Activation of c-Jun NH2-terminal kinase 1 increases cellular responsiveness to BMP-2 and decreases...

Activation of c-Jun NH2-Terminal Kinase 1 IncreasesCellular Responsiveness to BMP-2 and Decreases Bindingof Inhibitory Smad6 to the Type 1 BMP Receptor

Hui Liu,1,2 Yunshan Liu,1 Manjula Viggeswarapu,1 Zhaomin Zheng,2 Louisa Titus,1 and Scott D Boden1

1Atlanta Veterans Affairs Medical Center and Department of Orthopaedics, Emory University School of Medicine, Decatur, GA, USA2Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, People’s Republic of China

ABSTRACTBone morphogenetic protein 2 (BMP-2) plays a critical role in the differentiation of precursor cells and has been approved for clinical

application to induce new bone formation. To date, unexpectedly high doses of recombinant BMP-2 have been required to induce bone

healing in humans. Thus, enhancing cellular responsiveness to BMP-2 potentially has critically important clinical implications. BMP

responsiveness may be modulated in part by cross-talk with other signaling pathways, including mitogen-activated protein kinases

(MAPKs). c-Jun NH2-terminal kinase (JNK) is a MAPK that has been reported to be required for late-stage differentiation of preosteoblasts

and BMP-2-induced differentiation of preosteoblasts and pleuripotent cells. In this study we determined that MC3T3-E1-clone 24 cells

(MC-24) can be induced by BMP-2 to differentiate into mineralizing osteoblast cultures. Using this inducible system, we employed both

JNK loss-of-function and gain-of-function reagents to make three key observations: (1) JNK is required for phosphorylation of Smad1 by

BMP-2 and subsequent activation of Smad1 signaling and osteoblast differentiation, (2) JNK1, but not JNK2, is required for BMP-2-

induced formation of mineralized nodules, and (3) JNK1 activation decreases binding of inhibitory Smad6 to the type I BMP receptor

(BMPR-I) and reciprocally increases binding of Smad1, both observations that would increase responsiveness to BMP-2. Understanding

this and other pathways that lead to increased cellular responsiveness to BMPs could greatly aid more cost-effective and safe clinical

delivery of these important molecules. � 2011 American Society for Bone and Mineral Research.

KEY WORDS: C-JUN NH2-TERMINAL KINASE (JNK); MITOGEN-ACTIVATED PROTEIN KINASE (MAPK); BMP; SMAD; OSTEOGENESIS

Introduction

Bone morphogenetic proteins (BMPs) are members of the

transforming growth factor b (TGF-b) superfamily and exert

a wide range of biologic effects in musculoskeletal tissues.(1,2)

Several BMPs play an especially critical role in differentiation of

precursor cells to induce bone formation.(3) Of the BMPs, BMP-2

is the most well studied inducer of osteoblast differentiation

and bone formation.(4) BMP-2 is approved for clinical application

to induce new bone formation in spine and severe fracture

patients, but the amount of recombinant protein requiredmakes

its use prohibitively expensive.(5,6) In addition, several adverse

side effects, including edema, have been noted.(5,7) Our goal to

improve the understanding of cellular responsiveness to BMP-2

may help to reduce the dose required to induce bone formation

and prevent unwanted complications.

BMPs transduce signals through a unique receptor system

composed of heteromeric complexes of two related transmem-

brane serine/threonine kinase receptors known as type I receptor

(BMPR-I) and type II receptor (BMPR-II). Receptor Smads (R-

Smads) 1, 5, and 8 specifically mediate cellular responses to

BMPs. Once activated, R-Smads associate with Smad4 and

translocate to the nucleus to regulate gene expression.(8–10)

Inhibitory Smads6 and -7 potently antagonize the BMP/Smad

pathway by interacting with activated BMPR-I and preventing

activation of R-Smads or by competing with activated R-Smads

to form complexes with Smad4.(11–14)

BMP responsiveness may be modulated in part by complex

cross-talk with other signaling pathways, including mitogen-

activated protein kinases (MAPKs).(15,16) MAPKs are a group of

well-described serine/threonine kinases that transduce extra-

cellular signals to intracellular targets.(17) MAPKs can be activated

by osteoinductive factors such as BMPs and insulin-like growth

factor 1 (IGF-1) and play an important role in cellular responses to

these agents.(18–21) Three structurally related MAPKs have been

described in mammalian cells.(17) c-Jun NH2-terminal kinase

ORIGINAL ARTICLE JJBMR

Received in original form January 12, 2010; revised form September 30, 2010; accepted November 5, 2010. Published online November 18, 2010.

Address correspondence to: Louisa Titus, PhD, Emory University School of Medicine, Research Service 151, 1670 Clairmont Road, Decatur, GA 30033, USA.

E-mail: [email protected]

Additional Supporting Information may be found in the online version of this article.

Journal of Bone and Mineral Research, Vol. 26, No. 5, May 2011, pp 1122–1132

DOI: 10.1002/jbmr.296

� 2011 American Society for Bone and Mineral Research

1122

(JNK) is one of these MAPKs, and it contributes to multiple

cellular events such as proliferation, differentiation, and survival

in response to extracellular stimuli.(22) JNK has been reported

to be required for late-stage differentiation of preosteoblasts

and BMP-2-induced differentiation of preosteoblasts and

pleuripotent cells.(18,20,23,24) JNK is considered a critical regulator

of activating transcription factor 4, osteocalcin, and bone

sialoprotein expression in spontaneously differentiating osteo-

blasts.(21,23) These studies suggest that JNK plays an important

role in the induction of osteogenesis by BMP-2. However, the

exact mechanism of this interaction is still unclear.

In this study, we describe a novel role of JNK in Smad1

C-terminal phosphorylation and consequent osteogenic differ-

entiation induced by BMP-2 in MC3T3-E1 subclone 24

preosteoblasts that do not differentiate spontaneously.(25)

Specifically, activation of JNK suppresses binding of Smad6 to

BMPR-I and enhances binding and consequent phosphorylation/

activation of Smad1.

