Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation. 1999;99:2276-2282

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Eickelberg, O.
Right arrow Articles by Block, L.-H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Eickelberg, O.
Right arrow Articles by Block, L.-H.
Related Collections
Right arrow Cardiovascular Pharmacology
Right arrow Cell signalling/signal transduction

(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-{kappa}B in Primary Human Vascular Smooth Muscle Cells

Oliver Eickelberg, MD; Michael Roth, PhD; Rainer Mussmann, PhD; Jochen J. Rüdiger, MD; Michael Tamm, MD; André P. Perruchoud, MD; Lutz-Henning Block, MD

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.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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-{kappa}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-{kappa}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-{kappa}B activity.

Conclusions—The results demonstrate that CCB of all 3 subclasses are capable of activating NF-IL6 and NF-{kappa}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


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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 ({alpha}1, {alpha}2, ß, {gamma}, and {delta}), of which the {alpha}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 {alpha}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 {alpha}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 {alpha}1-subunits with G-protein, ß-{gamma} complexes, and protein kinase C (PKC) have been implicated in the control of cellular reactions.20 21 22 Although the {alpha}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-{kappa}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.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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-{kappa}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 1aDown). 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 1aDown). 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-{kappa}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-{kappa}B mutant, and a combined NF-IL6 and NF-{kappa}B mutant (Figure 1aDown).



View larger version (31K):
[in this window]
[in a new window]
  		Figure 1. Luciferase activity of IL-6 promoter deletion mutants in response to CCB in human VSMC. a, IL-6 promoter deletion mutants were generated by internal restriction sites. Mutants of the consensus sequences for NF-IL6 and/or NF-{kappa}B in pIL6-luc160 were generated by site-directed mutagenesis. b, VSMC were transiently transfected with the indicated constructs and stimulated with diltiazem (10-8 mol/L). Luciferase expression was analyzed at 36 hours. Values were compared with unstimulated cells transfected with the respective plasmids. Similar results were obtained using amlodipine and verapamil. Each bar represents the mean of 3 independent experiments made in quadruplicate in one cell line. Similar results were obtained with 2 different VSMC cell lines.

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 1Up). 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-{kappa}B (5'-AGTTGAGGGGACTTTCCCAGGC-3') were end-labeled with ({gamma}-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-{kappa}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.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
CCB Induce IL-6-Promoter Activity in VSMC
Figure 2aDown 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 2aDown through 2c).



View larger version (33K):
[in this window]
[in a new window]
  		Figure 2. IL-6 promoter activation by CCB in primary human VSMC. Dose-dependent effects of the 3 CCB, amlodipine (a), diltiazem (b), and verapamil (c), on pIL6-luc651 transfected human primary VSMC were analyzed by luciferase assays. Negative control: unstimulated cells (0.1% FCS); positive control: stimulation with PDGF-BB (10 ng/mL). Each bar represents the mean of 3 independent experiments made in quadruplicate in one cell line. Similar results were obtained with 2 different VSMC cell lines.

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 1aUp). 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-{kappa}B (Figure 1aUp).23 24 Figure 1bUp 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 {Delta}NF-IL6, 25% inhibition) or to the NF-{kappa}B site (pIL6-luc160 {Delta}NF-{kappa}B, 50% inhibition). Induction of the IL-6 promoter in response to CCB was reduced by 80% when both NF-IL6 and NF-{kappa}B DNA-binding sites were mutated (pIL6-luc160 {Delta}NF-IL6 {Delta}NF-{kappa}B) (Figure 1bUp).

Similar inhibition of CCB-induced IL-6 promoter activity was observed when mutations for NF-IL6, NF-{kappa}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-{kappa}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-{kappa}B, or AP-1. Characteristic EMSA show the time course of NF-IL6 (Figure 3Down), or NF-{kappa}B (Figure 4Down), 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 3Down). Binding specificity was confirmed by addition of unlabelled competitive oligonucleotides (Figure 3Down). 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 3Down).



View larger version (66K):
[in this window]
[in a new window]
  		Figure 3. Time course of the induction of the transcription factor NF-IL6. Representative EMSA demonstrating the time course of NF-IL6 activation on stimulation with amlodipine, diltiazem, and verapamil (at 10-8 mol/L each) at time points 0, 0.5, 1, 3, 6, and 12 hours. Negative control: unstimulated cells (0 hours). Specificity of interactions was analyzed by the addition of unlabelled competitor NF-IL6 oligonucleotide. Lanes are as follows: a, radiolabeled NF-IL6-oligonucleotide alone; b, positive control (HeLa cell nuclear extract); and c, HeLa in the presence of unlabelled competitor NF-IL6 oligonucleotides. All experiments were performed in triplicate in at least 3 different primary cell lines.



