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Cytokine
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CYTOKINE
Regulation of stimulus-induced interleukin-8 gene transcription in human adrenocortical carcinoma cells - Role of AP-1 and NF-KB
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Gerald Thielª,*, Myriam Ulrichª, Naofumi Mukaidab, Oliver G. Rösslerª
a Department of Medical Biochemistry and Molecular Biology, Saarland University Medical Faculty, D-66421 Homburg, Germany
b Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Kanazawa 920-1192, Japan
ARTICLE INFO
Keywords:
AP-1
Designer receptor Interleukin-1฿ Interleukin-8 NF-KB
ABSTRACT
Stimulation of H295R adrenocortical carcinoma cells with angiotensin II or cytokines induces the secretion of the chemokine interleukin-8 (IL-8). Here, we have analyzed the molecular mechanism of stimulus-induced IL-8 expression. IL-8 expression and IL-8 promoter activity increased in H295R cells expressing an activated Gaq- coupled designer receptor. H295R cells stimulated with either interleukin-1ß (IL-1ß) or phorbol ester also showed elevated IL-8 mRNA levels and higher IL-8 promoter activities. Deletion and point mutations of the IL-8 promoter revealed that the AP-1 binding site within the IL-8 promoter is essential to connect designer receptor stimulation with the transcriptional activation of the IL-8 gene. Expression of a constitutively active mutant of c- Jun, or expression of constitutively active mutants of the protein kinases MEKK1 and MKK6 confirmed that the IL-8 gene is a bona fide target of AP-1 in adrenocortical carcinoma cells. Upregulation of IL-8 expression in IL-1ß- treated H295R cells required NF-KB while the phorbol ester TPA used both the AP-1 and NF-KB sites of the IL-8 gene to stimulate IL-8 expression. These data were corroborated in experiments with chromatin-embedded AP-1 or NF-KB-responsive reporter genes. While stimulation of Gaq-coupled designer receptors increased the AP-1 activity in the cells, IL-1ß specifically stimulated NF-KB-regulated transcription. Stimulation of the cells with TPA increased both AP-1 and NF-KB activities. We conclude that stimulation of Gaq-coupled designer receptors or IL- 1 receptors triggers distinct signaling pathways in H295R cells leading to the activation of either AP-1 or NF-KB. Nevertheless, both signaling cascades converge to the IL-8 gene, inducing IL-8 gene transcription.
1. Introduction
Interleukin-8 (IL-8) is a proinflammatory cytokine that plays im- portant roles during inflammation. IL-8 functions as a potent che- moattractant and activator of immune cells, including neurotrophils and T-cells. IL-8 is the main mediator in neutrophil-mediated in- flammation, regulating neurophil chemotaxis and transmigration of neurotrophils across the endothelium [1]. Il-8 secretion has therefore been associated with various inflammatory diseases.
Although IL-8 expression and function has been initially detected and analyzed in immune cells such as monocytes and macrophages, it is now well appreciated that IL-8 is synthesized and secreted by a wide array of non-immune cells, for example endothelial and epithelial cells, smooth muscle cells, and fibroblasts. IL-8 overexpression and secretion has also been detected in various tumor cells, including lung and breast cancer cells, melanoma cells, glioblastoma cells, and adrenocortical carcinoma cells. The biological role of IL-8 involves tumor growth and
invasion, cell migration, metastasis, and angiogenesis [2-8].
Given the proinflammatory properties of IL-8, expression needs to be tightly regulated. IL-8 is expressed at low levels in many cell types, but expression can be robustly induced, sometimes more than 100-fold, in response to inflammatory stimuli such as interleukin-1ß (IL-1B), tumor necrosis factor, bacterial lipopolysaccharides, or virus infection. IL-8 gene transcription is regulated by stimulus-responsive transcription factors, in particular by nuclear factor-KB (NF-KB), activator protein-1 (AP-1), and C/EBPß [9]. The transcription factors STAT and Egr-1 have also been proposed to regulate IL-8 gene transcription via modulation of NF-KB activity [10,11].
