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Department of Biology, The University of York, Heslington, York YO10 5YW, United Kingdom
1Correspondence: Bone and Joint Biology Research Group, Department of Biology, The University of York, Heslington, York YO10 5YW, U.K. E-mail: tsg1{at}york.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN-) or IFN-
. Whereas IFN- induced a rapid,
transient induction of best5 expression peaking at 4–6
h poststimulation, IFN- elicited a more prolonged induction of
best5 expression, which remained elevated 48 h
poststimulation. A polyclonal antibody generated to a peptide derived
from the best5 coding region recognized a 27 kDa protein
on Western blot analysis of osteoblast lysates. We localized BEST5
protein in osteoblast progenitor cells and mature osteoblasts in
sections of rat tibiae and in sections of bones loaded in
vivo to induce adaptive bone formation. Best5
may therefore be a fundamental intermediate in the response of
osteoblasts to stimuli that modulate proliferation/differentiation,
such as interferons or mechanical loading. These findings highlight the
close interactions between the immune system and bone cells and may
open new therapeutic avenues in modulating bone mass—Grewal, T. S., Genever, P. G., Brabbs, A. C., Birch, M., Skerry, T. M. Best5: a novel interferon-inducible gene expressed
during bone formation.
Key Words: osteoblasts • differentiation • cytokines • osteoporosis • mechanical loading
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Among the cytokines implicated in these interactions are the
two distinct types of interferons (Type I: IFN-/ß, and Type II:
IFN-). The IFNs are responsible for a wide range of cellular
responses including regulation of cell growth, immunomodulation,
resistance to infectious agents, cell metabolism, and differentiation
(3)
. Although Type I and Type II IFNs are structurally
unrelated and operate via distinct receptors, they exhibit many common
biological effects mediated by the JAK-STAT pathway (for reviews, see
refs 4
, 5
). The JAK-STAT intracellular pathway is also
used by other cytokines, including those cytokines whose receptors
belong to the Type I cytokine receptor superfamily. Although the exact
role of the IFNs in bone is presently unclear, evidence for their
involvement in skeletal tissue biology is growing (6)
.
Interferon- is known to suppress the proliferation of
osteoprogenitor cells (7
, 8)
and to modify the expression
of a number of important cytokines in bone marrow cells (9
, 10
, 11)
. Interferon- inhibits bone resorption
(12
13
14)
and exhibits antiproliferative actions on
bone-derived cells (15
16
17)
. Overexpression of IFN- in
transgenic mice results in degenerative lesions in bone and cartilage
as well as reduced numbers of B cells in bone marrow, spleen, and lymph
nodes (18)
. The specific mechanisms involved in
inflammatory diseases that involve bone (such as osteoarthritis and
rheumatoid arthritis, RA) are not clear. However, further insight into
the molecular mechanisms of IFN action in these conditions could be of
important clinical significance, since both the mRNA and protein for
IFN- (19)
and IFN- (20
, 21)
have been
detected in RA synovial tissue. In addition, the ability of IFNs to
modulate the proliferation of osteoprogenitor cells indicates an
important role for these cytokines during normal and pathological bone
remodeling.
The best5 (bone-expressed sequence tag 5) gene was
originally identified as part of a differential display polymerase
chain reaction (PCR) project identifying genes regulated by estrogen in
whole bone samples. Here we report on the molecular characterization of
this novel cDNA and show that it is expressed predominantly in bone and
spleen. The temporal pattern of best5 expression in primary
rat osteoblasts indicates that it may play an important role in
osteoblast function or differentiation. We also show that the
best5 gene is regulated by both IFN- and IFN- and that
mechanical loading of bone in vivo, which induces
proliferation and differentiation of osteoblasts, results in rapid
induction of best5 protein expression. These data provide
evidence that best5 induction is part of the process of
osteoblast differentiation and bone formation, and increases links
between cytokines of the immune system and physiological regulation of
bone mass.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cloning of best5 cDNA
The best5 gene was initially identified as part of a
differential display PCR (DD-PCR) project investigating the expression
profile of genes in the tibiae of ovariectomized rats. The DD-PCR
experiments were performed using the Delta RNA Fingerprinting kit
(Clontech Laboratories U.K. Ltd., Basingstoke, U.K.) on DNase I-treated
total RNA from whole rat tibiae. The original 576 bp best5
DD-PCR product was cloned into pCR-Script SK(+) (Stratagene Ltd.,
Amsterdam, The Netherlands) and five individual clones were sequenced
using an ABI 377 system (Perkin Elmer, Warrington, U.K.). This sequence
information was used to design gene-specific PCR primers for the
cloning of the full-length best5 cDNA by the rapid
amplification of cDNA ends (RACE) technique (best5 RACE1
primer: 5'-TGAGGCCTGCATGATTGTTCTTGGACTACT-3' and best5 RACE2
primer: 5'-AGCTCGTGTTTTACCCTTTTCATGGACTGA-3'). The RACE reactions were
performed using the Marathon cDNA Amplification Kit (Clontech Ltd.).
Briefly, double-stranded cDNA was synthesized from 1.5 µg of
poly(A)-enriched mRNA purified from pooled total RNA (400 µg)
obtained from whole rat tibiae. The best5 RACE reactions
were performed, after adapter ligation, using a two-step touchdown PCR
protocol (94°C for 1 min; 5 cycles of 94°C for 5 s, 72°C for
4 min; 5 cycles of 94°C for 5 s, 70°C for 4 min; 20 cycles of
94°C for 5 s, 68°C for 4 min). The resulting 5' and 3' RACE
products were separated on a 1.2% agarose gel and the two major PCR
products were excised from the gel, purified with GeneClean II Spin kit
(BIO 101, La Jolla, Calif.), and cloned into the vector pCR 2.1 using
the TA Cloning Kit (Invitrogen BV, Groningen, The Netherlands). Four
individual clones for each of the RACE products were sequenced using
internal sequencing primers, as required, to obtain data along the full
length of the inserts from both DNA strands.
A set of gene specific PCR primers (best5-PCR1F: 5'-GGCCACCGGTACAGTTCAAA-3' and best5-PCR1R: 5'-CAATGGCAGCCTTATCCGGAGAG-3') were designed to amplify a 450 bp region of the best5 cDNA, including part of the potential coding region. Total RNA (10 µg) from whole rat tibiae were used for the synthesis of cDNA using the First-strand cDNA Synthesis kit (Life Technologies, Inc.-BRL) with oligo-d(T) as the primer. The cDNA reaction was diluted 1:10 and 1 µl used for PCR with the best5-PCR1F and best5-PCR1R primers (94°C for 1 min; 28 cycles of 94°C for 10 s, 58°C for 30 s, 72°C for 1 min; followed by a final extension of 72°C for 5 min). The resulting 450 bp best5-PCR1 product was cloned into the vector pCR 2.1 (Invitrogen BV) and five independent clones were sequenced using an ABI 377 (Perkin Elmer) to confirm the identity of the PCR inserts. The best5-PCR1 product was used as template for the generation of all subsequent probes for Northern blot analyses.
All PCR reactions were performed using a PTC-200 DNA Engine (GRI Ltd., Dunmow, U.K.) and Platinum Taq polymerase (Life Technologies, Inc.-BRL), except the RACE reactions, which were performed using Advantage Taq Polymerase mix (Clontech Ltd.).
Computer analysis of best5 cDNA
Sequence similarity database searches were performed using the
gapped BLAST (BLAST2) program (22)
(http://www.ncbi.nlm.nih.gov/BLAST) against nonredundant sequence
database ‘nr’. The significance of a match is assessed with an
E value, which measures the expected number of sequences in
the database that would achieve a given score by random. Protein domain
search was performed using search machinery within the Protein Domain
Database ProDom release 99.1
(http://protein.toulouse.inra.fr/prodom.html). This database has been
constructed from a procedure based on recursive searches using
PSI-BLAST (22
, 23)
.
Local sequence similarity between pairs of sequences was measured using
the program BESTFIT within Wisconsin Package Version 9.1 (GCG, Madison,
Wis.). Significance of the alignment was assessed with the Z
score that counted the number of standard deviations from the mean
score achieved after one of the sequences was randomized 100 times.
Multiple sequence alignment was calculated using program CLUSTAL W
(24)
(http://www2.ebi.ac.uk/clustalw/).
Cell culture
Human osteosarcoma cell line MG63 and primary rat osteoblasts
(ROBs) were maintained in DMEM supplemented with 10% fetal calf serum
(FCS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml
streptomycin (Life Technologies, Inc.-BRL).
Cultures of primary ROBs were obtained essentially as described
previously (25)
, with the following modifications.
Calvaria from 3-day-old neonatal rats were sequentially digested with 1
mg/ml collagenase in HBSS (Sigma, Poole, U.K.) and EDTA (4 mM). Cells
were seeded onto 75 cm2 tissue culture flasks
with DMEM plus 10% FCS. On gaining confluence, cells were split 1:3
(passage 1) in DMEM, 10% FCS. Primary cells at passage 2 (1:3 split)
were plated onto 25 cm2 flasks containing DMEM,
10% FCS and grown until reaching confluence (48 h). At this point
(referred to as day 0) the media was replaced with fresh DMEM
supplemented with 10% FCS, 10 nM dexamethasone, 5 mM ß glycerol
phosphate, and 100 µg/ml L-ascorbic acid phosphate. This
osteo-inductive media promotes the differentiation/maturation of cells
in the culture to the osteoblast phenotype. Fresh osteo-inductive media
was added every 4 days for the duration of the experiments. Total RNA
for Northern blot analysis was obtained from these cultures at
different time points (days, 0, 4, 12, 21, and 35) by washing cells
twice with phosphate-buffered saline (PBS), followed by the addition of
1.0 ml of TRIzol reagent per 25 cm2 flask.
Human osteosarcoma cells (MG63) were maintained in DMEM, 10% FCS until
reaching confluence in 25 cm2 flasks (48 h). The
cells were serum starved in 5 ml of DMEM, 0.5% FCS for 24 h prior
to treatment. Total RNA was extracted from control MG63 and
interferon-treated cells (hIFN- or hIFN- at 100 IU/ml media;
total of 500 IU per 25 cm2 flask) at 1, 2, 4, 8,
and 24 h poststimulation. Human interferon-(2b) (hIFN-) was
purchased from PeproTech EC Ltd. (London, U.K.);
hPTH1–34 was obtained from Calbiochem Ltd.
(Nottingham, U.K.) and 1,25-dihydroxy vitamin
D3 (Vit. D3) was a gift
from Roche (Welwyn Garden City, U.K.). Human interferon- (hIFN-),
interleukin 1ß (IL-1ß), IL-6, and IGF-1 were all purchased from
Boehringer Mannheim Ltd. (Lewes, U.K.).
Northern blot analysis
Total RNA samples (10 µg) were fractionated on 1% agarose
gels containing 6.7% formaldehyde and transferred by capillary
blotting onto Zeta-Probe GT membranes (Bio-Rad Laboratories Ltd., Hemel
Hempstead, U.K.) in 10x SSC. Probes for the Northern hybridizations
were prepared using 120 ng of the 450 bp best5-PCR1 product
as template in random-primed reactions (HighPrime Radiolabeling Kit;
Boehringer Mannheim Ltd.) containing 50 µCi of
32P -dCTP (3,000 Ci/mmol; Amersham Pharmacia
Biotech, Little Chalfont, U.K.). Hybridizations were carried out at
68°C for 1 h in QuickHyb (Stratagene BV) and blots were washed
three times for 10 min each in 2x SSC, 0.1% sodium dodecyl sulfate
(SDS) at room temperature, followed by two washes of 15 min each in
0.1x SSC, 0.1% SDS at 60°C.
Antisera production and assessment
A peptide (BEST5-PEP-1: amino acid residues
344-RGGKYVWSKADLKLDW-360 of the best5 coding region) was
synthesized and used for the production of polyclonal antisera in
rabbits (B1047 and B1062) after coupling to KLH carrier protein
(Genosys Biotechnologies Ltd., Cambridge, U.K.). Rabbit bleeds were
assessed for antibody production by ELISAs using 96-well plates coated
with the uncoupled BEST5-PEP-1 peptide (100 ng/well) and incubated with
doubling dilutions (1: 400 to 1:819,200) of antisera B1047 and B1062.
Antisera B1062 was chosen for all subsequent use in immunoblot analyses
and characterization studies.
Immunoblot analysis
Total cell lysates from primary ROBs and MG63 cells were
obtained by washing cells with PBS, followed by the addition of lysis
buffer (20 mM Tris HCl pH 7.5; 2 mM EDTA; 0.5 mM EGTA; 1 mM DTT; 2 mM
PMSF; 10 µg/ml aprotin; 0.3 M sucrose; 0.1% Triton X-100; 0.1%
SDS). Cells were scraped off the surface of culture dishes in Lysis
buffer and stored at -80°C. Total cell lysate samples were
centrifuged at 13,000 rpm for 10 min at 4°C, and 15 µl of the
resulting supernatant was mixed with 5 µl of 4x SDS-sample buffer
and boiled for 2 min. The resulting protein samples (5 µl) were
separated on 12% SDS-polyacrylamide gels with 4% staking gels
(Bio-Rad) using standard SDS-PAGE conditions. After electrophoresis,
proteins were electrophoretically transferred (Trans-Blot System;
Bio-Rad) to Protran BA85 nitro-cellulose membranes (Scheicher &
Schuell, Dassel, Germany) using standard methodology. Protein blots
were blocked in Tris-buffered saline (TBS) containing 0.1% Tween-20
(TBS-T) and 5% bovine serum albumin (BSA) for 1 h at room
temperature. The blots were incubated with B1062 antisera (rabbit
primary antibody) at a final dilution of 1:20,000 in TBS-T containing
1% BSA for 2 h at room temperature. After extensive washing in
TBS-T, blots were incubated for 1 h at room temperature with
secondary antibody (goat anti-rabbit IgG conjugated to horseradish
peroxidase; Sigma) at a dilution of 1:10,000 in TBS-T, 1% BSA. Unbound
antibodies were removed by washing blots in TBS-T. Specific
antigen-antibody complexes were detected using enhanced
chemiluminesence (ECL System, Amersham) with exposure times of 30 s to 2 min. Control immunoblot analyses were also performed as
described above except that either preimmune serum or antiserum B1062
(at 1:1,000 dilution in PBS) preincubated with 10 µg of BEST5-PEP-1
(antigen-blocking peptide) for 16 h at 4°C was used as primary
antibody (both at final dilutions of 1:20,000).
Immunohistochemistry
Tissues used for immunolocalization studies were dissected out,
dipped in 10% polyvinyl alcohol (PVA, Sigma) and immediately frozen in
chilled n-hexane (-70°C). The tissues were mounted in 10% PVA on
brass chucks and 5–7 µm sections cut using a Brights cryostat
(Brights Instrument Co., Huntington, U.K.). Sections were collected on
Vectorbonded slides (Vector Laboratories, Peterborough, U.K.) and
stored at -35°C until use. Prior to immunolocalization, the sections
were fixed in 4% paraformaldehyde for 5 min and endogenous peroxidase
activity was depleted with 3% hydrogen peroxide (Sigma) for 30 min. A
further preincubation was performed with 10% normal goat serum (Vector
Laboratories) for 30 min to block nonspecific antibody binding.
Sections were incubated with the primary antibody (B1062, rabbit
anti-BEST5 at 1:500 dilution) for 30 min, followed by biotinylated goat
anti-rabbit secondary antibody (Vector Laboratories; 1:200 dilution)
for 10 min and avidin-biotinylated-peroxidase reagent (ABC Elite,
Vector Laboratories, 1:50 dilution) for 15 min. Peroxidase activity was
visualized with 0.5 mg/ml diaminobenzidine (Sigma) with 0.3% hydrogen
peroxide as substrate. All dilutions were made up in phosphate-buffered
saline (PBS), pH 7.4 and incubations were performed at room temperature
with three PBS washes between each incubation. Negative control serial
sections received same concentrations of B1062 preincubated overnight
at 4°C with 10 µg of BEST5-PEP-1 (antigen-blocking peptide) in
place of primary antibody. Sections were counterstained with
hematoxylin prior to mounting in glycerol/PBS.
Mechanical loading in vivo
The left ulnae of five male Wistar rats (85–88g body weight)
were loaded in vivo under general anesthesia essentially as
described previously (26)
. Briefly, the elbow and flexed
carpus were placed in a computer controlled servo-hydraulic materials
test machine (Instron 8511/20) and loaded cyclically at 2 Hz to a peak
of 7 newtons for 3.3 min. This load induces strains of ~4000
microstrain, which are 30–50% higher than those imposed by normal
physiological activity. Loading was performed for 5 consecutive days,
after which the ulnae were removed for analysis. Undecalcified cryostat
sections were cut from a region 2–3 mm distal to the midshaft, where
we have previously demonstrated mineralized new bone formation in
response to a more prolonged period of daily loading (26)
.
The unloaded contralateral bones from each animal were used as
controls. Loaded and control bone sections were processed for BEST5
immunolocalization as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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BLAST2 search revealed two close homologs of best5: a human
homologue cig5 (27)
(accession number AF026941)
(E=e-166) and an uncharacterized
protein, VIG-1 (accession number AF076620), identified from rainbow
trout (Oncorhynchus mykiss;
E=e-141). The protein sequence
analysis performed using the program BESTFIT revealed that all three
proteins have very strong homology between their carboxyl-terminal
domains. Best5:68–360 vs. cig5:67–359 shows 91% sequence
identity, whereas best5:68–360 vs. VIG-1:56–348 has 79%
identity. Homology in the amino-terminal domain is much weaker:
best5:1–67 vs. cig5:1–66 shows 44% identity whereas
VIG-1:1–55 domain shows no significant homology with either
best5 or cig5. Multiple sequence alignment of all three
sequences is presented in Fig. 1
. In addition, 20% of the best5 protein shares weak sequence
homology to a domain characteristic to a family of mainly prokaryotic
proteins involved in the biosynthesis of metallo cofactors.
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Expression of best5 mRNA
The expression profile of best5 was investigated by
Northern blot analysis of total RNA obtained from various rat tissues.
The multiple-tissue Northern blot was probed with the
best5-PCR1 probe as described in Materials and Methods and a
single band of ~4 kb in size was detected as shown in Fig. 2A
. This multiple-tissue Northern blot shows that expression
of best5 occurs predominantly in bone marrow and spleen,
with none detectable in brain, liver, thymus, and muscle. The
best5 mRNA transcript was also evident in lung, heart,
cortical bone, and kidney, but at much lower levels of expression than
in either bone marrow or spleen.
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To clarify the potential role of best5 in bone formation, we
examined the profile of its expression in cultures of differentiating
primary rat osteoblasts. The primary ROBs can be induced to form large
numbers of alkaline phosphatase-positive cells and mineralized nodules
in vitro, which is phenotypic of mature osteoblasts.
Northern blot analysis of RNA obtained from these cultures at different
time points showed a temporal pattern of best5 expression
(Fig. 2B
). The best5 mRNA was not detectable
during the early stages of culture when the population consists mainly
of undifferentiated cells. However, by day 21, when the culture
consists of differentiating and mature osteoblasts, which are in the
process of depositing matrix that finally becomes mineralized, the
expression of best5 is clearly elevated. The expression of
best5 then declines to a much reduced level at 35 days in
culture when the nodules are fully mineralized.
The kinetics of best5 mRNA induction in MG63 cells by
hIFN- and hIFN- were investigated by Northern blot analysis.
MG-63 cells were treated with 100 IU/ml of either hIFN-, hIFN-,
or PBS (control) and total RNA was extracted at various time points
poststimulation. The expression of best5 mRNA was determined
by Northern blot analysis of these RNA samples (Fig. 3
). As shown in Fig. 3
, the kinetics of best5 mRNA induction
by the two types of IFNs were distinct. hIFN- induced a continued
and prolonged induction of best5 mRNA, which remained
elevated 24 h poststimulation. hIFN-, however, elicited a
transient induction of best5 mRNA, which peaked at 4 h
and returned to basal levels 24 h after stimulation. This distinct
response to IFN- and IFN- is common among the small subset of
interferon-stimulated genes responsive to both types of IFNs with a
resulting potentiation of induction in the presence of both IFNs. We
also investigated the effects of PTH, IL-1ß, IL-6, IGF-1, Vit.
