Eif

XY

H

Oii

Fp

Sd

RC

1

xpression characteristic and significance ofnterleukin-6, nuclear factor kappa beta, and boneormation markers in rat models of osteoporosis

IAOHU ZHU, JIE LUO, XIAPING CHEN, JUNHUA WANG, GANG WANG, HAIFENG LI,UANHONG XU, JINCAI FENG, and HANJUN TU

UBEI, CHINA

The goal of this study was to investigate the expression levels of interleukin 6 (IL-6),nuclear factor kappa beta (NF-��), bone-specific alkaline phosphatase (BALP),and bone osteocalcin (BGP) in rats with osteoporosis and their significance in thepathogenesis of osteoporosis. In all, 60 adult female SD rats were divided randomlyinto 3 groups of 20 rats each: normal control group (control), sham-operated group(sham), and ovariectomized group (OVX). In 2, 3, 4, 5, and 6 months after surgery,4 rats were randomized from each group for assays of BMD, IL-6, BALP, and BGP.Then, the rats were sacrificed for the detection of IL-6 and NF-�� expression levelsin bone tissue by quantitative real-time RT-PCR analysis. Compared with the sham(0.097 � 0.04 g/cm2, 0.097 � 0.01g/cm2, 0.095 � 0.07g/cm2) and control group(0.107 � 0.01 g/cm2, 0.103 � 0.07 g/cm2, 0.108 � 0.06 g/cm2), the BMD of rats in theOVX group was reduced remarkably in 4, 5, and 6 months (0.082 � 0.05g/cm2, 0.073� 0.02 g/cm2, 0.061 � 0.05g/cm2, respectively; P < 0.01); the serum IL-6 levelincreased significantly from 2 to 6 months after surgery (P < 0.01); and the serumlevels of BALP and BGP were greater at 4, 5, and 6 months (P < 0.05). The quanti-tative real-time RT-PCR analysis demonstrated that IL-6 and NF-�� mRNA levels inOVX group increased in a time-dependent manner. Moreover, the IL-6, NF-��,BALP, and BGP levels were correlated negatively with the BMD. Meanwhile, apositive correlation was observed between IL-6 and NF-��. In conclusion, theexpression levels of IL-6, NF-��, and bone formation markers may increase signif-icantly in the osteoporosis rats. These molecules could play a role in the pathogen-esis. (Translational Research 2008;152:18–23)

Abbreviations: BALP � bone-specific alkaline phosphatase; BGP � bone osteocalcin; BMD �bone mineral density; DEXA � dual-energy X-ray absorptiometry; IL-6 � interleukin 6; NF-�� �

nuclear factor kappa beta; OVX � ovariectomized

adcma

Sy

S2

1

©

steoporosis, which is a systemic diseasecharacterized by a decreased bone mineraldensity (BMD) and encountered frequently

n postmenopausal women, may cause an increasedncidence of vertebral and iliac fractures and is associ-

rom the Taihe Hospital of Yunyang Medicine College, Taihe Hos-ital Rehabilitation Medicine Center, Shiyan, Hubei, China.

upported by grant 2003AA301C56 from the Natural Science Foun-ation of Hubei Province in China.

eprint requests: HanJun Tu, Taihe Hospital of Yunyang Medicine

ollege, Taihe Hospital Rehabilitation Medicine Center, Renmin d

8

ted with a significant morbidity and mortality.1,2 Manyifferent kinds of therapeutics are available, such asalcium supplement (a fundamental therapy) and hor-one replacement therapy (a classic therapy), used

gainst bone loss and osteoporosis development in

outhRoad 32#, 442000 Shiyan, Hubei, China; e-mail: zhuxh0831@ahoo.com.cn.

ubmitted for publication March 16, 2008; revision submitted May0, 2008; accepted for publication May 22, 2008.