Stimulation of H295R adrenocortical carcinoma cells with either angiotensin II, cytokines or the tumor promoter 12-O-tetra- decanoylphorbol-13-acetate (TPA), a compound that irreversibly acti- vates protein kinase C, induces the secretion of IL-8 [12]. Expression of IL-8 has also been reported to occur in other analyzed adrenocortical carcinoma cells [2]. Our study has been aimed at elucidating the
Abbreviations: AP-1, activator protein-1; bZIP, basic region leucine zipper; IL-1}, interleukin-1}; IL-8, interleukin-8; TPA, 12-O-tetradecanoylphorbol-13-acetate
* Corresponding author at: Department of Medical Biochemistry and Molecular Biology, Saarland University, Building 44, D-66421 Homburg, Germany. E-mail address: gerald.thiel@uks.eu (G. Thiel).
molecular mechanism of stimulus-induced IL-8 gene transcription in H295R adrenocortical carcinoma cells. The results show that stimula- tion of Gaq-coupled designer receptors activates AP-1 in H295R adre- nocortical carcinoma cells, leading to an increase in IL-8 gene tran- scription via the AP-1 site within the IL-8 gene. In contrast, stimulation of the cells with IL-1ß activated NF-KB and IL-8 gene transcription via the NF-KB binding site of the IL-8 gene promoter. Stimulation of the cells with TPA induced IL-8 expression involving both AP-1 and NF-KB. Thus, different stimuli converge to the IL-8 gene, involving distinct genetic elements.
2. Materials and methods
2.1. Cell culture
H295R adrenocortical carcinoma cells were cultured in DMEM/F12 medium containing 5% NuSerum™ (BD Bioscience, Bedford, USA), 1% ITS™ universal culture supplement (BD Bioscience, Bedford, USA), 100 mg/ml streptomycin, and 100 units/ml penicillin [13]. Stimulation with clozapine N-oxide (CNO, 1 mM), interleukin-1ß (10 ng/ml), or TPA (10 ng/ml) was performed in medium containing 0.05% fetal bo- vine serum or 0.05% NuSerum™.
2.2. Lentiviral gene transfer
The lentiviral transfer vectors pFUW-Raq, pFUW-MEKK14, pFUW- MKK6E and pFUW-C2/c-Jun have been described elsewhere [14-17]. The viral particles were produced in HEK293T/17 or HEK293-TN cells by triple transfection of the cells with a gag-pol-rev packaging plasmid, an env encoding plasmid (pCMV-G) and a lentiviral transfer vector [18,19].
2.3. Reporter assays
Lentiviral transfer vectors expressing IL-8 promoter/luciferase re- porter genes have been described [20]. The lentiviral vectors pFW- Coll.luc, pFWHIVLTR.luc and pFWCYP11B2.luc have been described [13,21]. H295R cells were infected with a recombinant lentivirus containing a reporter gene and stimulated for 24h. Cell extracts of stimulated cells were prepared using reporter lysis buffer (Promega, Mannheim, Germany) and analyzed for luciferase activities. Luciferase activity was normalized to the protein concentration.
2.4. Western blots
Western blots were performed using the M2 monoclonal antibody directed against the FLAG epitope (Sigma-Aldrich, # F3165).
2.5. RT-PCR
Cells were stimulated for three hours. Total RNA was isolated from H295R cells and reverse transcribed. The PCR reaction was performed with Taq DNA Polymerase (# M0267S, New England Biolabs, Frankfurt, Germany, 1 U) using the primers, 5’-AAGCTGGCCGTGGCT CTCTT-3’ and 5’-TGGTGGCGCAGTGTGGTCCA-3’ to detect IL-8 mRNA. Primers specific for GAPDH (5’-TTCCAGGAGCGAGATCCCT-3’; 5’-CAC CCATGACGAACATGGG-3’) were used as a loading control. The PCR products were separated by agarose gel electrophoresis and visualized with ethidium bromide.
