BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 238, 528–533 (1997)
ARTICLE NO. RC977188

Bone Mass Loss Due to Estrogen Deficiency
Is Compensated in Transgenic Mice Overexpressing
Human Osteoblast Stimulating Factor-11
Haruchika Masuda,*,† Atsushi Tsujimura,* Makoto Yoshioka,‡ Yuji Arai,§ Yoshinori Kuboki,ØTsunehiro Mukai,§ Toshitaka Nakamura,\ Hajime Tsuji,†
Masao Nakagawa,† and Tamotsu Hashimoto-Gotoh*,2*Department of Biochemistry and Molecular Genetics, Research Institute for Neurological Diseases and Geriatrics, and†Second Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kamigyo-Ku, Kyoto 602,
Japan; ‡Department of Orthopedic Surgery, Matsushita Memorial Hospital, Moriguchi 570, Japan;§National Institute of Circulovascular Diseases, Suita 565, Japan; ØDepartment of Biochemistry,
School of Dentistry, Hokkaido University, Sapporo 060, Japan; and \Department of Orthopedics,
University of Occupational and Environmental Health, Hitakyushu 807, Japan

Received July 22, 1997

Osteoblast stimulating factor-1 (OSF-1) stimulates invitro proliferation and differentiation of osteoblastic
cells, and its gene is expressed in the bone and brain
tissues in mammals and amphibians. To evaluate
the in vivo function of OSF-1 in bone metabolism,
transgenic mice overexpressing the human osf-1 gene
driven by the osteocalcin promoter were generated.
Femoral bone mineral content was increased intransgenic mice relative to wild-type controls as estimated by ash assay, depending on the transgene copy
number per cell. In ovariectomized mice, bone massloss due to estrogen deficiency was observed in bothtransgenic and control mice but bone mass was still
higher in transgenic mice than in controls. Bone massin ovariectomized transgenic mice was comparable tothat in wild-type mice without ovariectomy. These observations indicate that OSF-1 may direct in vivo appositional bone formation by increasing osteoblast activity rather than decreasing osteoclast activity, suggesting a new way to treat osteoporosis with OSF-1.
q 1997 Academic Press

Bone is a complex tissue composed of cells, collagenous matrix and inorganic elements, mainly calcium,
1
The nucleotide sequence of the HPoc-Hosf-1 transgene will appear in the DDBJ databases under Accession No. AB004306.
2
To whom correspondence should be addressed at the ResearchInstitute for Neurological Diseases and Geriatrics, Department ofBiochemistry and Molecular Genetics, Kyoto Prefectural Universityof Medicine, Kamigyo-Ku, Kyoto 602, Japan. Fax: (81-75)-251-5799.
E-mail: e54093@sakura.kudpc.kyoto-u.ac.jp.
Abbreviations: DXA, dual energy X-ray absorptiometry; OSF-1,
osteoblast stimulating factor-1; VD3 , vitamin D3 .

0006-291X/97 $25.00

and is continuously remodeled during adulthood
through the resorption of old bone by osteoclast cells
and the subsequent formation of new bone by osteoblast cells [1]. cDNA clones encoding osteoblast stimulating factor-1 (OSF-1, formally called osteoblast specific factor-1 [2], and also named HB-GAM [3], PTN[4] and HBNF [5]) have been isolated from mouse andhuman osteoblastic cells on the assumption that atleast some proteins involved in growth and differentiation of bone tissues may be produced specifically inosteoblasts. OSF-1, a heparin binding Lys-rich 18 kDa
protein, purified from bovine bone powder has beenshown to stimulate both proliferation and differentiation of mouse osteoblastic cells in vitro without affecting cell shape unlike 1,25-(OH)2 vitamin D3 (VD3) or
TGF-b1 [6]. The transcription of osf-1 was not affected
by VD3 but was markedly suppressed by TGF-b1 in
cultured mouse osteoblastic cells [7]. Developmental
and differential expression of the amphibian X-ptf-a
and X-ptf-b corresponding to mammalian mk [8] andosf-1, respectively, all of which belong to the same family, have been studied in Xenopus laevis [9]. AlthoughX-ptf-a transcripts exist as maternal mRNA in oocytes
and are widely expressed throughout development invarious embryonic tissues, X-ptf-b transcripts become
evident only at the later stages of larval development
and are spatially restricted to the region from the hindbrain along with the spinal cord in tailbud embryos.
To elucidate the in vivo function of OSF-1 in mammalian bone metabolism, we generated transgenic miceexpressing the human osf-1 gene under the control of
the human osteocalcin promoter. Analyses of the bonemass evaluated by bone mineral content using ash

