risk factor for
osteoporotic incidence of vertebral fractures 18). These data
indicate that accumulation of AGEs may be involved in theloss of bone quality.
Bone remodeling is also important to maintain bonehomeostasis. Mature bone tissue is removed by osteoclast
and new bone tissue is formed by osteoblast, and about 10 %of bone material is renewed each year 19). The imbalance
between these two processes results in significant boneloss and decreased bone quality. In our previous report,
we demonstrated that AGEs, made from human serum
albumin (HSA) and glyceraldehyde, inhibited the receptor
activator of nuclear factor kappa-B ligand (RANKL)-induced
osteoclast differentiation (osteoclastgenesis) in RAW264.7
cells 20). Except for bone absorption, mature osteoclasts
secrete cytokines which regulate osteoblast differentiation(osteoblastgenesis) such as transforming growth factor beta
(TGF-β) and bone morphogenetic protein 2 (BMP2) 21), thus,
our previous data suggest that AGEs may delay bone remodelingvia inhibition both osteoclastgenesis and osteoblastgenesis,
leading to loss of bone quality.
BMP belongs to the large super family of TGF-ß and
BMPs, including BMP2, are well known to be implicated inosteoblastgenesis 22). BMPs induce osteoblastgenesis throughboth Smad-dependent or – independent signal pathways 23). In
Smad dependent pathways, when the secreted BMP2 binds totype II receptor, type I receptor kinase is phosphorylated by
the activated type II receptor kinase, and then, the signalingtransduces to Smad proteins. Phosphorylated Smad 1/5/8
regulate expression of osteoblast specific proteins such asalkaline phosphatase (ALP) and osteocalcin (OC) and theosteogenic transcription factor such as Runx2, osterix andDlx5 24-26).
Several studies have been demonstrated that AGEs
induced apoptosis in osteoblast cells via oxidative stress 27-29)
and McCarthy et al. reported that AGEs made from serum
bovine albumin (BSA) and glucose (AGE-BSA) inhibited
cell proliferation and ALP activity in two different linesof osteoblast-like cells after several days incubation 30). In
the present study, we are focusing on the effect of AGEs on
BMP2-induced osteoblastgenesis without cytotoxicity toclarify if AGEs alter osteoblastgenesis independent of cell
damage.

Materials and Methods
Materials

BMP2 Human/Rat/Mouse recombinant protein was
obtained from R&D systems ( Minneapolis, MN, USA).

Human serum albumin (HSA) was purchased from SigmaAldrich (St. Louis, MO, USA). All other chemicals wereobtained from Wako (Osaka, Japan) as analytical grade.

Cell culture
C2C12 cells, mouse myoblast, were purchased from
American Type Culture Collection (Manassas, VA, USA).
Cells were maintained in Dulbecco's Modified Eagle
Medium with high glucose (DMEM, Wako) containing 10%
fetal bovine serum (Sigma-Aldrich) and antibiotics (Wako),
and grown at 37 °C under an atmosphere of 5% CO 2 .

Preparation of glycated proteins
Glycated proteins were made by human serum albumin(HSA) and glyceraldehyde. HSA (8 mg/mL) and 33 mmol/L
glyceraldehyde in 50 mmol/L phosphate buffer (PB, pH 7.4)
were incubated at 60 °C for 40 h. We also prepared heated
HSA, by which HSA without glyceraldehyde were incubatedat 60°C for 40 h as a control. To remove remaining unreacted
glycating agents, reaction mixtures were ultrafiltrated using
centrifugal filter units (10K, Merck Millipore Burlington,
MS, USA) and were washed three times using distilled water.

Determination of Cell viability
To determine the cell viability, a WST- 8 assay was
performed using Cell Counting Kit-8 (Dojindo, Kumamoto,Japan) according to the manufacturer’s protocol. Data wereexpressed as percentage of water or vehicle control treatedcells.

