Receptor Activator of NF-
B and Osteoprotegerin Expression by
Human Microvascular Endothelial Cells, Regulation by Inflammatory
Cytokines, and Role in Human Osteoclastogenesis*
Patricia
Collin-Osdoby
§¶,
Linda
Rothe
,
Fred
Anderson
,
Maureen
Nelson
,
William
Maloney
, and
Philip
Osdoby
§
From the
Department of Biology, Washington
University, St. Louis, Missouri 63130, the § Division of
Bone and Mineral Metabolism, Washington University Medical School, and
the
Department of Orthopedics, Washington University Medical
School, St. Louis, Missouri 63110
Received for publication, November 7, 2000, and in revised form, March 22, 2001
 |
ABSTRACT |
The receptor activator of NF-
B (RANKL) is the
essential signal required for full osteoclast (OC) development,
activation, and survival. RANKL is highly expressed in areas of
trabecular bone remodeling and inflammatory bone loss, is increased on
marrow stromal cells or osteoblasts by osteotropic hormones or
cytokines, and is neutralized by osteoprotegerin (OPG), a soluble decoy
receptor also crucial for preventing arterial calcification. Vascular
endothelial cells (VEC) are critically involved in bone development and
remodeling and influence OC recruitment, formation, and activity.
Although OCs develop and function in close association with bone VEC
and sinusoids, signals mediating their interactions are not well known. Here, we show for the first time that human microvascular endothelial cells (HMVEC) express transcripts for both RANKL and OPG; inflammatory cytokines tumor necrosis factor-
and interleukin-1
elevate
RANKL and OPG expression 5-40-fold in HMVEC (with an early OPG peak that declines as RANKL rises), and RANKL protein increases on the
surface of tumor necrosis factor-
-activated HMVEC.
Cytokine-activated HMVEC promoted the formation, fusion, and bone
resorption of OCs formed in co-cultures with circulating human
monocytic precursors via a RANKL-mediated mechanism fully antagonized
by exogenous OPG. Furthermore, paraffin sections of human osteoporotic
fractured bone exhibited increased RANKL immunostaining in
vivo on VEC located near resorbing OCs in regions undergoing
active bone turnover. Therefore, cytokine-activated VEC may contribute
to inflammatory-mediated bone loss via regulated production of RANKL
and OPG. VEC-derived OPG may also serve as an autocrine signal to
inhibit blood vessel calcification.
 |
INTRODUCTION |
The receptor activator of NF-
B ligand
(RANKL),1 also known as
osteoprotegerin ligand (OPGL), osteoclast differentiation factor, or
TNF-related activation-induced cytokine (TRANCE), is a recently discovered transmembrane molecule of the tumor necrosis factor (TNF)
ligand superfamily that is highly expressed in lymphoid tissues and
trabecular bone, particularly in areas associated with active bone
remodeling or inflammatory osteolysis (1-4). RANKL is the essential
and final common signal required both in vitro and in
vivo for full osteoclastic (OC) differentiation from multipotential hematopoietic precursor cells into mature multinucleated bone-resorptive OCs in the presence of the permissive factor macrophage colony-stimulating factor (M-CSF) (1-7). RANKL expressed on the surface of osteoblasts (OB) or bone marrow stromal cells (BMSC) interacts with a cell surface receptor, RANK, present on pre-OC (induced by M-CSF) and mature OC to stimulate their fusion,
development, bone resorption, and cell survival (5-9). RANKL
expression increases during early OB development and is up-regulated in
OB and BMSC by various pro-resorptive stimuli such as parathyroid
hormone (PTH), 1,25-dihydroxyvitamin D3
(VD3), dexamethasone (Dex), prostaglandin E2, or interleukin-11 (IL-11) (6, 10-12). Recently, the
pro-resorptive inflammatory cytokines TNF-
and IL-1
were also
shown to elevate RANKL mRNA levels in human BMSC and MG63
osteosarcoma cells (13). Targeted ablation of RANKL in mice results in
suppressed osteoclastogenesis and an osteopetrotic phenotype (14),
whereas RANKL administration into normal adult mice elicits increased
OC size and activation (but not numbers) and systemic hypercalcemia
(5).
Osteoprotegerin (OPG, also known as osteoclastogenesis inhibitory
factor or OCIF) is a naturally occurring soluble member of the TNF
receptor superfamily that is widely expressed in multiple tissues and
binds to RANKL, thereby neutralizing its function (1-4). OPG therefore
acts as a secreted decoy receptor to negatively regulate OC
differentiation, activity, and survival both in vivo and
in vitro (15, 16). OPG production by OB and BMSC is
regulated by calcitropic hormones and cytokines, and the balance in the ratio of RANKL to OPG critically determines net effects on OC development and bone resorption (1-4, 11). In vivo
administration of OPG to normal rats reduces osteoclastogenesis and
increases bone density, OPG prevents estrogen deficiency-associated
bone loss in ovariectomized animals, and transgenic mice overexpressing OPG exhibit increased bone density and severe osteopetrosis (15). Conversely, mice deficient in OPG display increased OC development and
activity, early onset osteoporosis, and arterial calcification (17,
18). Thus, OPG has been proposed to regulate bone resorption both
locally and systemically, as well as to serve as a physiological suppressive signal of local calcification in blood vessels (2, 17).
Vascular endothelial cells (VEC) are intimately associated with pre-OC
and OC during both their formation and resorption of bone (19-22).
This close physical interaction permits VEC to influence directly and
convey local and systemic regulatory signals for OC development and
bone remodeling under normal or pathological conditions (19-22).
Recent studies indicate that many pre-OCs reside in the peripheral
circulation as well as in the bone marrow (23-26). Circulating pre-OC
may therefore also be exposed to and potentially activated by signaling
molecules displayed on the VEC surface during and following their
transmigration across the VEC layer of blood vessels in response to
local stimulatory signals to reach the bone microenvironment. Although
all OCs develop and function in close association with the VEC and
sinusoids of bone, specific signals mediating interactions between
these cells are not well known. Because VEC function as primary immune
response cells that are potently activated by TNF-
and IL-1 (27,
28), we investigated whether primary human VEC expressed RANKL and/or
OPG, and if such expression was regulated by these or other
pro-resorptive stimuli. The functional consequences of RANKL expression
and regulation in HMVEC were assessed relative to the in
vitro development and activity of multinucleated bone-resorptive
OCs from precursors present in human peripheral blood mononuclear cell preparations.
 |
EXPERIMENTAL PROCEDURES |
HMVEC Culture and Treatments--
Primary HMVEC of normal adult
female dermal tissue origin, media (essential growth
medium-microvascular, EGM-MV, and essential basal medium, EBM), and
media supplements (packaged as SingleQuots containing human recombinant
epidermal growth factor, hydrocortisone, gentamicin, bovine brain
extract, and fetal bovine serum, FBS) were obtained from Clonetics
Corp. (San Diego, CA). HMVEC were grown, subcultured by trypsin/EDTA,
and used within 4 passages as recommended. HMVEC expressed all the
hallmark characteristics of endothelial cells (morphology, tubule
formation, acetylated low density lipoprotein uptake, factor VIII
expression, PECAM-1, ICAM-1, VCAM-1, ELAM-1, P-selectin, ACE, vimentin,
and no smooth muscle
-actin). For molecular analyses, HMVEC were
cultured to near confluence in 24-well dishes in EGM-MV complete medium
(with supplements and 5% FBS); modulators were administered the next day in fresh medium, and the cells were incubated for the times indicated before RNA was harvested. For withdrawal experiments, the
modulator medium was removed after the induction period, the cells were
rinsed twice briefly with fresh medium, and the cells were cultured in
medium without modulator for various times before RNA was harvested.
Cytokine release by HMVEC was evaluated in cells grown to near
confluence in EGM-MV complete medium and switched to phenol red-free
EBM (lacking supplements) plus 5% FBS for 16-24 h before modulators
were administered in fresh medium. The conditioned medium (briefly
centrifuged) and cells were harvested after 24 h and stored at
80 °C until analyzed for cytokine and protein levels,
respectively. Modulators used were 1,25-dihydroxyvitamin D3
(VD3, a gift of Hoffmann-La Roche), dexamethasone (Dex),
and human parathyroid hormone-(1-34) (PTH-(1-34); both from Sigma), and human recombinant cytokines TNF-
, IL-1
, and M-CSF (all from R
& D Systems, Minneapolis, MN).
Primary Human Osteoblast and Bone Marrow Stromal Cell
Populations--
Primary human osteoblasts (HOB) were obtained as
cultured outgrowth cells from trabecular bone explants according to the
Robey/Termine method as described previously (29). Human bone marrow
stromal cells (HBMSC) were isolated from discarded thoracic rib
surgical specimens or bone obtained from accident victims and cultured in
-MEM with 10% FBS and 1% antibiotic/antimycotic (Life
Technologies, Inc.), with or without Dex (100 nM) and
VD3 (10 nM) to promote a more OB-like phenotype
as described previously (29, 30).
RNA Isolation and RT-PCR Analysis--
RNA was isolated from
cells using RNA STAT-60 (Tel-Test, Inc., Friendswood, TX).