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assay with femora and by bone mineral density using
dual energy X-ray absorptiometry (DXA) with vertebrae indicate that (i) bone mass was increased in femora of transgenic mice relative to negative controls depending on transgene copy number per cell, (ii) although bone mass loss due to estrogen deficiency was
found in both transgenic and negative control mice, thebone mass increase was more evident in transgenic
mice relative to negative controls when ovariectomized,and (iii) bone mass in ovariectomized transgenic micewas comparable to that in wild-type mice without
ovariectomy. These results suggest that OSF-1 may
play a role in vivo in stimulating osteoblasts resulting
in a bone mass increase without affecting osteoclast
activity unlike parathyroid hormone, interleukin-1 andtumor necrosis factor (TNF) [10].
MATERIALS AND METHODSTransgenic mice. Construction strategy and structure of thetransgenic human (H-) osf-1 gene are schematically shown in Fig.
1A and B, respectively. The HPoc-Hosf-1 transgene DNA fragment
was excised by EcoRI and HindIII digestion, purified and microinjected into fertilized eggs of BDF1 mice essentially as described [15].
The eggs were transferred into the oviducts of pseudopregnant ICR
female mice and the presence of the transgene in mouse chromosomes was confirmed by tail-tip Southern blotting using Probe I (Fig.
1B). Mice were kept at room temperature and had free access to a
standard diet, MF (Oriental Yeast Co. Ltd., Tokyo, Japan), containing 0.88 % phosphorus, 1.15 % calcium and 1.08 IU/g VD3 . The
mice used for mRNA analysis and bone mass evaluation were all
female. Ovariectomy or sham-operation was carried out at the ageof 14 weeks by the dorsal incision method under anesthesia inducedby the intrapenitoneal injection of pentobarbital sodium salt, andthe mice were allowed to survive for further 14 weeks.
Northern blotting analysis. Total RNA from the whole brain, calvaria, liver, muscle, spleen, kidney of 8- or 28-week-old mice wasisolated by the acid guanidinium isothiocyanate-phenol extraction
method [16], electrophoresed, transferred onto nylon membranes andhybridized with radiolabeled Probe II (Fig. 1B) as described [17].
Bone mass evaluation. Bone mineral content and density were
evaluated by ash assay and DXA using femoral and vertebral bones,
respectively. For ash assay, homozygous (hosf 6/hosf 6) and heterozygous (hosf 6/hosf 0) transgenic and wild-type (hosf 0/hosf 0) mice were
produced twice by independent crossing, where hosf 6 and osf 0 indicate the existance of 6 and 0 copies of human osf-1 transgene per
haploid genome, respectively. Bilateral femora were excised and soft
tissues including muscles surrounding the bones were completely
removed. After determining the wet weight of femora, the samples
were dried at 1007C for 48 hrs, treated with ethanol/ether (2:1) solution to remove fat, and ashed in a muffle (FM37, Yamato Co., Japan)
at 6507C for 12 hrs. The ashed samples were weighed to determinebone mineral weight. Bone mineral content was defined as percentage of ratio of bone mineral weight to wet weight. For DXA using
vertebrae, soft tissues and spinal nerves were removed and lumber
spines (L2-L4) were resected off as a block, of which bone mineraldensity expressed by mg/cm2 was determined using a DCS-600R instrument (Aloka Co., Japan) and bone radiodensity was photographed with soft X-rays using FR film (Fuji Co., Japan) at 30 V, 2
mA and 60 sec. The vertebral images were enhanced using an Epscan
1.40 (Epson Co, Japan) and Adobe Photoshop 2.5J (Adobe Systems
Incorporated, USA.) under the same conditions. The final pictures
were taken by Color Fast (GCC Technologies, USA). The precisionand stability of the measurements were evaluated periodically andthe coefficient of variation was õ 0.5%. The operators were unawareof sample identities during manipulation.