RNA extraction and Quantitative real-time
PCR analysis
C2C12 cells were seeded at 4 x 10 3 cells/well in a 24well plate. Twenty-four hours later, cells were stimulatedwith BMP2, glycated HSA for indicated concentration andtime period. Then, cells were washed by PBS once, then, 400
μL ISOGEN II (Nippon Gene, Tokyo, Japan) were used for
RNA extraction according to the manufacturer’s protocol.
Amount of RNA was measured by Nanodrop 2000 (Thermo
Fisher Scientific, Waltham, MA USA). A five-hundred ng
total RNA was reverse-transcribed with PrimeScript T M RT
Master Mix (Takara Bio Inc., Shiga, Japan) using Applied
Biosystems 2720 Thermal cycler (Thermo Fisher Scientific,MA). Quantitative real-time PCR (qPCR) was performed
with a Thunderbird T M SYBR qPCR mix (Toyobo Co., Osaka,
Japan) according to the manufacturer’s protocol with genespecific primers (Thermo Fisher Scientific). The primers
were used are as follows: Alkaline phosphatase (ALP), 5' GAT CAT TCC CAC GTT TTC AC - 3' (forward), 5' - TGC
GGG CTT GTG GGA CCT GC -3' (reverse); Osteocalcin(OC), 5' - CCT CTC GAC CCG ACT GCA GAT C - 3'
(forward), 5' - AGC TGC AAG CTC TCT GTA ACC ATG
AC - 3' (reverse); Osterix, 5' - CGT CCT CTC TGC TTG
AGG AA -3' (forward), 5' - GGG CTG AAA GGT CAG
CGT AT - 3' (reverse); Runx2, 5’- CAG TCC CAA CTT
CCT GTG CT -3' (forward), 5' - CCC ATC TGG TAC
CTC TCC GA -3' (reverse); GAPDH, 5' - TGA AGG TCG_ 143 _

Effect of Glycated Protein on Osteoblastgenesis

GTG TGA ACG GAT TGG C -3' (forward), 5' - CAT GTA
GGC CAT GAG GTC CAC CAC -3' (reverse); Receptor
activator of N F-к B ligand (RANKL), 5' - AGC CAT T TG
CAC ACC TCA CC -3' (forward), 5' -AAG CAA ATG ATT
GGC GTA CAG G -3' (reverse); Osteoprotegerin (OPG), 5' AGT GTG AGG AAG GGC GTT AC -3' (forward), 5' - AAT
GTG CTG CAG TTC GTG TG -3' (reverse).

ALP staining
C2C12 cells were seeded at 8 x 10 3 cells/well in a 12well plate. Twenty-four hours later, cells were stimulatedwith or without 400 ng/mL BMP2 and glycated HSA for 72 h.
To examine the alkaline phosphatase activity, Alkaline
Phosphatase Staining Kit (Cosmo Bio Ltd. Tokyo, Japan) wasused as according to the manufacturer’s protocol. Briefly,
cells were washed with PBS three times, then, fixed with
10 % formaldehyde for 10 min at room temperature. Afterwashing with distilled water, the cells were incubated with
ALP activity solution for 20 min at 37 °C. The cell images
were captured by inverted microscope (CKX41, OLYMPUS
corp. Tokyo, Japan) with digital camera (DP21, OLYMPUS
corp.) with Cell Sense software (OLYMPUS corp.).

Western blot analysis
C2C12 cells were seeded in a 12-well plate at a densityof 5 × 10 4 cells/well and incubated for 24 h. Then, they
were treated with or without 400 ng/mL BMP2, glycated orheated-HSA for the indicated time periods. The entire celllysate was extracted with RIPA buffer containing 50 mmol/L
Tris-HC l (pH 7.4), 150 mmol/L NaCl, 0.1% SDS, 1%
Triton X-100 with complete protease inhibitor (Wako) andphosphatase inhibitor (Roche Applied Science, Penzberg,
Germany). Cell lysates were electrophoresed by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) (8% polyacrylamide), then proteins were transferred
to a polyvinylidene difluoride (PVDF) membrane followedby blocking with a 5% skim milk solution in TBS-T. Then,the membranes were immunoblotted with each primary
antibody. The antibodies against Runx2, phospho-Smad
1/5/9 and Smad 1 were from cell signaling technology(Danvers, MA, USA), anti-GAPDH was purchased from
Abcam (Cambridge, UK), and anti-β-actin was obtained
from Sigma-Aldrich. The antigen- antibody complexes werevisualized with the appropriate secondary antibodies (Santa
Cruz Biotechnology, CA, USA) and chemiluminescencehorseradish peroxidase (HRP) substrate along with detection
system, as recommended by the manufacturer. The results
illustrated in each figure are representative of three
independent experiments. Image J was used to measure theoptical density of the protein bands.