For qRT-PCR analyses, total cellular RNA was extracted from 3 different passages of H295R cells by using the RNeasy Protect Mini Kit (Qiagen, Hilden, Germany). To avoid genomic DNA contamination the RNA was purfied with an on-column RNase-free DNase treatment (Qiagen, Hilden, Germany). The isolated RNA was eluted in 20 ul RNase-free water. 1 µg RNA was reverse transcribed with RevertAid Reverse Transcriptase (Thermo Fisher Scientific GmbH, Dreieich,
A
Gaq-coupled designer receptor Raq
CNO
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membrane
· Y 148 C
* A 238 G
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CYP11B2.luc
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CRE
AP2 CRE
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luciferase
-1521
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aldosterone synthase promoter
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CYP11B2 promoter activity (relative luciferase activity, fold induction)
CYP11B2.luc
8
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CNO [1 [M]
Germany). The cDNA was 1:5 diluted with PCR grade water and 2,5 ul of the diluted cDNA were used for the real time PCR reaction. Amplification of the cDNA was performed by using the Brilliant SYBR Green QPCR Master Mix (Stratagene, Agilent Technologies, Waldbronn, Germany) on an Mx3000P QPCR operator system (Stratagene). The reactions were run as follows for a total of 40 cycles: (10 min at 95 ℃, than denaturation at 95 ℃, 30 s, annealing at 63 ℃, 1 min; extension at 72 ℃, 30 s, denaturation at 95 ℃ for 1 min, and a final incubation at 55 ℃, 30 s. Control primers used to detect human GAPDH cDNA were 5’-GAAGGTGAAGGTCGGAGTC-3’ and 5’-GAAGATGGTGATGGGAT TTC-3’. The IL-8 primers were used at a final concentration of 250 nM and the GAPDH primers were used at a final concentration of 350 nM
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RT-PCR
[bp]
CNO
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IL-8
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TPA
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IL-8
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GAPDH
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qRT-PCR IL-8
fold gene expression 2(-44Ct)
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qRT-PCR IL-8
fold gene expression
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2(-44Ct)
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IL-16 [10 ng/ml]
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qRT-PCR IL-8
fold gene expression
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TPA [10 ng/ml]
| H295R cells | Fold induction average | SD | p-value |
|---|---|---|---|
| - CNO | 1,19 | 0,70 | 0,01794 |
| + CNO | 4,73 | 1,37 | |
| -IL-1B | 1,01 | 0,22 | 0,00028 |
| + IL-1฿ | 689,31 | 471,91 | |
| -TPA | 1,11 | 0,65 | 0,00051 |
| + TPA | 26,57 | 8,65 |
per reaction. Controls of each PCR run were a no template control (only water) for each primer set and for each sample a no RT control (non- reverse-transcribed mRNA). To ensure the specifity of the PCR products a melt curve analysis for each run and visualization of the products in an agarose gel were performed. The threshold cycle (Ct) value for each gene (IL-8 and GAPDH) and each sample was measured using the MxPro QPCR software from Stratagene. Fold induction of the relative gene expression of IL-8 was calculated by normalization to GAPDH expression and using the 2(-44Ct) method [22].
2.6. Statistics
Statistical analyses were done by using the two-tailed student’s t- test. Data shown are mean ± SD from at least three independent ex- periments. Statistical probability is expressed as *P < 0.05, ** P < 0.01, and *** P < 0.001. Values were considered significant when P < 0.05.