931-5244/$ – see front matter

2008 Mosby, Inc. All rights reserved.

oi:10.1016/j.trsl.2008.05.003

psacgscarnp

oafloNltpotttar

M

cpA

2rwt5r((

ifwaM

ort2loawnavgot

watmmstfsO

smw(BbWtak

Translational ResearchVolume 152, Number 1 Zhu et al 19

ostmenopausal women. However, long-term estrogenupplementation after menopause poses several risksnd is no longer recommended as a prevention ofhronic diseases.3 Additionally, because of the prolon-ation of life expectancy as the improvement of livingtandard, the number of postmenopausal women is in-reasing dramatically, and osteoporosis has constitutedgreat socioeconomic problem. Therefore, additional

ecognition of the pathogenesis is needed as well asew diagnostic and therapeutic strategies to attenuateostmenopausal bone loss at an early date.Bone-specific alkaline phosphatase (BALP) and bone

steocalcin (BGP) in serum are considered as specificnd sensitive markers of bone formation.4,5 The proin-ammatory cytokine interleukin 6 (IL-6) influencessteoclast function and stimulates bone resorption.6

uclear factor kappa beta (NF-��) is a family of re-ated, dimeric transcription factors that are readily ac-ivated in cells by signals associated with stress orathogens. Its inhibitors can induce apoptosis of maturesteoclasts and inhibit bone resorption by isolated os-eoclasts.7,8 The goal of this study was to investigatehe expression levels of IL-6, NF-��, and bone forma-ion markers (BALP and BGP) in rats with osteoporosisnd their significance in the pathogenesis of osteopo-

AT A GLANCE COMMENTARY

Background

Osteoporosis is a systemic disease characterizedby a decreased bone mineral density and encoun-tered frequently in postmenopausal women. Thisdisease may cause an increased incidence of ver-tebral and iliac fractures and is associated with asignificant morbidity and mortality. Hence, post-menopausal bone loss must be attenuated at anearly date.

Translational Significance

In the current study, our research group investi-gated the expression levels of interleukin 6 (IL-6),nuclear factor kappa beta (NF-��), bone-specificalkaline phosphatase (BALP), and bone osteocal-cin (BGP) in rats with osteoporosis and their sig-nificance in the pathogenesis of osteoporosis.Changes in the expression levels of these 4 mark-ers were correlated with osteoporosis progression,which suggests that these molecules could play arole in the pathogenesis of the disease.

osis. X

ATERIALS AND METHODS

Materials. Animals. Animals were housed in facilities ac-redited by International Guidelines, and studies were ap-roved by and conducted in accordance with the Institutionalnimal Care and Use committee.In all, 60 female Sprague-Dawley rats (5 months old,

20–250 g, Experimental Animal Center of Life science andesearch institute of Taihe Hospital, Shiyan, Hubei, China)ere housed 5 per cage with free access to food and water;

hey were kept in a constant environment (22 � 2 °C, 50 �% humidity, 12-h light/dark cycle). These rats were dividedandomly into 3 groups of 20 rats: normal control groupcontrol), sham-operated group (sham), and ovariectomizedOVX) group.

Chemicals and reagents. All chemicals were of analyt-cal reagent grade. Before the experiment, all vessels and tipsor pipetting were dipped in strong HNO3 for 24 h and thenashed with ultrapure water. The water used was purified inMilli-Q water purification system (Millipore, Bedford,ass).Methods. Preparation of osteoporosis rat model by

variectomy surgery. After the acclimatization period, theats in the OVX group were performed a bilateral ovariec-omy. For this procedure, the rats were anesthetized with5-mg/kg thiopental sodium by intraperitonal injected. Aongitudinal incision (0.5–1 cm) was made in the midline areaf the lower abdomen. A small peritoneal incision was made,nd the ovaries were removed. The rats in the sham groupere subject to sham surgery exposing, but the ovaries wereot removed. The success of the ovariectomy was confirmedt necropsy by failure to detect ovarian tissue and by obser-ation of marked atrophy of uterine horns. In the controlroup, the rats were performed with no management. Afterperation, penicillin was taken orally continuously for 3 dayso prevent infection.