Statistics.
The statistical analysis was performed by TukeyKramer’s multiple comparison test. Differences wereconsidered significant at p values less than 0.05.

Results
Osteoblastgenesis were induced by BMP2 intime-and dose-dependent manner in C2C12 cells.
First of all, we estimated the condition of
osteoblastgenesis in C2C12 cells. Cells were stimulatedwith 4 00 ng/mL BMP2 for the indicated time periods
and we performed qPCR analyses ( Fig. 1-a). Both mRNAlevels of alkarine phosphatase (ALP), early markers ofosteoblastgenesis and osteocalcin (OC), and markers ofmature osteoblasts were induced in a time-dependent
manner. Next, C2C12 cells were incubated with an indicatedconcentration of BMP2 for 72 h, then, qPCR analyses
were conducted. Both mRNA levels of ALP and OC were
induced in a dose-dependent manner ( Fig. 1-b). Especiallyover 300 ng/mL treatment, BMP2 induced those mRNAlevels significantly. Furthermore, to examine the effect of
BMP2 on ALP activity in C2C12 cells, we performed an
ALP staining assay. As shown in Fig. 1-c, ALP activity was
induced by 400 ng/mL BMP2 for 72 h. Thus, to investigate
the effect of glycated proteins on osteoblastgenesis, C2C12
cells were stimulated with 400 ng/mL BMP2 for 72 h.

The effect of glycated HSA on BMP2-induced
osteoblastgenesis.
Initially, we evaluated the cytotoxicity of glycatedproteins using WST-8 assay. Seventy-two hours treatmentof glyceraldehyde-derived glycated HSA (HSA-glycer) did
not induce cell death up to 400 μg/mL (Fig. 2-a). Next, toinvestigate the effect of HSA-glycer on osteoblastegenesis,
we treated with BMP2 and HSA-glycer at the same timein C2C12 cells. Cells were treated with 400 ng/mL BMP2
and indicated concentration of HSA-glycer for 72 h, then, we
performed qPCR analyses. Both mRNA levels of BMP2induced ALP and OC were inhibited by HSA-glycer (Fig. 2-b).
We also performed ALP staining assay, HSA-glycer inhibited
BMP2-induced ALP activity (Fig. 2-c). These data indicatedthat HSA-glycer mediate BMP2-induced osteoblastgenesis.

Glycated HSA altered the expression oftranscription factors that regulate osteoblastgenesis.
Several major transcription factors that regulate
osteoblastgenesis have been identified, such as runt relatedtranscription factor 2 (Runx2) and osterix 25). In thisexperiment, we investigated whether HSA-glycer mediate
the expression levels of Runx2 and osterix. As shown in
Fig. 3-a, 400 ng/mL BMP2 induced mRNA levels ofosterix and it was inhibited by 400 μg/mL HSA-glycer.
HSA-heated, by which HSA were incubated at 60 °C for
40 h without glyceraldehyde, did not affect BMP2-inducedmRNA expression of osterix. Also, BMP2-induced mRNAlevels of Runx2 were inhibited by HSA-glycer but not HSAheated (Fig. 3-b). We further determined the protein levelsof Runx2 and it was also inhibited by HSA-glycer, but not
HSA-heated (Fig. 3-c). These data indicated that HSAglycer affect the expression of transcription factors which
regulate osteoblastgenesis.