Materials and Methods

MC3T3-E1 subclone 24 cell culture

MC3T3-E1 subclone 24 (MC-24) cells, a mouse preosteoblast cell

line that can be induced to differentiate into osteoblasts and

produce mineralized nodules in vitro, was obtained from the

American Type Culture Collection (ATCC, Manassas, VA, USA) and

maintained in a-modified essential medium (a-MEM, Invitrogen/

Gibco, Grand Island, NY, USA) with 10% non-heat-inactivated

fetal bovine serum (FBS; Atlanta Biologicals, Norcross, GA, USA) at

378C in 5% CO2 humidified air. Differentiation of MC-24 cells was

induced by BMP-2 and the addition of 50mg/mL of ascorbic acid

(AA) and 5mM b-glycerophosphate (b-GP) in the culture

medium.

Fetal rat calvarial osteoblast cultures

Following approval by the Institutional Animal Care and Use

Committee, fetal Sprague-Dawley rats were removed at 20 days’

gestation, decapitated, and calvarial preosteoblasts were

obtained as described previously.(26) Cells were seeded in T-75

flasks (Corning, Inc., Corning, NY, USA) at 1� 106 cells/flask in

MEM supplemented with L-glutamine (Gibco/Invitrogen) plus

heat-inactivated 10% FBS (Hyclone, Logan, UT, USA) and ex-

panded after 1 week of culture. Seven days later, cells were

plated in 6-well plates at 5� 104 cells/well and grown to

confluence prior to initiation of differentiation with the addition

of 50mg/mL of AA with or without BMP-2 (100 ng/mL, reapplied

after 4 days) in the culture medium. During the second week of

differentiation, cultures were grown in Biggers, Gwathin, and

Judah (BGJb) medium, Fitton Jackson modification (Invitrogen/

Gibco), in the presence of 5mM b-GP.

RNA interference of Smad1 or JNK1/2

Using lipofectamine RNAiMAX transfection reagent (Invitrogen,

Carlsbad, CA, USA) according to the manufacturer’s instructions,

MC-24 cells were transfected with small interfering RNA (siRNA)

duplexes specific for mouse Smad1, JNK1, JNK2, or control siRNA

duplexes predesigned and purchased from Applied Biosystems/

Ambion (Austin, TX, USA) Briefly, cells were seeded at 5� 104/

cm2 in 6-well plates and incubated for 16 hours. Different

amounts of 20mM siRNA duplexes (to achieve final concentra-

tions of 10 or 50 pmol/mL) were mixed with 5mL/well of

transfection reagent and Opti MEM Reduced Serum Medium

(Invitrogen) to a total volume of 500mL and incubated for

20minutes at room temperature. The mixture then was applied

to cells and incubated for 16 hours at 378C in 5% CO2. Duplex

siRNA used were as follows: Smad1 sense: 5’-GGC AGU UGC UUA

CGA GGA Att-3’; Smad1 antisense: 5’-UUC CUC GUA AGC AAC

UGC Ctg-3’; JNK1 sense: 5’-CAA AGA UCC CGG ACA AGC Att-3’;

JNK1 antisense: 5’-UGC UUG UCC GGG AUC UUU Ggt-3’; and JNK2

sense: 5’-CCU AUG UGG UAA CUC GAU Att-3’; JNK2 antisense:

5’-UAU CGA GUU ACC ACA UAG Gga-3’. Control sense: 5’-UAA

CGA CGC GAC GAC GUA Att-3’; Control Antisense: 5’-UUA CGU

CGU CGC GUC GUU Att-3’.

Rat calvarial osteoblasts were treated 24 hours after plating

with 25 pmol/mL of siRNA duplexes for rat JNK1 or JNK2 (Sigma-

Aldrich, St Louis, MO, USA) using the same protocol as for MC-

24 cells. Duplex sequences were as follows: JNK1 sense: 5’-GAU

GCU UAU UCU UGA AAG AAtt-3’; JNK1 antisense: 5’-UUC UUU

CAA GAA UAG CAU Ctt-3’; and JNK2 sense: 5’-GCU AAC UUA UGU

CAG GUU Att-3’; JNK2 antisense: 5’-UAA CCU GAC AUA AGU UAG

Ctt-3’. The transfection reagent was removed after 16 hours, and

fresh medium was applied. Total RNA was harvested 48 hours

after transfection to establish the efficacy of each siRNA by real-

time reverse-transcriptase polymerase chain reaction (RT-PCR).

RNA isolation and real-time RT-PCR

Total RNA was isolated using the RNeasy Mini Kit (Qiagen,

Valencia, CA, USA) according to the manufacturer’s protocol.

After isolation, 2mg of total RNAwas reverse transcribed in a 100-

mL total volume containing 50mM KCl, 10mM Tris-HCl, pH 8.3,

5.5mM MgCl2, 0.5mM dNTPs, 0.125mM random hexamer, 40

units of RNase inhibitor, and 125 units of MultiScribe (Applied

Biosystems, Foster City, CA, USA). In control samples, the RNase

inhibitor and MultiScribe were omitted. The samples were

incubated for 10minutes at 258C, 30minutes at 488C, and

5minutes at 958C to inactivate the enzyme. Real-time PCR then

was performed on 5mL of the resulting cDNA in a total volume of

25mL containing 12.5mL of 2� SYBR Green PCR Master Mix

(Applied Biosystems) and 0.8mM of each primer. Real-time PCR

was performed using the 7500 Real-Time PCR System from

Applied Biosystems. Real-time PCR conditions were as follows:

508C for 2minutes, 958C for 10minutes, 958C for 15 seconds, and

628C for 1 minunte for 32 cycles. Data were normalized to

endogenous 18S rRNA levels using the Applied Biosystems DDCtmethod. The Smad1, alkaline phosphatase (Alp), osteocalcin (Ocn),

and 18S rRNA primer sets were designed and constructed by

Applied Biosystems. Primer sequences were as follows: mouse

Smad1: forward: 5’-ACC CTG TCT GAG GAG CGT GTA-3’; reverse:

5’-ACC AAA GCG TCC ACA GCT TT-3’; mouse Alp: forward: 5’-CGG

CCC TGA GTC TGA CAA AG-3’; reverse: 5’-CTC GTC ACA AGC AGG

GTCAA-3’; mouse osteocalcin: forward: 5’-TGA GTC TGA CAA AGC

CTT C-3’; reverse: 5’-CTG CTG TGA CAT CCA TAC TTG-3’; rat JNK1:

forward: 5’-CGG AAC ACC TTG TCC TGA AT-3’; reverse: 5’-TCG CCT

GAC TGG CTT TAA GT-3’; rat JNK2: forward: 5’-ATC ACA AAG CAC

JNK1 INCREASES CELLULAR RESPONSIVENESS TO BMP-2 Journal of Bone and Mineral Research 1123

CCC ATC TC-3’; reverse: 5’- TGG TGG CTT CTG TCA GTG -3’. The

18S rRNA primer sequence (Applied Biosystems) is proprietary.

