Angiotensin II induces interleukin-6 synthesis in osteoblaststhrough ERK1/2 pathway via AT1 receptor

Ling Guo a, Min Wang a,*, Zhi-yi Zhang a, Liang Hao a, Bei-yan Lou a, Xiao-yu Li a,Wings T.Y. Loo b, Lijian Jin b, Mary N.B. Cheung b

a State Key Laboratory for Oral Diseases and Department of Prosthodontics, West China Hospital of Stomatology, Sichuan University,

Renminnanlu 3rd Part 14#, Chengdu, PR Chinab Faculty of Dentistry, The University of Hong Kong, Hong Kong

a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 2 0 5 – 2 1 1

a r t i c l e i n f o

Article history:

Accepted 19 September 2010

Keywords:

Interleukin-6

Angiotensin II

Osteoblast

Extracellular signal-regulated kinase

a b s t r a c t

Background: Interleukin-6 (IL-6) is a potent stimulator of osteoclastic bone resorption.

Osteoblast secretion of IL-6 plays an important role in the regulation of bone metabolism.

Angiotensin II (Ang II) has been shown to regulate the expression of potent inflammatory

factors, including MCP-1 and IL-6, by stimulating endothelia cells, vascular smooth muscle

cells (VSMC) and monocytes. However, of the mechanism by which Ang II regulates IL-6

expression in osteoblasts is unknown.

Aims: The present study was designed to investigate the effect of Ang II on IL-6 expression in

osteoblasts isolated from mice. The receptor(s) required and the potential role of extracel-

lular signal-regulated kinase 1/2 (ERK1/2) activation in Ang II-induced IL-6 synthesis was

also examined in these cells.

Methods: The osteoblasts were isolated from the calvaria of mice and cultured in a-MEM

medium. IL-6 mRNA expression and protein synthesis was determined by qPCR and ELISA

analyses. ERK1/2 kinase activation was determined by western blot.

Results: The results indicate that Ang II induced IL-6 mRNA expression and protein synthe-

sis in cultured osteoblasts. However, these effects were abolished by pre-treatment with

Ang II type 1 (AT1) receptor antagonist, losartan, and the ERK1/2 inhibitor, U0126, inhibited

Ang II-mediated IL-6 expression and the phosphorylation of ERK1/2.

Conclusion: Ang II induces the synthesis of IL-6 in osteoblasts through activation of the ERK1/

2 pathway via the AT1 receptor.

Crown Copyright # 2010 Published by Elsevier Ltd. All rights reserved.

avai lab le at www.sc iencedi rec t .com

journal homepage: http://www.elsevier.com/locate/aob

1. Introduction

Interleukin-6 (IL-6) is a well-known multifunctional cytokine

that plays an important role in the regulation of various

biological processes, including hematopoiesis, the inflamma-

tory response, immunological reaction and neural develop-

ment.1–4 More specifically, IL-6 is a sensitive systemic

* Corresponding author. Tel.: +86 28 61153338; fax: +86 28 85582167.E-mail address: wtyloo@hkusua.hku.hk (M. Wang).

0003–9969/$ – see front matter . Crown Copyright # 2010 Published bdoi:10.1016/j.archoralbio.2010.09.016

indicator of inflammation and tissue damage.5,6 During

inflammatory processes, such as rheumatoid arthritis and

periodontal disease, leukocytes induce kinins, chemokines

and cytokines, which are known to enhance bone resorption.

Therefore, these cytokines been implicated in the pathogene-

sis of bone loss seen in the vicinity of inflammatory processes.

It has been reported that bone resorption is mediated by the

y Elsevier Ltd. All rights reserved.

a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 2 0 5 – 2 1 1206

increased local production of inflammatory cytokines, such as

tumour necrosis factor-a (TNF-a) and IL-6.7,8 IL-6-induces

osteoclastic formation from precursors and stimulates bone

resorption.9 It is well recognised as one of the most potent

osteoclastogenic factors in bone metabolism.10

The majority of serum IL-6 is predominately produced by

mononuclear macrophages. However, recent data have

demonstrated that other cell types, including epithelial cells

of the respiratory tract, fibroblasts and osteoblasts can also

produce IL-6.11–13 Recently, increasing evidence suggests that

IL-6 secreted by osteoblasts plays an important role in the

regulation of bone metabolism, the balance between bone

formation by osteoblasts and bone resorption by osteoclasts.