_ 14 4 _Glycative Stress Researcha)c)Relative
copy number
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number4［ / 104［］/GAPDH10 4 GAPDH］［ / 10 GAPDH ］

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Fig. 1. BMP2 increases the expression levels of ALP and OC in C2C12 cells.
(a, b) Sucrose (Suc). Time-dependent induction of ALP and OC, the markers of osteoblastgenesis, by BMP2. C2C12 cells weretreated with 400 ng/mL BMP2 for up to 72 h. Quantitative real-time PCR (qPCR) analyses were performed for (a) ALP and (b) OC.
All data obtained were normalized by GAPDH and shown as the mean ± SD (n = 6). ** p < 0.01 vs. Time 0. (c, d) Concentration-

dependent induction of ALP and OC by BMP2. C2C12 cells were treated with up to 400 ng/mL BMP2 for 72 h. The expression levelsof (c) ALP and (d) OC. All data obtained were normalized by GAPDH and shown as the mean ± SD (n = 6). * p < 0.05, ** p < 0.01
vs. 0 ng/mL BMP2. (e) BMP2 induced ALP activity in C2C12 cells. C2C12 cells were treated with 400 ng/mL BMP2 for 72 h. ALP
staining were performed to determine ALP activity. The images shown are representative of three independent experiments withsimilar results. Bar, 100 μm. BMP2, bone morphogenetic protein 2; ALP, alkaline phosphatase; OC, osteocalcin; PCR, polymerase
chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SD, standard deviation.

_ 145 _

Effect of Glycated Protein on Osteoblastgenesis

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Fig. 2. Glyceraldehyde-derived glycated HSA suppress BMP2-induced ALP and OC in C2C12 cells.
(a, b, c) C2C12 cells were treated with glyceraldehyde-derived glycated HSA (HSA-glycer) up to 400 μg/mL for 72 h with or without
400 ng/mL BMP2. (a) WST-8 assay was performed to determine cytotoxicity of HSA-glycer (n = 6). (b, c) Effect of HSA-glycer on
BMP-2 -induced mRNA expression of (b) ALP and (c) OC. All data obtained were normalized by GAPDH and shown as the mean ±SD (n=6). ** p < 0.01 vs. without BMP2. † p < 0.05, †† p < 0.01 vs. BMP2 without HSA-glycer. (d) Effect of glycated HSA on BMP2 -induced ALP activity. C2C12 cells were treated with 400 μg/mL HSA-glycer or HSA-heated and 400 ng/mL BMP2 for 72 h. ALP
activity was examined by ALP staining assay. The images shown are representative of three independent experiments with similar
results. Bar, 10 0 μm. HSA, human serum albumin; BMP2, bone morphogenetic protein 2; ALP, alkaline phosphatase; OC, osteocalcin;
WST- 8, 2- ( 2- methoxy- 4 - nitrophenyl) - 3 - (4 - nitrophenyl) -5 - ( 2, 4 - disulfophenyl)-2H - tetrazolium; GAPDH, glyceraldehyde
3-phosphate dehydrogenase; SD, standard deviation.

_ 14 6 _

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Fig. 3. Glyceraldehyde-derived glycated HSA inhibit the expression of transcription
factors regulate osteoblastgenesis.

Fold change

BMP2
C2C12 cells were treated with 400 μg/mL
HSA-glycer or HSA-heated and 400 ng/mL BMP2 for 48 h. qPCR analysis were performed for
1.5(a) Osterix and (b) Runx2. All data obtained were normalized
by GAPDH and shown as the mean ± SD (n = 4). * p < 0.05, ** p < 0.01
vs. no addition. † p < 0.05,
††
p
<
0.01
vs.
BMP2.
(c)
Samples(30 μg of proteins) of the crude extract were used for western blot analysis
BMP2using antibody against Runx2. The bars show the mean ± SD (n = 3) of the ratio against BMP2. ** p < 0.01 vs. BMP2. †† p < 0.01 vs.
1
BMP2
with HSA-heated.
HSA, humanserum albumin; glycer,
glyceraldehyde; BMP 2, bone morphogenetic protein 2; PCR, polymerase
no addition
HSA-heated
HSA-glycer＊＊
chain reaction; qPCR, quantitative real-time PCR; GAPDH, glyceraldehyde 3-phosphate dehydrogenase;SD, standard deviation.

Runx2
GAPDH

Glycated HSA inhibited BMP2-induced
phosphorylation of Smad in C2C12 cells.

