overexpression of receptor for hyaluronan-mediated motility (rhamm) in mc3t3-e1 cells induces...

ORIGINAL ARTICLE

Overexpression of receptor for hyaluronan-mediated motility(RHAMM) in MC3T3-E1 cells induces proliferationand differentiation through phosphorylation of ERK1/2

Hiroko Hatano • Hideo Shigeishi • Yasusei Kudo •

Koichiro Higashikawa • Kei Tobiume •

Takashi Takata • Nobuyuki Kamata

Received: 11 June 2011 / Accepted: 28 August 2011 / Published online: 27 September 2011

� The Japanese Society for Bone and Mineral Research and Springer 2011

Abstract Receptor for hyaluronan (HA)-mediated

motility (RHAMM) was first described as a soluble HA

binding protein released by sub-confluent migrating cells.

We previously found that RHAMM was highly expressed

and plays an important role in proliferation in the human

cementifying fibroma (HCF) cell line, which we previously

established. HCF is a benign fibro-osseous neoplasm of the

jaw and is composed of fibrous tissue containing varying

amounts of mineralized material. However, the pathogen-

esis of HCF is not clear. In this paper, we examined the

roles of RHAMM in osteoblastic cells. We generated

RHAMM-overexpressing MC3T3-E1 cells and examined

the cell proliferation and differentiation of osteoblastic

cells. In MC3T3-E1 cells, overexpressing RHAMM was

located intracellular and activated ERK1/2. Interestingly,

the ERK1/2 activated by RHAMM overexpression pro-

moted cell proliferation and suppressed the differentiation

of osteoblastic cells. Our findings strongly suggest

that RHAMM may play a key role in the osteoblastic dif-

ferentiation process. The rupture of balance from

differentiation to proliferation induced by RHAMM over-

expression may link to the pathogenesis of bone neoplasms

such as HCF.

Keywords Cementifying fibroma � RHAMM � HA �ERK � Proliferation

Abbreviations

RHAMM Receptor for hyaluronan-mediated motility

ERK Extracellular regulated kinase

HA Hyaluronan

MAPK Mitogen activated protein kinase

Introduction

Human cementifying fibroma (HCF) is a benign fibro-

osseous neoplasm of the jaw, and is composed of fibrous

tissue containing varying amounts of mineralized material

[1, 2]. However, the process of development consisting of

proliferation and differentiation is not clear. We previously

established immortalized cell lines from HCF of the jaw [3]

and found by microarray analysis that receptor for hyalu-

ronan (HA)-mediated motility (RHAMM) was highly

expressed in comparison with normal osteoblasts obtained

from normal human mandibular bone [4].

RHAMM was first described as a soluble hyaluronan

binding protein released by sub-confluent migrating cells

[5]. The protein, which is called HMMR or CD168, is

located intracellularly in the cytoplasm and the nuclei as

well as on the cell surface [6]. The RHAMM gene is

located on chromosome 5q33.2 and contains 18 exons.

The full-length RHAMM mRNA codes for a 84-kDa

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00774-011-0318-0) contains supplementarymaterial, which is available to authorized users.

H. Hatano � H. Shigeishi � K. Higashikawa � K. Tobiume �N. Kamata (&)

Department of Oral and Maxillofacial Surgery,

Division of Cervico-Gnathostomatology, Graduate School

of Biomedical Sciences, Hiroshima University,

1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan

e-mail: [email protected]

Y. Kudo � T. Takata

Department of Oral and Maxillofacial Pathobiology,

Division of Frontier Medical Science, Graduate School

of Biomedical Sciences, Hiroshima University, Hiroshima, Japan

123

J Bone Miner Metab (2012) 30:293–303

DOI 10.1007/s00774-011-0318-0

http://dx.doi.org/10.1007/s00774-011-0318-0

protein [7]. Injured, subconfluent, or neoplastic cultured

cells express some additional RHAMM proteins [8, 9].

These seem to be conformationally altered RHAMM and/

or have a cellular or intracellular localization different

from that of normal cells, which is known to be present

on the cell surface as a glycosylphosphatidylinositol-

linked receptor [10]. Surface and intracellular forms of

RHAMM are detected in multiple myeloma [11]. Over-

expression and modulation of the balance between solu-

ble, surface, and intracellular RHAMM may control the

regulation of key signaling molecules and the behavioral

characteristics of premalignant cells, leading to constitu-

tive stimulation and malignancy.