The time for measuring daily food consumption and bodyeight was the same during the entire period. At 2, 3, 4, 5,

nd 6 months after surgery, a longitudinal section of the rightibiae from rats of all groups was used for BMD measure-ent, and blood samples were withdrawn by the tail veinethod to assess IL-6, BALP, and BGP. The rats were then

acrificed by using ether anesthesia. After that, the femora,ibia, and lumbar vertebra were dissected and stored in areezer at –80°C until quantitative real-time RT-PCR analy-is. Their uteri were weighed to ensure the efficacy of theVX surgery.Measurement of BMD. After removing adherent soft tis-

ues, a longitudinal section of the right tibiae was prepared byanual grinding with whetstones. All longitudinal specimensere dehydrated by a stepwise application of 70% and 99.9%

vol/vol) ethanol solutions. These samples were used forMD measurements, which were quantitatively determinedy the dual-energy X-ray absorptiometry (DEXA, Madison,is). The radiographs of the longitudinal sections of the

ibiae were taken along with an aluminum step-wedge usingn X-ray apparatus set at 5 mA, 40 cm, 30 s, 50 V, and 55Vp. The densitometric pattern of the proximal tibia on an

-ray image was read by a personal computer (PC-9801;

Ndpad

efsckaTomtscTeea5

Tswbmm

efktesuwr6TC(5tS

tP17ssqrmd

dtuewcgCa

shblaa

R

Bmaa0ssP

efIgt

T

CSO

*†

‡

§

Translational Research20 Zhu et al July 2008

EC, Tokyo, Japan) using a software program for rat boneensity (Version 2.10A-M; Teijin Pharma, Ltd., Tokyo, Ja-an). The reproducibility of DEXA was 1.8%. BMD valuesre presented in g/cm2 and are expressed in standardeviations.

Biochemical assays of IL-6 and bone formation mark-rs. The serum IL-6, BALP, and BGP levels of rats in dif-erent groups were detected in 2, 3, 4, 5, and 6 months afterurgery. The level of IL-6 in serum was measured with theommercially available enzyme-linked immunosorbent assayit (Catalog #KAC1261; Biosource International, Inc, Cam-rillo, Calif) according to the manufacturer’s instructions.he sensitivity of the assay was 3 pg/mL, and the coefficientf variation was 2.6%. The level of BALP in serum waseasured with an enzyme immune assay method, according

o the manufacturer’s instructions (Alkphase-B; Metra Bio-ystems, Palo Alto, Calif). The intra-assay and interassayoefficients of variation were 4.1% and 5.2%, respectively.he level of BGP in serum was measured with a competitivenzyme immune assay method, according to the manufactur-r’s instructions (NovoCalcin; Metra Biosystems). The intra-ssay and interassay coefficients of variation were 4.0% and.3%, respectively.

RNA extraction. Total cellular RNA was prepared withrizol reagent (Life Technologies, Inc.) from parallel femoralamples (collected at 2, 4, and 6 months after OVX), fromhich bone marrow had been rapidly flushed and bones hadeen snap-frozen in liquid nitrogen, as recommended by theanufacturer, resuspended in DEPC water, and quantified byeasuring the A260/280 nm.Quantitative real-time RT-PCR analysis of IL-6 and NF-��

xpression levels. Quantitative RT-PCR analysis was per-ormed using the SYBR Green PCR Master Mix and RT-PCRit (Applied Biosystems, Foster City, Calif) as suggested byhe manufacturer. �-actin was used as an endogenous refer-nce, and the expression levels of markers in the control andham group were performed as controls. In all, 3 primers weresed in this section, which are primer specific for IL-6 (for-ard 5’-AAG TCG GAG GCT TAA TTA CAC ATG T-3’,

everse 5’-AAG TGC ATC ATC GTT GTT CAT ACA-3’,61bp), primer specific for NF-�� (forward 5’-ACG ATCGT TTC CCC TCA TC-3’, reverse 5’-TGC TTC TCT CCCAG GAA TA-3’, 221bp), and primer specific for �-actin

forward 5’-CTG TGC CCA TCT ATG AGG GT-3’, reverse’- CAG TGA GGC CAG GAT AGA GC-3’, 526bp). Reac-ions were performed in 20 �L with 20-ng RNA, 10 �L 2 �

able I. Quantitative BMD detection in different gro

Group n 2 months 3 months

ontrol 4 0.105 � 0.02 0.102 � 0.03ham 4 0.100 � 0.01 0.098 � 0.05VX 4 0.096 � 0.03 0.091 � 0.06

