Aluminum-fluoride complexes induce osteoclastogenesis

Eunho Yang

Department of Dental ScienceThe Graduate School, Yonsei University

Aluminum-fluoride complexes induce osteoclastogenesis

Directed by Professor Dong Min Shin

Eunho Yang

The Doctoral Dissertation Submitted to the Department of Dental Scienceand the Graduate School of Yonsei University

in partial fulfillment of the requirements for the degree of Doctor of Philosophy

June 2011

This certifies that the Doctoral Dissertation of Eunho Yang is approved.

Thesis Supervisor: Prof. Dong Min Shin

Thesis Committee: Prof. Syng-Ill Lee

Thesis Committee: Prof. Jeong Taeg Seo

Thesis Committee: Prof. Jung Goo Kang

Thesis Committee: Prof. Hyeok Namkoong

The Graduate School Yonsei University

June 2011

Acknowledgements

오랫동안 기다려 왔던 꿈이 실현되는 순간입니다. 남들보다는 늦은 나이지

만 학문의 최종과정이라고 생각하고 대학원에 첫발을 디뎠었는데, 이젠 멀어

만 보이던 그 끝을 앞에 두고 있습니다. 세월의 흐름을 알 수 있었던 치.의학

학문의 진보를 몸으로 체험하면서, 스스로 날로 새로워지는 걸 느끼며 지내왔

던 소중한 시간이었습니다. 학문은 끝이 없이 변하고 발전한다는 진리를 깨달

았으며, 앞으로도 평생 동안 항상 새로운 것을 배우고 준비하며 현재의 모습

에 안이하게 안주하지 않고 겸손하게 새로운 것을 받아드리도록 노력할 것입

니다.

항상 든든히 뒤에서 지켜봐 주시고 격려해주시는 이승일 교수님, 노심초사

하며 실험 결과를 위해 애쓰신 박보령선생님, 양유미선생님, 손아란선생님,

박순홍선생님, 외에도 생리학교실 선생님들께 감사 드립니다. 또한 앞선 경험

자로써 도와주신 강정구 외래교수님, 남궁혁 외래교수님. 이관우 외래교수님

외에 생리학 외래교수님 들에게도 감사 드립니다. 벗이자 학자로서 존경스러

운 서정택 교수님께도 감사 드립니다. 특히 오랜 대학원 생활 내내 용기를 잃

지 않도록 지도하시고 이끌어주신 신동민 지도교수님께 깊은 감사를 드립니다.

앞으로도 대학원 생활 동안 얻은 지혜와 용기를 가지고 주위에 봉사하며, 제

가 처한 위치에 맞는 역할을 해나가겠습니다.

항상 꿈을 잃지 말고 노력하라는 말씀으로 격려 해주신 아버님, 어머님 감

사합니다.

세상에서 또 하나의 나인 인성이, 정은이, 정민아, 고마워.

i

Contents

List of figures ............................................................................ iii

Abbreviations ............................................................................ iv

ABSTRACT ............................................................................... v

I. INTRODUCTION .................................................................... 1

II. MATERIALS AND METHODS ................................................ 6

1. Cell culture and reagents .................................................... 6

2. Preparation of bone marrow derived macrophages ............... 7

3. Analysis of bone density and skeletal morphology ................ 7

4. [Ca2+]i measurement .............................................................. 8

5. Western blotting ...................................................................... 9

6. In vitro osteoclastogenesis ..................................................... 9

7. Statistics ................................................................................ 10

ii

III. RESULTS ........................................................................ 11

1. Exposure to aluminum-fluoride complexes and bone density in

mice ......................................................................................... 11

2. [Ca2+]i oscillations induced by RANKL and aluminum-fluoride

complexes in osteoclast precursor cells ................................ 11

3. Dependence of [Ca2+]i oscillations on extracellular Ca2+ ....... 15

4. Activation of MAPK signaling in the presence of RANKL or

aluminum-fluoride complexes in osteoclast precursor cell .... 19

5. Enhanced RANKL-mediated osteoclastogenesis by aluminum-

fluoride complexes ................................................................. 19

IV. DISCUSSION ...................................................................... 24

V. CONCLUSIONS ................................................................... 29

VI. REFERENCES .................................................................... 30

ABSTRACK (IN KOREAN) ...................................................... 37

iii

List of Figures

Fig. 1. Schematic drawing showing the similarity mimic in Gα

Proteins ................................................................................ 4

