The Mouse Osteopontin Gene › content › 265 › 24 › 14432.full.pdfVol. 265, No. 24, Issue of August 25, pp. 14432-14438.1990 Printed in U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 265, No. 24, Issue of August 25, pp. 14432-14438.1990 Printed in U.S.A.

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

The Mouse Osteopontin Gene EXPRESSION IN MONOCYTIC LINEAGES AND COMPLETE NUCLEOTIDE SEQUENCE*

(Received for publication, January 30, 1990)

Yoshitaka Miyazaki, Mihoko Setoguchi, Seiji Yoshida, Yasunori Higuchi, Shin’ichiro Akizuki, and Shunsuke Yamamoto From the Department of Pathology, Medical College of Oita, Oita, Japan

Murine macrophage cell lines and resident macrophages showed various levels of expression of the murine osteopontin (OP) gene, and macrophage stimulating agents were found to enhance transcription of the gene with kinetics which are unique for each stimulator.

The organization of the murine OP gene was determined. The gene comprises six exons and five introns and spans approximately 4.8 kilobases. Exon 1 contains the 16 amino acids of the leader sequence. Exons 2,3,4,5, and 6 encode 12,27,14,94, and 129 amino acid residues, respectively. Exon 5 encodes regions containing 10 consecutive Asp amino acid residues and a Gly-Arg-Gly-Asp-Ser peptide. Exon 6 encodes the C- terminal half of OP and contains no 15- and 54-base pair nucleotide sequences which are deleted in murine OP cDNA compared to that of rat OP cDNA. Since Southern blot analysis indicated that the OP gene is a single copy, it is obvious that the murine OP cDNA has the sequence previously determined (Miyazaki, Y., Se- toguchi, M., Yoshida, S., Higuchi, Y., Akizuki, S., and Yamamoto, S. (1989) Nucleic Acids Res. 17, 3298). A comparison with the cDNA sequences reported previously suggested the presence of nucleotide sequence polymorphisms. The 5’ end of the murine OP gene was defined by primer extension and Sl nuclease mapping. Sequence analysis of the 5’-flanking DNA revealed the presence of many potential regulatory motifs.

Recent molecular investigations have clearly demonstrated that cell-matrix interactions are attributable to receptor-li- gand binding reactions. Moreover, the role of ligands containing the tripeptide Arg-Gly-Asp (RGD), which is thought to function as a crucial structural site that is recognized by cell surface receptors (l), has been widely accepted. As represent- ative RGD containing molecules, various proteins such as fibronectin (FN)’ (2, 3), vitronectin (4, 5), collagens (6), thrombospondin (7), fibrinogen (8), osteopontin (OP) (9), and von Willebrand factor (8) have been reported.

Adherence, spreading, migration, and phagocytosis are essential properties of macrophages which play important roles in self-defense mechanisms. Analyses of the molecular mech-

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X51834.

‘The abbreviations used are: FN, fibronectin; OP, osteopontin; NSl, P3/NSl/l-Ag-1; Pipes, piperazine-N,N’-bis(2-ethansulfonic acid); bp, base pair; kb, kilobase pair; UT, untranslated.

anisms of macrophage-matrix interactions, therefore, are important. It has been reported that macrophages produce FN (lo-12), bind FN with the FN receptor termed VLA-5 (13), and migrate to FN fragments (14), and that complement and Fc receptor-mediated phagocytosis by macrophages is enhanced by the interaction of macrophages with FN (15-17). The involvement of other RGD proteins, however, has yet to be documented.

OP is a sialoprotein of M, 69,000 which was originally found in bone cells. Molecular cloning and analysis of the cloned OP cDNA indicated that mature rat OP consists of 297 amino acids containing a Gly-Arg-Gly-Asp-Ser (GRGDS) sequence which is identical to the cell-binding sequence within the FN molecule (9, 18). It has been shown that rat osteosarcoma cells bind to and spread on surfaces coated with OP and that the binding is suppressed by synthetic GRGD peptides (9). Subsequently, expression of OP has been observed in various cells including epidermal cells, kidney, placenta, nerve cells, and T lymphocytes in rat and mouse systems (8, 19-23). Although the exact role of OP is still unclear, the expression of OP has been suggested to be associated with host resistance to infection by Rickettsia tsutsugamushi (24). Furthermore, it has been reported that OP plays a part in tumorigenicity (25) and adherence in kidney cells (26).

We have cloned several cDNAs from a murine peritoneal macrophage cDNA library (27-30) and have previously reported that the MS21 cDNA encodes a murine OP-like protein. Sequencing analysis, however, revealed that the cDNA contains a truncated coding sequence compared to that of rat OP (9, 31) and contains a number of differences between our murine OP-like cDNA sequence and the OP cDNA sequences from other groups (24, 25). Allelic polymorphisms of the OP gene have also been suggested by host resistance to Rickettsial infection and by Southern blot analysis of genomic DNA (24, 32). These results prompted us to investigate the expression of OP in macrophages. We herein present results on the expression of the murine osteopontin gene in macrophage cell lines and in macrophages compared with that in other cells. Furthermore, to examine the inter- and intra-species nucleotide sequence diversity in OP cDNA, and as a first step in determining the regulatory mechanisms of cell-type specific expression of the OP gene, we have isolated the OP gene from a murine genomic phage library and determined its complete nucleotide sequence.