D3, and dexamethasone on best5 gene
expression in MG63 cells. These agents did not induce any significant
change in best5 mRNA levels (data not shown), which
highlights the specificity of the IFN-stimulated induction of
best5 gene expression.
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Immunodetection of BEST5 protein expression
The rabbit polyclonal antiserum B1062 was raised against
BEST5-PEP-1 peptide and used in Western blot analysis and
immunolocalization studies. Western blot analysis of B1062 antiserum
using total cell lysates from primary rat osteoblasts detected a band
of 27 kDa. This 27 kDa band was confirmed to contain the BEST5-PEP-1
epitope by Western blot analysis with antiserum B1062 preincubated with
10 µg of BEST5-PEP-1 as a blocking peptide. As shown in Fig. 4
, preincubation of antiserum B1062 with BEST5-PEP-1 blocking peptide
results in the absence of the 27 kDa band that is present in the blot
incubated with B1062 alone.
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The antiserum B1062 was used in immunolocalization studies to determine
the nature of the cells expressing BEST5 protein in neonatal rat
tibiae. Figure 5
shows that BEST5 protein was specifically localized to cells of the
periosteum and osteoblasts lining forming bone surfaces on both
periosteal and endosteal surfaces. In addition, specific localization
was also present on articular surfaces of cartilage and in
perichondoral cells but was absent in chondrocytes (results not shown).
No specific BEST5 protein localization was evident in multi-nucleated
osteoclasts, osteocytes, muscle myoblasts, and in the majority of the
various stem and hematopoietic cells present in the bone marrow. Serial
sections incubated with BEST5-PEP-1 blocked B1062 antiserum served as
negative controls and showed little or no specific staining.
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Induction of BEST5 by mechanical loading in vivo
In sections from bones loaded in vivo, there was clear
induction of a formative response on the medial periosteal surface of
the ulna. In control bones, this surface undergoes resorption as a
result of the modeling drifts by which the bone acquires its adult
shape, and is characterized by a narrow band of cells between the
overlying muscle and the bone (26)
. Cells on this surface
have been shown to express high levels of the enzyme tartrate-resistant
acid phosphatase (26)
, which is a characteristic of
osteoclasts and their precursors. Loaded bones had a much broader band
of plump cuboidal osteoblastic cells that expressed high levels of
BEST5 (Fig. 6
). The highest levels of expression were seen in the cells most
peripheral to the bone surface.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although we first identified best5 in differential display
studies in order to study the effects of estrogen in bone, estrogen did
not regulate the gene in subsequent studies. However, at that stage
identification of the human cig5 gene by others (27)
,
which shares high homology within its coding region with
best5, gave us additional clues to the regulation of this
gene. Those workers had identified cig5 from virus-stimulated cells and
shown regulation by IFN-, so it was logical for us to investigate
interferon regulation of best5 gene expression in bone
cells.
Regulation of best5 gene expression by both Type I (IFN-)
and Type II (IFN-) interferons in the human MG63 cell line increased
the likelihood that best5 was involved in the process of
bone formation, particularly as the two types of interferon have
kinetically different effects. These findings point to IFN- as a
major physiological regulator of best5 expression in bone
cells in vivo. Other osteotropic factors so far tested (PTH,
IL-1ß, IL-6, IGF-1, Vit. D3, and dexamethasone)
were ineffective in modulating the expression of best5.
However, we cannot rule out the possibility that other as yet untested
osteotropic factors may share with interferons the ability to modulate
best5 expression. It is not clear yet whether the induction
of best5 by mechanical loading acts through an
interferon-mediated pathway.
The Northern analysis of best5 expression in IFN stimulated
MG63 cells (Fig. 3)
shows two distinct hybridization bands of 3.9 and
3.2 kb in size whereas the ROB Northern blots revealed only the
presence of the predominant 3.9 kb band. A number of reasons could
explain this observation. The bands may result from abnormal splicing
of the best5 mRNA in this particular human cell line.
Alternatively, it is possible that in rat, only the 3.9 kb
best5 transcript is expressed whereas human cells express
both 3.9 kb and 3.2 kb best5 transcripts. It is worth noting
that the cloned human cig5 cDNA is 3.2 kb in size whereas the rat
best5 cDNA is 3.9 kb.
The sequence analysis presented in this paper demonstrates that
best5 is part of a new family of genes that encompasses cig5
(human) and VIG-1 (trout). All members of this new family of genes have
been shown to be expressed in response to either viruses or IFNs,
suggesting that the high sequence homologies between the three genes is
matched by related functions. Since cig5 was first detected in response
to virus infection of human fibroblasts (27)
, the high
level of best5 expression seen in the bone and spleen
tissues could be suggested to be due to latent viral infection of the
animals during rearing. However, in primary cultures of neonatal rat
osteoblasts, the expression of best5 is not evident during
the early stages of culture, as would be expected if the cells were
obtained from virally infected animals. Regulated best5
expression is temporal only during the later stages of bone nodule
matrix formation and spatial during bone formation in vivo,
suggesting that its expression plays an important role in osteoblast
function.
Western blot analysis of total cell proteins from primary ROBs using
our polyclonal antibody confirmed that the 27 kDa band contains the
epitope present within the BEST5-PEP-1 blocking peptide. The predicted
molecular mass of the best5 coding region is 42 kDa, which
contrasts with the observed BEST5-PEP-1 containing band of 27 kDa. The
difference in the predicted molecular mass and the observed 27 kDa band
may possibly be due to posttranslational processing of the BEST5
protein. This potential processing event may possibly involve protease
cleavage of the BEST5 protein at the carboxyl-terminal domain (amino
acid 1–68), since this is the least conserved region of this new
family of proteins (Fig. 1)
.
The expression of best5 in osteoblasts both in
vivo and in vitro is consistent with the mechanical
loading experiments. In that study, a regimen of loading that would
subsequently induce formation of mineralized bone by 7 days
(26)
was (at 5 days) associated with periosteal
proliferation and increased levels of best5 expression.
Since the equivalent surfaces of the control contralateral bones were
undergoing resorption, there was no detectable best5
expression. This provides clear evidence that best5
expression is an early response to stimuli capable of modulating
osteoblast proliferation such as mechanical loading and interferons.
The main sites of BEST5 protein expression in rat tibiae are consistent
with the alterations in phenotype of transgenic mice overexpressing
IFN- (18)
. These mice have osteochondrodysplasia, with
abnormal growth/development of both cartilage and bone, and are
characterized by complex osteogenesis imperfecta-like lesions,
chondrodysplasia, and osteoarthritis, accompanied by reduced cortical
bone thickness and increased incidence of fractures. These defects
cannot adequately be explained by the known actions of the interferons
in bone and joint cells (12
, 28
29
30
31
32)
. These observations
indicate that the defects observed in the transgenic mice are not
solely due to inhibition of osteoclast formation/function and reduced
collagen synthesis by osteoblasts. This suggests that IFN- modulates
the function of osteoblasts, osteoclasts, and chondrocytes, resulting
in defects of both bone and cartilage. Further support for such a
hypothesis is demonstrated by the induction of best5 by
loading. It would be of interest to determine whether in IFN null mice
there was a normal response to loading and/or an induction of
best5 expression.
Our studies demonstrate that best5 is a member of a novel family of interferon-stimulated genes that is normally expressed at high levels by actively forming osteoblasts, whether that activity is part of normal growth or induced by an external stimulus such as mechanical loading. The fact that best5 is also induced by IFNs leads to the inference that the mechanism of action of these cytokines in regulating osteoblast differentiation and/or proliferation involves best5. If best5 is an intermediary that precedes osteoblastic bone formation, then it could provide a target for clinical manipulation of bone mass.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication January 5, 1998. Revised for publication October 8, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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