Refers to the comparison with the control group, P � 0.05.Refers to the comparison with the control group, P � 0.01.Refers to the comparison with the sham group, P � 0.05.Refers to the comparison with the control group, P � 0.01.

YBR Green PCR Master Mix, 6.25 U MultiScribe reverse g

ranscriptase, 10 U RNase inhibitor, and 0.1-�M primers.CR was performed for 36 cycles (94°C for 1 min, 55°C formin, 72°C for 1 min, and a final elongation step of 7 min at

2°C). The identity of all the PCR products was confirmed byequencing the bands and by comparison with publishedequences (NCBI; BLAST search). Amplified bands wereuantified by densitometric analysis, and the results plottedepresent the X� � s of triplicate determinations of 1 experi-ent; however, similar results were observed in 3 indepen-

ent experiments.We used a method for relative quantification, which was

escribed by Juarranz et al,9 and compared the amount ofarget normalized with an endogenous reference. The formulased was 2-��Ct, which represents the n-fold differentialxpression of a specific gene in a treated sample comparedith the control sample, where Ct is the mean of threshold

ycle. �Ct was the difference in the Ct values for the targetene and �-actin; ��Ct represents the difference between thet from the control and each datum. The correct size of themplified products was checked by electrophoresis.

Statistical analysis. Data obtained were expressed as X� �and analyzed by 1-way analysis of variance with the post

oc Tukey test applied for paired comparisons. A differenceetween means was considered significant if the P value wasess than 0.05. The Spearman correlation was calculatedmong the expression levels of IL-6, NF-��, BALP, BGP,nd BMD.

ESULTS

BMD determinations. As shown in Table I, the tibialMD of OVX rats decreased dramatically from 4onths after surgery, which (0.082 � 0.005) was 18%

nd 30% lower than that of the sham (0.097 � 0.004)nd control (0.107 � 0.001) groups (P � 0.05, P �.05). Moreover, at 6 months, the BMD value wasignificantly decreased, and the differences from that ofham and control groups became significant (P � 0.01,� 0.01).Biochemical assays of IL-6 and bone formation mark-

rs. The serum levels of IL-6 and markers of boneormation in different groups are summarized in TableI. Their levels in the sham group were only slightlyreater than the control group and did not reach statis-ical significance. The serum IL-6 level in the OVX

varying time points (X� � s, g/cm2)

4 months 5 months 6 months

.107 � 0.01 0.103 � 0.07 0.108 � 0.06

.097 � 0.04 0.097 � 0.01 0.095 � 0.07

.082 � 0.05*† 0.073 � 0.02†‡ 0.061 � 0.05†§

ups at

000

roup increased dramatically from 2 months to 6

mpa�Bwm

ttasOCN12Omsa

tN�net

D

ritb

itorsacwIBd

vseiobcbmlAfoccp

ilIUc

T

SO

CSO

CSO

*†

‡

§

Translational ResearchVolume 152, Number 1 Zhu et al 21

onths after surgery (20.93 � 5.68–25.96 � 3.69g/mL, P � 0.01), and its differences from the shamnd control groups were also significant (P � 0.05 or P

