Aluminum-fluoride complexes induce osteoclastogenesis · 2020. 6. 29. · Aluminum-fluoride...
Transcript of Aluminum-fluoride complexes induce osteoclastogenesis · 2020. 6. 29. · Aluminum-fluoride...
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
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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).
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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)
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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