View larger version (58K):
[in this window]
[in a new window]
  		Figure 4. Time course of the induction of the transcription factor NF-{kappa}B. Representative EMSA illustrating the time course of NF-{kappa}B activation in response to treatment with CCB amlodipine, diltiazem, and verapamil (10-8 mol/L each) at time points 0, 0.5, 1, 3, 6, and 12 hours. Negative control: unstimulated cells (0 hours). Specificity of interactions was analyzed by the addition of unlabelled competitor NF-{kappa}B oligonucleotides and by supershift analyses using an antibody directed against the p65-subunit of NF-{kappa}B. Lanes are as follows: a, radiolabeled NF-{kappa}B oligonucleotide alone; b, positive control (HeLa nuclear extract); c, HeLa in the presence of polyclonal antibody to NF-{kappa}B p65 subunit; and d, HeLa in the presence of unlabelled competitor NF-{kappa}B oligonucleotides. All experiments were performed in triplicate in at least 3 different primary cell lines.

Binding characteristics for NF-{kappa}B on stimulation with CCB are demonstrated in Figure 4Up. DNA binding specificity was demonstrated by addition of unlabelled competitor oligonucleotides (lane d). Supershift analyses with polyclonal antibody directed against p65 subunit of NF-{kappa}B led to a significant reduction of the specific band whereas the unspecific complex was unaffected (lane c). Time courses of NF-{kappa}B induction were similar to that of NF-IL6. DNA binding of NF-{kappa}B was maximal after 60 minutes, subsequently declining in the cases of diltiazem and amlodipine. In contrast, verapamil constantly increased DNA binding of NF-{kappa}B with a maximum at 6 hours (Figure 4Up). Concerning the drugs' efficacy, verapamil led to the highest induction of NF-IL6 and NF-{kappa}B DNA binding, whereas amlodipine had the latest onset of transcription factor activation (Figures 3Up and 4Up).

All CCB failed to affect the activity of AP-1 used to prove the specificity of CCB-induced NF-IL6 and NF-{kappa}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 1bUp).

Changes of Intracellular Calcium Do Not Affect Activation of NF-IL6 or NF{kappa}B
To analyze whether CCB-induced activation of NF-IL6 and NF-{kappa}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-{kappa}B (Figure 5Down, left), nor did it modulate CCB-induced NF-IL6 and NF-{kappa}B DNA binding activity (Figure 5Down, left). Similarly, BayK8644 (10-8 mol/L), did not effect NF-IL6 or NF{kappa}B binding (Figure 5Down, right). Thus in this experimental model, NF-IL6 and NF-{kappa}B activation is uncoupled from changes in intracellular calcium levels.



View larger version (80K):
[in this window]
[in a new window]
  		Figure 5. Effect of modulation of intracellular calcium on activation of NF-IL6 and NF-{kappa}B. Representative EMSA illustrating the effect of calcium-free medium (left) or BayK8644 (right) on the activation of NF-IL6 and NF-{kappa}B. Lanes are as follows: a, negative control, 32P-labeled oligonucleotides; b, positive control, HELA cell extract plus 32P-labeled oligonucleotides; c, HELA cell extract plus 32P-labeled oligonucleotides in the presence of cold oligos; 0, unstimulated VSMC nuclear extracts. In subsequent lanes, the effect of amlodipine, with or without calcium-free medium or BaK8644, respectively, was analyzed at time intervals indicated (1 and 3 hours). All experiments were performed in triplicate in at least 3 different primary cell lines.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
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-{kappa}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-{kappa}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-{kappa}B. Synergistic activation of the IL-6 promoter by NF-IL6 and NF-{kappa}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-{kappa}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{alpha}, IL-1ß, or TNF-{alpha}. 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-{kappa}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 {alpha}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 {gamma}{delta} 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-{kappa}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-{kappa}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.


*    Acknowledgments
 
We are indebted to Victoria Bruce for editorial help during preparation of this article.


*    Footnotes
 
Dr Eickelberg is currently at Yale University, New Haven, Conn.

