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Carcinogenesis Advance Access originally published online on July 6, 2005
Carcinogenesis 2006 27(1):43-52; doi:10.1093/carcin/bgi174
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Carcinogenesis vol.27 no.1 © Oxford University Press 2005; all rights reserved.

Production of chemokine CXCL1/KC by okadaic acid through the nuclear factor-{kappa}B pathway

Gong Feng 1, Yoshihiro Ohmori 3 and Pi-Ling Chang 1, 2, *

1 Department of Nutrition Sciences and 2 Comprehensive Cancer Center, University of Alabama at Birmingham, AL 35294-3360, USA and 3 Center for Molecular Biology, Meikai University School of Dentistry, Saitama 350-0283, Japan

* To whom correspondence should be addressed at: Department of Nutrition Sciences, 311 Susan Mott Webb Nutrition Sciences Building, 1675 University Boulevard, University of Alabama at Birmingham, Birmingham, AL 35295-3360. Tel: +1 205 975 6624; Fax: +1 205 934 7049; Email: plchang{at}uab.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
The murine chemokine CXCL1/KC is known as a chemoattractant for neutrophil infiltration and as a promoter of tumor growth. To determine its relevance in tumorigenesis, we first asked whether okadaic acid (OKA), a natural tumor promoter and a potent protein phosphatase 1 and 2A inhibitor, stimulates KC expression and if it does, through what pathway, in a promotable mouse epidermal-like JB6 cell line commonly used for studying molecules related to tumor promotion. We found that OKA stimulated the de novo synthesis of KC mRNA and protein in a dose- and time-dependent manner. To determine the mechanism by which OKA stimulated the expression of KC at the transcriptional level, transient transfection assays using serially deleted sections of KC promoter fused to luciferase reporter gene were performed. These studies showed that transactivation of KC promoter by OKA specifically involved the region between –104 and –59 containing the two nuclear factor-kappaB (NF-{kappa}B) response elements ({kappa}B1 and {kappa}B2). Further analyses using the mutated NF-{kappa}B response elements {kappa}B1 and {kappa}B2 indicated that both regions were required for optimum transactivation of KC by OKA with the former NF-{kappa}B response element playing a more significant role in regulating KC expression. Gel-shift and supershift analyses demonstrated the involvement of three NF-{kappa}B subunits, p65, p50 and c-Rel, with p65 as the major subunit in the NF-{kappa}B dimer complex. Additionally, immunohistochemistry and western blot analyses confirmed the presence of p65 in the nucleus with its transactivation domain phosphorylated at serine 536. In summary, this is the first report to show that the tumor promoter OKA can stimulate the de novo synthesis and secretion of KC, and that this stimulation is mediated through the NF-{kappa}B pathway in JB6 cells.

Abbreviations: AP-1, activator complex protein-1; CXC, chemokine; CXCR2, chemokine receptor 2; EMSA, electrophoretic mobility shift assay; EGF, epidermal growth factor; GRO{alpha}, growth-regulated oncogene alpha; HRP, horseradish peroxidase; IL-1{alpha}, interleukin-1{alpha}; MEM, Minimum Essential Medium Eagle; NF-{kappa}B, nuclear factor-kappaB; OKA, okadaic acid; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; RHD, Rel homology domain; TAD, transactivation domain; TNF{alpha}, tumor necrosis factor {alpha}; TPA, 12-O-tetradecanoylphorbol 13-acetate


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Okadaic acid (OKA) is a 38-carbon cyclic polyether fatty acid produced by dinoflagellates. It can accumulate in the digestive tracts of shellfish and also exists in minimum amount in all other fish (1,2). This fatty acid is one of the major diarrhetic shellfish toxins. It functions as an inhibitor of protein phosphatase 1 and 2A, with a much stronger effect on the latter enzyme (3,4). OKA binds to these phosphatases and acts as a non-competitive or mixed inhibitor (5–7). Consequently, OKA leads to hyperphosphorylation of phosphoserines and phosphothreonines, which are critical components of signaling cascades of a diverse range of cellular processes. The hyperphosphorylation effect by OKA is very similar to that of the skin tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), thus like TPA, OKA is also known as a potent tumor promoter (8–10). However, unlike TPA, which binds to protein kinase C and causes a rapid phosphorylation cascade, the effect of OKA on hyperphosphorylation is slower but longer lasting.

The mechanisms by which OKA stimulates tumor promotion are not clearly understood. OKA has been reported to activate transcription factor complexes such as activator complex protein-1 (AP-1) and/or nuclear factor kappa B (NF-{kappa}B) (11,12). The activation of these two transcription factors has been shown to mediate tumorigenic transformation in vitro (13,14) and in vivo (15–17). Recent findings have also demonstrated that OKA suppresses dephosphorylation of heterogenous nuclear ribonucleoprotein A1 and heterogenous nuclear ribonucleoprotein A2 and consequently prevents apoptosis (18). Therefore, in the context of tumor promotion, OKA could indirectly contribute to the survival of initiated cells.

In vivo tumor promotion studies using the two-stage mouse skin carcinogenesis system suggest that the tumor promotion effect of OKA is mediated through tumor necrosis factor {alpha} (TNF{alpha}) expression because TNF{alpha}-deficient mice showed delayed tumorigenic response to OKA (19–21). Thus, these studies implicate the possible role of TNF{alpha} as an endogenous tumor promoter. Additionally, TNF{alpha} induces the expression of a chemokine murine (CXCL1/KC), originally found to stimulate the migration of inflammatory cells and recently shown to modulate tumor growth and angiogenesis. These findings suggest that in addition to TNF-{alpha}, KC may play a critical role downstream of TNF-{alpha} in the tumor promotion stage.

KC is characterized as a glutamate–leucine–arginine (ELR) CXC chemokine family because it contains the ELR motif at the N-terminus and because the first two conserved cysteine residues are separated by one amino acid (22). KC has high affinity for chemokine CXC chemokine receptor 2 (CXCR2), a seven-transmembrane G protein-coupled receptor (23–25). The human homolog of mouse KC is the growth-regulated oncogene alpha (GRO{alpha}), also known as melanoma growth stimulatory activity alpha. Human GRO{alpha} and mouse KC share only 64% homology in amino acid sequence but 90% homology in conserved regions (26). Therefore, mouse KC has very similar function as GRO{alpha}. KC is secreted by activated monocytes and acts as an agent for neutrophil-specific chemotaxis (27,28). Besides its expression in activated monocytes, KC has been reported to act as an autocrine growth factor in stimulating the proliferation of epithelial cells and squamous cell carcinoma cells through a CXCR2-mediated pathway (29,30). Elevated KC expression has been shown to be associated with malignant transformation (31). Over expression of KC in slow growing squamous cell carcinoma results in increased tumor growth, metastasis and angiogenesis (30,31), suggesting its possible role in tumor progression.

Besides its potential role in tumor progression, KC may also contribute to tumor promotion since both the exogenous (TPA) and endogenous tumor promoters such as TNF{alpha}, epidermal growth factor (EGF) and interleukin-1{alpha} (IL-1{alpha}), have been shown to stimulate KC expression (28,32,33). Additionally, the human homolog of KC, GRO{alpha} when over expressed in melanocytes was shown to enhance tumor-forming capacity (34). Here, we investigate whether OKA, a natural tumor promoter, can also induce KC expression directly using the promotable epidermal-like JB6 Cl41.5a cell line, commonly used for tumor promotion studies. We report that OKA stimulates the de novo synthesis and secretion of KC in a time- and dose-dependent manner. OKA induction of KC expression is mediated through the NF-{kappa}B pathway. Transient transfection studies showed that KC induction by OKA specifically requires the activation of two NF-{kappa}B motifs in the promoter region of KC. Electrophoretic mobility shift assays (EMSAs) confirmed the binding of OKA-treated nuclear protein to these two NF-{kappa}B motifs. Furthermore, the NF-{kappa}B subunits involved in the activation of OKA-induced KC expression are p65, p50 and c-Rel with p65 as the major subunit involved in the NF-{kappa}B dimer transcription complex. Immunohistochemical studies and western blot analyses showed that p65 translocates to the nucleus and that the transactivation site of p65 at serine residue 536 is phosphorylated. Our findings are the first to show that OKA can induce de novo KC production which is mediated through the NF-{kappa}B pathway.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 References
 
Reagents
Minimum Essential Medium Eagle (MEM) and phosphate buffered saline (PBS) were obtained from Mediatech (Herndon, UT). Fetal bovine serum (FBS) was obtained from Hyclone (Logan, UT). The L-glutamine, trypsin/EDTA and antibiotics were purchased from Irvine (Santa Ana, CA). The bicinchoninic acid assay reagents were purchased from Pierce (Rockford, IL). Polyclonal antibodies against the conserved epitope of mouse origin NF-{kappa}B subunits (p65, Rel B, c-Rel, p50 and p52) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Luciferase report vector pGL2-Basic (Promega, Madison, WI) was obtained from Dr Amy Ridall (University of Texas, Dallas, TX). CMV-ß-galactosidase plasmid was obtained from Dr Wei-Chin Lin (University of Alabama at Birmingham, Birmingham, AL). FuGENE 6 and chlorophenolred-ß-D-galactopyranoside were obtained from Roche Diagnostic (Indianapolis, IN). Receptor lysis buffer, luciferase substrate and polydI-dC were obtained from Promega. Oligonucleotides were synthesized by Intergrated DNA Technologies (Skokie, IL). OKA sodium salt and all other reagents, unless otherwise stated, were purchased from Sigma (St Louis, MO).

Cell culture
JB6 Cl41.5a mouse epidermal cells (obtained from Dr Nancy Colburn, National Cancer Institute, Frederick, MD) were maintained in MEM Eagle supplemented with 5% heat-inactivated FBS, 2% glutamine and 0.5% Fungi–Bact. Cells were subcultured prior to confluence and checked monthly by DNA fluorochrome staining (35) to be sure that they were not contaminated with mycoplasma.

RNA extraction and northern blot analyses
JB6 Cl41.5a cells were seeded at 9000/cm2 cells in 60-mm dishes. The next day, cells were replaced with fresh medium for 4 h and treated with OKA or an equal volume of vehicle (H2O) as a negative control for a designated time period. Duplicate dishes were used for each treatment. Each experiment was repeated at least 2–3 times. Total cellular RNA was isolated from JB6 cells using a previously described procedure (36). Ten micrograms of total RNA was denatured, and loaded onto 1% agarose containing 2.2 M formaldehyde, and then transferred to nylon membranes (Oncor, Gaithersburg, MD). Blots were stained with methylene blue to determine efficiency of transfer and to ascertain RNA integrity on the basis of the prominence of 18S and 28S ribosomal RNA bands.

Northern blots were performed as described previously (36). Briefly, cDNA for mouse KC and human ß-actin (both from American Type Culture Collection, Manassas, VA) were labeled with [{alpha}-32P]dCTP using a random primer labeling kit, Prime-It® II (Stratagene, La Jolla, CA) and hybridized to mRNA on the nylon membrane overnight. Membranes were washed and then exposed to film (Scientific Imaging film XAR; Eastman Kodak, Rochester, NY) at –20°C and developed. Autoradiographs were analyzed using a densitometer (ChemiDoc XRS; Bio-Rad Laboratories, Hercules, CA). Quantization of KC and ß-actin mRNA was performed using the Quantity One software (Bio-Rad Laboratories). The relative amounts of KC mRNA were normalized to the levels of ß-actin mRNA.

Enzyme-linked immunosorbent assays (ELISAs)
Cells were seeded, maintained and treated by following the same procedure described above but using 12-well culture plates. Media were collected at different time points (quadruplicate samples/time point) after stimulation with OKA and stored at –70°C until assayed. Cells of each treatment were digested, and the number of cells was counted with a Z1S Coulter counter (Beckman Coulter, Fullerton, CA). The concentration of KC in the medium was determined with a commercially available sandwich ELISA (R&D Systems, Minneapolis, MN). In brief, microplates coated with an anti-KC polyclonal antibody were incubated with supernatants and washed, and followed with the addition of a horseradish peroxidase (HRP)-linked polyclonal antibody to which KC was then added. After incubation, the plates were washed to remove unbound antibody. Color substrate was added and allowed to react with HRP for 30 min. The reaction was stopped with hydrochloric acid solution. Absorbance values were detected at 450 nm and corrected using absorbance values at 550 nm. The assay has a sensitivity ranging from 15.6 to 1000 pg/ml of KC. The final KC concentration was normalized to 10 000 cells.

