The Function of Reactive Oxyegn Species in Bone Loss

골손실을 조절하는 활성산소종의 역할 규명

양미혜

*

^{,†·박효정†·이동석}

*

^·임미정#

숙명여자대학교 약학대학, *경북대학교 자연과학대학 생명공학부

(Received March 25, 2009; Revised May 11, 2009; Accepted May 21, 2009)

The Function of Reactive Oxyegn Species in Bone Loss Mihye Yang*, Hyojung Park, Dong-Seok Lee* and Mijung Yim

^#

College of Pharmacy, Sookmyung Women's University, Seoul 140-742, Korea

*College of Natural Sciences, Kyungpook National University, Daegu 702-701, Korea

Abstract

— We explored the role of reactive oxygen species (ROS) in LPS-induced bone loss. LPS was shown to increase the concentration of ROS in osteoclast precursors. The antioxidant decreased osteoclast formation by LPS. Furthermore, the antioxidant decreased NFATc1 expression by LPS, suggesting that ROS mediates NFATc1 expression in the regulation of LPS-induced osteoclast formation. Finally, the antioxidant decreased LPS-induced RANKL mRNA expression in osteo- blasts. Taken together, these data indicate that LPS mediates ROS to induce bone loss.

Keywords □

ROS, LPS, osteoclast, osteoblast, bone loss

최근인구고령화에따라노인성질환이급증하고있으며

,

이 로인해노인의료비도지난

10

년간

8

배이상폭발적으로증가 하고있다

.

^{노화에따른질환중대표적인것이골격계질환으로}

,

특히염증성골손실은류마티스관절염

,

치주염

,

골다공증등을 유발하여삶의질을저하시킨다

.

건강한뼈는일생동안뼈가지속적으로형성되고파괴되며

,

^이

를뼈의재형성이라한다

.

¹⁾뼈의재형성은새로운뼈를만드는 조골세포

(osteoblast)

와오래된뼈를파괴하는파골세포

(osteoclast)

에의해평형을유지한다

.

^{조골및파골세포는밀접한관계를맺}

고있어

,

파골세포의분화는조골세포에의해엄격하게조절된 다

.

^1,2)즉

,

^{조골세포는파골세포분화인자인}

M-CSF(Macrophage Colony-Stimulating Factor, or CSF-1)

^및

RANKL(Receptor Activator of Nuclear Factor-kappaB Ligand, OPGL, ODF, or

TRANCE)

^{을통해파골세포의분화를조절함으로써체내골형성}

과골파괴의동적인평형을유지한다

.

^2-5)

염증성골파괴의병인인자로

lipopolysaccharide(LPS)

를들수 있다

.

⁶⁾

LPS

^{감염에의한골파괴에는}

Toll-Like Receptor(TLR)

를매개한자연면역계가중요하다고생각되고있다

. TLR

^는지

금까지사람에게서

11

종류

,

마우스에서는

13

종류가보고된바있 다

.

⁷⁾

LPS

는

TLR family

중

TLR4

에 결합하여

IL-6, IL-12, TNF-

^{α등의염증성사이토카인이나케모카인의발현을유도한}

다

. TLR4

의세포내도메인에는

Toll/IL-1R

상동영역

(TIR

도메인

)

이존재하여

, TLR4

가

LPS

와결합하면

TIR

에

MyD88, TIRAP, TRIF, TRAM, SARM

^의

adaptor

^{분자가결합해세포내에신호}

를전달한다

.

특히

MyD88

은

TLR4

신호전달계에있어서매우 중요한역할을담당하고있다

.

⁸⁾

조골및파골세포도표면에

TLR4

^{를발현하고있다}

.

^9)조골세

포에서

LPS

는

TLR4

에결합해

MyD88

를매개해

Ca

²⁺

/PKC, ERK

^{의존적으로파골세포분화인자인}

RANKL

^{발현을유도한}

다

.

^10)또한

COX-2

^{를 활성화해}

PGE

2생성을 촉진하고그의

autocrine/paracrine

작용에의해파골세포분화억제인자인

OPG

발현을억제함이알려져있다

.

^{11)이기전에있어서}

MyD88-/-

^유

래의세포에서

LPS

^는

RANKL

^{발현을유도하지않는것으로부}

터

, TLR4

의세포내

adaptor

분자

MyD88

이

RANKL

생성에중 요함이밝혀졌다

.

^12)한편

LPS

^{는성숙한파골세포에직접작용해}

MyD88

^{를매개해골흡수기능및생존을촉진하며}

,

^{파골세포전}

구세포에작용해파골세포로의분화를촉진함이알려져있다

.

