(Circulation. 2000;102:2636.)
© 2000 American Heart Association, Inc.
Basic Science Reports |
Tumor Necrosis Factor-
Promotes In Vitro Calcification of Vascular Cells via the cAMP Pathway
From the Division of Cardiology, Departments of Medicine and Physiology, UCLA School of Medicine, Los Angeles, Calif.
Correspondence to Yin Tintut, Division of Cardiology, UCLA School of Medicine, 47-123 Center for the Health Sciences, 10833 Le Conte Ave, Los Angeles, CA 90095-1679. E-mail ytintut{at}ucla.edu
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Abstract |
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Background—Vascular calcification is an ectopic calcification that commonly occurs in atherosclerosis. Because tumor necrosis factor-
(TNF-), a pleiotropic cytokine found in atherosclerotic
lesions, is also a regulator of bone formation, we investigated the
role of TNF- in in vitro vascular calcification.
Methods and Results—A cloned subpopulation of bovine aortic
smooth muscle cells previously shown capable of osteoblastic
differentiation was treated with TNF-, and osteoblastic
differentiation and mineralization were assessed. Treatment of vascular
cells with TNF- for 3 days induced an osteoblast-like morphology. It
also enhanced both activity and mRNA expression of alkaline
phosphatase, an early marker of osteoblastic differentiation.
Continuous treatment with TNF- for 10 days enhanced matrix
mineralization as measured by radiolabeled calcium incorporation in the
matrix. Pretreatment of cells with a protein kinase A–specific
inhibitor, KT5720, attenuated cell morphology, the alkaline
phosphatase activity, and mineralization induced by TNF-.
Consistent with this, the intracellular cAMP level was elevated
after TNF- treatment. Electrophoretic mobility shift assay
demonstrated that TNF- enhanced DNA binding of osteoblast specific
factor (Osf2), AP1, and CREB, transcription factors that are important
for osteoblastic differentiation.
Conclusions—These results suggest that TNF- enhances in vitro
vascular calcification by promoting osteoblastic differentiation of
vascular cells through the cAMP pathway.
Key Words: muscle, smooth • signal transduction • atherosclerosis
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Tumor necrosis factor-
(TNF-), a pleiotropic
cytokine, has been shown to play a role in both vascular and
bone pathophysiology. TNF- is mainly secreted by macrophages
in response to factors such as oxidized LDL,1
acetylated LDL,2 physically damaged extracellular
matrix,3 or bacterial infection.4 TNF-
influences many aspects of atherogenesis,5 including
increasing permeability of endothelial
cells,6 promoting monocyte adhesion,7
inducing macrophage differentiation,8 and
promoting foam cell formation.9 In vivo, TNF- induces
arteriosclerosis-like lesions in coronary
arteries.10 TNF- also regulates bone turnover,
inhibiting osteoblastic function11 12 13 and stimulating
bone resorption.14 Vascular calcification is a pathological condition that occurs in many diseases, including atherosclerosis, diabetes, and uremia.15 We and others have provided evidence that atherosclerotic calcification resembles osteogenesis,16 17 18 and factors regulating bone mineralization have been demonstrated in calcified atherosclerotic plaques.16 17 19 20 21 22 In previous studies of vascular calcification, we cloned a subpopulation of vascular cells from the bovine aortic media.16 17 In long-term culture, these calcifying vascular cells (CVCs) express osteoblastic differentiation genes in the sequence reported for bone cells and form a mineralized matrix in vitro.23 Similar in vitro models have been developed by other investigators.18 24 25 26 Agents that are present in atherosclerotic arteries have been shown to promote the differentiation and mineralization of these vascular cells.17 27
Although TNF- has been detected in both human and mouse
atherosclerotic lesions,28 29 its contribution to vascular
calcification has not been assessed. We and others found TNF-
immunoreactivity in arteries with calcification in C57BL/6J mice fed an
atherogenic diet.29 In contrast, TNF- is not found in
normal vascular intima or fatty streaks.29 30 Additional
evidence that TNF- may regulate vascular calcification arises from
recent in vivo studies showing that mice lacking the osteoprotegerin
gene, a novel soluble member of the TNF- receptor superfamily,
develop vascular calcification in addition to premature
osteoporosis.31 Therefore, we hypothesized that TNF-
regulates calcification in atherosclerotic lesions, and we examined the
direct role of TNF- in in vitro vascular calcification.
In the present report, CVCs were treated with TNF- and the
effects of TNF- on differentiation and mineralization of CVCs were
determined. The results indicate that TNF- promotes CVC
mineralization by modulating the expression of genes important for
extracellular matrix formation and mineralization. We also showed that
the enhanced mineralization of CVCs by TNF- was mediated in part by
activation of the cAMP pathway and enhancement of the activity of
transcription factors that are important for osteoblastic
differentiation.
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Methods |
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Materials
Human recombinant TNF-
was purchased from Sigma Chemical
Co. [45Ca]CaCl2,
[-32P]dCTP,
[
-32P]ATP, and
[3H]thymidine were from Amersham.
Oligonucleotide probes containing AP1 and
cAMP-responsive element-binding protein (CREB) consensus sequences were
purchased from Santa Cruz Biotechnology, Inc.
Oligonucleotide probe containing Cbfa1 binding site
(OSE2)32 from osteocalcin promoter was synthesized
by GIBCO BRL. Pertussis toxin, chelerythrine chloride, phorbol
12-myristate 13-acetate (PMA), AACOCF3,
indomethacin, and KT5720 were purchased from
Calbiochem. Anti-TNF- antibody was obtained from BioSource
International. Anti-Fos and anti-Jun antibodies were purchased from
Santa Cruz Biotechnology, Inc. Antibody specific to
Cbfa1/Osf233 was a generous gift from Dr Gerald Karsenty.
Quantification of bacterial endotoxin in human recombinant TNF- was
tested with quantitative chromogenic limulus amebocyte
lysate (LAL; BioWhittaker, Inc).
Cell Culture
CVCs were isolated from bovine aortic media layer and were
identified as described previously.16 17 CVCs were grown
in Dulbecco’s modified Eagle’s medium (DMEM; Irvine Scientific)
containing 15% heat-inactivated FBS (Hyclone Labs) and
supplemented with sodium pyruvate (1 mmol/L), penicillin (100
U/mL), and streptomycin (100 U/mL), all from Irvine Scientific. Cells
(passages 12 to 17) were treated at 80% confluence with appropriate
agents in DMEM containing 5% FBS.
Alkaline Phosphatase Activity Assay
Cells were cultured in 24-well plates and treated with or
without test agents at 80% confluence for 2 to 3 days. Cells were
lysed and alkaline phosphatase (ALP) activity was measured as
previously described.27 ALP activity was normalized to
total protein determined with the Bio-Rad protein assay solution
(Bio-Rad Laboratories).
Reverse Transcription–Polymerase Chain Reaction
Total RNA (3 µg) extracted as mentioned above was
reverse-transcribed, and polymerase chain reaction (PCR) with ALP and
GAPDH-specific primers was performed as described
previously.23
45Ca Incorporation Assay
Cells were cultured in 24-well plates and treated at 80%
confluence with or without 10 ng/mL TNF-. In the experiments in
which there were pretreatments, cells were treated with agents 2 hours
before addition of TNF- to the media. After 3 to 4 days of
incubation, media were replaced with fresh media containing TNF-
(pretreated agents and TNF-), 4 mmol/L
CaCl2, and 5 mmol/L ß-glycerophosphate
(ßGP) and incubated for an additional 3 to 4 days. Cells were washed
3 times and incubated in fresh media containing TNF-, ßGP, and
[45Ca]CaCl2 (1.0
µCi/mL) for an additional 48 hours. The matrix-bound radiolabeled
calcium incorporation was performed as described
previously.23
cAMP Assay
Cells were plated in 6-well plates and treated at 80%
confluence with serum-free medium for 30 minutes before being treated
with 50 ng/mL TNF- in serum-free medium supplemented with 1
mmol/L 3-isobutyl-1-methylxanthine (IBMX; Calbiochem) for an additional
30 minutes. After incubation, cells were washed and scraped in 20
mmol/L phosphate buffer (pH 7.0) containing 20 mmol/L EDTA and
1 mmol/L IBMX. The extracts were sonicated briefly and boiled for
7 minutes to precipitate the cellular proteins. The extract was
clarified by centrifugation, and supernatant was
assayed for cAMP level with the cAMP EIA kit (Stratagene).
Gel Mobility Shift Assay
Nuclear extracts were prepared as described
previously.34 Three to 5 µg of nuclear extracts was
incubated with binding buffer containing 20 mmol/L Tris-HCl (pH
8), 10 mmol/L NaCl, 3 mmol/L EDTA, 0.05% Nonidet P-40,
5 mmol/L dithiothreitol, 5% glycerol, and 1 µg of poly(dIdC)
for 15 minutes, after which labeled probe was added and incubated for
an additional 15 to 20 minutes. The samples were subjected to
electrophoresis on a chilled 5% polyacrylamide gel at 400 V
for 14 minutes. In the supershift experiments, respective antibodies
were preincubated with nuclear extracts for 30 minutes before addition
of the labeled probe. In competition experiments, 100-fold excess of
the respective cold oligonucleotides was preincubated
with nuclear extracts for 15 minutes before addition of the labeled
probe.
Statistical Analysis
Data are expressed as mean±SD, and means were compared by 1-way
ANOVA. The Tukey-Fisher least significant difference (LSD) criterion
was used to judge statistical significance.
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Results |
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Effects of TNF-
on Markers of Osteoblastic Differentiation
and MineralizationTo determine the effects of TNF-
on onset of osteoblastic
differentiation, CVCs were treated for 3 days, and 2 established
markers of early osteoblastic differentiation, morphological
change35 and ALP activity,36 were assessed.
TNF- induced a morphological change from an elongated to a cuboidal
shape (Figure 1A
). Treatment of CVCs with
increasing concentrations of TNF- showed a dose-dependent increase
in ALP activity (Figure 1B). We compared the mean values with
1-way ANOVA. A significant increasing trend by dose was observed
(F=2117; P<0.00005). The effect of TNF- on ALP activity
was not due to an artifact of normalization, because even without
normalization for total protein, the ALP activity was increased 15-fold
by TNF- (10 ng/mL) (data not shown). Further tests were performed to
determine that the effects were specific to TNF-. Results from a
quantitative chromogenic LAL assay showed an endotoxin
(lipopolysaccharide) level of
4x10-5 pg/µL, which is
5 orders of magnitude below the level required for activity in this
system. In addition, treatment with TNF- antibody completely blocked
the induction of ALP activity (data not shown), indicating that TNF-
effect on ALP activity is not attributable to contamination with
bacterial endotoxin.
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To determine whether the induction of ALP by TNF- was at the level
of mRNA expression, total RNA was isolated from samples treated with
TNF- for 3 days, and semiquantitative reverse transcription-PCR
(RT-PCR) was performed. Results showed that TNF- also enhanced mRNA
expression of the ALP gene (Figure 1C
). TNF- did not enhance
expression of the internal control gene GAPDH. Treatment of CVCs with
TNF- for 3 days did not appreciably affect expression of other
osteoblastic genes, type I procollagen, and osteocalcin (data not
shown).
To determine whether enhanced ALP activity ultimately results in
increased mineralization, CVCs were treated with increasing
concentrations of TNF- for 9 to 10 days, and matrix calcium mineral
incorporation was assayed with 45Ca as reported
previously.23 The results showed that TNF- dose
dependently increased mineralization in these cells (Figure 1D). A significant increasing trend by dose was observed (F=123;
P<0.00005).
Second Messenger of TNF- in CVC Differentiation and
Mineralization
In various cell types, the pleiotropic effects of TNF- are
mediated by different intracellular signaling pathways. Therefore, we
examined the signaling pathway used by TNF- in induction of CVC
differentiation and mineralization. First, we assessed the role of
pertussis toxin–sensitive G protein, which mediates TNF- signaling
in other systems.6 37 CVCs were pretreated with pertussis
toxin for 2 hours before TNF- treatment. Results showed that
pertussis toxin did not inhibit TNF-–induced ALP activity (measured
2 days after TNF- treatment), indicating that TNF- does not
signal through a pertussis toxin–sensitive G protein in CVCs (Figure 2A). Instead, pertussis toxin induced ALP activity synergistically with
TNF- (Figure 2A). By 1-way ANOVA, the
difference between control and TNF- plus pertussis toxin (100 ng/mL)
was significant (P<0.05 level; LSD 83.8). We previously
showed that treatment with pertussis toxin alone (without subsequent
TNF- treatment) had relatively little effect on ALP
activity.23
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To determine whether TNF- activated phospholipase
A2 (PLA2) and
cyclooxygenase, as found in bone
cells,38 CVCs were pretreated with inhibitors
of cytoplasmic PLA2 (AACOCF3) or
cyclooxygenase (indomethacin) for 2
hours before addition of TNF-. ALP activity (measured 2 days after
treatment) showed that these agents also did not inhibit TNF-induced
ALP activity (Figure 2B). Instead, the ALP activity induced by
TNF- was further enhanced by the inhibitors. By 1-way
ANOVA, the difference between control and each higher-dose treatment
was significant (P<0.05 level; LSD 44.7). Treatments with
inhibitor (AACOCF3 or indomethacin) alone
had relatively little or no effect on ALP activity, CVC morphology, and
total protein level (data not shown).
We also examined the role of the protein kinase C (PKC)
pathway in TNF- effects in CVCs. Inhibition of the PKC pathway by
pretreatment with a PKC-specific inhibitor, chelerythrine
chloride (0.5 to 1.0 µmol/L), for 2 hours did not reverse the
TNF-–induced ALP activity (Figure 2C). Instead, as seen with
pertussis toxin and cyclooxygenase
inhibitors, ALP activity induction by TNF- was further
enhanced by the chelerythrine chloride. By 1-way ANOVA, the difference
between chelerythrine chloride–treated CVCs and the controls was
significant (P<0.05; LSD 65.6). The same response was
observed when PKC was downregulated by prolonged treatment (24 hours)
with 1 mmol/L PMA (data not shown). Treatment of CVCs with
chelerythrine chloride or PMA alone without subsequent TNF-
treatment had no effects on ALP activity or cell morphology (data not
shown).
Because we have previously shown that the cAMP pathway promotes
osteoblastic differentiation of CVCs,23 we also examined
the involvement of the cAMP/protein kinase A (PKA) pathway in response
to TNF-. CVCs were treated with a PKA-specific
inhibitor, KT5720.39 Treatment of CVCs for 2
hours with KT5720 before addition of TNF- dose dependently inhibited
the TNF-–induced morphological change (Figure 3A). KT5720 also inhibited
TNF-–induced ALP activity (measured 2 days after treatment) (Figure 3B) and mineralization (measured 9 days after treatment) (Figure 3C) in a dose-dependent manner. Consistently,
intracellular cAMP level was increased 2-fold in TNF-–treated cells
(Figure 3D).
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Effect of TNF- on Transcription Factors Cbfa1/Osf2, AP1,
and CREB
Because TNF- modulated the gene expression in CVCs as shown
above, we also examined the effects of TNF- on transcription factors
important in osteoblastic differentiation: Cbfa1/Osf2, AP1, and
CREB.36 40 41 Nuclear extracts from CVCs treated with
TNF- were prepared, and an electrophoretic mobility shift assay was
performed with probes containing Cbfa1/Osf2, AP1, or CREB binding
sites. Results showed that treatment of CVCs with forskolin or TNF-
enhanced the DNA binding of Cbfa1/Osf2 (Figure 4A, left). Dibutyryl cAMP also enhanced
Cbfa1/Osf2 DNA binding, whereas the same concentration of dibutyryl
cGMP had little or no effect (data not shown). Preincubation of
anti-Cbfa1 antibody33 with TNF-–treated nuclear
extracts before addition of labeled probe resulted in a supershifted
band and a decrease in the faster-migrating complex, indicating the
specificity of Cbfa1/Osf2 (Figure 4A, right). TNF- also
enhanced DNA binding of AP1 and CREB transcription factors (Figure 4 B and 4C). AP1 DNA binding was competed with 100-fold
excess of cold AP1 oligo probe (Figure 4B), and CREB DNA binding
was competed with 100-fold excess of cold CREB oligo probe (Figure 4C, left). Incubation of nuclear extracts from TNF-–treated
cells with anti-CREB antibody resulted in a supershifted complex
(Figure 4C, right). Incubation with anti-Fos or anti-Jun
antibodies, however, did not result in a supershifted complex,
indicating that AP1 family members other than c-fos and
c-jun were activated in response to TNF- in CVCs
(data not shown).
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Discussion |
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The present study showed that TNF-
enhanced in vitro
calcification of vascular cells, providing further evidence that
vascular and bone calcification share regulatory factors. TNF-
promotes CVC mineralization by increased expression and activity of
ALP, an enzyme that has been shown to be important for matrix
mineralization.36 42 The enhanced ALP may be a result of
enhanced DNA binding of transcription factors shown to play a role in
osteoblastic differentiation.36 40 41 These data, together
with the previous report associating TNF- with calcified vascular
lesions in mice,29 raise the possibility that TNF-
facilitates mineralization in atherosclerotic lesions.
TNF- has been shown to signal through different pathways in
different cell types. In hepatic stellate cells and in osteoblasts, the
TNF- receptor is coupled to a pertussis toxin–sensitive G protein,
leading to activation of PLA2 and de novo
synthesis of cyclooxygenase.6 37 38 In
cultured rat mesangial cells and human fibroblasts, TNF-
signals through increased intracellular cAMP.43 Our
results suggest that TNF- triggers CVC mineralization via the cAMP
pathway, based on the findings that PKA inhibition blocks and cAMP
elevation mimics23 the TNF-induced effects. In addition,
the finding that pertussis toxin synergizes with TNF- in ALP
induction further supports the possibility that TNF- signals via the
cAMP pathway in CVCs, because inhibition of Gi
by pertussis toxin enhances adenylate cyclase activity.
The results also suggested a form of cross talk between signaling
pathways in CVCs. Inhibition of the PKC pathway potentiated the TNF-
effect on ALP activity, which suggests that the PKC pathway may act as
an antagonist of PKA-mediated CVC differentiation. This may
be due to involvement of PKC in cellular
proliferation,44 45 the decline of which has been shown to
be necessary for the onset of osteoblastic
differentiation.36 The results also showed that similar
effects were seen with inhibitors of the
cyclooxygenase pathway, because activation of the
PKC pathway by products of the cyclooxygenase
pathway has been reported.46
Although CVCs share features with bone-derived osteoblasts in many
aspects, we previously showed that their responses to certain agents
such as oxidized lipids27 and active vitamin D are
directly opposite.47 We and other
investigators11 12 13 14 have found that TNF- inhibited bone
cell differentiation (unpublished data). The present results
suggest that TNF- has similar reciprocal effects (stimulation of
CVCs and inhibition of bone cell differentiation). The coexistence of
vascular calcification and osteoporosis has been reported recently by
Bucay et al31 in mice lacking the osteoprotegerin gene, a
novel soluble member of the TNF- receptor superfamily. These inverse
effects on bone and vasculature may be a result of different signaling
pathways induced by TNF- in the 2 cell types: stimulation of the
cAMP pathway in CVCs versus stimulation of the pertussis
toxin–sensitive G-protein–mediated pathway but not the cAMP pathway
in osteoblasts.38 These differential responses to the same
inflammatory agent may contribute in part to the paradoxical
coexistence of vascular calcification and osteoporosis in older
patients.
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Acknowledgments |
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This work was supported by NIH grant HL30568. We thank Dr Judith Berliner for suggestions; Dr Gerald Karsenty for providing us with anti-Cbfa1–specific antibody; H. Huynh and V. Le for technical assistance; and A. Han and A. Gasparyan for general assistance. We also thank the Atherosclerosis Research Unit Core A for LAL assay and Dr J. Gornbein for statistical analysis. The graphic illustrations were prepared in the Biomedical Technology Research and Instructional Productions (BTRIP) facility, and we also thank its staff for their assistance.
Received February 11, 2000; revision received June 21, 2000; accepted June 22, 2000.
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