Received July 23, 1998; revision received January 26, 1999; accepted February 4, 1999.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Triggle DJ, Rampe D. 1,4-dihydropyridine activators and antagonists: structural and functional distinctions. Trends Pharmacol Sci. 1989;10:507–511.[Medline] [Order article via Infotrieve]
  2. Catterall WA, Striessnig J. Receptor sites for Ca2+ channel antagonists. Trends Pharmacol Sci. 1992;13:256–262.[Medline] [Order article via Infotrieve]
  3. Triggle DJ. Sites, mechanisms of action, and differentiation of calcium channel antagonists. Am J Hypertens. 1991;4:422S–429S.[Medline] [Order article via Infotrieve]
  4. Triggle DJ. The classification of calcium antagonists. J Cardiovasc Pharmacol. 1996;27(suppl A):S11–S16.
  5. Hess P. Calcium channels in vertebrate cells. Annu Rev Neurosci. 1990;13:337–356.[Medline] [Order article via Infotrieve]
  6. Perez-Reyes E, Schneider T. Molecular biology of calcium channels. Kidney Int. 1995;48:1111–1124.[Medline] [Order article via Infotrieve]
  7. Soldatov NM. Molecular diversity of L-type Ca2+ channel transcripts in human fibroblasts. Proc Natl Acad Sci U S A. 1992;89:4628–4632.[Abstract/Free Full Text]
  8. Takahashi M, Seagar MJ, Jones JF, Reber BFX, Catterall WA. Subunit structure of dihydropyridine-sensitive calcium channels from skeletal muscle. Proc Natl Acad Sci U S A. 1997;84:5478–5482.
  9. Tsien RW, Ellinor PT, Zhang JF, Bezprozvanny I. Molecular biology of calcium channels and structural determinants of key functions. J Cardiovasc Pharm. 1996;27:S4–S10.
  10. Tang S, Yatani A, Bahinski A, Mori Y, Schwartz A. Molecular localization of regions in the L-type calcium channel critical for dihydropyridine action. Neuron. 1993;11:1013–1021.[Medline] [Order article via Infotrieve]
  11. Regulla S, Schneider T, Nastainczyk W, Meyer HE, Hofmann F. Identification of the site of interaction of the dihydropyridine channel blockers nitrendipine and azodipine with the calcium-channel {alpha}1 subunit. EMBO J. 1991;10:45–49.[Medline] [Order article via Infotrieve]
  12. Spedding M, Kenny B, Chatelain P. New drug binding sites in Ca2+ channels. Trends Pharmacol Sci. 1995;16:139–142.[Medline] [Order article via Infotrieve]
  13. Sieber M, Nastainczyk W, Zubor V, Wernet W, Hofmann F. The 165-kDa peptide of the purified skeletal muscle dihydropyridine receptor contains the known regulatory sites of the calcium channel. Eur J Biochem. 1987;167:117–122.[Medline] [Order article via Infotrieve]
  14. Walz G, Zanker B, Barth C, Wieder KJ, Clark SC, Strom TB. Transcriptional modulation of human IL-6 gene expression by verapamil. J Immunol. 1990;144:4242–4248.[Abstract]
  15. Roth M, Keul R, Emmons LR, Hörl W, Block LH. Manidipine regulates the transcription of cytokine genes. Proc Natl Acad Sci U S A. 1992;89:4071–4075.[Abstract/Free Full Text]
  16. Rödler S, Roth M, Nauck M, Tamm M, Block LH. Ca(2+)-channel blockers modulate the expression of interleukin-6 and interleukin-8 genes in human vascular smooth muscle cells. J Mol Cell Cardiol. 1995;27:2295–2302.[Medline] [Order article via Infotrieve]
  17. Block LH, Matthys H, Emmons LR, Perruchoud AP, Erne P, Roth M. Ca(2+)-channel blockers modulate expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and low density lipoprotein receptor genes stimulated by platelet-derived growth factor. Proc Natl Acad Sci U S A. 1991;88:9041–9045.[Abstract/Free Full Text]
  18. Wagner JA, Reynolds IJ, Snyder SH. Physiological and pharmacological correlates of calcium antagonist receptors. J Cardiovasc Pharmacol. 1987;10(suppl. 10):S1–S9.
  19. Triggle DJ, Janis RA. Recent development in calcium channel antagonists. Magnesium.. 1989;8:213–222.[Medline] [Order article via Infotrieve]
  20. Zamponi GW, Bourinet E, Nelson D, Nargeot J, Snutch TP. Crosstalk between G proteins and protein kinase C mediated by the calcium channel {alpha}1 subunit. Nature. 1997;385:442–446.[Medline] [Order article via Infotrieve]
  21. DeWaard M, Liu H, Walker D, Scott VE, Gurnett CA, Campbell KP. Direct binding of G-protein ß{gamma} complex to voltage-dependent calcium channels. Nature. 1997;385:446–450.[Medline] [Order article via Infotrieve]
  22. Schneider T, Igelmund P, Hescheler J. G protein interaction with K+ and Ca2+ channels. Trends Pharmacol Sci. 1997;18:8–11.[Medline] [Order article via Infotrieve]
  23. Akira S, Taga T, Kishimoto T. Interleukin-6 in biology and medicine. Adv Immunol. 1993;54:1–78.[Medline] [Order article via Infotrieve]
  24. Yamashita I, Katamine S, Moriuchi R, Nakamura Y, Miyamoto T, Eguchi K, Nagataki S. Transactivation of the human interleukin-6 gene by human T-lymphotropic virus type 1 tax protein. Blood. 1994;84:1573–1578.[Abstract/Free Full Text]