3. Results
3.1. Stimulation of a Gaq-coupled designer receptor increases transcription of a chromatin-embedded CYP11B2 promoter/luciferase reporter gene
Stimulation of pluripotent human H295R adrenocortical carcinoma cells with angiotensin II triggers the secretion of IL-8 [12]. To elucidate the molecular mechanism of stimulus-induced IL-8 gene transcription, we used H295R cells to compare our findings with the results published by Romero et al. [12]. It has been shown that expression of AP-1 pro- teins is induced in H295R cells that had been stimulated with either angiotensin II or TPA [23,24]. We observed that culturing H295R cells for several passages leads to a gradual loss of angiotensin II respon- siveness of the cells (G. Thiel, unpublished observations). As
A
IL8-133.luc
LTR
HIV-1_
0
AU3
flap
,
€
WPRE-
LTR
AU3
AP-1
-
0
0
TATA”
luciferase
-133
NF-KB
+44
B
IL-8 promoter activity (relative luciferase activity,
IL8-133.luc
5 -
fold induction)
4
3
2
1
0
+
+
Raq
+ CNO [1 [M]
-
C
IL-8 promoter activity (relative luciferase activity, fold induction)
IL8-133.luc
14 -
12
10
8
6
4
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IL-18 [10 ng/ml]
-
D
IL-8 promoter activity (relative luciferase activity, fold induction)
IL8-133.luc
28
-
24
20
16
12
8
4
0
+
TPA [10 ng/ml]
-
angiotensin II binds to a Gaq-coupled receptor, we expressed a Gaq- coupled designer receptor in H295R cells that could be stimulated with the compound clozapine N-oxide (CNO) [15]. The modular structure of the designer receptor is depicted in Fig. 1A. To demonstrate the bio- logical activity of the designer receptor in H295R cells, we analyzed the
activity of the CYP11B2 promoter, derived from the aldosterone syn- thase gene. Stimulation of H295R cells with angiotensin II had been shown to upregulate the transcription of a chromatin-embedded CYP11B2 promoter/luciferase reporter gene (Fig. 1B) [13]. Fig. 1C shows that stimulation of a Gaq-coupled designer receptor with its li- gand CNO increased transcription of the CYP11B2 promoter/luciferase reporter gene, indicating that the designer receptor initiates a signal transduction cascade comparable to the endogenous angiotensin II re- ceptor.
3.2. Stimulus-induced expression of interleukin-8 (IL-8) in H295R adrenocortical carcinoma cells
We measured the IL-8 mRNA level in H295R cells expressing an activated Gaq-coupled designer receptor. Fig. 2A shows that stimula- tion of the cells with CNO increased IL-8 mRNA level. CNO stimulation did not induce IL-8 expression in the absence of the designer receptors (Supplemental Fig. S1). IL-8 expression was increased in H295R cells stimulated with either IL-1ß or TPA (Fig. 2B). This effect was in- dependent of expression of the designer receptor (Supplemental Fig. S2). To quantify IL-8 expression we performed qRT-PCR experiments with the RNA extracted from stimulated H295R cells. Fig. 2C shows that stimulation of designer receptor-expressing H295R cells with CNO in- creased IL-8 expression almost 5-fold. Stimulation of the cells with IL- 1ß increased IL-8 expression more than 600-fold (Fig. 2D), indicating that IL-1ß is a very strong signaling molecule for activating IL-8 ex- pression. As a control, we analyzed TPA stimulated H295R cells. IL-8 expression was stimulated 26-fold in the presence of TPA (Fig. 2E). The quantitative data are summarized in Table 1.
3.3. Stimulation of H295R adrenocortical carcinoma cells activates transcription of a reporter gene controlled by the IL-8 promoter
The proximal Il-8 promoter, encompassing 133 nucleotides of 5’- upstream region, has been described as the important control region of the IL-8 gene [9,25]. To analyse IL-8 promoter activities in H295R cells, we therefore used a chromatin-embedded reporter gene under the control of the proximal IL-8 promoter (sequence -133 to +44, Fig. 3A). The reporter gene was integrated into the genome of H295R cells. This strategy ensured that the reporter gene was integrated into the nucleosomal context. Stimulation of a Gaq-coupled designer re- ceptor triggered the transcriptional activation of this IL-8 promoter- controlled reporter gene (Fig. 3B). Likewise, stimulation of the cells with either IL-1 or TPA induced transcription of the reporter gene (Fig. 3C, D). Similar results were obtained in the analyses of a reporter gene under the control of 1481 nucleotides of the IL-8 5’-upstream re- gion (Supplemental Fig. S3).
3.4. Stimulus-induced activation of the IL-8 promoter in H295R adrenocortical carcinoma cells - role of the AP-1 and NF-KB sites of the IL-8 gene
We analyzed mutated versions of IL-8 promoter/luciferase reporter gene with the aim to identify genetic response elements that mediate IL- 8 gene transcription following cellular stimulation. In the IL-8 promoter there are binding sites for the transcription factors AP-1 and NF-KB. We therefore analyzed IL-8 promoter reporter genes containing mutations of either the AP-1 or the NF-KB site (AAP-1 and ANF-KB, Fig. 4A). H295R cells expressing the designer receptor were stimulated with CNO. Fig. 4B shows that mutation of the AP-1 site significantly reduced reporter gene transcription, indicating that AP-1 is required to connect designer receptor stimulation with IL-8 gene transcription. Mutation of the NF-KB site had no significant effect on reporter gene transcription in H295R cells expressing activated designer receptors.
Mutation of the NF-KB binding site within the IL-8 promoter com- pletely attenuated transcription of an IL-8 promoter-controlled reporter
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IL8-133.luc
IL8-133.luc
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AU3
IL-8 promoter activity (relative luciferase activity, fold induction)
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IL-8 promoter activity (relative luciferase activity, fold induction)
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gene following stimulation of the cells with IL-10, while mutation of AP-1 site had no effect on the IL-1ß-induced gene transcription (Fig. 4C). Thus, the signaling cascade induced by IL-1ß used NF-KB to activate IL-8 gene transcription.
Fig. 4D shows that mutation of either the AP-1 or the NF-KB sites within the IL-8 promoter attenuated reporter gene transcription in TPA- stimulated H295R cells. These data indicate that the TPA-induced sig- naling cascade activated both AP-1 and NF-KB leading to IL-8 gene transcription in a concerted manner.
3.5. The IL-8 gene is regulated by AP-1 in H295R adrenocortical carcinoma cells
The preceding experiments showed that AP-1 is involved in the upregulation of IL-8 expression following stimulation of a Gaq-coupled receptor. While the regulation of the IL-8 gene by NF-KB has been re- ported to occur in many different cell types, the regulation of the IL-8 gene by AP-1 has been considered to be tissue-specific. We therefore examined whether AP-1 activates the IL-8 promoter in adrenocortical carcinoma cells. First, we expressed the constitutively active mutant of the protein kinase mitogen-activated protein kinase/extracellular signal regulated kinase kinase kinase-1 (MEKK1) that had been described as an IL-8 gene activator in HEK293 cells [26]. Fig. 5A shows that ex- pression of MEKK14, a truncated mutant of MEKK1, strongly stimulated transcription of a chromatin-embedded IL-8 promoter/luciferase re- porter gene. Second, we expressed a constitutively active mutant of MKK6, an activator of p38 protein kinase. The results show that IL-8 promoter activity was increased in H295R cells expressing the MKK6 mutant MKK6E (Fig. 5A). We recently showed that expression of these kinase mutants strongly stimulated AP-1 [17].
Next, we expressed a constitutively active mutant of the transcrip- tion factor c-Jun, C2/c-Jun. c-Jun is a major constituent of the AP-1 transcription factor. The C2/c-Jun mutant (Fig. 5B, C) contained a constitutively active transcriptional activation domain derived from the
bZIP protein CREB2. Fig. 5D shows that expression of C2/c-Jun sig- nificantly activated transcription of a IL-8 promoter-controlled reporter gene, while this effect was abolished following mutation of the AP-1 binding site within the IL-8 promoter. We conclude that the Il-8 gene is a bona fide target gene of AP-1 in adrenocortical carcinoma cells.
3.6. Stimulation of a Gaq-coupled designer receptor in H295R adrenocortical carcinoma cells leads to the activation of the AP-1 transcription factor
A collagenase promoter/luciferase reporter gene (Fig. 6A) was used as a measure for AP-1 activity as described [13,15,16,27-30]. The collagenase promoter contains an AP-1 binding site in the proximal promoter region that is also known as 12-O-tetradecanoylphorbol-13- acetate (TPA)-responsive element (TRE). Stimulation of a Gaq-coupled designer receptor resulted in an activation of AP-1 in H295R adreno- cortical carcinoma cells (Fig. 6B), as previously described for HEK293 cells [15]. Also stimulation of the cells with TPA activated AP-1 (Fig. 6D). In contrast, no activation of AP-1 was observed in IL-1ß-sti- mulated H295R cells (Fig. 6C).
3.7. Stimulation of H295R cells with IL-1ß activates NF-KB
We used a luciferase reporter gene under the control of the HIV-1 long terminal repeat (LTR), to measure the activity of NF-KB in H295R adrenocortical carcinoma cells (Fig. 7A). Most subtypes of HIV-1 have two copies of a NF-KB consensus motif in the LTR [31]. Fig. 7C and D show that stimulation of H295R cells with IL-1ß or TPA significantly activated transcription of the reporter gene controlled by HIV-1 LTR sequences. In contrast, NF-KB was not activated in H295R cells ex- pressing activated Gaq-coupled designer receptors (Fig. 7B).
A
IL8-133.luc
IL-8 promoter activity (relative luciferase activity,
32
fold induction)
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TGAGTCA-
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4. Discussion
Stimulation of the H295R adrenocortical carcinoma cells with an- giotensin II, cytokines or phorbol ester has been shown to induce se- cretion of IL-8 [12]. Expression of IL-8 is mainly regulated on the level of transcription. The objective of this study was to elucidate the mo- lecular mechanism of stimulus-induced IL-8 gene transcription in H295R adrenocortical carcinoma cells. These cells express angiotensin II receptors, synthesize steroids of the adrenal cortex and represent an
A
HIVLTR.luc
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flap
NF-KB
Sp1
TATA
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-120
+83
— GGACTTTCCG
GGACTTTCCA-
B
NF-KB activity (relative luciferase activity,
3
n.s.
fold induction)
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1
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Raq
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+ CNO [1 µM]
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NF-KB activity (relative luciferase activity,
3
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IL-1B [10 ng/ml]
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IL-8 promoter activation (relative luciferase activity, fold induction)
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excellent cellular model system to study the cell biology and genetics of adrenocortical carcinoma cells [32].
At the level of gene regulation, many signaling molecules that in- duce IL-8 expression, activate NF-KB. Thus, NF-KB has been viewed as the major transcription factor responsible for stimulus-induced
activation of IL-8 gene transcription in all cell types. The NF-KB tran- scription factor complex encompasses the Rel proteins RelA (p65), RelB, and c-Rel that can dimerize with the proteins p50 and p52. In the nucleus, the dimeric NF-KB transcription factor complex binds to the KB recognition site. In resting cells, NF-KB is sequestered in the cytoplasm through binding to IKB (inhibitor of KB). Upon stimulation of the cells, the IKB kinase phosphorylates IKB with the result that IKB is degraded by the proteasome. The liberated NF-KB dimer then translocates into the nucleus and activates gene transcription of KB-containing genes, such as the IL-8 gene with the KB site encompassing the sequence 5’-GGAATT TCCTC-3’. Accordingly, stimulation of various cell types with the clas- sical proinflammatory stimuli IL-1 or tumor necrosis factor was shown to activate NF-KB and to increase transcription of the IL-8 gene [25,26,33-35]. Transcriptional upregulation of the IL-8 gene via NF-KB has also been reported for gastrin stimulation of human gastric cancer cells, for stimulation of endothelial cells with activators of peroxisome proliferator-activated receptor 8 [36,37], and for resveratrol-stimulated HEK293 cells [38]. In this study, we showed that IL-1ß activated NF-KB in adrenocortical carcinoma cells, leading to a strong activation of IL-8 gene transcription via NF-KB. Quantitative RT-PCR analysis showed that IL-8 expression were stimulated more than 600-fold in IL-1-B treated H295R cells, indicating that this cytokine is a very strong reg- ulator of IL-8 gene transcription. Stimulation of adrenocortical carci- noma cells with IL-1ß did not activate AP-1, as shown with an AP-1- responsive reporter gene. In contrast, in human gastric carcinoma cells, it was shown that both AP-1 and NF-KB were involved in the regulation of IL-1ß-induced transcription of the IL-8 gene [35]. Thus, there are cell type-specific differences in the signaling pathway of IL-1B, but they nevertheless converge to the IL-8 gene.
The role of AP-1 in the regulation of IL-8 gene transcription is an issue of discussion. AP-1 has been viewed as dispensible for IL-8 ex- pression [39]. Alternatively, AP-1 has been suggested to play a role in either maintaining basal IL-8 transcription [40], or to function sy- nergistically with NF-KB to regulate IL-8 gene transcription [26,35,40-42]. However, there are stimuli that regulate IL-8 expression solely via AP-1, without any contribution from NF-KB. We recently showed that stimulation of the transient receptor potential channels TRPM3 and TRPC6 activated IL-8 gene transcription via the AP-1 binding site of the IL-8 gene promoter [20]. In human endothelial cells, proteasome inhibition induces IL-8 gene transcription through AP-1 [43]. In this study, we showed that stimulation of Gaq-coupled re- ceptors activated AP-1 in adrenocortical carcinoma cells, leading to an activation of IL-8 expression via AP-1. Quantitative RT-PCR experi- ments revealed that stimulation of Gaq-coupled receptors increased IL- 8 expression in the order of 5-fold. The stimulation was statistically significant and could be reproduced in three independent experiments. Experiments involving an NF-KB-responsive reporter gene clearly showed that NF-KB was not activated under these conditions. These data were corroborated by experiments showing that expression of constitutively active protein kinases MEKK14 and MKK6E strongly ac- tivated the IL-8 promoter. Likewise, expression of a constitutively ac- tive c-Jun transcription factor stimulated a reporter gene under the control of the IL-8 promoter. Transcriptional activation of the reporter gene was attenuated when the AP-1 site had been inactivated by mu- tation. Together, these data show that the IL-8 gene is a bona fide target gene for AP-1, connecting Gaq-coupled receptor signaling with IL-8 expression. Other AP-1 target genes in adrenocortical cells encode steroid 11-ß-hydroxylase (CYP11B1) and steroidogenic acute reg- ulatory protein [44,45].
Stimulation of adrenocortical carcinoma cells with TPA induced IL-8 expression involving both AP-1 and NF-KB, as shown with transcription factor-specific reporter genes. These data are in agreement with the known function of TPA to activate both AP-1 and NF-KB.
5. Conclusion
The chemokine IL-8 is expressed in H295R adrenocortical carci- noma cells following stimulation of Gaq-coupled receptors. Likewise, stimulation of the cells with the cytokine IL-16 or the tumor promoter TPA induced IL-8 expression and activated the IL-8 promoter. A mo- lecular dissection of the IL-8 promoter revealed that stimulation of Gaq- coupled receptors activated AP-1 and induced IL-8 expression involving the AP-1 site of the IL-8 gene promoter. Stimulation of the cells with IL- 1ß activated NF-KB, promoting IL-8 expression via the NF-KB binding site of the IL-8 promoter. The tumor promoter TPA activated both AP-1 and NF-KB, and triggered IL-8 expression involving both the AP-1 and NF-KB binding sites of the IL-8 gene. Thus, different signaling cascades converge to the activation of IL-8 expression. An important aim of fu- ture research will be the elucidation of the functional consequence of IL-8 expression in adrenal carcinomas. Expression of IL-8 in adreno- cortical cells has not been reported [46], suggesting that IL-8 expression may be a hallmark of transformed cells. The analysis of an IL-8 ex- pressing adrenocortical carcinoma revealed that the tumor was in- filtrated with neutrophils [2]. Thus, IL-8 expression may play an im- portant role in adrenocortical tumorigenesis, due to the chemotactic and angiogenic activity of IL-8. Accordingly, inhibition of IL-8 expres- sion may represent a therapeutic invention for cancer treatment.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influ- ence the work reported in this paper.
Acknowledgements
We thank Magali Cucchiarini and Jagadesh Kumar Venkatesan for their help in setting up the qRT-PCR experiments. We thank Libby Guethlein for critical reading of the manuscript. This study was sup- ported by the Saarland University (LOM-T201000492).
Appendix A. Supplementary material
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cyto.2019.154862.
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