Identification of osteopontin in isolated rabbit osteoclasts

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Pages 91 l-91 7

IDENTIFICATION

OF OSTEOPONTIN

IN ISOLATED

RABBIT

OSTEOCLASTS* Ken-ichi Tezukat , Takuya Sate’, Hiroshi Kamiokas, Peter J. Nijweide4, Kayo Tanaka’, Tetsu Matsuos, Mitsue Ohtas, Noriyoshi Kuriharaz, Yoshiyuki Hakedat , and Masayoshi Kumegawal Departments

of ‘Oral Anatomy and 2Periodontology, Meikai University of Dentistry, Sakado 350-02, Japan

sDepartment “Department sHikone

School of Dentistry,

of Cell Biology and Histology, Medical Faculty, University Leiden, Leiden, 2333 AA, The Netherlands

Research

sBio-organics

Received

of Orthodontics, Tokushima University Tokushima 770, Japan

School

Laboratories,

Research CIBA-GEIGY

June

10,

of

Maruho Co. Ltd., Hikone 522-02, Japan

Department, International (Japan) Ltd., Takarazuka

Research Laboratories, 665, Japan

1992

Summarv Bone remodeling is a complex process coupling bone formation and resorption. Osteoblasts, the bone-forming cells, are known to produce various bone matrix proteins and cytokines; however, little is known about protein factors produced by osteoclasts or bone-resorbing cells. A method utilizing the high affinity of osteoclasts for tissue culture dishes was developed to isolate a large number of pure osteoclasts from rabbit long bones. A cDNA library was then constructed from these isolated osteoclasts, and differential cDNA screening was performed between osteoclasts and spleen cells. Two clones representing osteoclast-specific clones, named OC-1 and OC-2, were isolated. By Northern blot analysis, OC-I was expressed in osteoclasts and in kidneys, whereas OC-2 was specific for osteoclasts. OC-1 was found to encode osteopontin from its nucleotide sequence, and therefore, osteopontin may have other functions for osteoclastic bone resorption besides osteoclast attachment to bone. 0 1992Academic Press,Inc. Osteoclasts are multinucleate giant cells derived from hematopoietic stem cells(l), and playing key roles in bone resorption. However, because of difficulty

of purification

information

of osteoclasts

from bone tissues, only limited

on them has been forthcoming

by mainly histological

and

cytochemical analyses. * Sequence data from this article have been deposited with the EMBUGenBank Data Libraries under Accesion No. D11411. Abbreviations : TRAcP, tartrate-resistant acid phosphatase; FBS, fetal bovine serum; PBS, phosphate-buffered saline; kb, ki:obase pairs. 0006-291X/92

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Up to now, limited number of proteins have been identified in osteoclasts. TRAcP, calcitonin receptor, and vitronectin receptor are used as markers of osteoclasts(2-4). osteoclasts(5), and cathepsin osteoclasts(6). performed

However, with isolated

other unidentified

Cathepsin L activity is also D is immunohistochemically

molecular osteoclasts,

proteins

biological

approach

and, therefore,

are produced

detected localized

has never

it is very possible

by osteoclasts

in in been that

and playing important

roles in bone resorption. Intending developed

osteoclasts

at the gene expression

a method to isolate a pure population

of Chambers isolation

to characterize

and Magnus(7),

of osteoclast-specific

and constructed cDNA

clones

library against spleen cells, and characterization

Materials

of osteoclasts

level, we

based on that

a cDNA library. Here, we report by differential

screening

of the

of them.

and Methods

Isolation of Osteoclasts. Unfractionated bone cells were isolated from tibiae, femora, humeri, ulnae, and radii of IO-day-old rabbits (Japan white, Saitama Experimental Animal, Saitama, Japan). After removal of connective tissues, these bones were minced into pieces in alpha-MEM (Flow Laboratories, McLean, VA). Cells were dissociated from bone fragments by vigorous vortexing. After removal of bone fragments by sedimentation under normal gravity, cells were collected from the supernatant by centrifugation. The cells were resuspended in alpha-MEM supplemented with 10% FBS and seeded into 90-mm tissue culture dishes (Falcon, Becton Dickinson, Lincoln Park, NJ) at a number of forty million living cells per dish. After cultivation for 3 hours, the medium was exchanged for fresh medium. After an additional 20 hours of culture, the cells were washed with PBS to remove non-adherent cells and then treated with PBS containing 0.001% pronase E (Sigma Chemical, St. Louis, MO) and 0.02% EDTA for 10 minutes at 37°C. Stromal cells were removed by washing of the dishes several times with PBS, and the remaining osteoclasts were used for further analyses. To examine the response to calcitonin, the cells were treated with 70-7 M chick calcitonin in alpha-MEM containing 10% FBS before enzyme treatment. For the TRAcP staining, the cells were stained with the reagents of a May-Grunwald-Giemsa acid phosphatase kit (Sigma Chemical) in the presence of 50mM L-tartrate. Monoclonal antibody 23C6 was incubated with the cells at a concentration of 1 Ol.rg/ml in alpha-MEM containing 15% FBS and visualized by use of horseradish peroxidase-conjugated rabbit anti-mouse antibody. Construction of a cDNA librarv. Total RNA was prepared from isolated osteoclasts as described previously(8). Poly A+ RNA was purified by oligo-dT cellulose column chromatography and double stranded cDNA was synthesized. Synthesized cDNA was cloned into hgtl0 cloning vector by cDNA cloning system 3,gtlO (Amersham, Buckinghamshire, England). The resulting library containing approximately 4x1 O4 independent clones were amplified and used for screening. Differential screenina. The screening of the library was performed as described previously(8). The cDNA library was screened with radioactive 912

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cDNA probes of osteoclasts and unfractionated spleen one after the other, and osteoclast-specific clones were picked up. Radioactive probe was prepared from the cDNA insert of each clone, and cross-hybridization was performed. DNA sequencing was performed by dideoxy chain termination method. The DNA sequence was subjected to homology search against GenBank nucleotide sequence database (release 70.0). Northern blottina. Total RNA was prepared from each tissue isolated from a lo-day-old rabbit. The RNA was blotted onto a nylon membrane after formaldehyde agarose gel electrophoresis. 32P-Labeled radioactive probes were prepared by the random primer labeling procedure and used for hybridization.

Results From observation at the light microscope level, more than 95% of the cells that remained attached to the dishes after enzymatic removal of other adherent cells were multinucleate (Fig.la). A trace number of mononuclear cells were also observed. All of these multinucleate cells and some of the mononuclear ones were positive for TRAcP activity (Fig.la,

TRAcP-positive

cells u. total cells was 351 vs. 356 [>98%]) and monoclonal antibody 23C6 (Fig.1 b) which recognizes vitronectin receptors(4). Alkaline phosphatase and

Fig,. 1. Light micrographs of isolated statned for TRAcP(a) and immunostained 913

osteoclasts from rabbit long bones with monoclonal antibody 23(X(b).

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12345678

a

- 28s -18s

- 28s -18s

Fia. 2. Northern blot analysis of OC-1 and OC-2. s2P-Labeled radioactive probes were prepared from cDNA inserts of OC-1 (a) and OC-2 (b), and human p-actin gene (c) by the random primer labeling procedure and used for hybridization. Lanes contained lpg (a,b) or 2pg (c) of total RNA isolated from following sources:l, brain; 2, gut; 3, kidney; 4, liver; 5, lung; 6, spleen; 7, thymus; 8, osteoclasts. 18s and 28s indicate the migratory positions of 18s and 28s ribosomal RNA, respectively.

non-specific

esterase

indicating shown).

the absence Moreover,

response

activities these

osteoclastic osteoclasts previously(8). osteoclasts

and

from this cell population, macrophages

cells changed

(data

not

their morphology

in

before enzyme treatment

we concluded

Approximately

To characterize

cells

multinucleate

to 10s7M chick calcitonin

were osteoclasts. rabbit.

totally absent

of osteoblastic

From these observations,

osteoclasts.

were

(data not shown).

that these isolated multinucleate

3x105 osteoclasts

the isolated osteoclasts

were

obtained

in comparison

cells

from one

with other non-

cells, we performed differential cDNA cloning between and unfractionated spleen cells in the same manner as described A cDNA

library

Five thousand and spleen

clones

was

constructed

were

screened

cells one after the other,

from with

5x10s cDNA

and seventy-five

isolated probes

of

clones

specific for osteoclasts were picked up. By cross hybridization analysis, two clones, designated OC-1 and OC-2, representing 35 and 16, respectively, of the picked up clones and not hybridizing with each other, were selected. The remaining 24 clones were not specific for osteoclasts by RNA dot blot analysis and may be screening artifacts. OC-I and OC-2 contained cDNA inserts of approximately 1.4kb and 1.6kb, respectively. The frequencies OC-2 in the library were calculated as 0.7% and 0.3%, suggesting osteoclasts

of OC-I and respectively,

that these genes are expressed in rather high amount in isolated and are not derived from contaminating non-osteoclastic cells. 914

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Human Mouse Rat Pig

BIOCHEMICAL

: 1 : 1 : 1 : 1:

1

oc-1

AND

BIOPHYSICAL

RESEARCH

MRIAVICFCLLGITCAIPVKQADSGSSEEKQLYNKYPDAVATWLNPDPSQ MRLAVICFCLFGIASSLPVKVTDSGSSEEK-LYSLHPDPIATWLVPDPSQ MRLAVVCLCLFGLASCLPVKVAEFGSSEEKAHYSKHSDAVATWLKPDPSQ MRIAVIAFCLWGFASALPVKQTNSGSSEEKLLSNKYTDAVATLLKPDPSQ MRIAVICFCLLGMAYALPVKHADSGSSEEKQLYHKHPDALATWLNPDPSQ

Human Mouse Rat Pig oc-1

51 50

Human Mouse Rat Pig

99 100 100 99 99

: : : : :

149 134

: VVPTVDTYDGRGDSVVYGL-RSKSKKFRRPDATDEDITSHMESEE : IVPTVDVPNGRGDSLAYGL-RSKSRSFQVSDEQYPDATDEDLTSHMKSGE : IAPTVDVPDGRGDSLAYGL-RSKSRSFPVSDEQYPDATDEDLTSRMKSQE : AVPTGDPNDGRGDSVVYGL-RSKSKKFRRSEAQQLDATEEDLTSHVESEE : VVPTVETYDGRGDSVAYRLKRSKSKMFHVSNAQYPGASEEDLSSHVDSED

oc-1

Human Mouse Rat pig oc-1

Human Mouse Rat Pig oc-1 Human Mouse Rat Pig

51 51

51

134 144 145 198

183 183 193 195

: KQNLLAPQNAVSSEETNDFKQETLPSKSNESHDHMDDMDDEDDD--DHVD : KQNLLAPQNAVSSEEKDDFKQETLPSNSNESHDHMDDDDDDDDDDGDHAG : KQNLLAPQNSVSSEETDDFKQETLPSNSNESHDHMDDDDDDDDD-GDHAE : KQTFLAPQNTISSEETDDFKQETLPSKSNESPEQTDDVDDDDDE--DHVD : KQNLLTPQNAMSSEEKDDLKQETLPSKSIESHDHMDDIDEDEDD--DHVD SQDSIDSNDSDDVDDTDDSHQSDESHHSDESDELVTDFPTDLPATEVFTP SEDSVD---SDE---------SDESHHSDESDE--TV--TASTQADTFTP SEDSVN---SDE---------SDESHHSDESDE--S-F-TASTQADVLTP SRD---T-DSEEADHADDADRSDESHHSDESDELVTDFPTDTPATDV-TP NRD---SNESDDADHPDDSHHSDESHQSDESDE-VTVYPTEDAATTVFTE

: LNGAYKAIPVAQDLNAPSDWDSRGKDSYETSQLDDQSAETHSHKQSRLYK : SKESLDVIPVAQLLSMPSDQDNNGKGSHESSQLDEPSLETHRLEHS---: SDEAIKVIPVAQRLSVPSDQDSNGKTSHESSQLDEPSVETHSLEQSKEYK : TDGTPKAILVAQRLHVASDLDSQEKDSQETSQPDDRSVETRSQEQSKEYT : LDDTPRAIPVAQHLNVPSDWDSQEKDSHDVSQVDDHSVETQSHEQARQYK

oc-1

233 243 245

: RKANDESNEHSDV------------------IDSQELSKVSREFHSHEFH : -KESQESADQSDV------------------IDSQASSKASLEHQSHKFH : QRASHESTEQSDAIDSAEKPDAIDSAERSDAIDSQASSKASLEHQSHEFH : IKTYDGSNEHSNV------------------IESQENPKVSQE-----FH : REANDNSVEHSHS------------------IDSQESSKVSQESQSREFR

Human

280

:

Mouse Rat Pig

260 283 270 277

: : : :

oc-1

240 229

COMMUNICATIONS

SHEDMLVVDPKSKEEDKHLKFRISHELDSASSEVN SHKDKLVLDPKSKEDDRYLKFRISHELIZSSSSEVN SHEDKLVLDPKSKEDDRYLKFRISHELESSSSEVN SHEDKLVPDSKS-EEDKHLKLRVSHELESASSEIN SHEDKLAIEPKSEEDEEHRQLRVSHELDSTSSEIN

Fia. 3. Comparison of the cDNA-deduced amino acid sequence of OC-1 with human, mouse, rat, and pig osteopontin. Amino acids are indicated by smgleletter codes, and numbers indicate the position number of the first amino acid in each line. Hyphens indicate gaps.

Tissue

specificity of X-1

and OC-2 was examined by Northern blot

analysis with total RNA prepared from various tissues of a lo-day-old

rabbit

(Fig.2). OC-I was expressed in osteoclasts and in kidneys, whereas OC-2 was specific for osteoclasts only. The nucleotide sequences of the cDNA inserts of these clones were determined, performed.

and a homology search was

OC-1 contained an open reading frame specifying 311 amino

acids; and, as seen in Fig.3, it shared significant homology throughout the sequence with cDNA-deduced sequences reported for human, mouse, rat, and pig osteopontin(9-12).

The amino acid sequence of OC-1 was 68.6%,

57.6%, 55.2%, and 62.8% identical with those of human, mouse, rat, and pig respectively. Therefore, OC-1 is supposedly the rabbit osteopontin, counterpart of this gene. OC-2 did not show any significant homology in this search, and would thus seem to represent an unknown gene. The structure of OC-2 will be described elsewhere. 915

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Discussion The production of osteopontin has been reported in various cell types such as osteoblastic cells, kidney cells, nerve cells, and macrophages(l3-18). Mark &t a. (13) found that some osteoclasts and bone matrix adjacent to them are immunohistochemically positive for osteopontin; however, they could not be certain whether or not the osteopontin was produced by osteoclasts. Osteopontin released

produced by osteoblasts accumulates in bone matrix and is when

osteoclasts

resorb

bone.

For this reason, immunohistochemical techniques are not suitable for investigating whether osteoclasts produce osteopontin or not; and in bone tissues, osteopontin has long been believed to be produced by osteoblastic cells. However, our present

results

obtained

with isolated

osteoclasts

from

bone tissue

demonstrate that isolated osteoclasts express a large amount of osteopontin mRNA.

Thus, this paper represents

the first direct

demonstration

of

osteopontin in osteoclasts. Osteopcntin contains the cell-binding Arg-Gly-Asp sequence, and is thought to play an important role in adhesion of osteoclasts to bone surfaces(l9).

Moreover, Miyauchi et al. (20) recently reported that

osteopontin reduced the cytosolic calcium concentration

in osteoclasts via

vitronectin receptors. This fact indicates that osteopontin may also participate in regulation of osteoclast functions. Therefore, the osteopontin produced by osteoclasts may have important roles in osteoclastic bone resorption by not only participating

in binding of osteoclasts

to bone surfaces

but also

controlling some yet unspecified osteoclastic function(s).

Acknowledament: We thank Dr. Michael A. Horton for kindly providing monoclonal antibody 2366 and helpful discussions.

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9. Kiefer, M.C., Bauer, D.M., and Barr, P.J. (1989) Nucleic Acids Res. 17, 3306.

IO. Craig, A.M., Smith, J.H., and Denhardt, D.T. (1989) J. Biol. Chem. 264, 9682-9689. 11. Oldberg, A., Franzen, A., and Heinegard, D. (1986) Proc. Natl. Acad. Sci. USA 83, 881 g-8823. 12. Wrana, J.L., Zhang, Q., and Sodek, J. (1989) Nucleic Acids Res. 17, 10119. 13. Mark, M.P., Prince, C.W., Oosawa, T., Gay, S., Bronckers, A.L.J.J., and Butler, W.T. (1987) J. Histochem. Cytochem. 35, 707-715. 14. Yoon, K., Buenaga, R., and Rodan, G.A. (1987) Biochem. Biophys. Res. Commun. 148, 1129-l 136. 15. Mark, M.P., Prince, C.W., Gay, S., Austin, R.L., and Butler, W.T. (1988) Cell Tissue Res. 251, 23-30. 16. Nomura, S., Wills, A.J., Edwards, D.R., Heath, J.K., and Hogan B.L.M. (1988) J. Cell Biol. 106, 441-450. 17. Mark, M.P., Butler, W.T., Prince, C.W., Finkelman R.D., and Ruth, J.-V. (1988) Differentiation 37, 123-l 36. 18. Miyazaki, Y., Setoguchi, M., Yoshida, S., Higuchi, Y., Akizuki, S., and Yamamoto, S. (1990) J. Biol. Chem. 265, 14432.14438. 19. Reinholt, F.P., Hultenby, K., Oldberg, A., and Heinegard, D. (1990) Proc. Natl. Acad. Sci. USA 87, 4473-4475. 20. Miyauchi, A., Alvarez, J., Greenfield, E.M., Teti, A., Grano, M., Colucci, S., Zambonin-Zallone, A., Ross, F.P., Teitelbaum, S.L., Cheresh, D., and Hruska, K.A. (1991) J. Biol. Chem. 266, 20369-20374.

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