Western blot analysis

Cells were lysed to obtain total protein using Mammalian Protein

Extraction Reagent (Pierce Biotechnology, Rockford, IL, USA) or

lysed to obtain nuclear protein using NE-PER Nuclear and

Cytoplasmic Extraction Reagents (Pierce Biotechnology) accord-

ing to the manufacturer’s protocol. Each sample (10mg of

protein) was mixed with NuPage loading buffer (Invitrogen) for a

total volume of 20mL and boiled for 5minutes. The proteins were

separated by electrophoresis under denaturing conditions on

NuPage Bis-Tris Pre-Cast Gels (Invitrogen) for 60minutes at 200 V

and transferred onto nitrocellulose membranes (Invitrogen) for

60minutes at 30 V. After the transfer, the membranes were

incubated in 25mL of blocking buffer [5% nonfat dry milk in Tris-

buffered saline (TBS)] for 1 hour at room temperature. After

blocking, membranes were washed three times for 5minutes

each in 15mL of TBS with 0.1% Tween-20 (TBST). Washed

membranes were incubated with various primary antibodies in

TBST overnight at 48C as follows: anti-phospho-Smad1/5/8

(1:1000), anti-Smad1 (1:1000), anti-phospho-JNK (1:1000), anti-

JNK (1:1000), anti-Smad6 (1:1000), or anti-BMPR-I (1:500). The

membranes also were incubated with anti-b-actin (1:2500) or

anti-lamin B (1:2000) to verify the equal loading of proteins in

each lane. Anti-BMPR-I, anti-Smad6 (for coimmunoprecipitation),

and anti-lamin B antibodies were purchased from Santa Cruz

Biotechnology, Inc. (Santa Cruz, CA, USA); other antibodies were

purchased from Cell Signaling Technology (Beverly, MA, USA).

After incubation with primary antibody, membranes were

washed three times for 5minutes each with 15mL of TBST.

Washed membranes were incubated with horseradish perox-

idase–conjugated anti-rabbit or anti-mouse secondary antibo-

dies as indicated (1:2000; Cell Signaling Technology in 10mL of

blocking buffer with gentle agitation for 1 hour at room

temperature. After incubation with secondary antibodies,

membranes were washed three times for 5minutes each with

15mL of TBST. Washed membranes were incubated with 5mL of

SuperSignal West Pico Western blot substrate (Pierce Biotech-

nology) with gentle agitation for 4minutes at room temperature.

Membranes were wrapped in plastic wrap and exposed to X-ray

films.

Coimmunoprecipitation

Coimmunoprecepitation (co-IP) was performed using the Pierce

Biotechnology Co-immunoprecepitation Kit following the man-

ufacturer’s instruction. Briefly, cells were lysed with co-IP lysis

buffer, and the immune complexes from 1mg of lysate were

collected using an optimized amount (10 to 20mg) of resin-

immobilized BMPR-I or Smad6 antibody with overnight incuba-

tion. After incubation and washes, the complexes were eluted

from the resin and analyzed by Western blot using Smad6 and

BMPR-I antibodies as described.

Transient transfection and luciferase reporter assay

A Smad1-responsive luciferase reporter that contains the Smad1

binding element (9�GCCG) was used to determine the effect of

JNK inhibitor or constitutively active JNK1 (caJNK1) on BMP-2-

induced Smad1 transcriptional activity, as described pre-

viously.(27) caJNK1 was purchased from Addgene (Cambridge,

MA, USA) and was constructed previously as a MAP kinase kinase

7 (MKK-7)–JNK1 fusion protein by Dr Roger Davis.(28) Cells were

plated at a density of 1� 104 cells/cm2, grown overnight, and

transfected for 4 hours either with a luciferase reporter construct

plasmid mixture of 1.45mg of pcDNA3.1 empty vector, 1.45mg of

Smad1-responsive reporter, and 0.1mg of pGL4.74 Renilla

luciferase control vector or a luciferase reporter construct

plasmid mixture of 1.45mg of caJNK1 construct, 1.45mg of

Smad1-responsive reporter, and 0.1mg of pGL4.74 Renilla

luciferase control vector. Superfect tranfection reagent (Qiagen,

Inc., Valencia, CA, USA) was used for the transfection. Empty

pGL3-BASIC vector (Promega, Madison, WI, USA) was used as the

control. After 4 hours, the transfection reagent was removed, and

the cells were washed with cold PBS and grown overnight. Cells

then were treated with various doses of BMP-2 or the JNK

inhibitor SP6001225 (10mM) plus BMP-2 for 24 hours. Luciferase

activity was measured using the Luciferase Assay Kit (Promega)

according to the manufacturer’s instructions on a luminometer

(Victor X Light; PerkinElmer Life and Analytical Sciences,

Waltham, MA, USA). Data were normalized to Renilla luciferase

activity, and relative luciferase units (RLU) were calculated as

firefly luciferase activity/Renilla luciferase activity.