0.01). The large differences in the serum levels ofALP and BGP between OVX and the other 2 groupsith statistical significance occurred at 4, 5, and 6onths after surgery (P � 0.05 or P � 0.01).mRNA expression of IL-6 and NF-��. Quantitative real-

ime RT-PCR analysis has been performed to examinehe local expression levels of IL-6 and NF-�� in 2, 4,nd 6 months after OVX surgery. From 2 months afterurgery, the expression levels of IL-6 and NF-�� in theVX group increased in a time-dependent manner.ompared with the control and sham groups, IL-6 andF-�� mRNA expressed in the OVX group increased.5–2 fold in 2 months after OVX; they showed a–3-fold and 4–5-fold increase in 4 and 6 months afterVX, respectively. The differences of IL-6 and NF-��RNA expression levels among the 3 groups were

tatistically significant at the same time point (P � 0.01nd P � 0.05, respectively, Fig 1).Furthermore, the Spearman correlation indicated that

he expression levels of IL-6 (rs � –0.58, P � 0.023),F-�� (rs � –0.59, P � 0.020), BALP (rs � –0.43, P

0.031), and BGP (rs � –0.49, P � 0.028) wereegatively correlated with the BMD; meanwhile, thexpression level of IL-6 was positive correlated withhat of NF-�� (rs � 0.66, P � 0.025, respectively).

ISCUSSION

Bone is a dynamic organ system that is related di-ectly to calcium and phosphor metabolism. Imbalancen these 2 parameters during aging or menopause leadso osteoporosis, which is the most frequent metabolic

able II. Serum IL-6, BALP, and BGP levels of rats in

Group n 2 months 3 months

IL-6 (XControl 4 18.66 � 8.09 18.59 � 8.11

ham 4 19.01 � 7.89 19.20 � 7.72VX 4 20.93 � 5.68*‡ 21.15 � 5.07*‡

BALPontrol 4 72.33 � 1.9 72.68 � 7.1ham 4 72.42 � 2.1 73.91 � 6.2VX 4 72.50 � 4.6 88.06 � 10.1*‡

BGP (ontrol 4 12.25 � 0.2 12.37 � 0.7ham 4 12.36 � 0.5 12.41 � 0.3VX 4 12.43 � 0.2 12.75 � 0.4

Refers to the comparison with the control group, P � 0.05.Refers to the comparison with the control group, P � 0.01.Refers to the comparison with the sham group, P � 0.05.Refers to the comparison with the control group, P � 0.01.

one disease. Osteoporosis may lead to skeletal fragil- w

ty and increasing susceptibility to fractures because ofhe rapid loss of BMD and a high rate of bone turn-ver.10-15 Additionally, aging individuals with osteopo-osis have an increasing risk of developing diseases,uch as high blood pressure, which is associated withbnormalities in calcium metabolism and leads to in-reased calcium loss, cancer, and diabetes.16-18 Hence,e herein are dedicated to investigate the changes in

L-6, NF-��, and bone formation marker (BALP andGP) expression in vivo during osteoporosis to assisteveloping its diagnostic and therapeutic measurement.OVX rats have been a well-accepted model for in-

estigating postmenopausal osteoporosis for 2 rea-ons19,20: First, rats may cause a negative bone remod-ling balance that augments the loss of bone mass andncreases the incidence of osteopenia occurring aftervariectomy. Second, the similar pathophysiologies ofone loss with osteoporosis of postmenopausal womenould be caused by ovariectomy in a rat model. On theases mentioned above, we have used this animalodel for our research, the results of which have shown

ow BMD from 4 months after OVX surgery (Table I).s reported recently, reduced BMD is a useful marker

or bone loss and risk of fracture. Combining with thebservation of pathological section, which includes de-reased bone density and thinned trabecula, we couldonfirm that the rat models of osteoporosis had beenrepared successfully.To our knowledge, many proinflammatory cytokines

nvolved in stimulating osteoclastic activity and regu-ation of bone resorption, including TNF-�, IL-1, andL-6, fall under the control of NF-�B transcription.nder a normal condition, estrogen can inhibit the

ombination of NF-�� with IL-6 promoter by binding

t groups at varying time points

4 months 5 months 6 months

/mL)8.87 � 6.28 18.29 � 3.65 18.72 � 3.379.36 � 5.40 19.61 � 4.76 19.83 � 2.582.98 � 6.33†‡ 24.01 � 4.24†§ 25.96 � 3.69†§

U/L)3.87 � 4.2 76.21 � 10.4 80.23 � 2.75.77 � 5.1 79.28 � 11.7 82.37 � 5.98.01 � 2.6†§ 137.69 � 10.9†§ 156.88 � 7.3†§

/mL)2.42 � 0.3 12.67 � 0.2 12.90 � 1.12.58 � 0.1 12.96 � 0.7 13.07 � 0.86.07 � 0.8†‡ 18.59 � 1.2†§ 20.17 � 0.3†§

differen

� � s, pg112

(X� � s,77

11X� � s, ng

111

ith its receptor to repress the transcription and expres-

sIlgdttt

obbpaeiidnindcqer

groiprtpuecOcrtBmtel

eaHg

Hb

R

FR2esNdstX

Translational Research22 Zhu et al July 2008

ion of IL-6 in osteoblast and stroma cells.21 Therefore,L-6 gene expression may be out of control, and itsevel may increase because of downregulation of estro-en levels in some abnormal conditions. Much evi-ence from in vitro experiments have already indicatedhat NF-�� may repress oestrogen receptor-�-mediatedransactivation and increase IL-6 expression to enhance

ig 1. mRNA expression of IL-6 and NF-�� detected by quantitativeT-PCR analysis. A–C, Expression of mRNA for IL-6 and NF-�� in, 4, and 6 months after OVX surgery and corrected by mRNAxpression for �-actin in each sample (see Materials and Methodsection). Two months after surgery, the expression levels of IL-6 andF-�� in the OVX group increased in a time-dependent manner, theifferences of which with the control group and the sham group weretatistically significant (*P � 0.05 and **P � 0.01, comparing withhe expression levels in the sham group, respectively). Results are the� � s of 3 separate experiments.

he bone resorption and accelerate the bone turn-

ver.22-24 Additionally, enhanced IL-6 levels have alsoeen observed in several other conditions characterizedy excessive loss of bone, such as rheumatoid arthritis,eriodontal disease, Paget’s disease, multiple myeloma,nd hyperparathyroidism.25-28 However, some previousfforts to determine whether IL-6 expression is alteredn osteoporotic patients or animal models have yieldednconsistent results. For example, the observation fromifferent laboratories has detected either an increase oro change in circulating IL-6 protein in OVX mice bymmunoassay or semiquantitative RT-PCR, which mayot have been able to detect subtle but significantifferences in mRNA expression.29,30 Therefore, in theurrent study, we combined biochemical assay anduantitative real-time RT-PCR together to detect thexpression levels of IL-6 and NF-�� in osteoporosisats, so as to improve the confidence of our results.

Our study clearly demonstrated the significantlyreater serum levels of IL-6, BALP, and BGP in OVXats compared with sham and control (Table II). More-ver, we also found that IL-6 and NF-�� mRNA levelsn the OVX group were increased dramatically com-ared with the sham group. The possible reason for thisesult may be as follows. It has been well documentedhat estrogen may impair IL-6 expression by preventingrotein binding on the NF-�� site.31 The rats thatnderwent sham operation could excrete much morestrogen than those performed ovariectomy, whichould explain why IL-6 and NF-�� mRNA levels inVX group were upregulated. These data are also

onsistent with the ability of IL-6 to promote boneesorption through stimulating osteoclastogenesis. Fur-hermore, these results parallel those of Poli et al32 andellido et al,33 who demonstrated that IL-6–deficientice are protected from gonadectomy-induced os-

eopenia. The Spearman correlation indicated that thexpression levels of these 4 markers also were corre-ated negatively with the changes of BMD.

In conclusion, the above observations have stronglymphasized the changes of IL-6, NF-��, and BALPnd BGP expression in osteoporosis progression.ence, these molecules could play a role in the patho-enesis.

We appreciate the help given by Dr. Kenepell and Dr. Ralferrmann of Berufsgenossenschaftliches Unfall Krankenhaus Ham-urg (BUKH) to our research.