RESULTS
Generation of human osf-1 (H-osf-1) transgenic mice.
Three founder mice (F0) harboring the H-osf-1
transgene were identified by Southern blotting analysis of tail DNA samples and the F1 heterozygotes wereanalyzed for mRNA at 8 weeks of age (Fig. 1C). In twoof the three lines, the transgene mRNA was detected
specifically in the calvaria, whereas endogenous mouse
osf-1 transcripts were detected in the brain and calvaria but not in other tissues examined (data notshown). One of the two lines, HPoc-Hosf1-6, carrying
6 transgene copies per haploid cell, was used for further
studies. No significant differences were found in external appearance, body weight or behavior between thetransgenic and wild-type BDF1 mice. No apparent
pathological conditions such as osteosarcoma and osteopetrosis were observed by X-ray photography intransgenic mice irrespective of homozygosity (hosf 6/
hosf 6) or heterozygosity (hosf 6/hosf 0) for the transgene(data not shown).
Ash assay using femora. Transgenic littermateswith hosf 6/hosf 6 and hosf 6/hosf 0 genotypes and wildtype BDF1 mice, all of which were bred under the same
conditions from parental mating to sacrifice at 30
weeks of age, were examined to determine their bonemineral content by ash assay using bilateral femora as
described in Materials and Methods. Significant bonemineral increases were found in hosf 6/hosf 6 mice relative to hosf 6/hosf 0 littermates and wild-type mice(põ0.05), but no significant bone mass difference wasfound between hosf 6/hosf 0 and wild-type mice (pú0.05)(Fig. 2A). Subsequently, the possible influence of estrogen deficiency was examined on the bone mineral increase in transgenic mice with or without ovariectomy.
Homozygous transgenic and wild-type mice were ovariectomized or not operated at 14 weeks of age and sacrificed at 28 weeks of age. As shown in Fig. 2B, femoral
bone mineral content was decreased by ovariectomy
in both transgenic and wild-type mice, but was again
higher in transgenic mice than in wild-type controls
irrespective of ovariectomy or non-ovariectomy (Fig.
2B). In the ovariectomized transgenic mice, bone mineral content was comparable to that in wild-type mice
without ovariectomy.
Dual energy X-ray absorptiometry (DXA) using vertebrae. To shuffle possible unknown gene(s) which might
have been responsible for the bone mass increase in thetransgenic mice, hosf 6/hosf 6 females were back-crossed towild-type BDF1 males. The heterozygous offspring werepaired and hosf 6/hosf 6, hosf 6/hosf 0 and hosf 0/hosf 0 pups
were produced. As shown in Fig. 3, bone mass increase intransgenic mice was evident, depending on the transgene

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FIG. 1. Construction strategy and structure of the H-osf-1 transgene and mRNA expression in transgenic mice. (A) The coding regionfor H-osf-1 cDNA cloned between EcoRI and BamHI sites on pKF4 [11], pHosf1, was excised, blunt-ended and recloned at blunt-ended
EcoRI site flanked by the 5*- and 3*-untranslated regions (UTRs) of the rabbit b-globin gene on pLG1 [12], pLGHosf1. Then, the BamHI
and XhoI fragment containing the H-osf-1 coding region flanked by the rabbit 5*- and 3*-UTRs was excised and placed under the controlof the human osteocalcin promoter (H-Poc) [13] between BamHI and SalI sites on pKF18k [14], pHPoc-Hosf1. Shadowed and open boxes
represent H-Poc promoter and H-osf-1 coding region, respectively. Arrows inside plasmid circles indicate transcriptional direction in H-Poc
and H-osf-1. R-bg5* and R-bg3* indicate the 5*- and 3*-UTRs. (B) The H-osf-1 transgene structure is shown schematically with representative
restriction sites. The small hatched box in the H-Poc promoter indicates the approximate position of the vitamin D responsive element(VDRE). The horizontal arrows labeled Probe I and Probe II indicate approximate extents of probe sequences (2.6 kbp and 570 bp) used
for Southern and Northern hybridization experiments, respectively. The vertical arrow marked with polyA indicates the approximate
position of the polyA signal sequence. Other symbols are as in (A). (C) Northern blotting analysis of total RNA isolated from the brain andcalvaria of a F1 heterozygote. Total RNAs (10 mg/lane) isolated from mouse brain and calvaria at 8 weeks of age were electrophoresed andhybridized with probe II as described in Materials and Methods. ‘‘Wt’’ and ‘‘He’’ indicate wild-type and transgenic heterozygote, respectively.