Semi-quantitative RT-PCR amplification for RANKL was performed using
forward and reverse primers (designed by Drs. N. Weitzmann and L. Rifas, Washington University, St. Louis, MO) to the extracellular
region of OPGL/TRANCE/RANKL (nucleotides 4-735) of cloned human TRANCE
(nucleotides 1-738, GenBankTM accession number AF013171)
and Amersham Pharmacia Biotech Ready-to-Go RT-PCR beads.
Oligonucleotide primers were as follows: forward,
5'-GCTCTAGAGCCATGGATCCTAATAGAAT-3', and reverse
5'-ATCTCGAGTCACTATTAATGATGATGATGATGATGATCTATATCTCGAACTTTAAAAGCC-3'. RT-PCR amplification for OPG was performed using forward and reverse primers to cloned human OPG (GenBankTM accession number
U94332) as follows: forward, 5'-GGGGACCACAATGAACAAGTTG-3' (nucleotides
85-106), and reverse, 5'-AGCTTGCACCACTCCAAATCC-3' (nucleotides
473-493) (31). Parallel reactions were performed for every assay using
primers designed to amplify human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as described previously (32). Initial trials were
performed using RNA obtained from unstimulated cultures as well as each
modulator treatment to establish optimal cycle numbers and RNA amounts
for routine use so that RT-PCRs would yield RANKL, OPG, and GAPDH
amplifications within an exponentially linear range over the amount of
input RNA used. Thus, cycle numbers were varied from 20 to 35 (OPG), 30 to 35 (RANKL), and 15 to 35 (GAPDH), and RNA amounts from 0.15 ng to 1.5 µg (OPG), 25 ng to 12 µg (RANKL), and 1 pg to 4 µg
(GAPDH), in up to 6 replicate trials each. No products were
obtained in controls lacking either RNA or first strand primers, and
amplified products were not eliminated or reduced by DNase treatment of
the RNA samples. Conditions were chosen that consistently provided
mid-linear range amplification of each PCR product for all further
studies. Therefore, PCRs were run at 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 2 min for 35 (RANKL), 26 (OPG), or 20 (GAPDH)
cycles. Products were separated by agarose gel electrophoresis,
visualized by ethidium bromide staining, photographed using a Polaroid
camera, and quantified in a scanner (Scanjet II, Hewlett-Packard)
computer linked to a Quantimet image analysis system (Leica, United
Kingdom). RANKL or OPG signals were normalized to GAPDH signals
determined in parallel for each sample, and data were expressed as a
percentage of the RANKL/GAPDH or OPG/GAPDH ratio for untreated HMVEC
measured in the same trial. The 731-bp RANKL and 408-bp OPG amplicons
generated by RT-PCR from HMVEC (as well as HOB and HBMSC) were directly sequenced using an ABI Prism Cycle Sequencing kit (PerkinElmer Life
Sciences), and DNA sequences were compared with published sequences to
confirm their identity using computation performed at the NCBI and the
BLAST network service.
Quantification of Cytokine Secretion by HMVEC--
Cytokine
levels in conditioned medium were measured using specific enzyme-linked
immunoassay kits (Quantikine kits, R & D Systems, Minneapolis, MN) for
human IL-1
, TNF-
, and M-CSF as recommended. Standard curves were
run with each assay, and control medium was analyzed for background
levels of each cytokine (which were insignificant), and modulators were
tested in at least 3 separate culture wells per trial for 2-8
independent HMVEC cultures. Results were normalized for cell protein
using the bicinchoninic acid protein assay (Pierce) and bovine serum
albumin (BSA) as a standard (33), and data were expressed as the
mean ± S.E. ng/ml of cytokine released per mg of cell protein
during 24 h of culture.
Immunodetection of RANKL Protein Expression on
HMVEC--
HMVEC were cultured in EGM-MV complete medium on glass
coverslips in 24-well tissue culture dishes to near confluency, TNF-
(1 nM) was administered in fresh medium for 24 h, and
the cells were fixed and immunostained (34). Briefly, HMVEC were
rinsed, fixed in 3% paraformaldehyde/HBSS (15 min), rinsed, blocked
for 1 h with 1% BSA and 10% horse serum in phosphate-buffered
saline (PBS), and reacted for 1 h with (or without) primary
antibodies diluted in block. Mouse monoclonal antibodies (mAb) specific
for human ICAM-1 or VCAM-1 (Serotec, Raleigh, NC, each at 1:200
dilution), mAb to the angiogenesis-related integrin
v
3 (LM 609, Chemicon International,
Temecula, CA, 1:100 dilution), and goat polyclonal antibody (pAb)
raised to a C-terminal extracellular peptide region of human RANKL
(Santa Cruz Biotechnology, Santa Cruz, CA, 1:100 dilution) were used.
Primary antibody binding was detected using a secondary goat anti-mouse
fluorescein isothiocyanate conjugate (Life Technologies, Inc.; 1:200)
for mAbs or a biotinylated donkey anti-goat antibody (Santa Cruz
Biotechnology; 1:200) followed by a streptavidin-Texas Red conjugate
(Life Technologies, Inc.; 1:1000) for RANKL pAb. In some cases, HMVEC
were simultaneously immunostained for both ICAM-1 and RANKL to
visualize their co-localization on the HMVEC plasma membrane.
Coverslips were mounted on glass microscope slides in glycerol-buffered
mounting medium (Becton Dickinson, Cockeysville, MD), and images were
viewed and digitally captured using a Leica scanning laser
(argon/krypton/He) confocal microscope (TCS-SP-2) equipped with a 20×
phase objective. Wavelengths for excitation and emission for
fluorescein isothiocyanate were 494 and 518 nm, respectively, and those
for Texas Red were 595 and 615 nm, respectively.
RANKL Immunodetection in Paraffin-embedded Sections of Human
Osteoporotic Bone--
Human osteoporotic bone was obtained from
fractured femoral heads discarded during hip replacement surgery and
briefly held at 4 °C in
-MEM before fixation in 10% buffered
formalin. Samples were decalcified, paraffin-embedded, and sectioned by
standard procedures. Sections were prepared for immunostaining by
deparaffinization in xylene, hydration through 100% EtOH, 95% EtOH,
and water, and heating (97 °C for 20 min) in an antigen
unmasking Target Retrieval Solution (Dako, Carpinteria, CA). Cooled
sections were PBS-rinsed, endogenous peroxidase activity was quenched
in Dako Peroxidase Blocking reagent (15 min), rinsed sections were
blocked with Dako serum-free Protein Block (10 min), and sections were
reacted overnight at 4 °C with the pAb to human RANKL described
above (diluted 1:500 to 1:1000 in PBS + 1.5% Dako Protein Block).
Sections were rinsed, incubated with biotinylated donkey anti-goat pAb
(Santa Cruz Biotechnology, 1:100 dilution in PBS/block, 45 min)
followed by Dako streptavidin-peroxidase (1:300 in PBS, 15 min),
reacted with Dako diaminobenzidine solution (5 min), briefly
counterstained using Dako hematoxylin solution, and mounted on glass
slides with Permount. Immunostained sections were viewed by light
microscopy, and images were digitally captured using a computer-linked
Olympus microscope.
OC Development in Human Peripheral Blood Mononuclear Cells (PBMC)
Co-cultured with HMVEC--
HMVEC were cultured in 24-well dishes in
EGM complete medium to near confluency, two-thirds of the wells were
treated for 48 h with either 1 nM TNF-
or IL-1
to maximally induce RANKL (while allowing stimulated OPG levels to
decline), and the cells were washed (3 times) to remove cytokines just
prior to the addition of human PBMC for co-culture. Human PBMC were
prepared from heparinized blood obtained from the American Red Cross
(St. Louis, MO). Mononuclear cells were isolated by Ficoll-Paque (26,
32, 33), resuspended in
-MEM plus 10% FBS and 1%
antibiotic/antimycotic, and added (1.6 × 106
PBMC/well) to the 24-well dish containing unactivated or
cytokine-pre-activated HMVEC. Some wells also received 100 ng/ml
recombinant human OPG:Fc fusion peptide (Alexis Corporation, San Diego,
CA). The next day (day 1) all wells were treated with 10 nM
VD3 and 25 ng/ml M-CSF, and OPG:Fc was readministered to
the wells originally receiving this treatment. On day 5 the cells were
refed with M-CSF, with or without OPG:Fc, and the cells were harvested
on day 7, rinsed, fixed in 3% PF/HBSS, rinsed, and stained for TRAP
activity (34-36). Cells were co-stained with DAPI (Molecular Probes,
Eugene, OR) to label nuclei, and the numbers of mononuclear and
multinucleated TRAP+ cells, as well as the number of nuclei per TRAP+
cell, were counted (encompassing ~2000-7000 TRAP+ cells per
condition per trial) across a constant number of sequential random
fields using an Olympus light and fluorescent microscope. To evaluate
simultaneously OC formation and acquisition of bone-resorbing
capability, HMVEC were cultured in 24-well dishes in EGM complete
medium to near confluency, half the wells were pre-activated with
TNF-
(1 nM, 21 h), HMVEC were washed, and human
PBMC were added (1.6 × 106 PBMC/well) and initially
co-cultured with VD3 and M-CSF, with or without OPG:Fc, as
above. Every 4th day, cells were refed with M-CSF with or without
OPG:Fc, a small circular disc of ivory was added to each well on day 9, and the cells and ivory pieces were harvested on day 16, rinsed, fixed
in 1% PF/HBSS, rinsed, and stained for TRAP activity (34-36). Cells
in the wells were co-stained with DAPI and analyzed for the numbers of
TRAP+ cells formed and nuclei per cell. Ivory was subjected to
resorption pit analysis (below). Parallel human PBMC were cultured
alone (without HMVEC) with VD3 and M-CSF, in the presence
or absence of human recombinant RANKL (75 ng/ml, Alexis Corp., San
Diego, CA) and/or OPG:Fc fusion peptide (100 ng/ml), according to the
same feeding regimen used for the co-cultures, and the cells and ivory
were harvested on day 16 for analysis as in the co-cultures.