††

0.5
0

d
osteoblastgenesis.
When eweargue the bone remodeling,
r
te
on
iti
ea
lyca key incident. Osteoblasts secrete
osteoclastgenesis
is
also
d
h
g
ad
AA(OPG)and receptor activator of NF- кB
The BMP2 signaling pathway is partially regulated by no osteoprotegerin
HS
HS

the Smad pathway 31). In canonical BMP signaling cascade,BMP receptor I and II form dimer and type II receptor
phosphorylates and activates the type I receptor. Then, thetype I receptor phosphorylates Smad 1/5/8 (Smad 8 is also
known as Smad 9). In this experiment, we investigated theeffect of HSA-glycer on BMP2-induced Smad pathway.
Twenty-four hours treatment of 400 ng/mL BMP2 induced
phosphorylation of Smad 1/5/9 and it was significantly
inhibited by co-treatment of 400 μg/mL HSA-glycer (Fig. 4 ).
These data indicate that AGEs, which were contained in
HSA-glycer, may affect early stages of osteoblastgenesis.

Effect of glycated proteins on BMP2-induced
cytokines regulate bone remodeling.
Ourdata suggests that

HSA-glycer

inhibits

ligand (RANKL) which are cytokines that play an importantrole for BMP2
the osteoclastgenesis. The binding of RANKL to its
receptor RANK in osteoclast precursors cells triggers theoccurrence of osteoclastgenesis, and OPG acts as a decoy
receptor, preventing the binding of RANKL on RANK.
Because of the opposite effects of OPG and RANKL
on osteoclastgenesis, the RANKL/OPG ratio is a major
indicator of bone turnover. We examined qPCR analyses todetermine the effect of glycated HSA on the expression levelsof OPG and RANKL. Cells were treated with 400 ng/mL
BMP2 and 400 μg/mL HSA-glycer for 48 h, then qPCR
analysis were performed. As shown in Figs. 5-a and b, HSAglycer down-regulated the mRNA levels of RANKL (Fig.
5-a), but not OPG (Fig. 5-b). Thus, HSA-glycer decreased
RANKL/OPG ratios means HSA-glycer may also delay
osteoclastgenesis.

_ 147 _

no

d
ad

iti

on

H

Relative copy
［ / 10 6 GAP††

60

Smad 1 ＊＊40

Effect of Glycated Protein on Osteoblastgenesis

ß -actin

20
0

25

+ +
+ - +

-

-

-

Fold change

BMP2
HSA-glycer

Relative copy number［ / 10 6 GAPDH ］

pSmad 1/5/9
Smad 1
ß -actin

20 n o

a

i
dd

n

at

e
＊＊
-h

HS

15

ed

† HS

A

A

l
-g

yc

er

＊＊

BMP2

10
2500
5
2000
0
1500

BMP2

1000

HSA-glycer

25

tio

-

-

+

+

-

+

-

+

500

Fig. 4. Effect of glyceraldehyde-derived glycated HSA on BMP2-induced Smad phosphorylation.
＊＊0†

Fold change

C2C12 20
cells were treated with 400 μg/mL HSA-glycer and 400 ng/mL BMP2 for 24 h. Samples (20 μg of proteins) of the crude
extract were used for western blot analysis using antibody against phosphor-Smad 1/5/9, Smad
1 and β-actin. The
d bars show the e r＊＊teon
iti BMP2. HSA, humanmean ±15SD (n = 3) of the ratio against no addition. ** p < 0.01 vs. no addition. † p < 0.05 vs.
ea serum albumin;
l yc
d
h
-g
d
a
A
A
glycer, glyceraldehyde; BMP2, bone morphogenetic protein 2; SD, standard deviation. o
HS
HS
n
10

c)

Relativecopy
copynumber
numberRelative
106 6GAPDHGAPDH］］［［/ /10

5
100
100

0

80BMP280

HSA-glycer
60
60

-

-

+

-

+

-

＊＊
＊＊

20
20

BMP2
2
1.5

††

＊＊

1
0.5
0

on
ittiio ndiadd dn oo anRelativecopy
copynumber
numberRelative
106 6GAPDHGAPDH］］［［/ /10

††
+ ††

40
40

0
0

b)

+

Fold changea)edat e dh eeat
A -h
HSSA
H

er
yc e r
gllyc
A -g
HSSA
H

no

d
ad

iti

on
HS

h
A-

ea

te

d
HS

g
A-

l yc

er

BMP2

BMP2
BMP2
2500
2500
2000
2000
1500
1500
1000
1000
500
500
0
0
n
tio n
diitioda ddn oo aned
at e dh eeat
A -h
HSSA
H

er
yc e r
gllyc
A -g
HSSA
H

BMP2
BMP2
2

FoldchangechangeFold

2Fig. 5. Glyceraldehyde-derived
glycated proteins inhibited BMP2-induced RANKL, but not OPG.

