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Biochemical and Biophysical Research Communications 279, 258–264 (2000)

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NF-a-Dependent Activation of NF-kB in Humansteoblastic HOS-TE85 Cells Is Repressed

n Vector-Averaged Gravity Using Clinostat Rotation

enji Kobayashi,*,† Fukushi Kambe,*,1 Kazutoshi Kurokouchi,† Tadahiro Sakai,*,†aoki Ishiguro,† Hisashi Iwata,† Kazuo Koga,‡ Raphael Gruener,§ and Hisao Seo*

Department of Endocrinology and Metabolism, Division of Molecular and Cellular Adaptation, and ‡Space Medicineesearch Center, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan;Department of Orthopedics, Nagoya University School of Medicine, Nagoya, Japan; andDepartment of Physiology, University of Arizona College of Medicine, Tucson, Arizona

eceived November 7, 2000

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Effects of vector-averaged gravity on tumor necrosisactor (TNF)-a-dependent activation of nuclear factorappa B (NF-kB) in human osteoblastic HOS-TE85ells were investigated by culturing the cells usinglinostat rotation (clinorotation). Cell cultures wereotated for 72 h at 40 rpm in a clinostat. At the end oflinorotation, the cells were treated with TNF-a for 30in under stationary conditions. Electrophoretic mo-

ility shift assays revealed that TNF-a-dependent ac-ivation of NF-kB was markedly reduced in the clino-otated cells when compared with the cells in controltationary cultures or after horizontal rotation (mo-ional controls). The NF-kB-dependent transactivationas also impaired in the clinorotated cells, as evi-enced by a transient transfection assay with a re-orter plasmid containing multimerized NF-kB sites.onsistent with these findings, the TNF-a-dependent

nduction of endogenous NF-kB-responsive genes105, IkB-a, and IL-8, was significantly attenuated inlinorotated cells. These results demonstrate thatector-averaged gravity inhibits the responsiveness ofsteoblasts to TNF-a by repressing NF-kB activation.2000 Academic Press

Key Words: TNF-a; NF-kB; clinostat; vector-averagedravity; osteoblasts; microgravity.

A number of paracrine and autocrine factors regu-ate proliferation, differentiation and gene expressionn osteoblasts. Among them, tumor necrosis factorTNF)-a, a cytokine synthesized in the bone microen-

1 To whom correspondence should be addressed at Department ofndocrinology and Metabolism, Research Institute of Environ-ental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Na-

oya 464-8601, Japan. Fax: 181-52-789-3891. E-mail: [email protected].

258006-291X/00 $35.00opyright © 2000 by Academic Pressll rights of reproduction in any form reserved.

n osteoblasts by affecting synthesis of DNA and col-agen (1), osteocalcin (2), and proteolytic enzymes suchs metalloproteinases (3). In altered gravity, however,ither during space flights or after clinostat rotationclinorotation; vector-averaged gravity conditions) theesponses of osteoblasts to such hormones and cyto-ines are markedly affected. For example, human os-eosarcoma MG-63 cells cultured for 9 days in spaceight showed reduced induction of collagen, alkalinehosphatase and osteocalcin mRNA expression in re-ponse to 1,25-dihydroxyvitamin D3 or transformingrowth factor b2 (4). Similarly, reduction in both basallkaline phosphatase activity and its induction byreatment with 1,25-dihydroxyvitamin D3 was re-orted in human osteoblastic HuO9 cells which under-ent clinorotation for 4 days (5). However, effects oficrogravity on responses of osteoblasts to TNF-a have

ot been studied to date.TNF-a has been shown to exert its action, in various

ell types, by binding to a membrane receptor (TNF-R)ith subsequent activation of a dimeric transcription

actor NF-kB consisting of the Rel family proteins suchs p65 (RelA), p50, p52, c-Rel and RelB. In unstimu-ated cells, NF-kB is sequestered in the cytoplasmound to an inhibitory protein IkB. Several isoformsuch as IkB-a and IkB-b have been identified (6, 7).NF-a induces the activation of NF-kB by promotinghe phosphorylation and degradation of IkB. NF-kB,hich is released from IkB, translocates into the nu-

leus, binds to the distinct regulatory element of thearget genes and controls their transcription (8, 9).lthough the signaling pathway of TNF-a has not yeteen fully defined in osteoblasts, we recently demon-trated that TNF-a increases expression of interleukinIL)-6 and intercellular adhesion molecule-1 genes

through activation of NF-kB consisting of p50-p65 het-e

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Vol. 279, No. 1, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

rodimer in osteoblast-like cells (10).The clinostat is an effective, ground-based tool to

imulate microgravity (11–14). Clinorotation mimicsertain aspects of the microgravity environment byulling the gravitational vector through continuousveraging. In the present study, we utilized this sys-em to investigate whether the vector-averaged grav-ty, generated by clinorotation, affects TNF-a-depen-ent activation of NF-kB in human osteoblastic HOS-E85 cells.

ATERIALS AND METHODS

Cell culture. Human osteoblastic HOS-TE85 cells [ATCC CRL-543] which express osteoblast-like phenotypes (15, 16) were cul-ured in Dulbecco’s modified Eagle medium (DMEM) supplementedith 10 mM Hepes (pH 7.4) and 10% fetal bovine serum. After the

ells grew to about 50% confluency, in 12.5-cm2 flasks, the mediumas removed and the flasks were completely filled with fresh me-ium and sealed with paraffin to eliminate air bubbles thus reducingurbulence and shear forces during clinorotation. The cells wereivided into three groups: (i) stationary (control condition), (ii) hor-zontal rotation (motional control for turbulence, shear forces andibrations) in which the flasks were rotated around the vertical axis,nd (iii) clinorotated cultures in which the flasks were rotatedround the horizontal axis to produce a vector-averaged environ-ent. Details of the clinostat apparatus were previously described

17). All cultures were placed in an incubator at 37°C. After 72-hotation at 10 or 40 rpm, the cells were treated with 50 U/ml recom-inant human TNF-a (2.5 3 103 U/mg; Asahi Chemical Industry Co.td., Tokyo, Japan) for 30 min in stationary culture. The cells werehen harvested, and nuclear extracts were prepared for electro-horetic mobility shift assay as described previously (18). For North-rn blot analysis, the cells were treated with TNF-a and 100 nMhorbol 12-myristate 13-acetate (PMA; Sigma Chemical Co., St.ouis, MO) for 6 h and 1 h, respectively, and total RNA was extractedy the acid guanidium thiocyanate-phenol-chloroform-extractionethod (19). The cell proliferation was assessed by WST-1 assay

Cell Counting Kit; Dojindo Laboratories, Tokyo, Japan) after 24-hnd 72-h rotation.

Electrophoretic mobility shift assay (EMSA). Details of theMSA procedure were described previously (20). The nuclear ex-

racts (10 mg protein) were subjected to EMSA. The kBwt oligonu-leotide (59-TCGAGCAGAGGGGACTTTCCGAGAGTCGA-39) con-aining a canonical NF-kB binding site identified in mouse Igknhancer (underlined) (21) was labeled with [32P]dCTP and used asprobe. The kBmu oligonucleotide (59-TCGAGCAGAGCTCACTT-

CCGAGAGTCGA-39) in which a mutation was introduced in theF-kB binding site (underlined) (22) and the unlabeled kBwt oligo-ucleotide were used as competitors for displacement analysis with0-fold molar excess. The supershift analysis employed anti-p65ntibody (Santa Cruz Biotechnology, Santa Cruz, CA) and preim-une rabbit serum.

Transfection assay. The construction of a luciferase reporterlasmid pGL3pro-3kB which is driven by a tandem repeat of threeF-kB-binding sites was described previously (18). The pGL3pro-

kB plasmid (1 mg/flask) together with a a-galactosidase-expressinglasmid (0.2 mg/flask; pSV-b-galactosidase control vector driven byimian virus 40 early promoter and enhancer segments, Promegaorporation, Madison, WI) were transfected into HOS-TE85 cellssing the calcium-phosphate method as described previously (23).fter a 16-h transfection, the flasks were filled with fresh mediumnd subjected to clinorotation for 48 h. The cells were then treatedith 50 U/ml TNF-a for 4 h in stationary culture. The luciferase and

259

uminometer (LB9501, Berthold, Germany). The enzyme activitiesere normalized to the protein contents of cell lysates determinedith a microassay kit (BIO-RAD Laboratories, Hercules, CA).

Northern blot analysis. The detailed procedure was describedreviously (24). After fractionation of 20 mg total RNA on 0.8%garose gel, the RNA was transferred onto a Gene Screen Plusembrane (New England Nuclear, Boston, MA). The membrane wasybridized with human IkB-a, p105, IL-8, GAPDH (glyceraldehyde-phosphate dehydrogenase) and c-fos cDNA probes labeled with

32P]dCTP. After hybridization, the membrane was subjected touantitative analysis using BAS 2000 bioimage analyzing systemFuji film Co., Tokyo, Japan) and then exposed to X-ray film. Human105 and IkB-a cDNAs were synthesized by RT-PCR using mRNArepared from TNF-a-treated FRTL-5 cells and HOS-TE85 cells,espectively. The primers for p105 were 59-GATGGCCCATACC-TCAAATAT-39 and 59-GGCTTTACTGTCATAGATGGCG-39 (25).he primers for IkB-a were 59-ATGTTCCAGGCGGCCGAGCG-CCCCA-39 and 59-TCATAACGTCAGACGCTGGCCTCC-39 (6). Themplified PCR product was ligated into pGEM-T-easy plasmid (TAloning system; Promega). The authenticity of cDNA was verified byNA sequencing. The preparations of IL-8, GAPDH and c-fos cDNAsere described in our previous reports (26, 27)

Enzyme-linked immunosorbent assay (ELISA) of IL-8. During2-h clinorotation at 40 rpm, the cells were continuously exposed to0 U/ml TNF-a. The concentrations of IL-8 in the media were deter-ined by a human IL-8 ELISA kit (R&D systems Minneapolis, MN)sing a microplate reader (BIO-RAD).

Western blot analysis. The preparation of whole cell lysates androcedure for Western blot analysis were described in our previouseport (28). The protein contents in the whole cell lysates wereeasured by a microassay kit. Cell lysates containing 80 mg proteinere denatured and subjected to SDS–12% polyacrylamide gel elec-

rophoresis. The proteins in the gel were transferred onto Hybond-Cembrane (Amersham Life Sciences, Arlington Heights, IL). The

ands for IkB-a, IkB-b, tumor necrosis factor-receptor 1 (TNF-R1),nd tumor necrosis factor-receptor 2 (TNF-R2) were visualized bysing each specific antibody (Santa Cruz) diluted at 1:500 and theecond antibody conjugated with alkaline phosphatase (Zeimed, Sanrancisco, CA) diluted at 1:1000.

Statistical analysis. Results from different treatment groupsere subjected to one-way analysis of variance with Bonferroni test.P value smaller than 0.05 was considered to demonstrate statis-

ically significant differences.

ESULTS

linorotation Represses the Activation of NF-kBInduced by TNF-a

Figure 1A shows, by using the EMSA assay, thatreatment of HOS-TE85 cells in stationary culturesith TNF-a induced a single protein/kBwt complex

lane 2). This complex was displaced with excessmount of unlabeled kBwt oligonucleotide (lane 4), butot with kBmu oligonucleotide in which the NF-kB-inding site was mutated (lane 5). Anti-p65 antibodyupershifted the complex (lane 7). However, pre-mmune rabbit serum did not affect the mobility of theomplex (lane 6). These results indicate that TNF-anduces the activation of NF-kB containing p65 in con-rol HOS-TE85 cells.

Quantified DNA-binding activity of NF-kB was in-reased fivefold by TNF-a, over basal levels, in station-

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ry cultures (Fig. 2B). However, after culturing cellsor 72 h under clinorotation at 10 or 40 rpm, the mag-itude of TNF-a-dependent increase in NF-kB bindingctivity was attenuated in a rotation speed-dependentanner. Approximately 50% reduction in the binding

ctivity was measured in cells which underwent clino-otation at 40 rpm. In contrast, the extent of TNF-a-ependent increase in the binding activities in the cellsfter horizontal rotation (motional controls) at either0 or 40 rpm were similar to that observed in thetationary control culture (Fig. 2C). These results dem-nstrate that clinorotation represses the TNF-a-ependent activation of NF-kB.

FIG. 1. Effects of clinorotation on TNF-a-dependent activationOS-TE85 cells treated with or without 50 U/ml TNF-a for 30 min i

ligonucleotide as a probe. Displacement analysis was performed by) oligonucleotides as competitors. Subunits of NF-kB were charactlane 7) and preimmune rabbit serum (pre, lane 6). (B and C) Thlinorotation at 10 rpm and 40 rpm for 72 h, or (iii) under horizontar without TNF-a (50 U/ml). After a 30-min stationary incubation, thesing a kBwt oligonucleotide as a probe. The experiment was perforAS 2000 system. The data are presented as percentage of the levelsean 6 range. Similar results were obtained from two separate exp

FIG. 2. Effects of clinorotation on TNF-a-dependent transactivatiGL3pro-3kB containing a luciferase reporter gene driven by multimpSV-b-gal). After a 16-h transfection, the cells underwent the clinor8 h, and then the cells were treated with or without TNF-a (50 Uctivities in cell lysates were determined, and normalized by the protevels of the untreated cells in stationary culture, and expressed asxperiment. *P , 0.05 vs horizontal rotation with TNF-a.

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linorotation Decreases NF-kB-DependentTransactivation

We next examined whether NF-kB-dependent genexpression is altered by clinorotation. We performed aransient transfection assay using a reporter plasmidontaining a luciferase gene driven by multimerizedF-kB sites (pGL3pro-3kB). As shown Fig. 2A, the

uciferase activity in the cells transfected withGL3pro-3kB was markedly increased by TNF-a byore than threefold in stationary cultures. A similar

nduction was also observed in cultures subjected toorizontal rotation. In contrast, after 40-rpm clinoro-

f NF-kB in HOS-TE85 cells. (A) Nuclear extracts prepared fromtationary culture were subjected to EMSA using a 32P-labeled kBwtng 50-fold molar excess of unlabeled kBwt (lane 4) and kBmu (laneed by supershift analysis employing antibody directed against p65ells were cultured in (i) stationary conditions for 72 h, (ii) undertation at 10 rpm and 40 rpm for 72 h. They were then treated withlls were harvested, and the nuclear extracts were subjected to EMSA

in duplicate. The radioactivity of the bands were measured by thethe untreated cells in stationary culture. The data are expressed asments.

of NF-kB in HOS-TE85 cells. Cells were transiently transfected withized NF-kB sites, together with b-galactosidase-expressing plasmidtion (40 rpm), horizontal rotation (40 rpm) or stationary culture forl) for 4 h in stationary culture. The luciferase and b-galactosidasecontents of the lysates. The data are presented as percentage of the

ean 6 SD (n 5 3). Similar results were obtained from a separate

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ation, a significant decrease in the induction wasound in comparison to that of stationary or horizontalotation controls. While the basal level under clinoro-ation was somewhat lower than stationary or horizon-al controls, this difference was not statistically signif-cant. These results demonstrate that the reduction inNA-binding activities of NF-kB consequent to clino-

otation leads to the reduced expression of the reporterene. This effect appears to be specific, because thexpression of b-galactosidase reporter gene (which isot driven by NF-kB sites) did not differ among thehree culture conditions (Fig. 2B).

ffects of Clinorotation on Expression of EndogenousTarget Genes for NF-kB

It has been reported that the IkB-a, p105 (a precur-or of p50), and IL-8 genes are targets of NF-kB. In-eed, as shown in Fig. 3, Northern blot analysis re-ealed that TNF-a markedly increased these threeRNA levels in stationary cultures (Figs. 3A–3C). In

he cells under horizontal rotation, the basal levels andhe magnitude of their increase by TNF-a were similaro those observed in stationary cultures. However, in

FIG. 3. Clinorotation suppresses the induction of endogenous NFotation (40 rpm) or stationary culture, the cells were treated with orlot analysis using p105 (A), IkB-a (B), IL-8 (C) and GAPDH (D)adioactivity of the bands were measured by the BAS 2000 system, thxpressed as a percentage of the level of the stationary untreatedorizontal rotation with TNF-a. Similar results were obtained from

261

ultures subjected to clinorotation, the magnitude ofhe increase in these mRNAs was significantly de-reased. In contrast, GAPDH mRNA levels were notffected by TNF-a after clinorotation (Fig. 3D).Consistent with the change in the mRNA levels,

oncentrations of IL-8 in the medium were markedlyncreased by TNF-a in stationary cultures, and thisncrease was significantly attenuated in cells whichnderwent clinorotation (Fig. 4). These results clearlyhow that down-regulation of NF-kB also resulted inhe decreased expression of its endogenous targetenes.

ffects of Clinorotation on c-fos Expression,Degradation of IkB-a, and Cell Growth

We next examined the effects of clinorotation onMA-dependent induction of c-fos gene. HOS-TE85ells that underwent clinorotation at 40 rpm for 72 here treated with 100 nM PMA for 1 h, and Northernlot analysis was performed. As shown in Fig. 5A, PMAnduced a marked increase in c-fos mRNA levels in theells under stationary and horizontal cultures. Similarnduction was also observed in the cells under clinoro-

target genes by TNF-a. After 72-h clinorotation (40 rpm), horizontalhout TNF-a (50 U/ml) for 6 h. Total RNA was subjected to NorthernAs as probes. Experiment was performed in triplicate. After the

RNA levels were normalized by the GAPDH mRNA levels, and thenls. The data are expressed as mean 6 SD (n 5 3). *P , 0.05 vseparate experiment.

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To elucidate possible mechanisms involved in theeduced activation of NF-kB by TNF-a under clinostatonditions, we studied the expression of TNF receptor(TNF-R1), TNF-R2, IkB-a and IkB-b. Western blot

nalysis showed that the cellular levels of these pro-eins were not changed by 72-h clinorotation (data nothown). Also, degradation of IkB-a in response to thereatment with TNF-a for 30 min after clinorotationas not altered when compared to those after station-ry and horizontal cultures (Fig. 5B). Cell proliferationates were assessed after 24-h and 72-h clinorotationy WST-1 assay. Percent changes in cell number weres follows: 24 h after stationary culture 100 6 16.8%mean 6 SD), 24 h after clinorotation 95.69 6 19.1;2 h after stationary culture 100 6 6.26%, 72 h afterlinorotation 95.74 6 11.9%. These results indi-ated that cell proliferation rates were not altered bylinorotation.

ISCUSSION

The present study demonstrates for the first timehat cells subjected to vector-averaged gravity (simu-ated microgravity) conditions using clinostat rotationepresses the TNF-a-dependent activation of NF-kB inuman osteoblastic HOS-TE85 cells. Consistent withhe impairment of NF-kB activation, the NF-kB-ependent transactivation of a luciferase reporter genend the endogenous genes, IkB-a, p105, and IL-8, werelso significantly suppressed by clinorotation, but noty horizontal rotation which serves as a motional con-rol. The effect of clinorotation can be attributed to theector-averaged gravity condition, because cells under

FIG. 4. Clinorotation represses TNF-a-dependent IL-8 secretionn HOS-TE85 cells. At the beginning of 72-h clinorotation (40 rpm),NF-a (50 U/ml) was added to the culture medium. The concentra-ions of IL-8 in the media were determined by ELISA. The datare expressed as mean 6 SD (n 5 4). *P , 0.05 vs stationaryontrol with TNF-a. Similar results were obtained from a separatexperiment.

262

hear forces, and vibrations, responded to TNF-a to aimilar extent found in stationary control cultures.The clinostat simulates the microgravity of space by

oing two things: (i) it produces a vector-averagedravity environment. When the “g” vector is averaged,ts value becomes theoretically zero, and (ii) thelinostat, working as a low level centrifuge, produces amall centrifugal force. This force (which could cancelut the vector-averaged environment, if it were largeompared to “g” the gravity force) can be calculatedsing an estimation of the cell mass (in the 10212 3 gange), the distance from the axis of rotation (less than5 mm) and the speed of rotation (less than 100 rpm).he calculated value of the centrifugal force, underhese conditions, is about 1024 to 1023g (29–31). Thus,he expected “g” force value during clinorotation be-omes less than 1023g (note that the “g” force valuetself while still existing on earth has a vectorial valuef zero due to the continuous averaging produced in thelinostat by the rotation of the cultured cells). Thisalue is within the definition of the “microgravity” ofpace where the gravity vectorial force is reduced tobout 1026g (32).It should be noted that vector-averaged gravity did

ot affect the b-galactosidase activity in the transfec-ion experiments. The b-galactosidase gene in pSV-b-al plasmid is driven by simian virus 40 earlyromoter, which was shown to be activated by abiquitous transcription factor Sp-1 (33). It is thusonsidered that Sp-1 activity is not affected by vector-

FIG. 5. Effects of clinorotation on c-fos expression and degrada-ion of IkB-a. After 72-h clinorotation (40 rpm), horizontal rotation40 rpm) or stationary culture, HOS-TE85 cells were treated with00 nM PMA for 1 h (A) or 50 U/ml TNF-a for 30 min (B). Total RNAxtracted from the PMA-treated cells was subjected to Northern blotnalysis using human c-fos cDNA as a probe. Whole cell lysates fromNF-a-treated cells were subjected to Western blot analysis usingnti-IkB-a antibody.

averaged gravity. Similarly, GAPDH mRNA levelswetie

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ere also unaffected by clinorotation. In addition, thexpression of c-fos, another inducible transcription fac-or, was not affected by clinorotation. These resultsndicate that vector-averaged gravity exerts a specificffect on TNF-a-dependent activation of NF-kB.Repression of TNF-a-dependent activation of NF-kB

y clinorotation could be caused by reduction in TNF-Rumber or by an increase in IkB content. We thusxamined the amounts of TNF-R1, TNF-R2, IkB-a andkB-b, and the degradation of IkB-a in response toNF-a after clinorotation. However, these parametersere not altered. Previous reports have demonstrated

hat actual microgravity (space flight) and its simula-ion via clinorotation are associated with alterations tohe cytoskeleton of osteoblasts (17, 34). Such an alter-tion may provide a clue to the mechanism involved inhe findings we report here. Thus, the altered cytoskel-tal network may impair the translocation of NF-kB tohe nucleus or the trafficking of NF-kB/IkB kinases inhese cells.

In summary, we show here that the TNF-a-ependent induction of several endogenous NF-kB tar-et genes was suppressed in osteoblastic cells culturednder vector-averaged gravity conditions. Consideringhe wide variety of target genes of NF-kB (8, 9), im-airment of the NF-kB activation would affect variousunctions of osteoblasts such as cell growth and differ-ntiation, as well as responses to hormones and cyto-ines, which in turn might disturb normal bone remod-ling and lead to the development of bone atrophyhich has been reported to take place in humans andnimals after space flight (35–37).

CKNOWLEDGMENTS

This study was funded as a part of the “Ground Research for Spacetilization”, promoted by Japan Space Forum, and was supported inart by a Grant-in-Aid for Scientific Research from the Ministry ofducation, Science, Sports, and Culture, Japan and by Researchrants for Longevity Sciences from the Ministry of Health andelfare of Japan.

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tnf-α-dependent activation of nf-κb in human osteoblastic hos-te85 cells is repressed in...

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