EFERENCES

1. O’Brien C, Jilka RL, Fu Q, Stewart S, Weinstein RS, ManolagasSC. IL-6 is not required for parathyroid hormone stimulation ofRANKL expression, osteoclast formation, and bone loss in mice.Am J Physiol Endocrinol Metab 2005;289:E784–93.

2. Hidaka S, Okamoto Y, Uchiyama S, et al. Royal jelly prevents

osteoporosis in rats: beneficial effects in ovariectomy model and

1

1

1

1

1

1

1

1

1

1

2

2

2

2

2

2

2

2

2

2

3

3

3

3

Translational ResearchVolume 152, Number 1 Zhu et al 23

in bone tissue culture model. Evid Based Complement AlternatMed 2006;3:339–48.

3. Halici Z, Borekci B, Ozdemir Y, Cadirci E, Suleyman H. Pro-tective effects of amlodipine and lacidipine on ovariectomy-induced bone loss in rats. Eur J Pharmacol 2008;579:241–5.

4. Eren E, Yilmaz N. Biochemical markers of bone turnover andbone mineral density in patients with b-thalassaemia major.J Clin Pract 2005;59:46–51.

5. Kataoka C, Egashira K, Ishibashi M, et al. Novel anti-inflam-matory actions of amlodipine in a rat model of arteriosclerosisinduced by long-term inhibition of nitric oxide synthesis. Am JPhysiol Heart Circ Physiol 2004;286:768–74.

6. Chen SU, Chou CH, Lee H, Ho CH, Lin CW, Yang YS. Lyso-phosphatidic acid up-regulates expression of interleukin-8 and -6granulosa-lutein cells through its receptors and NF-�B dependentpathways: implications for angiogenesis of corpus luteum andovarian hyperstimulation syndrome. J Clin Endocrin Metab2008;93:935–43.

7. Xing L, Carlson L, Story B. Expression of either NF-kappaB p50or p52 in osteoclast precursors is required for IL-1 induced boneresorption. J Bone Miner Res 2003;18:260–9.

8. Franzoso G, Carlson L, Xing L, et al. Requirement for NF-kB inosteoclast and B-cell development. Gene Devel 1997;11:3482–96.

9. Juarranz Y, Abad C, Martinez C, et al. Protective effect ofvasoactive intestinal peptide on bone destruction in the collagen-induced arthritis model of rheumatoid arthritis. Arthritis ResTher 2005;7:R1034–45.

0. O’Brien CA, Jia D, Plotkin LI, et al. Glucocorticoids act directlyon osteoblasts and osteocytes to induce their apoptosis andreduce bone formation and strength. Endocrinology 2004;145:1835–41.

1. Özmen B, Kirmaz C, Aydin K, Kafesciler SO, Guclu F, Hekim-soy Z. Influence of the selective oestrogen receptor modulator(raloxifene hydrochloride) on IL-6, TNF-a, TGF-b and boneturnover markers in the treatment of postmenopausal osteoporo-sis. Eur Cytokine Netw 2007;18:148–53.

2. Yousefzadeh G, Larijani B, Mohammadirad A. Determination ofoxidative stress status and concentration of TGF-beta in theblood and saliva of osteoporotic subjects. Ann N Y Acad Sci2006;1091:142.

3. NIH Consensus Development Panel on Osteoporosis Prevention.Diagnosis and Therapy. Osteoporosis prevention, diagnosis, andtherapy. JAMA 2001;285:785.

4. Sarkar S, Reginster JY, Crans GG. Relationship between changesin biochemical markers of bone turnover and BMD predictvertabral fracture risk. J Bone Miner Res 2004;19:394.

5. Hansdöttir H, Franzson L, Prestwood K. The effect of raloxifeneon markers of bone turnover in older women living in long-termcare facilities. J Am Geriatric Soc 2004;52:779.

6. Walsh BW, Cox DA, Sashegy A. Role of tumor necrosis factor-aand interleukin-6 in the effect of hormone replacement therapyand raloxifene on C-reactive protein in postmenopausal women.Am J Cardiol 2001;88:825.

7. Lan JM, Russell L, Kan SN. Osteoporosis. Clin Orthop 2000;372:139–50.

8. Todhunter CE, Sutherland-Craggs A, Bartram SA, et al. Influ-ence of IL-6, COL1A1, and VDR gene polymorphisms on bone

mineral density in Crohn’s disease. Gut 2005;54:1579–84.

9. Zhang Q, Badell IR, Schwarz EM, et al. Tumor necrosis factorprevents alendronate-induced osteoclast apoptosis in vivo bystimulating Bcl-xL expression through Ets-2. Arthritis Rheum2005;52:2708–18.

0. Pereda CA, Hannon RA, Naylor KE, Eastell R. The impact ofsubcutaneous oestradiol implants on biochemical markers ofbone turnover and bone mineral density in postmenopausalwomen. BJOG 2002;109:812–20.

1. Abrams J, Broxmeyer H, Manolagas SC. Estrogen loss upregu-lates hematopoiesis in the mouse: a mediating role of IL-6. ExpHematol 1995;23:500–6.

2. Han JH, Choi SJ, Kurihara N, Koide M, Oba Y, Roodman GD.Macrophage inflammatory protein-1a is an osteoclastogenic fac-tor in myeloma that is independent of receptor activator ofnuclear factor kB ligand. Blood 2001;97:3349–53.

3. Mann J, Oakley F, Johnson PWM, Mann DA. CD40 Inducesinterleukin-6 gene transcription in dendritic cells. J Biol Chem2002;277:17125–38.

4. Cappellen David, Luong-Nguyen NH, Bongiovanni S, Grenet O,Wanke C, Mira S. Transcriptional program of mouse osteoclastdifferentiation governed by the macrophage colony-stimulatingfactor and the ligand for the receptor activator of NF kB. J BiolChem 2002;277:21971–82.

5. Juarranz Y, Abad C, Martinez C, et al. Protective effect ofvasoactive intestinal peptide on bone destruction in the collagen-induced arthritis model of rheumatoid arthritis. Arthritis ResTher 2005;7:R1034–45.

6. Jimi E, Aoki K, Saito H, DÀcquisto F, May MJ, Nakamura I, etal. Selective inhibition of NF-kB blocks osteoclastogenesis andprevents inflammatory bone destrction in vivo. Nature Med2004;10:617–24.

7. Ikeda F, Nishimura R, Matsubara T, et al. Critical roles of c-Junsignalling in regulation of NFAT family and RANKLregulatedosteoclast differentiation. J Clin Invest 2004;114:475–84.

8. Buxton EC, Yao W, Lane NE. Changes in serum receptor acti-vator of nuclear factor- kB ligand, osteoprotegerin, and interleu-kin-6 levels in patients with glucocorticoid-induced osteoporosistreated with human parathyroid hormone. J Clin EndocrinolMetab 2004;89:3332–6.

9. Taketa T, Sakai A, Tanaka S, et al. Selective cyclooxygenase-2inhibitor prevents reduction of trabecular bone mass in collagen-induced arthritic mice in association with suppression ofRANKL/OPG ratio and IL-6 mRNA expression in synovialtissues but not in bone marrow cells. J Bone Miner Metab2008;26:143–51.

0. Evans BA, Elford C, Pexa A, et al. Human osteoblast precursorsproduce extracellular adenosine, which modulates their secretionof IL-6 and osteoprotegerin. J Bone Miner Res. 2006;21:228–36.

1. Galien R, Garcia T. Estrogen receptor impairs interleukin-6expression by preventing protein binding on the NF-kB site.Nucleic Acids Res 1997;25:2424–9.

2. Poli V, Balena R, Fattori E, et al. Interleukin-6 deficient mice areprotected from bone loss caused by estrogen depletion. EMBO J1994;13:1189–96.

3. Bellido T, Jilka RL, Boyce BF, et al. Regulation of interleukin-6,osteoclastogenesis and bone mass by androgens. The role of the

androgen receptor. J Clin Invest 1995; 95:2886–95.