EXPERIMENTAL PROCEDURES

Materials-Restriction enzymes were purchased from Takara, TO- yobo, Bethesda Research Laboratories, and Wako Pure Chemicals. Agarose, ultrapure DNA grade, was from Takara. DNA ligation kit, reverse transcrintase, and mung bean nuclease were obtained from Takara, and bo;ine alkaline phoiphatase was obtained from Boehrin- ger Mannheim. Radioactive nucleotides [u-32P]dCTP (3000 Ci/mM) and [T-~‘P]ATP (6000 Ci/mM) were obtained from Du Pont-New

14432

Expression and Structure of the Murine Osteopontin Gene 14433

England Nuclear. Chemicals used for DNA sequencing were obtained from Toyobo. X-ray film (XAR-351) was obtained from Kodak.

Cell Lines and Tissues-The HINSB3 cell line was produced by fusion between the 8-azaguanine-resistant mouse myeloma cell line P3/NSl/l-Ag-1 (NSl) and human peripheral blood monocytes and the CANS-20 line was the result of fusion between NSl and mouse pleura1 macrophages, as previously described (27, 33-35). These hybrids initially lack macrophage functions, but they later express them. Early HINSB3 and CANS-20 cells do not have macrophage properties, whereas aged HINSB3 and CANS-20 cells manifest macrophage properties. Mouse macrophage cell lines, RAW264 and 5774, mouse mveloma cell line. NSl, mouse T lymphoma cell line, EL4, mouse mast cell tumor cell line, P815, mouse fibroblast cell line, L929, and mouse osteoeenic cell line. MC3T3-El (36) kindlv sunolied bv Dr. Y. Amagai (Degartment of Physiology, Tohoku Denial University) were also used. Resident mouse peritoneal macrophages were obtained as described previously (33). Mouse liver, kidney, placenta (9.5 days postcoitum). and brain were obtained from BALB/c mice.

Cell Cultures-75-cm2 flask cultures of aged HINSB3 cells were arown in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum. For time course studies for stimulator-induced enhancement of OP expression in aged HINSB3 cells, the culture medium was replaced when the cells were subconfluent, and each stimulator was added.

Preparation of RNA and Northern Blot Hybridization-For each cell line, cells in one flask were quickly disrupted with 6 M guanidine isothiocyanate, and total RNA was separated by CsCl centrifugation (37). Northern blot hybridization was performed as previously described (32). Briefly, total RNA was electrophoresed through a 1.5% agarose, 6% (v/v) formaldehyde gel, and blotted onto nylon membranes. Those membranes were exposed to ultraviolet light for 7 min and then hybridized with a 32P-1abe1ed cDNA probe according to the method of Rigby et al. (38).

Production of Genomic Libraries and Isolation of OP-associated Clones-The bacteriophage EMBLS murine liver library was produced as described previously (29, 39). Briefly, genomic DNA prepared from murine liver was partially digested with MboI, followed by size fractionation via sucrose gradient ultracentrifugation. The DNA fragments in the size range of 12 to 20 kb were inserted into the BamHI site of EMBLS arms. The annealed DNA was packaged by using a commercial packaging extract (Strategene Cloning Sys- tems, La Jolla, CA), and the phage was grown in Escherichia coli Q359 and screened as described. Radioactive probes were prepared by “‘P labeling of cDNA inserts from an MS21 clone (31) using nick translation (38), with a specific activity of 10s cpm/wg of DNA. Filters containing recombinant-plaques were-screened according to the pro- cedure of Benton and Davis (40). nrehvbridized at 68 “C in 6 X SSC (1 X SSC = 0.15 M NaCl, ‘15”mM sodium citrate, pH 7.0), 5 X Denhardt’s solution for 4 h, and hybridized with lo6 cpm/ml 32P- labeled insert at 68 “C for 20 h. The filters were exposed to Kodak XAR-5 film with a Fuji intensifying screen at -80 “C for 2 days. Phage clones were subsequently purified by repeated cycles of screening. Large-scale phage DNA preparations and minipreps were carried out as described-by Maniatiset al. (41).

Restriction Enzyme and Southern Blotting Analvsis-DNA digested with restriction endonucleases was subjected to electrophoresis on 1% agarose gels in TEA buffer (40 mM Tris, 1 mM EDTA, adjusted to pH 8.2 with glacial acetic acid). After electrophoresis, gels were stained with ethidium bromide and photographed, alkali-denatured, neutralized, and soaked in 6 X SSC. The DNA was transferred to nylon membranes according to Southern (42). Hybridization with “‘P-labeled DNA probes was carried out under the same conditions as the phage library screening.