C2C12 cells were treated with 400 μg/mL HSA-glycer and 400 ng/mL BMP2 for 48 h. qPCR analyses were performed for (a) RANKL and
1.5 (c) The ratio of RANKL /OPG. All data obtained were normalized by GAPDH and shown as the mean ± SD (n = 4). ** p < 0.01(b) OPG.
1.5
vs. BMP2. †† p < 0.01 vs. BMP2 with heated-HSA. HSA, human serum albumin; BMP2, bone morphogenetic protein 2; RANKL, receptor
GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SD, standard deviation.
activator of NF- кB ligand; OPG, osteoprotegerin;＊＊
1
＊＊
1

††
††

0.5
0.5

_ 14 8 _

0
0
n
io n

d
te d

r
ce r

Glycative Stress Research

Discussion
Osteoporosis is caused by a decrease in bone strength
due to bone mass reduction and deterioration of bone quality,and furthermore leads to fractures and lowers quality oflife in the elderly. Since osteoporosis is prevalent in diabetic
patients as well as in postmenopausal women, studies havebeen conducted on the relationship between osteoporosis andglycative stress, a condition in which a hyperglycemic statecontinues and AGEs are produced in large quantities. For the
maintenance of bone quality, the balance between osteoblasts
and osteoclasts is important for smooth remodeling. In
this study, we elucidated the effects of glycation stress onbone quality using C2C12 cells, focusing on the process ofosteoblast differentiation.
C2C12 is an immortalized mouse myoblast cell line,
and Katagiri T. et al. reported that BMP2 differentiate
C2C12 cells to osteoblast cells 32). As shown in Fig. 1, BMP2dependent calcification and expression of ALP and OC,
osteoblast markers, was observed in C2C12 cells. Under these
conditions, the effects of glycated proteins were verified.
The AGE-related substances used this time areglycelaldehyde 33) generated during the process of fructose
metabolism and glycelaldehyde-derived glycated protein(HSA-glycer) generated from HSA, which is the mostabundant protein in human blood. As a result, HSA-glycer
suppressed BMP2-induced osteoblast differentiation markers
and ALP activity (Fig. 2).
Other than our study, it has been reported thatglyceraldehyde-derived AGEs reacted with bovine serum
albumin (BSA) suppresses OC mRNA expression, ALP
activity, and calcification in ST2 cells and stromal cells
isolated from mouse bone marrow 34). Also, with regard toglycolaldehyde, Notsu M. et al. 35) have reported that AGEs
obtained by reacting with BSA suppresses calcificationand ST mRNA expression in ST2. These reports indicatethat AGEs derived from reaction between serum albumin
and glyceraldehyde / glycolaldehyde induce osteoblast
differentiation, although the cells used were different fromthose used in this study.
AGEs derived from glyceraldehyde and glycolaldehyde
are also called toxic AGEs and have been reported to exhibit
cytotoxicity 36, 37). In ST2 cells, AGEs were shown to be
involved not only in osteoblast differentiation but also in
induction of cell death 34, 35), while the glycated protein usedin this experiment did not show cytotoxicity (Fig. 2-a). Thismay be mainly due to the different conditions in the glycative
reaction. Our results suggest that the effect of cytotoxicity on
the suppression of osteoblast differentiation is small.
Runx2 and osterix are known as transcription factors
essential for osteoblast differentiation. Runx2 has beenreported to be involved in the regulation of OC and collagentype I expression 38). Osterix is ​​known to be regulated by
Runx2 39), and is, in the report of Matsubara T. et al. 40), ​​also
expressed in mesenchymal cells isolated from Runx2deficient mice. These findings indicate that the osterix
expression mechanism has both Runx2-dependent and-independent pathways.
In this study, we examined the effect of HSA-glycer
on Runx2 and osterix expression (Fig. 3). As a result,Runx2 expression was weakly inhibited by about 20 %due to glyceraldehyde-derived AGEs in both mRNA andprotein (Fig. 3-b, c). However, osterix mRNA expression was
suppressed by more than 50% (Fig. 3-a). Therefore, it ispossible that AGEs may regulate the induction mechanism of
Runx2-independent osterix expression.
Miranda, C. et al. 41) also conducted clinical trials inpatients with both type 2 diabetes and osteoporosis, in which
diabetic patients have been observed to suppress theexpression of Runx2 and osterix compared to healthy subjects.
This also suggests that AGEs, which are accumulating
in diabetic patients, may affect bone metabolism throughabnormalities of these transcription factors.
In this study, it was shown that HSA-glycer suppresses
osteoblast differentiation by suppressing the phosphorylationof Smad generated through the activation of BMP2 receptors(Fig. 4). Regarding glycative stress and Smad signals, sofar, Sassi-Gaha, S. et al. 42) have reported that AGEs derived
from 3-deoxyglucosone (3-DG), an intermediate in glycative
reaction, suppress the expression of collagen type I andincrease the expression of Smad 7 which suppresses the
Smad pathway. However, there are few reports on the effects
of glycation stress on Smad expression. For this question,
we have shown that, although the level of inhibition of SSA
phosphorylation by HSA-glycer was about 30 % (Fig. 4 ),the experimental results showed a marked inhibition of
osteoblast differentiation (Fig. 2). These differences willneed further verification in the future.
We have shown that HSA-glycer suppresses osteoblast
differentiation, and in order to further evaluate the balanceof bone metabolism, the RANKL /OPG ratio, an index ofbone metabolism 40, 43, 44), was verified (Fig. 5 ). RANKL is
an osteoclast differentiation promoting factor produced
from osteocytes and osteoblasts and OPG produced fromosteoblasts is an osteoclast differentiation inhibitory factor.
Ideally, a constant RANKL/OPG ratio would be required
to maintain a balance of bone modeling between osteoblastand osteoclast, however, our study showed that the ratio of
RANKL/OPG was reduced by the addition of HSA-glycer(Fig. 5-c). It is supposed that, when the value of the RANKL/OPG ratio is low, bone resorption is slowed by inhibiting
osteoclast differentiation, thus resulting in the slowing ofbone metabolism. Low bone turnover is thought to cause adeterioration in bone quality and a decrease in bone strength.
In fact, a decrease in the RANKL/OPG ratio in diabetic
patients suffering from osteoporosis has been noted 41, 43).
Finally, this study showed that HSA-glycer suppresses
osteoblast differentiation. We have reported that the same
HSA-glycer also suppresses RANKL-induced osteoclastdifferentiation of mouse macrophage-like cells RAW264.7 45).
Coupled with these findings, it is suggested that glyceraldehydederived glycated protein inhibits both osteoblast differentiationand osteoclast differentiation and, as a result, promotes
low turnover bone metabolism. So far, our laboratory has
discovered that more than 500 kinds of plants that have the_ 14 9 _Effect of Glycated Protein on Osteoblastgenesis

effect of inhibiting AGE production. Nowadays, various
osteoporosis drugs are on the market. We plan to study
the effects of plants that inhibit AGE production from theviewpoint that AGEs are involved in the development of
osteoporosis by causing abnormal bone remodeling.

Conclusion
This study demonstrated that glyceraldehyde-derived
glycated protein inhibits BMP2-induced osteoblastgenesis
via inhibition of Smad pathways. It also might be associatedwith low bone turnover, leading to low bone quality.

Acknowledgement
This work was partially supported by the JapaneseCouncil for Science, Technology and Innovation, SIP (Project
I D 14533567), “ Technologies for creating next-generation
agriculture, forestry and fisheries” (funding agency: Biooriented Technology Research Advancement Institution,
NARO).

Conflict of Interest Statement
The authors claim no conflict of interest in this study.

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