Determination of culture mineralization

For alizarin red staining of MC-24 cultures, cultures were washed

three times with PBS and fixed in 70% ethanol for 30minutes at

48C. After three washes with distilled water and air drying, the

cells were stained with 40mM alizarin red S (Sigma-Aldrich)

solution (pH 4.1) for 10minutes to visualize matrix calcium

deposition. The remaining dye was washed with five washes of

distilled water, and the stained cells were photographed. For

quantification, the calcium deposits were destained with 10%

cetylpyridinium chloride in 10mM sodium phosphate (pH 7.0),

the extracted stain was transferred to a 96-well plate, and the

absorbance of the samples was measured at 570 nm using a

microplate reader (Tecan, Salzburg, Austria). A standard curve

was produced using known concentrations of alizarin red. The

amount of alazirin red per culture was determined according to

the standard curve and expressed as micromoles per square

centimeter.

For evaluation of nodules in rat calvarial cultures, cultures were

fixed with 70% ethanol for 30minutes at 378C and air dried

overnight. von Kossa silver stain (Sigma-Aldrich) was applied to

detect the calcium phosphate–containing nodules for 30min-

utes. After three washes with distilled water and air drying, a

semiautomated computerized video imaging system (Optomax

5; Optomas, Hollis, NH, USA) was used to quantify the nodules in

each well, as described previously.(26)

Statistical analysis

Results are reported as the mean of determinations, with

error bars representing the SEM. Statistical significance was

calculated with a one-way analysis of variance (ANOVA) with

Levene’s homogeneity of variance test and, subsequently,

1124 Journal of Bone and Mineral Research LIU ET AL.

Bonferroni’s post-hoc test or Dunnett’s T3 post-hoc test based

on the comparison to be made and the statistical indication of

each test. Analysis (including correction of p values when

Bonferroni was used) was performed using Statistical Products

for Social Sciences, Version 13.0 for Windows (SPSS, Inc, Chicago,

IL, USA). Statistical probability of p< .05 was considered

significant.

Results

BMP-2 induces preosteoblast differentiation andmineralization in a dose-dependent manner

To first establish the cell model of BMP-2-induced osteoblast

differentiation, MC-24 cells were treated with various doses of

BMP-2. Cells were fixed and stained to detect calcium deposition,

as measured by alizarin red staining on days 3, 11, and 17. RNA

was isolated to detect changes in the levels of mRNA for

osteogenic markers with real-time RT-PCR on days 1, 3, 5, and 7.

BMP-2 induced nodule formation in a dose- and time-dependent

manner (Fig. 1A). Quantification indicated that BMP-2 at 100 ng/

mL increased alizarin red deposition to 14mmol/cm2 on day 3,

337mmol/cm2 on day 11, and 540mmol/cm2 on day 17. In

addition, BMP-2 at 200 ng/mL increased alizarin red deposition

to 25mmol/cm2 on day 3, 528mmol/cm2 on day 11, and

819mmol/cm2 on day 17 (Fig. 1B). On both days 11 and 17,

200 ng/mL of BMP-2 induced more alizarin red deposition than

BMP-2 at 100 ng/mL (p< 0.05). Thus, in subsequent experiments,

day 11 was selected as the time point to detect mineralization.

Increased Alp and OcnmRNA levels also were induced by BMP-2

in a dose- and time-dependent manner. Alp was increased by

100 ng/mL of BMP-2 4-fold, 83-fold, 216-fold, and 116-fold on

days 1, 3, 5, and 7, respectively, and also was increased by


days 1, 3, 5, and 7, respectively (Fig. 1C). Ocn was increased by


days 1, 3, 5, and 7, respectively, and was further increased by


days 1, 3, 5 and 7, respectively (Fig. 1D). On day 3, both Alp and

Ocn had significantly different mRNA levels induced by BMP-2 at

both doses tested (p< .05; Fig. 1E, F). Thus, in subsequent

experiments, day 3 was selected as the time point for detecting

changes in mRNA levels.

Smad1 is essential for BMP-2-induced preosteoblastdifferentiation and mineralization

To clarify that the Smad pathway is essential for BMP-2-induced

osteogenic differentiation in MC-24 cells and that JNK does not

regulate BMP-2-induced osteogenic differentiation in a Smad-

independent manner, RNA interference of Smad1 was per-

formed. Application of Smad1 siRNA and successful knockdown

of Smad1 mRNA (p< .05; Fig. 2C) rendered BMP-2 unable to

inducemineralization on day 11 (Fig. 2A, B). Furthermore, Alp and

OcnmRNA levels did not increase with BMP-2 treatment (Fig. 2D,

E). These data suggest that Smad1 is essential for BMP-2 induced

osteogenic differentiation in this cell line.

JNK activation is required for BMP-2 inducedpreosteoblast differentiation and mineralization

To determine whether JNK activation is required for BMP-2-

induced osteoblast differentiation and mineralization, JNK-

specific inhibitor (SP600125) and Jnk1 and Jnk2 siRNA were

applied. JNK inhibitor suppressed BMP-2-induced mineralization

in a dose-dependent manner (Fig. 3A). SP600125 at 5, 10, and

25mM decreased the BMP-2-induced alizarin red from 369 to

345, 131 (p< .05), and 72mmol/cm2 (p< .05), respectively

(Fig. 3B). The Alp increase induced by BMP-2 was suppressed

from about 360-fold to 301-fold, 102-fold (p< .05), and 71-fold

(p< .05) by SP600125 at 5, 10, and 25mM, respectively. Also, the

BMP-2-induced increase in Ocn mRNA levels was suppressed

from 91-fold to 80-fold, 32-fold (p< .05), and 10-fold (p< .05) by

SP600125 at 5, 10, and 25mM, respectively (Fig. 3C, D). We

confirmed the importance of JNK in BMP-2-induced MC-24 cell

differentiation and extended these observations by applying

specific siRNA for Jnk1 or Jnk2. siRNA application (10 and

50 pmol/mL) specifically and dose-dependently reduced Jnk1 or

Jnk2 mRNA levels 85% to 90% compared with the level in cells

treated with control siRNA (Fig. 3E). BMP-2-induced alizarin red

deposition was suppressed significantly by Jnk1 siRNA compared

with the BMP-2-induced deposition in the presence of control

siRNA (Fig. 3F, G). However, with Jnk2 siRNA application, BMP-2-

induced alizarin red staining was not reduced significantly.