Construction of luciferase reporter plasmids
Various lengths of KC promoter fused to CAT constructs were constructed as described previously (37). The different fragment lengths (–1500, –474, –146, –104, –75 and –59) or {kappa}B site-directed mutants (m{kappa}B1 and m{kappa}B2) of KC promoter were cleaved out of the CAT vector using restriction enzymes and inserted directionally into the pGL2-Basic. The recombined plasmids containing the shorter KC promoter fragment were verified by sequencing, while the larger fragments were confirmed by restriction enzyme digestion.

Transient transcriptional assays
Transient transfection assays were performed similarly to a previously described procedure (38) with modifications. JB6 cells were seeded at 7000 cells/cm2 in 60-mm dishes (triplicate dishes per treatment). The next day, cells were cotransfected with 1 µg of luciferase reporter plasmid fused to different lengths of KC promoter or mutants and 1 µg of CMV-ß-galactosidase (control plasmid) using 3 µl of FuGENE 6 for 24 h. Prior to initiation of drug treatment, cells were incubated in low serum medium (0.2% FBS) for 24 h and then treated with 50 nM OKA or vehicle for another 24 h. Cells were then rinsed with PBS containing calcium and magnesium, and reporter lysis buffer was then added. Scraped cell lysates were centrifuged to remove cell debris. To determine luciferase activity, 10 µl of cell lysates were added to 100 µl of substrate (Promega) and the light emitted was detected using a Turner 20/20 luminometer (Turner Design, Sunnyvale, CA). The luciferase activity was normalized against ß-galactosidase activity. ß-Galactosidase activity was assayed by incubating 10 µl of cell lysates for 20 min at 37°C in 200 µl of reaction mixture containing 2 mM chlorophenolred-ß-D-galactopyranoside of substrate in 80 mM sodium phosphate buffer (pH 7.3), 102 mM 2-mercaptoethanol and 9 mM MgCl2. The absorption of the resulting color product was measured at 550 nm using the Microplate Reader Model 680 (Bio-Rad Laboratories).

Electrophoretic mobility shift assays (EMSAs)
JB6 cells were seeded at 9000 cell/cm2 and changed to media with 0.2% media for 24 h prior to the addition of drugs for a designated time (see figure legends). Nuclear extracts were prepared by a modified method of Dignam et al., (39) as previously described (37). Briefly, cells were washed with ice-cold PBS three times, collected by scraping, and resuspended in 300 µl of buffer A [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride and 10 µg/ml proteinase inhibitors (leupeptin, antipain, aprotinin and pepstatin)] and put on ice for 5 min. Cells were then lysed in 0.6% NP-40 by vortexing for 10 s and then centrifuged at 5000 r.p.m. for 3 min. Pellets (nuclei) were washed with 300 µl of buffer A once, resuspended in buffer C [20 mM HEPES (pH 7.9), 25% glycerol, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride and 10 µg/ml proteinase inhibitors], and briefly sonicated on ice. Supernatant containing nuclear extracts was obtained by centrifugation at 16 000x g for 8 min. Protein concentrations were determined using the bicinchoninic acid method following manufacturer's instructions.

The oligonucleotides containing the NF-{kappa}B consensus sequence used in EMSA were KC {kappa}B1 sense strand 5'-GAT CTA CTC CGG GAA TTT CCC TGG CC-3', KC {kappa}B1antisense strand 3'-ATG AGG CCC TTA AAG GGA CCG GCT AG-5', KC {kappa}B2 sense strand 5'-GAT CTT GCA GGG AAA CAC CCT GTA CT-3', and KC {kappa}B2 antisense strand 3'-AACG TCC CTT TGT GGG ACA TGA CTA G-5'. The sense strands were annealed with their antisense strands by a fill-in reaction prior to labeling with [{alpha}-32P]dCTP using Klenow fragment of DNA polymerase I (New England Biolabs, Beverly, MA).

For binding reactions, 10 µg of nuclear extracts were incubated with the reaction mixture [12.5 µl of total volume containing 20 mM HEPES (pH 7.9), 50 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 5% glycerol, 200 µg/ml bovine serum albumin (BSA), and 2.5 µg poly[dI–dC] for 15 min and then with 1x105 c.p.m. 32P-labeled double-stranded oligonucleotide for 15 min at room temperature. In competition reaction, 1 µl of 1x, 5x or 25x of unlabeled NF-{kappa}B oligonucleotides (1, 5 or 25 ng/µl) was added to the system. Reaction products were analysed on 4% polyacrylamide gel with 0.25x tris–borate–EDTA buffer. The separated bands in the gel were visualized by autoradiography.

To verify the binding specificity to NF-{kappa}B, supershift assays were also performed using purified polyclonal antibodies to mouse NF-{kappa}B subunits (p65, Rel B, c-Rel, p50 and p52). The binding reaction mixture was incubated with various antibodies to NF-{kappa}B subunits at room temperature for 45 min. Radiolabeled double-stranded oligonucleotide was then added to the mixture by following the procedure as described.

Immunohistochemical analyses
JB6 cells were seeded and treated with the same procedure used in the transient transfection assay. Cells were then fixed with 100% methanol and incubated with 3% H2O2 to block endogenous peroxidase. Cells were incubated for 2 h at room temperature with polyclonal antibodies to NF-{kappa}B p65 and subsequently rinsed in TBS prior to the addition of biotinylated secondary antibody. This procedure was followed by the addition of HRP conjugated to streptavidin, which binds to the biotin on the secondary antibody. The presence of NF-{kappa}B subunit was detected by the addition of the substrate 3'3'tetra-diamino-benzidine catalysed by HRP to produce a brown precipitate.

Western blot analyses
JB6 cells were seeded, treated with drugs and harvested for nuclear protein as described in the EMSA procedure above. Protein concentration was determined using the bicinchoninic acid method. Equal amounts of protein (20 µg) were incubated in SDS sample buffer [62.5 mM Tris–HCl (pH 6.8) containing 2% w/v SDS, 10% glycerol, 50 mM dithiothreitol and 0.1% w/v bromophenol blue], separated on 10% SDS–PAGE, and then transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA). Prestained markers (Amersham, Piscataway, NJ) were run alongside samples to determine molecular weights and to verify the efficiency of transfer. Membranes were blocked with 5% non-fat milk in 0.1% Tween-20 for 2 h at 4°C and then were incubated with antibody to phosphoserine 536 of p65 (Cell Signalling, Beverly, MA) in Tris–buffered saline [50 mM Tris–HCl (pH 7.4) containing 150 mM NaCl, 0.5% Tween-20, and 3% BSA] overnight at 4°C. Primary antibody-binding sites were detected using secondary antibody coupled to HRP. Membranes were reprobed with total p65 (Santa Cruz Biotechnologies). Bands were visualized by chemiluminescence using HRP substrate from Amersham.


   Results
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 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 References
 
OKA induces the expression of a steady-state level of KC mRNA in dose- and time-dependent manner
Studies have shown that endogenous (TNF{alpha}, EGF and IL-1) and exogenous (TPA) tumor promoters stimulate the expression of KC. Here, we investigate whether OKA can also induce KC expression in the mouse epidermal-like, promotable, preneoplastic JB6 Cl41.5a cell line commonly used as an in vitro model to study tumor promotion (14,40,41) and if it does, through what molecular pathway.

Prior to determining whether OKA stimulated KC expression, preliminary studies were performed to determine the optimum dose of OKA that does not lead to toxicity in JB6 Cl41.5a cells. We found that OKA at 50 and 75 nM showed no cellular toxicity by 24 h of treatment at a density of 7000–10 000 cells/cm2, while OKA at 100 nM showed slight toxicity after 24 h (data not shown). Thus, JB6 cells were treated with 5–75 nM of OKA or its vehicle (H2O) for 24 h, and KC mRNA expression was determined by northern blot analyses. Figure 1A shows that OKA stimulated the expression of steady-state KC mRNA in a dose-dependent manner, with 75 nM of OKA having the highest induction capability. Additionally, in vehicle-treated cells no basal level of KC mRNA expression was observed (see Figure 1A, lane 1 vehicle treatment).



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  		Fig. 1. Dose- and time-dependent induction of murine KC by OKA. (A) Dose-dependent induction of steady-state KC mRNA expression by OKA. JB6 Cl41.5a cells were treated with 5, 10, 25, 50 and 75 nM of OKA or H2O as vehicle for 24 h. Total RNA was isolated, separated on agarose gel, and transferred to nylon membrane following the methods described under Materials and methods. Top panel: northern blot, where the membrane was probed with radiolabeled KC and ß-actin cDNA. Bottom panel: plot of adjusted KC mRNA expression versus dose. Densitometric analyses of the steady-state level of KC mRNA induced by OKA from the data in the top panel. Values of KC mRNA were normalized to values of ß-actin mRNA. Data are representative of two repeated experiments. (B) Kinetics of steady-state KC mRNA expression by OKA. Cells were treated with 50 nM OKA or vehicle (H2O) at different time points from 1 to 24 h. Top panel: northern blot analysis of steady-state KC mRNA expression. Bottom panel: plot of KC mRNA expression versus time. Densitometric analyses of KC mRNA from the top panel. The amount of KC mRNA was normalized to that of ß-actin mRNA. Data are representative of three repeated experiments. (C) Synthesis and secretion of murine KC protein by OKA. Cells were treated with 50 and 75 nM of OKA or vehicle (H2O) for 8, 14, and 22 h. KC concentration from conditioned media was determined by ELISA, average concentration ± SD, n = 4 quadruplicate samples per time point. The final KC concentration was normalized to cell number as pg/10 000 cells.

 
To determine the kinetics of KC mRNA expression by OKA, we used 50 nM of OKA because we wanted to examine the earliest time point at which the lower concentration of OKA would induce KC mRNA expression. OKA stimulated the expression of steady-state KC mRNA by as early as 4 h, and its level increased to a maximum by 24 h of treatment (Figure 1B). From 12 to 24 h of OKA incubation, an ~2-fold increase of KC mRNA expression was observed. Although the ß-actin expression appears to be suppressed by OKA, repeated experiments showed that this is due to loading effect (see also Figure 1A, ß-actin mRNA expression is affected by OKA).

The dose- and time-dependent synthesis and secretion of KC protein by OKA correlates with its mRNA expression
Because OKA-induced KC mRNA expression in a time- and dose-dependent manner, we also examined whether KC protein was expressed. JB6 cells were treated with 50 nM or 75 nM of OKA for 8, 14 and 22 h. The amount of KC protein secreted in the medium was determined by ELISA and normalized to cell number. OKA-induced KC protein in a dose- and time-dependent manner, which correlated with the level and time of KC mRNA appearance (Figure 1C). The amount of KC secreted by 75 nM of OKA was 2.3-fold higher than that of KC produced by 50 nM of OKA at 24 h. Additionally, the dramatic increase (7.5-fold) in KC protein from 14 to 24 h with 50 nM of OKA is consistent with the observed 2-fold increase of the steady-state KC mRNA level from 12 to 24 h (Figure 1B).

The regulation of KC transcription by OKA is mediated through two NF-{kappa}B response elements
The observation that OKA stimulated mRNA expression by as early as 4 h with no KC transcript detected in vehicle-treated cells at all the time points studied (Figure 1B), showed that OKA stimulated the de novo synthesis of KC at the level of transcription in JB6 cells. Thus, we investigated the specific response elements involved in regulating OKA-induced KC mRNA expression. Transient transfection studies using various lengths of KC promoter (Figure 2A) fused to the reporter luciferase gene were performed. Cells were cotransfected with luciferase reporter plasmid containing different fragments of KC promoter and the ß-galactosidase plasmid and treated with 50 nM of OKA for 24 h. OKA-activated KC promoter from –1500 to –104 with similar induction levels (12.9–16.1-fold; average 14.7-fold) (Figure 2B). However, when transient transfection assays were performed with shorter KC promoter fragments containing only one (–75) or no NF-{kappa}B responsive element (–59), a dramatic reduction (48 and 84%, respectively) in the transactivation activity was observed (Figure 2B). Thus, serial deletion of the KC promoter indicated that OKA induction of KC mRNA expression is specifically mediated through the two NF-{kappa}B responsive elements, {kappa}B1 and {kappa}B2, localized in the region between –104 and –59 of KC promoter. The third NF-{kappa}B responsive element, {kappa}B3 (Figure 2A), located between –847 and –644 of KC promoter, appears to be not involved in the transactivation of KC promoter because deletion of this region did not reduce the luciferase activity by OKA.



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  		Fig. 2. Transcriptional regulation of murine KC by OKA is mediated through the two NF-{kappa}B responsive elements {kappa}B1 and {kappa}B2. (A) Schematic diagram of serial deletions of KC promoter. SRE, serum response element; LUC, luciferase gene. (B) OKA-responsive region is localized between –104 and –59 of KC promoter. The luciferase reporter plasmids containing the various lengths of KC promoters shown in (A) were transiently cotransfected with CMV-ß-galactosidase plasmid overnight into JB6 Cl41.5a cells using FuGENE 6. Cells were then serum depleted and treated with 50 nM OKA or H2O as control for 24 h, as described under Materials and methods. Luciferase activity was normalized to ß-galactosidase activity. The relative luciferase activity (mean of triplicate samples ± SD) is indicated as fold induction by OKA over that of control (***, significantly different from KC –1500 to KC –104, P < 0.001; •••, significantly different from KC –75, P < 0.001). The data are representative of two repeated experiments. (C) Schematic diagram of mutants on two {kappa}B sites from –146 fragment of KC promoter. (D) Mutation of the {kappa}B sites significantly impaired the response of KC promoter to OKA induction. Cells were transiently cotransfected with each of the plasmids shown in C and the plasmid CMV-ß-galactosidase, followed by treatment with OKA as described in A. Luciferase activity (mean of triplicate samples ± SD) was normalized to ß-galactosidase activity and indicated as fold induction over that of vehicle-treated cells (••• luciferase activity is significantly different from that of non-mutated promoter KC –146, P < 0.001; *** luciferase activity is significantly different from KC –146 m{kappa}B2, P < 0.001). Luciferase activity was normalized to ß-galactosidase activity. The data are representative of two repeated experiments.

 
To further confirm the involvement of these two NF-{kappa}B response elements in transactivation of KC promoter by OKA, additional transient transfection assays were performed using KC promoters (–146) containing mutated NF-{kappa}B response elements (Figure 2C). A change in 1 bp in the {kappa}B1 site (–146 m{kappa}B1) resulted in 88% reduction in the transactivation response of KC promoter, whereas a 4-bp alteration in the {kappa}B2 site and an additional change of 2 bp next to this site (–146 m{kappa}B2) resulted in only a 61% reduction of luciferase activity when compared with that of the KC –146 with no mutated NF-{kappa}B sites (Figure 2D). These data suggest that both {kappa}B sites are necessary for optimum transactivation of KC promoter by OKA and that the {kappa}B1 site plays a more important role in KC expression by OKA. Furthermore, these studies implicate the involvement of OKA activation of NF-{kappa}B binding to these two {kappa}B sites.

OKA-activated nuclear protein binds to both NF-{kappa}B response elements {kappa}B1 and {kappa}B2
To determine whether NF-{kappa}B subunits bind to the two {kappa}B sites shown, EMSAs were performed to first determine whether nuclear protein extracts from OKA-treated JB6 cells will bind to these response elements. JB6 cells were incubated with OKA at 50 nM for 20 and 24 h prior to nuclear protein extraction. Cells were also treated with 25 U/ml of TNF{alpha} for 1 h as a positive control, because it has been shown to stimulate KC mRNA expression through the NF-{kappa}B pathway in other cell lines (42). Nuclear proteins from cells treated with vehicle only did not bind to DNA fragments containing {kappa}B1 or {kappa}B2 (Figure 3A, lanes 1 and 9, respectively). However, cells treated with OKA for 20 h showed substantial binding to {kappa}B1 and minimal binding to {kappa}B2 (Figure 3A, lanes 2–4 and lane 10, respectively). The minimal binding to {kappa}B2 is consistent with the transient transfection studies, which showed that the {kappa}B1 response element plays a more important role in OKA-induced KC expression. Cells treated with OKA for 24 h showed less binding ability to the two response elements when compared with cells at 20 h.



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  		Fig. 3. OKA dose-dependently induces nuclear protein binding to the NF-{kappa}B response elements in murine KC promoter. (A) Detection of OKA-induced nuclear protein binding activity. Cells were treated with 50 nM OKA or vehicle (H2O, as negative control) for 20 or 24 h and then harvested for nuclear protein extraction. As a positive control, cells were treated with 25 U/ml of TNF-{alpha} for 1 h. Binding activity of two {kappa}B sites to nuclear protein extract was determined by EMSA using double-stranded radiolabeled oligonucleotides containing {kappa}B1 or {kappa}B2 sequence. Binding capacity of the nuclear protein was also tested using increasing concentration of potassium chloride 0, 25 and 50 mM (lanes 2, 3 and 4, respectively, in cells treated with OKA for 20 h and lanes 6, 7 and 8, respectively, in cells treated with TNF-{alpha}) in the binding reaction mixture. For lanes 1, 5 and 9–12, 50 mM of potassium chloride was used. Arrow head indicates the shifted band produced by the binding of the nuclear extracts to radiolabeled {kappa}B1 and {kappa}B2 from OKA and TNF{alpha}-treated cells. The dash on the right side of the panel indicated the second shifted band produced by TNF{alpha}-treated cells. (B) Binding nuclear protein to NF-{kappa}B response elements is dose-dependently activated by OKA. JB6 Cl41.5a cells were treated with 50 or 75 nM of OKA or H2O for 20 h, and then nuclear proteins were prepared from the cells. In this experiment and all the following EMSA experiments 50 mM of potassium chloride was added in the binding reaction mixture. Data of (A) and (B) are representative of two repeated experiments.

 
Nuclear protein extracts from TNF{alpha} treated cells, used as a positive control, also showed greater binding to {kappa}B1 compared with {kappa}B2 (Figure 3A, lanes 6–8 and lane 12). Unlike OKA-treated cells, the EMSA data from TNF{alpha} treated cells showed a second prominent retarded band (C2) located below the C1 band. Although, there appears to be a faint C2 band in OKA-treated cells, this was not consistently observed in our repeated experiments (see Figures 3A and B, and 4B). Furthermore, the addition of potassium chloride (0, 25 and 50 mM) in the binding reaction mixture was used to determine whether it could increase or decrease the binding capacity of the protein nuclear extract to the response element. Figure 3A (lanes 2–4 and lanes 6–8, respectively) showed that the presence of increasing concentrations of potassium chloride did not affect the binding capacity of OKA or TNF-{alpha}-induced nuclear protein binding to {kappa}B1. Therefore, in the subsequent EMSA or supershift experiments, 50 mM of potassium chloride was added to the binding reaction mixture.



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  		Fig. 4. OKA induces nuclear protein binding preferentially to {kappa}B1 site over that of {kappa}B2 site and the nuclear proteins are composed of NF-{kappa}B subunits p65, p50 and c-Rel. (A) Preferential binding of nuclear protein to {kappa}B1 site over that of {kappa}B2 site. Nuclear protein extracts of JB6 Cl41.5a cells treated with H2O or 50 nM OKA for 20 h were incubated with double-stranded radiolabeled {kappa}B1 or {kappa}B2 oligonucleotides with/without 1-, 5- or 25-fold excess unlabeled oligonucleotides of {kappa}B1 or {kappa}B2, respectively. Additionally, nuclear proteins were also incubated with radiolabeled {kappa}B1 oligonucleotides and unlabeled {kappa}B2 oligonucleotides or vice versa to test for specificity of binding. CT, control. (B) Antibodies against p65, p50 and c-Rel supershift the nuclear protein binding to the NF-{kappa}B response elements. Cells were treated with 75 nM of OKA for 20 h, and the nuclear protein extracted was incubated with antibodies against NF-{kappa}B subunits p65, c-Rel, RelB, p50 and p52 (1.6 µg each) prior to incubation with double-stranded radiolabeled {kappa}B1 or {kappa}B2 oligonucleotides. Normal rabbit IgG was used as negative control. Arrows indicate supershifted bands. Unpurified double-stranded radiolabeled oligonucleotides were used in these experiments. Data of (A) and (B) are representative of two repeated experiments.

 
Prior to determining the specificity and the NF-{kappa}B subunits involved in binding to its response elements in the KC promoter, we examined whether a higher dose of OKA (75 nM) than 50 nM (Figure 3B) would induce greater binding of the nuclear protein to both NF-{kappa}B response elements {kappa}B1 and {kappa}B2 and therefore provide a more sensitive dose for performing supershift assays. Figure 4B shows that, compared with OKA at 50 nM, OKA at 75 nM has greater induction on nuclear protein binding to both {kappa}B sites, with, again, stronger binding to {kappa}B1 of KC promoter.

OKA-activated NF-{kappa}B subunits p65, p50 and c-Rel bind to both NF-{kappa}B response elements
To verify the specificity of nuclear protein binding to the two NF-{kappa}B promoter regions, gel-shift assays were performed with excess (1-, 5- and 25-fold) unlabeled {kappa}B1 or {kappa}B2. The unlabeled {kappa}B1 oligonucleotides were able to dose-dependently compete with labeled {kappa}B1 DNA fragments for nuclear protein binding (Figure 4A). This binding is not suppressed by unlabeled {kappa}B2 oligonucleotides, thus the binding of nuclear proteins to the {kappa}B1 promoter site is specific. For the {kappa}B2 promoter site, although very low nuclear protein binding was observed, unlabeled {kappa}B2 oligonucleotides also dose-dependently suppressed nuclear protein binding to labeled {kappa}B2 promoter site. However, unlike the {kappa}B1 binding reaction, the addition of unlabeled {kappa}B1 oligonucleotides appears to totally block the nuclear protein binding to {kappa}B2 DNA fragment (Figure 4A), suggesting that OKA-induced nuclear proteins may preferentially bind to {kappa}B1 promoter when both sites are available. Further studies, however, will be necessary to verify this.

To determine the NF-{kappa}B subunits involved in binding to these two response elements using supershift assay, cells were treated with 75 nM of OKA for 20 h prior to extraction of nuclear protein. Antibodies specific to five NF-{kappa}B subunits (p65, c-Rel, RelB, p50 and p52) or the negative control IgG were incubated separately with nuclear protein extract followed by the addition of radiolabeled {kappa}B1 and {kappa}B2 DNA fragments. Antibodies specific to p65, c-Rel, and p50 shifted the binding bands to a higher molecular weight in both {kappa}B sites (Figure 4B). However, due to the weaker binding of nuclear proteins to {kappa}B2, many of the supershifted bands are only faintly visible (note Figure 4B, the X-ray film has been overexposed to try to show the faint bands). These three antibodies also shifted the binding bands with different intensity. Antibody to p65 totally shifted the binding band in both {kappa}B sites. Antibodies to c-Rel and p50 only partially shifted the binding bands, but antibody to p50 appears to have a stronger effect because the supershifted band is more prominent than that of the c-Rel antibody. Neither pre-immune IgG nor antibodies to RelB and p52 had any effect on the mobility of the binding band. These results suggest that p65 protein is the major subunit and that c-Rel or p50 is the other minor partner in the heterodimeric complex of NF-{kappa}B mediating the transactivation of KC expression by OKA.

OKA promotes p65 translocation from cytoplasm to nucleus and stimulates the phosphorylation of p65
It is known that, in resting cells, hetero- or homodimer NF-{kappa}B binds to I{kappa}B and remains inactive (43). The complex can transport into and out of the nucleus but is mainly sequestered in the cytoplasm when cells are not activated (44). Once stimulated, I{kappa}B is phosphorylated; this phosphorylation releases NF-{kappa}B to translocate into the nucleus, and the phosphorylated form of I{kappa}B is degraded through the proteasome pathway (43). Thus, cellular localization of NF-{kappa}B can provide further evidence of the NF-{kappa}B activity status. Our studies using transient transfection assay and EMSA indicate the involvement of two NF-{kappa}B responsive elements (Figure 2) and confirm the binding of NF-{kappa}B subunits (p65, c-Rel and p50) to these response elements (Figure 4B). However, these findings are derived from in vitro experiments. Therefore, to further provide evidence that the major NF-{kappa}B subunit (p65) is involved in OKA-induced KC expression, we performed immunohistochemical studies to confirm the translocation of NF-{kappa}B to the nucleus. Cells were incubated with 50 nM OKA or its vehicle (H2O) for 0, 1/4, 1/2, 1, 2, 4, 8, 12, 16 and 20 h prior to fixation and staining for NF-{kappa}B subunit p65. In cells not treated with OKA, p65 was localized in the cytoplasm (Figure 5A). Stimulation with OKA did not produce nuclear localization of p65 until around 12 h; increased localization was observed up to 20 h. However, unlike stimulation by TNF{alpha} (data not shown), we did not observe 100% of the cells with complete nuclear localization of p65.



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  		Fig. 5. OKA stimulates the translocation of NF-{kappa}B p65 from the cytoplasm to the nucleus and induces the phosphorylation of the transactivation domain of p65. (A) Nuclear localization of p65 in OKA-treated cells. Cells were seeded at 7000/cm2 in two-well chamber slides and serum depleted prior to treatment with 50 nM OKA (right panels) or vehicle (H2O, as negative control, left panels) for 20 h and then fixed. Slides were incubated with polyclonal antibody to NF-{kappa}B subunit p65 or pre-immune IgG. NF-{kappa}B subunit p65 was visualized using immunohistochemical assay as described under Materials and methods. Slides incubated with pre-immune IgG show negative staining (data not shown). (B) Western blot analyses of nuclear p65. Cells were treated with OKA at 50, 75 or 100 nM for 20 h or at 50 and 75 nM for 24 h. As a positive control for nuclear localization of p65, cells were also treated with 25 U/ml of TNF-{alpha} for 1 h. The top panel represents the phosphorylated p65 using antibody specific to serine 536 of p65. The bottom panel represents total p65 in the nucleus (lower band). The upper band is non-specific. Data from (A) and (B) are representative of two repeated experiments. See online Supplementary material for a colour version of this figure.

 
In addition to nuclear localization of p65, it has been reported that the phosphorylation of the serine residues such as Ser-276 in the Rel homology domain (RHD) and Ser-529/Ser-536 in the C-terminal transactivation domain (TAD) play a key role in the transcriptional activation (45–50). Because OKA inhibits dephosphorylation of phosphoserines and phosphothreonines, it is possible that all the phosphoserine residues of p65 are phosphorylated in the nucleus of OKA-treated JB6 cells. Thus, we selected one of the serine residues 536 of p65 to determine whether it remains phosphorylated in the nucleus of OKA-treated cells. Western blot analyses of nuclear extracts were performed to confirm nuclear localization of p65 and whether OKA induces p65 phosphorylation at serine 536. Figure 5B shows that various doses of OKA-treated cells induced p65 nuclear localization. Furthermore, compared to control cells, OKA-treated nuclear extract at 20 (50, 75 and 100 nM) and 24 h (50 and 75 nM) showed phosphorylated p65 at Ser-536 in a dose-dependent manner.


   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 References
 
OKA known as a non-phorbol ester tumor promoter is an inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) with a stronger effect on the latter enzyme. The resultant effect of OKA function is an accumulation of phosphorylated proteins. We found that OKA has the capacity to stimulate KC expression in the promotable epidermal-like JB6 cells. This finding is significant, because OKA, found commonly in shellfish and minimally in many other types of fish (1), could potentially stimulate the expression of this inflammatory chemokine in epithelial cells during the tumor promotion stage in vivo. Furthermore, over expression of KC has been shown with stimulate cell proliferation (29,30); thus, KC could play an important role in promoting the proliferation of initiated cells.

The induction of KC mRNA expression and its protein by OKA in JB6 cells is time- and dose- dependent. The absence of detectable KC protein or its mRNA in control cells indicated that OKA stimulates the de novo synthesis of KC. OKA induction of KC expression appears to be slow because its steady-state mRNA level is minimal at 4 h and is not prominent until after 12 h of OKA treatment.

The slow onset and prolonged effect of OKA on KC mRNA expression is similar to its induction on other genes such as the proto-oncogene c-fos, primary response genes, gap-junctional protein—connexin, and the matrix protein—osteopontin (9,11,51,52). Two possible factors could explain the slow onset of KC expression by OKA. Firstly, the low cell permeability of OKA could increase the time required for OKA to reach equilibrium within the cell (53–55). Secondly, the induction of KC by OKA could involve the activation of intermediate biochemical events with or without the additional requirement of protein synthesis.

It is not likely that mediation of KC mRNA induction by OKA requires protein synthesis such as the production of TNF{alpha}, which can also stimulate KC (37), because the addition of the protein synthesis inhibitor cycloheximide did not suppress KC mRNA expression (data not shown). Instead, we observed that cycloheximide at 10 µg/ml in the presence of OKA resulted in superinduction of KC mRNA expression, which has also been previously reported in platelet-derived growth factor-induced KC expression (56). The mechanism of superinduction of KC by cycloheximide remains to be determined.

Instead of the involvement of protein synthesis in the mechanism of OKA-induced KC expression, we postulate that the slow onset of KC expression by OKA is contributed by both the low permeability of OKA and followed by the activation of intermediate biochemical events without the requirement of protein synthesis. One of the possible intermediate biochemical events is the activation of AP-1 mediating OKA-induced KC expression. AP-1 is an immediate early transcription factor complex consisting of the homo- or heterodimeric complex of the jun and fos gene superfamily. AP-1 is normally stimulated within 15–30 min and rapidly degraded; thus, if AP-1 mediates OKA induction of KC, we should expect to see KC mRNA appearing as early as 1 h of treatment. However, this early appearance was not observed. Additionally, our transient transfection studies showed that deletion of the putative AP-1 response element (located between –847 and –644) in the KC promoter did not significantly suppress the transactivation of KC promoter. Therefore, OKA-induced KC expression is not mediated through the activation of AP-1. Instead, it is more likely that the intermediate events involve the activation of NF-{kappa}B, because PP2A, the enzyme inhibited by OKA, has been reported to bind directly to the NF-{kappa}B subunit RelA or p65 and induce dephosphorylation of p65 (12).

Studies from transient transfection assay using different lengths of KC promoter fused to luciferase reporter further support the involvement of NF-{kappa}B. There are actually three NF-{kappa}B binding sites ({kappa}B1, {kappa}B2 and {kappa}B3) on mouse KC promoter. {kappa}B1 site is highly conserved in mouse KC, and in rat and human GRO-{alpha}, but {kappa}B2 site exists only in mouse and rat KC promoter (37). Sequential deletion assays of KC promoter indicate that the {kappa}B3 sequence upstream of –474 KC promoter does not mediate OKA activation and that the promoter sequence between positions –104 and –59, which contains the two NF-{kappa}B sites ({kappa}B1 and {kappa}B2), is required for optimal induction of KC expression by OKA. Deletion of these two NF-{kappa}B sites resulted in an almost complete loss of OKA-induced gene expression. Independent mutations of each NF-{kappa}B site showed a >50% decrease in OKA inducibility and further showed that {kappa}B1 is more responsive to OKA-induced transactivation than the {kappa}B2 site. This differential response is also observed in previous studies of KC induction by lipopolysaccharide and TNF{alpha} (37).

The differential transcriptional activation responses induced by OKA on the two NF-{kappa}B sites is further supported from the EMSA studies showing that OKA-induced higher binding capacity of nuclear proteins to the {kappa}B1 site compared with that of the {kappa}B2 site. Supershift analyses indicated that OKA-induced nuclear proteins regulating KC expression consist of the three NF-{kappa}B subunits, p65, c-Rel and p50. Total shift of binding band by antibody to p65 at {kappa}B1 and {kappa}B2 sites indicates that p65 is the major subunit binding to these sites, while the other subunits, p50 and c-Rel, appear to be present in minimal amounts. Because p65 and c-Rel subunits are involved in the transactivation of targeted genes, while p50 does not contain the transactivation region, we postulate that the following combination of active NF-{kappa}B complexes exists: p65/p65, p65/p50 and p65/c-Rel (57). The most common NF-{kappa}B complex found in several cell systems is p65/p50. Further co-immunoprecipitation studies will be necessary to verify the actual NF-{kappa}B complex formed in JB6 cells.

The involvement of these three NF-{kappa}B subunits is also observed in lipopolysaccharide stimulation of KC gene transcription in RAW264.7 cells. Like OKA induction of KC in JB6 cells, lipopolysaccharide induction of KC is mediated through greater affinity of these nuclear proteins to the {kappa}B1 site than to the {kappa}B2 site. However, the major difference between OKA- and lipopolysaccharide-induced KC expression is that all three subunits in the latter stimulus appear to be present in similar levels (37), suggesting that the amount and specificity of NF-{kappa}B subunits binding to the two conserved {kappa}B sites are dependent on stimulus and cell type.

Immunohistochemical study of nuclear localization of NF-{kappa}B subunit p65 (shown to be the major component in binding to the two {kappa}B1 and {kappa}B2 response elements of KC promoter upon OKA stimulation) confirmed the involvement of this subunit, as shown from the supershift assay. Moreover, the finding that no significant nuclear localization of p65 was observed until at or after 12 h of OKA treatment further supported the observed lag time in the increased steady-state level of OKA-induced KC mRNA and protein expression. The unexpected finding was that we did not see a complete nuclear localization of p65 in JB6 cells treated with OKA by 20 h compared with the TNF{alpha} treated cells after 1 h (data not shown).

Western blot analyses of the total p65 also confirmed its nuclear localization in OKA-treated cells. Compared with OKA treatment, the level of total nuclear p65 is higher in TNF{alpha}-treated cells, which is consistent with the observed immunohistochemistry data (not shown) and the higher binding capacity of TNF{alpha}-treated nuclear protein to the two NF-{kappa}B sites. Furthermore, in addition to nuclear localization of p65, OKA-treated cells show a persistent level of phosphorylated p65 at serine 536. This is probably due to OKA inhibiting the activity of possibly a nuclear PP2A involved in dephosphorylation of the p65 (12).

The minimally phosphorylated nuclear p65 in TNF{alpha}-treated JB6 cells after 1 h is consistent with previously reported findings and further support the presence of a nuclear phosphatase. It has been shown in HeLa cells that TNF{alpha} induces a rapid phosphorylation of p65 by 5 min, but the level of phosphorylation is decreased by 20 min. Additionally, this rapid dephosphorylation of p65 can be prevented by the addition of calyculin A, which, like OKA, is also a PP2A inhibitor (58). Interestingly, the lack of phosphorylated nuclear p65 in TNF{alpha}-treated JB6 cells after 1 h does not appear to inhibit its binding to the NF-{kappa}B sites. Thus, the exact function of the phosphorylated p65 at serine 536 although postulated to play an important role in NF-{kappa}B transactivation remains to be studied in more detail. Additionally, whether OKA also induces the phosphorylation of I{kappa}B-{alpha} or other upstream regulators in JB6 cells remains to be determined. However, previous studies have shown in HeLa cells that preventing the dephosphorylation of I{kappa}B-{alpha} at serines 32 and 36 by OKA resulted in its degradation and subsequent activation of NF-{kappa}B (59).

In summary, we show for the first time that OKA induces the de novo synthesis and secretion of KC. The induction by OKA is mediated through the activation of the three NF-{kappa}B subunits, p65, p50 and c-Rel, with p65 as the major subunit in the NF-{kappa}B dimer complex. These NF-{kappa}B subunits bind to both NF-{kappa}B response elements; however, {kappa}B1 is the response element which showed greater nuclear protein binding and transactivation response. Additionally, OKA treatment induced nuclear localization of p65 subunit with persistent phosphorylation at serine 536, which is postulated to mediate the transactivation of gene expression.


   Supplementary material
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
References
 
Supplementary material is available at: http://www.carcin.oxfordjournals.org/


   Acknowledgments
 
We thank Patricia Hicks for technical assistance. This work was supported by US National Institutes of Health grant R01 CA90920 (P.-L.C.) and by a Sigma Xi Grant-in-Aid of Research Program award (G.F.).

Conflict of Interest Statement: None declared.


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 

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