copy number per cell, in cases with ovariectomy. In shamoperated mice, consistent results with the data by ash
assay in non-ovariectomized mice were obtained, although the bone mass increase in homozygous mice wasless significant in vertebrae (Fig. 3). The bone mass difference in ovariectomized mice was consistently demonstrated by X-ray radiography using vertebrae, wherebone density increases were visible in ovariectomized
transgenic mice (Fig. 4).
DISCUSSION
A number of factors are known to be involved in development of osteoclasts, including interleukin-1, interleu-

kin-3, interleukin-6, interleukin-11, tumor necrosis factor(TNF), granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, leukemia inhibitory factor, stem-cell factor [1]. Including inhibitors
against such factors, substances that decrease or suppress osteoclast activity may contribute to retention ofhigher bone mass in vivo. In fact, anti-osteoporosis reagents currently in clinical use are all included in thiscategory. Recently, Simonet et al. [18] and Ammann etal. [19] reported bone mass increases in transgenic mice
overproducing authentic and artificially manipulated soluble receptors, respectively, belonging to the TNF receptor family. In the former study, bone mineral increase or

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FIG. 1—Continued

osteopetrosis was observed without ovariectomy, whereas
in the latter no bone mineral increase was observed before ovariectomy. They both found that bone mass wasincreased dependent on the soluble TNF receptors intheir animals even after ovariectomy. Their results may
be explained in terms of suppression of osteoclast development through TNFa inactivation by the soluble receptors. On the other hand, few factors, if any, involved inosteoblast development are known thus far. Bone mass
increase observed in our transgenic mice relative to negative controls without ovariectomy was not as much asthat in osteopetrosis observed by Simonet et al. [18]. The
increase was significantly detected without ovariectomy
only when evaluated by ash assay using femora but not
by DXA or X-ray radiodensity. With ovariectomy, however, bone mass increase in transgenic mice was evident
by either evaluation using femora or vertebrae (Figs. 2B,
3 and 4). It should be stressed that bone mass loss due
to estrogen deficiency was detected in our transgenic mice(Figs. 2B and 3) in contrast to their reports describedabove. This suggests that the increase of osteoclast activity after ovariectomy was undergoing in our mice but

appositional bone formation may still progress due to thetransgene overexpression as to keep a high bone mass in
our transgenic mice relative to that in wild type mice
after ovariectomy. Consequently, bone mass in the ovariectomized transgenic mice was comparable to that in negative control mice without ovariectomy. Thus, we concluded that the H-osf-1 transgene could compensate forbone mass loss due to osteoclast activation by estrogen
deficiency. In fact, we observed H-osf-1 expression in addition to that of mouse endogenous osf-1 in bone tissues,
including vertebrae, femora and calvaria, of ovariectomized transgenic mice at the time of sacrifice by Northernblotting analysis (data not shown). The role of OSF-1 inbone formation should be different from that of TGFb related proteins such as bone morphogenetic proteins(BMPs), because the latter proteins trigger endochondralbone formation or ectopic bone morphogenesis [20] andfunction primarily at the early stages of embryonic development in regulation of dorsoventral pattern formation
not only in vertebrates [21] but also in invertebrates [22].
It may be noteworthy to point out that Xenopus osf-1
homologue (X-ptf-b) transcripts, are not present in unfer-