Cell-surface RHAMM, which is not an integral

membrane protein, partners with CD44 and, in the

presence of hyaluronan, activates ERK1/2, which results

in the expression of genes that are required for motility

and invasion [12, 13]. Extracellular expression of cyto-

plasmic proteins, such as RHAMM, results from a

redistribution of intracellular pools to the extracellular

compartment that may be associated in part with

increased synthesis or stability of mRNA or protein. On

the other hand, intracellular RHAMM binds to actin

filaments, centrosomes, microtubules, and mitotic spin-

dles [14, 15] and plays important roles in several cellular

processes like signaling [16], mitosis, tumorigenesis, and

cell proliferation [17, 18]. The cell growth mediated by

RHAMM is considered to occur via signaling events

leading to the phosphorylation of several intracellular

proteins [19]. In our previous reports, we found the

EGFR–RHAMM/ERK signaling pathway to be impli-

cated in the growth of HCF cells.

HCF usually consists of multiplied fibroblasts that pro-

duce extracellular collagen fibers and osteoblastic cells on

the surface of spherules–bone/cementum granules. We

found that RHAMM protein expression could be observed

in most of the several cementifying fibroma cases exam-

ined. Moreover, expression of RHAMM protein was

detected in cells from the fibrous region of the tissues. We

assumed that ectopic-overexpressing RHAMM may be

linked to the characterization of HCF: the promotion of

proliferation and the suppression of differentiation. How-

ever, the biological behavior of ectopic-overexpressing

RHAMM has not been fully investigated. To clarify the

mechanism involved in RHAMM leading to HCF deviating

from normal differentiation, we have further focused on the

proliferation and differentiation of normal osteoblasts.

Moreover, recent studies showed that ERK activity is

associated with anti-osteogenesis in osteoblasts [20–22]. In

this paper, we report the identification of a specific

mechanism of growth and differentiation by RHAMM and

highlight the novel signaling through RHAMM/ERK

interaction in osteoblastic cells.

Materials and methods

Cell culture

Immortalized human cementifying fibroma (HCF) cell lines

were established by co-transfection with simian virus-40

(SV40) T-antigen and hTERT [3]. HCF cells were main-

tained in a-modified Eagle medium (a-MEM; Sigma) sup-

plemented with penicillin/streptomycin and 10% heat-

inactivated fetal bovine serum (FBS; Invitrogen) under 5%

CO2 in air at 37�C. MC3T3-E1 cells were provided by the

Japanese Collection of Research Bioresources Cell Bank.

They were maintained in a-MEM supplemented with peni-

cillin/streptomycin and 10% heat-inactivated FBS under 5%

CO2 in air at 37�C. The culture medium was changed every

4 days. They were then cultured with the same medium and

used for the following analyses. For the proliferation assay,

5.0 9 104 cells were plated on 6-well plates (Falcon), and

trypsinized cells were counted by Cell Counter. To examine

the differentiation, 2.0 9 106 cells were plated on 6-well

plates, and after reaching confluence the medium was

changed to the osteogenic medium containing L-ascorbic

acid (vitamin C) (50 lg/ml), b-glycerophosphate (10 mM),

and dexamethasone (100 nm). In addition to the prior

medium, the treated group was incubated with synthesized

160-kDa HA (Hyalose) at 10 lg/ml, anti-CD44 (Thermo

Fisher Scientific), and the commercial phosphorylation of

ERK1/2 inhibitor PD98059 at 50 lM final concentration.

Generation of RHAMM-overexpressing MC3T3-E1

cells

The recombinant vector was produced by Origene Tech-

nologies. The pCMV6-RHAMM-GFP or pCMV6-GFP

plasmid was introduced into MC3T3-E1 cells, and the stable

clones were obtained by G418 selection (500 lg/ml, Life

Technologies) in the culture medium. We obtained pool and

stable clones. Cell transfection was done using FuGENE 6

HD (Roche) according to the manufacturer’s instruction.

Small interfering RNA (siRNA)

ERK1 siRNA, ERK2 siRNA, and negative control siRNA

were purchased from Santa Cruz Biotechnology. Cells

were transiently transfected with the indicated combina-

tions of the siRNAs using LipofectamineTM

2000 transfec-

tion reagent (Invitrogen), according to the manufacturer’s

recommendations.

Quantitative reverse transcription-PCR

Total RNA was isolated from cultures of confluent cells

using the RNeasy mini kit (Qiagen). Preparations were

294 J Bone Miner Metab (2012) 30:293–303

123

quantified, and their purity was determined by standard

spectrophotometric methods. cDNA was synthesized from

1 lg total RNA using the ReverTra Dash kit (Toyobo

Biochemicals). The quantification of mRNA levels was

carried out using a real-time fluorescence detection

method. The fluorescence was detected using a ABI7000

(Applied Biosystems) by measuring the binding of a fluo-

rescence dye, SYBR Green I, to double-stranded DNA.