Plasmid Subcloning and Sequencing-DNA fragments originating from the genomic DNA inserts of phage clones were subcloned into pUCll8. The pUCll8 vector was linearized, and ligation was carried out for 12 h at 16 “C using a ligation kit purchased from Takara. The ligated DNA was used to transform MV1184 cells. Bacteria1 colonies that were white in color were selected. Nucleotide sequences were determined by the dideoxy chain termination method (43) using single stranded plasmid DNA with modifications as described in the Se- quenase’” technical manual (Toyobo). Nucleotide sequences were determined on DNA subcloned in pUC118 vectors using pUC primers. Other oligonucleotides used as primers for sequencingwere-synthe- sized by an automated DNA synthesizer (Biosearch mode1 8700). All DNA fragments were sequenced on both sides of the DNA, as shown in Fig. 4.

Primer Extension Analysis-The probe for primer extension analy-

sis was a synthetic 20-base single stranded oligomer (3’-CCGTCAC- TAAACGAAAACGG-5’) corresponding to base pairs +205 to +224 of the first exon of the OP gene. Total cellular RNA was isolated from aged HINS-B3 cells and murine placenta. The primer was annealed to the 100 gg of total RNA by heating the reaction mixture for 15 min at 80 “C in a 20-~1 volume containing 80% formamide, 400 mM NaCl, 40 mM Pipes (pH 6.4), and 1 mM EDTA. The resulting DNA-RNA hvbrid was ethanol precipitated and dissolved in reverse transcriptasebuffer (50 mM Tris-HCl, pH 8.0, 20 mM 2-mercaptoeth- anol. 100 mM KCl. 10 mM MaCb) in the presence of la-32PldCTP (3000 Ci/mmol, Du Pont-New England Nuclear), 1 mM deoxynucleo- tides, 100 units of reverse transcriptase, and 60 units of RNase inhibitor. After 90 min at 40 “C, the DNA-RNA hybrids were phenol extracted, ethanol precipitated, dissolved in loading buffer (95% formamide, 20 mM EDTA, 0.05% bromphenol blue, 0.05% xylene cyan01 FF), heated to 90 “C, and resolved on a 6% acrylamide, 8 M urea sequencing gel.

Sl Nuclease Mapping-S1 nuclease mapping was carried out according to the method of Berk and Sharp (44). First, 50 gg of total RNA from HINSB3 cells was hybridized with an end-labeled RsaI- MspI fragment (Fig. 7) in 40 mM Pipes (pH 6.4), 0.4 M NaCI, 1 mM EDTA. and 80% formamide for 16 h at 50 “C. Following hvbridization, the reaction was diluted lo-fold with SI nuclease buffer (300 mM NaCl, 30 mM sodium acetate, pH 4.5, and 3 mM ZnClz) and 10 pg of salmon sperm DNA. Sl nuclease (130 units) was added and the reaction mixture was incubated for 1 h at 37 “C. The reaction mixture was terminated by the addition of termination buffer (2.5 M ammo- nium acetate and 50 mM EDTA), and the DNA-RNA hybrids were extracted with phenol, precipitated with ethanol, resuspended in 80% formamide, heated to 90 “C, and resolved on an 8% acrylamide, 7 M urea sequencing gel.

RESULTS AND DISCUSSION

Expression of the OP Gene-A cDNA clone MS21 encoding mouse OP has been isolated from cDNA libraries constructed using mRNA from macrophage cell lines and macrophages (27, 31). Recent reports have also demonstrated the expression of OP in macrophages (24, 26). The expression of the OP gene in macrophage cell lines, in macrophages, and in other cells and tissues, therefore, was analyzed by Northern blot hybridization. As shown in Fig. lA, a 32P-labeled MS21 cDNA probe detected mRNA from all macrophage cell lines and resident macrophages. Expression in 5774 and HINS-B3 was high and comparable to that of MC3T3-El (Fig. 1B). L929 and kidney cells also showed significant levels of expression, as did placenta tissue. The most abundant transcripts had an approximate size of 1.5 kb, although various sizes of transcripts among cells and tissues were observed. Next, we ex- amined the effect of macrophage stimulating agents on OP gene expression. For time course studies of stimulator-induced enhancement of the OP gene expression in HINSB3 cells, the culture medium was replenished when the cells were subconfluent, and each stimulator was added. Lipopolysac- charide (1 pg/ml) induced immediate and prolonged enhancement of the OP gene expression (Figs. 1C and 2A), whereas crude lymphokines (concanavalin A-stimulated lymphocyte culture supernatant, 10%) caused delayed enhancement (Figs, 1D and 2B).

