(Circulation. 1999;99:2276-2282.)
© 1999 American Heart Association, Inc.
Clinical Investigation and Reports |
Calcium Channel Blockers Activate the Interleukin-6 Gene Via the Transcription Factors NF-IL6 and NF-
B in Primary Human Vascular Smooth Muscle Cells
From the Department of Research and Internal Medicine, University Hospital Basel, Switzerland (O.E., M.R., J.J.R., M.T., A.P.P.); Division of Molecular Biology, The Netherlands Cancer Institute (R.M.); and Department of Internal Medicine IV, University Hospital Vienna, Austria (L.H.B.).
Correspondence to L.-H. Block, University Hospital Vienna, Department of Internal Medicine IV, Währinger Gürtel 18-20, A-1090 Vienna, Austria.
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Abstract |
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Background—Calcium channel blockers (CCB) of all subclasses: the dihydropyridines, benzothiazepines, and phenylalkylamines, at nanomolar concentrations, have been shown to up-regulate interleukin-6 (IL-6) mRNA. We investigated the underlying molecular mechanism responsible for IL-6 induction in response to the CCB amlodipine, diltiazem, and verapamil in primary human vascular smooth muscle cells (VSMC).
Methods and Results—All 3 CCB directly activated
transcription of the human IL-6 gene in primary human VSMC in a time-
and dose-dependent manner, as demonstrated by luciferase reporter gene
assays using a 651-bp fragment of the human IL-6 gene promoter.
Deletion analysis of the IL-6 promoter revealed that CCB
inducible promoter activity was localized to a 160-bp fragment directly
upstream of the transcriptional start site of the IL-6 gene. Known
transcription factor consensus sequences within this fragment include a
NF-IL6 and a NF-
B site. Site-directed mutagenesis suggested that
both transcription factors had positive regulatory activity and
cooperatively transmitted induction of the IL-6 gene by CCB. The data
are confirmed by electrophoretic mobility shift analyses using
nuclear extracts from CCB-stimulated and control primary VSMC. CCB of
all subclasses increased DNA binding of NF-IL6 and NF-B as early as
30 minutes after stimulation with the drugs. This effect was
independent of intracellular calcium concentrations because
calcium-free medium did not increase NF-IL6 or NF-B activity.
Conclusions—The results demonstrate that CCB of all 3 subclasses
are capable of activating NF-IL6 and NF-B. CCB may thus directly
regulate cellular functions by affecting the activity of transcription
factors independent of changes of intracellular calcium concentrations,
an observation that is of interest considering the biological effects
induced by CCB.
Key Words: calcium channels • interleukins • pharmacology • signal transduction • muscle, smooth
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Introduction |
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Calcium channel blockers (CCB) are a heterogeneous class of drugs used in the treatment of coronary heart disease and hypertension. According to their chemical structure, they are divided into 3 subclasses: dihydropyridines, phenylalkylamines, and benzothiazepines.1 2 3 4 CCB are presumed to exert their biological effects by decreasing intracellular calcium concentrations achieved through inhibition of high voltage gated-type calcium channels.1 2 3 4 These channels are expressed in skeletal and vascular smooth muscle cells (VSMC), nerve cells, and fibroblasts.4 5 6 7 The L-type calcium channel consists of 5 subunits (
1, 2, ß, 
, and 
), of which the
1-subunit forms the ion pore responsible for channel activity and
CCB binding.6 7 8 9 Dihydropyridines and
phenylalkylamines have been shown to bind specifically to the
1-subunit of the L-type calcium channel, with
dihydropyridines binding extracellularly at domain
IV,2 9 10 11 12 and phenylalkylamines binding intracellularly
at domain IV.2 13 Benzothiazepines are also presumed to
bind to the 1-subunit but their distinct receptor site has not been
as completely analyzed.2 3 4
Several reports have demonstrated that CCB induce gene expression at
nanomolar concentrations.13 14 15 16 17 These concentrations of
CCB correspond to their KD value for
binding to their cognate receptor sites (0.2 to 0.4
nmol/L),18 19 but do not change intracellular calcium
levels.14 15 Significant changes in intracellular calcium
concentrations could only be observed with CCB used at the micromolar
range. This suggests a potent pharmacodynamic effect of CCB at
nanomolar concentrations, which is independent from changes in
intracellular calcium levels. In this regard, we have previously shown
that members of all 3 subclasses of CCB increased expression of
interleukin 6 (IL-6) and the immediate early genes
c-fos and c-jun on mRNA and protein
levels.15 16 However, the molecular mechanisms by
which CCB increase IL-6 gene expression is unknown. Direct association
of signal transducer elements with membrane calcium channels has
recently been demonstrated in neuronal N and P/Q calcium channels,
where interaction of 1-subunits with G-protein, ß-
complexes, and protein kinase C (PKC) have been implicated in the
control of cellular reactions.20 21 22 Although the
1-subunit of the L-type calcium channel, which serves as
the receptor site for CCB, possesses several intracellular
phosphorylation sites,2 6 9
receptor-specific signaling pathways resulting in the activation of
transcription factors have not yet been characterized.
We describe the molecular mechanism by which the 3 subclasses of CCB,
at nanomolar concentrations, lead to activation of the human IL-6 gene
promoter. Using a 651-bp fragment of the human IL-6 gene promoter
linked to a luciferase reporter gene vector, we demonstrate the
involvement of the transcription factors NF-IL6 and NF-B in the
induction of IL-6 gene expression by CCB. These results are confirmed
by electrophoretic mobility shift assays (EMSA) displaying rapid
activation of these transcription factors, both of which are known to
be involved in the regulation of the human IL-6 gene.23 24
Furthermore, we confirm that these effects are calcium-independent.
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Methods |
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Smooth Muscle Cell Culture
Primary cultures of human lung vascular smooth muscle cells (VSMC) were established from pulmonary arteries obtained during lung surgery.16 17 VSMC were cultivated in Dulbecco's minimal essential medium (Serotec) supplemented with 10% FCS (Gibco), 5% colostrum (Chemie Brunschwick), 8 mmol/L L-glutamine (Serotec), and 20 mmol/L HEPES buffer (Serotec). Subconfluent cultures (80% confluence) were used between passages 3 and 5. Before stimulation with amlodipine, diltiazem, or verapamil, cells were serum-deprived for 48 hours (0.1% FCS).
To evaluate the involvement of intracellular calcium concentrations on
the activation of NF-IL6 and NF-B, subconfluent cells were incubated
with either calcium-free minimal essential medium (Sigma) or BayK8644
(1x10-8 mol/L) with or without CCB as
indicated.
Plasmid Construction and Site-Directed Mutagenesis
A plasmid containing a 651-bp fragment of the human IL-6 gene
promoter located directly upstream of the transcriptional start site
was provided by Shigeru Katamine (Nagasaki University, Nagasaki,
Japan).24 This 651-bp insert was subcloned (5'
Kpn I, 3'-Xho I) into pGL3 luciferase reporter
gene vector (Promega Corp) to give the parental pIL6-luc651 clone
(Figure 1a
). Subclones were made by
5'-deletion of fragments containing the consensus sequence for
transcription factor AP-1 (pIL6-luc220) or CREB (pIL6-luc160) using
internal restriction sites for NHE I (pIL6-luc220) or AAT II
(pIL6-luc160; Figure 1a). Using site-directed mutagenesis, the
NF-IL6 consensus sequence (position -157 to -145,
5'-CACATTGCACAAT-3') within pIL6-luc160 was mutated to
5'-CACACCGTTCAAT-3'. The NF-B consensus sequence (position -75 to
-64, 5'-GTGGGATTT-3') was mutated to 5'-GTCTCATTT-3'. Mutant sequences
of the IL-6 promoter were verified by DNA sequencing. Three different
mutants of pIL6-luc160 were achieved through use of a single NF-IL6
mutant, a single NF-B mutant, and a combined NF-IL6 and NF-B
mutant (Figure 1a).
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Cell Transfection and Luciferase Assays
Transfections were performed in 24-well plates
(1x104 cells/well) precoated with 1% gelatin.
After 24 hours, cells were serum-deprived for 24 hours and transfected
with the cationic lipid Tfx-50 (Promega Corp) at a DNA to lipid ratio
of 1:3 (1 µg of plasmid per well). Cells were then overlaid with
low-serum medium with or without the respective CCB (Figure 1
).
After 36 hours, cells were harvested and lysates were analyzed
for firefly luciferase expression. In brief, 20 µL aliquots of
cell lysates were mixed with 100 µL of luciferase reagent buffer
(Promega Corp) and luminescence of the samples was integrated over a
period of 10 seconds in a LUMAC Biocounter M1500P (Landgraaf). To
assess transfection efficiency, a SV40 promoter driven Renilla
luciferase vector was used (Dual Luciferase Assay, Promega Corp).
Preparation of Nuclear Extracts and Electrophoretic Mobility
Shift Assay
Nuclear and cytosolic extracts from VSMC were prepared as
described earlier.25 Cells were washed twice and harvested
in 1 mL of PBS, centrifuged (1 minute 6.000g), and
resuspended in 50 µL low-salt buffer (20 mmol/L Hepes, pH 7.9,
10 mmol/L KCl, 0.1 mmol/L NaVO4, 1
mmol/L EDTA, 1 mmol/L EGTA; 0.2% NP-40, 10% Glycerol,
supplemented with a set of proteinase inhibitors
[Complete, Boeringer Mannheim AG]). After 10 minutes of incubation on
ice, samples were centrifuged (10 minutes 13.000g)
and supernatant taken as cytosolic extract. Nuclei were resuspended in
high salt buffer (20 mmol/L Hepes, pH 7.9, 420 mmol/L NaCl,
10 mmol/L KCl, 0.1 mmol/L NaVO4, 1
mmol/L EDTA, 1 mmol/L EGTA, 20% glycerol, supplemented with
Complete) and nuclear proteins were extracted by shaking on ice for 30
minutes.
EMSA were performed as described earlier.25
Oligonucleotides comprising the consensus sequences for
the transcription factors AP-1 (c-jun)
(5'-CGCTTGATGAGTCAGCCGGAA-3'), NF-IL6 (5'-TAAGATTGTACAATGT-3'), or
NF-B (5'-AGTTGAGGGGACTTTCCCAGGC-3') were end-labeled with
(-32P)-ATP (Amersham) using
T4-polynucleotide-kinase (Promega Corp). Nuclear extracts
(1 µg) were incubated with labeled consensus
oligonucleotides under binding conditions (4%
glycerol, 1 mmol/L MgCl2, 0.5 mmol/L
EDTA, 0.5 mmol/L dithiotreitol, 50 mmol/L NaCl, 10
mmol/L Tris-HCl, pH 7.5, 50 µg/mL poly(dI-dC)) in a total volume of
10 µL and incubated for 30 minutes at room temperature. Protein-DNA
complexes were analyzed on a 4% polyacrylamide gel.
Identity of transcription factors was confirmed by addition of
competitive unlabelled consensus-sequence
oligonucleotides, polyclonal antibodies to p65 of
NF-B (Santa Cruz Biotechnology, Inc), or polyclonal antibody to
NF-IL6 (C/EBP-ß, Santa Cruz Biotechnology, Inc). The protocol for
establishing primary human cell cultures from biopsies was approved by
the ethical committee of the Faculty of Medicine, University Hospital
Basel (approval # M75/97). For statistical analysis,
Student's t test and ANOVA were performed.
P<0.05 was considered significant.
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Results |
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CCB Induce IL-6-Promoter Activity in VSMC
Figure 2a
through 2c demonstrates
the stimulatory effect of the CCB amlodipine, diltiazem, or
verapamil on transcriptional activity of the human IL-6
promoter-luciferase construct pIL6-luc651 in primary human VSMC. After
36 hours, CCB significantly increased expression of luciferase protein
compared with controls (P<0.005): amlodipine by 214%,
diltiazem by 292%, and verapamil by 292%. PDGF-BB, a
known inducer for IL-6, at 10 ng/mL, stimulated promoter activity to
325%. The stimulatory effect of CCB on promoter activity was
dose-dependent with a maximal effect obtained at
10-8 M for diltiazem and verapamil,
and at 10-7 M for amlodipine. Dose dependency of
CCB-induced IL-6 promoter activity was similar between the 3
subclasses. Concerning the drugs' efficiency, verapamil
and diltiazem were more potent than amlodipine (Figure 2a
through 2c).
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Deletion Analysis of the Promoter Construct
pIL6-luc651
To identify CCB-inducible regulatory regions in the 651-bp
fragment of the human IL-6 promoter, we generated 5' deletion mutants
of the parental plasmid, pIL6-luc651 (Figure 1a). The basic
construct, pIL6-luc651, comprises the sequence of the IL-6 promoter
from nt -644 to nt +1, containing consensus sequences for
transcription factors AP-1, CREB, NF-IL6, and NF-B (Figure 1a).23 24 Figure 1b demonstrates that
deletion of the AP-1 binding site (pIL6-luc220), or the AP-1 and CREB
sites (pIL6-luc160), did not reduce promoter activity in response to
CCB compared with pIL6-luc651. Additionally, inactivation of the
DNA-binding sites for AP-1 and CREB by site-directed mutagenesis did
not affect promoter inducibility in response to CCB (data not shown).
Reductions in luciferase activity were observed when mutations were
introduced to the NF-IL6 site (pIL6-luc160 
NF-IL6, 25% inhibition)
or to the NF-B site (pIL6-luc160 NF-B, 50% inhibition).
Induction of the IL-6 promoter in response to CCB was reduced by 80%
when both NF-IL6 and NF-B DNA-binding sites were mutated
(pIL6-luc160 NF-IL6 NF-B) (Figure 1b).
Similar inhibition of CCB-induced IL-6 promoter activity was observed
when mutations for NF-IL6, NF-B, or for both binding sites were
introduced to the parental pIL6-luc651 (data not shown), thus excluding
the possibility that the minimal promoter of pIL6-luc160 lacked
intrinsic activity.
CCB Increase DNA Binding of the Transcription Factors NF-IL6
and NF-B
Inducibility of the IL-6 promoter activity in CCB-stimulated VSMC
was characterized by EMSA in nuclear extracts using
oligonucleotides, comprising the consensus sequences
for NF-IL6, NF-B, or AP-1. Characteristic EMSA show the time course
of NF-IL6 (Figure 3), or NF-B (Figure 4), activation on stimulation with
amlodipine, diltiazem, or verapamil. CCB increased binding
of NF-IL6 to its consensus sequence as early as 30 minutes after
stimulation with the drugs (Figure 3). Binding specificity was
confirmed by addition of unlabelled competitive
oligonucleotides (Figure 3). DNA binding of
NF-IL6 was maximal at 3 hours and subsequently declined in the cases of
diltiazem and verapamil, whereas amlodipine-induced NF-IL6
binding continuously increased reaching a maximum at 6 hours (Figure 3).
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Binding characteristics for NF-B on stimulation with CCB are
demonstrated in Figure 4. DNA binding specificity was
demonstrated by addition of unlabelled competitor
oligonucleotides (lane d). Supershift analyses
with polyclonal antibody directed against p65 subunit of NF-B led to
a significant reduction of the specific band whereas the unspecific
complex was unaffected (lane c). Time courses of NF-B induction were
similar to that of NF-IL6. DNA binding of NF-B was maximal after 60
minutes, subsequently declining in the cases of diltiazem and
amlodipine. In contrast, verapamil constantly increased DNA binding of
NF-B with a maximum at 6 hours (Figure 4). Concerning the
drugs' efficacy, verapamil led to the highest induction of
NF-IL6 and NF-B DNA binding, whereas amlodipine had the latest onset
of transcription factor activation (Figures 3 and 4).
All CCB failed to affect the activity of AP-1 used to prove the
specificity of CCB-induced NF-IL6 and NF-B activation (data not
shown). This observation is in agreement with data obtained by
luciferase reporter gene analyses demonstrating that deletion
of the AP-1 consensus sequence within the human IL-6 promoter did not
affect activation of the human IL-6 promoter by CCB (Figure 1b).
Changes of Intracellular Calcium Do Not Affect Activation of NF-IL6
or NFB
To analyze whether CCB-induced activation of NF-IL6 and
NF-B was dependent on decreased intracellular calcium
concentrations, we incubated VSMC in calcium-free medium with and
without CCB. Calcium-free medium did not induce activation of NF-IL6 or
NF-B (Figure 5, left), nor did it
modulate CCB-induced NF-IL6 and NF-B DNA binding activity (Figure 5, left). Similarly, BayK8644 (10-8
mol/L), did not effect NF-IL6 or NFB binding (Figure 5, right). Thus in this experimental model, NF-IL6 and NF-B activation
is uncoupled from changes in intracellular calcium levels.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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All 3 subclasses of CCB increased IL-6 promoter activity as shown by luciferase reporter gene analyses. Amlodipine, diltiazem, and verapamil, at nanomolar concentrations, stimulated IL-6 promoter activity to 214%, 292%, and 292% of unstimulated controls, respectively. Deletion analysis suggested that the effect of CCB is mediated by the activation of transcription factors NF-IL6 and NF-
B. These consensus sequences are
located within a 160-bp fragment directly upstream of the
transcriptional start site of the human IL-6 gene promoter. Data were
confirmed by EMSA demonstrating that IL-6 gene induction coincided with
rapid activation of NF-IL6 and NF-B, but not AP-1, in nuclear
extracts of cells stimulated with CCB. Neither modulation of
intracellular calcium levels nor extracellular calcium depletion
affected the activation of both transcription factors.
CCB have been demonstrated to up-regulate IL-6 mRNA and protein
levels.14 15 16 17 These investigations confirmed that IL-6
induction by CCB was independent of intracellular calcium
concentrations, suggesting that CCB affect gene expression by a
different, calcium-independent mechanism. We thus aimed at
characterizing the transcription factors responsible for the increase
in CCB-induced IL-6 gene expression. Here, we demonstrate that CCB
directly induce IL-6 gene transcription and that this is mediated by
the cooperative action of NF-IL6 and NF-B. Synergistic activation of
the IL-6 promoter by NF-IL6 and NF-B has been documented before and
seems to be essential in the transcriptional induction of this
cytokine.27
Concerning inflammatory mechanisms associated with cardiovascular diseases, the biological significance of IL-6 induction by CCB remains to be elucidated. IL-6 has primarily been recognized as a proinflammatory mediator of the acute phase response and a regulator of the host defense.23 Expression of IL-6 in atherosclerotic plaques seems to be elevated compared with normal intima.28 29 It remains uncertain whether increased IL-6 in pathological states is a marker for injury rather than for recovery. It has recently been demonstrated that IL-6 essentially participates in the control of cell proliferation.23 30 Regarding vascular tissue remodeling, IL-6 is known to induce vascular endothelial growth factor31 and tissue inhibitors of metalloproteases (TIMP).23 32 Atherosclerosis is hypothesized as an inflammatory process associated with increased protease expression, decreased antiprotease expression, and endothelial denudation.32 CCB are known to counteract or ameliorate atherosclerotic progression in animal models.33 It is therefore reasonable to assume that local induction of IL-6 in the arterial vessel wall by CCB may essentially reduce atherosclerotic progression by inducing TIMPs and/or vascular endothelial growth factor. This notion is further substantiated by the observation that IL-6 directly induces migration of endothelial cells, thereby facilitating reendothelialization of injured plaques.34
Similar to CCB treatment, hypoxia also induces IL-6, involving
the activation of NF-IL635 and NF-B.36 The
increase of IL-6 in response to low oxygen can be detected in various
human cell culture models37 but also in sera of hypoxic
individuals transferred to elevated heights.38 In the
latter model, this was independent of a concomitant increase of
IL-1, IL-1ß, or TNF-. This finding suggested that IL-6 was not
purely a marker of injury but rather an indicator of adaptation and
recovery.38
A major result of our investigations is the discovery of the activation
of transcription factors resulting from CCB treatment. In
general, CCB are postulated to exert their biological effects by
decreasing the intracellular concentration of calcium
ions.1 2 3 4 Experimentally, this effect is usually achieved
at micromolar concentrations of the drugs. However, accumulating
evidence suggests that CCB, used at therapeutically effective doses
(ie, at the nanomolar range), activate calcium-independent
signal transduction pathway(s) altering gene
expression.14 15 16 17 Here, we show that CCB directly
activate the transcription factors NF-IL6 and NF-B in human
VSMC, independent of intracellular calcium levels. This is supported by
the existence of multiple regulatory regions within the intracellular
part of the L-type calcium channel. It remains to be
investigated, however, along which signal transduction pathway this
action of CCB occurs. Domains within the 1-subunit have been
characterized as substrates for cAMP-dependent protein
kinase,39 40 calmodulin-dependent protein
kinase,41 PKC,40 41 and G-protein
subunits.20 21 22 In this respect, earlier studies from our
group demonstrated specific modulatory effects of CCB on the activation
of calcium-independent PKC isoforms.42 These kinases may
contribute to the activation of NF-IL6 or NF-B in VSMC stimulated
with CCB.
In conclusion, our data present evidence for a novel signal
transduction pathway activated by CCB, a class of drugs that is
widely used in the treatment of cardiovascular
disorders. CCB stimulation of VSMC, the target cell type for CCB
treatment, leads to direct activation of the transcription factors
NF-IL6 and NF-B. This molecular mechanism may thus be responsible
for CCB-induced effects that result in increased gene transcription
associated with long-term remodeling of vascular tissue.
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Acknowledgments |
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We are indebted to Victoria Bruce for editorial help during preparation of this article.
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Footnotes |
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Dr Eickelberg is currently at Yale University, New Haven, Conn.
Received July 23, 1998; revision received January 26, 1999; accepted February 4, 1999.
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