  25. Dignam JD, Lebovitz RM, Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983;11:1475–1489.[Abstract/Free Full Text]
  26. Sen R, Baltimore D. Inducibility of kappa immunoglobulin enhancer-binding protein Nf-kappa B by a posttranslational mechanism. Cell. 1986;47:921–928.[Medline] [Order article via Infotrieve]
  27. Matsusaka T, Fujikawa K, Nishio Y, Mukaida N, Matsushima K, Kishimoto T, Akira S. Transcription factors NF-IL6 and NF-{kappa}B synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc Natl Acad Sci U S A. 1993;90:10193–10197.[Abstract/Free Full Text]
  28. Seino Y, Ikeda U, Ikeda M, Yamamoto K, Misawa Y, Hasegawa T, Kano S, Shimada K. Interleukin 6 gene transcripts are expressed in human atherosclerotic lesions. Cytokine. 1994;6:87–91.[Medline] [Order article via Infotrieve]
  29. Rus HG, Vlaicu R, Niculescu F. Interleukin-6 and interleukin-8 protein and gene expression in human arterial atherosclerotic wall. Atherosclerosis. 1996;127:263–271.[Medline] [Order article via Infotrieve]
  30. Roth M, Nauck M, Tamm M, Perruchoud AP, Ziesche R, Block LH. Intracellular interleukin 6 mediates platelet-derived growth factor-induced proliferation of nontransformed cells. Proc Natl Acad Sci U S A. 1995;92:1312–1316.[Abstract/Free Full Text]
  31. Cohen T, Nahari D, Cerem LW, Neufeld G, Levi BZ. Interleukin 6 induces the expression of vascular endothelial growth factor. J Biol Chem. 1996;271:736–741.[Abstract/Free Full Text]
  32. Lotz M, Guerne PA. Interleukin-6 induces the synthesis of tissue inhibitor of metalloproteinases-1/erythroid potentiating activity (TIMP-1/EPA). J Biol Chem. 1991;266:2647–2651.[Abstract/Free Full Text]
  33. Ross R. Cell biology of atherosclerosis. Annu Rev Physiol. 1995;57:791–804.[Medline] [Order article via Infotrieve]
  34. Rosen EM, Liu D, Setter E, Bhargava M, Goldberg ID. Interleukin-6 stimulates motility of vascular endothelium. EXS. 1991;59:194–205.[Medline] [Order article via Infotrieve]
  35. Yan SF, Tritto I, Pinsky D, Liao H, Huang J, Fuller G, Brett J, May L, Stern D. Induction of Interleukin-6 (IL-6) by hypoxia in vascular cells. Central role of the binding site for nuclear factor-IL-6. J Biol Chem. 1995;270:11463–11471.[Abstract/Free Full Text]
  36. Muraoka K-I, Kouichi S, Sun X, Zhang YK, Tani T, Hashimoto T, Yagi M, Miyazaki I, Yamamoto K-I. Hypoxia, but not reoxygenation, induces Interleukin-6 gene expression through NF-{kappa}B activation. Transplantation. 1997;63:466–470.[Medline] [Order article via Infotrieve]
  37. Yamauchi-Takihara K, Ihara Y, Ogata A, Yoshizaki K, Azuma J, Kishimoto T. Hypoxic stress induces cardiac myocyte-derived interleukin-6. Circulation. 1995;91:1520–1524.[Abstract/Free Full Text]
  38. Klausen T, Olsen NV, Poulsen TD, Richalet JP, Pedersen BK. Hypoxemia increases serum interleukin-6 in humans. Eur J Appl Physiol. 1997;76:480–482.
  39. Sculptoreanu A, Rotman E, Takahashi M, Scheuer T, Catterall WA. Voltage-dependent potentiation of the activity of cardiac L-type calcium channel alpha 1 subunits due to phosphorylation by cAMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1993;90:10135–10139.[Abstract/Free Full Text]
  40. Calkhoven CF, Ab G. Multiple steps in the regulation of transcription-factor level and activity. Biochem J. 1996;317:329–342.
  41. Imagawa T, Leung AT, Campbell KP. Phosphorylation of the 1,4-dihydropyridine receptor of the voltage-dependent Ca2+ channel by an intrinsic protein kinase in isolated triads from rabbit skeletal muscle. J Biol Chem. 1987;262:8333–8339.[Abstract/Free Full Text]
  42. Curtis BM, Catterall WA. Phosphorylation of the calcium antagonist receptor of the voltage-sensitive calcium channel by cAMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1985;82:2528–2532.[Abstract/Free Full Text]
  43. Block LH, Keul R, Crabos M, Ziesche R, Roth M. Transcriptional activation of low density lipoprotein receptor gene by angiotensin-converting enzyme inhibitors and Ca(2+)-channel blockers involves protein kinase C isoforms. Proc Natl Acad Sci U S A. 1993; 90: 4097–4101.The molecular mechanism of IL-6 induction by calcium channel blockers (CCB) was investigated. Luciferase assays demonstrated induction of the IL-6 gene by CCB; inducible promoter activity was localized 160 bp upstream of the transcriptional start site of the IL-6 gene. Site-directed mutagenesis of the known NF-IL6 and NF-kB site suggested that both factors cooperatively transmitted CCB-induced gene expression. Mobility shift analyses demonstrated that all CCB increased DNA binding of NF-IL6 and NF-kB independent of intracellular calcium concentrations. CCB thus directly regulate cellular functions by affecting activity of transcription factors. These findings are of interest for the biological effects induced by CCB.