Bone Pit Resorption Analysis--
The number of TRAP+ cells was
determined for a constant number (40) of random fields per ivory piece,
the cells were then removed, and bone pit resorption within these same
fields was quantified using a computer-linked dark-field reflective
light microscopic image analysis system (34-36). TRAP+ cell counts on the ivory include both MNC and mononuclear cells (since DAPI nuclear staining cannot be used on ivory to distinguish between them). The
total area (µm2) of ivory resorbed, the number of pits
formed, and the size of each excavation were assessed. Results were
also normalized to TRAP+ cell numbers in order to compare the mean area
of bone resorbed per TRAP+ cell (area/TRAP+ cell) and number of pits
formed per TRAP+ cell (pits/TRAP+ cell). More than 2000 TRAP+ cells and
their associated resorption pits were evaluated per co-culture trial. Data shown are presented as means ± S.E. from a representative co-culture trial performed in triplicate.
Statistical Analysis--
Data are presented as the mean ± S.E. of 3-12 independent trials, typically having at least 3 replicates per condition and assayed at least in duplicate. Differences
between treatments were analyzed using single factor analysis of
variance. For simultaneous comparisons between multiple treatments,
significant differences were determined using the post-analysis of
variance Bonferroni test. Differences were considered significant for
p < 0.05.
 |
RESULTS |
Primary HMVEC Express mRNA for Both RANKL and OPG--
RT-PCR
using specific primers to cloned human OPGL/TRANCE/RANKL (hereafter
referred to as RANKL) yielded a single amplicon product of the expected
size (731 bp) from primary HMVEC, as well as from primary HOB and HBMSC
(Fig. 1A). Complete nucleotide
sequencing of the products obtained from HMVEC and HBMSC demonstrated
their identity to sequences reported previously for cloned human OPGL (GenBankTM accession number AF053712), RANKL (accession
number AF019047), and TRANCE (accession number AF013171). RT-PCR using
specific primers to cloned human OPG also yielded a single amplicon
product of the expected size (408 bp) from each of these three primary human cell types (Fig. 1B), and complete nucleotide
sequencing of these three products showed that they matched the
sequence reported for cloned human OPG (GenBankTM accession
number U94332). Therefore, HMVEC express mRNA transcripts for both
RANKL and OPG.

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Fig. 1.
HMVEC, HOB, and HBMSC express mRNA
transcripts for RANKL and OPG. RT-PCR using total RNA isolated
from human primary HMVEC, HOB, or HBMSC and specific primers to the
extracellular region of cloned human RANKL (A) or cloned
human OPG (B) was performed as described under
"Experimental Procedures." A and B, single
amplicon products of the expected sizes were obtained for both RANKL
(731 bp) and OPG (408 bp) from each of the three primary human cell
types, and complete nucleotide sequencing of the products generated
from HMVEC and HBMSC confirmed their identity to RANKL or OPG.
Amplified RANKL and OPG products were RNA-dependent since
they were not obtained in the absence of input RNA or oligonucleotides
for reverse transcription into cDNA, and they were not eliminated
by DNase pretreatment of RNA samples before RT-PCR (not shown).
|
|
HMVEC Differ from HBMSC in Their Hormonal Regulation of RANKL and
OPG--
Because RANKL and OPG mRNA levels are regulated during OB
differentiation and stimulated by PTH or VD3 (alone or in
combination with Dex) in OB or stromal cells, the modulatory actions of
these hormones on RANKL and OPG mRNA expression were studied in
HMVEC using semi-quantitative RT-PCR. Treatment of HMVEC for 6, 24, or
72 h with PTH-(1-34) (500 nM) or VD3 (10 nM) and Dex (100 nM) had no effect on either
RANKL or OPG mRNA expression levels normalized to GAPDH in
comparison with untreated HMVEC cultured for the same times (Fig.
2, A and C). By
contrast, HBMSC differentiated with VD3 and Dex over 8 days
of culture exhibited a 1.5-fold increase in RANKL/GAPDH mRNA levels
(Fig. 2B), and a remarkable 10-fold decrease in OPG/GAPDH
mRNA levels (Fig. 2D), in comparison with untreated
HBMSC. Although RANKL/GAPDH mRNA expression was higher in HOB than
in VD3/Dex-differentiated HBMSC (Fig. 2B),
OPG/GAPDH mRNA expression was also significantly higher in HOB
(Fig. 2D). Consequently, VD3/Dex differentiation
of HBMSC raised the relative GAPDH-normalized ratio of RANKL to OPG
mRNA expression (in arbitrary densitometric units) from 0.04 in
untreated HBMSC to 0.63 in VD3/Dex-treated HBMSC, a value
close to the RANKL/OPG mRNA expression ratio associated with
primary HOB (0.86) and HMVEC (set at 1.0). Therefore, RANKL and OPG
mRNA levels are regulated by these calcitropic hormones in HBMSC
but not in HMVEC under the conditions tested.

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Fig. 2.
Calcitropic hormones modulate RANKL and OPG
mRNA expression in HBMSC but not in HMVEC. Semi-quantitative
RT-PCR was performed as described under "Experimental Procedures"
to amplify RANKL, OPG, and GAPDH in a mid-linear range, and the signals
for RANKL or OPG were each normalized to the GAPDH signals determined
in parallel. All data were expressed as the mean ± S.E.
percentage of the RANKL/GAPDH or OPG/GAPDH mRNA expression
determined in control untreated HMVEC (set at 100%). A and
C, primary HMVEC cultured with PTH-(1-34) (500 nM) or VD3 (10 nM) in combination
with Dex (100 nM) for 6, 24, or 72 h did not exhibit
any significant change in their GAPDH-normalized steady state mRNA
expression of either RANKL or OPG. The data shown in the bar
graph represents 2 independent cultures of HMVEC incubated for
24 h in the presence or absence of these hormones and analyzed for
RANKL (A) and OPG (C) mRNA expression. HMVEC
cultured with or without these hormones for 6 or 72 h yielded
identical findings (not shown). B and D, primary
HBMSC were cultured in the presence or absence of VD3 (10 nM) and Dex (100 nM) for 8 days and analyzed
from 2 independent cultures by semi-quantitative RT-PCR for mRNA
expression of RANKL/GAPDH (B) and OPG/GAPDH (D).
Primary isolated trabecular HOB (cultured without any modulator
treatments) were similarly analyzed. In each graph (A-D),
the inset shows results from a representative RT-PCR trial
for RANKL or OPG (top bands) and GAPDH (lower
bands in each set) corresponding to the 3 conditions
represented by the bars.
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|
TNF-
Increases Both RANKL and OPG mRNA Levels in HMVEC but
with Temporally Different Kinetics--
HMVEC are highly responsive to
inflammatory signals such as TNF-
and IL-1
, and each of these
potent immune activators increases RANKL and OPG mRNA levels in
HBMSC. TNF-
and IL-1
were therefore tested for their potential
effects on RANKL and OPG mRNA expression in HMVEC. TNF-
significantly and dose-dependently increased both RANKL and
OPG mRNA levels in HMVEC by 24 h as measured by
semi-quantitative RT-PCR (Fig. 3,
A and C). Although maximal induction of both
RANKL and OPG were achieved with 1-10 nM TNF-
, OPG
mRNA levels were increased further (25-fold) than RANKL mRNA
levels (5-fold) in relation to their expression levels in untreated
HMVEC.

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Fig. 3.
TNF- stimulates both
RANKL and OPG mRNA expression in HMVEC. A and
C, HMVEC were cultured for 24 h with or without various
concentrations of TNF- , and semi-quantitative RT-PCR analysis was
performed to determine the relative mRNA expression of RANKL/GAPDH
(A) and OPG/GAPDH (C) as described under
"Experimental Procedures." All data were expressed as the mean ± S.E. percentage of the RANKL/GAPDH or OPG/GAPDH mRNA expression
determined in control untreated HMVEC. Data shown in the bar
graphs were compiled from at least 3 independent HMVEC
experiments, each of which displayed dose-dependent
increases in both RANKL and OPG mRNA expression in response to
TNF- . RANKL/GAPDH and OPG/GAPDH were significantly
(p < 0.05) increased by TNF- concentrations of 0.01 nM and higher or 0.1 nM and higher,
respectively, in comparison to unstimulated HVMEC. Insets
show the results from a representative RT-PCR trial for RANKL or OPG
(top bands) and GAPDH (lower bands in each
set) corresponding to the same conditions represented by the
bars. B and D, HMVEC were treated with
or without 1 nM TNF- for 1-72 h and were subsequently
analyzed by semi-quantitative RT-PCR for the GAPDH-normalized temporal
profile of RANKL and OPG mRNA expression. Data were obtained from
at least 3 independent experiments and were expressed as the mean ± S.E. percentage of the RANKL/GAPDH or OPG/GAPDH mRNA expression
determined at time 0 in control untreated HMVEC. Open
symbols connected by dashed lines represent the results
obtained following the withdrawal of TNF- for 24 or 48 h from
24-h stimulated HMVEC.
|
|
Analysis of the temporal kinetics of RANKL mRNA expression by
TNF-
(1 nM) revealed that stimulation was first apparent
at 10 h post-treatment, rose to a maximal 3-6-fold elevation over untreated HMVEC by 24 h, and was sustained over at least 48-72 h
of culture in the continuous presence of this cytokine (Fig. 3B). Following TNF-
(1 nM) withdrawal from
24-h stimulated HMVEC cultures, induced RANKL mRNA levels began to
decline (Fig. 3B). However, even after 48 h of culture
in the absence of TNF-
they remained elevated 2-fold over the levels
originally observed in unstimulated HMVEC cells. A similar temporal
pattern of RANKL mRNA induction in HMVEC was observed in response
to either 0.1 or 10 nM TNF-
(data not shown).
In contrast to RANKL, OPG mRNA levels in HMVEC were more rapidly
and transiently increased in response to TNF-
(Fig. 3D). Stimulated OPG mRNA levels were apparent within 1 h, reached a maximum by 10 h, declined to less than half their peak values by
24 h, and thereafter declined more modestly over 72 h of
culture in the continued presence of TNF-
. However, OPG mRNA
levels at 72 h were still 10-fold elevated over those in untreated
HMVEC (Fig. 3D). Following TNF-
withdrawal from 24-h
stimulated HMVEC, OPG mRNA levels rapidly returned to the levels
associated with unstimulated HMVEC (Fig. 3D). Together,
these results indicate that TNF-
increases both RANKL and OPG
mRNA levels in HMVEC and furthermore that their regulation exhibits
a nearly reciprocal temporal relationship. OPG mRNA levels rapidly
increase in response to TNF-
and then begin to decline, whereas
RANKL mRNA levels rise more slowly and are sustained at their peak
over at least 48 h of culture. Moreover, within 24 h of
TNF-
withdrawal from HMVEC cultures, OPG mRNA expression returns
to basal levels, whereas RANKL mRNA expression remains 3-fold above
unstimulated levels.
IL-1
Increases Both RANKL and OPG mRNA Levels in HMVEC in a
More Complex Manner Than TNF-
--
Treatment of HMVEC for 24 or
48 h with IL-1
also significantly and
dose-dependently increased both RANKL and OPG mRNA
levels measured by semi-quantitative RT-PCR (Fig.
4, A and C).
Maximal induction of RANKL or OPG at either 24 or 48 h was
achieved with 1-10 nM IL-1
. Unlike TNF-
, IL-1
(1 nM, 48 h) elicited nearly equivalent ~10-fold
increases in both OPG and RANKL mRNA expression over the
corresponding levels in untreated HMVEC (Fig. 4, A and C). Dose-response curves for RANKL and OPG mRNA
expression after 48 h of IL-1
(1 nM) treatment
paralleled, but were much higher than, RANKL and OPG mRNA
stimulation measured after only 24 h of exposure to this cytokine
(Fig. 4, A and C).

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Fig. 4.
IL-1 stimulates both
RANKL and OPG mRNA expression in HMVEC. A and
C, HMVEC were cultured for either 24 (open bars)
or 48 h (hatched bars) with or without various
concentrations of IL-1 , and semi-quantitative RT-PCR analysis was
performed to determine the relative mRNA expression of RANKL/GAPDH
(A) and OPG/GAPDH (C) as described under
"Experimental Procedures." All data were expressed as the mean ± S.E. percentage of the RANKL/GAPDH or OPG/GAPDH mRNA expression
determined in control untreated HMVEC. Data for the bar
graphs were compiled from at least 3 independent HMVEC
experiments, each of which exhibited dose-dependent
increases in RANKL and OPG mRNA expression in response to IL-1 .
RANKL/GAPDH and OPG/GAPDH were each significantly (p < 0.05) increased at 24 h by IL-1 concentrations of 0.01 nM or higher or 0.001 nM or higher,
respectively, and at 48 h by IL-1 concentrations of 0.1 nM or higher (for both RANKL and OPG), in comparison to
unstimulated HVMEC. Insets show results from a
representative RT-PCR trial for RANKL or OPG (top bands) and GAPDH
(lower bands in each set) corresponding to the
48-h conditions represented by the bars. B and
D, HMVEC were treated with or without 1 nM
IL-1 for 1-72 h and were subsequently analyzed by semi-quantitative
RT-PCR for the GAPDH-normalized temporal profile of RANKL and OPG
mRNA expression. Data were obtained from at least 3 independent
experiments and were expressed as the mean ± S.E. percentage of
the RANKL/GAPDH or OPG/GAPDH mRNA expression determined at time 0 in control untreated HMVEC. Open symbols connected by
dashed lines represent the results obtained following the
withdrawal of IL-1 for 24 or 48 h from 24- or 48-h-stimulated
HMVEC. Note the differences in scales for RANKL/GAPDH and OPG/GAPDH
graphs between Figs. 3 and 4.
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Analysis of the temporal kinetics of RANKL mRNA expression induced
by IL-1
(1 nM) showed an early increase first observed 3-6 h post-treatment that rose to a reproducible level 3-4-fold over
untreated HMVEC by 10 h (Fig. 4B). This initial
stimulation of RANKL mRNA levels was comparable to that elicited by
TNF-
(Fig. 3B). However, it was immediately followed by a
second phase of induction in response to IL-1
, to levels 8-10-fold
over untreated HMVEC, that peaked within 48 h and was sustained
over at least 72 h of culture in the continuous presence of this
cytokine (Fig. 4B). Following the withdrawal of IL-1
(1 nM) from either 24- or 48-h-stimulated HMVEC cultures,
induced RANKL mRNA levels declined within the next 24-h period to
the low levels seen in unstimulated HMVEC (Fig. 4B). This
contrasts with the more moderately elevated, but sustained, levels of
RANKL expression maintained following TNF-
induction and withdrawal.
The continued presence of IL-1
beyond the first 24 h was
essential for achieving the second higher peak of RANKL mRNA
expression in cultured HMVEC because this did not occur if IL-1
was
withdrawn immediately after the initial 24-h period. A similar bimodal
temporal pattern of RANKL mRNA level induction in HMVEC was also
observed in response to 10 nM IL-1
(data not shown).
OPG mRNA levels rose more rapidly in response to IL-1
than did
RANKL mRNA levels (Fig. 4D). However, like RANKL, OPG
mRNA levels always exhibited a bimodal temporal pattern of
IL-1
-stimulated expression (Fig. 4D). Elevated OPG
mRNA levels were detectable by 1 h, reached an initial maximum
by 6 h, and then partially declined briefly, but reproducibly,
before rising again to maximal levels 10-fold over unstimulated HMVEC
by 24 h (Fig. 4D). Thereafter, OPG mRNA expression
declined over 72 h of culture in the continued presence of
IL-1
, although OPG mRNA levels at 72 h were still 4-fold
elevated over those in untreated HMVEC (Fig. 4D). Compared with TNF-
, maximal levels of OPG expression elicited by IL-1
were
4-fold lower than peak OPG expression induced by TNF-
and were
equivalent to the naturally declining levels of OPG expression seen
after 72 h of continuous TNF-
exposure (Fig. 3, D
and 4D). However, like TNF-
, IL-
withdrawal from 24-h
stimulated HMVEC led to the rapid return within the next 24 h of
OPG mRNA levels to the lower levels associated with unstimulated
HMVEC (Fig. 4D). If IL-1
withdrawal was delayed until
48 h, when OPG mRNA levels were already naturally declining,
little further acceleration in the rate of decline occurred (Fig.
4D). Together, these results indicate that IL-1
increases
both RANKL and OPG mRNA levels in HMVEC and that their regulated
expression exhibits an apparent reciprocal relationship analogous to
what had been observed for RANKL and OPG in response to TNF-
.
However, in contrast to TNF-
, IL-1
-stimulated RANKL and OPG
mRNA increases were more similar in magnitude and exhibited more
complex temporal expression profiles. Thus, prolonged stimulation of
HMVEC with IL-1
caused a greater induction of RANKL and a lesser
induction of OPG expression than was elicited by TNF-
. However,
following 24-48 h of cytokine withdrawal, RANKL expression remained
partially elevated in TNF-
-treated HVMEC, whereas it declined to
basal levels in IL-1
-treated HMVEC, and OPG expression returned to
basal levels in both.
When HMVEC were simultaneously treated with TNF-
(1 nM)
and IL-1
(1 nM) for 24 h, a small additive
stimulation of RANKL mRNA expression over that evoked by either
cytokine alone was typically observed (Fig.
5A). Additive increases were
also seen using 0.01 nM of each cytokine in combination
(data not shown). In contrast, RANKL mRNA expression was not raised
beyond the maximal levels induced by either cytokine alone after
48 h of combined TNF-
(1 nM) and IL-1
(0.01, 0.1, or 1.0 nM) treatment (Fig. 5B). Like RANKL,
OPG mRNA levels were higher in HMVEC concurrently treated for
24 h with both TNF-
and IL-1
in comparison with the levels
induced by either cytokine alone (Fig. 5C). However, OPG
mRNA levels at 48 h were generally no higher (except with TNF-
in combination with 0.01 nM IL-1
) in the
co-treated cells than in those treated with TNF-
alone, although
these levels were significantly greater than the stimulation of OPG
mRNA by IL-1
alone (Fig. 5D).

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Fig. 5.
TNF- and
IL-1 act additively to increase RANKL and OPG
mRNA expression at 24 h, but not 48 h, in HMVEC.
HMVEC were cultured for either 24 (A and C) or
48 h (B and D) in the presence or absence of
TNF- (1 nM), IL-1 (0.01 to 1 nM), or a
combination of these cytokines and were subsequently analyzed by
semi-quantitative RT-PCR to determine the relative mRNA expression
of RANKL/GAPDH (A and B) and OPG/GAPDH
(C and D) as described under "Experimental
Procedures." The results shown were compiled from 2 to 5 independent
trials, and data were expressed as the mean ± S.E. percentage of
the RANKL/GAPDH or OPG/GAPDH mRNA expression determined in control
untreated HMVEC at 24 or 48 h. Significant differences from
unstimulated HMVEC are denoted by *, p < 0.05, from
TNF- -stimulated HMVEC by +, p < 0.05, and from
IL-1 -stimulated HMVEC by #, p < 0.05.
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TNF-
Increases RANKL Protein Expression in HMVEC--
Changes
in mRNA steady state levels are not always accompanied by
corresponding changes in protein expression. Therefore, immunostaining
was employed to learn whether RANKL protein expression increased in
parallel with RANKL mRNA levels following cytokine activation of
HMVEC. By using a pAb to the C-terminal extracellular region of human
RANKL, a low basal level of specific immunostaining was detected in
unstimulated HMVEC (Fig. 6A)
that was markedly increased in HMVEC cultured with TNF-
(1 nM) for 24 h (Fig. 6B). RANKL
immunostaining was primarily associated with the plasma membrane of
TNF-
-activated HMVEC and was intense throughout the sample when
viewed by optical sectioning confocal microscopy. Similarly,
unstimulated HMVEC exhibited a low level of specific basal
immunostaining using a mAb to the cell surface integrin adhesion
molecule ICAM-1 (Fig. 6C), and such staining was greatly enhanced and predominantly cell surface-associated in HMVEC stimulated with 1 nM TNF-
for 24 h (Fig. 6D). HMVEC
co-stained for both RANKL and ICAM-1 exhibited strong overlapping
signals for these two molecules all over the cell surface (not shown).
TNF-
-treated HMVEC also exhibited specific increases in
immunostaining for the cell surface adhesion molecules VCAM-1 and
v
3 (not shown). Therefore, TNF-
stimulation of HMVEC increases expression of RANKL mRNA in addition
to RANKL protein, which is primarily located on the surface plasma
membrane of activated HMVEC.

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Fig. 6.
TNF- increases RANKL
and ICAM-1 protein expression on the HMVEC cell surface. HMVEC
were cultured for 24 h in the presence or absence of 1 nM TNF- , fixed, immunostained using antibodies specific
to human RANKL or ICAM-1, and the labeled cells viewed in an optical
sectioning confocal microscope as described under "Experimental
Procedures." A and B, RANKL protein
expression was immunodetected using a pAb to the C terminus of human
RANKL together with a biotinylated secondary antibody and a
streptavidin-Texas Red conjugate. Unactivated HMVEC (A)
exhibited a low basal level of RANKL protein immunostaining, whereas a
markedly stronger signal was associated with HMVEC that had been
exposed to TNF- (B). C and D,
ICAM-1 protein expression was immunodetected using a mAb to human
ICAM-1 and a fluorescein isothiocyanate-conjugated secondary antibody.
As for RANKL, unactivated HMVEC (C) displayed a low basal
level of ICAM-1 protein immunostaining, whereas an intense signal was
detected in TNF- -activated HMVEC (D). No staining was
apparent in parallel samples (±TNF- ) developed in the absence of
either primary antibody (data not shown). A D,
magnification × 200.
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TNF-
and IL-1
Stimulate the Release of M-CSF, as Well as One
Another, from HMVEC--
Because M-CSF is an essential permissive
factor required for OC differentiation promoted by RANKL, the effects
of TNF-
and IL-1
on the production of M-CSF by HMVEC were also
investigated. HMVEC cultured for 24 h with either IL-1
or
TNF-
(1 or 10 nM) released 2-4-fold more M-CSF into the
culture medium than did unstimulated HMVEC (Table
I). These pro-inflammatory cytokines also
stimulated the release of one another from HMVEC, although IL-1
(1 or 10 nM) elicited a somewhat greater increase in TNF-
release (up to 8-fold) than TNF-
(only at 10 nM) did in
IL-1
release (3-fold) from HMVEC (Table I).
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Table I
Cytokine production by HMVEC
HMVEC were cultured for 24 h in the presence of IL-1 or TNF-
(1 or 10 nM), after which the conditioned media were
harvested and analyzed using cytokine-specific immunoassays for the
levels of IL-1 , TNF- , and M-CSF released as described under
"Experimental Procedures." Results obtained from duplicate wells of
at least three independent trials were each normalized for cell
protein, and the data were expressed as the mean ± S.E. ng/ml
cytokine released per mg cell protein.
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Inflammatory Cytokine Activation of HMVEC Promotes in Vitro
Osteoclastogenesis in Co-cultured Human PBMC by a
RANKL-dependent Mechanism Antagonized by OPG--
Whether
cytokine stimulation of RANKL mRNA and protein expression in HMVEC
enhanced their ability to promote OC formation and development was
evaluated in HMVEC co-cultured with M-CSF and human PBMC as a source of
OC precursors. Because TNF-
and IL-1
can themselves influence OC
development, bone resorption, and/or survival, these cytokines were
withdrawn from activated HMVEC before their co-culture with human PBMC
so that biological effects mediated by cytokine-induced RANKL and OPG
could be distinguished from potential direct actions of TNF-
or
IL-1
. HMVEC that were pre-activated with either TNF-
or IL-1
(1 nM, 48 h), and then washed prior to the addition of
human PBMC, caused a significant 1.5-fold increase in the number of
TRAP+ multinucleated cells (MNC) having 3 or more nuclei that formed in
the co-cultures after 1 week compared with the number generated from
human PBMC co-cultured with unstimulated HMVEC (Fig.
7, A and B). These
increases were attributed in each case to a cytokine-mediated induction
of RANKL in HMVEC, and not other soluble or cell-surface factors (eg.
ICAM-1, VCAM-1), because inclusion of an inhibitory OPG fusion peptide that directly binds and neutralizes RANKL completely blocked the stimulatory effects obtained with either TNF-
(Fig. 7A)
or IL-1
(Fig. 7B). These data also suggest that
endogenous OPG production by TNF-
or IL-1
pre-activated HMVEC in
the co-cultures was below the inhibitory levels achieved through
exogenous addition of 100 ng/ml OPG fusion peptide. In contrast to its
suppression of MNC formation promoted by cytokine-stimulated HMVEC, OPG
did not diminish the 2-5-fold greater MNC formation that occurred in
human MN co-cultured with (untreated) HMVEC (Fig. 7, A and
B) compared with human MN cultured in the absence of HMVEC
(data not shown). Thus, OPG restrained TRAP+ MNC formation stimulated
by either TNF-
- or IL-1
-activated HMVEC via a
RANKL-dependent mechanism but not that promoted by unactivated HMVEC in a RANKL-independent pathway.

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Fig. 7.
TNF- - or
IL-1 -activated HMVEC promote in
vitro osteoclastogenesis by day 7 in co-cultured human PBMC
via a RANKL-dependent mechanism antagonized by OPG.
HMVEC were pre-activated for 48 h with 1 nM TNF- or
IL-1 , washed (to eliminate potential direct effects of the
cytokines), and co-cultured with human PBMC (containing OC cell
precursors) in the presence or absence of a RANKL-neutralizing
recombinant human OPG fusion peptide (100 ng/ml) for 7 days, and the
development of TRAP+ multinucleated cells was evaluated as described
under "Experimental Procedures." A and B, the
number of TRAP+ multinucleated cells (containing 3 or more nuclei)
formed in co-cultures containing TNF- - (A) or IL-1
(B)-pre-activated HMVEC were counted for each well (totaling
over 25,000 MNC per trial), and the data were expressed as the
mean ± S.E. total number of TRAP+ MNC formed per well.
Significant differences from control co-cultures of human PBMC and
unactivated HMVEC in the absence of the OPG fusion peptide are denoted
by *, p < 0.05, and from TNF- or IL-1 activated
HMVEC co-cultured with human PBMC in the absence of OPG by +,
p < 0.05.
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Inflammatory Cytokine Activation of HMVEC Promotes the Fusion of OC
Precursors to Form Larger, More Multinucleated TRAP+ MNC--
In
vitro osteoclastogenesis was examined in further detail using
TNF-
-pre-activated HMVEC (1 nM, 24 h) co-cultured
with human PBMC for 16 days. Again, TNF-
-activated HMVEC caused a
significant 2-fold increase in the number of TRAP+ MNC (with 3 or more
nuclei) that formed in the co-cultures compared with co-cultures with unstimulated HMVEC, and OPG fusion peptide fully blocked such induction, thereby implicating a RANKL-dependent mechanism
(Fig. 8A). The proportion of
total TRAP+ cells that became MNC was also significantly increased by
TNF-
pre-activation of HMVEC (Fig. 8A). However, the
overall number of mononuclear or total (mononuclear plus MNC) TRAP+
cells in the co-cultured human PBMC population was not affected by
TNF-
pretreatment of HMVEC, suggesting that the increase in TRAP+
MNC formation in the presence of TNF-
pre-activated HMVEC likely
involved the stimulated fusion, rather than proliferative expansion, of
TRAP+ precursor cells (Fig. 8B). Consistent with this,
microscopic examination of human PBMC co-cultured with
TNF-
-activated HMVEC revealed formation of not only more numerous
but also generally larger TRAP+ MNC than were obtained in co-cultures
of human PBMC with unstimulated HMVEC (Fig. 8C). Inclusion
of the OPG fusion peptide prevented these increases in TRAP+ MNC
numbers and size, without affecting basal co-culture TRAP+ MNC
formation (Fig. 8C). MNC size changes were confirmed by
quantifying the number of nuclei contained within individual TRAP+ MNC
(Table II). Thus, TNF-
-activated HMVEC
day 16 co-cultures contained fewer small MNC (with 3 nuclei/MNC) and
more numerous large MNC (with
4 nuclei/MNC). These were the only
cultures to contain MNC with as many as 11-16 nuclei/MNC, and the
addition of OPG fusion peptide abrogated the stimulated formation of
larger MNC from human PBMC (Table II). Similarly, TRAP+ MNC sizes were
increased in day 7 co-cultures containing TNF-
(Table II) or IL-1
(not shown) pre-activated HMVEC, and reduced in the presence of the OPG
inhibitory peptide. Thus, inflammatory cytokine-activated HMVEC
stimulated both the number and multinuclearity of TRAP+ MNC
formed in co-cultures with human PBMC, in each case via a
RANKL-dependent pathway antagonized by OPG.

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Fig. 8.
TNF- -activated HMVEC
promote the fusion of OC precursors to form larger, more multinucleated
TRAP+ MNC via a RANKL-dependent mechanism antagonized by
OPG. HMVEC were pre-activated for 24 h with TNF- (1 nM), washed, co-cultured with human PBMC (containing OC
cell precursors) in the presence or absence of a RANKL-neutralizing
recombinant human OPG fusion peptide (100 ng/ml) for 16 days, and the
development of TRAP+ multinucleated cells was evaluated as in Fig. 7.
A, the number of TRAP+ multinucleated cells (containing 3 or
more nuclei) were counted in each co-culture well (totaling over 5000 MNC), and the data were expressed as the mean ± S.E. total number
of TRAP+ MNC formed per well (open bars) or proportion of
TRAP+ MNC relative to total TRAP+ cells formed (hatched
bars). Significant differences from control co-cultures of human
PBMC and unactivated HMVEC in the absence of the OPG fusion peptide are
denoted by *, p < 0.05, and from TNF- -activated
HMVEC co-cultured with human PBMC in the absence of OPG by ++,
p < 0.01, and +++, p < 0.001. B, the mean ± S.E. total number of TRAP+ mononuclear
cells (open bars) or TRAP+ mononuclear plus MNC cells
(hatched bars) per well was not influenced by TNF-
pre-activation of HMVEC. Although a trend toward higher numbers of
TRAP+ mononuclear cells was seen in the co-culture wells containing the
OPG fusion peptide, this did not reach statistical significance.
C, microscopic examination of the co-cultures fixed and
stained for TRAP activity on day 16. Note the formation of more
numerous and generally larger TRAP+ MNC in wells containing TNF-
pre-activated HMVEC, and the loss of this stimulation upon addition of
the RANKL neutralizing OPG fusion peptide. Magnifications, × 50 (upper panel) and 100 (lower panel).
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TNF-
Activation of HMVEC Promotes in Vitro Formed OC Bone Pit
Resorption by a RANKL-dependent Mechanism--
Increased
MNC formation was also accompanied by parallel changes in bone pit
resorption activity (Fig. 9). Human PBMC
co-cultured with TNF-
-pre
activated HMVEC exhibited substantial
increases in the overall area of ivory resorbed and the total number of pits formed, effects that were completely abolished in the presence of
the OPG fusion peptide (Fig. 9, A and B).
Although such increased resorption activity could simply reflect the
greater numbers of TRAP+ MNC formed under these conditions,
TNF-
-stimulated HMVEC also activated individual TRAP+ cells formed
to resorb more ivory. Thus, the mean area resorbed per TRAP+ cell (Fig.
9D), the number of pits formed per TRAP+ cell (Fig.
9E), and the size of lacunae formed (Fig. 9F)
were all increased in the co-cultures containing TNF-
-stimulated
HMVEC, and elevations in these resorption parameters were completely
prevented by the presence of the OPG fusion peptide. Therefore, overall
the TNF-
activation of HMVEC promoted the developmental formation
and fusion of TRAP+ mononuclear cells into MNC and activated their bone
pit-resorptive function via RANKL-dependent mechanisms that
were antagonized by OPG.

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Fig. 9.
TNF- -activated HMVEC
stimulate the bone pit resorptive activity of OCs formed in co-cultures
with human PBMC via a RANKL-dependent mechanism antagonized
by OPG. Ivory chips harvested from the co-culture wells of Fig. 8
were stained for TRAP activity and analyzed over a constant number of
random fields (40) for the overall amount of bone pit resorption
activity (A and B), number of TRAP+ cells
(C), and resorptive activity per TRAP+ cell
(D-F) as described under "Experimental Procedures."
Data were obtained from 3 independent wells per condition of a
representative co-culture resorption trial (totaling over 2000 TRAP+
cells and their associated resorption pits) and were expressed as the
mean ± S.E. for each resorption parameter. A and
B, TNF- -pre-activated HMVEC significantly increased the
mean overall area (µm2) of ivory resorbed (A)
and the mean total number of pits formed (B) by TRAP+ MNC
formed in the co-cultures. The OPG fusion peptide completely prevented
such stimulation. C, TNF- -pre-activated HMVEC did not
significantly increase the total number of TRAP+ cells attached to the
ivory chips (consistent with the data in Fig. 8 demonstrating that the
number of TRAP+ MNC, but not total TRAP+ cells, is increased by this
co-culture treatment). D-F,
TNF- -pre-activated HMVEC significantly increased the mean area
(µm2) of ivory resorbed per TRAP+ cell formed
(D), the mean number of pits formed per TRAP+ cell
(E), and the mean size (µm2) of pits excavated
(F). The OPG fusion peptide completely prevented this
activation of the resorption activity of TRAP+ cells formed in the
TNF- -stimulated HMVEC co-cultures. Significant differences from
control co-cultures of human PBMC and unactivated HMVEC in the absence
of the OPG fusion peptide are denoted by *, p < 0.05, and from TNF- -activated HMVEC co-cultured with human PBMC in the
absence of OPG by +, p < 0.05, and ++,
p < 0.01.
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RANKL Is Highly Expressed by Bone VEC Located Near Resorbing OC in
Areas of Active Human Bone Turnover in Vivo--
Because developing
and resorbing OCs are situated close to blood vessels and capillaries
in bone, and circulating OC precursors must transmigrate through a VEC
barrier to reach the bone marrow microenvironment, inflammatory
regulated expression of RANKL and OPG by VEC may impact on and
contribute to the development, survival, and resorptive activity of OCs
at sites of localized bone loss. Therefore, to validate further the
physiological relevancy of our in vitro findings, we
examined whether RANKL protein was detectable on VEC within human bone
tissue, particularly in areas undergoing bone turnover.
Immunohistochemical staining of paraffin-embedded sections of
human osteoporotic bone derived from femoral head fractures was
performed using a pAb raised to human RANKL and showed RANKL protein
expression consistently displayed on blood vessels or capillaries
located in regions associated with OC lacunar resorption and active
bone remodeling (Fig. 10,
A-D). RANKL signals on VEC appeared comparable in intensity
to that of other RANKL-expressing cells in these regions, including
OB-like cells located along the bone surface (Fig. 10, B and
D). RANKL appeared to be expressed in a polarized fashion on
the basolateral surface of VEC facing the bone marrow microenvironment,
where it could presumably interact with circulatory cells emigrating
into the bone tissue as well as with pre-OC and resorbing OC in the
bone marrow via close physical interactions between VEC and such cells
(Fig. 10D). By contrast, blood vessels and capillaries
proximal to newly forming osteoid or quiescent bone surfaces within
these same bone sections evidenced no detectable specific RANKL protein
staining (Fig. 10E). Control sections incubated without
primary pAb also showed no peroxidase signals in any cell type,
including VEC (Fig. 10F).

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Fig. 10.
Blood vessels or capillaries of human
osteoporotic bone exhibit RANKL immunostaining in areas associated with
OC resorption and bone remodeling. Paraffin-embedded sections of
human osteoporotic bone derived from fractured femoral heads were
immunostained using a pAb to human RANKL, developed using a
peroxidase/diaminobenzidine protocol, counterstained with hematoxylin,
and viewed and photographed as detailed under "Experimental
Procedures." RANKL protein was consistently immunodetected on VEC
situated close to resorbing OCs and regions of bone remodeling;
conversely, no RANKL immunostaining was exhibited by VEC that were not
located in such areas. A, immunodetection of RANKL protein
on VEC (arrows) adjacent to OCs (arrowhead)
engaged in the resorption and remodeling of bone. Note that other cells
within the bone marrow and along the bone surface also exhibit positive
signals for RANKL protein. OCs were RANKL-negative. B,
immunodetection of RANKL protein on VEC (arrows) and
OB-related cells (indicated by asterisks below the cells) in
a region of active bone remodeling. C, RANKL immunostaining
associated with a bone capillary in which a portion has been sectioned
parallel to the length of the capillary (arrow). Note the
relatively homogeneous distribution of the RANKL signal along the
capillary surface. D, RANKL immunostaining in VEC-
(arrows) and OB-related cells (asterisks) in a
region undergoing bone remodeling. Note that RANKL signals detected on
the bone capillary appear to be generally polarized to the outer VEC
surface. E, RANKL protein is not immunodetected on VEC
(arrows) associated with a region of newly forming bone
osteoid (star) in a different area of the bone section shown
in B. F, control section demonstrating no
peroxidase signals associated with VEC (arrow) near
resorbing OCs (arrowhead) in the absence of the primary pAb
to human RANKL. Magnification, × 600 (A) and 400 (B-F).
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|
 |
DISCUSSION |
This study has demonstrated for the first time that primary HMVEC
express both RANKL and OPG, that the pro-inflammatory cytokines TNF-
and IL-1
induce elevated RANKL and OPG mRNA levels in HMVEC according to differing temporal expression profiles, and that TNF-
- or IL-1
-stimulated levels of RANKL mRNA and protein in HMVEC function to promote the in vitro development and
activation of bone pit-resorptive OCs that form in co-cultures with
human PBMC. In vivo, RANKL protein expression is increased
on blood vessels or capillaries in the vicinity of resorbing OCs and
regions of active bone remodeling within sections of human osteoporotic bone. Therefore, microvascular endothelial cells may have an important role in regulating localized bone loss through their stimulated expression of RANKL and OPG, as well as M-CSF, the key factors involved
in controlling OC development, survival, and bone resorption both
in vitro and in vivo (1-4).
Although it has long been known that the vasculature of bone plays an
essential role in the development, dynamic remodeling, and repair of
bone, it has only recently become clear that the vascular endothelium
functions as more than a permeability barrier and passive conduit of
cells and endocrine signals, but also as a vital secretory and
immunologic organ (19, 22-27). OC hematopoietic precursor cells are
present within both the peripheral circulation and the bone marrow, and
in all cases they develop into mature functional OC within the bone
tissue while in close spatial proximity to the microvasculature and
sinusoids (19-23, 37). Thereafter, OCs remain intimately associated
with microvascular endothelial cells during their active resorption of
bone (19-23, 38, 39). Whereas OB and BMSC are well documented to
regulate OC formation and function through both soluble and cell
contact-mediated mechanisms, much less is known regarding how
microvascular endothelial cells may interact with developing and mature
OC. Recently, RANKL mRNA was detected in the metaphyseal vessels of
bone (5), RANKL protein in the small blood vessels of the skin (40),
and OPG, RANK, and RANKL mRNA in the calcified arteries of
OPG-deficient mice but only OPG mRNA in normal adult mouse arteries
(41). Here, we have shown that primary HMVEC express mRNA
transcripts for both RANKL and OPG in a regulated manner. Unlike BMSC
or OB, HMVEC did not respond to PTH or VD3/Dex with any
changes in RANKL or OPG mRNA levels, although VEC have been
reported to respond in other ways to such signals (42-44). This
contrasts with numerous studies in which human or mouse BMSC or OB have
responded to these and other osteotropic hormonal signals by
up-regulating RANKL mRNA and reducing (or not altering) OPG
mRNA levels (1-6, 10, 11, 45-48). A reciprocal pattern of RANKL
and OPG mRNA expression also occurs during OB-like development,
with consequent effects on osteoclastogenesis (1-6, 11, 46-50). Our
findings therefore suggest that VEC may not directly contribute to the
increased OC formation and resorptive activity associated with the
complex actions of these osteotropic hormones.
In contrast, VEC may have a prominent role in regulating the
recruitment and development of bone-resorptive OCs at localized sites
of inflammation, thereby contributing to the osteopenia associated with
rheumatoid arthritis, periodontal disease, and other inflammatory
disorders. Because the pro-inflammatory cytokines TNF-
and IL-1
potently activate VEC, stimulate OC-mediated bone resorption in
vivo and in vitro, and are elevated and known to play
an important role in pathological conditions associated with bone loss,
their potential regulation of RANKL and OPG in HMVEC was investigated.
TNF-
or IL-1
activation of HMVEC led to the dose- and
time-dependent stimulation of both RANKL and OPG mRNA levels, with OPG mRNA levels rising rapidly and then declining at
about the time that RANKL mRNA levels were achieving maximal sustained levels. Compared with TNF-
, IL-1
evoked a similar initial but greater maximal rise in RANKL mRNA expression (to levels comparable to HBMSC), a lesser stimulation of OPG mRNA levels, and more complex biphasic kinetics of RANKL and OPG mRNA expression. Recently, TNF-
and IL-1
were also reported to
increase RANKL and/or OPG mRNA levels in primary HBMSC, HOB, and
human osteosarcoma MG-63 cells (13, 48, 50, 51). As in HMVEC, RANKL
mRNA levels were stimulated 2-4-fold in HBMSC by nanomolar concentrations of the cytokines; peak RANKL mRNA levels were
reached by 12 h and maintained for at least 24 h, and IL-1
elicited a greater increase than did TNF-
in RANKL mRNA levels,
whereas the opposite was true for OPG mRNA induction (13). However, no temporal analysis of OPG mRNA induction was reported (13). In
MG-63 and HOB, OPG mRNA levels increased in response to TNF-
or IL-1
within 2 h, peaked by 4-8 (MG-63) or 16 h (HOB),
and thereafter declined (MG-63) by 24 h (50, 51). However,
parallel changes in RANKL mRNA levels were not examined. To our
knowledge, the present study is the only direct demonstration that a
co-stimulation of RANKL and OPG mRNA levels can involve a
reciprocal temporal expression pattern, with an early rise in OPG
followed by its decline in parallel with a delayed and sustained rise
in RANKL. Because the net effects on OC formation, survival, and bone
resorption activity are critically determined by the ratio of RANKL to
OPG, the temporal nature of their mRNA regulation and the actual
levels of RANKL and OPG protein produced are key parameters that govern their physiological effects (1-4, 41).
RANKL protein levels assessed by immunostaining were increased along
with RANKL mRNA levels in TNF-
-activated HMVEC. Enhanced RANKL
expression on the surface of HMVEC proved physiologically capable of
inducing the in vitro formation of OCs when HMVEC were pre-activated with either TNF-
or IL-1
and then directly
co-cultured with human PBMC containing OC precursors. Whereas M-CSF
stimulates the early stages of OC recruitment and development
(including RANK and TRAP expression), RANKL affects the later stages of
OC cell fusion and differentiation into mature functional OCs (1-4). Consistent with this, all of the co-cultures that received M-CSF contained TRAP+ cells, and the total number of TRAP+ cells was not
influenced by TNF-
pre-activation of HMVEC. However, either TNF-
or IL-1
pre-activation of HMVEC caused a remarkable
1.5-3-fold greater number of TRAP+ MNC to form in the co-cultures, as
well as an increase in the size and multinuclearity of such TRAP+ MNC. Addition of an anti-RANKL neutralizing inhibitory OPG fusion peptide completely abolished this stimulation of TRAP+ MNC number, size, and
multinuclearity, thereby establishing that the mechanism by which
TNF-
or IL-1
activated HVMEC increased OC formation was via a
RANKL-dependent pathway. The fact that TNF-
or IL-1
stimulated HMVEC each caused a similar level of
RANKL-dependent human OC development by day 7 in the
co-cultures, even though these cytokines induced RANKL/GAPDH and
OPG/GAPDH mRNA levels in HMVEC according to somewhat different
temporal profiles, may potentially be explained by the kinetics of
their effects on these molecules (Figs. 3 and 4). Thus, following
TNF-
or IL-1
induction and withdrawal from stimulated HMVEC (as
was performed before the addition of human PBMC to avoid potential
confounding effects due to direct actions of these cytokines on human
OC formation or survival), RANKL/GAPDH mRNA levels remained
moderately elevated in TNF-
-stimulated HMVEC but declined from
highly elevated to basal levels in IL-1
-stimulated HMVEC, whereas
OPG/GAPDH levels returned to basal levels within 24 h in both
TNF-
- and IL-1
-stimulated HMVEC. Therefore, human PBMC might
be exposed to higher initial, but briefer, elevated RANKL levels in the
co-cultures containing IL-1
pre-activated HVMEC versus
more moderate, but sustained, elevated RANKL levels in the co-cultures
containing TNF-
pre-activated HMVEC. Only low basal OPG levels
should be present in these co-cultures after 1 or 2 days due to the
rapid fall in OPG expression following cytokine withdrawal, the labile
nature of OPG, and its removal through medium changes. These effects
might balance out overall to produce similar levels of in
vitro human OC formation. Although additional experiments would be
required to confirm and dissect this further, the key message derived
from the current studies is that both TNF-
and IL-1
prove capable
of activating HMVEC to promote human OC formation in vitro
via a RANKL-dependent mechanism. Greater OC formation was
accompanied by enhanced bone pit resorption activity, both overall as a
result of increased OC numbers and on a per cell basis reflecting
activation of these OCs for resorption. Thus, the total area resorbed,
the number of pits formed, the area resorbed per cell, the resorption
sites initiated per cell, and the mean lacunar pit size excavated by
OCs formed in the TNF-
-activated HMVEC co-cultures were all
significantly higher than in the other co-culture conditions. A
RANKL-mediated pathway was again implicated since the OPG inhibitory
peptide fully suppressed this activation of OCs for resorption. All of
these actions elicited by TNF-
- or IL-1
-activated HMVEC to
promote pre-OC cell fusion, multinucleation, differentiation, and
activation of OCs for bone pit resorption match those reported
previously to be caused by RANKL and antagonized by OPG (1-4).
In vivo, TNF-
and IL-1
tend to be co-produced at sites
of localized inflammation, elicit many similar biological responses, may function in concert with one another, and often remain elevated if
the inflammatory condition does not resolve and progresses into a
chronic disorder. Many inflammatory cytokines, including TNF-
and
IL-1, that act to initiate and maintain inflammation and to promote
bone resorption also stimulate angiogenesis, a hallmark characteristic
and a vital component of the pathology of inflammation,
tumor-associated osteolysis, osteoporosis, and other skeletal disorders
(19, 27, 28, 36, 52-54). Increased angiogenesis enables greater
recruitment of circulating OC precursors to localized sites of
inflammation, and the close contact that VEC share with transmigrating
cells and OC precursors already residing within the bone marrow allows
for their direct exposure to RANKL, M-CSF, and other regulatory
molecules expressed by activated VEC that could initiate their
development into bone-resorptive OCs. Consistent with this, we found
that sections of human osteoporotic bone prepared from femoral head
fractures exhibited RANKL protein expression on VEC that were located
proximal to resorbing OCs and regions of active bone remodeling
in vivo. In contrast, negligible RANKL signals were detected
on VEC associated with newly forming osteoid or quiescent regions of
these same human bone samples. Osteolysis in such fractured femoral
heads is known to be associated with increased OC numbers, trabecular
scalloping, and locally elevated levels of pro-inflammatory cytokines
including TNF-
and IL-1. Therefore, the in vivo
immunohistochemical findings strongly support the in vitro
HMVEC studies and suggest that activated VEC may contribute to
promoting the development, activity, and survival of bone-resorptive
OCs at inflammatory sites through their regulated expression of RANKL
and OPG. Prolonged exposure of VEC in vivo to TNF-
or
IL-1
may provoke a relatively persistent increase in RANKL
expression, together with a rise and subsequent fall in OPG expression,
based on our in vitro findings with continuously stimulated
HMVEC. Furthermore, in contrast to our in vitro co-culture studies wherein cytokines were withdrawn after HMVEC activation, osteoclastogenic effects by VEC in vivo might be more
pronounced in response to IL-1
versus TNF-
since
continuous IL-1
stimulation caused greater RANKL induction and
lesser OPG induction than did TNF-
in HMVEC. Moreover, the effects
we have measured in vitro may be further magnified in
vivo since OPG is produced as a secreted factor whose local
concentrations might be dissipated systemically through circulatory
flow, thereby effectively increasing the potency of transmembrane RANKL
locally expressed on activated VEC. These issues will require further
investigation. Such VEC-related mechanisms likely interface with
similar and additional important soluble and
contact-dependent regulatory signals received from BMSC,
OB, and other cells present in the bone marrow microenvironment.
The regulated production of RANKL and OPG by VEC also has broader
implications that extend beyond control of OC-mediated bone development
and remodeling. Thus, RANKL expression by activated VEC potentially
allows the vasculature to participate directly, in a spatial and
temporal manner, in various important RANKL-mediated developmental and
immune-related processes, such as lymph node organogenesis, lymphocyte
development, and T cell/dendritic cell interactions (1-4, 14).
Conversely, VEC-derived OPG could provide a counterbalancing signal for
such processes, in addition to its role in calcium homeostasis and as a
local and systemic inhibitor of OC formation and bone resorption
(1-4). Due to the enormous surface area represented by the endothelium
throughout the body, VEC could represent a major source of the
circulating OPG found in serum and contribute to the increased serum
OPG levels reported for aging healthy men and women and in
postmenopausal osteoporotic women (a condition associated with
increased vascularity and levels of IL-1 and TNF-
) (55). Because OPG
functions as an important physiological suppressor of vascular
calcification, mice deficient in OPG exhibit arterial calcification in
addition to early onset osteoporosis (which are both prevented by
transgenic OPG expression), and OPG acts as an
v
3-induced survival factor for VEC OPG
production by VEC may also serve as a key autocrine signal to inhibit
blood vessel calcification (1-4, 17, 41, 56).
In conclusion, inflammatory cytokine activation of HMVEC caused a
sustained up-regulation of RANKL and a more transient expression of
OPG, the key molecules involved together with M-CSF in controlling OC
formation, survival, and bone resorption in vivo and
in vitro. Functionally, this led to an increased ability of
HMVEC to stimulate the in vitro co-culture development and
activity of mature bone pit-resorptive OCs from circulating human
monocytic precursors. In vivo, RANKL expression was
up-regulated on VEC only in the vicinity of resorbing OCs and areas of
active bone remodeling. Therefore, we surmise that
inflammatory-activated VEC may help promote localized bone loss via
their RANKL-mediated effects on pre-OC and OC to increase the number of
sites and/or rates of bone remodeling. Therapeutic intervention aimed
either at preventing an increase in RANKL or further enhancing OPG
production by activated VEC may therefore help to alleviate the
osteopenia seen in various inflammatory diseases, metabolic bone
disorders, malignancy-related osteolyses, or certain immune disorders.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Len Rifas for providing primary
HOB cells, Drs. Len Rifas and Neil Weitzman for primer design and
advice in setting up the RT-PCRs for RANKL, and Dr. Teresa Sunyer for
valuable advice on initially performing and analyzing the RANKL RT-PCR studies.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants DK46547 (to P. C. O.) and AR32927 (to P. O.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence and reprint requests should be
addressed: Dept. of Biology, Box 1229, Washington University, St.
Louis, MO 63130. Tel.: 314-935-5304; Fax: 314-935-5134; E-mail:
collin@biology.wustl.edu.
Published, JBC Papers in Press, March 23, 2001, DOI 10.1074/jbc.M010153200
 |
ABBREVIATIONS |
The abbreviations used are:
RANKL, receptor
activator of NF-
B;
TNF-
, tumor necrosis factor-
;
TRANCE, TNF-related activation induced cytokine;
OPGL, osteoprotegerin ligand;
OPG, osteoprotegerin;
RANK, receptor activator of NF-
B;
IL-1
, interleukin-1
;
M-CSF, macrophage colony-stimulating factor;
VD3, 1,25-dihydroxyvitamin D3;
Dex, dexamethasone;
PTH, parathyroid hormone;
OC, osteoclast;
VEC, vascular
endothelial cells;
HMVEC, human microvascular endothelial cells;
(H)OB, (human) osteoblast;
(H)BMSC, (human) bone marrow stromal cells;
PBMC, peripheral blood mononuclear cell;
MNC, multinucleated cell;
EGM-MV, essential growth medium-microvascular;
EBM, essential basal medium;
-MEM,
-minimal essential medium;
FBS, fetal bovine serum;
HBSS, Hanks' balanced salt solution;
PF, paraformaldehyde;
BSA, bovine serum
albumin;
mAb, monoclonal antibody;
pAb, polyclonal antibody;
TRAP, tartrate resistant acid phosphatase;
RT-PCR, reverse
transcriptase-polymerase chain reaction;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
bp, base pair;
DAPI, 4,6-diamidino-2-phenylindole.
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