Bone resorption promoters, such as TNF-a, interleukin-1 (IL-1)

and parathyroid hormone, have been reported to stimulate IL-

6 production in cultured osteoblasts.14,15

Angiotensin II (Ang II), a biologically active octapeptide, is a

primary effector of the renin angiotensin system (RAS). Ang II

mediates hemodynamic, growth, inflammation and metabolic

response innumeroustissues, including the heart, liver, kidney,

and arteries.16–19 Additionally, Ang II has been shown to affect

the expression of inflammatory factors, such as MCP-1 and IL-6,

by stimulating endothelia cells, vascular smooth muscle cells

(VSMC) and monocytes.20–22 Ang II acts through two distinct

subtypes of cell-surface receptors, Ang II type 1 (AT1) and type 2

(AT2) receptors. Osteoblasts express AT1, a member of the

seven transmembrane-spanning G protein-coupled receptor

superfamily.23 It has been reported that Ang II induces bone

resorption, suppresses alkaline phosphatase activity in vitro,

and stimulates the proliferation of osteoblasts.24–26 Further-

more, recent clinical studies report the benefit of angiotensin-

converting enzyme (ACE) inhibitors in reducing the risk of

fractures and improving bone mineral density.27,28 Thus, Ang II

is currently recognised as playing a crucial role in bone

metabolism, however, the exact mechanism by which Ang II

affects bone metabolism has not yet been fully clarified.

Extracellular signal-regulated kinases (ERK1/2) are well

recognised to be essential mediators in intracellular signalling

for various agonists.29,30 It has been shown that ERK1/2 are

activated by several agonists in osteoblasts, including basic

fibroblast growth factor and oestrogen.31,32 The present study

was designed to investigate the effect of Ang II on IL-6

expression in osteoblasts. We show that Ang II may stimulate

IL-6 synthesis through the ERK1/2 pathway via the AT1

receptor in these cells.

2. Materials and methods

2.1. Materials

Ang II was obtained from Sigma (St. Louis, MO, USA). Foetal

bovine serum (FBS) and a-modified minimal essential medium

(a-MEM) were purchased from GIBCO (Grand Island, NY).

Losartan (AT1 receptor antagonist) was obtained from LKT

Laboratories (Paul, USA) and PD123319, the selective AT2

receptor antagonist was obtained from Sigma (St. Louis, MO,

USA). U0126 a selective ERK1/2 inhibitor, was purchased from

Sigma (St. Louis, MO, USA). Mouse IL-6 enzyme-linked

immunosorbent assay (ELISA) kit was got from R&D systems

(Minneapolis, MN, USA). Anti-ERK1/2 and anti-phospho-ERK1/

2 antibodies were obtained from Cell Signaling Technology

(Beverly, MA, USA).

2.2. Cell culture from animals

The animal study was approved by the Sichuan University

Ethics Committee. Suckling mice (24 h old) were obtained from

the Sichuan University Animal Center. Mice were euthanised,

the calvaria removed and the skin excised to release the

skullcap. Brain tissue was removed and the skullcap washed

three times in sterile Hank’s balanced salt solution (HBSS)

pH = 7.4 with penicillin (200 U/mL), and streptomycin (200 mg/

mL). Skullcaps were collected and the parietal bones harvested,

well clear of the developing sagittal suture,and digested inHBSS

containing collagenase (1 mg/mL) and 0.25% trypsin/0.53 mM

EDTA at 37 8C for 10 min. The solution was removed to a fresh

sterile tube (fraction 1)and mixed with freshmedium tostopthe

digestion reaction. This procedure was repeated with fresh

collagenase five more times (fractions 2–6). Fractions 1–6 were

combined and the cells pelleted by centrifugation at 2000 rpm

for 10 min. Cells were seeded (6 � 105) in 75 cm2 culture flasks

(Corning, USA) in a-MEM supplemented with 10% foetal bovine

serum (GIBCO, Grand Island, NY), penicillin (100 U/mL), and

streptomycin (100 mg/mL) at 37 8C in a 5% CO2 humidified

environment.33 The medium was changed every 3 days

throughout the experiments.33

2.3. IL-6 enzyme-linked immunospecific assay (ELISA)

ELISA was performed using the osteoblast culture super-

natants and following the manufacturer’s instructions from

the IL-6 ELISA kit (R&D systems, Minneapolis, MN). Cell

culture supernatants (100 mL) were pipeted into the provided

96-well plate and incubated for 2 h followed by three washes

with washing buffer. Wells were dried and 200 mL of

substrate (tetramethylbenizidine) was added into each well

for 20 min in the dark at room temperature. The plate was

then read at 450 nm wavelength using a Universal Micro-

plate Reader (Bio-Tek Instruments Inc., Winooski, VT). The

levels of IL-6 in the samples were determined by compari-

son with the standard curve generated from standards

supplied by the manufacturer.