Fig. 2. Micro-CT images of tibiae and femurs of mice exposed to

normal tap water or aluminum-fluoride complexes treated

tap water ............................................................................... 12

Fig. 3. Histology of tibiae from mice ............................................. 14

Fig. 4. Ca2+ signaling in osteoclast precursor cells induced by

Receptor activator of NF-kB ligand (RANKL) ................... 16

Fig. 5. [Ca2+]i oscillations evoked by AlF4- in osteoclast precursor

cells ..................................................................................... 17

Fig. 6. Effect of removal of extracellular Ca2+ on [Ca2+]i

oscillations .......................................................................... 18

Fig. 7. Effect of RANKL or AlF4- on MAPK signaling ................. 21

Fig. 8. TRAP staining of osteoclast from RAW 264.7 cells and

BMMs .................................................................................. 22

iv

Abbreviations

[Ca2+]i : intracellular concentration of calcium

BMMs : Bone marrow-derived macrophages

RANKL: Receptor activator of NF-κB ligand

M-CSF : macrophage-colony stimulating factor

PSS : physiologic salt solution

MAPKs : mitogen-activated protein kinases

JNK : c-Jun-NH2-terminal kinase

ERK : extracellular signal-regulated kinase

TRAP : Tartrate-Resistant Acid Phosphatase

v

ABSTRACT

Aluminum-fluoride complexes

induce osteoclastogenesis

Eunho Yang

Department of Dental Science

The Graduate School, Yonsei University

(Directed by Professor Dong Min Shin)

Receptor activator of NF-kB ligand (RANKL)-induced

osteoclastogenesis is accompanied by Ca2+ signaling which sequentially

activates calcineurin and NFATc1 and triggers osteoclastogenesis.

However, it is not known whether RANKL-independent Ca2+ signaling

could be induced and activates differentiation of osteoclasts during

osteoclastogenesis. In the present study, the effect of aluminum-fluoride

complexes, a G-protein activator, on Ca2+ signaling and

vi

osteoclastogenesis in mouse leukemic macrophage cell line, RAW 264.7

cells, and primary cultured mouse bone marrow derived macrophages

was investigated. Using micro-CT and toluidine blue staining, decreased

bone density was observed in the group of mice exposed to aluminum-

fluoride complexes in drinking water for eight weeks. Aluminum-fluoride

complexes induced the generation of extracellular Ca2+-dependent

intracellular Ca2+concentration ([Ca2+]i) oscillations. Moreover,

aluminum-fluoride complexes enhanced the generation of RANKL-

mediated TRAP positive osteoclasts and induced the activated MAPK

signaling such as ERK and JNK proteins. These results suggest that G-

protein activation by aluminum-fluoride complexes could induce [Ca2+]i

oscillations and osteoclast differentiation in osteoclast precursor cells.

Key words: osteoclastogenesis, MAPK, TRAP, RANKL, Aluminum-fluoride, Ca2+ signaling.

１

Aluminum-fluoride complexes

induce osteoclastogenesis

Eunho Yang

Department of Dental Science

The Graduate School, Yonsei University

(Directed by Professor Dong Min Shin)

I. INTRODUNCTION

As fluoride was believed to have a beneficial effect of strengthening

teeth and preventing decay, water fluoridation began in the United States

in 1940s and was introduced in many countries. However, many studies

have been reported that fluoride has influences on the activity of a

variety of enzymes and the controversy about fluoride arises from safety

concerns regarding the fluoridation of public water supplies. People now

２

get fluoride unconsciously from various sources, including foods and

beverages prepared with fluoridated water, fluorinated drugs, fluorine-

containing volatile anesthetics, pesticide and fertilizer residues, dental

treatments and products. Fluoride has been used to increase bone mass

in osteoporosis patient on the other hand, there are conflicting reports on

the effect of fluoride on trabecular bone formation and bone strength

(Ostergren et al., 2004). Consumed fluoride through medicine, dietary

food or water can form aluminum-fluoride complexes spontaneously in

aqueous solutions with even small amount of aluminum.

Aluminum is one of the most abundant elements in the Earth’s crust

and is a nonessential element to humans. These days people are

constantly exposed to aluminum because of industrialization and health-

related technology. Aluminum is found in foods, water, air, and soil.