The reaction mixture contained 1.0 lg of cDNA, 10 ll of

SYBR Green PCR Master Mix (Toyobo Biochemicals),

and 10 pmol of each pair of oligonucleotide primers. The

primer sequences were: human RHAMM: 50-TCTAAAC

AAAATCTTAATGTTGACAAA-30 (sense), 50-TCTTTCT

CTAATATCTTCAAATCTTTA-30 (antisense) [8]; mouse

G3PDH: 50-CACCATGGAGAAGGCCGGGG-30 (sense),

50-GACGGACACATTGGGGGTAG-30 (antisense); mouse

ALP: 50-GTTGCCAAGCTGGGAAGAACA-30 (sense),

50-CCCACCCCGCTATTCCAAAC-30 (antisense); mouse

bone sialoprotein (BSP): 50-ACCCCAAGCACAGACTT

TTGA-30 (sense), 50-CTTTCTGCATCTCCAGCCTTCT-30

(antisense). The PCR program was as follows: initial

melting at 95�C for 60 s followed by 40 cycles at 95�C for

15 s, 58�C for 40 s. The quantification of mRNA expres-

sion relative to an internal control, G3PDH, was performed

by the DCt method [23].

Protein extraction

For extracting total lysate, cultured cells were extracted

using TBSN(?) buffer consisting of 20 mM Tris–HCl (pH

8.0), 150 mM NaCl, 1 mM EDTA (pH 8.0), 5 mM EGTA

(pH 8.0), 0.5 mM Na3VO4, 20 mM p-nitrophenyl phos-

phate, 1 mM PMSF, 0.5% NP-40, various protease inhib-

itor cocktails (Sigma-Aldrich), and various phosphatase

inhibitor cocktails (Sigma-Aldrich). Each sample was fro-

zen in liquid nitrogen and thawed at 4�C, and then vortexed

and centrifuged. The supernatant was then used for the

immunoblot experiments.

For the preparation of the nuclear extracts, cells were

suspended in 10–20 volume of ice-cold hypotonic buffer N,

10 mM HEPES pH 7.5, 2 mM MgCl2, 25 mM KCl, 1 mM

DTT, various protease inhibitor cocktails (Sigma-Aldrich),

and various phosphatase inhibitor cocktails (Sigma-

Aldrich), centrifuged at 1500 rpm for 10 min and the

supernatant discarded. The pellet was resuspended in

10–20 volume of hypotonic buffer N and incubated on ice

for 30 min. Cells were transferred to a Dounce homoge-

nizer and lysed with 15 strokes. Cell lysis was confirmed

by the addition of trypan blue and examination under a

microscope. When cells were lysed, 125 ll of 2 M sucrose

solution per milliliter of lysate was added, mixed well by

inversion, and centrifuged at 1000 rpm for 10 min. After

decanting supernatant, the pellet containing nuclei was

resuspended in 10–20 volume of ice-cold buffer N; 10 mM

HEPES pH 7.5, 250 mM sucrose, 2 mM MgCl2, 25 mM

KCl, 1 mM DTT, various protease inhibitor cocktails, and

various phosphatase inhibitor cocktails, centrifuged at

1000 rpm for 10 min, and the supernatant discarded. The

pellet was resuspended in one volume of buffer N.

For extraction of membranous and cytoplasmic proteins,

cells were suspended in TM-PEK Extraction Buffer 1

containing Protease Inhibitor Cocktail SET III from TM-

PEK (Merck KGaA) for 10 min. Membranous and cyto-

plasmic proteins were separated into pellet and supernatant

fractions, respectively, by centrifuging at 1000g. Membra-

nous protein was incubated for 45 min at 22�C in TM-PEK

Reagent. After centrifuging at 15000g, the supernatant was

transferred to fresh tubes which were enriched with integral

membrane protein.

Western blot analysis

Protein concentrations were measured using Protein Assay

Reagent (BIO-RAD). Protein samples (10 lg) were solu-

bilized in sample buffer by boiling, subjected to sodium

dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–

PAGE) followed by electrotransfer onto Protran� Nitro-

cellulose Membranes (Whatman). We detected the band of

Western blotting using enhanced chemiluminescence (ECL)

Western blotting reagent (GE Healthcare). Images were

captured with a cooled CCD camera system (LAS-4000)

from Fujifilm (Japan). Anti-GFP mouse monoclonal anti-

body (Wako), anti-RHAMM mouse monoclonal antibody

(Monosan), anti-G3PDH mouse monoclonal antibody

(Millipore), anti-CENP rabbit polyclonal antibody (Santa

Cruz Biotechnology), anti-tubulin rabbit monoclonal anti-

body, anti-cadherin rabbit monoclonal antibody, anti-Raf

rabbit polyclonal antibody, anti-phosphorylated-Raf rabbit

polyclonal antibody, anti-MEK1/2 rabbit polyclonal anti-

body, anti-phosphorylated-MEK1/2 rabbit polyclonal anti-

body, and anti-ERK1/2 rabbit polyclonal antibody, and anti-

phosphorylated-ERK1/2 rabbit polyclonal antibody (all Cell

Signaling Technology) were used. For detecting the phos-

phorylation of ERK1/2 correlated with RHAMM, we per-

formed an immunoprecipitation assay. Antibodies were

allowed to bind to protein A-Sepharose (Sigma-Aldrich) and

then incubated with equal amounts of protein (0.5 mg of

total protein in 400 ll) for 12 h at 4�C. Beads were washed

three times with PBS. Each pellet was boiled in 20 ll of

SDS–PAGE sample buffer at 95�C for 3 min, and the entire

volume was loaded onto a gel for Western blotting.

Cell cycle analysis

The distribution of cells at different stages in the cell cycle

was estimated by flow cytometric analysis. Briefly,

J Bone Miner Metab (2012) 30:293–303 295

123

5.0 9 105 cells were incubated at 37�C. To synchronize

them in G0, the cells were starved by serum deprivation for

48 h. After restimulation with medium containing 10%

FBS, cells passed through the cell cycle synchronously and

were incubated for 0, 6, 12, 18, 24, or 30 h. Cells from the

different conditions were trypsinized, washed in PBS, fixed

in 70% ethanol, and stored at 4�C for 2 h. An aliquot

(1 ml) of the fixed cell suspension was washed twice in

PBS. The fixed cells were treated for 30 min at 4�C in the

dark with 40 lg of propidium iodide and 0.1 mg of RNase

A, and then analyzed by flow cytometry. The percentage of

cells in each cell cycle phase (G0/G1, S, or G2/M) was

calculated by using ModFit LT software (Becton–

Dickinson).

Immunofluorescence microscopy

Cells were seeded onto glass Lab-Tek II Chamber Slides

(Thermo Fisher Scientific) at a density of 5.0 9 104 cells/

well and incubated for one day. The growth medium was

then removed, and cell monolayers were washed three

times with a 10% PBS solution and fixed with 3.5%

paraformaldehyde for 10 min at room temperature. Cells

were washed three times with PBS and permeabilized by

0.2% Triton X-100 for 10 min at room temperature.

Nonspecific binding sites were blocked by treatment at

room temperature for 30 min with PBS containing 1%

BSA. The cells were washed three times with PBS and

incubated with anti-RHAMM mouse monoclonal anti-

body, anti-GFP mouse monoclonal antibody, and anti-

phosphorylated-ERK1/2 rabbit polyclonal antibody in

PBS with 1% BSA, for 60 min at room temperature.

RHAMM and GFP staining was revealed by incubation

with an Alexa-Fluor dye-labeled goat anti-mouse antibody

(Invitrogen) for 60 min at room temperature. Phospho-

ERK staining was revealed by incubation with an Alexa-

Fluor dye-labeled goat anti-rabbit antibody (Invitrogen)

for 60 min at room temperature. After three rinses in PBS,

the slides were mounted in Vectashield (Vecto Labora-

tories) and examined using a Leica TCS STED (Leica

Microsystems).

Alkaline phosphatase activity

Cells were plated in a 6-well plate, then the alkaline

phosphatase (ALP) activity was evaluated, as described

below. The confluent cells were grown in osteogenic

medium for 7 days. The ALP activity of the lysate was

determined using p-nitrophenyl phosphate (pNPP; Wako)

using the Lowry method. After 30 min incubation at 37�C,

b

a

0

0.005

0.01

0.015

0.02

0.025

0 6 12 18 24 30

mRN

A e

xpre

ssio

n(R

HA

MM

/G3P

DH

)

hours a�er res�mula�on

hours a�er res�mula�on60 1812 24 30

RHAMM

G3PDH

c hours a�er res�mula�on60 1812 24 30

Fig. 1 RHAMM mRNA and

protein expression in HCF cells

during the cell cycle. HCF cells

were serum-starved for 48 h to

synchronize them in the G0

phase of the cell cycle. At

different time points before and

after restimulation with serum,

cells were harvested and total

RNA as well as protein were

isolated. a RHAMM levels were

determined by real-time RT-

PCR in duplicates and are

displayed relative to mRNA

levels. b RHAMM as well as

G3PDH protein expression

during the cell cycle was

detected by Western blot

analysis. c In parallel, DNA

content of the cells, stained with

propidium iodide, was

monitored by FACS analysis to

determine their distribution in

different phases of the cell cycle


123

absorbance of pNPP at 405 nm was measured using a

Multiskan JX microplate reader (Thermo Fisher Scientific).

Staining for mineralization

The mineralization of MC3T3-E1 cells was determined in

6-well plates using von Kossa staining and Alizarin red

staining, respectively. The confluent cells were grown in

osteogenic medium for 3 weeks, and the cells were fixed

with 95% ethanol and stained with AgNO3 by the Von

Kossa method to detect phosphate deposits in bone nod-

ules. At the same time, the other plates were fixed with ice-

cold 70% ethanol and stained with Alizarin red S (Sigma)

to detect calcification.

Statistical methods

The statistical analysis was performed using one-way

ANOVA and Student’s t test. P values less than 0.05 were

regarded as statistically significant.

Results

Cell-cycle-dependent expression of RHAMM in HCF

cells

To investigate the expression pattern of RHAMM in HCF

cells, RHAMM mRNA and protein levels were analyzed by

DAPI

GFP-RHAMM

phosho-ERK

merge

prop

hase

met

apha

se

anap

hase

inte

rpha

se

IB: phospho-ERK

IP: RHAMM

IB:ERK

IB: RHAMM

pare

nt

cont

rol

#1 #2 #3

RHAMM-GFP

d

ba

G3PDH

ERK

phosho-ERK

phosho-MEK

MEK

phosho-Raf

Rafpa

rent

cont

rol

#1 #2 #3

RHAMM-GFP

pare

nt

cont

rol-G

FP

#1 #2 #3

G3PDH

GFP

RHAMM-GFP

RHAMM

IB: phospho-ERK

IP: RHAMM

IB:ERK

IB: RHAMM

pare

nt

cont

rol

#1 #2 #3

RHAMM-GFP

c

Fig. 2 Generation of RHAMM-overexpressing cells and localization

of RHAMM correlated with the phosphorylation of ERK1/2.

a MC3T3-E1 cells were engineered to overexpress RHAMM by

transfection with pCMV6-AC-RHAMM-GFP. We obtained three

stable clones of RHAMM-overexpressing cells. Expression of

RHAMM protein was done by Western blot analysis. RHAMM-

overexpressing cells expressed RHAMM at a high level. Ectopic

expression of RHAMM was examined by immunoblotting with anti-

GFP antibody. G3PDH expression was used as a loading control.

b RHAMM correlates with the phosphorylation of ERK1/2 more than

Raf and MEK. Expression of phosphorylated-ERK1/2 protein was

done by immunoblotting and immunoprecipitation. c Phosphorylated-

ERK1/2 protein was precipitated from lysates with the use of

monoclonal antibodies specific to RHAMM and immunoblots were

probed with RHAMM, ERK1/2 and phosphorylated-ERK1/2 anti-

bodies. IP immunoprecipitaion, IB immunoblotting. d We confirmed

the different localization of the exogenous and endogenous RHAMM

by immunofluorescence microscopy of RHAMM and phospho-ERK

in RHAMM-overexpressing MC3T3-E1 cells. Exogenous RHAMM

was detected by GFP antibody. Total RHAMM expression was

observed in the cytoplasm and nuclei, while exogenous RHAMM

expression was observed in the nuclei. The phosphorylation of ERK1/

2 was proportional to the expression of RHAMM in the nuclei


123

real-time RT-PCR and Western blotting at various time points

after re-entering the cell cycle (Fig. 1a, b). In parallel, DNA

content of the cells was monitored by fluorescence-activated

cell sorting (FACS) analysis to determine their cell cycle

distribution (Fig. 1c). The results show that RHAMM

expression increased in the G2/M cells, indicating that

RHAMM was involved in the accumulation of G2/M cells and

has an important role in cell cycle progression in HCF cells.

Overexpressing RHAMM promotes

the phosphorylation of ERK1/2 in nuclei

To elucidate the involvement of RHAMM in the patho-

genesis of HCF, we generated RHAMM-overexpressing

osteoblastic cells (MC3T3-E1). We transfected RHAMM

into MC3T3-E1 cells that showed low expression of

RHAMM. We obtained three stable clones (#1, #2 and #3)

of RHAMM-overexpressing cells (Fig. 2a). RHAMM is

thought to control ERK1/2, which has a significant role in

cell motility and proliferation [24]. We examined the

activation of ERK1/2 signaling in RHAMM-overexpress-

ing osteoblastic cells. Interestingly, overexpressing

RHAMM promotes the phosphorylation of ERK1/2

(Fig. 2b) by binding to phospho-ERK1/2 (Fig. 2c) more

directly than to Raf and MEK1/2. These results suggest that

RHAMM binds with ERK1/2, and this interaction may

play an important role in the phosphorylation of ERK1/2 in

nuclei.

a

c

b

d

Num

ber

of th

e ce

lls

days

RHAMM-GFP

0

1000000

2000000

3000000

4000000

5000000

0 1 2 3 4

parent

control

#1

#2

#3

pare

nt

cont

rolm

RNA

exp

ress

ion

(ALP

/G3P

DH

)

cont

rol

cont

rol m

ediu

m

0

0.0005

0.001

0.0015

0.002

#1 #2 #3

RHAMM-GFP

mRN

A e

xpre

ssio

n (B

SP/G

3PD

H)

pare

nt

cont

rol

cont

rol

cont

rol m

ediu

m

0

0.0002

0.0004

0.0006

0.0008

0.001

0.0012

0.0014

#1 #2 #3

RHAMM-GFP

ALP

ac�

vity

pare

nt

cont

rol

cont

rol

cont

rol m

ediu

m

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

#1 #2 #3

RHAMM-GFP

pare

nt

cont

rol-G

FP

#1 #2 #3cont

rol-G

FPco

ntro

l med

ium

RHAMM-GFP

Alizarin Red

Von Kossa

Fig. 3 RHAMM promotes

proliferation and suppresses

differentiation. a Cell

proliferation of RHAMM-

overexpressing MC3T3-E1

cells. Cells were plated on

6-well plates, and trypsinized

cells were counted by Cell

Counter up to 4 days.

b Expression of osteogenic

markers was measured by RT-

PCR. Cells were placed on the

6-well plates and analyzed after

7 days. ALP alkaline

phosphatase, BSP bone

sialoprotein. c ALP activity in

lysates was measured using

pNPP as substrate and

absorbance at 405 nm was

measured as previously

described. Cells were placed on


7 days. d Alizarin red and von

Kossa staining for bone nodules

in control and RHAMM-


cells. Extracellular matrix

calcium deposits for matrix

mineralization were measured

using Alizarin red S dye which

binds with calcium and silver

nitrate dye which binds with

phosphorus in the cell layer

matrix. Cells were placed on the


3 weeks


123

RHAMM promotes proliferation and suppresses

differentiation

We compared proliferation and differentiation between

control and RHAMM-overexpressing MC3T3-E1 cells.

RHAMM overexpression promoted proliferation (Fig. 3a).

In contrast, ALP and BSP mRNA expression (Fig. 3b),

ALP activity (Fig. 3c), and mineralization (Fig. 3d) were

lower than in control cells. Thus, we found that RHAMM

overexpression promoted proliferation and suppressed

differentiation in osteoblastic cells.

The function of phosphorylated-ERK1/2 induced

by overexpressing RHAMM is not inhibited

by anti-CD44

CD44 is thought to function as a receptor for HA and to

have a major role in HA-mediated signaling [25, 26]. CD44

is a major receptor for HA and can activate the Raf–MEK–

ERK signaling pathway, and the phosphorylation of Raf,

MEK1/2, and ERK1/2 are inhibited by anti-CD44 antibody

[4]. We examined whether the phosphorylation of ERK1/2

was inhibited by anti-CD44 antibody in ectopic RHAMM-

b

d

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

ALP

ac�

vity

RHAMM-GFPcontrol-GFP

an�-CD44HA

c

0

0.0005

0.001

0.0015

0.002

0.0025

0.003

mRN

A e

xpre

ssio

n (A

LP/G

3PD

H)


an�-CD44HA

mRN

A e

xpre

ssio

n (B

SP/G

3PD

H)


an�-CD44HA

0

0.0005

0.001

0.0015

0.002

0.0025

0.003

a

e

Alizarin Red

Von Kossa

control-GFP RHAMM-GFP

HA

an�-CD44


HAan�-CD44

G3PDH

phosho-ERK

ERK


Num

ber

of th

e ce

lls a

t 3da

ysan�-CD44HA

0

1000000

2000000

3000000

4000000

Fig. 4 Anti-CD44 could inhibit

the function of phosphorylated-

ERK1/2 only by HA-induced

RHAMM. a The

phosphorylation of ERK1/2

with or without HA and anti-

CD44. b Cell proliferation of

MC3T3-E1 control-GFP and

RHAMM-GFP cells with or

without HA and anti-CD44.

Cells were plated on 6-well

plates, and trypsinized cells

were counted by Cell Counter

after 3 days. b Expression of

osteogenic markers was

measured by RT-PCR. Cells

were placed on the 6-well plates

and analyzed after 7 days. ALPalkaline phosphatase, BSP bone

sialoprotein. c The ALP activity

in lysates was measured using












mineralization were measured





matrix. Cells were placed on


3 weeks


123

overexpressing cells, or not. In our previous findings,

RHAMM binds to ERK and phosphorylates in the presence

of HA [4]. As in our previous findings, levels of phos-

phorylated ERK1/2 were increased in the presence of HA

and the phosphorylation was inhibited by anti-CD44 anti-

body (Fig. 4a). Ectopic overexpressing RHAMM-induced

phosphorylation was not inhibited by anti-CD44 antibody

(Fig. 4a). We further examined proliferation and differen-

tiation under this treatment. Correlated with the levels of

phosphorylation of ERK1/2, the presence of HA promoted

proliferation, and it was inhibited by anti-CD44 antibody

(Fig. 4b). Ectopic overexpressing RHAMM also promoted

proliferation but it was not inhibited by anti-CD44 anti-

body (Fig. 4b). Correlated with the levels of phosphory-

lation of ERK1/2, the presence of HA suppressed

osteogenesis, as shown by ALP and BSP mRNA expres-

sion (Fig. 4c), ALP activity (Fig. 4d), and mineralization

(Fig. 4e), and it was inhibited by anti-CD44 antibody.

Ectopic overexpressing RHAMM also suppressed osteo-

genesis, but it was not inhibited by anti-CD44 antibody

(Fig. 4c–e).

The function of phosphorylated-ERK1/2 induced

by overexpressing RHAMM is inhibited by ERK

inhibitor

To examine the role of RHAMM-induced ERK1/2 phos-

phorylation on proliferation and differentiation, we used

the ERK inhibitor, PD98059. We examined whether the

effects of PD98059 were the same as siRNAs for ERK1

b

c

0

0.0005

0.001

0.0015

0.002

0.0025

0.003

0.0035

mRN

A e

xpre

ssio

n (A

LP/G

3PD

H)


PD98059HA

mRN

A e

xpre

ssio

n (B

SP/G

3PD

H)


PD98059HA

0

0.0002

0.0004

0.0006

0.0008

0.001

0.0012

d

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

ALP

ac�

vity


PD98059

HA

a

e

Alizarin Red

Von Kossa


HA

PD98059


HA

PD98059

G3PDH

phosho-ERK

ERK


Num

ber

of th

e ce

lls a

t 3da

ysPD98059

HA

0

1000000

2000000

3000000

4000000Fig. 5 ERK inhibitor could

inhibit the proliferation and

differentiation induced by both

HA-induced and overexpressing

RHAMM. a The

phosphorylation of ERK1/2

with or without HA and ERK

inhibitor PD98059. b Cell

proliferation of MC3T3-E1

control-GFP and RHAMM-GFP

cells with or without HA and

PD98059. Cells were plated on

6-well plates, and trypsinized

cells were counted by Cell

Counter after 3 days.

b Expression of osteogenic

markers was measured by RT-

PCR. Cells were placed on


7 days. ALP alkaline

phosphatase, BSP bone

sialoprotein. c ALP activity in

lysates was measured using












mineralization was measured





matrix. Cells were placed on


3 weeks


123

and ERK2 (Electronic Supplementary Material Figures).

PD98059 treatment inhibited the phosphorylation of

ERK1/2 induced by HA and ectopic overexpression of

RHAMM (Fig. 5a). We further examined proliferation and

differentiation under treatment with PD98059. Correlated

with the levels of phosphorylation of ERK1/2, the presence

of HA and ectopic overexpression of RHAMM promoted

proliferation, and it was inhibited by PD98059 (Fig. 5b). In

contrast, correlated with the levels of phosphorylation of

ERK1/2, the presence of HA and ectopic overexpression of

RHAMM suppressed osteoblast differentiation, as shown

by ALP and BSP mRNA expression (Fig. 5c), ALP activity

(Fig. 5d), and mineralization (Fig. 5e), and it was inhibited

by PD98059.

Discussion

We previously identified RHAMM as a proliferation factor

of HCF [4]. Previous studies also show the overexpression

of RHAMM in tumor development and the prognostic

significance of its expression [27–29]. On the other hand,

the signaling mediated by mitogen-activated protein kina-

ses (MAPKs) has been shown to have critical roles in the

regulation of cell growth and differentiation [30]. In

addition, recent studies have raised the possibility that

RHAMM could regulate MAPKs such as ERK [24]. In

HCF cells, we found the EGFR–RHAMM/ERK signaling

pathway to be implicated in the regulation of growth [4].

For the normal development of the jaw, the balance of

proliferation and differentiation of osteoblasts is important,

but the mechanism of its regulation is not yet eluci-

dated. We assumed that ectopic RHAMM overexpression

make the balance incline to proliferation in HCF, and it

may be linked to its pathogenesis. To prove this hypothe-

sis, we generated ectopic RHAMM overexpression in

osteoblastic cells, MC3T3-E1, that showed low expression

of RHAMM, and examined the proliferation and its

mechanism.

Interestingly, ectopic overexpressing RHAMM is

expressed intracellularly. As in previous reports, the

RHAMM localized to nuclei interacted with the phos-

phorylation of ERK1/2 more directly. It may be that the

increased proliferation associated with cancer cells facili-

tates the secretion of intracellular protein and provides for

outside-in and inside-out control of genetic stability. As we

expected, RHAMM overexpression promoted cell prolif-

eration and suppressed cell differentiation to osteoblasts of

MC3T3-E1 cells. We also found that RHAMM overex-

pression activated ERK1/2.

To elucidate the mechanism of RHAMM for cell

proliferation and differentiation, we compared HA-

induced RHAMM and overexpressing RHAMM func-

tions. Both HA-induced RHAMM and overexpressing

RHAMM induced the phosphorylation of ERK1/2. How-

ever, the phosphorylation of ERK1/2 correlated with HA-

induced RHAMM was inhibited by anti-CD44, whereas

that of overexpressing RHAMM was not inhibited. ERK

inhibitor suppressed the phosphorylation of ERK1/2 by

both HA-induced RHAMM and overexpressing RHAMM,

indicating that HA-induced RHAMM activates ERK

through the Raf–MEK–ERK signaling cascade but

RHA

MM

P

cell prolifera�oncell differen�a�on

an�-CD44 an�body

PD98059

CD44

RHAMM

Raf-1

MEK1/2

ERK1/2

endogenous RHAMM

P

ectopic RHAMM

ectopic RHAMMectopic

RHAMM

ectopic RHAMM

ectopic RHAMM

ectopic RHAMM

extracellular RHAMMextracellular RHAMM

intracellular RHAMM

intracellular RHAMM

Fig. 6 Proposed model of

overexpressing RHAMM.

Endogenous RHAMM at

normal levels is stimulated by

HA and localized in the cell

surface. Cell surface RHAMM,

which is not an integral

membrane protein, partners with

CD44 and, in the presence of

hyaluronan, activates ERK1/2,

indicated as phosphorylated

ERK1/2, which results in the

promotion of proliferation and

the suppression of

differentiation. On the other

hand, exogenous RHAMM or

overexpressing RHAMM like

HCF cells are redistributed to

intracellular pools and the

extracellular compartment.

Intracellular RHAMM activates

ERK1/2 more directly, which

results in the promotion of

proliferation and the

suppression of differentiation


123

overexpressing RHAMM activates ERK directly (Fig. 6).

Proliferation was promoted and differentiation was sup-

pressed in proportion to the levels of phosphorylation of

ERK1/2, the.

Our present study suggests that ectopic overexpression

of RHAMM breaks down the balance of cell proliferation

and differentiation. Above all, we showed that the locali-

zation of overexpressing RHAMM to nuclei may be

important in the pathogenesis of disease. Further studies

are required to clarify the mechanism of up-regulation of

RHAMM. However, our results provide the possibility of

controlling HCF by regulating the RHAMM protein

localized to nuclei. In conclusion, our studies have revealed

a critical role for RHAMM in cell proliferation and dif-

ferentiation in osteoblastic cells. These findings provide

new and important information on the metabolism of bone

and pathogenesis of HCF.

Acknowledgments This work was supported by a Grant-in-Aid for

JSPS fellows (No. 22-6035) from the Japan Society for the Promotion

of Science (JSPS).

Conflict of interest The authors declare no conflict of interest.

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overexpression of receptor for hyaluronan-mediated motility (rhamm) in mc3t3-e1 cells induces...

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