2.4. Real-time quantitative polymerase chain reaction(RT-qPCR)

The levels of IL-6 mRNA were quantitatively measured by RT-

qPCR. Total cellular RNA was isolated using TRIzol reagent

(Invitrogen Life Technologies, Carlsbad, CA). Total RNA from

cells was reverse-transcribed using PrimeScriptTM RT reagent

kit (Takara, Osaka, Japan) in the presence of an oligo-(dT)

primer at 42 8C for 1 h to produce cDNA. Briefly, the cDNA

synthesised from total RNA was amplified in a 10 mL volume

with SYBR1 Premix Ex TaqTM II (Takara, Osaka, Japan), 0.1 mM

dNTPs, 0.4 mM each primer, and 1 U Taq DNA polymerase

(Takara, Osaka, Japan) using an ABI Prism 7300 sequence

detection PCR system (Applied Biosystems, Foster City, CA)

according to the manufacturer’s protocol. The primer

sequences were:

[()TD$FIG]

Fig. 1 – Effects of Ang II treatment on IL-6 mRNA expression

in osteoblasts. (A) Osteoblasts were treated with Ang II

(100 nM) for 3, 6, 12 and 24 h. Relative IL-6 mRNA levels

were determined by qPCR. (B) Osteoblasts were pre-treated

with the AT1 receptor antagonist (losartan, 10 mM), the

AT2 receptor antagonist (PD123319, 10 mM) or the MEK

inhibitor (U0126, 10 mM) for 1 h followed by Ang II

treatment for 12 h. Relative IL-6 mRNA levels were

determined by qPCR. Results are mean W SEM, n = 3/group,

*P < 0.05 vs. control; #P < 0.05 vs. Ang II.

a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 2 0 5 – 2 1 1 207

IL-6: sense (50-ACA-AAG-CCA-GAG-TCC-TTC-AGA-G-30),

antisense (50-CCT-TAG-CCA-CTC-CTT-CTG-TGA-CT-30),

149 bp;

b-actin: sense (50-AGA-GCA-AGA-GAG-GTA-TCC-TGA-

CC-30),

antisense (50-CAC-ACG-CAG-CTC-ATT-GTA-GAA-G-30),

113 bp.

Reaction conditions were as follows: 95 8C for 5 s, followed

by 40 PCR cycles at 95 8C for 5 s, 60 8C for 30 s. In each PCR, a

nuclease-free water tube was set as a control. The specificity of

RT-qPCR products was confirmed both by melting curves and

agarose gel electrophoresis. Quantification of relative gene

expression was calculated by the comparative Ct method

(2�DDCt), as recommended by the manufacturer.34

2.5. Protein isolation and western blot analysis

Cultured osteoblasts were washed with ice-cold PBS and lysed

with ice-cold Lysis Buffer (1% Triton X-100, 20 mM HEPES

(pH = 7.5), 5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM

dithiothreitol, 1 mM phenylmethylsulphonyl fluoride and

1 mg/mL each of leupeptin, aprotinin and pepstatin) for

30 min. Total cell lysates were centrifuged at 12,000� g for

10 min at 4 8C, and the supernatant was collected and stored at

�80 8C until use. Equal amounts of protein (30 mg) were diluted

with SDS sample buffer (0.125 M Tris–HCl, pH = 6.8, 10%

glycerol, 2% b-mercaptoethanol, 2% SDS and 0.1% bromophenol

blue) and boiled for 5 min. Samples were then resolved by SDS-

PAGE and electrotransferred to PVDF membrane. Nonspecific

protein binding was blocked by incubating the membrane with

5% non-fat dry milk, washing with TBST, and then incubating

with anti-ERK1/2 or anti-phospho-ERK1/2 antibodies (Cell

Signaling Technology, Beverly, MA) at 4 8C overnight. Mem-

branes were incubated with HRP-conjugated secondary anti-

bodies at room temperature. Following washes with TBST, the

protein bands were visualised using the ECL detection system

(Pierce, USA) according to the manufacturer’s instructions.

2.6. Statistical analysis

The data were analysed by ANOVA of 16.0 SPSS (USA) for

multiple comparisons between pairs, and a p < 0.05 was

considered significant. All data are presented as the mean-

s � SEM of triplicate determinations.