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suppresses the gene expression of the latter [7]. In thiscontext, it is of interest to point out that osteoblast-specific overexpression of TGF-b2 in transgenic mice resulted
in progressive bone mass loss resembling human osteoporosis [23]. In addition, OSF-1 did not exhibit in vivo activity for ectopic bone formation like BMPs (data not shown).
OSF-1 could be one of the bone mass determinants which
positively controls appositional bone formation occurringat bone remodeling sites through osteoblast activation

FIG. 2. Bone mineral content (BMC) in femora of H-osf-1 transgenic
mice. BMC values were evaluated by ash assay as described in Materials and Methods. Means of groups were compared by ANOVA andsignificance of differences was determined by post hoc test using Scheffe’s method. (A) and (B) are independent experiments. (A) Mean values{SEM (%) are 39.534{3.965, 40.497{2.694 and 43.049{1.481 for
Ne, He and Ho, respectively, where nÅ5-9. ‘‘Ne’’ indicates wild-type
negative control mice. ‘‘He’’ and ‘‘Ho’’ indicate heterozygous and homozygous transgenic littermates, respectively. (B) Means values{SEM (%)
are 31.530{0.523, 35.274{1.236, 28.255{0.177 and 30.550{0.868 for
Ne/non-OVX, Ho/non-OVX, Ne/OVX and Ho/OVX, respectively, wherenÅ2-4. ‘‘Ne’’ and ‘‘Ho’’ are as in (A). ‘‘OVX’’ and ‘‘non-OVX’’ indicate
ovariectomy and no operation, respectively.

tilized eggs and in the early stages of larval development[9]. Furthermore, TGF-b and OSF-1 should function in
opposite directions in bone metabolism since the former

FIG. 3. Bone mineral density (BMD) in vertebrae oftransgenic and negative littermates. The transgenic heterozygous
offspring were paired separately and their pups from each hosf 6/
hosf 6, hosf 6/hosf 0 or hosf 0/hosf 0 genotype were taken independently and divided into the 3 groups of 8 pups. In each group,
pups were divided into two subgroups for ovariectomy and shamoperation, which were carried out at 14 weeks of age. The animals
were sacrificed for BMD evaluation by DXA using vertebral bones(L2-L4) at 28 weeks of age as described in Materials and Methods.
Means of groups were compared by ANOVA and significance ofdifferences was determined by post hoc test using Bonferroni’s
method. Mean values{SEM of BMD (mg/cm2) are 34.875{1.303,
29.800{1.507, 35.450{0.417, 30.150{0.659, 37.775{1.436 and35.375{1.108 for Ne/Sham, Ne/OVX, He/Sham, He/OVX, Ho/
Sham and Ho/OVX, respectively, where nÅ4. ‘‘He’’, ‘‘Ho’’, ‘‘Ne’’
indicate heterozygous (hosf 6/hosf 0) and homozygous (hosf 6/hosf 6)transgenic and negative (hosf 0/hosf 0) littermates, respectively.
‘‘OVX’’ and ‘‘Sham’’ are as in Fig. 2B.

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FIG. 4. Vertebral bone radiodensity of the sham-operated orovariectomized heterozygous and homozygous transgenic and negative littermates. The vertebrae (L2-L4) of ovariectomized or shamoperated littermates were photographed by soft X-rays as describedin Materials and Methods. ‘‘Sham’’, ‘‘OVX’’, ‘‘Ne’’, ‘‘He‘‘ and ‘‘Ho’’ are
as in Fig. 3.

but not osteoclast suppression [24]. OSF-1 may be usefulto stimulate isotopic bone formation in treatment of postmenopausal osteoporosis in addition to use of osteoclast
suppresser-type substances.
ACKNOWLEDGMENTS
We thank Messrs N. Matsumoto and T. Aramaki for their support
to RINDG, Profs. K. Kuriyama, A. Iwashima and J. Imanishi for
their encouragement during this work, Dr. J. Miyazaki for providing
us with pLG1, Dr. T. Kobayashi for X-ray photography, Dr. M. Tamura for DXA. This work was supported by grants from Kyoto Prefectural Government and the Japan Osteoporosis Foundation.

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