The OP amino acid sequence deduced from the nucleotide sequence predicts that OP is a secreted protein on the basis of the following: it has a hydrophobic signal peptide, is rich in Asp and Glu residues, is highly hydrophilic, and has neither a transmembrane region nor membrane anchoring structures (31). Rat OP was shown to promote the attachment of rat osteosarcoma cells via RGDS tetrapeptides (9). Recent investigations of murine OP also indicated that OP is a secreted protein (24,26). Binding to matrices or ligands is an essential function of macrophages, and movement and phagocytosis by macrophages are achieved through this function. Macro- phages produce FN (10-12) and possess FN receptors (12,45, 46). The present experiments clearly demonstrate that the

14434 Expression and Structure of the Murine Osteopontin Gene

(A) (B) kb 7 8 9 IO 11 12 13 14 2 15 16

7.5 - 4.4 -

2.4 - 1.4 -

(0 KJ)

1234567 1234567

FIG. 1. Expression of murine OP mRNA in cell lines and tissues. Total RNA (30 fig) was electrophoresed on a 1.5% formaldehyde gel, blotted onto a nylon membrane, and hybridized with a “P-labeled MS21 cDNA probe. Lanes contained RNA from the following sources: A, 1, early HINS-B3; 2, aged HINS-B3; 3, early CANS-20; 4, aged CANS-20; 5, RAW264; 6, 5774; 7, NSl; 8, EL4; 9, P815; IO, L929; 11, mouse peritoneal macrophages; 12, mouse liver; 13, mouse brain; 14, mouse kidney. B, 2, aged HINS-B3; 15, MC3T3- El; 16, mouse placenta. C, lanes contained RNA extracted from HINS-B3 stimulated with lipopolysaccharide (1 pg/ml) for the indicated times: 1, 0 min; 2, 30 min; 3, 90 min; 4, 3 h; 5, 6 h; 6, 12 h; 7, 24 h. D, lanes contained RNA extracted from HINS-B3 stimulated with lymphokines (10%) for the indicated times; 1, 0 min; 2, 30 min; 3, 3 h; 4, 12 h; 5, 24 h; 6, 48 h; 7, 72 h. The lower parts of C and D show the same blots rehybridized with a cDNA probe for &a&in to demonstrate approximately equal amounts of RNA in each line.

I (1 91 17h -rgl. Ah

FIG. 2. Densitometry profiles of expression of murine OP mRNA in HINS-B3 cells after stimulation with lipopolysaccharide or lymphokines. A, lipopolysaccharide; B, lymphokines.

murine OP gene is expressed in macrophage cell lines and macrophages, as well as in an osteogenic cell line, kidney, and placenta. Expression of the OP gene in resident peritoneal macrophages suggests that OP plays a role in the intrinsic properties of macrophages. In addition, expression of the OP gene is significantly enhanced by macrophage stimulators, suggesting that OP may also be involved in host defense mechanisms.

FN fragments have been reported to mediate chemotactic responses of macrophages, and the interaction of macrophages with FN enhances the phagocytosis of opsonized particles by

these cells (47). FN is a macromolecule consisting of two disulfide-linked subunits, each having a molecular mass of approximately 230 kDa, and contains GRGDS and binding sites for a number of proteins and ligands, including collagen, fibrin, and heparin (2). On the other hand, OP is a small protein (-69 kDa) when compared to FN. This may enable OP to play different roles in macrophage-matrix interactions. For example, OP may modulate cell-matrix interactions by binding to FN, since the nonphosphorylated form of OP has been shown to form complexes with FN (26).

Mouse OP Gene Isolation and Characterization-Genomic DNA from HINS-B3, NSl, and MC3T3-El cells containing MS21 cDNA were digested with PstI, X6a1, or BamHI and subjected to Southern blot analysis using a 32P-labeled MS21 cDNA probe. As shown in Fig. 3, no differences were detected among the DNA samples extracted from different cell types, suggesting that the murine OP gene is most likely a single copy gene. To obtain murine OP genomic clones, 32P-labeled MS21 cDNA (31) was used to screen a murine liver genomic library. Two positive clones were identified, one of which, 12G26, was subjected to restriction mapping and successive hybridization with probes from the 5’ and 3’ regions of the cDNA. This analysis revealed that the clone contained the entire 4.8-kb region of the murine OP gene in addition to the 4.0-kb 5’-flanking and 9.4-kb 3’-flanking sequences (Fig. 4). The restriction enzyme map of the mouse OP gene region was in accordance with the results of the Southern blot analysis (Fig. 3).

The nucleotide sequence of a portion of the insert from 12G26 was determined by the sequencing strategy as shown in Fig. 4. The nucleotide sequence of the entire gene and approximately 0.17 kb of the 3’-flanking region of the mouse OP gene are shown in Fig. 5. The gene spanning 4866 bp is split into 6 exons by 5 introns. The exons were determined by colinearity of the genomic sequences with the nearly full length sequence of mouse OP cDNA clone MS21 (Fig. 5). The sites of both exons and introns vary considerably: exons range between 36 and 821 bp, and introns range between 98 and 1075 bp in length. All the mRNA splicing takes place at both ends of the codons. The first exon is 250 bp long and has an untranslated region of 196 bp. A single ATG initiation codon is present in exon 1; the previously described murine osteopontin-like cDNA clone begins at position +16 with respect

(A) (B) (C) ka 1 2 3 1 2 3 1 2 3

21.2-/ I ,I

2.3- 2.0-

FIG. 3. Southern blot analysis of the murine OP gene. Ge- nomic DNA samples were extracted from cell lines, digested with PstI (A), XbaI (B), and BamHI (C), electrophoresed on 1% agarose gels, and blotted onto nylon membranes. The membranes were hybridized with a “P-labeled MS21 probe. Lanes contained restriction enzyme- digested DNA from the following sources: I, NSl; 2, HINS-B3; 3, MC3T3-El.