3. Results

3.1. Ang II induces IL-6 mRNA expression in osteoblastsvia the AT1 receptor

In order to define the concentration of Ang II that would

induce IL-6 expression, osteoblasts were treated with various

concentrations (1–100 nM). It was determined that treatment

with 100 nM Ang II resulted in the highest increase in IL-6

mRNA expression (data not shown). Osteoblasts were cultured

with Ang II (100 nM) for various times. The results indicate that

Ang II induces IL-6 mRNA levels as early as 3 h and the levels of

IL-6 mRNA level reached a plateau at 12 h post-stimulation

(Fig. 1A).

It is known that the effects of Ang II are mediated via

activation of the AT1 or AT2 receptors. Therefore, the role of

the AT1 and AT2 receptors in Ang II-mediated induction of IL-6

mRNA expression was investigated. Osteoblasts were treated

with losartan (10 mM) and PD123319 (10 mM), selective AT1 and

AT2 receptor antagonists, respectively, 1 h before the addition

of Ang II to the cells. As shown in Fig. 1B, losartan significantly

inhibited the AngII-induced increase in IL-6 mRNA. However,

PD123319 did not alter the levels of IL-6 mRNA induced by Ang

II. These results suggest that the Ang II-induced IL-6 expres-

sion is mediated by the activation of AT1 receptors.

3.2. Ang II induces IL-6 protein secretion in osteoblastsvia AT1 receptor

To determine whether Ang II also induces IL-6 secretion from

osteoblasts, serum deprived cells were treated with Ang II for

various times (3, 6, 12, and 24 h) and secreted IL-6 levels were

[()TD$FIG]

Fig. 2 – Effects of Ang II on IL-6 secretion in osteoblasts. (A)

Osteoblasts were treated with Ang II (100 nM) for 3, 6, 12

and 24 h, and levels of IL-6 the supernatant was determined

by ELISA. (B) Osteoblasts were pre-treated with the AT1

receptor antagonist (losartan, 10 mM), the AT2 receptor

antagonist (PD123319, 10 mM) or the MEK inhibitor (U0126,

10 mM) for 1 h followed by Ang II treatment. Levels of IL-6 in

osteoblast supernatants were determined by ELISA after

Ang II stimulation for 24 h. Results are mean W SEM, n = 3/

group, *P < 0.05 vs. control; #P < 0.05 vs. Ang II.

[()TD$FIG]

Fig. 3 – Effect of Ang II treatment on ERK1/2 activation in

osteoblasts. Osteoblasts were stimulated with Ang II for 1,

2, and 3 h. Upper panel: western blot analysis was

performed using anti-phospho-ERK1/2 and anti-ERK1/2

(total). Lower panel: quantification of western blot for

phospho-ERK1/2. Results are mean W SEM, n = 3/group,

*P < 0.05 vs. control.

a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 2 0 5 – 2 1 1208

measured by ELISA. The concentration of IL-6 in culture

medium progressively increased in a time dependent manner

(between 3 h and 24 h post-treatment) (Fig. 2A). Ang II-

induced IL-6 secretion as early as 3 h and maximal effects

were observed by 24 h.

Osteoblasts were also pre-incubated for 1 h with losartan

(10 mM) or PD123319 (10 mM), subsequently treated with Ang II

(100 nM) for 24 h and secreted IL-6 levels measured. Pre-

treatment with losartan, but not PD123319, abolished Ang II-

induced IL-6 secretion (Fig. 2B). Osteoblasts were also

pretreated for 1 h with U0126 (10 mM) followed by stimulation

with Ang II (100 nM). Inhibition of ERK1/2 significantly reduced

Ang II-mediated IL-6 secretion (Fig. 2B). These findings

indicate that Ang II-induces IL-6 production through activa-

tion of the AT1 receptor and ERK1/2.

3.3. ERK1/2 is required for Ang II-induced IL-6 mRNAexpression and secretion in osteoblasts

Activation of ERK1/2 was analysed by determining the levels of

p-ERK1/2 during Ang II treatment of osteoblasts. ERK1/2

activation was increased between 1 h and 3 h after Ang II

treatment (Fig. 3). However, total ERK1/2 protein levels did not

differ during treatment. Pre-treatment with the ERK1/2

inhibitor, U0126 (10 mM), decreased the Ang II-induced

activation of ERK1/2 (Fig. 4). Importantly, pre-treatment with

losartan inhibited Ang II-induced activation of ERK (Fig. 4).

Pre-treatment with U0126 and losartan significantly inhib-

ited Ang II-induced IL-6 expression and phosphorylation of

ERK1/2 (Figs. 1B, 2B and 4). Furthermore, treatment with

losartan inhibited Ang II-induced activation of ERK1/2.

Collectively, these data demonstrate that Ang II engages the

AT1 receptor, inducing ERK1/2 activation and, subsequently,

IL-6 synthesis.

4. Discussion

In the present study, we show that Ang II significantly induces

IL-6 synthesis in osteoblasts. However, this effect was

abolished by pre-treatment with the AT1 receptor antagonist,

losartan, but not the AT2 receptor antagonist, PD123319. It has

been demonstrated that the activation of MAPKs is important

in the regulation of IL-6 expression by mediating the activation

of NF-kB and TNF-a.35,36 In our experiments, the ERK1/2

inhibitor, U0126, significantly inhibited Ang II-induced IL-6

synthesis in osteoblasts.

Cytokines are potent regulators of bone resorption and

have been involved in diseases characterised by excess bone

loss, such as osteoporosis, rheumatoid arthritis and periodon-

tal disease.37,38 Osteoblast-derived cytokines are known to

[()TD$FIG]

Fig. 4 – Inhibition of the AT1 receptor or ERK1/2 activation

in osteoblasts inhibits Ang II-induced IL-6 synthesis.

Osteoblasts were pretreated with losartan (10 mM) and

U0126 (10 mM) for 1 h, followed by treatment with Ang II

for 1, 2, and 3 h. Upper panel: western blot of phospho-

ERK1/2 levels in osteoblasts after 1 h Ang II treatment.

Lower panel: quantification of western blot for phospho-

ERK1/2. Results are mean W SEM, n = 3/group, *P < 0.05 vs.

control; #P < 0.05 vs. Ang II.

a r c h i v e s o f o r a l b i o l o g y 5 6 ( 2 0 1 1 ) 2 0 5 – 2 1 1 209

regulate both osteoclast differentiation from monocyte line-

age precursors and the activity of mature osteoclasts.39

Osteoblast secretion of IL-6 plays an indispensable role in

bone metabolism, which is regulated by a balance between

bone formation by osteoblasts and bone resorption by

osteoclasts. IL-6 is recognised to act as a bone resorbing

factor, inducing osteoclast formation and stimulating bone

resorption. Bone resorptive agents, such as TNF-a, IL-1 and

parathyroid hormone, were reported to stimulate IL-6 pro-

duction in cultured osteoblasts.14,40

Ang II is a biologically active octapeptide, and a primary

effector RAS. Data have shown that Ang II mediates

hemodynamic, growth, the inflammatory process, and the

metabolic response in numerous tissues, including the heart,

arteries, liver, and kidney.16–19 Meanwhile, it has been proven

that Ang II affects the expression of potent inflammatory

factors, such as MCP-1 and IL-6, by stimulating VSMCs and

monocytes. Interestingly, treatment with an AT1 receptor

antagonist decreases plasma levels of IL-6 in patients with

chronic heart failure.41 These findings suggest that IL-6 is

required for Ang II effects in some biological processes.

However, the mechanism by which Ang II induces IL-6

expression has not been delineated. In this paper, we first

investigated whether Ang II induces IL-6 secretion in osteo-

blasts. We have shown that Ang II treatment induces IL-6

secretion in a time-dependent manner in osteoblasts.

Ang II acts through two distinct subtypes of cell-surface

receptors, the AT1 and AT2 receptors. Osteoblasts express the

AT1 receptor, a member of the seven transmembrane-

spanning G protein-coupled receptor superfamily. Our further

investigations confirmed that the AT1 receptor is required for

Ang II-induced IL-6 secretion in osteoblasts.

It has been reported that Ang II rapidly activates ERK1/2 in

VSMC and fibroblasts.42,43 In addition, ERK1/2 has been shown

to play a role in IL-6 expression in osteoblasts and it was

demonstrated that Ang II induces ERK1/2 activation via the

AT1 receptor.44,45 Our data show that Ang II requires ERK1/2

phosphorylation to induce IL-6 expression and secretion in

osteoblasts. These results suggest that Ang II exhibits pro-

inflammatory properties, mediating this process via activation

of the ERK1/2 pathway resulting in IL-6 secretion.

In conclusion, these results demonstrate that Ang II

stimulates the synthesis of IL-6 in osteoblasts through the

ERK1/2 pathway via the AT1 receptor.

Acknowledgements

This study was supported by the Public Welfare Foundation of

the Sichuan Science and Technology Department of China

(No. 2008FN0174).

Funding: None.

Competing interests: None declared.

Ethical approval: Not required.

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