¹³⁾

그러나이러한일련의보고에도불구하고

, LPS

^{가골손실을유}

발하는기전은아직충분히밝혀지지않았고

,

^{이는치료제개발}

에필요한타겟분자의발굴을어렵게하고있다

.

이에따라염

#본논문에관한문의는저자에게로

(

^전화

) 02-710-9572 (

^팩스

) 02-710-9871 (E-mail) [email protected]

†양미혜와박효정은본논문에대해동일하게기여하였습니다

.

뚜렷한치료제가없는실정이다

.

^{이에본연구자는항산화제를}

이용해 활성산소종

(Reactive Oxygen Species, ROS)

이

LPS

에 의한골세포조절에미치는영향을조사하였기에보고하는바이다

.

실험 방법

파골세포의분화유도

마우스

macrophage cell line

인

Raw 264.7

세포

2×10

³

cells/

well

^을

RANKL 50 ng/m

^l

(peprotech)

^{존재하에서}

36

^{시간배양하}

여파골전구세포를생성하였다

.

파골세포형성을위해추가적 으로

300 ng/m

l

LPS(Sigma, Germany)

와 표기된 양의

N- Acetyl-L-cysteine(NAC, Fluka, Germany)

^{을처리하여}

5

^일간배

양하였다

.

배양이끝난세포는

10% formalin

으로

10

분간고정한 후

ethanol-aceton(1 : 1)

로

1

분간 재고정하여

TRAP(tartrate- resistant acid phosphatase) staining

^을했다

. 3

^{개이상의핵을가}

진

TRAP+

세포를다핵파골세포로판정했다

.

Western blot분석

파골전구세포를

LPS(1

µ

g/m

l

)

와

NAC(20 mM)

을처리하여일 정시간배양후

, lysate buffer

^{로용해하고원심분리하였다}

.

^단

백질농도는

BSA(bovine serum albumin)

^{을표준화하여}

protein assay kit(Bio-Rad)

를사용하여정량하였다

. 20

µ

g

의단백질을

SDS-PAGE(poly acrylamide gel electrophoresis)

^{로변성분리하}

고

,

^이를

80 v

^에서

1

^시간

45

^{분처리하여}

PVDF membrane

^에이

전시켰다

.

이를

5% skim milk

가 함유된

PBST

용액으로

blocking

^하고

, 1

^{차항체로서}

anti-NFATc1(1 : 1000, Santa cruiz), COX-2(1 : 2000, R&D systems), I

κ

B(1 : 2000, Cell signaling),

또는 β

-actin(1 : 4000, Sigma)

항체와각각반응시켰다

. PBST

로

5

^{회세정하고}

HRP(Horseradish peroxidase)

^가결합된

2

^차항체

와반응시킨후

ECL Advance(Amersham. CO.)

^{로발색시켜분}

석하였다

.

마우스조골세포의초기배양

생후

0~1

일의

ICR mouse

로부터두개골피부를벗긴후두 개골을적출하였다

.

^{이때후두골근육부착부분은제거하였다}

.

부착된근육

,

혈구등을제거한후 α

-MEM

으로가볍게세척하 였다

. 0.1% collagenase

와

0.2% dispase

혼합효소용액에넣어

37

^o

C

^에서

5

^{분간진탕시킨후상등액을버리고새로운효소용액}

을가하였다

. 37

^o

C

에서약

10

분간진탕하여상등액을모으는조 작을

4

회반복하였다

.

원심분리한후

10% FBS

함유 α

-MEM

으로약

3~5×10

^4세포

/100 mm plastic dish

^{가되도록접종하였}

다

. 5% CO

2

, 37

^o

C

에서

3~4

일간배양한세포를이후조골세포 로실험에사용하였다

.

Total RNA

^추출은

easy-Blue(iNtRON Bio technology co., Ltd, Korea)

를 이용하였다

. cDNA

는

1

µ

g

의

total RNA

를

oligodT primer, 10 mM dNTP, 1 unit RNase inhibitor

^그리고

4 unit Script reverse transcriptase(Fermentas, Life science)

로

42

^o

C

에서

60

분처리하여합성한후

, 70

^o

C

에서

10

분가열함으로 써 반응을 중지 시켰다

. Polymerase chain reaction(PCR)

^은

RANKL

에 특이적인 서열

(forward: 5'-ccagccatttgcacacctc-3', reverse: 5'-agcagggaagggttggaca-3')

을사용하여

94

^o

C 3

분간처 리한후

, 94

^o

C 30

^초

, 58

^o

C 45

^초

72

^o

C 1

^{분의과정을}

32

^회반복

하였다

. PCR

결과물은전기영동으로확인하였다

.

ROS생성능측정(DCF법)

ROS

생성능을측정하기위해

DCFH-DA(2',7'-dichlorofluorescin- diacetate)

를사용하였다

. Raw 264.7

세포를

RANKL

이포함된 배지에서

36

^{시간동안배양하고}

, 0.5% FBS

^{가포함된배지에서}

12

시간동안배양하였다

. 0.05 mM DCFH-DA

를

30

분동안처 리하고

, PBS

^로

washing

^{한후시간대별로}

LPS(300 ng/m

^l

)

^를처

리하였다

.

^생성된

DCF

^{형광의변화는}

excitation, 488 nm

^및

emission, 515~540 nm

에서

Fluorescence Microplate Reader

로 측정하였다

.

통계처리

실험결과는평균

+

^{표준편차로표기하였고}

, Student's t-test

^로

분석하여 p값이

0.05

^{미만일때통계적으로유의하다고판단하}

였다

.

실험 결과 및 고찰

LPS에의한파골전구세포내활성산소종의증가

먼저

LPS

^{에의한파골세포분화에관여하는활성산소종의역}

할을규명하기위해

,

파골전구세포를

LPS

로처리하였을때세포 내활성산소종의변화를시간대별로조사하였다

.

^{파골전구세포}

로는마우스

macrophage cell line

인

Raw 264.7

세포를파골세 포필수분화인자인

RANKL

로

36

시간처리하여형성된세포를 사용하였다

.

^{파골전구세포는}

LPS

^{로처리하여}

5

^{일간배양하면다}

핵의성숙파골세포로분화한다

(data not shown).

파골전구세포 내활성산소종의농도는

300 ng/m

l의

LPS

처리후

10

분부터유 의적으로증가하였으며

2

^{시간에서최고치를나타냈다}

(Fig. 1).

^그

후점차적으로감소하였으나

,

처리후

6

시간에서도비교적높은 세포내활성산소종농도를유지하였다

.

이상의결과로

LPS

가파 골전구세포내에서활성산소종농도를증가시킴을알수있으며

,

이는활성산소종이

LPS

에의한파골세포분화에관여할가능성 을보여주는것이라할수있다

.

항산화제에의한LPS유도성파골세포분화의억제

LPS

^{유도성파골세포분화에있어서활성산소종의기능을규}

명하기위해

, LPS

^{와항산화제를함께처리하였을때파골세포로}

의분화변화를추적하였다

.

항산화제로는

N-Acetyl-L-cysteine (NAC)

^{을사용하였다}

.

^{파골전구세포는}

300 ng/m

^l의

LPS

^처리로

TRAP+

다핵파골세포로분화되었으며

,

이는

10 mM NAC

처 리로유의성있게억제되었다

(Fig. 2A).

이로써

LPS

는파골전구 세포내활성산소종을매개하여파골세포로의분화를촉진함이 밝혀졌다

.

파골세포분화에관여하는많은유전자가밝혀졌으며

,

그중 에서 특히

Nuclear Factor of Activated T cells(NFAT)c1

^은파

골세포의분화를조절하는중요한전사인자로알려져있다

.

¹⁴⁾항

산화제의처리가

LPS

에의한

NFATc1

의발현을변화시키는지

여부를항

NFATc1

^{항체를이용한}

western blotting assay

^로조

사하였다

.

파골전구세포를

LPS

로

48

시간처리하였을때

NFATc1

의발현은증가하였으며

,

^이는

NAC

^{처리로감소하는것으로나}

타났다

(Fig. 2B),

^{이는항산화제가}

LPS

^{에의한파골세포분화를}

억제하는것과일치하는결과로

,

활성산소종은

NFATc1

발현을 조절함으로써

LPS

에의한파골세포분화를매개하는것으로추 정되었다

.

LPS

에의한파골세포분화에

cycloxygenease-2(COX-2)

¹⁵⁾와

nuclear factor kappa B(NF-

^κ

B)

^{16)가관여함이보고된바있다}

.

따라서

LPS

^{에의해유발된활성산소종이}

COX-2

^와

NF-

^κ

B

^를조

절하는지의여부를항산화제처리에의한

western blotting assay

로조사하였다

. LPS

^{는파골전구세포에서}

COX-2

^{발현을증가시}

Fig. 1 −

Effects of LPS on the concentration of ROS in osteoclast precursor cells. Raw 264.7 cells were cultured with 50 ng/

m l RANKL for 36 hrs to produce osteoclast precursor cells.

After starvation with 0.5% FBS for 12 hrs, cells were treated with 300 ng/m l LPS for indicated times. The concentration of ROS were measured as described in Materials and methods. The experiments were performed 3 times, and the reproducibility was confirmed. Values are the mean±SD of triplicate cultures in a representative experiment. *: p <0.05, significantly different from 0 hr.

Fig. 2 −

The involvement of ROS in LPS-induced osteoclast for-

mation. A, Osteoclast precursor cells were further cultured

with 300 ng/m l LPS for 5 days in the absence or presence

of 10 mM NAC pretreatment. Cells were then fixed and

stained for TRAP. TRAP-positive (+) multinucleated cells

(MNCs) were counted. The experiments were performed 3

times, and the reproducibility was confirmed. Values are

the mean±SD of triplicate cultures in a representative

experiment. Veh: vehicle, *: p <0.05, significantly different

from LPS. B, Osteoclast precursor cells were cultured with

300 ng/m l LPS for 2 days in the absence or presence of

20 mM NAC pretreatment. The expression of NFATc1 was

determined by western blotting assay. Western blot images

are representative of 3 experiments with similar results.

켰으나

,

항산화제처리는아무런변화를보이지않았다

(Fig. 3A).

또한

LPS

^는

I

^κ

B

^{분해를촉진하여}

NF-

^κ

B

^{를활성화시키는것으}

로나타났으나

,

^{항산화제는이에대해아무영향을미치지않았}

다

(Fig. 3B).

이상의결과는

LPS

에의해증가된활성산소종이

COX-2

^또는

NF-

^κ

B

^{의신호전달경로와는무관하게파골세포분}

화를조절함을시사한다

.

향후추가적인실험을통해활성산소

종이매개하는

LPS

유도성파골세포분화기전이보다명확하

게규명되기를기대한다

.

항산화제에의한LPS유도성RANKL발현의억제

LPS

는파골전구세포에직접작용하여파골세포분화를촉진 할뿐아니라

,

^{조골세포내}

RANKL

^{발현을유도함으로써파골세}

포분화를간접적으로도촉진하는것이알려져있다

.

¹⁶⁾따라서

LPS

에의한조골세포내

RANKL

발현이활성산소종을매개하는 지규명하고자하였다

.

^{이를위해항산화제처리에의한조골세}

포내

RANKL

발현변화를

RT-PCR

로조사하였다

.

조골세포로 는마우스의두개관에서분리한초기배양조골세포를사용하였 다

. LPS

^{처리는조골세포내}

RANKL

^{발현을촉진하였으며}

,

^그

발현촉진효과는항산화제처리로다소감소하는것으로나타 났다

(Fig. 4).

따라서활성산소종은

LPS

의파골전구세포에대한 직접적인작용뿐아니라조골세포에대한간접적인작용도매개 하는것으로밝혀졌다

.

본연구를통해

LPS

에의한파골세포분 화촉진을매개하는활성산소종의기능이처음으로밝혀졌으며

,

이는체내

LPS

^{에의한염증성골손실의새로운발병기전을밝}

히는것과동시에

,

항산화제의염증성골손실치료제로서의가 능성을제시해주는것이라할수있다

.

결 론

LPS

^{는파골전구세포내활성산소종의농도를증가시켰으며}

,

^활

성산소종은

LPS

에의한파골세포분화촉진을매개함이밝혀졌 다

.

^{활성산소종은파골세포분화에필수적인전사인자}

NFATc1

Fig. 4 −

The involvement of ROS in LPS-induced RANKL expres- sion. Mouse calvarial osteoblasts were treated with 300 ng/

m l LPS in the absence or presence of 20 mM NAC pretreatment for 3 hrs. The mRNA expression of RANKL were determined by RT-PCR. Band images are representative of 3 experiments with similar results.

Fig. 3 −

The involvement of ROS in LPS-activated signaling path-

ways. Osteoclast precursor cells were cultured with 300

ng/m l LPS for 24 hrs (COX-2) or 30 min (I

^κ

B) in the

absence or presence of 20 mM NAC pretreatment. The

expression of COX-2 (A) or I

κ

B (B) was determined by

western blotting assay. Western blot images are re-

presentative of 3 experiments with similar results.

을조절함으로써

LPS

^{의작용을매개하는한편}

, COX-2

^와

NF-

^κ

B

의조절과는무관한것으로밝혀졌다

.

^{또한활성산소종은}

LPS

^에

의한조골세포내

RANKL

발현도매개하는것으로나타나

,

파골 전구및조골세포에대한

LPS

^{의작용에모두관여하는것으로}

추정되었다

.

감사의 말씀

본연구는보건장학회의지원으로수행되었으므로이에감사 드립니다

.

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