Aluminum tends to accumulate in the body, especially in the brain and the

bones. The toxicity of aluminum has been reported continually and it can

cause several clinical disorders such as anemia, bone diseases and

neurotoxicity (Del Pilar Martinez et al., 2011). In bone tissue aluminum

has an effect on inhibition of bone cell proliferation, hydroxyapatite

formation and growth and suppression of bone cell activity (Malluche,

2002). These effects lead to the inhibition of bone mineralization,

decreased bone formation and lower bone mass.

３

Aluminum-fluoride complexes act as an analog of a phosphate group

because of their atomic and molecular similarities. Aluminum-fluoride

complexes combine with α-subunit in G-proteins and forms Gα

۰GDP۰Aluminum-fluoride complex (Fig. 1). Thus, α-subunit is

separated from β/γ-subunit, because the complex acts like GTP-

bound G-proteins (Bigay et al., 1987). Furthermore, because the

complex has more powerful activities which of α-subunit activate

GTPase or GTPase activating proteins than GTP, the complex elevates

G-proteins reacting rates when adequate agonists exist (Chen and

Penington, 2000). Therefore, aluminum-fluoride complexes can be used

as useful tools investigating the signal pathways following G-proteins

activation. Aluminum-fluoride complexes stimulate G-protein and mimic

the action of many neurotransmitters, hormones, and immune system

(Struneckáa and Patočkab, 1999). And it also affects bone cells, Ca2+

influx and mobilization, ion transport, cell growth and differentiation and

protein phosphorylation. It was reported that the effects of fluoride and

aluminum on levels of the second messenger molecules are dependent on

the type of cells or tissues (Strunecka et al., 2002).

In osteoclasts, aluminum-fluoride complexes decrease cAMP level but

increase the level of inositol phosphates and cytosolic Ca2+ (Moonga et

al., 1993). It is known that oscillations in intracellular concentration of

４

Fig. 1. Schematic drawing showing the similarity mimic in Gα Proteins.

Aluminum fluoride binds to the α-subunit of G-proteins and activates

the downstream of it.

５

calcium ([Ca2+]i) by Receptor activator of NF-kB ligand (RANKL)

stimulation play important roles in mediating osteoclastogenesis

(Takayanagi et al., 2002). These oscillations are important for the

activation and amplification of NFATc1. NFAT activation is tightly

regulated by phosphorylation, which is controlled by the Ca2+-dependent

phosphatase, calcineurin. Dephosphorylated NFAT is allowed to

translocate into the nucleus and induces a number of genes involved in

differentiation of cells like osteoclasts. RANKL stimulates TRAF6 and

activates mitogen activated protein kinases (MAPKs) like extracellular

signal-regulated kinase (ERK), c-Jun-N-terminal kinase (JNK) and

p38 (Lee et al., 2002). In osteoclasts ERK activation is involved in

osteoclast survival and JNK and p38 play essential roles in osteoclast

differentiation (Tanaka et al., 2006),(Nishio et al., 1991) and

intracellular Ca2+ dynamics (Satoh et al., 1999).

From these reports, it was postulated that Ca2+ signaling induced by

aluminum-fluoride complexes may affect osteoclast differentiation. In

the present work, the investigation was undertaken to study the effects

of aluminum fluoride complexes (AlF4-) on mice bone density in vivo and

on Ca2+ signaling and osteoclasts differentiation in mouse leukemic

marcrophage cell line, RAW 264.7 cells, and primary cultured mouse

bone marrow derived marcrophages (BMMs).

６

MATERIALS AND METHODS

1. Cell culture and reagents

The mouse monocyte cell line RAW264.7 cells (KCLB) were cultured

in Dulbecco's modified eagle medium (DMEM; Gibco/BRL, Grand Island,

NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco/BRL,

Grand Island, NY, USA). Bone BMMs were cultured in α-minimum

essential medium(α-MEM; Gibco/BRL, Grand Island, NY, USA)

supplemented with 10% FBS and M-CSF(10ng/ml). Both cells were

maintained at 37°C in a humidified atmosphere of 5% CO2. Culture media

were changed every other day. RANKL and macrophage-colony

stimulating factor (M-CSF) were purchased from KOMA Biotech (Seoul,

Korea). AlCl3 and NaF were purchased from Sigma Aldrich (St. Louis,

MO, USA) and Fluka (Buchs, Switzerland), respectively. Fura-2/AM was

purchased from Teflabs (Austin, TX, USA). Pluronic acid was obtained

from Invitrogen (Eugene, Oregon, USA). Monoclonal antibodies against

ERK, phospho-ERK, JNK and phospho-JNK were purchased from Santa

Cruz Biotechnology (SantaCruz, CA, USA).

７

2. Preparation of bone marrow derived macrophages

The femurs and tibiae were removed from 4-week-old ICR male mice

(KOATECH, Kyung-gi-Do, Korea) and the flushed out cells were

cultured in α-MEM containing 10% FBS and 10ng/ml M-CSF for 24h.

Non adherent cells were isolated and seeded in 35 mm dish or 96-well

plates and treated with M-CSF (10ng/ml). After 2-3 days the adherent

cells were used as BMMs.

3. Analysis of bone density and skeletal morphology

4-week-old male mice of the ICR strain were acclimatized for a week

in plastic cages under hygienic conditions and were separated 2 groups.

The animals of group A were provided with standard feed and tap water.

The animals of group B were exposed to aluminum (as AlCl3 300 ppm)

and fluoride (as NaF 100ppm) in drinking tap water. At the end of 8

weeks, femurs and tibiae were removed from ICR mice. Bone density

was measured with a three-dimensional Micro-CT (SkyScan-1076 high

resolution in vivo micro-CT system; SkyScan, Aartselaar, Belgium). For

histological examination femurs and tibiae from mice were fixed in 4%

neutral phosphate-buffered formalin. After decalcification using 10%

ethylene diamine tetra acetic acid (EDTA) solution paraffin sections

８

were made following conventional methods. 5μm-sections were cut and

stained with toluidine blue staining. Stained sections were examined and

photographed under Axio Imager microscopy (Carl Zeiss, Jena,

Germany)

4. [Ca2+]i measurement

The cells were seeded on cover glass in a 35 mm dish (5x104 RAW

264.7 cells/dish or 1x105 BMM s/dish) and treated with RANKL

(50ng/ml) or aluminum-fluoride complex (2.5 mM NaF + 50 nM AlCl3

for RAW 264.7 cells or 1.25 mM NaF + 25 nM AlCl3 for BMMs). [Ca2+]i

was determined in cells by fura-2/AM in an extracellular physiologic salt

solution (PSS), the composition of which was as follows : 140 mM NaCl,

5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, and 10 mM glucose,

titrated to pH 7.4 with NaOH. The osmolarity of the PSS was 310 mOsm.

Ca2+-free medium contained 1 mM EDTA and 1mM ethylene glycolbis-

(β-amino ethylether)-N, N, N′, N′-tetra acetic acid (EGTA) in PSS.

The RAW 264.7 cells and BMMs were loaded with 3μM fura-2/AM for

40 min and after washing with standard solution, [Ca2+]i was measured

by alternately illuminating the cells at wave lengths 340 and 380 nm, and

the emitted light was passed through a 510 nm cut off filter and was

９

collected with a CCD camera and analyzed with a Meta Fluor system

(Universal Imaging Co., Downingtown, PA, USA). The 340/380 fura-2

ratio was taken as a measure of [Ca2+]i and fluorescence images were

obtained at 2s intervals.

5. Western blotting

Whole cell extracts were prepared in lysis buffer (20 mM Tris, pH 7.4,

250 mM NaCl, 2 mM EDTA, pH 8.0, 0.1% Triton-X100, 0.01 mg/ml

aprotinin, 0.005 mg/ml leupeptin, 0.4 mM PMSF, and 4 mM NaVO4), and

then spun at 13,000 rpm for 15 min to remove insoluble material. 20μ

g/well proteins were loaded on 10% SDS-PAGE, and then were

separated by size. Separated proteins were electro-transferred to

nitrocellulose membranes, blocked with 5% BSA in TBS, and probed with

antibodies against phospho-ERK (1:1000), total-ERK (1:1000),

phospho-JNK (1:1000) and total-JNK (1:1000). Thereafter, blots were

washed, exposed to HRP-conjugated secondary antibodies for 1 h, and

finally detected by chemiluminescence (ECL, Amersham Pharmacia

Biotech).

１０

6. In vitro osteoclastogenesis

RAW 264.7 cells were seeded in 96-well plates at a density of 2 x 103

cells/well with a medium containing RANKL (50 ng/ml) and/or AlF4-

(1.25 mM NaF + 25 nM AlCl3). RANKL (50 ng/ml) and/or AlF4- (1.25

mM NaF + 25 nM AlCl3) were also added to BMMs (4x105 cells/well)

cultured in 96-well plates with α-MEM containing M-CSF (10ng/ml).

Six days later osteoclastogenesis was confirmed by Tartrate-Resistant

Acid Phosphatase (TRAP) staining to evaluate TRAP-positive

multinucleated osteoclast formation. Naphthol 3-hydroxy-2-naphtho-

2,4-xylidide (AS-MX) phosphate (Amresco, Cleveland, OH, USA) was

used as the substrate and fast red violet 5–chloro–4–benzamido–2-

methylbenzenediazoniumchloride, hemi [zinc chloride] (LB) salt (Sigma

Aldrich, St. Louis, MO, USA) as the diazonium salt.

7. Statistics

Results are expressed as means ± SDs from at least 3 independent

experiments. The statistical significances of differences between groups

were determined using the Student t-test. (+P<0.05,*P<0.01,

**P<0.005)

１１

II. RESULTS

1. Exposure to aluminum-fluoride complexes and bone density

in mice

It was predicted that the aluminum-fluoride complexes have effects on

bone density. Therefore, to confirm the role of aluminum-fluoride

complexes, in vivo experiment with two groups of mice using micro-CT

and toluidine blue staining were conducted. As shown in micro-CT

images (Fig. 2A and B), lower trabecular bone volume observed in the

group of mice exposed to aluminum-fluoride complexes for eight weeks

as compared with the group of mice provided with normal tap water.

Histological analysis of mice tibiae using toluidine blue also confirmed the

reduced bone density in the group of mice exposed to aluminum-fluoride

complexes (Fig. 3).

2. [Ca2+]i oscillations induced by RANKL and aluminum-

fluoride complexes in osteoclast precursor cells

It is known that RANKL induces [Ca2+]i oscillations in RAW264.7 cells

(Kuroda et al., 2008) and BMMs (Takayanagi et al., 2002) and this

RANKL-evoked Ca2+ signaling is essential for osteoclastogenesis.

１２

A

B

１３

Fig. 2. Micro-CT images of tibiae and femurs of mice exposed to normal

tap water or aluminum-fluoride complexes treated tap water. A, whole

tibia and femur micro-CT scan images. B, cross-sectional micro-CT

images of mouse tibia and femur. (tap water: The group of mice provided

with standard feed and tap water. AlF4- : The group of mice exposed to

aluminum (as AlCl3 300 ppm) and fluoride (as NaF 100 ppm) in drinking

tap water.)

１４

Fig. 3. Histology of tibiae from mice. Toluidine blue staining (scale bar,

50 μm) of tibia from mice exposed to normal tap water (left panel) or

aluminum-fluoride complexes (right panel).

１５

To confirm the Ca2+ signaling, [Ca2+]i oscillations were analyzed in the

presence of RANKL in RAW 264.7 cells and BMMs. As shown in Fig. 4,

[Ca2+]i oscillations were observed about 24-72 hours after RANKL

treatment in RAW 264.7 cells (Fig. 4A), and BMMs (Fig. 4B).

Furthermore, Ca2+ signaling induced by AlF4

- was confirmed in both cells.

As shown in Fig. 5, AlF4- stimulation also produced [Ca2+]i oscillations

after 24-72 hours treatment in RAW264.7 cells (Fig. 5A) and BMMs

(Fig. 5B).

3. Dependence of [Ca2+]i oscillations on extracellular Ca2+

A previous work reported that the RANKL-induced [Ca2+]i oscillations

are dependent on the presence of extracellular Ca2+ (Kim et al., 2010).

The effect of extracellular Ca2+ removal on the RANKL or AlF4--evoked

Ca2+ oscillations was examined. As expected, the RANKL-induced

[Ca2+]i oscillations were abolished by removal of extracellular Ca2+ in

RAW 264.7 cells and BMMs (Fig. 6 left panel). In addition, the [Ca2+]i

oscillations in RAW 264.7 cells and BMMs by AlF4- also showed

dependency on extracellular Ca2+ (Fig. 6 right panel).

１６

Fig 4. Ca2+ signaling in osteoclast precursor cells induced by

Receptor activator of NF-kB ligand (RANKL). A, Induced

intracellular calcium concentration ([Ca2+]i) oscillations in RAW

264.7 cells. B, Induced [Ca2+]i oscillations in BMMs (Bone

marrow-derived macrophages).

B

A

１７

Fig 5. [Ca2+]i oscillations evoked by AlF4

- in osteoclast precursor cells.

A, Induced [Ca2+]i oscillations by AlF4- in RAW 264.7 cells. B, Induced

[Ca2+]i oscillations by AlF4- in BMMs.

A

B

１８

Fig 6. Effect of removal of extracellular Ca2+ on [Ca2+]i oscillations.

Altered RANKL-induced (left panel) or AlF4--induced (right panel)

[Ca2+]i oscillations in RAW 264.7 cells and BMMs by removal of

extracellular Ca2+.

１９

4. Activation of MAPK signaling in the presence of RANKL or

aluminum-fluoride complexes in osteoclast precursor cell

RANKL is known to activate MAPK signaling by phosphorylation of

ERK (Ang et al., 2011) and JNK (Darnay et al., 1998) during

osteoclastogenesis. To test the expression level of these proteins,

Western blot analysis was performed on lysates of RAW 264.7 cells

stimulated with RANKL (50 ng/ml) or AlF4- (50nM AlCl3 + 2.5 mM NaF)

in a time-dependent manner. As reported, activated ERK and JNK (Fig.

7A) were observed at 1 or 30 min after RANKL treatment. Moreover, in

RAW 264.7 cells incubated with AlF4-, enhanced phospho-ERK protein

and phospho-JNK protein were also detected (Fig. 7B).

5. Enhanced RANKL-mediated osteoclastogenesis by

aluminum-fluoride complexes

TRAP is expressed by the osteoclast during bone resorption and

secretion correlates with resorptive behavior TRAP-positive

multinucleated cells. (Athanasou and Quinn, 1992). To confirm an

osteoclastogenesis derived from RAW 264.7 cells and BMMs by RANKL

(50 ng/ml) and/or AlF4- (1.25 mM NaF + 25 nM AlCl3), TRAP staining

２０

were conducted. TRAP-positive cells were observed in RAW 264.7 cells

and BMMs incubated with RANKL and/or AlF4- for six days. In addition,

both cells showed enhanced osteoclast formation in the presence of

RANKL together with AlF4- (Fig. 8A and B).

２１

Fig 7. Effect of RANKL or AlF4- on MAPK signaling. A, phosphorylation

of ERK and JNK by RANKL (50 ng/ml). B, phosphorylation of ERK and

JNK by AlF4- (50 nM AlCl3 + 2.5 mM NaF).

A

B

２２

A

B

２３

Fig 8. TRAP staining of osteoclast from RAW 264.7 cells and BMMs. A,

Osteoclast formation in RAW264.7 murine macrophage cells by RANKL

(50 ng/ml) and/or AlF4- (1.25 mM NaF + 25 nM AlCl3). B, Osteoclast

formation in BMMs by RANKL (50 ng/ml) and/or AlF4- (1.25 mM NaF +

25 nM AlCl3).

２４

Ⅲ. DISCUSSION

There has been a continuous water fluoridation debate because fluoride

is a known toxin that accumulates in tissues and causes damage. Some

studies suggested that fluoride can cause bone damages (Schlesinger et

al., 1956) and (Czerwinski et al., 1977). Fluoride plays different role on

different region of the bone. In trabecular bone, it enhances the bone

mineral density, but it decreases the cortical bone density (Strunecká et

al., 2004). It is known that traces of aluminum can form aluminum-

fluoride complex (mostlikely AlF4-)(Strunecka et al., 2002) easily in

aqueous solutions and make a synergistic action of fluoride to activate G

proteins (Sternweis et al., 1981). G protein signaling plays important

roles in bone development, remodeling and disease (Wu et al., 2010).

Moonga (Moonga et al., 1993) reported that exposure of osteoclasts to

AlF4- resulted in an increased secretion of TRAP but inhibited bone

resorption in a concentration-dependent fashion. Fluoride and aluminum

stimulated osteoclastic activity that leads to bone reabsorption and

skeletal changes (Chen et al., 1997). Aluminum has a role as an antidote

to protect against fluoride accumulation on bone of rabbits and rats but

aluminum levels in bone of rabbits are increased by the addition of

fluoride to the drinking water (Ahn et al., 1995). There are possibilities

２５

that excess usage of aluminum may cause hyperactivity of parathyroid

gland (Franke, 1985) and some osteotoxicity associated with high

fluoride levels in bone may be due to the accumulation of aluminum or

aluminum-fluoride complexes (Ahn et al., 1995).

On the basis of previous findings, I hypothesized that (1) AlF4- affects

bone density in vivo, and (2) that activated G-protein by AlF4- generate

Ca2+ signaling and promotes osteoclastogenesis. In this report, I

demonstrated the effects of AlF4- on bone density using ICR mouse and

on Ca2+ signaling in preosteoclastic RAW 264.7 cells and in primary

BMMs. Five week old ICR male mice were exposed to AlF4- (AlCl3 300

ppm and NaF 100 ppm) in drinking water for 56 days for the purpose of

understanding the effects of AlF4- on bone density in vivo. It is known

that the absorbed aluminum by intestines can be transported into bone

and inhibits mineralization and bone cell growth and activity (Malluche,

2002) and the deleterious effects of aluminum on bone density could be

predicted in some aspects. However, the effects of aluminum-fluoride

complexes on bone were unclear because it seems that the effects of

fluoride on bone quality are dependent on the balance between the

positive effects and negative effects, which are influenced by daily doses

and the duration of treatment, and actual content of fluoride in the bone

(Strunecká et al., 2004).

２６

In the result using mycro CT and toluidine blue staining, tibia and femur

of mice showed decreased bone density after exposure to AlF4- for eight

weeks (Fig. 2 and 3). A typical symptom which occurred in RANKL-

induced osteoclastogenesis is the generation of [Ca2+]i oscillations.

RANKL-induced [Ca2+]i oscillations have been known as an essential

factor for triggering terminal stage of differentiation into osteoclasts

(Takayanagi, 2005). If [Ca2+]i oscillations are induced artificially, it could

act as good tools to regulate differentiation rate into osteoclasts. Under

this hypothesis, AlF4- was used to activate Ca2+ signal pathways. The

[Ca2+]i oscillations were generated by RANKL and AlF4- (Fig. 4 and 5) in

both RAW 264.7 cells and BMMs. These results suggest that AlF4- may

transmit differentiation-related signals to downstream like RANKL does.

It is known that RANKL-induced [Ca2+]i oscillations, which transmit a

series of differentiation-related signals, are dependent on extracellular

Ca2+ influx and this influx is important to maintain the [Ca2+ ]i oscillations,

which activate calcineurin and NFATc1 sequentially (Masuyama et al.,

2008). The dependence on extracellular Ca2+ of [Ca2+]i oscillations

induced by AlF4- was identified (Fig. 6), suggesting that AlF4

- induced

[Ca2+ ]i oscillations also may transmit a series of differentiation-related

signals.

Activation of MAPK, such as ERK and JNK, has been known a typical

２７

phenomenon, while osteoclast precursors differentiate into osteoclasts

(Leibbrandt and Penninger, 2008). Activated and activated JNK (Tanaka

et al., 2003) is critical for the bone-resorbing function of osteoclasts

(Miyazaki et al., 2000). In addition, activated ERK and JNK transmit

various signals to downstream effectors, including AP-1, NFATc1 and

NF-κB (Ghayor et al., 2011). It was necessary to identify the activation

of MAPK whether the signals from AlF4- are similar with them from

RANKL or not. As shown in Fig. 6 and 7, activated ERK and JNK were

observed in RAW 264.7 cells incubated with RANKL (Fig. 7A) and AlF4-

(Fig. 7B). These findings suggest that AlF4- can cause the

phosphorylation of ERK and JNK like RANKL and that such

phosphorylation leads to osteoclast differentiation.

TRAP stain is widely used as a criterion to confirm the differentiation

into osteoclasts (Han et al., 2005). To clarify whether AlF4- stimulation

may induce TRAP expression or not, TRAP staining was performed after

6 days in the presence of AlF4- and/or RANKL in RAW264.7 cells (Fig.

8A) and BMMs (Fig. 8B). This is done at several different concentrations

of AlF4- because its toxicity to cell survival (data not shown). External

feature of AlF4--induced TRAP-positive cells were similar with it of

RANKL-induced TRAP-positive cells. Moreover, AlF4- enhanced the

generation of RANKL-mediated TRAP positive osteoclasts.

２８

In conclusion, these results suggest that expose to AlF4- cause low

bone density and G-protein activation by AlF4- could induce [Ca2+]i

oscillations and osteoclast differentiation in osteoclast precursor cells.

AlF4- activated ERK and JNK signaling like RANKL. Aluminum-fluoride

complexes act as a messenger of false information (Strunecka et al.,

2002) and have been often used in many laboratories to investigate for

their effects on various cells and tissues. Further studies are needed to

clarify the potential risks for human health of long-term exposure to

AlF4- .

２９

Ⅳ. CONCLUSIONS

In summary, the effects of aluminum fluoride (AlF4-) on bone density

and Ca2+ signaling and osteoclastogenesis in osteoclast precursor cells

(RAW 264.7 cells and BMMs) were investigated.

1. Mice exposed to AlF4- in drinking water for two months

showed the decreased bone density.

2. AlF4- induced the generation of extracellular Ca2+-dependent

intracellular Ca2+ concentration ([Ca2+]i) oscillations.

3. AlF4- activated ERK and JNK signaling.

4. AlF4- enhanced the generation of RANKL-mediated TRAP

positive osteoclasts.

Therefore, G-protein activation by AlF4- could induce [Ca2+]i

oscillations and osteoclast differentiation in osteoclast precursor cells.

３０

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３７

국문요약

알루미늄-불소 복합체의 파골세포 분화기전 연구

<지도교수: 신 동 민>

연세대학교 대학원 치의학과

양 은 호

불소의 경우 수돗물의 불소화 사업으로 그 유해여부가 논쟁중이며 미량의

알루미늄과 만나게 되면 G-단백을 활성화시켜 이하 신호전달계가 활성화됨

이 보고된바 있으며, 골세포에 미치는 영향에 대하여는 피질골밀도를 증가시

킨다는 보고가 있는 반면, 뼈골절률을 높인다는 이중적인 보고가 있어 현재까

지 논란을 일으키고 있다. 특히, 의약품과 식음료에 다양한 용도로 사용되고

있는 불소와 식품첨가제 또는 다양한 방법으로 인체에 흡수될 수 있는 알루미

늄이 만날 경우 자발적으로 생길 수 있는 알루미늄-불소 복합체가 골세포에

미치는 영향에 대하여는 잘 알려져 있지 않다. 파골세포 분화에서 칼슘신호는

순차적인 NF-kB 신호전달을 동반하며 NFATc1의 활성화를 유도하는 것으

로 알려졌다. 본 연구에서는 알루미늄-불소 복합체가 G-단백의 활성을 유도

하여 칼슘신호를 일으키고 이에 따라 파골세포 분화를 전개시킬 수 있는 지를

알아보고자 하였다.

일정량의 알루미늄-불소 복합체를 복용시킨 생쥐의 골밀도가 일반 수돗

３８

물을 마신 생쥐보다 낮은 것을 생쥐 대퇴골 경골의 CT촬영과 톨루이딘 염색

을 통해 확인할 수 있었다. 파골전구세포인 RAW 264.7세포와 생쥐의 골수세

포를 이용하여 RANKL에 의한 칼슘 신호전달을 확인한 결과 예상과 마찬가

지로 칼슘의 주기적 변동을 관찰할 수 있었으며 이는 G-단백의 활성을 유도

하는 물질인 알루미늄-불소 복합체에 의해서도 발생하는 것으로 관찰되었다.

또한 알루미늄-불소 복합체에 의한 칼슘신호가 RANKL에서와 같이 외부 칼

슘의 유입에 의존적임을 확인하였다. 파골 세포 분화 과정에서 나타나는

MAPK 활성(ERK와 JNK의 단백 발현)을 살펴본 결과 RANKL뿐만 아니라

알루미늄-불소 복합체에 의해서도 활성화되는 것을 확인하였다. 특히, 알루미

늄-불소 복합체는 RANKL 유도성 TRAP 양성세포의 형성을 촉진시켰다.

파골세포전구체에서 RANKL 신호 전달 경로 외에 알루미늄-불소 복합

체만으로 G-단백을 활성화하여 발생하는 칼슘신호전달 만으로도 일련의 파

골세포 분화과정을 일으키는 것을 확인하였고, 알루미늄-불소 복합체가

RANKL 신호 전달경로를 더욱 활성화시키는 것으로 보아 골세포에서 불소와

알루미늄에 대한 인체 내 영향에 대한 연구가 필요하다고 판단된다.

핵심되는 말: 파골세포, 알루미늄-불소 복합체, RANKL, MAPK, TRAP