To confirm the requirement for JNK1 in BMP-2-induced

differentiation of nontransformed, freshly isolated preosteo-

blasts, we applied siRNA for Jnk1 or Jnk2 to rat calvarial

preosteoblast cultures treated with BMP-2. The siRNA duplexes

specifically reduced the targeted Jnk mRNA by more than 75%

but did not significantly affect the nontargeted Jnk mRNA (data

not shown). As with the MC-24 cell line, JNK1 was required for

BMP-2 stimulation of nodule formation, whereas JNK2 was not

required (Supplemental Fig. S1A, B). BMP-2 stimulated miner-

alized nodules from 60� 1 to 158� 4/well (p< .05). Nodule

number in the cultures treated with Jnk1 siRNA prior to BMP-2

treatment was reduced to 78� 1/well (p< .05), whereas nodule

number in the cultures treated with Jnk2 siRNA was slightly

increased. These data suggest that JNK, especially JNK1, is

required for BMP-2-induced osteoblast differentiation and

mineralization.

Constitutively active JNK1 increases BMP-2-inducedpreosteoblast differentiation and mineralization

To determine whether JNK1 activation enhances BMP-2-induced

osteoblast differentiation and mineralization, a constitutively

active JNK1 (caJNK1) construct was transfected into MC-24 cells

prior to treating the cells with or without BMP-2. Constitutively

active JNK1 alone did not increase mineralization, but it

significantly increased the response of MC-24 cells to BMP-2

(Fig. 4A). With caJNK1 overexpression, alizarin red induced by

BMP-2 was increased from 419 to 1083mmol/cm2 (p< .05)

(Fig. 4B). The increase in AlpmRNA levels induced by BMP-2 was

elevated from 170-fold to 405-fold (p< .05); the increase in Ocn

mRNA levels induced by BMP-2 was elevated from 60-fold to

186-fold (p< .05; Fig. 4C, D). These data suggest that JNK1


constitutive activation increases BMP-2-induced osteoblast

differentiation and mineralization.

Inhibition of JNK suppresses but activation of JNK1increases pSmad1 nuclear accumulation induced byBMP-2

To explore the role of JNK in BMP-2-induced osteogenesis, we

determine whether JNK regulated BMP-2-induced osteogenesis

by interacting with the Smad pathway. MC-24 cells were

transfected with empty pcDNA3.1 control plasmid or caJNK1

plasmid. After transfection, the cells were pretreated with DMSO

(control) or JNK inhibitor (SP600125, 25mM) for 60minutes and

then treated with BMP-2 (100 ng/mL) for 30minutes. Nuclear

protein was isolated, and Western blots were used to determine

pSmad1 nuclear accumulation induced by BMP-2. JNK inhibitor

or caJNK1 alone had no effect on Smad1 nuclear accumulation;

however, JNK inhibitor suppressed but caJNK1 increased

Fig. 1. BMP-2 induces osteoblast differentiation and mineralization in a dose-dependent manner. (A) Nodule formation by MC-24 cells induced by BMP-2

at 100 or 200 ng/mL. Cells were stained with alizarin red on days 3, 11, or 17. The image presented is from one representative experiment out of three

independent experiments performed in duplicate. (B) Alizarin red was quantified as described under ‘‘Materials and Methods.’’ Data are presented as

mean� SEM from three independent experiments performed in duplicate. (C, D) Time course of increased Alp and OcnmRNA levels in response to BMP-2

treatment. Cells were treated with the indicated doses of BMP-2 for 1, 3, 5, or 7 days. The mRNA levels of Alp and Ocnwere detected and expressed as the

fold change over untreated cells on day 1. Data are presented as mean� SEM from three independent experiments performed in duplicate. (E, F) BMP-2

induced Alp andOcnmRNA levels on day 3. Cells were treated with the indicated doses of BMP-2 for 3 days. ThemRNA levels of Alp andOcnwere detected

and expressed as fold change over untreated cells on day 3. Data are presented as mean� SEM from three independent experiments performed in

duplicate. ap< .05.


pSmad1 nuclear accumulation (Fig. 5A). A luciferase reporter

assay was used to detect Smad1 transcriptional activity, as

described under ‘‘Materials and Methods.’’ The Smad1 transcrip-

tional activity was increased by BMP-2 in a dose-dependent

manner (Fig. 5B). BMP-2 at 2.5 ng/mL was selected for

subsequent experiments. JNK inhibitor or caJNK1 alone did

not have any effect on Smad1 transcriptional activity. However,

JNK inhibitor significantly suppressed and caJNK1 dramatically

increased BMP-2-induced Smad1 transcriptional activity. These

data suggest that JNK affects BMP-2-induced osteogenesis by

interaction with the Smad pathway.

Inhibition of JNK suppresses but activation of JNK1increases Smad1 C-terminal phosphorylation

To further explore regulation of the Smad pathway by JNK, we

determined whether JNK has any effect on BMP-2-induced

Smad1 C-terminal phosphorylation, which is a direct down-

stream effect of BMP-2 stimulation. First, we determined the

profile of Smad1 C-terminal phosphorylation induced by BMP-2.

MC-24 cells were treated with BMP-2 for 15, 30, 60, 90, and

120minutes. Western blots were used to detect Smad1 C-

terminal phosphorylation as well as JNK phosphorylation. BMP-2

induces both Smad1 C-terminal phosphorylation and JNK

phosphorylation in a time-dependent manner (Fig. 6A). Based

on these results, 15, 30, and 90minutes were the time points

selected for subsequent experiments. MC-24 cells were trans-

fected with empty pcDNA3.1 control plasmid or caJNK1 plasmid

for 16 hours. After transfection, the cells were pretreated with

DMSO (control) or JNK inhibitor (SP600125, 25mM) and then

treated with BMP-2 for 15, 30, or 90minutes. Western blots were

used to detect Smad1 C-terminal phosphorylation as well as JNK

phosphorylation. BMP-2-induced Smad1 C-terminal phosphor-

ylation was suppressed by JNK inhibitor but enhanced by caJNK1

significantly at 15, 30, and 90minutes. These data suggest that

JNK affects BMP-2-induced osteogenesis by modulating Smad1

C-terminal phosphorylation.

Inhibition of JNK increases but activation of JNK1suppresses Smad6 competition with Smad1 binding toBMPR-I

To further explore themechanism by which JNK affects Smad1 C-

terminal phosphorylation, the direct interaction of Smad1 and its

upstream kinase, BMPR-I, was compared with the interaction of

BMPR-I with inhibitory Smad6, the endogenous inhibitor of the

BMP-2 pathway. Cells were transfected with empty pcDNA3.1

control plasmid or caJNK1 plasmid as described under ‘‘Materials

and Methods.’’ After transfection, cells were pretreated with

DMSO or JNK inhibitor for 1 hour and then treated with BMP-2

(100 ng/mL) for 30minutes. After BMP-2 treatment, total protein

was isolated, and BMPR-I antibody or Smad6 antibody was used

to immunoprecipitate BMPR-I or Smad6, respectively. Interaction

of Smad1 or Smad6with BMPR-I was determined byWestern blot

of the proteins coimmuoprecipitating with BMPR-I. Inhibition of

JNK decreased Smad1 binding to BMPR-I, whereas constitutive

activation of JNK1 suppressed Smad1 binding to BMPR-I (Fig. 7A).

Similarly, inhibition of JNK increased but activation of JNK1

suppressed binding of BMPR-I with Smad6 (Fig. 7B). These data

Fig. 2. Smad1 is essential for BMP-2-induced preosteoblast differentia-

tion and mineralization. (A) BMP-2-induced nodule formation by MC-

24 cells with or without pretreatment with Smad1 interference RNA. Cells

were transfected with control or Smad1 siRNA (10pmol/mL) as described

under ‘‘Materials and Methods,’’ treated with BMP-2 (100 ng/mL), and

stained with alizarin red on day 11. The image presented is from one

representative experiment out of three independent experiments per-

formed in duplicate. (B) Alizarin red was quantified as described under

‘‘Materials and Methods.’’ Data are presented as mean� SEM from three

independent experiments performed in duplicate. (C) Interference RNA

for Smad1. Cells were transfected with control or Smad1 siRNA (10 pmol/

mL) as described under ‘‘Materials and Methods’’ and treated with BMP-2

(100 ng/mL) for 3 days. The mRNA level of Smad1 was detected and

expressed as the fold change from the level in cells treated with control

siRNA. Data are presented as mean� SEM from three independent

experiments performed in duplicate. (D, E) BMP-2-induced Alp and

Ocn mRNA levels on day 3 in cells treated with control or Smad1 siRNA.

Cells were transfected with control or Smad1 siRNA (10 pmol/mL) as

described under ‘‘Materials and Methods’’ and then treated with BMP-2

(100 ng/mL) for 3 days. The mRNA levels of Alp and Ocn were detected

and expressed as the fold change from the level in cells treated with

control siRNA. Data are presented as mean� SEM from three indepen-

dent experiments performed in duplicate. ap< .05.


Fig. 3. JNK activation is required for BMP-2-induced osteoblast differentiation andmineralization. (A) Nodule formation by MC-24 cells induced with BMP-

2 in the presence or absence of JNK inhibitor. Cells were pretreated with DMSO or the indicated doses of JNK inhibitor, SP600125, for 1 hour, treated with

BMP-2 (100 ng/mL) for 3 days, and stained with alizarin red on day 11. The image presented is from one representative experiment out of three

independent experiments performed in duplicate. (B) Alizarin red deposition was quantified as described under ‘‘Materials and Methods.’’ Data are

presented asmean� SEM from three independent experiments performed in duplicate. (C,D) BMP-2-induced Alp andOcnmRNA levels on day 3 from cells

treated with or without the inhibitor of JNK. Cells were pretreated with DMSO or the indicated doses of the JNK inhibitor, SP600125, for 1 hour and then

treated with BMP-2 (100 ng/mL) for 3 days. ThemRNA levels of Alp andOcnwere detected and expressed as the fold change from cells treated with control

siRNA. Data are presented as mean� SEM from three independent experiments performed in duplicate. (E) siRNA reduced Jnk1 or Jnk2 mRNA levels

specifically and dose dependently. Cells were transfected with control (cont), Jnk1, or Jnk2 siRNA (10 or 50 pmol/mL) as described under ‘‘Materials and

Methods.’’ The mRNA level of Jnk1 or Jnk2 was detected and expressed as the fold change from the level in cells treated with control siRNA. (F, G) Nodule

formation and alizarin red deposition were significantly reduced using Jnk1 siRNA but not using Jnk2 siRNA. The image and analysis presented are from

one representative experiment performed three times in duplicate. ap< .05.


suggest that JNK1 regulates Smad1 phosphorylation and BMP-2

induction of osteogenesis by regulating Smad6 competition with

Smad1 binding to BMPR-I.

Discussion

Enhancing cellular responsiveness to BMP-2 potentially has

critically important clinical implications. To date, unexpectedly

high doses (concentrations) of recombinant BMP-2 have been

required to induce bone healing in humans compared with that

required in rodents and cell culture.(5,6,29) BMPs bind specific

receptors, resulting in the formation of heteromeric receptor

complexes containing type I (BMPR-I) and type II (BMPR-II)

receptors. The type I receptor then phosphorylates receptor

Smad1, -5, and -8 (R-Smads) as the next step in transmitting the

BMP-2 signal. Phosphorylated R-Smads bind Smad4 and

Fig. 4. Constitutively active JNK1 increases BMP-2-induced osteoblast

differentiation and mineralization. (A) BMP-2-induced nodule formation

by MC-24 cells transfected with empty plasmid (cont) or constitutively

active JNK1 (caJNK1) plasmid. Cells were transfected with plasmids for

4 hours, grown overnight in fresh medium, and treated with BMP-2

(100 ng/mL) for 3 days. Cells were fixed and stained with alizarin red

on day 11. The image presented is from one representative experiment

out of three independent experiments in duplicate. (B) Alizarin red was

quantified as described under ‘‘Materials and Methods.’’ Data are pre-

sented as mean� SEM from three independent experiments performed

in duplicate. (C, D) BMP-2-induced Alp and Ocn mRNA levels 3 days after

transfection with control or caJNK1 plasmid. Cells were transfected with

plasmids for 4 hours, grown overnight in fresh medium, and treated with

BMP-2 (100 ng/mL) for 3 days. Alp and Ocn mRNA were detected and

expressed as fold change over cells treatedwith noncoding plasmid. Data

are presented as mean� SEM from three independent experiments

performed in duplicate. ap< .05.

Fig. 5. Inhibition of JNK suppresses but activation of JNK1 increases

BMP-2-induced pSmad1 nuclear accumulation and Smad1 transcrip-

tional activity. (A) Cells were transfected with empty vector (cont) or

caJNK1 plasmid for 4 hours and grown overnight in fresh medium. Then

the cells were pretreated with DMSO or JNK inhibitor for 1 hour, followed

by BMP-2 (100 ng/mL) treatment for 30minutes. Nuclear protein was

extracted and the protein level of pSmad1 in nuclear extracts was

determined by Western blot. Lamin B antibody was used to show equal

protein loading. The image presented is from one representative experi-

ment out of three independent experiments. (B) BMP-2-induced Smad1-

responsive reporter activity. A Luciferase reporter assay was used to

assess Smad1 transcriptional activity. The reporter construct was trans-

fected into cells as described under ‘‘Materials andMethods.’’ Transfected

cells then were treated with the indicated doses of BMP-2 for 24 hours.

Luciferase activity was determined as described under ‘‘Materials and

Methods’’ and normalized to Renilla luciferase activity as relative lucifer-

ase units (RLUs). Data from two independent experiments performed in

triplicate are presented as mean� SEM of the fold change in RLUs

compared with the untreated control. (C) BMP-2-induced Smad1-respon-

sive reporter activity change in cells with inhibition or activation of JNK.

Cells were cotransfected with the Smad1-responsive reporter and the

noncoding or caJNK1 plasmid as described under ‘‘Materials and Meth-

ods.’’ Then cells were pretreated with DMSO or JNK inhibitor for 1 hour.

After pretreatment, cells were treated with BMP-2 (100 ng/mL) for

24 hours. Luciferase activity was normalized to Renilla luciferase activity

as relative luciferase units (RLUs). Data from two independent experi-

ments performed in triplicate are presented as mean� SEM of the fold

change in RLU compared with the control cells treated with DMSO and

the control plasmid. ap< .05.


translocate to the nucleus, where they regulate gene expres-

sion.(8,9)

The experimental study of BMP-2 signaling and BMP

responsiveness is challenging and is complicated by at least

two issues: (1) cross-talk with numerous other pathways(15,16)

and (2) lack of synchronization of cellular differentiation in

spontaneously differentiating heterogeneous preosteoblast

cultures.(25) Several studies have demonstrated that MAPKs

are required for BMP-2-induction of the osteoblastic phenotype

and mineral deposition in vitro.(18,21,30) In particular, the MAPK

JNK is reported to be required for both late-stage spontaneous

differentiation of osteoblasts and BMP-2-induced differentiation

of preosteoblasts and pleuripotent cells in vitro.(20,21,23) However,

in those previous studies, the points of intersection of the JNK

and BMP-2 pathways were not investigated.

In addition to cross-talk, study of BMP signaling is further

complicated by virtue of the fact that most osteoblast cell culture

systems result in spontaneous differentiation of preosteoblasts

that are at varying stages of lineage commitment.(31,32) Since

BMP signaling and cross-talk are likely to be time-depen-

dent,(32,33) we felt that it might be helpful to study BMP

responsiveness in a model where preosteoblasts were more

synchronized in their differentiation toward the mature

osteoblast phenotype. To this end, we used subclone 24 of

MC3T3-E1 cells (MC-24), which were characterized originally by

Franceschi as a nondifferentiating subclone of preosteoblasts.(25)

While not undergoing spontaneous differentiation, like the more

commonly used subclone 14 of MC3T3-E1 cells,(25) we show for

the first time that this MC-24 subclone can be induced by BMP-2

to differentiate into osteoblasts that produce mineralized

nodules.

We used the unique property that the MC-24 preosteoblasts

required a stimulus to initiate osteoblast differentiation to

facilitate the measurement of BMP responsiveness in the

presence of various inhibitors of cross-talk signaling pathways.

A major limitation of these transient gain- or loss-of-function

studies arises whenmeasuring amore physiologic endpoint such

as mineralized nodules, which requires 11 to 17 days in culture.

There is often uncertainty as to whether and when to reapply an

inhibitory agent if the positive BMP-2 stimulus requires repeated

Fig. 6. Inhibition of JNK suppresses but activation of JNK1 increases

Smad1 C-terminal phosphorylation. (A) Time course of BMP-2-induced

phosphorylation of Smad1, -5, and -8. Cells were treated with BMP-2

(100 ng/mL), and protein levels of pSmad1, -5, and -8, Smad1, pJNK, and

JNK were determined by Western blot. The image presented is from one

representative experiment out of three independent experiments. (B)

Inhibition of JNK suppresses but activation of JNK1 increases Smad1 C-

terminal phosphorylation. Cells were transfected with noncoding or

caJNK1 plasmids for 4 hours and grown overnight in fresh medium.

Then cells were pretreated with DMSO or JNK inhibitor for 1 hour,

followed by treatment with BMP-2 (100 ng/mL) for 15, 30, or 90minutes.

Total protein was extracted, and the protein levels of pSmad1, -5, and -8,

Smad1, and pJNK were determined by Western blot. b-Actin antibody

was used to show equal protein loading. The image presented is from

one representative experiment out of three independent experiments.

Fig. 7. JNK modulates binding of Smad1 to BMPR-I by regulating recep-

tor binding of the inhibitor Smad6. Cells were transfected with noncod-

ing or caJNK1 plasmids and grown overnight in fresh medium. Then cells

were pretreated with DMSO or JNK inhibitor for 1 hour, followed by

treatment with BMP-2 (100 ng/mL) for 30minutes. Total protein was

isolated, and BMPR-I (A) or Smad6 (B) antibodies were used for coim-

munoprecipitation studies. Interaction of Smad6 or Smad1 with BMPR-I

was determined by Western blot with specific antibody for each protein.

The image presented is from one representative experiment out of three

independent experiments.


applications every few days, which is frequently recommended

in other systems. We addressed this potential confounder and

ensured more synchronized differentiation by optimizing the

protocol to require only a single administration of BMP-2 protein

rather than multiple reapplications. This single BMP-2 admin-

istration protocol allowed us to test inhibitors of pathways

known to interact with the BMP-2 signaling pathway without

concern over the need or timing of reapplication of the inhibitor.

Having validated an inducible osteoblast culture system, we

made three key observations that provide new insight into

modulation of BMP-2 signaling by JNK. First, we made the novel

observation that JNK is required for phosphorylation of Smad1,

the first intracellular event in the canonical BMP-2 signaling

cascade that can be observed within 15minutes of treatment.

Second, we found that JNK1, but not JNK2, was required for BMP-

2-induced formation of mineralized nodules. This important

observation was confirmed in freshly isolated rat calvarial

osteoblast cultures, implying that it is not an artifact of

transformed cell lines. Third, we found that JNK1 activation

results in decreased binding of the inhibitory Smad6 to BMPR-I

and, reciprocally, increased binding of Smad1, both observations

that would increase BMP responsiveness.

To understand the relative importance of isoforms of JNK

expressed in skeletal tissues, we used specific siRNA and found

that JNK1, but not JNK2, was required for BMP-2-induced nodule

formation. This observation is contrary to at least one previously

published work stating that JNK2, but not JNK1, is required for

late-stage osteoblast differentiation of spontaneously differen-

tiating MC3T3 cells.(23) We interpret this discrepancy as suggest-

ing that JNK1 is required for the initial stages of differentiation by

preosteoblasts but less important when cells are more

committed to the osteoblast lineage.

The observation that JNK1 activation suppresses Smad6

association with BMPR-I and enhances Smad1 association was

further confirmed by the observation that inhibition of JNK1/2

enhances binding of Smad6 to BMPR-I, whereas it decreases

Smad1 binding. These early-event findings are consistent with

our observations of downstream endpoints that JNK1 activation

enhances BMP-2-induced Smad1 activation, osteoblast differ-

entiation, and nodule formation in vitro, whereas inhibition of

JNK1/2 has the opposite effect.We hypothesize two possible

ways in which JNK might modulate Smad6 or Smad1 binding to

BMPR-I. Either JNK directs posttranslational modification of

Smad1 or Smad6 or JNK modifies a Smad1 or Smad6 binding

partner to alter Smad availability to bind the receptor.

Phosphorylation within the linker region of Smad1 by MAPKs

has emerged as an important mechanism by which growth

factors desensitize cells to BMP-2.(34,35) Intense study of linker-

region phosphorylation has led to the realization that many

kinases, in addition to MAPKs, contribute to linker-region

phosphorylation. Further, the E-3 ligase Smurf1 has been shown

to bind preferentially to Smad1 that is phosphorylated in the

linker region, targeting it for degradation.(36) Recently, an

additional level of complexity in understanding the importance

of Smad1 linker-region phosphorylation has been added with

the identification of R-Smad linker phosphatases that have site

specificity.(37) Less is known about linker-region phosphorylation

of Smad6. These complex modifications are of considerable

interest when considering the mechanism by which JNK

activation enhances Smad1 binding to the BMPR-I or reduces

the binding of Smad 6 to the receptor. However, to date, no

evidence has emerged indicating that MAPK-induced linker-

region phosphorylation would affect the relative association of

these molecules with the BMPR-I. Future studies will be needed

to address specifically what cellular changes JNK elicits to

modulate Smad6 and Smad1 binding to BMPR-I.

One potential limitation of this study is its dependence on the

specificity of the pJNK and pSmad1, -5, and -8 antibodies to

detect only the phosphorylated molecule without cross-reacting

with unphosphorylated forms. Further, we cannot evaluate the

relative contribution of pSmad1, pSmad5, or pSmad8 in the

detected species. However, these antibodies have been used by

other investigators in many studies to evaluate pSmad1, -5, and -

8 levels. More specific antibodies have not been developed

because of the sequence homology in the region. Also, our

conclusion regarding Smad1 or Smad6 binding to BMPR-I

assumes complete immunoprecipation of Smad6 and its bound

proteins. The consistency of our findings in three independent

experiments and the fact that our coimmunoprecipitation

studies are consistent with the physiologic outcomes measured

independently suggest that our conclusions accurately reflect

cellular events.

In summary, we have made the novel observation that JNK1 is

required for BMP-2 activity and moreover can modulate cellular

responsiveness to BMP-2 in an osteoblastic cell line and in freshly

isolated calvarial osteoblasts. JNK1 activation results in

decreased binding of inhibitory Smad6 and increased binding

of Smad1 to the BMP receptor, resulting in increased cellular

response to BMP-2, as measured within 30minutes by increased

levels of phosphorylated Smad1 and at later points by expression

of the mature osteoblast phenotype, including increased

formation of mineralized nodules. Understanding this and other

pathways that lead to increased cellular responsiveness to BMPs

could greatly aid more cost-effective and safe clinical delivery of

these important molecules.

Disclosures

SDB has received compensation in the past as a consultant for

Medtronic Sofamor Danek and for intellectual property. The

terms of this arrangement were reviewed and approved by

Emory University in accordance with its conflict-of-interest

policies. All the other authors state that they have no conflicts of

interest.

Acknowledgments

This work was supported in part by a VAMerit Award to SDB. This

work was performed in facilities at the Atlanta Veterans Affairs

Medical Center (Decatur, GA, USA). HL is partially supported by

the International Program of Project 985, Sun Yat-sen University.

We would like to thank Dr Sreedhara Sangadala for helpful

discussions and Mesfin Teklemariam for real-time RT-PCR, plas-

mid purification, and Western blot assistance.


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