11 sx E XP x 5 X P H

12G26 IV 1 II I I I 1 I

FIG. 4. Schematic diagram of the murine OP gene and mRNA. Exons are boxed. Filled boxes are coding regions. Open boxes are untranslated regions. Ab- breviations for restriction sites: B, BarnHI; S, SacI; X, XbaI; E, EcoRI; P, &I; H, HindIII. Arrows indicate the direction and extent of sequencing. Only sequenced regions are shown.

----A---e-

- - - - - - - - - -

- - - - - - - - -

- __ - - - - - - - - -

FIG. 5. Sequence of the murine OP gene. The numbering indicates the nucleotide position relative to the ultimate 5’ transcriptional site which is designated +l. The amino acid sequence is shown under the nucleotide sequence in a single letter code. Amino acid numbers are given below the amino acid sequence; +l was assigned to the N-terminal Asp residue of the mature protein; amino acids of the signal peptide were given negative numbers. Exons are indicated on the right margin. Asterisks indicate stop codon. Putative polyadenylation signals are underlined.

to the transcription initiation site (31). The first exon encodes sequence and does not permit such insertions. The colinearity the full length of the signal peptide. Exon 5 encodes regions of genomic and cDNA sequences ends with the poly(A) point of the 10 consecutive aspartic amino acids and Gly-Arg-Gly- at nucleotide +4866. The nucleotide sequences of exons reveal Asp-Ser peptides. Exon 6 is the largest exon and encodes the a few differences between our murine OP gene and the MS21 C-terminal half of OP and contains a 3’ noncoding region of cDNA sequences in 3’-UT (31) (Table I). Allelic polymor- 434 bp. A comparison of the nucleotide sequences between phism in the murine OP gene has been suggested (24, 32), rat and murine OP cDNA shows that the sequences have 89% which may be due to the genomic sequence originating from homology at the nucleotide level, and 86% homology at the the BALB/c library, whereas MS21 cDNA is from ICR mice. amino acid level, but the rat OP sequence has 15- and 54-bp A comparison with the cDNA sequence reported previously insertions at positions 4241 and 4280, respectively. The nu- (24, 25) reveals a number of differences. The OP cDNAs cleotide sequence of exon 6 clearly demonstrates that the reported by other groups were isolated from cDNA libraries coding sequence completely matches the murine OP cDNA using mRNA from cell lines of BALB/c origin (24,25, 48,49)


and differences are found between these cDNA sequences (Table I). Such differences may be attributable to artifacts that could have been responsible for sequencing errors. Fur- thermore, 53 nucleotides of the most 5’-UT reported by Craig et al. (25) are completely different from those reported by Miyazaki et al. (31) and Patarca et al. (24).

All of the intron donor and acceptor sites conform to the consensus rules (24, 25, 31, 50). The noncoding region contains two potential polyadenylation signals (31) starting at nucleotides +4706 and +4846 (Fig. 4). The poly(A) point of murine OP cDNA is located 16 bp downstream of the end of the second of these polyadenylation signals (position +4867).

Identification of the 5’ End of OP mRNA-The longest cloned murine OP 5’-untranslated region, a length of 198 bp, was reported by Patarca et al. (24). However, no additional evidence, such as nuclease mapping, was presented showing this to be the full length of the 5’-untranslated region. To

TABLE I Differences in nucleotide sequence between the previously published

murine osteopontin cDNA and that presented in Fig. 5 Blanks indicate sequences not available. Hyphens indicate absence

of the corresponding nucleotide(s).

Nucleotide cDNA position Region Gene

(from Fig. 5) 1” 26 3

5’ Upstream Exon 1 Exon 3 Exon 5 Exon 6 Exon 6 3’-UT

-2 9-12 1497 3159 4072 4170 4501 4611 4648 4669 4702 4725 4729 4737 4759 4827 4836 4859

4862-4866

3’.UT 3’-UT 3’-UT 3’-UT 3’.UT 3’-UT 3’-UT 3’-UT 3’-UT 3’-UT 3’-UT 3’-UT

T AAGG

T T A A c c A A G G

c c A A T T C C E C T

T -

AATGT -

TTCC C T

6 A C

c A T

T C T C : A - T - T T -

AATGT

” Miyazaki et al. (31). h Patarca et al. (24). ’ Craig et al. (25).

define the transcription initiation site of the OP gene, primer extension analyses were performed, as shown in Fig. 6A. A 20-mer complementary to mRNA located near the 5’ end of the coding region was used for primer extension analysis. This oligonucleotide was annealed to 50 pg of total cellular RNA derived from HINSB3 cells and extended (see “Experimental Procedures”). The primer extension product from this analysis revealed OP messages of various lengths, ranging from 133 to 204 nucleotides. The ultimate 5’ transcriptional site was

(A) (B) TGCA123

FIG. 6. Primer extension and S 1 nuclease mapping analysis of murine OP mRNA. A, total RNA from HINSB3 or murine placenta was annealed to a 20-base oligonucleotide specific for a 5’ region of murine OP mRNA. The oligomers were extended and analyzed as described under “Experimental Procedures.” Dideoxy- nucleotide sequencing reactions were electrophoresed in parallel as markers. Lane I, HINSB3 RNA with reverse transcriptase; lane 2, placenta RNA with reverse transcriptase; lane 3, HINSB3 RNA without reverse transcriptase. Arrow indicates the ultimate 5’ transcriptional site (nucleotide +l, see Fig. 7). B, a 395-bp RsaI-MspI fragment end-labeled at the MspI site (Fig. 7) was hybridized to total RNA from HINS-B3. After digestion of single stranded RNA with Sl nuclease, the resultant protected fragments were analyzed by electrophoresis. Dideoxynucleotide sequencing reactions were electrophoresed in parallel as markers. Lane 1, products of Sl nuclease digestion in the presence of total RNA from HINS-B3. Arrow indicates the site corresponding to that of the most predominant band in primer extension (nucleotide +56, see Fig. 7).

FIG. 7. Nucleotide sequence of the 5’ region of the murine OP gene. Nucleotides are numbered relative to the ultimate 5’ transcriptional site which is designated +l. The bold arrow marks the major site of transcription initiation determined on the basis of primer extension; other arrows mark minor sites of transcription initiation determined on the basis of primer extension; the filled triangle indicates the longest cloned murine OP 5’- untranslated region (24); open triangles below the sequence represent start sites determined on the basis of Sl protection; lines above the sequence indicate TATA-like sequences; consensus interferon regulatory factor-l binding sequences are underlined with double lines; double lines above the sequence indicate consensus GF-1 binding sequences; a dotted line above the sequence marks a glucocorticoid-responsive element; the inverted octamer sequence is boxed, the interferon inducible gene is underlined; CT repeats are underlined with dotted lines; ATT repeats are underlined with dashes.


the nearest to the longest cloned murine OP 5’-untranslated region and was numbered mRNA residue +l (Fig. 7). The predominant band, however, corresponded to the A residue at +56 which is the preferred initiation site of RNA polymerase II (51) and placenta RNA only produced a shorter band at +69. To confirm the start sites identified by primer extension, an Sl nuclease protection experiment was next performed. A 395-nucleotide RsaI-MspI fragment that contains the entire sequence of the first exon was end-labeled at the MspI site (Fig. 7) and hybridized with HINS-B3 RNA. Digestion with Sl nuclease, however, resulted in two protected fragments of 195 and 211 bp (Fig. 6B), the former of which corresponded to the most predominant fragment produced by primer extension. The 209-bp band had no corresponding primer extension product, and may indicate incomplete digestion of single stranded RNA by Sl nuclease or heterogeneity in transcriptional initiation. Since the longest murine OP 5’-untranslated region cloned was derived from a T-lymphocyte library (24), it is possible to speculate that the transcription initiation sites among cell types are different, and that monocytic cells mainly use the A residue at +56 as the transcription initiation site.

Identification of Potential cis-Acting Regulatory Elements- A nucleotide sequence of approximately 0.75 kb of the 5’- flanking regions of the OP gene was determined. Analysis of the DNA flanking the 5’ side of the OP gene revealed several TATAA-like elements approximately 20-40 bases upstream of each transcription start site situated in three regions, but the other common regulatory consensus element CCAAT was not observed (Fig. 7). The elements that were located are TAAAGTTT and ATGAATA sequences located upstream of the region of the ultimate 5’ transcriptional site, a TAGAAAA sequence located upstream of the predominant transcription initiation site, and AAATATT located upstream of the ultimate 3’ transcriptional site. In addition, several potential regulatory sequence elements were located upstream. Inter- feron regulatory factor-l binding sequences AAATGT, AAGTGG, and AAATGG (52-54) were shown to be located at positions -589 to -584, -570 to -565, and -207 to -202, respectively. The inverted immunoglobulin octamer enhancer sequence ATGCAAAT (52-57), which shares part of the interferon regulatory factor-l binding sequence, was located at position -211 to -204. An erythroid-specific factor (GF-1) binding motif, AGATA (58), was found at positions -512 to -508, -265 to -261, and -171 to -167. Interestingly, the AGATA at position -265 to -261 is a palindromic sequence with an inverted GF-1 binding motif TATCT at position -262 to -258. The glucocorticoid-responsive element AGAACA (59) is located at position -287 to -282. A regulatory consensus sequence for interferon inducible genes GGAAA (60) is located at position -203 to -199. Whether these elements, however, are functional remains to be determined. The 5’ upstream sequence contains ATT and CT stretches. Although such repeated stretches appear in numer- ous data base entries, their significance is unknown at present. Various short direct and inverted repeats, which may have regulatory potential, are found in 5’ upstream regions and within exon 1.

