Vol. 137, No. 11
Printed in U.S.A.
0013-7227/96/$03.00/O
Copyright
&996
by The Endocrine
Society
Establishment
Conditionally
Adult Human
PETER
V. N. BODINE,
and Hormonal
Regulation
Transformed
Preosteocytic
Bone*
STEVEN
Women’s Health Research Institute
Radnor, Pennsylvania
19087
K. VERNON,
(P.V.N.B.,
B.S.K.)
AND
BARRY
and Discovery
of a
Cell Line from
S. KOMM
Research
(S.K.V.),
Wyeth-Ayerst,
ABSTRACT
Osteocytes
are differentiated
forms of osteoblasts
that arise upon
entrapment
within
the bone matrix.
In this report,
we describe
the
establishment
and hormonal
regulation
of the first
conditionally
transformed
human
preosteocytic
cell line. Primary
adult bone cells
were obtained
from protease
digestion
of cancellous
chips. The cells
were infected
with adenovirus-oriSV40 tsA 209, which
encodes
for
a temperature-sensitive
large T-antigen.
After
immortalization,
we
isolated
a clone designated
HOB-01X1.
This cell line expressed
the
mutant
T-antigen
and proliferated
at the permissive
temperature
(34
C) but stopped
dividing
at the nonpermissive
temperature
(39-40
C).
Electron
microscopy
of cells incubated
at 39 C demonstrated
the
presence
of preosteocytic
cellular
processes,
some of which
appeared
to form gap junctions
or were rich in microfilaments.
The clone expressed arl type (I) procollagen
messenger
RNA (mRNA)
and secreted
type I procollagen
C peptide at both temperatures,
and this expression
was elevated
1.6-fold to 1%fold
at 40 C. The cells expressed
very low
basal levels of alkaline
phosphatase
activity
(-0.02
nmol/mirmg),
which
was increased
2- to B-fold in a dose-dependent
manner
by
0.1-100
nM &25dihydroxyvitamin
D, (vitamin
D,) at both temper-
atures.
Vitamin
D, also increased
osteocalcin
secretion
in a dosedependent
manner
when the clone was maintained
at 34 C (-6-fold),
and this stimulation
was enhanced
>5-fold
at 40 C. In contrast
to the
low expression
of alkaline
phosphatase,
the cells secreted
high
amounts
of osteocalcin
in response
to vitamin
D, (-15
ng/mg cell
protein);
this biochemical
profile also resembled
that of preosteocytes.
Alizarin
red-S histochemical
staining
demonstrated
that these cells
rapidly
produced
mineralized
nodules
at both temperatures.
PTH (10
and 100 nM) had no effect on the intracellular
accumulation
of CAMP
at 34 C but stimulated
a 14- to l&fold
increase
in the production
of
this second messenger
at 40 C. In contrast,
100 nM prostaglandin
E,
and 1 ELM forskolin
stimulated
CAMP synthesis
better at 34 C. Western
blot analysis
indicated
that the cells expressed
CD44,
a putative
osteocytic
marker,
at both temperatures.
Finally,
interleukin-lp
and
tumor
necrosis
factor-a
(l-1000
PM) stimulated
dose-dependent
increases
in the secretion
of interleukin-6
and monocyte
chemoattractant protein-l
at 34 C and 40 C. We conclude
that the HOB-01-Cl
cell
line has a preosteocytic
phenotype.
Moreover,
these cells respond
to
calcitropic
hormones
and bone resorbing
cytokines.
(Endocrinology
137: 4592-4604,
1996)
0
ative levels of expression of marker proteins such as type I
collagen, alkaline phosphatase, and osteocalcin. Work from
other investigators indicates that bone sialoprotein is produced at the late stage of osteoblast maturation, whereas
biglycan is expressed by both osteoblasts and osteocytes (7).
From in situ studies of subperiosteal human bone, Gehron
Robey et al. (2) have also mapped the expression of marker
proteins throughout the different stages of osteoblast differentiation. In these studies, preosteoblasts (i.e proliferative
phase) incorporated [3H]-thymidine and expressed alkaline
phosphatase as well as several bone matrix proteins but did
not produce osteocalcin. Mature osteoblasts no longer incorporated [3H]-thymidine but did express alkaline phosphatase, bone matrix proteins, and low levels of osteocalcin.
Osteoid-osteocytes (preosteocytes or mineralization phase)
did not incorporate [3H]-thymidine and expressed only low
IeveIs of alkaline phosphatase; on the other hand, these cells
synthesized high levels of osteocalcin and continued to produce some bone matrix proteins. Finally, mature osteocytes
did not incorporate [3H]-thymidine, no longer expressed alkaline phosphatase, and produced only low levels of osteocalcin and other bone matrix proteins. These cells were also
embedded in mineralized matrix.
Osteocytes have other phenotypic characteristics as well
(8). One of the defining morphologic features of these cells
is the presence of cytoplasmic processes, which pass through
STEOBLASTS synthesize and mineralize the bone matrix (l-3). They develop from mesenchymal stem cells
and undergo further differentiation to either lining cells or
osteocytes (l-5). On the basis of in vi&o studies with primary
rat osteoblasts, Stein and Lian (4) have divided osteoblast
differentiation into three phases: proliferation, maturation,
and mineralization. Proliferation is characterized by high
level expression of marker proteins such as c-fos, histone H4,
transforming growth factor (TGF)$l, and type I collagen. As
the cells stop proliferating and enter the maturation phase,
the levels of these proteins decline, whereas the expression
of alkaline phosphatase, osteopontin, and other marker proteins increases. As the cells continue to differentiate and enter
the mineralization phase, the levels of the proteins associated
with maturation decline, and the expression of osteocalcin
and the formation of hydroxyapatite become evident. Thus,
based on these studies, the stage of differentiation
of an
osteoblast in vitro can be estimated by determining the relReceived
April 12, 1996.
Address
all correspondence
and reprint
requests
to: Dr. Peter V. N.
Bodine, Women’s
Health Research
Institute,
Wyeth-Ayerst,
145 King of
Prussia
Road,
Radnor,
Pennsylvania
19087. E-mail:
bodinepawar.
wyeth.com.
* Portions
of this work were presented
in abstract
form at the 17th
Annual Meeting
of the American
Society for Bone and Mineral
Research
in Baltimore,
Maryland,
1995.
4592
CONDITIONALLY
TRANSFORMED
channels or canaliculi in the mineralized bone (8). In preosteocytes, these protrusions are tubular or finger-like (9), but
in mature osteocytes, the structures are stellate or dendritic
(10). When mature osteocytes are entombed within mineralized bone, the cellular extrusions contained within the
canaliculi form a network of intercellular contacts through
gap junctions (8,9). This network is formed in vivo not only
among mature osteocytes but also between these cells and
preosteocytes, osteoblasts, and lining cells (1, 8,9). Moreover,
osteocytes isolated from chick calvaria form a similar elaborate network in vitro (10). Although the function of the
osteocyte network is not completely understood, it has been
proposed to play a role in regulating bone turnover, in ion
exchange, and to act as a mechanosensory system for physical stress adaptation (8, 11-13). In addition to a specific
morphology and location in mineralized bone, osteocytic
cells have also been shown to express CD44 (14, 15), high
extracellular levels of casein kinase II and ecto5’-nucleotidase (16), and estrogen receptors (17). Ironically, although
osteocytes are the most abundant cell type in mature bone
(11, 12), little is known about the biological properties of
these cells. A major limitation to the study of osteocytes is
that there are no immortalized clonal cell lines that faithfully
exhibit this phenotype in vitro. Instead, research on osteocytes has typically been done either with isolated mammalian bone samples or with primary cultures of chick or rat
calvaria-derived cells (8-16).
We have developed a series of conditionally transformed
adult human bone cell lines that appear to contain representatives of each stage of osteoblast differentiation. These
cells were immortalized with adenovirus-ori- SV40 tsA 209
(18, 19). This hybrid virus encodes for a temperature-sensitive mutant form of the large T-antigen (20) and was designed for optimal conditional transformation of human cells
(18). Cells immortalized with this large T-antigen mutant
proliferate and express a transformed phenotype at the permissive temperature of 34 C when the mutant protein is
active but revert to a nontransformed phenotype at the nonpermissive temperature of 39-40 C when the mutant protein
has been inactived (18-20). Using this approach, we previously reported the development of the HOB-OZCl cells, an
osteoblastic cell line in the maturation stage of differentiation
(21). In this communication, we describe the establishment
and hormonal regulation of the first conditionally transformed bone cell line with a preosteocytic phenotype: the
HOB-Ol-Cl cells (human osteoblast-isolation 01-clone 1).
Materials
and Methods
Materials
Except
where
noted, tissue culture
reagents
were purchased
from
GIBCO-BRL
(Grand
Island,
NY); other reagents
and chemicals
were
obtained
from Sigma (St. Louis, MO) or VWR (Philadelphia,
PA). Aqueous reagents
were prepared
with biotechnology
grade water produced
by a Millipore
Milli-Q
UF Plus System
(Marlborough,
MA).
Development
and maintenance
of the cell line
Normal
cancellous
bone fragments
were obtained
from a femoral
neck of an 82-yr-old
woman
who had undergone
hip replacement
surgery. Primary
bone cell cultures
were established
as follows.
The fragments were dissected
to remove
soft tissue and cortical
bone and were
HUMAN PREOSTEOCYTIC
CELLS
4593
minced
into small pieces (-3 X 3 X 1 mm). The trabecular
bone chips
were washed
with HBSS and subjected
to two 20-min
digestions
at 37
C with 2 mg/ml
of collagenase
solution
containing
0.25% (wt/vol)
trypsin
(in HBSS); this was followed
by a third ZO-min digestion
in the
absence
of trypsin.
Cells isolated
from the last two digestions
were
pooled,
pelleted
by centrifugation,
resuspended
in isolation
medium
[D-MEM/F-12
containing
10% (vol/vdl)
heat-inactivated
FBS, 630
pg/ml
Penicillin
G, 50 pg/ml
Gentamycin
sulfate, and 0.3 pg/ml
Fun@zone],
and plated into a T-75 flask. After an overnight
incubation
at 37
C in a 5% CO,/95%
humidified
air incubator
(Forma Scientific,
Marietta,
OH), the cells were washed
with HBSS, trypsinized
(0.25%, wt/vol),
and
seeded at approximately
200,000 cells per flask into T-25 flasks. After the
cells had settled, the flasks were incubated
at 34 C with isolation
medium
containing
O-75 plaque forming
units (PFU) per cell of adenovirus-ariSV40 tsA 209, as previously
described
for the tsA 209 SV40 virus (22).
Transformed
cells were incubated
at 34 C in a 5% CO,/95%
humidified
air incubator,
and colonies
were detectable
after about 6 weeks. Although
nontransformed
cells did proliferate
at 34 C, they did not form
colonies
(i.e. they exhibited
contact inhibited
growth).
During
the transformation
process,
many of the cells did not survive,
and the remaining
colonies
th‘at were well separated
and appeared
to arise from a single ten
were isolated
with alass cloning
cvlinders
(Bellco,
Vineland,
NT) and
trypsinized.
Each cotbny was tra&&red
to a’well of a 24.well plate and
incubated
at 34 C.
The HOB-Ol-Cl
cells were cloned from a flask that had been infected
with 75 PFU / cell of virus. Immunocytochemistry
for the T-antigen
indicated
that essentially
all of the cells expressed
the tsA 209 mutant
protein
at both middle
and late passages (21,23,24),
which implied
that
the cells were derived
from a single transformed
cell (data not shown).
Cultures
were routinely
maintained
in vented T-75 or T-175 flasks at 34
C using growth
medium
[D-MEM/F-12
containing
10% (vol/vol)
heatinactivated
FBS, 1% (vol/vol)
Penicillin-Streptomvcin
and 2 mM GlutaMAX-I].
The cells were pa&aged
at a ratidof
l:i once a week using
a solution
of trypsin-ethylenediaminetetraacedic
acid (0.05%, wt / vol,
and 0.53 mM, respectively).
Frozen stocks of cells (l-2 X 106/ml)
were
maintained
at I -150 C in growth
medium
containing
20% (vol/vol)
heat-inactived
FBS and 10% (vol/vol)
dimethylsulfoxide.
Cell viability
from the frozen stocks was estimated
to be >60%. The HOB-Ol-Cl
cells
proliferated
at 34 C for 15-20 passages
before reaching
crisis, at which
point the cells stopped
dividing.
Consequently,
all experiments
were
performed
with precrisis
cells. Before crisis, the cells appeared
to retain
a stable phenotype
(as determined
by the basal expression
and hormonal
regulation
of osteoblastic/osteocytic
markers).
Crisis is a common
phenomenon
with SV40-immortalized
human
cells, and transformation
typically increases
the life span of a cultured
human
cell by only 20-30
population
doublings
(25).
For comparison,
control
experiments
were performed
with explant
cultures
of nontransformed
human
osteoblasts
isolated
as previously
described
(21,26)
from cancellous
bone samples
obtained
from either a
77-yr-old
or a 30-yr-old
woman.
Electron
microscopy
Cells in growth
medium
were seeded
at 500,000
cells/well
onto
22-mm
diameter
Thermanox
coverslips
(Nunc)
in 6-well
plates
(-53,00O/cm*,
high density
to maximize
cell number)
and incubated
at
34 C for a day. The medium
was changed,
and the cells were incubated
at 39 C for 2 days. The coverslips
were washed
on ice with cold PBS, and
the cells were fixed sequentially
with 3% (vol/vol)
glutaraldehyde
in 0.1
M sodium
cacodylate,
pH 7.4, and 1% (wt/vol)
0~0, in the same buffer.
The cells were stained
with one-half
saturated
uranyl
acetate in 50%
(vol/vol)
ethanol
during
dehydration
in a graded
ethanol
series. The
coversliDs
were embedded
in Polv/Bed
812-Araldite
502 (Polvsciences,
Warriniton,
PA). After being re&ounted
on cylindrical
s&b;,
the cells
were sectioned
parallel
to the coverslip
surface,
and thin sections
(~90
run) were stained with Reynolds’
lead citrate (27). Grids were examined
in a JEOL 1OOCX electron
microscope
(Peabody,
MA) using accelerating
voltages
of 60 or 80 kV.
Measurement
Cells in growth
plates (-5000/cm’,
of cellular
proliferation
medium
were
low density
seeded at 10,000 cells/well
into 24-well
to optimize
cell proliferation)
and in-
CONDITIONALLY
TRANSFORMED
cubated for 2 days at 34 C. Four wells in each plate were washed
with
calcium/magnesium-free
PBS, pH. 7.4, trypsinized
(0.05%, wt/vol),
and
counted with a hemacytometer.
The remaining
cells were then incubated
at either 34 C or 40 C for up to 17 days. Cell number
was determined
periodically,
and the medium
in the remaining
wells was changed
twice
a week. Cell viability
after trypsinization
was >70% at 34 C and >60%
at 40 C (determined
by trypan blue dye exclusion).
Proliferation
studies
were also performed
with a Coulter
Multisizer
IIe system
(Coulter
Corporation,
Miami,
FL) as previously
described
(21). For these experiments, cells in growth
medium
were seeded at 50,000 cells/well
into
six-well
plates (-5000
/cm’).
RNA analysis
T-75 flasks containing
approximately
60,000 cells/cm’
(high density
to maximize
cell number)
were incubated
for 72 h in growth
medium
at
either 34 C or 40 C. The flasks were rinsed with PBS, and total cellular
RNA was isolated
using TRIzol
according
to the manufacturer’s
instructions
(GIBCO-BRL)
(28), but with modifications
described
previously (29). Five micrograms
of each RNA sample
were analyzed
by
Northern
hybridization
as previously
described
(21). The blots were
quantified
with a Molecular
Dynamics
Phospholmager
(Sunnyvale,
CA).
Alkaline
phosphatase
and protein
assays
Cells in experimental
medium
[phenol
red-free
DMEM/F-12
containing 10% (vol/vol)
heat-inactivated
charcoal-stripped
FBS (HyClone,
Logan
UT), 1% (vol/vol)
Penicillin-Streptomycin,
and 2 mM GlutaMAXwere seeded at 75,000 cells/well
into 24-well plates (-38,000/
cm’, moderate
density)
or 15,000 cells/ well into 96-well plates (-46,000/
cm’) and incubated
at 34 C overnight.
Seeding density
did not appear
to have a significant
effect on the phenotype
of these cells (this was also
true for the HOB-OZCl
cells; 21). The cells were washed
with PBS, BSA
medium
[phenol red-free
DMEM/F-12
containing
0.25% (wt/vol)
BSA
(Pentex crvstallized
BSA, Miles Laboratories,
Kankakee,
IL), 1% (vol/
vol) Penicillin-Streptomycin,
2 mM GlutaMAX-I,
50 pg/ ml ascorbicacid,
and 10 nM menadione
sodium
bisulfite
(vitamin
Ks)] was added to each
well (1 m1/24-well
or 0.2 m1/96-well),
and the cells were incubated
at
either 34 C or 40 C for 24 h. The medium
was changed,
and the cells were
treated with either vehicle
(ethanol,
O.l%, vol/vol),
or 0.1-100 nM l~u,25dihydroxyvitamin
Da (vitamin
Ds) (Calbiochem,
La Jolla, CA) for 48 h
at either 34 C or 40 C. At the end of hormonal
treatment,
the conditioned
media were saved and stored at -80 C. The cells were then washed
with
PBS and processed
for alkaline
phosphatase
activity
determination
as
previously
described
(21). The remaining
lysate was used for protein
determination
with bicinchoninic
acid (BCA) as described
(21, 30).
HUMAN PREOSTEOCYTIC
CELLS
Endo. 1996
Vol 137 . No 11
staining
as previously
described
(21,33). To quantify
the level of alizarin
red-S histochemical
staining,
cells in growth
medium
were seeded at
20,000 cells/well
into 96-well plates (-63,000
/cm’)
and allowed
to proliferate
at 34 C overnight.
The medium
was changed
to one of the five
listed above, and the cells were either returned
to 34 C or incubated
at
40 C for 2 days. At the end of the treatments,
quantitative
alizarin
red-S
histochemical
staining
was determined
as previously
described
(21,32).
Replicate
wells were washed
with PBS before protein
determination.
Measurement
of intracellular
CAMP
Cells in experimental
medium
were seeded at 75,000 cells/ well into
24-well plates (-38,OOO/cm’)
and incubated
at 34 C overnight.
The cells
were washed
with PBS, BSA medium
was added to each well, and the
cells were incubated
at either 34 C or 40 C for 48 h. The medium
was
changed,
and the cells were pretreated
with 0.5 mM isobutylmethylxanthine
(IBMX)
for 5 min at 37 C. The cells were then treated
in the
presence
of IBMX with either vehicle (ethanol,
O.l%, vol/vol),
l-100 nM
human
PTH (hPTH)
[fragment
l-341,100
nM prostaglandin
E, (PGE,),
or 1 PM forskolin
for 10 min at 37 C. These treatments
were performed
at 37 C to ensure
that enzyme
activity
was constant.
The cells were
washed
with cold PBS, and intracellular
CAMP was extracted
and measured as previously
described
(21,23). Protein was measured
in the dried
extracted
cellular
pellets using BCA.
Western
blot analysis
HOB-Ol-Cl
cells in growth
medium
were seeded at 4 million
cells/
dish into 150-mm
plates (-23,00O/cm*)
and allowed
to proliferate
to
high density
(to maximize
cell number)
for a week at 34 C. The medium
was changed,
and the cells were either returned
to 34 C or incubated
at
40 C for 2 more days. Confluent
loo-mm
dishes of Ishikawa
cells (a
human
endometrial
adenocarcinoma
cell line) incubated
at 37 C, and
two other HOB cell lines (HOB-02-Cl
and HOB-03-C5)
incubated
at 34
C or 40 C for 2 days, were also used (21). The cells were washed
with
PBS, and whole
cell extracts
were prepared
using 1% (vol/vol)
SDS.
Thirty-five
micrograms
of each sample were resolved
by electrophoresis
on 7.5% (wt/vol)
reduced
SDS-PAGE
and analyzed
by Western
blot
using a 1:2000 dilution
of a monoclonal
antibody
to human
CD44H
(hemopoietic
isoform,
R & D Systems)
as previously
described
(34). The
secondary
antibody
(protein
A/ G-horse radish peroxidase;
Pierce, Rockford, IL) was diluted
1:4000, and the blot was developed
using an ECL
chemiluminescence
kit (Amersham,
Arlington
Heights,
IL). SDS-PAGE
mol wt protein
markers
were obtained
from Bio-Rad
(Melville,
NY).
Cytokine
assays
Bone matrix
proteins
were measured
in the conditioned
media using
commercially
available
enzyme-linked
immunosorbant
assays (ELlSAs)
for human
type I procollagen
C peptide
(an indirect
measurement
of
collagen;
PanVera,
Madison,
WI) and intact full-length
human
osteocalcin (Biomedical
Technologies,
Stoughton,
MA). Procedures
were performed
as described
by the manufacturer,
but with modifications
as
previously
described
(21). Protein
was measured
in the PBS washed
cellular
extracts
using BCA.
Cells in growth
medium
were seeded at 15,000 cells/well
into 96-well
plates (-46,OOO/cm’)
and incubated
at 34 C overnight.
The cells were
washed
with PBS, 200 ~1 of BSA medium
was added to each well, and
the cells were incubated
at either 34 C or 40 C for another
24 h. The
medium
was changed,
and the cells were treated with l-1000 PM human
interleukin
(IL)-lp,
or human
tumor
necrosis
factor
(TNF)-(U
(R & D
Systems,
Minneapolis,
MN) for 24 h at either 34 C or 40 C. Cytokines/
chemokines
were measured
in the conditioned
media using- commerciallv available
ELISA kits from R & D Svstems.
Procedures
were oerformed
as described
by the manufacturer
but with modification’s
as
previously
described
(21). Protein was measured
in the washed
cellular
extracts
using BCA.
Determination
Statistical
Bone matrix
protein
assays
of mineralized
nodules
Cells in growth
medium
were seeded at 250,000 cells/ well into 6-well
plates (-27,OOO/cm’)
and allowed
to proliferate
to high density
at 34 C
for a week; this allowed
the cells to establish
a collagen
matrix
for
subsequent
mineralization.
The medium
was changed
to one of the
following:
growth
medium;
growth
medium
plus 50 yglml
of ascorbic
acid; growth medium
plus ascorbic acid and 5 mM P-glycerol
phosphate;
growth
medium
plus ascorbic
acid and 100 nM dexamethasone;
growth
medium
plus ascorbic
acid, P-glycerol
phosphate,
and dexamethasone
(21, 26, 24, 31, 32). The cells were either returned
to 34 C or incubated
at 40 C for 6 days. The medium
was changed
twice during
the course
of the experiment.
At the end of the treatments,
the formation
of in vitro
mineralized
nodules
was determined
by alizarin
red-S histochemical
analysis
The results
are presented
as means
? SD or SEM of three or
determinations
per experiment
and are representative
of two to
similar
experiments
(noted in the figure legends).
The data were
lyzed for statistical
significance
(P < 0.05) by the Behren-Fisher
one-way
ANOVA
using the Dunnett’s
test, or by paired ANOVA
the Tukey-Krammer
test.
more
three
anat test,
using
Results and Discussion
Development
of the cell line
The HOB-Ol-Cl cell line was derived from a primary culture of normal adult human bone cells. One day after iso-
CONDITIONALLY
TRANSFORMED
lation, these cells were infected with adenovirus-ori-
SV40
fsA 209 (19, 20). This hybrid virus contained the origin-defective (ori-) SV40 fsA 209 genome in place of the early type
5 adenoviral genes; consequently, the immortalized cells do
not produce virus (19,21). As shown in Fig. 1, the HOB-Ol-Cl
cells failed to form a monolayer even when grown to high
density at 34 C. This pattern was maintained when the cells
were incubated at 39-40 C, when they were plated on glass
instead of plastic, or when they were plated at high density
(data not shown). In contrast, most other osteoblastic cells,
including normal human osteoblasts (26), HOS-TE85 human
osteosarcoma cells (35), and the conditionally immortalized
human FOB (24) and HOB-OZCl (21) cell lines, form monolayer cultures at high density. For the hFOB and HOB-02-Cl
1. HOB-Ol-Cl
cells do not form monolayer
cultures.
Cells were
seeded with growth
medium
at a density
of 10,000/cm2
into T-75
flasks and allowed
to proliferate
at 34 C. A, Flask of cells after 6 days
in culture;
the cell number
was approximately
30,00O/cm’.
B, Flask
of cells after 12 days in culture;
the cell number
was approximately
60,000/cm2.
The cells in both panels were photographed
under phasecontrast
at 100 X magnification.
HUMAN PREOSTEOCYI’IC
CELLS
4595
cells, a monolayer phenotype is maintained at both the permissive and nonpermissive temperatures (21, 24). Finally,
when similar studies were performed with nonvirally transformed human osteoblasts, these cells also formed monolayers at 34 C, 37 C, and 40 C (data not shown; Table 1). As
described in the following discussion, the HOB-Ol-Cl cells
have a morphology and phenotype resembling a preosteocytic (i.e. osteoid-osteocyte-like) cell.
Formation
of cellular
processes
and gap junctions
A defining characteristic of osteocyte morphology is the
formation of cytoplasmic processes or extrusions (8). In
preosteocytic cells, these structures are tubular or finger-like
(9), whereas in the mature osteocyte they are stellate or
dendritic (10). When osteocytes become embedded within
the bone, these cellular protrusions form canaliculi as mineralized matrix is deposited around them (8,9). In turn, the
cellular processes within the canaliculi form a network of
intercellular contacts by way of gap junctions (1, 8, 9). To
determine if the HOB-Ol-Cl cells formed these processes,
transmission electron microscopy (TEM) was performed on
cultures of cells that had been incubated for 48 h at the
nonpermissive temperature (39 C). Figure 2A shows an electron micrograph of a single HOB-Ol-Cl cell that formed
many tubular-looking cellular processes that emanated in a
radial fashion from the cell. These cells also revealed a high
nucleus to cytoplasm ratio, which is characteristic of the
osteocytic phenotype (8). The dimensions of this cell were
approximately 21 X 32 pm. Figure 2B depicts two adjacent
cells that have contacted each other through long cellular
processes that spanned a distance of 7-8 pm. These processes
formed a side-to-side contact (identified by the bracket in B
and magnified in C, which was suggestive of structures
observed between preosteocytes and osteocytes in newborn
rabbit bone (9). As demonstrated in Fig. 2, D and E, the
cellular processes between some cells appeared to form gap
junctions (designated by the arrows); the length of the gap
junction shown in E was approximately 0.2 pm. Additional
examples of gap junction formation between cytoplasmic
processes were also observed (data not shown). Osteoblasts
also form gap junctions with each other (l), but these do not
occur between cellular processes. As shown in Fig. 2F, some
of these cytoplasmic extrusions were rich in microfilamentlike structures, which is another characteristic of osteocyte
protrusions (8). The width of this cellular process was about
0.35 pm, and it appeared to contain many longitudinal microfilaments. Finally, the presence of extracellular collagen
fibrils was also observed (data not shown). From these results, it appeared that the HOB-Ol-Cl cells exhibited a morphology that was consistent with that of a preosteocytic cell
(9). TEM studies of the HOB-02-Cl cell line (21) demonstrated that these mature osteoblasts did not posses long
cellular processes like the ones observed with the HOB-Ol-Cl
cells (data not shown).
FIG.
Temperature
control
of cellular
proliferation
Cell growth experiments were performed to determine if
the fsA 209 mutant T-antigen was functioning properly and
to characterize this aspect of the bone cell phenotype. As
CONDITIONALLY
4596
TABLE
1. Summary
of HOB-Ol-Cl
cell and
TRANSFORMED
nontransformed
HOB-01X1
Property
34 c
Monolayer
T-antigen
Division
TIPCCP
AP+VD,
oc+vD,
AR-s+vc
cAMP+PTH
No
-100%
-5 days
LO-fold
2.1-fold
5.7-fold
3.3-fold
l.O-fold
human
HUMAN
PREOSTEOCYTIC
osteoblast
No
-100%
No division
1.6-fold
4.7-fold
32.5fold
3.9-fold
18.3-fold
Nontransformed
Ratio1
34 c
HOB
Cells
37 c
Yes
0%
-7 days
ND
1.4-fold
2.8-fold
ND
ND
1.0
0.0”
1.6”
2.2
5.7”
1.2
18.3”
Endo. 1996
Vol 137 . No 11
properties
Cells
40 c
CELLS
40 c
Yes
0%
-6 days
ND
2.1-fold
4.1-fold
ND
ND
Ratio’
Yes
0%
-7 days
ND
1.5-fold
3.9-fold
ND
ND
0.9
ND
1.5
1.5
ND
ND
Monolayer:
Phenotype
of the cultures
as determined
by light microscopy.
T-Antigen:
SV40 tsA 209 mutant
T-antigen
expression
as the
percentage
of cells stained
with a monoclonal
antibody
for the wild-type
protein
(Materials
and Methods).
Diuision:
Doubling
time of the cells
(Fig. 3). TIPCCP,
Fold increase
in type 1 procollagen
C-peptide
secretion
after 48 h (Fig. 4). AP+VD,,
Fold increase
in alkaline
phosphatase
activity
after 48 h of 100 nM vitamin
D, treatment
(Fig. 5). OC+VD,,
Fold increase
in osteocalcin
secretion
after 48 h of 100 nM vitamin
D,
treatment
(Fig. 5). AR-S+VC,
Fold increase
in quantitative
alizarin
red-S staining
after 48 h of 50 pg/ml
ascorbic
acid treatment
(Fig. 6).
cAMP+PTH,
Fold increase
in intracellular
CAMP levels after 10 min of 10 nM parathyroid
hormone
treatment
(Fig. 7). HOB, Human
osteoblast;
Ratio?
40 C/34 C; Ratio2, 37 C/34 C; ND, not determined;
a, statistically
significant
difference
(refer to the corresponding
figures).
shown in Fig. 3, the HOB-Ol-Cl
cell line exhibited
exponential proliferation
at 34 C with a doubling
time of about 4-6
days (A). However,
no growth was detected after the cells
were incubated
at 40 C. When the cells were placed at 40 C
for 9 days and then returned
to 34 C for 6 more days, no
additional
proliferation
was observed
(B); this result suggested that inactivation
of the tsA 209 T-antigen
initiates an
irreversible
process. When cellular proliferation
experiments
were performed
with nonvirally
transformed
human osteoblasts, the growth
rates of these cells were essentially
the
same at 34 C, 37 C, or 40 C (Table 1). From these experiments,
it appeared that the temperature-sensitive
T-antigen
mutant
was working
as expected and that the HOB-Ol-Cl
cells resembled a preosteocyte
(2) because cell growth ceased at the
nonpermissive
temperature.
Temperature
regulation
of type I collagen
expression
Type I collagen is the major secretory product
from cells
of the osteoblast / osteocyte lineage and comprises 90% of the
bone organic matrix (l-3). As depicted in Fig. 4A, Northern
hybridization
of total RNA indicated
that the HOB-Ol-Cl
cells expressed a1 type (I) procollagen
mRNAs at both 34 C
and 40 C (36-38). When these results were normalized
to
glyceraldehyde
phosphate
dehydrogenase
message to correct for variations
in loading, the levels of type I procollagen
mRNA appeared
to increase about l&fold
after the cells
were incubated
at the nonpermissive
temperature
for 72 h.
This observation
was corroborated
by ELISA studies that
showed that the secretion of type I procollagen
C peptide into
the culture medium
increased
60% when the cells were incubated at the higher temperature
for 48 h (Fig. 48). Thus,
collagen expression was enhanced at the nonpermissive
temperature. Although
the HOB-Ol-Cl
cells expressed moderate
levels of type I procollagen
mRNA
(Fig. 4A), only trace
amounts of type III procollagen
message were detected (<
0.5% of the (Ye type (I) 5.0 kb mRNA; data not shown). By
comparison,
about 5% of the collagen
released by bonederived fibroblasts
is type III (26). The level of type I procollagen C peptide secreted by the HOB-Ol-Cl
cells at 40 C
was 60-70% less than that observed for the maturation
stage
HOB-02-Cl
cell line (-2250 ng/ mg cell protein/
48 h at 40 C)
(21). This observation
was therefore
consistent
with
the HOB-Ol-Cl
differentiation.
cells
Vitamin
D, regulation
osteocalcin expression
being
of alkaline
in
an
advanced
phosphatase
stage
of
and
Another
marker for the osteoblast / osteocyte phenotype
is
the expression
of alkaline phosphatase,
and the levels of this
enzyme are increased by vitamin
D, (l-3). Consequently,
the
effect of this seco-steroid on alkaline phosphatase production
by
the HOB-Ol-Cl
cells was analyzed.
As shown
in Fig. 5A, basal
levels
of
cellular
enzyme
activity
were
very
low
(0.01-0.03
nmol/mimmg
cell protein) and did not vary significantly
when
the cells were incubated at either of the two temperatures.
This
basal level of enzyme activity was approximately
100 times less
than that reported for the HOB-02-Cl
cells (l-2 nmol/mirmg
cell protein at 40 C; 21), approximately
500 times less than that
observed for nonvirally
transformed
human osteoblasts (4-5
nmol/min
. mg cell protein at 37 C; data not shown), and approximately
1000 times less than that reported for a wild-type
large T antigen-transformed
adult human osteoblastic cell line
(HOBIT; 23). Vitamin D, (0.1-100 nM) up-regulated
cellular alkaline phosphatase
activity in a dose-dependent
manner after
48 h of treatment
at both 34 C and 40 C. The stimulation
of
enzyme
activity
by
100
nM
vitamin
D,
appeared
to be greater
when the cells were maintained
at the nonpermissive
temperature (4.7- ZIS.2.1-fold), but this result may have been a function
of decreased basal expression at 40 C.
Osteocalcin
is an abundant
noncollagenous
bone matrix
protein, and vitamin D, regulation
of its expression
is one of
the hallmarks
of the osteoblast/osteocyte
lineage (l-3). As
depicted
in Fig. 5B, 0.1-100 nM vitamin
D, stimulated
the
secretion
of full-length,
intact osteocalcin
from the HOBOl-Cl cells in a dose-dependent
manner after 48 h of treatment at both temperatures.
When the cells were maintained
at 34 C, 100 nM vitamin
D, affected a moderate
5- to 6-fold
increase in osteocalcin
secretion
into the culture medium.
However,
this effect was dramatically
enhanced
when the
cells were incubated
at 40 C. At the nonpermissive
temperature, 100 nM vitamin D, treatment resulted in a 32- to 33-fold
increase in osteocalcin
secretion from the cells. Furthermore,
the level of this bone matrix protein in the cell conditioned
medium
was 14-15 times greater at the higher temperature
CONDITIONALLY
TRANSFORMED
HUMAN PREOSTEOCYTIC CELLS
4597
(-14.5 vs. -1 ng/ mg cell protein
with 100 nM vitamin Ds).
This level of osteocalcin secretion was comparable with that
observed for the HOB-02-Cl cell line (-18 ng/mg cell protein/48 h with 100 nM vitamin D, at 40 C; 21), and was two
times greater than that obtained by nonvirally transformed
human osteoblasts (-8 ng / mg cell protein/ 48 h with 100 nM
vitamin D, at 37 C; data not shown). When similar experiments were performed with nonvirally transformed human
osteoblasts, no significant differences were observed in the
secretion of osteocalcin from cells treated with vitamin D, at
34 C, 37 C, or 40 C (Table 1).
When compared with those obtained with the HOB-02-Cl
cell line (21), these biochemical results indicated that the
HOB-Ol-Cl cells were in an advanced stage of differentiation. Although the HOB-Ol-Cl cell line expressed very low
basal levels of alkaline phosphatase, these cells secreted high
amounts of osteocalcin in response to vitamin D, treatment.
Primary cultures of fetal chick calvaria-derived osteocytes
were also reported to express lower alkaline phosphatase
levels than the corresponding osteoblasts (39), whereas osteocytes isolated from newborn rat calvaria did not appear
to express this enzyme (16). According to the in vitro
primary rat osteoblast differentiation
model of Stein and
Lian (6), the HOB-Ol-Cl cells appeared to be in the mineralization stage of differentiation.
Alternatively,
when
compared with the human bone in situ studies of Gehron
Robey et al. (2), these cells would be characterized as
preosteocytic or osteoid-osteocyte-like.
Formation
FIG. 2. HOB-01-U
cells display
finger-like
cellular
processes
that
form gap junctions
and are rich in microfilaments.
Cells were incubated at 39 C (nonproliferative
temperature)
for 2 days and processed
for transmission
electron
microscopy
as described
in Materials and
Methods.
The bar in each panel is equal to a distance
of 1 pm unless
noted otherwise.
A, Single cell with many finger-like
protrusions.
B,
Two adjacent
cells contacting
each other through
long cellular
processes. C, A higher
magnification
photomicrograph
of the cellular
processes
in B forming
a side-to-side
contact
(bar, 0.1 pm). D, Two
adjacent
cells contacting
each other through
short cellular
processes.
E, Higher
magnification
photomicrograph
of the cellular
processes
in
D forming
a gap junction
(bar,
0.1 pm). F, Higher
magnification
photomicrograph
of a cellular
protrusion
that is rich in microfilamentlike structures
(bar, 0.1 pm). N, Nucleus;
Nu, nucleolus;
M, mitochondrion;
R, rough endoplasmic
reticulum.
of mineralized
nodules
Cells of the osteoblast / osteocyte lineage not only synthesize the bone matrix, but mineralize it as well (26, 31). One
method for analyzing mineralized nodule formation in vitro
is alizarin red-S histochemical staining (21, 32, 33). Alizarin
red-S is a dye that selectively binds to calcium salts (32,33).
For these studies, cultures of HOB-Ol-Cl cells were incubated at 34 C or 40 C in the presence of ascorbic acid (50
pg/ml) (21,26,24,31,32)
and then stained for the formation
of mineralized nodules. As shown in Fig. 6, A and C, mineralization was detected at both temperatures after 6 days in
culture, and these cells produced intensely stained nodules
(indicated by the UTYOWS).
To quantify the alizarin red-S histochemical staining, the
experiments presented in Fig. 6, B and D, were performed
(21,32). For these studies, the effect of ascorbic acid, /3-glycerol phosphate, and dexamethasone on the rate of mineralization was also examined (21, 26, 24, 31, 32). Significant
levels of mineralized matrix were detected after a l-day
incubation at 34 C (-200 nmol dye/mg cell protein on day
0). After only 2 additional days in culture at either 34 C or
40 C, mineralized matrix formation increased 2- to 4-fold.
When the cells were maintained at the permissive temperature, the most mineralization occurred in the presence of
ascorbic acid, P-glycerol phosphate, and dexamethasone; on
the other hand, ascorbic acid alone was sufficient to produce
the best level of mineralization when the cells were incubated
at the nonpermissive temperature. However, mineralization
was detected at either temperature in the presence or absence
of ascorbic acid, P-glycerol phosphate, and dexamethasone.
CONDITIONALLY
4598
TRANSFORMED
HUMAN PREOSTEOCYTIC
55000
55000
50000
50000-
45000
45000-
40000
40000-
00
35000
35000-
30000
30000-
25000
25000-
20000
20000-
s
,o
I
c
f
iG
6
t
15000
15000-
10000
'OfJOO-
5000
CELLS
Endo. 1996
Vd 137 l No 11
I
-B
__
7
.
/
--o-
,+-----
40/340(:
_____
'
--
*
--;--;
50004f
!
0
2
4
6
DAYS
8
10
12
14
16
18
of . I . I . I.
0
2
4
IN CULTURE
6
DAYS
I * 1 . I . I . I.
8
10
12
14
16
c
!
18
IN CULTURE
cellsproliferate at the permissivetemperature, but stop dividing at the nonpermissive temperature. Cells were seededwith
medium and allowed to proliferate for 2 days at 34 C. The cells were then incubated
at either 34 C or 40 C for 15 more days (A) or were
tncubated at 40 C for 9 days and then returned
to 34 C for 6 additional
days (B). Cell number
was determined
periodically
as described
in
3. HOB-Ol-Cl
growth
MaterialsandMethods.TheresultsarepreSentedasthe
mean?
SD,n=$
*, P < 0.005-0.001
from
the corresponding
34 C cells
(Behren’s-Fisher
t test). The cellsexhibited an exponential growth rate at 34 C with a doubling time of approximately 5 days (r = 0.965 for the fit to an exponential
curve, i.e. the solid line in A). The data are representative of three similar experiments.
Similar results were also obtained with the HOB-O2-Cl cells
(21). In addition, UMR 106-01 BSP cells have been reported
to form mineralized matrix in the absence of ascorbic acid
(32), whereas hFOB cells mineralized in nonsupplemented
growth medium (24). On the other hand, in vitro mineralization has been reported to be facilitated by these agents in
cuItures of primary mammahan osteoblasts (6, 26, 31). To
validate this method, we performed quantitative ahzarin
red-S histochemical staining with HOS-TB85 human osteosarcoma cells (35); as expected, these cells did not form a
mineralized matrix in the presence or absence of ascorbic
acid, p-glycerol phosphate, or dexamethasone (data not
shown).
Because ahzarin red-S binds approximately 2 mol calcium/mol dye (32), the results presented in Fig. 6 indicated
that the HOB-Ol-Cl cells concentrated about 800-1600 run01
calcium per mg of cellular protein after 2 days in culture. This
level of matrix mineralization was two to three times higher
than that reported for the HOB-02-Cl cell line (400 - 600 nmol
of caIcium/mg of cell protein/48 h at 40 C; 21). This observation was therefore consistent with the hypothesis that the
HOB-Ol-Cl cells were in the mineralization stage of osteoblast differentiation (6).
Temperature
control
of PTH
responsiveness
Another characteristic of the osteoblastl osteocyte phenotype is the expression of PTI-I receptors, and PTH stimulates
the production of cAMI’ in these cells (l-3). As shown in Fig.
7, a IO-min treatment at 37 C with l-100 m4 hPTH (fragment
l-34) failed to stimulate an increase in intracellular cAMP
levels when the HOB-Ol-Cl cells were maintained at the
permissive temperature of 34 C. However, 10 and 100 IZM
hPTH (l-34) stimulated an l&fold and 14fold increase, respectively, in cAMP production after the cells were preincubated at the nonpermissive temperature for 48 h. Conversely, 100 IIM PGE, or 1 w forskohn stimulated a 3 to
11-fold increase in CAMP production when the cells were
preincubated at either 34 C or 40 C. In fact, the fold stimulation by these agents was actually better when the cells were
maintained at the lower temperature, although this may have
been due in part to increased basal cAMP production at the
higher temperature. Primary fetal chick osteocytes were also
reported to respond to PTH (39), and in situ studies of rat
osteocytes indicated that these cells possess PTH binding
sites (40). A summary of the results obtained thus far with the
HOB-01-Cl cells is presented along with a comparison to
nontransformed human osteoblasts in Table 1.
CD44 expression
CD44, or the hyaluronate receptor, has been proposed to
be a marker of the osteocytic phenotype (14,15). CD44 is a
37-kDa transmembrane protein that contains both N- and
O-linked carbohydrates; consequently, it migrates on SDSPAGE with an apparent mass of SO-100 kDa (41,42). CD44
is expressed by a variety of tissues and cell types incIuding
osteocytes and osteoclasts (14, 15, 41, 42). The function of
CD44 in bone cells is unknown, but it may be involved in
cellular attachment to hyaluronan, type I collagen, and fibronectin (15). Hyahnonan is a very large (-1,400 kDa) nonsulfated glycosaminoglycan that is synthesized by human
osteoblasts and is a component of human bone (2,43). Although its role in bone is also unknown, hyaluronan may be
involved in regulating cellular proliferation and differentiation (2).
CONDITIONALLY
TRANSFORMED
5.8 KB
5.0 KB
(I) PROCOLLAGEN
GAPDH
1AKB
I
PREOSTEOCYTIC
CELLS
4599
the maturation stage HOB-02-Cl cells (21) and the proliferative stage HOB-03-C5 cells (work in progress). As shown in
Fig. SB, the three HOB cell lines expressed varying levels of
CD44H at both the permissive and nonpermissive temperatures. HOB-02-Cl cells appeared to contain the highest
amount of this protein whereas HOB-03-0 cells synthesired very low levels of CD44H. These results suggested that
CD44 expression in vitro was not limited to cells with an
osteocytic phenotype. This conclusion is supported by in situ
A
a 1 TYPE
IIUMAN
I
34
&‘I-FOLD
40
TEMPERATURE
NORMALIZED:
1.0 1.8
2.1-FOLD
(“c)
l.WWD
*
T-
_
*
_
T
I
[VITAMIN
4. HOB-Ol-Cl cells express more type I procollagen at the nonpermissive temperature. A, Confluent T-75 flasks of cells were incubated at either 34 C (proliferative temperature) or 40 C (nonproliferative temperature) for 72 h. Total RNA was isolated from the cells
and electrophoresed on agarose gels. Northern hybridizations
were
performed with [3zPldCTP-labeled cDNA probes for rat al type (I)
procollagen (36-38) and rat glyceraldehyde phosphate dehydrogenase (GAPDH) as described in Muterids
and Methods.
The blots were
quantified using a Molecular Dynamics PhosphoImager SI, and the
type I procollagen results were normalized to GAPDH (shown at the
bottom of A). The normalizations
of the two al messages were the
same; the ratio of the 5.8~kb mRNA to the LO-kb mRNA was approximately 1:180. B, Cells were incubated in BSA medium at either 34
C or 40 C for 48 h. Secreted type I procollagen C-peptide was measured
in the conditioned media, and cellular protein was determined, as
described in Materids
and Methods.
The results are presented as the
mean 5 SD, n = 7; *, P < 0.001 from the 34 C cells (Dunnett’s ANOVA
test). The data are representative of two similar experiments.
Dd
(nM)
FIG.
As shown in Fig. SA, Western blot analysis of whole cell
extracts demonstrated that HOB-01-Cl cells expressed the
hemopoietic isoform of CD44 (CD44H) as an approximately
%&Da protein. This isoform is associated with mesenchymal-derived cells (42), and it was present in HOB-Ol-Cl cells
incubated at either 34 C or 40 C. In contrast, Ishikawa cells
(a human endometrial adenocarcinoma cell line) did not
express CD44H.
To test the hypothesis that CD44 expression is a specific
marker of osteocytic cells (14,15), Western blot analysis was
performed with extracts from two additional
cell lines:
HOB
0
0.1
1
(VITAMIN
10
D$
100
(nM)
5. Vitamin Dz regulation of cellular alkaline phosphatase activity and osteocalcin secretion is enhanced at the nonpermissive
temperature. HOB-Ol-Cl cells were treated with BSA medium contaiuing either vehicle (ethanol, O.l%, vol/vol) or 0.1-100 no la,25
dibydroxyvitamin
D, (vitamin D,) for 48 h at either 34 C (proliferative
temperature) or 40 C (nonproliferative
temperature). Cellular alkaline phosphatase activity (A), secreted intact, full-length osteocalcin
(ES), and protein we= measured as described in Materials
and Methods. The results are presented as the mean + SD, n = 4; *, P <
0.05-0.001 from the 34 C or 40 C cells that did not receive treatment
(Behron’s-Fisher
t test). The data are representative
of two to three
similar experiments.
FIG.
CONDITIONALLY
4600
TRANSFORMED
HUMAN PREOSTEOCYTIC CELLS
Endo l 1996
Voll37 l No 11
E
*z ilOO5
hlooo-
*B
4.2.FOLD
*
3.9.FOLD
8
3
E
S
v)
x
600
600
440
300
&
z
200
3
100
=j'ii
4
0
0
DAYS IN CULTURE (34%)
0
2
DAYS IN CULTURE (40°C)
6. HOB-Ol-Cl cells form mineralized nodules at both temperatures. A and C, Cells were incubated in growth medium plus 50 &ml of
ascorbic acid at either 34 C (proliferative temperature) (A) or 40 C (nonproliferative
temperature) (C) for 6 days. Mineralized nodule formation
was determined by alizarin red-S histochemical staining as described in Materials and Methods. The cells in both panels were photographed
under phase-contrast at 200 X magnification. B and D, Cells were incubated at either 34 C (B) or 40 C (D) for 2 days in one of the following
media: growth medium (GM), growth medium plus 50 pglml of ascorbic acid (GM/W), growth medium plus ascorbic acid and 5 mM P-glycerol
phosphate (GM/VC/pGP), growth medium plus ascorbic acid and 100 nM dexamethasone (GMAKYDEX), growth medium plus ascorbic acid,
P-glycerol phosphate, and dexamethasone (GMAKY~GPIDEX).
Q uantitative alizarin red-S histochemical staining was performed as described
in Materials and Methods. The results are presented as the mean ? SEM, n = 4-47; *, P < 0.01-0.001 from the day 0 (34 C) cells (Dunnett’s
ANOVA test). No significant differences were observed among the various conditions of the day 2-40 C group; however, among the day $34
C group, the GM/VC@GP/DEX treatment was different from the GMYVUDEX and GMiVCI~GP treatments (P < 0.05, Tukey-Krammer ANOVA
test). The data are representative of two similar experiments.
FIG.
of rat tibia, which showed that osteoblasts express
CD44 immunoreactivity
on their cytoplasmic surfaces,
whereas osteocytes express this antigen as a plasma membrane protein (15). However, because the HOB-02-Cl cell line
was derived from an explant culture (21), it is also conceivable that these cells originated from osteocytes that dedifferentiated to osteoblasts (44). Consequently, these cells may
have retained the expression of CD44. In contrast, both the
HOB-Ol-Cl and HOB-03-C5 cell lines were obtained from
primary cultures and may have been immortalized before a
subsequent alteration in phenotype.
studies
IL-l/3 and TIVF-a stimulation
secretion
of cytokine
and chemokine
Osteoblasts produce a variety of growth factors, cytokines,
and chemokines (1,4,45). These include TGF+l, IL-l/3, IL-6,
TNF+, and monocyte chemoattractant protein (MCP)-1 (1,4,
21, 29, 37, 45-52). These factors act in an autocrine or paracrine manner to regulate osteoblast and/ or osteoclast differentiation and activity (1,4,45). For example, TGF+l stimulates the differentiation and activity of osteoblasts but has
the opposite effect on osteoclasts (45,52). On the other hand,
CONDITIONALLY
TRANSFORMED
HUMAN PREOSTEOCY’I’IC CELLS
A
450
kg
ox
l&3-FOLD
HOB-01 -Cl
*
T
400
ISHI
4601
2OOKD.
*
14.0-FOLD
116KD-
- CD44H
97KD.
66KD-
- NS (20 Ab)
45 KD-
37w
hPTH
(l-34)
PGE2
B
FOR
7. PTH responsiveness
is enhanced
at the nonpermissive
temperature.
HOB-Ol-Cl
cells were incubated
in BSA medium
at either
34 C (proliferative
temperature)
or 40 C (nonproliferative
temperature) for 48 h. The cells were pretreated
in fresh medium
with 0.5 mM
IBMX for 5 min at 37 C and then treated
in the presence
of IBMX with
either
vehicle
(ethanol,
O.l%,
vol/vol),
l-100
nM hPTH
(fragment
134) [hPTH
(l-34)],
100 no PGE,, or 1 PM forskolin
(FOR) for 10 min
at 37 C. Intracellular
CAMP was extracted
and measured
as described
in Materials
and Methods;
cellular
protein
was also determined.
The
results
are presented
as the mean 2 SD, n = 3-4; *, P < 0.05-0.005
from the 34 C or 40 C cells that did not receive
treatment
(Behren’sFisher
t test). The data are representative
oftwo similar
experiments.
HOB-OI-Cl
34w
HOB-OP-Cl
4ow
HOB-O3-C5
FIG.
116KD-
- CD44H
97KD66KD-
- NS (2O Ab)
34w
IL-lp, IL-6, and TNF-a are potent stimulators of osteoclast
differentiation or activity (4,45). In addition, IL-1 and TNF-(U
stimulate osteoblasts to secrete TNF-a, IL-6, MCI’-1, and
other factors that enhance bone resorption (4, 45, 47-50).
MCP-1 is a chemoattractant for monocytes and macrophages
and is thought to be an important mediator for the recruitment of these cells to inflamed bone (49-51). Although much
is known about the role of osteoblasts in the autocrine/
paracrine regulation of bone metabolism, very little is understood about the function of osteocytic cells in this process.
Consequently, the levels of TGF-fll, IL-6, TNF-CK,and MCI’-1
in HOB-Ol-Cl cell conditioned medium were determined. In
addition, the ability of these cells to respond to IL-l/3 and
TNF-a was also measured.
The HOB-Ol-Cl cells secreted relatively low levels of
TGF-Pl after 48 h at either temperature (-5-10 pM/mg cellular protein; data not shown). The basal concentration of this
peptide was about 50% less than that observed for normal
human osteoblasts (29,46) but was similar to the HOB-OZCl
cell line (21).
As shown in Fig. 9, A and B, treatment of the cells for 24 h
with l-1000 PM IL-lp stimulated a dose-dependent increase
in IL-6 and MCI’-1 secretion at either 34 C or 40 C: IL-lp
affected a 121- to 512-fold increase in IL-6 secretion and a 38to 57-fold increase in MCI’-1 secretion. The up-regulation of
cytokine/chemokine
secretion by IL-l/3 was better when the
cells were maintained at the permissive temperature, although this was due primarily to increased basal production
at the nonpermissive temperature. The secretion of IL-6 and
MCP-1 from the cells in response to IL-lp treatment reached
40%
34w
4wc
34%
40%
8. HOB cell lines express
CD44 at both temperatures.
A and B,
HOB-Ol-Cl
cells were incubated
at either
34 C (proliferative
temperature)
or 40 C (nonproliferative
temperature)
for 2 days. Whole cell
extracts
were then processed
for SDS-PAGE
and Western
blot analysis using a monoclonal
antibody
to human
CD44H
(hemopoietic,
-95
kDa) as described
in Materials
and Methods.
A, Ishikawa
cells incubated at 37 C were also processed
and analyzed
for this antigen.
B,
Two other HOB cell lines (HOB-02-Cl,
maturation
stage; 21); HOB03-C5, proliferative
stage) were incubated
at 34 C or 40 C for 2 days,
and then processed
and analyzed
for CD44H
expression.
The migration positions
of the SDS-PAGE
mol wt protein
markers
are indicated
on the left sides of the figures.
ISHI,
Ishikawa
cells; NS (2”Ab),
nonspecific
bands due to the secondary
antibody.
The data are representative
of two similar
experiments.
FIG.
a plateau at 100 PM. Similar results were obtained when the
.cells were treated with IL-la, except that the dose-response
patterns were linear up to 1000 PM (data not shown). The cells
produced large amounts of IL-6 in response to IL-l/3 treatment (1000-1400 pM/mg cell protein24 h at 100 pM IL-lp;
A). This level of IL-6 secretion was in the range of that
reported for normal human osteoblasts (47, 48). In contrast,
this cell line synthesized very low levels of TNF-(Y in response
to IL-lp (350-450 fM/mg cell protein24 h at 100 PM IL-lp;
data not shown). Finally, when compared with IL-6 production, IL-lp stimulated an even higher high rate of MCP-1
secretion (1600-2000 p~/mg cell protein-24 h at 100 pM
IL-lp; B), although this rate was only about half of that
reported for normal human osteoblasts (50). Using in situ
immunohistochemistry
of inflamed murine mandibular
bone, Rahimi ef al. (51) recently reported that osteoblastic
lining cells and osteoid-osteocytes expressed MCP-1; on the
CONDITIONALLY
4602
A
HUMAN PREOSTEOCYTIC
CELLS
Endo . 1996
Vol 137. No 11
1800
1600
IifQ
ij
ii
TRANSFORMED
I
IZl-FOLD
*
:[
1400
lz
1000
1200
Q
ii
E
800
Ji
=a
1000
8
r
800
3
9
600
9
A
400
1
a
5
0
I-
600
400-
200
0
IL-1P (PM)
TNF-cx (PM)
12.9-FOLD
3%FOLD
*
*
.
-I-
r
2250IiT
5(u
ri:
2
a
z
0
2000-
z
0)
1800
1760-
5
&
1600
3
1500-
s
z
0
E"
1250-
1400
1200
1000
F
3
IOOO750-
52
4
2
800
4
r
500
P
400
I
P
600
200
250
0
0
TNF-ol (PM)
IL-lb (PM)
9. Interleukin-16
and tumor
necrosis
factor-o
stimulate
interleukin-6
and monocyte
at both temperatures.
HOB-Ol-Cl
cells were treated
in medium
for 24 h at either
34 C
temperature)
with O-1000
PM IL-16
(A and B) or TNF-ol
(C and D). Secreted
IL-6 (A and C)
media, and cellular
protein
was determined,
as described
in Materials and Methods. The
(A) or as the mean 5 SD, n = 8-16 (B-D);
*, P < 0.001 from the 34 C or 46 C cells that did
data are representative
of two to three similar
experiments.
FIG.
other hand, osteocytes embedded within the mineralized
bone matrix did not appear to produce this chemokine.
As depicted in Fig. 9, C and D, treatment of the HOB-Ol-Cl
cells for 24 h with l-1000 PM TNF-a! also stimulated a dosedependent increase in IL-6 and MCP-1 secretion at either
temperature: TNFa! affected a 7- to 13-fold increase in IL-6
secretion and a 13- to 19-fold increase in MCI’-1 secretion. As
with IL-l& the up-regulation of MCI’-1 secretion by TNF-a!
was better when the cells were maintained at 34 C due to
increased basal production at 40 C (D). In addition, the levels
of MCI’-1 production attained with TNF-a treatment were
comparable to that observed with IL-1P treatment.
In summary, these results indicated that the HOB-Ol-Cl
chemoattractant
protein-l
secretion
from the cells
(proliferative
temperature)
or 40 C (nonproliferative
or MCP-1
(B and D) were measured
in the conditioned
results
are presented
as the mean i SEM, n = 8-24
not receive
treatment
(Dunnett’s
ANOVA
test). The
cells have the capacity to respond to potent bone resorbing
cytokines. Furthermore, the cells synthesized high amounts
of a cytokine and chemokine previously reported to be involved in the stimulation of bone resorption and repair.
Conclusions
From the results presented in this report, we conclude that
the HOB-Ol-Cl cell line has a phenotype consistent with a
preosteocyte and that these cells are responsive to both calcitropic hormones and bone resorbing cytokines. Morphologically, the cells have finger-like cellular processes that are
characteristic of preosteocytes and form gap junctions or are
CONDITIONALLY
TRANSFORMED
rich in microfilaments. In agreement with previous in situ
and in vitro studies of osteocytes, the HOB-Ol-Cl cell line
does not proliferate at the nonpermissive temperature, expresses very low levels of alkahne phosphatase activity but
secretes high amounts of osteocalcin in response to vitamin
D, treatment. These cells also synthesize moderate levels of
type I collagen, rapidly produce mineralized nodules, and
express CD44. Moreover, the HOB-Ol-Cl cells are responsive
to vitamin D, PTH, PGE,, IL-l& and TNF-cr. The cells also
secrete high levels of IL-6 and MCP-1. Finally, vitamin Da and
PTH responsiveness are enhanced at the nonpermissive temperature when the T-antigen mutant is inactivated. Thus,
these in vitro studies lend support to the hypothesis that
osteocytic cells may be involved in the hormonal regulation
of bone remodeling. We anticipate that this new clonal cell
line will become an important model for additional investigations of human preosteocyte biology. For example,
these cells can be used to identify preosteocyte-specific
genes or to study the molecular mechanisms of mechanosensory stimulation.
HUMAN PREOSTEOCYTIC
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