This article has been cited by other articles:


Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
M. L. Sheu, F. M. Ho, R. S. Yang, K. F. Chao, W. W. Lin, S. Y. Lin-Shiau, and S.-H. Liu
High Glucose Induces Human Endothelial Cell Apoptosis Through a Phosphoinositide 3-Kinase-Regulated Cyclooxygenase-2 Pathway
Arterioscler. Thromb. Vasc. Biol., March 1, 2005; 25(3): 539 - 545.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
R. ZIESCHE, V. PETKOV, C. LAMBERS, P. ERNE, and L.-H. BLOCK
The calcium channel blocker amlodipine exerts its anti-proliferative action via p21(Waf1/Cip1) gene activation
FASEB J, October 1, 2004; 18(13): 1516 - 1523.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
C. B Jones, D. C Sane, and D. M Herrington
Matrix metalloproteinases: A review of their structure and role in acute coronary syndrome
Cardiovasc Res, October 1, 2003; 59(4): 812 - 823.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
O. EICKELBERG, A. PANSKY, E. KOEHLER, M. BIHL, M. TAMM, P. HILDEBRAND, A. P. PERRUCHOUD, M. KASHGARIAN, and M. ROTH
Molecular mechanisms of TGF-{beta} antagonism by interferon {gamma} and cyclosporine A in lung fibroblasts
FASEB J, March 1, 2001; 15(3): 797 - 806.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
U. Ikeda, T. Ito, K. Shimada, O. Eickelberg, M. Roth, J. J. Rudiger, M. Tamm, A. P. Perruchoud, L.-H. Block, and R. Mussmann
Calcium Channel Blockers Activate the Interleukin-6 Gene Via the Transcription Factors NF-IL6 and NF-{kappa}B in Primary Human Vascular Smooth Muscle Cells Response
Circulation, May 9, 2000; 101 (18): e192 - e192.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Eickelberg, O.
Right arrow Articles by Block, L.-H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Eickelberg, O.
Right arrow Articles by Block, L.-H.
Related Collections
Right arrow Cardiovascular Pharmacology
Right arrow Cell signalling/signal transduction

Circulation Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search
Copyright © 1999 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited.