Full text of DOI:10.1359/jbmr.2002.17.1.111

JOURNAL OF BONE AND MINERAL RESEARCH
Volume 17, Number 1, 2002
© 2002 American Society for Bone and Mineral Research
Identification of the Functional Domain of Osteoclast
Inhibitory Peptide-1/hSca
MASANORI KOIDE, NORIYOSHI KURIHARA, HIDEFUMI MAEDA, and SAKAMURI V. REDDY
ABSTRACT
Osteoclast (OCL) activity is controlled by local factors produced in the bone microenvironment. We previ-
ously identified a novel inhibitor of OCL formation that is produced by OCLs (osteoclast inhibitory peptide-
1/human Sca [OIP-1/hSca]). OIP-1/hSca is a glycosylphosphatidylinositol (GPI)-linked membrane protein (16
kDa) that is cleaved from the OCL surface. Immunocytochemical staining further confirmed the expression
of OIP-1/hSca in OCL formed in mouse bone marrow cultures. However, the structure/function mechanisms
responsible for the inhibitory effects of OIP-1/hSca on OCL formation are unknown. Therefore, we expressed
deletion mutants of OIP-1 in 293 cells and tested their effects on OCL formation. These studies indicated that
the carboxy-terminal peptide (c-peptide) region is critical for OIP-1/hSca activity. A 33 amino acid OIP-1
c-peptide (10–100 ng/ml) significantly inhibited 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced OCL for-
mation and pit formation capacity of OCL on dentine slices in human bone marrow cultures. Furthermore,
the c-peptide (10–100 ng/ml) significantly inhibited early human OCL precursor (granulocyte-macrophage
colony-forming unit [GM-CFU]) colony formation in methylcellulose cultures. The polyclonal antibody
against the OIP-1 c-peptide neutralized the inhibitory effect of OIP-1 c-peptide on OCL formation in mouse
bone marrow cultures in vitro. These results show that the OIP-1 c-peptide is the functional domain of OIP-1
and that availability of neutralizing antibody specific to the OIP-1 c-peptide should provide important
mechanistic insights into OIP-1/hSca inhibition of osteoclastogenesis in the bone microenvironment. (J Bone
Miner Res 2002;17:111–118)
Key words: osteoclast inhibitory peptide-1/human Sca, osteoclast, c-peptide, inhibitors, granulocyte-
macrophage colony-forming unit
thymocytes and thymic epithelial cells.(4,5) OIP-1/hSca is a
glycophosphatidylinositol (GPI)-linked membrane protein (16
INTRODUCTION
kDa) containing a 79 amino acid extracellular peptide and a 32
amino acid carboxy-terminal GPI-linked peptide (c-peptide).
Previously, we have shown that OIP-1/hSca messenger RNA
(mRNA) is highly expressed in osteoblastic cells, OCLs, liver,
and bone marrow cells. High levels of Sca-2 expression in
murine bone marrow cells and spleen also were reported.(6)
STEOCLASTS (OCLS) are primary bone-resorbing cells
that produce factors that stimulate or inhibit OCL for-
O
mation and activity.(1,2) By screening an OCL-like cell
complementary DNA (cDNA) expression library, we re-
cently have cloned and identified OC inhibitory peptide
1/human stem cell antigen (OIP-1/hSca) as a novel inhibitor
of OCL formation and bone resorption.(3) OIP-1/hSca also Recently, it also was shown that Sca-2 is expressed in human
is termed retinoic acid–induced gene expression (RIG-E) or lymphoid tissues as well as various nonlymphoid tissues.(7)
human thymic shared antigen-1 (TSA-1)/Sca-2 and is an
We also have shown that OIP-1/hSca is cleavable from
Ly-6–related differentiation antigen expressed on immature the OCL surface and inhibits the OCL formation.(3) It is
Department of Medicine/Hematology, University of Texas Health Science Center, San Antonio, Texas, USA.
111

112
KOIDE ET AL.
well known that the GPI-anchored proteins are released products were subcloned into pcDNA3.1/V5/His-TOPO
easily from the cell surface by phosphatidylinositol-specific vector in frame with the V5 epitope(14) tag following the
phospholipase C purified from bacteria. In mammals, the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA).
phospholipase D cleaves the inositol phosphate linkage of The resulting plasmid constructs OIP-1CDS5–3 native and
GPI-anchored proteins.(8) Sca-2 is a useful marker in early OIP-1(-GPI) 5–3 deletion mutant were expressed transiently
T cell development and T cell activation and seems to play in 293 cells by calcium phosphate method (Stratagene).
a regulatory role in thymocyte differentiation.(9) However, Serum-free conditioned media collected from these cells
the functions of Sca-2 remain largely obscure. Previously, it were used to assay for its capacity to inhibit OCL formation
has been shown that Sca-2 functions as a modulator of a T in murine marrow cultures in vitro as described previously.(16)
cell receptor signaling pathway.(10) An anti–Sca-2 monoclo-
nal antibody (MAb) inhibited tyrosine phosphorylation of
Western blot analysis
CD3 ␨-chains and interleukin-2 (IL-2) production induced
by anti-CD3 stimulation in T cell hybridomas,(11) suggest-
Two hundred ninety-three cells transiently transfected
ing that a signal via Sca-2 regulates early and late events in with OIP-1CDS5–3 native and OIP-1(-GPI) 5–3 deletion
T cell receptor signaling. Furthermore, in vivo injection of mutant plasmid constructs as described previously were
Sca-2 MAb completely blocked anti–CD3-mediated apo- lysed in a buffer containing 20 mM of Tris, pH 7.4, 150 mM
ptosis of thymocytes.(12) However, because Sca-2 is a GPI- of NaCl, 1% Triton X-100, 10% glycerol, 1.5 mM of
anchored membrane protein and thus does not have trans- MgCl2, 1mM of EGTA, 200 ␮M of sodium vanadate, 1 mM
membrane and cytoplasmic regions, it is not known how of phenylmethylsulfonyl fluoride (PMSF), and 1 mg/ml of
Sca-2 transmits signals into the cytoplasm of the cell. Re- aprotinin. Then, samples were subjected to sodium dodecyl
cently, it has been reported that Sca-2 is associated physi- sulfate–polyacrylamide gel electrophoresis (SDS-PAGE),
cally and functionally with CD3 ␨-chains of the T-cell using 15% gels (BioRad, Hercules, CA, USA). For immu-
receptor (TCR) complex.(13)
noblot analysis, proteins were transferred from SDS gels
However, the molecular mechanisms responsible for the onto a NitroBind nitrocellulose membrane (Micron Separa-
inhibitory effects of OIP-1/hSca on OCL formation are tions, Inc., Westboro, MA, USA). After blocking with 5%
unknown. Therefore, in this study we determined structure/ nonfat dry milk in 150 mM of NaCl, 50 mM of Tris, pH 7.2,
function characteristics of OIP-1/hSca that are critical for its and 0.05% Tween 20 (TBST) buffer, the membrane was
OCL inhibitory activity.
incubated for 1 h with the mouse MAb (immunoglobulin
G2a [IgG2a]) against V5 epitope diluted 1:5000 in 5%
nonfat dry milk–TBST. Then, the blots were incubated for
1 h with horseradish peroxidase–conjugated rabbit anti-
mouse IgG (Sigma Chemical Co.) diluted 1:10,000 in 5%
nonfat dry milk–TBST and developed using an enhanced
chemiluminescence (ECL) system (Amersham, Arlington
Heights, IL, USA).
MATERIALS AND METHODS
DNA transfection kits were obtained from Stratagene
(LaJolla, CA, USA). Polymerase chain reaction (PCR) kits
were obtained from Perkin Elmer Cetus Corp. (Foster City,
CA, USA). All restriction enzymes used were from New
England Biolabs, Inc. (Beverly, MA, USA) and chemicals
were from Sigma Chemical Co. (St. Louis, MO, USA).
Human embryonic kidney epithelial cell line (293 cells) was
obtained from American Type Culture Collection (ATCC;
Rockville, MD, USA). The 23c6 antibody is a generous gift
from Dr. Michael Horton (UK).
OCL precursor culture
Nonadherent marrow mononuclear cells were cultured
in methyl cellulose to form GM-CFU as described pre-
viously.(15) Recombinant human GM-CSF was used as a
source of colony-stimulating factor in cultures at a final
concentration of 100 pg/ml. The cells (4 ϫ 105/ml) were
plated in triplicate and incubated for 8 days in the presence
or absence of c-peptide (0–100 ng/ml) or vehicle at 37°C in
OIP-1/hSca cDNA deletion mutant constructs and
expression in 293 cells
OIP-1 complete coding sequence and c-peptide region a humidified atmosphere of 5% carbon dioxide air. Cultures
deletion mutant cDNA were PCR amplified using an were scored for colonies (aggregates Ͼ 50 cells) using an
RIG5CDS sense primer 5Ј-ACCATGAAGATCTTCTTGC- Olympus dissecting microscope (Olympus Optical Co., To-
CAGTG-3Ј, an RIG3CDS antisense primer 5Ј-GGGG- kyo, Japan) at 40ϫ magnification. The results are reported
CCAAACCGCAGCAGGGCCGG-3Ј, and RIG(-GPI) anti- as the mean Ϯ SD for triplicate cultures.
sense primer 5Ј-ATTGCACAGAAAGCTCTGGCAGCA-
3Ј, respectively. The PCR reaction mixture of 100 ␮l con-
taining 2 mmol/liter of MgCl2, 50 mmol/liter of KCl, 10
OCL-like cell formation
mmol/liter of Tris-HCl (pH 8.3), 0.2 mmol/liter of each
Bone marrow from C57 black mouse tibias that had been
deoxynucleoside triphosphate (dNTP), 2.0 U of Ampli Taq removed aseptically was obtained by flushing the tibias with
DNA polymerase, and 0.1 ␮mol/liter of sense and antisense 1 ml of ␣-minimum essential medium (␣-MEM) using a
primers. The PCR reaction was carried out by incubating the tuberculin syringe fitted with a 27.5G needle. The bone
samples at 94°C for 1 minute followed by 35 cycles of 94°C marrow-derived cells were washed twice and resuspended
for 1 minute and 60°C for 1 minute, with a final extension in ␣-MEM–10% fetal calf serum, and the cells were de-
for 5 minutes at 60°C. The PCR-amplified Sca-2 cDNA pleted of cells adherent to plastic by incubating the marrow

OSTEOCLAST INHIBITORY PEPTIDE 1/hSca
113
FIG. 1. (A) RT-PCR analysis of OIP-1/hSca
mRNA expression in OCL lineage cell types.
Total RNA was isolated from human bone
marrow–derived OCL early precursors (GM-
CFU), late precursors that are anti-vitronectin
23c6 antibody positive, and non-OCL cell types
that are 23c6(Ϫ) in the left panel. Right panel
shows the ␤-actin amplification in all the cell
types analyzed as positive control for RT-PCR
analysis. (B) Immunocytochemical staining for
OIP-1/hSca expression in OCL-like cells formed
in mouse bone marrow cultures. Bone marrow
cells were cultured in the presence of recombi-
nant human RANKL (rhRANKL; 50 ng/ml) and
mouse macrophage colony-stimulating factor
(M-CSF; 25 ng/ml). At the end of a 6-day culture
period, the MNCs formed were fixed in 4%
formaldehyde and immunostained for OIP-1/
hSca expression using rat anti-mouse TSA/Sca-2
antibody (PharMingen) at a concentration of 1
␮g/ml as described in the Materials and Methods
section. Photomicrographs were taken at magni-
fication ϫ200. Multinucleated OCL-like cells
that were positively stained with anti-Sca-2 an-
tibody were pointed by arrows. No immuno-
staining was detected with a control rat IgG1 as
shown in the left panel.
cell suspension in sterile 10-cm tissue culture dishes for 2 h.
The nonadherent marrow cells were collected and cultured
in quadruplicate for 8 days in 48-well plates at a density of
5 ϫ 105 cells/well in ␣-MEM–10% fetal calf serum sup-
plemented with 10Ϫ9 M of 1,25-dihydroxyvitamin D3 [1,25
(OH)2D3] as described by Takahashi et al.(16) The cultures
were fixed with 4.5 mM of citric acid, 2.25 mM of sodium
citrate, 3 mM of sodium chloride, 3% formaldehyde, and
acetone and washed twice in distilled water. Then, the
cultures were stained for tartrate-resistant acid phosphatase
(TRAP) using an acid phosphatase staining kit (Sigma
Chemical). The TRAPϩ multinucleated cell (MNC) con-
taining three or more nuclei was counted with an inverted
microscope.
Human bone marrow–derived mononuclear cells (106/
ml) were cultured in a 96-well plate for 3 weeks in the
presence of 1,25(OH)2D3 (10Ϫ9 M) to form OCL-like cells.
At the end of the culture period, anti-vitronectin receptor
antibody 23c6 positive OCL-like MNCs were scored as
described.(17) Pit formation capacity of OCL on sterile den-
tin slices in human bone marrow cultures was determined as
described previously.(15)
Immunostaining of OIP-1 expression in OCL-like cells
Mouse marrow cells were cultured to form OCL-like
MNCs as described previously.(16) At the end of culture
period, the cells were fixed in 4% formaldehyde for 30

114
KOIDE ET AL.
FIG. 2. (A) Western blot analysis of OIP-1/
hSca c-peptide deletion mutant protein expres-
sion in 293 cells conditioned media. Two hun-
dred ninety-three cells were transiently transfected
with the (1) ⌬ c-peptide deletion mutant and (2)
full length OIP-1/hSca expression constructs.
After 48 h, the serum-free conditioned media
concentrates were subjected to SDS-PAGE
(15%) and blot-transferred onto a nitrocellulose
membrane. OIP-1/hSca protein was detected by
immunoprobing the blot with mouse MAb
against V5 epitope as described in the Materials
and Methods section (M, :Molecular weight
marker). (B) Inhibitory effect of OIP-1/hSca and
⌬ c-peptide mutant protein on OCL formation in
murine bone marrow cultures. Conditioned me-
dia obtained from 293 cells transiently trans-
fected with OIP-1/hSca and ⌬ c-peptide mutant
constructs were assayed for OCL inhibitory ac-
tivity in mouse bone marrow cultures in vitro.
Conditioned media obtained from 293 cells that
were mock-transfected with empty vector were
served as control for these experiments. Mouse
bone marrow cells were cultured with
1,25(OH)2D3 (10Ϫ9 M) in the presence of con-
ditioned media (1% and 5%) samples as de-
scribed. TRAPϩ MNC formed at the end of the
culture period were scored (*p Ͻ 0.05).
minutes. Cells were permeabilized by adding cold
phosphate-buffered saline (PBS) containing 0.5% bovine
serum albumin (BSA) and 0.1% Triton X-100 for 15 min-
utes. After preincubation in 1% BSA, rat anti-mouse Sca
MAb (PharMingen, San Diego, CA, USA) was added (di-
luted at 1 ␮g/ml in PBS and 1% BSA) and continued
incubation for 1 h at room temperature. The cells were
washed and then incubated with biotinylated rabbit anti-
mouse IgG (1:200) for 1 h and stained for OIP-1 expression
using an ABC kit from Vector Laboratories (Burlingame,
CA, USA) following the manufacturer’s protocol.
RESULTS
OIP-1/hSca gene expression in OCL lineage
Previously, we identified the expression of OIP-1/hSca in
OCL-like cells formed in long-term human bone marrow
cultures by in situ hybridization.(3) We now further analyzed
the expression of OIP-1 in OCL cell lineage types using
RT-PCR analysis and immunocytochemistry. As shown in
Fig. 1A, RT-PCR analysis of total RNA isolated from early
precursors (GM-CFU) and committed OCL precursors
(23c6 anti-human vitronectin antibody-positive cells)
showed expression of OIP-1/hSca mRNA. Furthermore,
RT-PCR analysis also indicated OIP-1 mRNA expression in
non-OCL lineage cell types (23c6 antibody-negative cells)
present in the human bone marrow cultures. ␤-actin mRNA
was amplified in all the samples analyzed as an internal
control for the RT-PCR. Furthermore, immunocytochemical
staining using rat MAb against mSca showed positive stain-
ing of OCL-like cells and mononuclear cells present in
mouse bone marrow cultures (Fig. 1B).
Neutralization of OIP-1/hSca c-peptide activity
OIP-1/Sca c-peptide (NFSAADGGLRASVTLLGAGL-
LLSLLPALLRFGP) was synthesized by Genemed Synthe-
sis, Inc. (San Francisco, CA, USA). Polyclonal antibody
was raised in rabbits against cysteine keyhole limpet hemo-
cyanin (KLH)-conjugated immunogenic region of OIP-1/
hSca c-peptide [(C)NFSAADGGLRASVT] (Zymed Labo-
ratories, Inc., San Francisco, CA, USA). OIP-1 c-peptide–
specific polyclonal antiserum was added to mouse bone
marrow culture at 1:200 dilution concentration in the pres-
ence of 10 ng/ml of concentration of OIP-1 c-peptide and
10Ϫ9 M 1,25(OH)2D3 as described previously.
Mapping of OIP-1/hSca functional domain
Native and c-peptide deletion mutant forms of OIP-1/
hSca were expressed transiently in 293 cells, and condi-

OSTEOCLAST INHIBITORY PEPTIDE 1/hSca
115
tioned media was collected after 48 h. The expression of
OIP-1/hSca protein in 293 cell lysates was confirmed by
Western blot analysis as shown in Fig. 2A. The effect of
hSca conditioned media was assayed on murine bone mar-
row cultures to test their capacity to inhibit OCL-like cell
formation in vitro. As shown in Fig. 2B, conditioned media
obtained from 293 cells expressing native OIP-1/hSca sig-
nificantly inhibited OCL-like cell formation. However, con-
ditioned media obtained from 293 cells expressing the
c-peptide deletion mutant form of OIP-1 did not inhibit
OCL-like cell formation compared with mock transfected
cells. These data suggest that OIP-1 c-peptide is critical for
the OCL inhibitory activity of OIP-1.
Molecular properties of OIP-1/hSca c-peptide
A 33 amino acid peptide corresponding to OIP-1/hSca-c-
peptide was synthesized chemically. Peptide analysis using
the ProtParam database search indicates OIP-1 c-peptide
showed molecular weight of 3.2 kDa and isoelectric point
(pI) 9.6. The peptide contains 30% leucine and 15% alanine
residues. The estimated in vivo half-life of the c-peptide
following the N-end (Asn residue) rule is 1.4 h.(18) Kyte-
Doolittle(19) analysis of c-peptide shows hydrophobicity and
poorly immunogenic nature of the peptide. Therefore, syn-
thetic c-peptide was solubilized in isopropanol (1 mg/50 ␮l)
and reconstituted in sterile PBS for working concentrations.
FIG. 3. (A) Inhibitory effect of OIP-1/hSca on OCL precursor GM-
CFU colony formation. Human bone marrow cells cultured with hGM-
CSF (100 pg/ml) in methyl cellulose (0.7%) to form GM-CFU colonies
in the presence or absence of OIP-1 c-peptide. At the end of a 1-week
culture period, GM-CFU colonies formed in these cultures were scored
using a light microscope (*p Ͻ 0.05). (B) Effect of OIP-1/hSca
c-peptide on OCL-like MNC formation in long-term human bone
marrow cultures. Synthetic OIP-1 c-peptide was added to human bone
marrow cultures at different concentrations (1–100 ng/ml) in the pres-
ence of 10Ϫ9 M of 1,25(OH)2D3. Vehicle used to solubilize the
c-peptide alone served as control. At the end of a 3-week culture
period, anti-human vitronectin receptor antibody 23c6-positive MNC
formed in these cultures were scored (*p Ͻ 0.05).
Effect of synthetic OIP-1/hSca c-peptide on OCL
precursors and OCL development
Human bone marrow mononuclear cells were cultured in
methyl cellulose with GM-CSF in the presence of OIP-1
c-peptide at 0–100 ng/ml concentration as described in the
Materials and Methods section. OIP-1 c-peptide signifi-
cantly inhibits GM-CFU colony formation at 10–100 ng/ml
concentration (Fig. 3A). The effect of synthetic c-peptide
also was examined on OCL-like cell formation in human
bone marrow cultures. OIP-1 c-peptide at a concentration of
10 ng/ml significantly inhibited 1,25(OH)2D3 (10Ϫ9 M)-
stimulated OCL-like cell formation in human bone marrow
cultures (Fig. 3B). We also tested the effect of OIP-1
c-peptide on bone resorption capacity of the OCL formed in
human bone marrow cultures. Human early OCL precursors
(GM-CFU) were cultured on dentine slices to form OCL in
the presence or absence of OIP-1 c-peptide. As shown in
Fig. 4A, OIP-1 c-peptide at a concentration of 10 ng/ml
significantly decreased the number and size of resorption
lacunae formed on dentine slices. Furthermore, quantifica-
tion of these data using a light microscope indicated that
there was a 2-fold decrease in number of resorption lacunae
formed on dentine slices derived from cultures in the pres-
ence of c-peptide compared with control cultures in the
absence of c-peptide (Fig. 4B).
rabbit polyclonal antibody (1:200 dilution) raised against
OIP-1 c-peptide was added to murine bone marrow cultures
in the presence of OIP-1 c-peptide (10 ng/ml), OCL inhib-
itory activity of c-peptide was neutralized completely. In-
terestingly, anti–OIP-1 c-peptide antiserum significantly in-
creased TRAPϩ MNC formation in control cultures in the
absence of OIP-1 c-peptide (Fig. 5). In contrast, a nonspe-
cific control IgG at 1:100 dilution did not significantly affect
TRAPϩ MNC formation in these cultures. This further
confirms the specificity of OIP-1 c-peptide antiserum to
neutralize the OCL inhibitory activity of c-peptide. There-
fore, these data suggest that c-peptide is critical for the OCL
inhibitory activity by OIP-1.
Neutralization of OIP-1/hSca c-peptide activity to
inhibit OCL formation
DISCUSSION
We examined the neutralization capacity of OIP-1
c-peptide–specific antiserum to block c-peptide inhibition of
Ly-6 family molecules are low molecular weight GPI-
OCL formation in mouse bone marrow cultures. When linked peptides with a distinctive cysteine-rich domain and

116
KOIDE ET AL.
FIG. 4. OIP-1 c-peptide inhibits dentine re-
sorption by OCL formed in human marrow cul-
tures. OCL precursors GM-CFU were cultured
over sterile dentine slices with RANKL (80 ng/
ml) and macrophage colony-stimulating factor
(M-CSF; 10 ng/ml) to form OCL-like MNC as
described.(15) (A) Resorption lacunae formed on
dentine slices derived from human bone marrow
cultures in the presence of OIP-1 c-peptide (10
ng/ml) and control cultures in absence of
c-peptide. (B) Quantification of the number of
resorption lacunae formed on dentine slices. Re-
sults represent the mean Ϯ SEM for quadrupli-
cate determinations for a typical experiment.
Similar results were seen in three independent
experiments (*p Ͻ 0.05).
O-linked carbohydrate. Expression of OIP-1/hSca and re-
lated Ly6 gene family members in osteoblastic cells(20)
suggested a potential role for GPI-linked proteins on bone
cell function. We recently reported that OIP-1/hSca mRNA
being highly expressed in OCL may function as an
autocrine/paracrine inhibitor of OCL formation and bone
resorption activity.(3) In this study, immunocytochemical
staining of OCL further confirmed the expression of OIP-1
protein in mature OCL. Furthermore, OIP-1 mRNA expres-
sion in OCL early and more committed precursors suggest
that OIP-1/hSca may play an important role in OCL forma-
tion. OIP-1/hSca has no significant homology with the re-
cently described OCL inhibitor, osteoprotegerin, expressed
in osteoblastic cells.(21) However, the effects of OIP-1 on
receptor activator of NF-␬B–receptor activator of NF-␬B
ligand (RANK-RANKL) signaling mechanism in osteoclas-
togenesis is yet unclear.(22)
FIG. 5. Neutralization of OIP-1/hSca c-peptide activity to inhibit
OCL formation. The ability of rabbit polyclonal antiserum against
c-peptide to neutralize the inhibitory effect of OIP-1 c-peptide on OCL
formation was assayed in mouse bone marrow cultures. Bone marrow
cells were cultured with 10Ϫ9 M of 1,25(OH)2D3 to form OCL-like
cells. c-Peptide antibody was added at 1:200 dilution to these cultures
in the presence or absence of OIP-1 c-peptide(10 ng/ml). Nonspecific
rabbit IgG at 1:200 dilution was used as control (*p Ͻ 0.05).
In this study, c-peptide deletion mutant forms of OIP-1
did not inhibit OCL formation, suggesting that the c-peptide
is critical for OCL inhibitory activity. The synthetic OIP-1
c-peptide inhibited OCL precursors, GM-CFU growth in
methyl cellulose cultures, suggesting that OIP-1 inhibits the
proliferative phase of osteoclastogenesis. In addition, the c-peptide to block the effects of OIP-1 further suggests that
c-peptide inhibited OCL formation by OCL precursors GM- the inhibitory activity of OIP-1 is mediated by the c-peptide
CFU. The capacity of an antibody specific to the OIP-1 region. The OIP-1 c-peptide antibody significant increase in

OSTEOCLAST INHIBITORY PEPTIDE 1/hSca
117
OCL formation in the absence of c-peptide suggests that cell activation, has been shown to inhibit OCL formation
OIP-1 may be present in significant amounts in bone mar- through increased production of GM-CSF by these cells.(33)
row cultures. Furthermore, the OIP-1 c-peptide does not However, changes in cytokines/growth factors produced in
contain an RGD motif, suggesting that the inhibitory activ- bone marrow culture that may affect OCL formation in the
ity is not mediated through blocking adhesion molecules presence of OIP-1 c-peptide are yet to be elucidated.
such as the vitronectin receptor.(23) Previously, we have
In summary, OIP-1/hSca is a potent inhibitor of OCL
shown that OIP-1 does not display toxicity to various types formation, which is expressed in multinucleated OCLs and
of hematopoietic cells. precursors. We have identified that the c-peptide region of
The molecular mechanisms responsible for OIP-1/hSca OIP-1/hSca is critical for inhibition of OCL formation.
inhibition of OCL formation is unknown. Biochemical and Furthermore, anti–OIP-1 c-peptide–specific antiserum neu-
molecular genetic analyses have confirmed that the Ly-6 tralized completely the OCL inhibitory activity of OIP-1
multigene family is independently regulated and expressed c-peptide. Therefore, availability of neutralizing antiserum
on different hematopoietic cell lineages.(4) The 9804 gene, against the functional domain of OIP-1/hSca may provide
which encodes a human Ly-6 protein most similar to mouse more insights into the molecular mechanisms associated
differentiation antigen TSA-1/Sca-2,(24) has been reported with OIP-1 to inhibit OCL formation and control of the
recently. It has been suggested that GPI-anchored proteins normal bone-remodeling process.
transmit signals to the cell interior by interacting with
nonreceptor-type tyrosine kinases p56lck and p59fyn (25)
.
GPI-anchored proteins are released easily from the cell
surface by phosphatidylinositol-specific phospholipase C–
and D–type activities.(8) Bastisch et al.(26) have developed
GPI-deficient Jurkat T cells, which are defective in PIG-A
gene as a model to study functions of GPI-anchored pro-
teins. This study shows that the OIP-1 c-peptide, which
lacks the GPI anchor, has the capacity to inhibit OCL
formation, suggesting that the N-terminal portion is not
required for OCL inhibition. However, the relative roles of
the GPI anchored membrane-bound and -soluble forms of
OIP-1/hSca on osteoclastogenesis remain unclear. Ly-6
members have been implicated in cellular activation during
hematopoiesis from multipotential stem cells to lineage-
committed precursor cells.(27) Furthermore, Ly-6 family
members have been shown to be induced transcriptionally
by interferons in distinct cell types.(28) Interferons have
been shown to inhibit osteoclastogenesis.(29,30) More re-
cently, interferon-␥ has been shown to inhibit osteoclasto-
genesis through rapid degradation of TNF receptor associ-
ated factor-6 (TRAF6), which results in strong inhibition of
RANKL-induced NF-␬B and c-Jun kinase activity.(31)
Therefore, further studies will have to determine the regu-
lation of OIP-1/hSca expression and activity by cytokines/
growth factors that modulate OCL formation and bone
resorption.
In this study, we have detected expression of OIP-1/hSca
OCL in non-OCL lineage mononuclear cell types present in
bone marrow cultures. TSA-1/Sca-2 has been shown to be
expressed on B220, Thy-1, CD4, and Gr-1 antibody-
positive cell types present in the bone marrow(6) and virtu-
ally all peripheral B cells but is not expressed on peripheral
T cells. However, Sca-2 is a unique marker for T cell
activation and may affect cytokine production by T cells.(10)
It has been postulated that CD8ϩ T cells may be involved in
osteoclastogenesis because depletion of this cell type results
in increased OCL formation.(32) Bone marrow and OCL
precursors GM-CFU cultures may contain low numbers
(Ͻ5%) of T lymphocytes. Because T cells have been im-
plicated in OCL formation,(32) it is possible that Sca-2
signaling through TCR may play an important role in T
cell–mediated regulation of osteoclastogenesis in the bone
microenvironment. IL-18, which has profound effects on T
ACKNOWLEDGMENTS
We thank Dr. David Roodman for his helpful discussions.
This work was supported by the National Institutes of
Health (NIH) grant DE12603.
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Address reprint requests to:
Sakamuri V. Reddy, Ph.D.
Division of Hematology/Oncology
Department of Medicine
University of Pittsburgh
3500 Fifth Avenue, Suite 206
Pittsburgh, PA 15213, USA
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human chromosome 8q24.3 of Ly-6 related gene 9804 encod-
ing an apparent homologue of mouse TSA-1. J Immunol Received in original form February 7, 2001; in revised form June
160:197–208. 17, 2001; accepted August 3, 2001.