Medicarpin, a legume phytoalexin, stimulates osteoblast differentiation and promotes peak bone mass...

RESEARCH ARTICLE

Identification of kaempferol-regulated proteins in rat

calvarial osteoblasts during mineralization

by proteomics

Avinash Kumar1, Anand K. Singh2, Abnish K. Gautam1, Deepak Chandra3, Divya Singh1,Bendangla Changkija1, Mahendra Pratap Singh2 and Ritu Trivedi1

1 Division of Endocrinology, Central Drug Research Institute, Chattar Manzil Palace, Lucknow, India2 Toxicogenomics and Predictive Toxicology Division, Indian Institute of Toxicology Research,

Mahatma Gandhi Marg, Lucknow, India3 Department of Biochemistry, Lucknow University, Lucknow, India

Received: September 22, 2009

Revised: January 7, 2010

Accepted: January 20, 2010

Kaempferol, a flavonoid, promotes osteoblast mineralization in vitro and bone formation in vivo;

however, its mechanism of action is yet unknown. We adopted proteomic approach to identify

the differential effect of kaempferol on rat primary calvarial osteoblasts during mineralization.

The primary rat calvarial osteoblasts were treated with kaempferol (5.0mM) for 9 days under

mineralizing condition that resulted in significant increase in alkaline phosphatase activity and

mineralization of the cells. Further, 2-D analysis of the kaempferol-treated osteoblast lysates

revealed 18 differentially expressed proteins (nine upregulated and nine downregulated) on the

basis of 4/o2.0-fold as cut-off (po0.01) that were then identified by MALDI-TOF MS. These

included cytoskeletal proteins, intracellular signaling protein, chaperone, extracellular matrix

protein, and proteins involved in glycolysis and cell–matrix interactions. Proteomics data were

confirmed by Western blotting and quantitative real-time PCR by randomly selecting two

upregulated and two downregulated proteins. Western blot analysis confirmed upregulation of

HSP-70 and cytokeratin-14 levels, and downregulation of aldose reductase and caldesmon

expression. We further demonstrated that kaempferol treatment inhibits aldose reductase

activity in osteoblasts indicating an altered cellular metabolism by decelerating polyol pathway

that was associated with the kaempferol-induced osteoblast mineralization. In conclusion, this is

a first comprehensive study on the differential regulation of proteins by kaempferol in primary

osteoblast, which would further help to elucidate the role of the identified proteins in the process

of osteoblast mineralization.

Keywords:

Aldose reductase / Animal proteomics / Caldesmon / Cytokeratin-14 / HSP-70 /

Osteoblast

1 Introduction

Osteoblast differentiation is the crucial event for bone

formation [1]. Primary calvarial cells from neonatal rats or

mice can form mineralized matrix in vitro, when induced by

L-ascorbate and b-glycerophosphate [2–4]. Robust demand

for energy is placed on osteoblasts during the production of

mineralized matrix of bone [5, 6]. Studies show that

mineralization at the time of bone formation involves tightly

regulated expression of several proteins [7–9]. Expression of

these proteins follows temporal patterns during successive

developmental stages. For example, cell cycle genes, histone

H3 and H4, and ribosomal S6 exhibit higher expression

levels during proliferative stage, while type I collagen,

fibronectin, tumor growth factor-b and osteonectin levels are

higher during matrix maturation stage [10]. However, little

is known about the molecular mechanism by which

Abbreviations: ALP, alkaline phosphatase; AR, aldose reductase;

K, kaempferol; PDI, disulfide isomerase; qPCR, quantitative real-

time PCR; SDH, sorbitol dehydrogenase

Correspondence: Ritu Trivedi, Division of Endocrinology, Central

Drug Research Institute, Chattar Manzil Palace, Post Box 173,

Lucknow 226 001, India

E-mail: [email protected]

Fax: 191-522-2623938

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

1730 Proteomics 2010, 10, 1730–1739DOI 10.1002/pmic.200900666

mineralization of bone matrix (osteoid formation) takes

place.

Osteoblast-‘‘like’’ cell lines including MC3T3-E1 [11, 12],

MG-63 [13] and SaOS-2 [14, 15] have been extensively used

to understand osteoblast differentiation; however, these

immortalized lines are phenotypically different from the

primary cultures. Indeed, an in-depth proteomic study using

human mature osteoblasts and SaOS-2 (human osteogenic

sarcoma cell line) revealed significant difference between

them [16]. We, therefore, used primary rat neonatal calvarial

cells to study changes in expression profile of proteins

during osteoblast mineralization as a way to identify the

proteins linked to osteoblast mineralization [17].

Various polyphenolic compounds derived from natural

source have been shown to promote bone health possibly by

stimulating osteoblast functions [18–21]. We have recently

shown that kaempferol (K), a flavonol, promotes differentia-

tion and mineralization of rat calvarial osteoblasts and has

osteogenic effect in vivo [20]. These results encouraged us to

study the differentially regulated proteins during osteoblast

mineralization. Long-term culture of neonatal calvarial osteo-

blasts required for the induction of mineralization in vitroexhibits maturation sequences and developmental stages that

are comparable with those in neonatal bone [22]. We have

applied comparative proteomic approach to identify the

proteins involved in osteoblast mineralization stimulated by K.

The differentially expressed protein spots in osteoblasts, trea-

ted with or without K under mineralizing condition, were

identified by 2-DE. Protein spots were excised from gel, in-gel

digested, and the extracted proteins were further identified by

MALDI-TOF MS. The differential expression of proteins was

validated by Western blotting quantitative real-time PCR

(qPCR) and enzyme assay (in case of aldose reductase, AR).

Identified proteins could provide an insight into the mechan-

ism of osteoblast mineralization.

2 Materials and methods

2.1 Reagents and chemicals

Cell culture media and supplements were purchased from

Invitrogen (Carlsbad, CA). All fine chemicals, including K, were

purchased from Sigma Aldrich (St. Louis, MO). HPLC grade

ACN was obtained from Merck India (Mumbai, India).

2.2 Culture of calvarial osteoblasts

Rat calvarial osteoblasts were isolated following our previously

published protocol of sequential digestion [23]. Briefly, calvaria

from 10–12 1- to 2-day-old Sprague Dawley rats (both sexes)

were pooled. After surgical isolation from the skull and the

removal of sutures and adherent mesenchymal tissues, calvaria

were subjected to five sequential (10–15 min) digestions at 371C

in a solution containing 0.1% dispase and 0.1% collagenase P.

Cells released from the second to fifth digestions were collected,

centrifuged, resuspended and plated in T-25 cm2 flasks in

a-MEM containing 10% FCS and 1% penicillin/streptomycin

(complete growth medium).

2.3 Mineralization of calvarial osteoblasts

Calvarial osteoblasts cultured until 80% confluence were

trypsinized and plated in the differentiation medium (25 000

cells/well in 12-well plate), consisting of complete growth

medium with ascorbic acid (50 mg/mL) and b-glyceropho-

sphate (10 mM). The medium was changed every alternate

day up to 9 days. A 10 mM stock solution of K was made in

DMSO. Treatment with K at 5.0 mM was given in such a

manner that the final concentration of DMSO did not

exceed 0.01%. The treatment group contained osteoblast

differentiation medium with K (5.0 mM). At the end of the

experiment, cells were washed with PBS and fixed with 4%

paraformaldehyde in PBS for 15 min. The fixed cells were

stained with 40 mM (pH 4.5) Alizarin Red S for 30 min

followed by washing with distilled water [24].

2.4 Measurement of osteoblast alkaline

phosphatase activity

Calvarial osteoblasts (1200 cells/96well plate) were plated in

osteoblast differentiation medium and cultured in the

presence or absence of K for 2 days. The cells were then

freeze thawed by first keeping them at �701C for an hour

and then bringing them to room temperature to determine

the ALP activity. The assay is based on colorimetric esti-

mation of p-nitrophenol formed after breakdown of

p-nitrophenol-phosphate by alkaline phosphatase (ALP) [25].

Absorbance was read at 405 nm using a pre-programmed

semi-automatic photometer (model 5010, Boehringer

Mannheim, Germany).

2.5 2-D PAGE

2.5.1 Sample preparation

The cell pellet, obtained following harvesting and centrifu-

gation, was suspended in lysis buffer (urea: 7 M; thiourea:

2 M; Tris-HCl: 20 mM; CHAPS: 4% w/v; DTT: 10 mM;

EDTA: 1 mM; pPMSF: 1 mM and protease inhibitor cock-

tail). The mixed content was centrifuged and protein was

quantified [26].

2.5.2 IEF

The IPG strips (11 cm) were rehydrated with proteins

(0.5 mg), mixed in rehydration buffer (8 M urea, 2% w/v

Proteomics 2010, 10, 1730–1739 1731


CHAPS, 130 mM DTT, 0.002% bromophenol blue) and IPG

buffer (10mL) in a total volume of 150mL before subjecting

to IEF [27]. The rehydrated strips (pH 4.0–7.0) were focused

in a multiphor-II electrophoresis unit up to 14 000 V h at

201C (500 V for 30 min linear gradient, 1000 V for 10 min,

8000 V for 3 h, 8000 V for 6 h and 8000 V for 7 h).

2.5.3 SDS-PAGE and visualization of proteins

After IEF, the strips were kept in equilibration buffer

(50 mM Tris-HCl pH 8.8, 6 M urea, 30% v/v glycerol, 2%

SDS, 65 mM DTT, 2.5% iodoacetamide and 0.002%

bromophenol blue) for 20 min prior to SDS-PAGE. The pre-

focused and equilibrated IPG strips were placed over the

resolving gels, covered with sealing gel (0.5% agarose in

SDS electrophoresis buffer) and SDS-PAGE was carried out.

Markers were run in parallel to the strips during second

dimension to calculate the approximate molecular weight of

the separated proteins. SDS-PAGE was performed at

constant current (10 mA/gel) using running buffer (25 mM

Tris-HCl, 192 mM glycine and 0.1% SDS). The proteins

were visualized by silver staining, as reported previously

[27]. Equal amount of proteins was loaded in each experi-

ment and similar procedure for staining was used to mini-

mize the variations arising due to sample loading, staining

procedures and run-to-run variability.

2.5.4 2-DE gel image analysis

The 2-DE gel images were analyzed using Image

Master 2D-Platinum software (Gene Bio and Amersham

Biosciences). The gels were aligned properly and the inte-

grated intensity was normalized. The differentially displayed

protein spots were determined on the basis of spot

volume and spot density. Each spot was quantified using

total spot volume normalization method multiplied by the

total area of all the spots. The differentially displayed

proteins that showed significant alteration were selected for

MS.

2.5.5 Sample preparation for MS

The sample for MS was prepared using standard procedure

[26] with slight modifications. In brief, differentially

expressed silver-stained spots were excised, cut into pieces

and washed with 500mL water with agitation on a vortex

mixer. To remove silver stain, the excised spots were incu-

bated with 250 mL of 50 mM sodium thiosulfate and 15 mM

potassium ferricyanide for 5 min and washed twice with

500 mL water to remove reducing agents. In a tube, excised

spot and 500 mL of 100 mM NH4HCO3 and 45 mM DTT was

added and incubated at 601C for 30 min. On cooling, 500mL

of 100 mM iodoacetamide was added and further incubated

for 30 min in dark at room temperature. Excised spots were

sliced and equilibrated with 500mL of 50 mM ammonium

bicarbonate in 50% ACN, dehydrated with 500mL of 100%

ACN for 20 min and air dried [26]. Further the spots were

rehydrated in 30 mL of a solution containing 0.02 mg/mL

trypsin and 50 mM ammonium bicarbonate at 41C for

60 min. The supernatant was discarded and 30–50 mL of

50 mM ammonium bicarbonate was added in excised spots,

incubated at 371C for 16–18 h and supernatant was taken.

To the pellet, 25–50mL of 1% TFA in 60% ACN was added

and sonicated for 10 min. The supernatant was collected

following centrifugation at 12 000� g for 30 s. Both the

supernatants were mixed, freeze dried and concentrated by

centrifugal evaporation to dryness.

2.5.6 MALDI-TOF and LC-MS

Mass spectrometric analysis of samples was carried out at

The Centre for Genomic Applications, New Delhi. In brief,

trypsinized peptide samples were dissolved and mixed with

matrix, namely CHCA. Following drying, the peptides were

spotted on ground steel plate and subjected to Bruker

Ultraflex MALDI-TOF/TOF and 2D Nano LC-ESI-Trap

(Agilent) for mass spectrometric identification. The MALDI-

TOF/TOF instrument was equipped with a pulsed nitrogen

laser. Peptide fragment peaks produced by auto-digestion

of trypsin were used as an internal standard for every

peptide sample to ensure high mass accuracy and to control

possible variations arose due to protein extraction, trypsini-

zation, reconstitution and suppression of ionization by

highly abundant species and incomplete/non-homogeneous

crystallization of proteins and peptides during matrix

preparation.

2.5.7 Analysis of peptide sequences

The analyses of peptide sequences were performed as

reported elsewhere [26, 27]. In brief, trypsinized peptides

were used to generate peptide mass fingerprints. The

MASCOT search engine based on NCBI and Swiss-Prot

protein database were used to identify peptide mass

fingerprints. Probability based MOWSE scores were esti-

mated as ion scores �10� log10 (P), where P is the prob-

ability. The MASCOT search engine was used to identify the

observed peptide mass fingerprints.

2.6 Western blotting

Cells were grown to 60–70% confluence following, which

they were treated with K for 9 days. The cells were lysed in

Mammalian Cell Lysis buffer (Sigma). Lysate were centri-

fuged at 12 000� g for 10 min and supernatant collected.

Protein concentration was determined by the Bradford

1732 A. Kumar et al. Proteomics 2010, 10, 1730–1739


assay. Fifty micrograms of total protein was loaded on 10%

SDS-PAGE gel. After electrophoresis, proteins were trans-

ferred onto PVDF membranes (Immobilon-P, Millipore).

The membranes were probed with AR, cytokeratin, HSP-70

and caldesmon (Santa Cruz Biotechnology) and then incu-

bated with anti-rabbit antibodies conjugated with HRP (Cell

Signaling); and visualized using an enhanced chemilumi-

nescence kit (Amersham Pharmacia Biotech).

2.7 Determination of AR activity

At the end of treatment of K at days 3, 6 and 9 for 9 days,

osteoblasts were harvested by trypsinization and resus-

pended in ice-cold buffer (100 mM phosphate buffer, pH

7.4). Cells were sonicated and after centrifugation super-

natants were kept on ice and assayed in a reaction mixture

containing 0.15 mM NADPH, 10 mM glyceraldehyde,

10 mM HEPES buffer (pH 7.4) and enzyme (protein lysate)

in a total volume of 1 mL. The reaction was initiated by the

addition of substrate (glyceraldehydes) and was

followed at 340 nm for 0, 60, 120 and 180 s at room

temperature with a UV spectrophotometer. As a control,

absorbance at 340 nm was monitored in the presence of

treatment.

2.8 qPCR

PCR was performed using a light cycler (Roche@480). SYBR

Green PCR kit (Roche) and cDNA synthesized from 1.0 mg

total RNA in a 20mL reactions were subjected to PCR

according to the manufacture’s instructions.

2.9 Statistics

Data were presented as the mean7SD of results from three

or four cultures and the significance of differences was

analyzed by Student’s t-test. Groups were analyzed via t-tests

0d 3d 6d 9d0d 3d 6d

0.03

0.04

0.05

. [40

5]n

m

**

0

0.01

0.02

5C μM K

mea

n O

.D.

C 5μM K

A

B

C

Figure 1. K-promoted miner-

alization of calvarial osteo-

blasts. (A) Treatment of

osteoblasts with K (5.0 mM)

induced characteristic

morphological alterations of

osteoblast differentiation as

function of time. Magnification

20� . (B) Osteoblast ALP activ-

ity in the presence of vehicle

(control) or K (5.0 mM) for 2

days. Data show mean7SD of

three independent experi-

ments; ��po0.01. (C) Osteo-

blasts at a density of 104 cells

were cultured in the presence

of vehicle (control) or K

(5.0 mM) in 12 well plates for 9

days. After 9 days, cells were

fixed and stained with Alizarin

Red-S to assess nodule forma-

tion. Photomicrograph show-

ing increased mineralized

nodules in osteoblasts treated

with K for 9 days compared

with control.

Proteomics 2010, 10, 1730–1739 1733


(two-sided). A p-value of o0.05 was considered to be

statistically significant.

3 Results

3.1 K increased ALP activity and mineralization in

primary osteoblasts

Cells extracted from neonatal calvariae are predominantly

preosteoblasts with proliferating ability [28]. When these

preosteoblasts were treated with K (5.0mM) in the presence of

osteoblast differentiation medium, cell morphology became

more cuboidal with the passage of time that was characteristic

of osteoblast differentiation (Fig. 1A). By day 9, cells displayed

more developed intercellular networks (Fig. 1A). Determina-

tion of ALP activity (measure of osteoblast differentiation) on

day 2 demonstrated significantly higher ALP levels (Fig. 1B). K

treatment continued for 9 days, resulted in increased forma-

tion of mineralized nodules compared with vehicle-treated

control osteoblasts (Fig. 1C).

3.2 Proteomic analysis of osteoblasts treated with K

Based on the data described above, osteoblasts treated with

or without K for 9 days were subjected to 2-DE analysis.

Three independent experiments were performed under

similar conditions and only those spots having altered

expressions in all the three experiments were selected

(Fig. 2). Cut-off criterion for changes in the expression levels

was 2.0-folds. By applying this criterion, a total of 18 spots

were identified as differentially expressed proteins by K

treatment. Nine of these proteins were overexpressed and as

many proteins were underexpressed (Table 1).

3.3 Confirmation of proteomic data

We randomly selected caldesmon, AR, HSP-70 and cytokera-

tin-14 from the proteomic data and performed western blotting

to confirm changes in their protein levels following K treat-

ment of osteoblasts. Our 2-DE data revealed marked increases

in cytokeratin-14 and HSP-70 spots in K-treated osteoblasts.

Western blotting data show that K treatment significantly

increased levels of both of these proteins in osteoblasts

(Figs. 3A and B). Cytokeratin-14 levels were increased more

robustly by K treatment compared with HSP-70 levels. In

addition, K treatment reduced levels of AR and caldesmon, as

assessed by Western blotting (Figs. 4A and B).

In addition, we confirmed 2-DE data by qPCR analysis of

mRNA levels of caldesmon, AR, cytokeratin-14 and HSP-70. K

treatment increased cytokeratin-14 mRNA levels in osteoblasts

(Fig. 3C), confirming Western blotting data. In contrast, HSP-

70 mRNA levels were decreased despite increased protein

levels by K treatment of osteoblasts (Fig. 3D), thereby

suggesting discordant regulation of mRNA and protein levels

of HSP-70. mRNA levels of AR and caldesmon were both

decreased by K treatment of osteoblasts (Figs. 4C and D),

further corroborating western blotting data.

200 kDa

C 5 μM K

6868 18 18

2

7

13

412

17

1

3

7

2

12

16

17

4

10

11

14

15

16

910

11

14

15

9

5

13

5

pH 4.0 pH 7.0

20kDa

13

Figure 2. Differential protein expression in

calvarial osteoblasts during mineralization

induced by K. Osteoblasts were plated at a

density of 104 cells in T-75 cm2 flasks, and

treated with 5.0 mM of K for 9 days. On ninth

day, cells were lysed in the protein lysis

buffer as described in Section 2. Only the

spots that showed altered expression (with a

minimum of 2.0-fold change in either direc-

tion) in three independent experiments

under similar conditions were defined as

differentially expressed proteins. The spots

from 2-D silver-stained gels were cut out and

digested, and the resulting peptides were

extracted, purified and identified by MALDI-

TOF. List of differentially expressed proteins

are given in Table 1.



3.4 K modulates the AR activity in osteoblasts

Having observed that K inhibits AR production in osteo-

blasts, we next investigated the effect of K on AR activity. AR

activity was studied at various time points during the course

of osteoblast mineralization (Fig. 5). Data show that induc-

tion of mineralization by ascorbate and b-glycerophosphate

had no effect on AR activity (Fig. 5). K treatment resulted in

modest but significant increase in AR activity at day 3 but

was significantly inhibited at day 9 of the treatment (Fig. 5).

Inhibition of AR activity at day 9 of K treatment demon-

strated that K not only inhibits AR production but also its

activity in osteoblasts.

4 Discussion

Acquisition of bone matrix and mineralization is a complex

phenomenon and is a hallmark of osteoblast function.

Mechanism of osteoblast mineralization and matrix acqui-

sition remains poorly understood. Based on the studies

using a murine calvarial osteoblast cell line, MC3T3-E1,

collagen, osteonectin and osteocalcin represent proteins

associated with bone matrix accumulation and mineraliza-

tion [10]. Given a variety of MC3T3-E1 clones being used by

the researchers and that these clones are phenotypically

different from each other [29, 30], studies on

osteoblast mineralization in MC3T3-E1 do not truly repre-

sent the process of osteoblast mineralization in primary

osteoblasts.

We undertook proteomic study, using K as mineraliza-

tion stimulus in rat primary osteoblasts. Our data indicate

that the expression patterns of proteins, which are differ-

entially regulated by K treatment of osteoblasts during

mineralization, are unique to primary culture system

compared with cell lines [31]. Authenticity of proteomic data

was confirmed independently by western blotting and qPCR

of randomly selected proteins. We observed that protein

levels of HSP-70 and cytokeratin-14 were increased and that

of caldesmon and AR were reduced in osteoblasts treated

Table 1. Identification of differentially expressed proteins in osteoblasts whose expression is modulated by K treatment

S.no.

Proteins Totalscore

Matchedpeptides

Accession no. Description K treatment

Downregulated Upregulated

1. Ubiquitin ligase Nedd4-rat 80 3 S70642 Protein rafficking,signal transduction

4.18

2. RATER60PNID 276 21 BAA09695 2.23. Ribophorin I 38 1 Q6P7A7_RAT Uncharacterized protein 2.644. Chaperonin containing TCP1,

subunit 2 (b)142 7 Q5XIM9_RAT Chaperone 2.13

5. Cytokeratin-14 67 3 K1C14_RAT Cytoskeletal protein 3.316. Caldesmon 60 9 A55887 Intracellular cell-

signaling protein2.0

7. Stress 70 protein 48 3 GRP75_RAT Cell stress and actinorganization

2.0

8. AR 43 4 ALDR_HUMAN Glycolysis 2.139. Procollagen, type XI, a-2-rat 30 4 Q6MGB2_RAT Cell–matrix interaction 2.13

10. Prohibitin 91 6 A39682 Intracellular proteinblocks DNAsynthesis

2.01

11. Vimentin rat 284 17 VIME_RAT Cytoskeletal protein 2.7312. Reticulocalbin 3, EF hand calcium

binding domain137 10 Q5XIG3_RAT Intracellular cell-

signaling protein2.24

13. Translation initiation factor eIF-5Ahuman

65 9 FIHUA 4.64

14. K1 intermediate/smallconductance Ca21- activatedchannel

20 2 Q3S914_RAT Membrane protein 2.02

15. Mitochondrial import stimulationfactor S1-chain rat

159 12 JC2502 3.33

16. Fibronectin precursor rat 29 2 S14428 Extracellular matrixglycoprotein

2.01

17. PDI 54 8 A28807 Oxidoreductase 4.3818. Rho GDP dissociation inihibitor

GDI-a68 3 Q5X173 2.13

Proteomics 2010, 10, 1730–1739 1735


with K by Western blotting, which corroborate 2-DE data.

qPCR further validated Western blotting data with the

exception of HSP-70. mRNA levels of HSP-70 were reduced,

whereas its proteins were increased by K treatment,

suggesting discordant regulation between the transcript and

the protein product of HSP-70.

In addition to these four proteins, our data show that

several proteins are regulated by K treatment, which

might be involved in proper organization of extracellular

matrix. According to functional classification of our

proteomic data, the identified proteins can be grouped into

those involved in signal transduction, glycolysis, extra-

cellular matrix formation, maintenance of cellular structure,

cellular motility, protein degradation, anti-oxidative defense

and chaperones. Analysis of the results reveals that most of

the proteins have functions that have not been investigated

in osteoblasts. We have attempted to form hypothesis

regarding how K stimulates osteoblast mineralization by

Cytokeratin - 14

β - actin β - actin

***

Rel

ativ

e fo

ld c

han

ge

Rel

ativ

e fo

ld c

han

ge

HSP 70

KC KC

C KC KC K

A B

DC

Figure 3. K increased the protein and

mRNA levels of cytokeratin-14 and HSP-70

in osteoblasts during mineralization.

Experiments were performed using the

same aliquots of cell lysates as described

in Fig. 4. Western blotting was performed

using cytokeratin-14 or HSP-70 antibody.

(A) Densitometric analysis of cytokeratin-

14 immunoreactive bands (upper panel)

and representative Western blot of cyto-

keratin-14 (lower panel). (B) Densitometric

analysis of HSP-70 immunoreactive bands

(upper panel) and representative Western

blot of HSP-70 (lower panel). b-Actin

served as protein-loading control for

Western blotting experiments. qPCR

analysis of cytokeratin-14 (C) and HSP-70

(D) mRNA levels in osteoblasts after 9

days of K (5.0mM) or vehicle treatment.

Results are pooled data from three inde-

pendent experiments and presented as

mean7SEM; ��po0.01 and ��po0.001

compared with vehicle control.

AR

β - actin β - actin

Caldesmon

C K

C K

C K

C K

Rel

ativ

e fo

ld c

han

ge

Rel

ativ

e fo

ld c

han

ge

A B

C D

Figure 4. K reduced the protein and mRNA

levels of AR and caldesmon in osteoblasts

during mineralization. Osteoblasts were

treated with K (5.0 mM) or vehicle for 9

days and cell lysates were obtained as

described in Section 2. Aliquots of 50.0 mg

of total cellular protein were fractionated

in SDS-PAGE and probed with either AR

or caldesmon or b-actin antibody. (A)

Densitometric analysis of AR immunor-

eactive bands (upper panel) and repre-

sentative Western blot of AR (lower

panel). (B) Densitometric analysis of

caldesmon immunoreactive bands (upper

panel) and representative Western blot of

caldesmon (lower panel). b-Actin served

as protein loading control for Western

blotting experiments. qPCR analysis of AR

(C) and caldesmon (D) mRNA levels in

osteoblasts after 9 days of K (5.0mM) or

vehicle treatment. Results are pooled data

from three independent experiments and

presented as mean7SEM; ��po0.001

compared with vehicle control.



reviewing the functions of the K-regulated proteins in

osteoblast mineralization with their known functions in

other cell types.

For example, we observed that K treatment during

osteoblast mineralization upregulated prohibitin, whose

function in osteoblast has never been studied. Prohibitin has

been shown to block DNA synthesis in normal fibroblasts

and HeLa cells [32]. It is surmised that by increasing the

levels of prohibitin in osteoblasts, K signals termination of

cellular proliferation, which could be crucial for the

commencement of osteoblast differentiation leading to its

mineralization. On the contrary, caldesmon, which is

downregulated by K treatment in osteoblast, is known to

inhibit mitosis in non-muscle cells [33]. As end of mitotic

cell growth signals, induction of osteoblast differentiation

caldesmon may have important role in osteoblast differ-

entiation.

Mineralization is characterized by deposition of both

collagenous and non-collagenous matrix proteins. We

observed that K increases fibronectin protein levels in

osteoblasts. Fibronectin is maximally produced in rat

calvarial osteoblasts during the matrix maturation stage

[34, 35]. It would be interesting to understand the effect

of fibronectin in osteoblast mineralization in general and

K-stimulated mineralization in particular. Cellular archi-

tecture during mineralization is required to be maintained

for optimum osteoblast function, particularly during the

transformation of osteoblasts to osteocytes. Our data show

that cytokeratin-14 levels are increased by K treatment. Like

in other cells [36], increased expression of cytokeratin-14 in

osteoblasts could result in alterations in cellular morphol-

ogy, adhesion and motility. Regarding cytoskeletal protein

levels being altered by K treatment, our 2-DE data also show

that vimentin levels are reduced in osteoblasts. Intermittent

PTH treatment to rats, which is known to exert osteogenic

effect, has been shown to decrease vimentin levels in bone

marrow cells [37, 38]. As K too has osteogenic effect in vivo,

decrease in vimentin levels by K treatment of osteoblasts

may favor osteoblast mineralization.

Mineralization involves deposition of nascent calcium in

the form of hydroxyapatite crystals. Intracellular calcium

homeostasis is expected to play a vital role in the process of

mineralization by osteoblasts, although nothing is known

about intracellular calcium homeostasis in mineralized

osteoblasts. Our data show that reticulocalbin 3-EF, a

calcium-binding protein [39] is upregulated in K-treated

osteoblasts. Reticulocalbin has no significant sequence

homology with any other proteins except the EF hand

motifs; however, the overall structure of reticulocalbin is

similar to that of calbindin D28K and calretinin [40, 41].

Although there are reports on the structure and localization

of reticulocalbin [42, 43], exact function of this protein

remains unknown. Separate studies are required to learn

whether or not reticulocalbin regulates K-stimulated osteo-

blast mineralization. Endoplasmic reticulum plays an

essential role in intracellular calcium homeostasis by being

an intracellular calcium store. The lumen of endoplasmic

reticulum contains a number of soluble proteins including

protein disulfide isomerase (PDI) [44]. Our 2-DE data show

that PDI levels are significantly increased upon K treatment

of osteoblasts. PDI is involved in the initial stages of

maturation of newly synthesized secretary proteins through

folding and formation of disulfide bonds [45, 46]. In addition

to HSP-70, K treatment increased TCP1 subunit beta, a

molecular chaperone [47], in osteoblasts. HSP-70 has high

affinity for non-native proteins and interacts with hydro-

phobic residues exposed to incompletely folded nascent

polypeptides. Therefore, it appears that increased levels of

reticulocalbin, PDI and TCP1 by K treatment of osteoblasts

could result in nascent polypeptide modification of proteins

that may be important for osteoblast mineralization, where

maturation of matrix protein is required.

There is considerable energy demand during the process

of osteoblast differentiation and mineralization that is met

not only by increasing activities of enzymes involved in

energy metabolic process such as glycolysis but also by their

increased production [48, 49]. AR catalyzes glucose to

sorbitol conversion, which is known as sorbitol/polyol

pathway. Sorbitol is the intermediate step of glucose to

fructose conversion that is catalyzed by sorbitol dehy-

drogenase (SDH). Therefore, by catalyzing glucose to

sorbitol conversion, AR bypasses the control points of

hexokinase and phosphofructokinase in glucose metabo-

lism. It is now well recognized that diabetes is a disease of

impaired glycolysis due to increased glucose flux via AR. As

a result, AR inhibitor is considered as therapeutic candidate

for diabetes and other metabolic disorders [50, 51]. Role of

polyol pathway in osteoblast mineralization is hardly

0.75C

T

0.6 **

**

0.3

0.45

OD

(34

0nm

)

9630

Days post treatment

Figure 5. K inhibits AR activity in osteoblasts. Effect of K (5.0 mM)

on AR activity in osteoblasts was studied as described in Section

2. Cells were cultured in 6-well plates in the presence or absence

of K at days 0 (control), 3, 6 and 9. Lysates containing equal

amounts of protein were sonicated and AR activity was assayed

in the reaction mixture (containing NADPH, glyceraldehydes and

HEPES buffer). The reaction was initiated by adding glycer-

aldehydes and was followed at 340 nm at various time points till

180 s. Results are pooled from six independent experiments with

similar results. Values are expressed as mean7SD. ��po0.01

versus their respective controls in the corresponding groups.

Proteomics 2010, 10, 1730–1739 1737


understood. Osteoblast differentiation and formation of

mineralized nodules have been correlated with increase in

osteoblast glycolysis [48]. In addition, intracellular sorbitol

accumulation is thought to be involved in the development

of osteoblast dysfunction [51, 52]. Furthermore, AR inhibitor

has been shown to attenuate development of galactose-

induced diabetic osteopenia [50]. Our data show that treating

osteoblasts with K resulted in reduced mRNA and protein

levels of AR. SDH mRNA levels were also reduced by K

treatment (data not shown). As K promotes osteoblast

mineralization, inhibition of AR and SDH expressions by K

suggest that activation of polyol pathway negatively regu-

lates osteoblast function.

In conclusion, our study has led to the identification of

proteins not hitherto known for their roles in promoting

osteoblast mineralization. Future studies are required to

demonstrate their specific roles either in elevated or reduced

levels as observed with K treatment in the formation of

mineralized matrix in osteoblasts derived from calvaria

as well as bone marrow. It would also be interesting to

determine whether these proteins are specifically linked

to K-stimulated osteoblast mineralization or other differ-

entiation promoting agents such as bone morphogenetic

proteins.

The authors thank Dr. Sanghamitra Bandhopadhyay forcritical reading of the manuscript. Grant support from Ministryof Health and Family Welfare is acknowledged.

The authors have declared no conflict of interest.

5 References

[1] Harada, S., Rodan, G. A., Control of osteoblast function and

regulation of bone mass. Nature 2003, 423, 349–355.

[2] Beck, G. R., Jr., Sullivan, E. C., Moran, E., Zerler, B.,

Relationship between alkaline phosphatase levels,

osteopontin expression, and mineralization in differentiat-

ing MC3T3-E1 osteoblasts. J. Cell. Biochem. 1998, 68,

269–280.

[3] Aubin, J. E., Regulation of osteoblast formation and func-

tion. Rev. Endocr. Metab. Disord. 2001, 2, 81–94.

[4] BeckJr., G. R., Zerler, B., Moran, E., Gene array analysis of

osteoblast differentiation. Cell. Growth Differ. 2001, 12,

61–83.

[5] Malaval, L., Modrowski, D., Gupta, A. K., Aubin, J. E.,

Cellular expression of bone-related proteins during in vitro

osteogenesis in rat bone marrow stromal cell cultures.

J. Cell. Physiol. 1994, 158, 555–572.

[6] Owen, T. A., Aronow, M., Calhoun, V., Baronet, L. M. et al.,

Progressive development of the rat osteoblast phenotype

in vitro: reciprocal relationships in expression of genes

associated with osteoblast proliferation and differentiation

during formation of the bone extracellular matrix. J. Cell.

Physiol. 1990, 143, 420–430.

[7] Yoon, K., Buenaga, R., Rodan, G. A., Tissue specificity and

developmental expression of rat osteopontin. Biochem.

Biophys. Res. Commun. 1987, 148, 1129–1136.

[8] Weinreb, M., Shinar, D., Rodan, G. A., Different pattern of

alkaline phosphatase, osteopontin, and osteocalcin

expression in developing rat bone visualized by in situ

hybridization. J. Bone Miner. Res. 1990, 5, 831–842.

[9] Sandberg, M., Autio-Harmainen, H., Vuorio, E., Localization

of the expression of types I, III, and IV collagen, TGF-beta 1

and c-fos genes in developing human calvarial bones. Dev.

Biol. 1988, 130, 324–334.

[10] Choi, J. Y., Lee, B. H., Song, K. B., Park, R. W. et al.,

Expression patterns of bone-related proteins during osteo-

blastic differentiation in MC3T3-E1 cells. J. Cell. Biochem.

1996, 61, 609–618.

[11] Kumegawa, M., Hiramatsu, M., Hatakeyama, K., Yajima, T.

et al., Effects of epidermal growth factor on osteoblastic

cells in vitro. Calcif. Tissue Int. 1983, 35, 542–548.

[12] Sudo, H., Kodama, H. A., Amagai, Y., Yamamoto, S., Kasai,

S., In vitro differentiation and calcification in a new clonal

osteogenic cell line derived from newborn mouse calvaria.

J. Cell. Biol. 1983, 96, 191–198.

[13] Prouillet, C., Maziere, J. C., Maziere, C., Wattel, A. et al.,

Stimulatory effect of naturally occurring flavonols

quercetin and kaempferol on alkaline phosphatase

activity in MG-63 human osteoblasts through ERK and

estrogen receptor pathway. Biochem. Pharmacol. 2004, 67,

1307–1313.

[14] Myung, J. K., Afjehi-Sadat, L., Felizardo-Cabatic, M., Slavc,

I., Lubec, G., Expressional patterns of chaperones in ten

human tumor cell lines. Proteome Sci. 2004, 2, 8.

[15] Im, G. I., Qureshi, S. A., Kenney, J., Rubash, H. E., Shanb-

hag, A. S., Osteoblast proliferation and maturation by

bisphosphonates. Biomaterials 2004, 25, 4105–4115.

[16] Spreafico, A., Frediani, B., Capperucci, C., Chellini, F. et al.,

A proteomic study on human osteoblastic cells proliferation

and differentiation. Proteomics 2006, 6, 3520–3532.

[17] Bellows, C. G., Aubin, J. E., Heersche, J. N., Antosz, M. E.,

Mineralized bone nodules formed in vitro from enzymati-

cally released rat calvaria cell populations. Calcif. Tissue Int.

1986, 38, 143–154.

[18] Hsu, Y. L., Chang, J. K., Tsai, C. H., Chien, T. T., Kuo, P. L.,

Myricetin induces human osteoblast differentiation through

bone morphogenetic protein-2/p38 mitogen-activated

protein kinase pathway. Biochem. Pharmacol. 2007, 73,

504–514.

[19] Trivedi, R., Kumar, A., Gupta, V., Kumar, S. et al., Effects of

Egb 761 on bone mineral density, bone microstructure, and

osteoblast function Possible roles of quercetin and

kaempferol. Mol. Cell. Endocrinol. 2009, 302, 86–91.

[20] Trivedi, R., Kumar, S., Kumar, A., Siddiqui, J. A. et al.,

Kaempferol has osteogenic effect in ovariectomized adult

Sprague-Dawley rats. Mol. Cell. Endocrinol. 2008, 289,

85–93.

[21] Trivedi, R., Mithal, A., Chattopadhyay, N., Anabolics in

osteoporosis: the emerging therapeutic tool. Curr. Mol.

Med. 2010, 10, 14–28.



[22] Stein, G. S., Lian, J. B., Owen, T. A., Relationship of cell

growth to the regulation of tissue-specific gene expression

during osteoblast differentiation. FASEB J. 1990, 4, 3111–3123.

[23] Wong, G. L., Cohn, D. V., Target cells in bone for para-

thormone and calcitonin are different: enrichment for each

cell type by sequential digestion of mouse calvaria and

selective adhesion to polymeric surfaces. Proc. Natl. Acad.

Sci. USA 1975, 72, 3167–3171.

[24] Prabhakar, U., James, I. E., Dodds, R. A., Lee-Rykaczewski,

E. et al. A novel human bone marrow stroma-derived cell

line TF274 is highly osteogenic in vitro and in vivo. Calcif.

Tissue Int. 1998, 63, 214–220.

[25] Akcakaya, H., Aroymak, A., Gokce, S., A quantitative

colorimetric method of measuring alkaline phosphatase

activity in eukaryotic cell membranes. Cell Biol. Int. 2007,

31, 186–190.

[26] Patel, S., Sinha, A., Singh, M. P., Identification of differen-

tially expressed proteins in striatum of maneb-and para-

quat-induced Parkinson’s disease phenotype in mouse.

Neurotoxicol. Teratol. 2007, 29, 578–585.

[27] Sinha, A., Srivastava, N., Singh, S., Singh, A. K. et al.,

Identification of differentially displayed proteins in cere-

brospinal fluid of Parkinson’s disease patients: a proteomic

approach. Clin. Chim. Acta 2009, 400, 14–20.

[28] Chattopadhyay, N., Yano, S., Tfelt-Hansen, J., Rooney, P. et al.,

Mitogenic action of calcium-sensing receptor on rat calvarial

osteoblasts. Endocrinology 2004, 145, 3451–3462.

[29] Wenstrup, R. J., Fowlkes, J. L., Witte, D. P., Florer, J. B.,

Discordant expression of osteoblast markers in MC3T3-E1

cells that synthesize a high turnover matrix. J. Biol. Chem.

1996, 271, 10271–10276.

[30] Bellows, C. G., Sodek, J., Yao, K. L., Aubin, J. E., Phenotypic

differences in subclones and long-term cultures of clonally

derived rat bone cell lines. J. Cell. Biochem. 1986, 31,

153–169.

[31] Miyake, M., Arai, N., Ushio, S., Iwaki, K. et al., Promoting

effect of kaempferol on the differentiation and mineraliza-

tion of murine pre-osteoblastic cell line MC3T3-E1. J. Biosci.

Biotechnol. Biochem. 2003, 67, 1199–1205.

[32] Nuell, M. J., Stewart, D. A., Walker, L., Friedman, V. et al.,

Prohibitin, an evolutionarily conserved intracellular protein

that blocks DNA synthesis in normal fibroblasts and HeLa

cells. Mol. Cell. Biol. 1991, 11, 1372–1381.

[33] Yamashiro, S., Yamakita, Y., Yoshida, K., Takiguchi, K.,

Matsumura, F., Characterization of the COOH terminus of

non-muscle caldesmon mutants lacking mitosis-

specific phosphorylation sites. J. Biol. Chem. 1995, 270,

4023–4030.

[34] Moursi, A. M., Damsky, C. H., Lull, J., Zimmerman, D. et al.,

Fibronectin regulates calvarial osteoblast differentiation.

J. Cell. Sci. 1996, 109, 1369–1380.

[35] Globus, R. K., Doty, S. B., Lull, J. C., Holmuhamedov, E.

et al., Fibronectin is a survival factor for differentiated

osteoblasts. J. Cell. Sci. 1998, 111, 1385–1393.

[36] Lee, C. H., Coulombe, P. A., Self-organization of keratin

intermediate filaments into cross-linked networks. J. Cell.

Biol. 2009, 186, 409–421.

[37] Kim, S. H., Jun, S., Jang, H. S., Lim, S. K., Identification of

parathyroid hormone-regulated proteins in mouse bone

marrow cells by proteomics. Biochem. Biophys. Res.

Commun. 2005, 330, 423–429.

[38] Steinert, P. M., Roop, D. R., Molecular and cellular biology of

intermediate filaments. Annu. Rev. Biochem. 1988, 57, 593–625.

[39] Ozawa, M., Muramatsu, T., Reticulocalbin, a novel endo-

plasmic reticulum resident Ca(21)-binding protein with

multiple EF-hand motifs and a carboxyl-terminal HDEL

sequence. J. Biol. Chem. 1993, 268, 699–705.

[40] Rogers, J. H., Immunoreactivity for calretinin and other

calcium-binding proteins in cerebellum. Neuroscience

1989, 31, 711–721.

[41] Tiwari, S., Gupta, S. K., Kumar, K., Trivedi, R., Godbole,

M. M., Simultaneous exposure of excess fluoride and

calcium deficiency alters VDR, CaR, and calbindin D 9 k

mRNA levels in rat duodenal mucosa. Calcif. Tissue Int.

2004, 75, 313–320.

[42] Booth, C., Koch, G. L., Perturbation of cellular calcium indu-

ces secretion of luminal ER proteins. Cell 1989, 59, 729–737.

[43] Kelly, R. B., Cell biology Tracking an elusive receptor.

Nature 1990, 345, 480–481.

[44] Freedman, R. B., Protein disulfide isomerase: multiple roles

in the modification of nascent secretory proteins. Cell 1989,

57, 1069–1072.

[45] Ferrari, D. M., Soling, H. D., The protein disulphide-

isomerase family: unravelling a string of folds. Biochem.

J. 1999, 339, 1–10.

[46] Kemmink, J., Darby, N. J., Dijkstra, K., Nilges, M.,

Creighton, T. E., The folding catalyst protein disulfide

isomerase is constructed of active and inactive thioredoxin

modules. Curr. Biol. 1997, 7, 239–245.

[47] Brackley, K. I., Grantham, J., Activities of the chaperonin

containing TCP-1 (CCT): implications for cell cycle

progression and cytoskeletal organization. Cell Stress

Chaperones 2009, 14, 23–31.

[48] Komarova, S. V., Ataullakhanov, F. I., Globus, R. K., Bio-

energetics and mitochondrial transmembrane potential

during differentiation of cultured osteoblasts. Am. J.

Physiol. Cell. Physiol. 2000, 279, C1220–C1229.

[49] Schirrmacher, K., Lauterbach, S., Bingmann, D., Oxygen

consumption of calvarial bone cells in vitro. J. Orthop. Res.

1997, 15, 558–562.

[50] Inaba, M., Terada, M., Nishizawa, Y., Shioi, A. et al.,

Protective effect of an aldose reductase inhibitor against

bone loss in galactose-fed rats: possible involvement of the

polyol pathway in bone metabolism. Metabolism 1999, 48,

904–909.

[51] Inaba, M., Effect of aldose reductase on the abnormality of

calcium metabolism in diabetic patients. Clin. Calcium 2006,

16, 1360–1365.

[52] Takizawa, M., Suzuki, K., Matsubayashi, T., Kikuyama, M. et al.,

Increased bone resorption may play a crucial role in the

occurrence of osteopenia in patients with type 2 diabetes:

possible involvement of accelerated polyol pathway in its

pathogenesis. Diabetes Res. Clin. Pract. 2008, 82 119–126.

Proteomics 2010, 10, 1730–1739 1739


Medicarpin, a legume phytoalexin, stimulates osteoblast differentiation and promotes peak bone mass...

Documents

Transcript of Medicarpin, a legume phytoalexin, stimulates osteoblast differentiation and promotes peak bone mass...