Acknowledgments-We thank Dr. Y. Amagai for providing the MC3T3-El cell line, Dr. M. Yamamoto for p-a&in cDNA, Dr. N. Nasu for his technical assistance, and T. Iwao for performing the photographic procedures.

REFERENCES

1. Ruoslahati, E., and Pierschbacher, M. D. (1987) Science 238, 491-497

2. Kornblihtt, A. R., Umezawa, K., Vibe-Pedersen, K., and Baralle, F. E. (1985) EMBO J. 4, 1755-1759

3. Pierschbacher, M. D., and Ruoslahati, E. (1984) Nature 309,30- 33

4. Hayman, E. G., Pierschbacher, M. D., Ohgren, Y ., and Ruoslahati, E. (1983) Proc. N&l. Acod. Sci. U. S. A. 80.4003-4007

5. Suzuki, S., Oldberg, A., Hayman, E. G., Pierschbacher, M. D., and Ruoslahati, E. (1985) EMBO J. 4,2519-2525

6. Dedhar, S., Ruoslahati, E., and Pierschbacher, M. D. (1987) J. CellBiol. 104, 585-593

7. Lawler, J., and Hynes, R. 0. (1986) J. Cell Biol. 103,1635-1648 8. Plow, E., Pierschbacher, M. D., Ruoslahati, E., Marguerie, G. A.,

and Ginsberg, M. H. (1985) Proc. N&l. Acad. Sei. U. S. A. 82, 8057-8061

9. Oldberg, A., Franzen, A., and Heinegard, D. (1986) hoc. N&l. Acad. Sci. U. S. A. 83, 8819-8823

10. Alitalo, K., Hovi, T., and Vaheri, A. (1980) J. Exp. Med. 151, 602-613

11. Johansson, S., Rubin, K., Hook, K., Ahlgren, T., and Seljelid, R. (1979) FEBS I&t. 105, 313-316

12. Villiger, B., Kelley, D. G., Engleman, W., Kuhn, C., III, and McDonald. J. A. (1981) J. Cell Biol. 90. 711-720

13. Holers, V. MI, Ruff,‘T. G:, Parks, D. L., McDonald, J. A., Ballard, L. L., and Brown, E. J. (1989) J. Exp. Med. 169, 1589-1605

14. Norris, D. A., Clark, R. A. F., Swigart, L. M., Huff, J. C., Weston, W. L., and Howell, S. E. (1982) J. Immunol. 129, 1612-1618

15. Bohnsack, J. F., Takahashi, T., and Brown, E. J. (1986) J. Zmmunol. 136,3793-3798

16. Pommier, C. G., Inada, S., Fries, L. F., Takahashi, T., Frank, M. M., and Brown, E. J. (1983) J. Ezp. Med. 157, 1844-1854

17. Wright,, S. D., Craigmyle, L. S., and Silverstein, S. C. (1983) J. Exp. Med. 158,1338-1343

18. Franzen, A., and Heinegard, D. (1985) Biochem. J. 232, 715-724 19. Craig, A. M., Nemir, M., Mukherjee, B. B., Chambers, A. F., and

Denhardt, D. T. (1988) Biochem. Biophys. Res. Commun. 157, 166-173

20. Mark, M. P., Prince, C. W., Gay, S., Austin, R. L., and Butler, W. T. (1988) Cell Tissue Res. 251, 23-30

21. Nomura, S., Willis, A. J., Edwards, D. R., Heath, J. K., and Hogan, B. L. M. (1988) J. Cell Biol. 106,441-450

22. Smith, J. H., and Denhardt, D. T. (1987) J. Cell. Biochem. 34, 13-22

23. Yoon, K., Buenaga, R., and Rodan, G. A. (1987) Biochem. Bio- phys. Res. Commun. 13,1129-1136

24. Patarca, R., Freeman, G. J., Singh. R. P., Wei, F.-Y., Durfee, W. T., Biattner, F., Regnier, D. ?.; Kozak, C.’ A., Mock, B.. A., Morse. H. C.. III. Jerrells. T. R.. and Cantor. H. (1989) J. EXD. Med. i70,145-i61 '

,.I ‘

25. Craig, A. M., Smith, J. H., and Denhardt, D. T. (1989) J. Biol. Chem. 264,9682-9689

26. Nemir, M., DeVouge, M. W., and Mukherjee, B. B. (1989) J. Biol. Chem. 264,18202-18208

27. Setoguchi, M., Yoshida, S., Higuchi, Y., Akizuki, S., and Yama- moto, S. (1988) Somat. Cell Mol. Genet. 14,427-438

28. Miyazaki, Y., Setoguchi, M., Higuchi, Y., Yoshida, S., Akizuki, S., and Yamamoto, S. (1989) Nucleic Acids Res. 16, 10373

29. Setoguchi, M., Higuchi, Y., Yoshida, S., Nasu, N., Miyazaki, Y., Akizuki, S., and Yamamoto, S. (1989) Mol. Cell. Biol. 9, 4515- 4522

30. Setoguchi, M., Nasu, N., Yoshida, S., Higuchi, Y., Akizuki, S., and Yamamoto, S. (1989) Biochim. BioDhvs. Acta. 1008. 213- _ . 222

31. Miyazaki, Y., Setoguchi, M., Yoshida, S., Higuchi, Y., Akizuki, S., and Yamamoto, S. (1989) Nucleic Acids Res. 17, 3298

32. Fet, V., Dickinson, M. E., and Hogan, B. L. M. (1989) Genomics 5,375-377

33. Higuchi, Y., Setoguchi, M., Yoshida, S., Akizuki, S., and Yama- moto, S. (1988) Oncogene 2, 515-521

34. Higuchi, Y., and Yamamoto, S. (1984) Immunogenetics 20, 95- 102

35. Yamamoto, S., and Higuchi, Y. (1984) Immunogenetics 19, 519- 526

36. Sudo. H., Kodama, H.-A.. Amagai. Y.. Yamamoto. S.. and Kasai. S. (1983) J. CellBiol. 9k, 191-198

,

37. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18,5294-5299

38. Rigby, P. W. J., Dieckmann, M., Rhodes, C., and Berg, P. (1977) J. Mol. Biol. 113, 237-251


39.

40. 41.

42. 43.

44.

45.

46.

47.

48.

49.

Frischauf, A.-M., Lehrach, H., Poustka, A., and Murray, N. (1983) J Mol. Biol. 1'70, 827-842

Benton, W. D., and Davis, R. W. (1977) Science 196, 180-182 Maniatis, T., Fritsch, F. F., and Sambrook, J. (1982) Molecular

Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

Southern, E. (1975) J. Mol. Biol. 98,503~517 Sanger, F., Nicklen, S., and Coulson, A. R. (1977) hoc. Natl.

Aead. Sci. U. S. A. 74,5463-5467 Berk. A. J., and Sharp, P. A. (1978) Proc. Natl. Acad. Sci. U. S.

A. +5,1274-1278 -. Bevilacqua, M. P., Amrani, D., Mosesson, M. W., and Bianco, C.

(1981) J. Exp. Med. 153,42-60 Brown, E. J., and Goodwin, J. L. (1988) J. Exp. Med. 167, 777-

793 Gudewicz, P. W., Molnar, J., Lai, M. Z., Beezhold, D. W., Siefing,

G. E.. Jr.. Credo. R. B.. and Lorand. L. (1980) J. Cell Biol. 87, 427-433 ’

Colburn, N. H., Former, B. F., Nelson, K. A., and Yuspa, S. H. (1979) Nature 281,589-591

Clayberger, C., Dekruyff, R. H., Aisenberg, J., and Cantor, H.

(1983) J. Enp. Med. 157,1906-1919 50. Breathnach, R., Benoist, C., O’Hare, K., Gannon, F., and Cham-

bon, P. (1978) Proc. Natl. Acnd. Sci. U. S. A. 75, 4853-4857 51. Baker. C. C.. and Ziff. E. B. (1981) J. Mol. Biol. 149.189-221 52. Fujita: T., Ohno, S., Yasumitsu, Hi, and Taniguchi, T: (1985) Cell

41,489-496 53. Ryals, J., Dierks, P., Ragg, H., and Waissmann, C. (1985) Cell

41,497-507 54. Cohen, B., Peretz, D., Vaiman, D., Benech, P., and Chebath, J.

(1988) EMBO J. 7, 1411-1419 55. Falkner, F. G., Mocikat, R., and Zachau, H. (1986) Nucleic Acids

Res. 14,8819-8827 56. Falkner. F. G.. and Zachau, H. G. (1984) Nature 310, 71-74 57. Palslow; T. G.: Blair, D. L., Murphy, W. J., and Granner, D. K.

(1984) hoc. Natl. Acad. Sci. I/. S. A. 81, 2650-2654 58. Martin,’ D. I. K., Tsai, S.-F., and Orkin, S. M. (1989) Nature

338,435-438 59. Jantzen, H.-M., Strahle, U., Gloss, B., Stewart, F., Schmid, W.,

Boshart, M., Miksicek, R., and Schutz, G. (1987) Cell 49, 29- 38

60. Porter, A. C. G., Chernajovsky, Y., Dale, T., Gilbert, C. S., Stark, G. R., and Kerr, I. M. (1988) EMBO J. 7,85-92

Documents

The Mouse Osteopontin Gene › content › 265 › 24 › 14432.full.pdfVol. 265, No. 24, Issue of August 25, pp. 14432-14438.1990 Printed in U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY