SEARCH

SEARCH BY CITATION

Keywords:

  • age-related macular degeneration;
  • AP-1;
  • cholesterol;
  • ERK;
  • interleukin-8;
  • MEK;
  • NF-κB;
  • oxycholesterol;
  • oxysterol;
  • PI3K

Abstract.

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Purpose:  This study aimed at elucidating the molecular mechanisms involved in the regulation of IL-8 production by several oxysterols in retinal pigment epithelium (RPE) cells.

Methods:  A human cell line from RPE (ARPE-19) was used to test the role of cholesterol and several oxysterols (25-OH, 7-KC and 7β-OH) in the expression and secretion of IL-8. Expression of IL-8 was assessed by real-time PCR, while IL-8 secretion was evaluated by ELISA. PI3K-, MEK1/2-, ERK1/2- and NF-κB-specific inhibitors were used to assess the specific role of the several players on the regulation of IL-8 production by oxysterols. A gene-reporter assay for AP-1 activity was also conducted to evaluate the putative role of this transcription factor on IL-8 expression induced by oxysterols.

Results:  Here, we demonstrate that 25-OH specifically increases transcription and secretion of the cytokine IL-8 in ARPE-19 cells. Indeed, treatment of ARPE-19 with 25-OH, but not with 7-KC, 7β-OH or cholesterol, induced the secretion of IL-8 from cells. 25-OH also induced the activation/phosphorylation of ERK1/2 through a mechanism dependent on MEK, ERK1/2 and PI3K kinase activity. Real-time PCR and ELISA experiments demonstrated that 25-OH increased transcription and secretion of IL-8 through a mechanism that is dependent on ERK1/2 and PI3K activity. Furthermore, 25-OH triggered the activation/phosphorylation of the AP-1 component c-Jun and, consistently, increased the transcriptional activity of AP-1. Additionally, we also found that 25-OH decreases the levels of IκB and increases the nuclear levels of NF-κB p65 subunit and that inhibition of NF-κB activity partially prevents the increased secretion of IL-8 induced by 25-OH.

Conclusions:  The results presented in this study suggest a role for 25-OH in inducing IL-8 production through pathways that are likely to involve AP-1 and NF-κB in ARPE-19 cells. Our data may also provide new molecular targets for the treatment of AMD.


Introduction

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

AMD is the leading cause of blindness in the world among the elderly (Gehrs et al. 2006). The development of new preventive and therapeutic strategies for AMD depends largely on the understanding of the cellular and molecular mechanisms underlying its pathogenesis.

AMD is a very complex disease, as several environmental, demographic and genetic risk factors contribute to the disease onset and progression. The development of AMD involves both a neovascular and an inflammatory component, and it affects primarily the photoreceptors, retinal pigment epithelium (RPE), Bruch’s membrane and choriocapillaries.

An imbalance between several chemokines and cytokines has been reported to occur in AMD and was shown to be the driving force for the inflammatory component of the disease. Genetic polymorphisms such as IL-8 (interleukin-8) A251T, which affect IL-8 production, were shown to be strongly associated with the risk of developing AMD (Goverdhan et al. 2008). Importantly, IL-1β C511T, IL-6 C174G and IL-10 G1082A polymorphisms showed no significant association with AMD (Goverdhan et al. 2008). Additionally, IL-8 levels appear to be correlated with choroidal neovascularization and macular oedema in AMD (Roh et al. 2009a,b). This is likely to be of great significance as IL-8 is a potent chemoattractant and activator of neutrophils, which are primarily involved in the initiation and amplification of acute inflammatory reactions and in chronic inflammatory processes (Kanda et al. 2008). These and other evidences suggest that IL-8 may have a crucial role in the development of AMD.

Progressive lipid and cholesterol accumulation in the Bruch’s membrane beneath the RPE has also been identified as a contributing factor to AMD (Rattner & Nathans 2006; Lakkaraju et al. 2007). Cholesterol and cholesterol esters are abundant in both drusen and other sub-RPE deposits in AMD eyes (Malek et al. 2003; Curcio et al. 2005; Li et al. 2005; Lakkaraju et al. 2007), suggesting compromised cholesterol homeostasis in the RPE. A plausible cause for the massive accumulation of these compounds in the retina is most likely associated with the impairment of cholesterol metabolism in RPE cells owing to the age-associated accumulation of the highly reactive lipofuscin fluorophore A2E (Lakkaraju et al. 2007). Considering that the retina is constantly subjected to different forms of physical and chemical oxidative stress (Beatty et al. 2000; Liang & Godley 2003), these conditions are likely to promote the spontaneous oxidation of cholesterol into oxysterols. Moreover, there is evidence for the presence of the oxysterol 7-ketocholesterol (7-KC) in lipid deposits in the primate retina (Moreira et al. 2009).

Oxysterols have important biological functions, and several oxysterols have already been demonstrated to be enzymatically synthesized. Oxysterols are involved in the regulation of cholesterol homeostasis and are also produced as intermediates in bile acid synthesis in the liver. They have also emerged as potential modulators of gene expression with specific oxysterol forms functioning as ligands for nuclear receptors (Javitt 2008). However, the excess formation and accumulation of oxysterols can also lead to pathological effects in cells and tissues. For example, oxysterols are known to have pro-inflammatory properties (Vejux & Lizard 2009). Indeed, several reports have suggested a link between accumulation of oxysterols and increased IL-8 production and secretion by RPE, as well as by other non-related retinal cell types (Bai et al. 2005; Erridge et al. 2007; Lemaire-Ewing et al. 2009; Larrayoz et al. 2010). Thus, we aimed at elucidating the molecular mechanisms involved in the regulation of IL-8 production by several oxysterols in RPE cells. In this work, we found that 25-hydroxycholesterol (25-OH) is a potent inducer of IL-8 expression and secretion in ARPE-19 cells, in opposition to 7-KC, 7β-hydroxycholesterol (7β-OH) and cholesterol. We also found that the 25-OH-induced IL-8 production is dependent on the PI3K and ERK pathways, and that the increased production of IL-8 is most likely due to the increased activity of the transcription factors AP-1 and NF-κB.

Material and Methods

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cell culture and treatments

The human RPE cell line ARPE-19 (LGC Promochem, Teddington, UK) was cultured in Ham’s F12/Dulbecco’s modified Eagle’s medium (DMEM) (1:1) supplemented with 10% foetal bovine serum, antibiotics/antimycotics (100 U/ml penicillin, 100 μg/ml streptomycin and 250 ng/ml amphotericin B and GlutaMax (1x)). The media, GlutaMax and antibiotics were purchased from Invitrogen (Carlsbad, CA, USA). Cells were treated with different compounds including 25-hydroxycholesterol (25-OH), 7-ketocholesterol (7-KC), 7β-hydroxycholesterol (7β-OH), cholesterol (Chol), the PI3K inhibitor LY294002, the MEK1/2 inhibitor U0126, the ERK1/2 inhibitor FR180204 and the NF-κB inhibitor Bay 11-7082. The cholesterol-derived compounds were purchased from Sigma-Aldrich (St. Louis, MO, USA), while the kinase inhibitors were purchased from Calbiochem (San Diego, CA, USA). The cholesterol-derived compounds were solubilized in ethanol, and the control of experiments refers to treatment with the vehicle. Ethanol per se had no effect on the obtained results.

Western blot

After the treatments, cells were washed twice in PBS, denatured with 2× Laemmli buffer, boiled at 95°C and sonicated. Whole-cell extracts were resolved by SDS–PAGE and electrophoretically transferred onto PVDF membranes. The membranes were blocked with 5% w/v non-fat milk in TBS-T (20 mm Tris, 150 mm NaCl, 0.2% Tween 20, pH 7.6) and probed for several proteins, using specific primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies. The antibodies used for this work were mouse anti-actin clone C4 1:1000 (Millipore-Chemicon, Billerica, MA, USA), rabbit anti-ERK1/2 1:1000 (Cell Signaling, Beverly, MA, USA), rabbit anti-phospho-ERK1/2 (Thr202/Tyr204) (197G2) 1:1000 (Cell Signaling), rabbit anti-phospho-c-Jun (Ser63) II 1:500 (Cell Signaling), rabbit anti-IκB-α 1:500 (Cell Signaling), rabbit anti-NF-κB p65 (C20) 1:1000 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-Lamin-B (Ab-1) 1:200 (Oncogene, La Jolla, CA, USA), HRP-conjugated secondary goat anti-mouse and goat anti-rabbit 1:10000 (Bio-Rad Laboratories, Hercules, CA, USA). Immunoreactive bands were visualized with an ECL system (enhanced chemioluminescence) (GE Healthcare Bio-Sciences, Uppsala, Sweden).

Nuclear fractionation

Cells were washed twice in PBS and lysed with four packed volumes of Buffer A (10 mm HEPES, 10 mm KCl, 0.1 mm EDTA, 0.4% NP-40, 1 mm DTT, 2 mm PMSF and 1× protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA)) and incubated on ice for 30 min. Cell lysates were centrifuged at 16 000 g for 5 min at 4°C. Nuclear pellets were resuspended in 3.5 packed volumes of Buffer B (20 mm HEPES, 0.4 m NaCl, 1 mm EDTA, 10% Glycerol, 1 mm DTT, 2 mm PMSF and 1× protease inhibitor cocktail), incubated for 1 hr on ice and briefly sonicated. After centrifugation at 16 000 g for 5 min at 4°C, supernatants containing the nuclear proteins were used to perform immunoblots.

MTT cell viability assay

After the treatments, 0.5 mg/ml of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Invitrogen) was added to ARPE-19 cells seeded onto 24-well plates and incubated for 2 hr at 37°C in a cell incubator. Subsequently, supernatants were removed, and the precipitated dye was dissolved in 300 μl 0.04 m HCl (in isopropanol) and quantified at a wavelength of 570 nm, with wavelength correction at 620 nm, using a Biotek Synergy HT spectrophotometer (Biotek, Winoosky, VT, USA).

LDH cell viability assay

ARPE-19 cells seeded onto 24-well plates were treated for 24 hr, after which the medium was recovered and used for LDH activity analysis. 7 μl of each sample was diluted into 36 μl of 9.76 mm pyruvate (dissolved in 81.3 mm Tris–HCl, 203.3 mm NaCl, pH 7.2). Reactions were carried out at 30 °C and initiated by the injection of 177 μl of 0.24 mm NADH (dissolved in 81.3 mm Tris–HCl, 203.3 mm NaCl, pH 7.2). The depletion of NADH was followed during 1 hr at a wavelength of 340 nm using a Biotek Synergy HT spectrophotometer (Biotek), after which LDH activity was quantified.

ELISA

The concentrations of diffusible human IL-8 in the cell culture supernatants were measured by Quantikine enzyme-linked immunosorbent (ELISA) assay kits using specific antibodies directed against human IL-8, according to the manufacturer’s protocol (R&D Systems, Minneapolis, MN, USA). The cell culture medium was replaced by fresh medium before the beginning of the treatments. The IL-8 levels secreted to the culture media are plotted as relative levels in the graphs, assuming the control as the baseline condition.

RT-PCR

Following the treatments, total RNA from ARPE-19 cells was purified according to the manufacturer’s specifications of Qiagen RNeasy mini kit (Qiagen, Valencia, CA, USA) and quantified at 260 nm according to the following formula: (total RNA) = 44 μg/ml × Abs260 nm × dilution factor. Total RNA samples were subsequently treated with RNase-free DNase I (GE Healthcare Bio-Sciences), to avoid genomic DNA contamination. SuperScript II reverse transcriptase (Invitrogen) and random hexadeoxynucleotide primers were used to synthesize the first strand of cDNA. The SYBR Green PCR master mix reagent was used to amplify the cDNA and perform the real-time PCR, according to the manufacturer’s protocol. 18S rRNA was used as the endogenous control for the quantification of IL-8 gene expression. The following sets of primers were used for cDNA amplification of IL-8 and 18S rRNA: forward IL-8 5′-AAACCACC GGAAGGAACCAT-3′; reverse IL-8 5′-CCTTCACACAGAGCTGCAGAA A-3′; forward 18S rRNA 5′-GTCT GCCCTATCAACTTTC; reverse 18S rRNA 5′-TTCCTTGGAT GTGGTA GC-3′. Control reactions, in which no reverse transcription took place, were used to exclude the occurrence of genomic DNA contamination.

Gene-reporter assay

ARPE-19 cells were plated in 12-well plates at 80% confluency and transfected with pAP-1-Luc and pMCS-Luc (kindly provided by Jonathan Ham from UCL, London, UK), using 1 μg of plasmid DNA and 3 μl of Lipofectamine 2000 (Invitrogen) diluted in Opti-MEM I Reduced Serum Medium (Invitrogen), according to the manufacturer’s specifications. Plasmid DNA was purified using the QIAprep Spin Miniprep Kit (Qiagen). The plasmid pAP-1-Luc contains multiple copies of the AP-1 enhancer fused to a TATA-like promoter region upstream to the firefly luciferase gene from Photinus pyralis. The pMCS-Luc vector was used as a negative control for the assay. Twenty-four hours after transfection, cells were treated and assayed for luciferase activity. Briefly, cells were washed twice with PBS and lysed with 100 μl of a lysis buffer containing 8 mm MgCl2, 1 mm DTT, 1 mm EDTA, 15% Glycerol, 1% Triton X-100 (v/v) and 25 mm Tris-phosphate, pH 7.6. The cell lysates were incubated for 30 min on ice and then centrifuged at 16 000 g for 10 min. Protein concentration of the samples was determined through the BCA method, and the concentration of the samples was normalized for the same value. For evaluation of the luciferase activity, an ATP buffer (8 mm MgCl2, 1 mm DTT, 1 mm EDTA, 15% Glycerol, 2 mm ATP, 25 mm Tris-phosphate, pH 7.6) and a 170 μm D-luciferin (Sigma-Aldrich) solution were used, both automatically injected (100 μl of each) by the luminometer injector system into the wells of a white 96-well plate, each one containing 30 μg of protein lysates. Measurements were performed using a Biotek Synergy HT System (Biotek). The tested treatments had no effect on the readout of the assay.

Statistical analysis

Data are reported as the mean ± standard deviation (SD) of at least three independent experiments. Statistical analyses between multiple groups were performed by the one-way analysis of variance test (anova) with the Tukey’s multiple comparison test, using the GraphPad Prism 5.0 software (GraphPad Software, La Jolla, CA, USA). In all cases, p < 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

25-hydroxycholesterol, but not cholesterol, induces an increase in IL-8 secretion in ARPE-19 cells

Oxysterols are known to induce the production of cytokines in several cell types, including ARPE-19 cells. Therefore, we first assessed whether this effect could be reproduced in our experimental model. In addition, we also assessed whether the effect of 25-OH was specific when compared to non-oxidized cholesterol. To investigate this, ARPE-19 cells were incubated with either cholesterol, 25-OH, 7-KC or 7β-OH. The concentrations used were similar to those used in other oxysterol studies (Rodriguez et al. 2004; Lemaire-Ewing et al. 2009; Moreira et al. 2009). To assess the levels of secreted IL-8, the culture medium was collected and analysed by ELISA. As shown in Fig. 1A, treatment of ARPE-19 cells with 25-OH led to a substantial increase in the levels of IL-8 that were secreted into the medium, reaching the concentration of 1560 ± 383.7 pg/ml (as compared to 532 ± 125.3 pg/ml in the control condition). Notably, cholesterol itself does not induce any changes in the secretion of IL-8 (Fig. 1A). Treatment with 7-KC or 7β-OH also showed no effect upon IL-8 secretion (Fig. 1B). In contrast to these results, Larrayoz et al. (2010) showed that in serum-free media, 7-KC is able to induce IL-8 expression. To assess whether this discrepancy could be explained by the presence of serum in the media, ARPE-19 cells were incubated with either 25-OH or 7-KC in the presence or absence of serum, and the levels of IL-8 analysed by ELISA. As shown in Fig. 1C, the presence of serum in the media by itself induces an increase in the release of IL-8. However, treatment with oxysterols induced similar effects on the release of IL-8 regardless of the presence or absence of serum in the media. 7-KC was shown to have no significant effect in the release of IL-8, while 25-OH induced a robust increase in the levels of IL-8. These results indicate that 25-OH specifically affects the release of IL-8 by ARPE-19 cells. Several authors have shown that 25-OH and 7-KC have cytotoxic effects on ARPE-19 cells (Ong et al. 2003; Rodriguez et al. 2004; Larrayoz et al. 2010). Thus, we investigated whether 25-OH and 7-KC induced cytotoxicity in our experimental model. No significant differences were detected in the metabolic activity and viability of cells as assessed by MTT assay (Fig. 1D). Similarly, no significant differences were detected in the release of lactate dehydrogenase between cells treated in the presence of serum; however, there was a significant increase in LDH release in cells treated with 7-KC in the absence of serum in the medium (Fig. 1E).

image

Figure 1.  25-OH increases the expression and secretion of IL-8. (A) ARPE-19 cells were incubated with 25-OH or cholesterol (20 μg/ml) for 24 hr. (B) ARPE-19 cells were incubated with 25-OH, 7-KC or 7β-OH (20 μg/ml) for 24 hr. (C, D and E) ARPE-19 cells were incubated with 25-OH or 7-KC (20 μg/ml) for 24 hr in the presence or absence of serum in the medium. (F) ARPE-19 cells were incubated with 25-OH (20 μg/ml) for 24 hr. The levels of IL-8 secreted to the culture medium were determined by ELISA (A, B and C). Cell viability was assessed by MTT (D) and LDH (E) assays. IL-8 mRNA levels were determined by real-time PCR (F). The results represent the mean ± SD of at least three independent experiments. **p < 0.01 and ***p < 0.001, significantly different from the control condition; ###p < 0.001, significantly different from the no serum control condition (one-way anova with the Tukey’s multiple comparison test).

Download figure to PowerPoint

To assess whether the increase in IL-8 secretion induced by 25-OH reflects an increase in IL-8 transcription, ARPE-19 cells were incubated with 25-OH before being harvested and the levels of IL-8 mRNA were evaluated by real-time PCR. As depicted in Fig. 1F, 25-OH induces a substantial increase in the levels of IL-8 mRNA, suggesting that the increased secretion of IL-8 is due, at least in part, to the upregulation of IL-8 transcription.

25-hydroxycholesterol induces the activation of ERK1/2 in a time-dependent manner

An important pathway for IL-8 induction involves the activation of the MEK/ERK kinase signalling cascade. In addition, it has been previously reported that 25-OH can induce the activation of the MEK/ERK kinase pathway in several cell types (Lemaire-Ewing et al. 2009; Palozza et al. 2010), while others have shown that MEK inhibitors could prevent the 25-OH-dependent induction of IL-8 secretion in ARPE-19 cells (Dugas et al. 2010). Thus, we decided to investigate whether treatment with 25-OH could induce the activation of ERK1/2 in ARPE-19 cells. Cells were treated with 25-OH for different time points and subsequently analysed by Western blot using antibodies directed against ERK1/2 and also antibodies specific to the activated form of the proteins, phospho-ERK1/2. Data presented in Fig. 2A clearly shows an activation of ERK1/2 12 hr after the treatment with 25-OH. Although this activation appears to be more pronounced for the ERK2 isoform, it is also possible to detect the activation of the ERK1 isoform (Fig. 2B). Non-oxidized cholesterol showed no effect on the activation of ERK1/2 (Fig. 2C).

image

Figure 2.  25-OH induces phosphorylation and activation of ERK in a PI3K- and ERK-dependent manner. (A) ARPE-19 cells were incubated with 25-OH (20 μg/ml) for 0, 1, 3, 6 and 12 hr. (B) ARPE-19 cells were incubated with 25-OH (20 μg/ml) for 12 hr. (C) ARPE-19 cells were incubated either in the presence or the absence of 25-OH or cholesterol (20 μg/ml) for 12 hr. (D) ARPE-19 cells were pre-incubated with FR180204 or U0126 (10 μm) for 1 hr and subsequently treated with 25-OH (20 μg/ml) for 12 hr. (E) ARPE-19 cells were pre-incubated with LY294002 (10 μm) for 1 hr and subsequently treated with 25-OH (20 μg/ml) for 12 hr. In all the experiments, the endogenous levels of phosphorylated ERK1/2, total ERK1/2 and actin were detected by Western blot.

Download figure to PowerPoint

Inhibition of MEK or PI3K prevents ERK1/2 activation by 25-hydroxycholesterol

The canonical pathway for ERK1/2 activation requires its prior phosphorylation by MEK. Thus, we investigated whether the ERK1/2 activation induced by 25-OH required MEK activity. As shown in Fig. 2D, treatment of cells with the specific MEK inhibitor U0126 prevented ERK1/2 activation by 25-OH, as revealed by the loss of phosphorylated forms of the protein.

Interestingly, treatment of cells with the specific ERK1/2 inhibitor FR180204 also prevented 25-OH from inducing the activation of ERK1/2 (Fig. 2D), suggesting that the kinase activity of ERK1/2 is required for the accumulation of activated forms of the protein, most likely through autophosphorylation.

The MEK/ERK pathway is not the only signalling pathway capable of inducing the transcription of IL-8. The PI3K/AKT pathway has also been shown to induce the transcription and release of IL-8 (Newcomb et al. 2005; Kim et al. 2006a,b; Fernandes et al. 2009). Furthermore, several reports suggest that there is a crosstalk between the PI3K/AKT and MEK/ERK pathways (Zhuang et al. 2004; Byun et al. 2006). Thus, we also tested whether inhibition of PI3K could influence the activation of ERK1/2 induced by 25-OH. As depicted in Fig. 2E, treatment of cells with the specific PI3K inhibitor LY294002 also prevented the 25-OH-induced activation of ERK1/2. Taken together, these data suggest that the activities of both MEK and PI3K are required to activate ERK1/2 in response to 25-OH treatment in ARPE-19 cells.

25-hydroxycholesterol induces IL-8 secretion and mRNA transcription through a mechanism that requires ERK1/2 and PI3K activity

Having shown that the ERK1/2 activation induced by 25-OH requires the activity of both MEK and PI3K, we decided to investigate whether ERK1/2 and PI3K were required for 25-OH to induce the secretion of IL-8. ARPE-19 cells were first pre-incubated with either ERK1/2 or PI3K inhibitors, followed by a further incubation with 25-OH. The cell medium was then recovered and the levels of released IL-8 evaluated through ELISA. Data presented in Fig. 3 clearly demonstrate that inhibition of either ERK1/2 or PI3K (Fig. 3A) prevented the increase in IL-8 secretion induced by 25-OH treatment.

image

Figure 3.  Expression and secretion of IL-8 induced by 25-OH are prevented by PI3K- and ERK-specific inhibitors. (A) ARPE-19 cells were pre-incubated with LY294002 or FR180204 (10 μm) for 1 hr and subsequently treated with 25-OH (20 μg/ml) for 24 hr. The IL-8 levels secreted to the culture medium were determined by ELISA. (B) ARPE-19 cells were pre-incubated with LY294002 or FR180204 (10 μm) for 1 hr and subsequently treated with 25-OH (20 μg/ml) for 12 hr. The IL-8 mRNA levels were determined by real-time PCR. The results represent the mean ± SD of at least three independent experiments. **p < 0.01 and ***p < 0.001, significantly different from the control condition; #p < 0.05, ##p < 0.01 and ###p < 0.001, significantly different from the 25-OH condition (one-way anova with the Tukey’s multiple comparison test).

Download figure to PowerPoint

To evaluate whether these effects were also reflected in the levels of IL-8 mRNA, cells were treated with either ERK1/2 or PI3K inhibitors, followed by a further incubation with 25-OH. Cells were then harvested, and the levels of IL-8 mRNA evaluated by real-time PCR. Consistent with the results obtained for IL-8 secretion, inhibition of either ERK1/2 or PI3K (Fig. 3B) prevented the increase in IL-8 mRNA transcription induced by 25-OH. Taken together, these data further confirm that 25-OH induced the transcription and secretion of IL-8, through a mechanism that is mediated by ERK1/2 and PI3K.

25-hydroxycholesterol modulates the activation of the transcription factors NF-κB and AP-1

Transcriptional activation of the IL-8 gene can be achieved through the activity of several different transcription factors. Among these, NF-κB and AP-1 appear as major regulators of IL-8 gene transcription (DeForge et al. 1993; Roebuck 1999; Wolf et al. 2001; Kim et al. 2006a,b). Furthermore, both the ERK/MEK and the PI3K/AKT pathways are involved in the modulation of these transcription factors (Defoe & Grindstaff 2004; Steelman et al. 2004, 2008; Yang et al. 2009). Having demonstrated that 25-OH treatment increases IL-8 mRNA levels and that this event can be prevented by inhibiting components of either the MEK/ERK or PI3K/AKT pathways, we attempted to ascertain whether 25-OH was able to induce the activation of these transcription factors.

The regulation of NF-κB-mediated gene expression can occur through several mechanisms. For example, IκB binding to NF-κB sequesters the transcription factor in the cytoplasm, preventing its translocation into the nucleus. Phosphorylation of IκB by IKK induces the proteasomal degradation of the protein, releasing NF-κB and allowing it to translocate to the nucleus where it binds DNA and activates gene transcription. Both the MEK/ERK and PI3K/AKT pathways are capable of activating NF-κB (Zhu et al. 2004). Thus, we investigated whether 25-OH is capable of modulating the activation of NF-κB. ARPE-19 cells incubated with 25-OH were analysed by Western blot using antibodies directed against either IκB or the NF-κB subunit p65. As depicted in Fig. 4A (left panels), treatment with 25-OH induced a decrease in the levels of IκB when compared to control cells. Conversely, 25-OH treatment also induced an increase in the levels of p65. Activated NF-κB is translocated into the nucleus where it activates gene transcription. As such, we also investigated whether 25-OH induced the translocation of p65 into the nucleus. As depicted in Fig. 4A (right panels), treatment of ARPE-19 cells with 25-OH induced an increase in p65 levels in the nuclear fraction of cells treated with 25-OH, suggesting an activation of the NF-κB transcription factor. Taken together, both the decrease in the levels of the inhibitor IκB and the increase in the nuclear levels of the NF-κB subunit p65 suggest that NF-κB activity is likely to increase following 25-OH treatment. To further confirm this, we investigated the role of NF-κB activity on the secretion of IL-8 induced by 25-OH. Cells were pre-incubated with BAY 11-7082, an inhibitor of NF-κB that prevents IκB phosphorylation and subsequent degradation, followed by a further incubation with 25-OH. The cell medium was then collected and the levels of released IL-8 evaluated by ELISA. As shown in Fig. 4B, inhibition of NF-κB activity partially inhibited the increased secretion of IL-8 induced by 25-OH. These results suggest that NF-κB activity is upregulated in the presence of 25-OH and that this transcription factor is, at least, partially involved in IL-8 production upon 25-OH treatment.

image

Figure 4.  25-OH increases activation of both NF-κB and AP-1. (A) ARPE-19 cells were incubated with 25-OH (20 μg/ml) for 24 hr, and the levels of IκB, p65, actin and Lamin-B were determined by Western blot. Left panels: total extracts; right panels: nuclear fraction. (B) ARPE-19 cells were pre-incubated with Bay 11-7082 (5 μm) for 1 hr and subsequently treated with 25-OH (20 μg/ml) for 24 hr. The IL-8 levels secreted to the culture medium were determined by ELISA. (C) ARPE-19 cells were pre-incubated with LY294002 or FR180204 (10 μm) for 1 hr and subsequently treated with 25-OH (20 μg/ml) for 12 hr. Levels of phospho-c-Jun and actin were determined by Western blot. (D) ARPE-19 cells, transiently transfected with the pAP-1 Luc vector, were pre-incubated with LY294002 or FR180204 (10 μm) for 1 hr and subsequently treated with 25-OH (20 μg/ml) for 12 hr. Luciferase activity, which is proportional to AP-1 activation, was monitored. The treatments had no effect on the readout of the luciferase reporter assay, as monitored by transfection of pMCS-Luc. The results represent the mean ± SD of at least three independent experiments. *p < 0.05 and ***p < 0.001, significantly different from the control condition; #p < 0.05 and ###p < 0.001, significantly different from the 25-OH condition (one-way anova with the Tukey’s multiple comparison test).

Download figure to PowerPoint

AP-1 is a dimeric transcription factor comprising proteins from several families, with the Jun and Fos subfamilies being the major components. Among these proteins, c-Jun has been reported to be involved in AP-1-driven transcription of IL-8 (Abdel-Malak et al. 2008). c-Jun is tightly regulated both at the levels of expression and activity. Phosphorylation of c-Jun, a modification that is catalysed by JNK, is required for its transcriptional activity and also to protect the protein from ubiquitination and subsequent proteasomal degradation (Fuchs et al. 1996). Furthermore, ERK1/2 has been reported to modulate c-Jun, both by inducing the expression of c-Jun mRNA and by increasing JNK activity (Lopez-Bergami et al. 2007). Additionally, PI3K may also regulate c-Jun stability through its ability to upregulate the MEK/ERK pathway (Zhuang et al. 2004). Thus, we investigated whether 25-OH could modulate the activation of c-Jun and whether this effect was dependent on ERK1/2 or PI3K activity. Cells were first pre-incubated with either ERK1/2 or PI3K inhibitors, followed by a further incubation with 25-OH. Cell lysates were then analysed by Western blot using antibodies specific to the activated/phosphorylated form of c-Jun. As depicted in Fig. 4C, 25-OH treatment induced the accumulation of phosphorylated c-Jun. Furthermore, inhibition of either ERK1/2 or PI3K prevented the accumulation of phosphorylated c-Jun induced by 25-OH. To confirm that this accumulation of phosphorylated c-Jun is consistent with an increase in AP-1 transcription activity, an AP-1 luciferase gene-reporter assay was conducted. Data presented in Fig. 4D show that treatment with 25-OH induces a substantial increase in the transcriptional activity of AP-1. Concurrently, treatment with either ERK1/2 or PI3K inhibitors reduced 25-OH-induced upregulation of AP-1 transcriptional activity. Taken together, these results suggest that 25-OH modulates the expression and secretion of IL-8, through pathways that are likely to involve activation of both NF-κB and AP-1 activity.

Discussion

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In this work, we found that 25-hydroxycholesterol (25-OH) is a potent inducer of IL-8 secretion in ARPE-19 cells, while 7-KC, 7β-hydroxycholesterol (7β-OH) and cholesterol had no effect on IL-8 secretion by these cells. We also found that the 25-OH-induced IL-8 production is dependent on the PI3K and ERK pathways, as specific inhibitors of these kinases prevent increased production of IL-8 induced by 25-OH. Moreover, increased secretion of IL-8 is accompanied by an increased expression of the IL-8 gene, which is, most likely, due to the increased activity of the transcription factors AP-1 and NF-κB. Consistent with this hypothesis, we observed an increase in the levels of phospho-c-Jun (an AP-1 subunit) and nuclear p65 (an NF-κB subunit), as well as a decrease in the levels of IκB (NF-κB inhibitor), upon treatment with 25-OH.

In most cell types, oxysterols often accumulate as a result of cholesterol metabolism or absorption through the diet (Hodis et al. 1991; Vine et al. 1997; Tomoyori et al. 2002; van Reyk et al. 2006; Otaegui-Arrazola et al. 2010; Steck & Lange 2010). At the retina, RPE cells phagocyte the outer segment of the photoreceptors that contain cholesterol-rich membranes and internalize low-density lipoprotein and oxidized low-density lipoprotein, which contain high levels of oxysterols, from the plasma. During ageing, there is also deposition of cholesterol in drusens, which may provide an additional source of oxysterols in the retina. In addition, the high metabolic rate of the retina and its continuous exposure to light are two important factors that contribute to the generation of a high oxidative environment that can favour the oxidation of cholesterol to oxysterols (Beatty et al. 2000; Liang & Godley 2003; Fernandes et al. 2008). An increasing body of literature has suggested a tight link between the accumulation of oxysterols and the development of an inflammatory response, which appears to be triggered by a variety of cytokines, such as IL-8 (Vejux & Lizard 2009). Our cell culture model consistently showed that oxysterols are indeed able to increase the expression and secretion of IL-8 by ARPE-19 cells. However, this effect seems to be specific to 25-OH, as other oxysterols, such as 7-KC or 7β-OH, had no effect on the induction of IL-8 expression and secretion. These results, however, are not without precedent. Indeed, other works showed that 25-OH is a very potent oxysterol in the induction of IL-8 expression, both in RPE and other non-related cell types, such as macrophages and colon carcinoma cells (Bai et al. 2005; Erridge et al. 2007; Joffre et al. 2007; Dugas et al. 2010). These results can presumably be ascribed to the intrinsic properties of each type of oxysterol. Of significance, it is known that 25-OH is a non-cytotoxic oxysterol (as we also observed), while 7-KC or 7β-OH induce cytotoxic effects on cells, usually triggering cell death processes (Lemaire-Ewing et al. 2009; Dugas et al. 2010).

We also observed that cholesterol oxides, but not cholesterol, induce IL-8 expression and secretion, which may explain the increase in IL-8 levels upon ageing, which is characterized by increased levels of oxidative stress, including accumulation of pro-oxidants, such as the lipofuscin fluorophore A2E (Lakkaraju et al. 2007). Indeed, we have previously shown that A2E-mediated photo-oxidation results in increased expression and secretion of IL-8 by a mechanism dependent on the MAPK pathways (Fernandes et al. 2008).

Another important finding of this work was that 25-OH induces IL-8 expression and secretion by a mechanism dependent on the ERK pathway, which is in accordance with a variety of other studies (Lemaire-Ewing et al. 2009; Dugas et al. 2010). Of great significance is the fact that IL-8 expression and secretion in the presence of 25-OH also appears to be dependent on the PI3K pathway and that 25-OH-induced activation of the ERK pathway is sensitive to PI3K inhibitors. Canonically, it is accepted that PI3K and ERK pathways are virtually independent and parallel pathways that are activated upon common stimuli, sharing similar downstream effectors, such as AP-1 and NF-κB (Defoe & Grindstaff 2004; Steelman et al. 2004, 2008; Yang et al. 2009). However, some studies showed that both pathways may crosstalk, with PI3K having a role on ERK phosphorylation and activation. Indeed, activation of ERK upon specific stimuli is blocked by PI3K inhibitors, such as LY294002 and wortmannin (Zhuang et al. 2004; Byun et al. 2006).

A detailed analysis of the literature indicates that IL-8 expression is mainly regulated by the transcription factors AP-1 and NF-κB (DeForge et al. 1993; Roebuck 1999; Wolf et al. 2001; Kim et al. 2006a,b). Indeed, in this work, we found that 25-OH-induced expression and secretion of IL-8 is likely to be dependent on both of these transcription factors. Data from Larrayoz et al. showed that oxysterols other than 25-OH, such as 7-KC, are able to induce IL-8 expression through NF-κB activation (Larrayoz et al. 2010), while Lemaire-Ewing et al. showed that 7β-OH and 25-OH both induce IL-8 secretion via the AP-1 pathway (Lemaire-Ewing et al. 2009). Significantly, our work showed that the 25-OH-induced secretion of IL-8 is partially prevented by a NF-κB inhibitor and that the 25-OH-dependent increase in the activity of AP-1 was prevented by ERK and PI3K inhibitors, similarly to what happened with IL-8 expression and secretion. Moreover, 25-OH decreased the levels of the NF-κB inhibitor IκB, which presumably would activate NF-κB (as suggested by the increased accumulation of the p65 subunit in the nucleus), and also increased phosphorylation of c-Jun (AP-1), an indicator of increased AP-1 activity, which was also prevented by PI3K and ERK inhibitors. Thus, based on these findings, we propose that NF-κB and AP-1 are most likely the transcription factors involved in the increase in IL-8 expression upon 25-OH treatment, which is consistent with data from other groups (Rydberg et al. 2003; Lemaire-Ewing et al. 2009; Larrayoz et al. 2010). Interestingly, Lemaire-Ewing et al. observed that oxysterols induce expression and secretion of IL-8 through calcium-dependent activation of AP-1 (c-fos) via the ERK signalling pathway in monocytic cells (Lemaire-Ewing et al. 2009). Some oxysterols are known to be incorporated in lipid rafts of the plasma membrane leading to opening of the Trpe1 Ca2 + entry channel (Berthier et al. 2004). This mechanism may contribute for a rise in the intracellular Ca2 + levels, leading to activation of the MAPK ERK1/2 pathway, which subsequently culminates in AP-1 activation. Indeed, calcium channel inhibitors, nifedipine and verapamil, appear to prevent oxysterol-induced IL-8 expression and secretion (Lemaire-Ewing et al. 2009). However, one may not exclude the role of other putative transcription factors and mechanisms on the 25-OH-induced expression of IL-8. A detailed study of this issue may open new avenues for a better understanding of the cellular and molecular mechanisms underlying 25-OH-induced inflammation, which appears to have a critical role on the pathogenesis of AMD and other age-related diseases.

Acknowledgements

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We would like to thank Dr Jonathan Ham (from Molecular Haematology and Cancer Biology Unit, UCL Institute of Child Health, London, UK) for providing the pAP-1-Luc and pMCS-Luc plasmids. We also would like to thank FCT (Fundação para a Ciência e a Tecnologia, Portugal) for providing financial support (grant PTDC/SAU-OSM/67498/2006).

References

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Abdel-Malak NA, Srikant CB, Kristof AS, Magder SA, Di Battista JA & Hussain SN (2008): Angiopoietin-1 promotes endothelial cell proliferation and migration through AP-1-dependent autocrine production of interleukin-8. Blood 111: 41454154.
  • Bai B, Yamamoto K, Sato H, Sugiura H & Tanaka T (2005): Combined effect of 25-hydroxycholesterol and IL-1beta on IL-8 production in human colon carcinoma cell line (Caco-2). Inflammation 29: 141146.
  • Beatty S, Koh H, Phil M, Henson D & Boulton M (2000): The role of oxidative stress in the pathogenesis of age-related macular degeneration. Surv Ophthalmol 45: 115134.
  • Berthier A, Lemaire-Ewing S, Prunet C et al. (2004): Involvement of a calcium-dependent dephosphorylation of BAD associated with the localization of Trpc-1 within lipid rafts in 7-ketocholesterol-induced THP-1 cell apoptosis. Cell Death Differ 11: 897905.
  • Byun HJ, Hong IK, Kim E et al. (2006): A splice variant of CD99 increases motility and MMP-9 expression of human breast cancer cells through the AKT-, ERK-, and JNK-dependent AP-1 activation signaling pathways. J Biol Chem 281: 3483334847.
  • Curcio CA, Presley JB, Malek G, Medeiros NE, Avery DV & Kruth HS (2005): Esterified and unesterified cholesterol in drusen and basal deposits of eyes with age-related maculopathy. Exp Eye Res 81: 731741.
  • Defoe DM & Grindstaff RD (2004): Epidermal growth factor stimulation of RPE cell survival: contribution of phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways. Exp Eye Res 79: 5159.
  • DeForge LE, Preston AM, Takeuchi E, Kenney J, Boxer LA & Remick DG (1993): Regulation of interleukin 8 gene expression by oxidant stress. J Biol Chem 268: 2556825576.
  • Dugas B, Charbonnier S, Baarine M et al. (2010): Effects of oxysterols on cell viability, inflammatory cytokines, VEGF, and reactive oxygen species production on human retinal cells: cytoprotective effects and prevention of VEGF secretion by resveratrol. Eur J Nutr 49: 435446.
  • Erridge C, Webb DJ & Spickett CM (2007): 25-Hydroxycholesterol, 7beta-hydroxycholesterol and 7-ketocholesterol upregulate interleukin-8 expression independently of Toll-like receptor 1, 2, 4 or 6 signalling in human macrophages. Free Radic Res 41: 260266.
  • Fernandes AF, Zhou J, Zhang X, Bian Q, Sparrow J, Taylor A, Pereira P & Shang F (2008): Oxidative inactivation of the proteasome in retinal pigment epithelial cells. A potential link between oxidative stress and up-regulation of interleukin-8. J Biol Chem 283: 2074520753.
  • Fernandes AF, Bian Q, Jiang JK, Thomas CJ, Taylor A, Pereira P & Shang F (2009): Proteasome inactivation promotes p38 mitogen-activated protein kinase-dependent phosphatidylinositol 3-kinase activation and increases interleukin-8 production in retinal pigment epithelial cells. Mol Biol Cell 20: 36903699.
  • Fuchs SY, Dolan L, Davis RJ & Ronai Z (1996): Phosphorylation-dependent targeting of c-Jun ubiquitination by Jun N-kinase. Oncogene 13: 15311535.
  • Gehrs KM, Anderson DH, Johnson LV & Hageman GS (2006): Age-related macular degeneration – emerging pathogenetic and therapeutic concepts. Ann Med 38: 450471.
  • Goverdhan SV, Ennis S, Hannan SR, Madhusudhana KC, Cree AJ, Luff AJ & Lotery AJ (2008): Interleukin-8 promoter polymorphism -251A/T is a risk factor for age-related macular degeneration. Br J Ophthalmol 92: 537540.
  • Hodis HN, Crawford DW & Sevanian A (1991): Cholesterol feeding increases plasma and aortic tissue cholesterol oxide levels in parallel: further evidence for the role of cholesterol oxidation in atherosclerosis. Atherosclerosis 89: 117126.
  • Javitt NB (2008): Oxysterols: novel biologic roles for the 21st century. Steroids 73: 149157.
  • Joffre C, Leclere L, Buteau B et al. (2007): Oxysterols induced inflammation and oxidation in primary porcine retinal pigment epithelial cells. Curr Eye Res 32: 271280.
  • Kanda A, Abecasis G & Swaroop A (2008): Inflammation in the pathogenesis of age-related macular degeneration. Br J Ophthalmol 92: 448450.
  • Kim KS, Rajagopal V, Gonsalves C, Johnson C & Kalra VK (2006a): A novel role of hypoxia-inducible factor in cobalt chloride- and hypoxia-mediated expression of IL-8 chemokine in human endothelial cells. J Immunol 177: 72117224.
  • Kim YM, Reed W, Wu W, Bromberg PA, Graves LM & Samet JM (2006b): Zn2+induced IL-8 expression involves AP-1, JNK, and ERK activities in human airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 290: L1028L1035.
  • Lakkaraju A, Finnemann SC & Rodriguez-Boulan E (2007): The lipofuscin fluorophore A2E perturbs cholesterol metabolism in retinal pigment epithelial cells. Proc Natl Acad Sci USA 104: 1102611031.
  • Larrayoz IM, Huang JD, Lee JW, Pascual I & Rodriguez IR (2010): 7-ketocholesterol-induced inflammation: involvement of multiple kinase signaling pathways via NFkappaB but independently of reactive oxygen species formation. Invest Ophthalmol Vis Sci 51: 49424955.
  • Lemaire-Ewing S, Berthier A, Royer MC et al. (2009): 7beta-Hydroxycholesterol and 25-hydroxycholesterol-induced interleukin-8 secretion involves a calcium-dependent activation of c-fos via the ERK1/2 signaling pathway in THP-1 cells: oxysterols-induced IL-8 secretion is calcium-dependent. Cell Biol Toxicol 25: 127139.
  • Li CM, Chung BH, Presley JB et al. (2005): Lipoprotein-like particles and cholesteryl esters in human Bruch’s membrane: initial characterization. Invest Ophthalmol Vis Sci 46: 25762586.
  • Liang FQ & Godley BF (2003): Oxidative stress-induced mitochondrial DNA damage in human retinal pigment epithelial cells: a possible mechanism for RPE aging and age-related macular degeneration. Exp Eye Res 76: 397403.
  • Lopez-Bergami P, Huang C, Goydos JS et al. (2007): Rewired ERK-JNK signaling pathways in melanoma. Cancer Cell 11: 447460.
  • Malek G, Li CM, Guidry C, Medeiros NE & Curcio CA (2003): Apolipoprotein B in cholesterol-containing drusen and basal deposits of human eyes with age-related maculopathy. Am J Pathol 162: 413425.
  • Moreira EF, Larrayoz IM, Lee JW & Rodriguez IR (2009): 7-Ketocholesterol is present in lipid deposits in the primate retina: potential implication in the induction of VEGF and CNV formation. Invest Ophthalmol Vis Sci 50: 523532.
  • Newcomb DC, Sajjan U, Nanua S, Jia Y, Goldsmith AM, Bentley JK & Hershenson MB (2005): Phosphatidylinositol 3-kinase is required for rhinovirus-induced airway epithelial cell interleukin-8 expression. J Biol Chem 280: 3695236961.
  • Ong JM, Aoki AM, Seigel GM, Sacerio I, Castellon R, Nesburn AB & Kenney MC (2003): Oxysterol-induced toxicity in R28 and ARPE-19 cells. Neurochem Res 28: 883891.
  • Otaegui-Arrazola A, Menendez-Carreno M, Ansorena D & Astiasaran I (2010): Oxysterols: a world to explore. Food Chem Toxicol 48: 32893303.
  • Palozza P, Simone R, Catalano A et al. (2010): Lycopene prevention of oxysterol-induced proinflammatory cytokine cascade in human macrophages: inhibition of NF-kappaB nuclear binding and increase in PPARgamma expression. J Nutr Biochem 22: 259268.
  • Rattner A & Nathans J (2006): Macular degeneration: recent advances and therapeutic opportunities. Nat Rev Neurosci 7: 860872.
  • van Reyk DM, Brown AJ, Hult’en LM, Dean RT & Jessup W (2006): Oxysterols in biological systems: sources, metabolism and pathophysiological relevance. Redox Rep 11: 255262.
  • Rodriguez IR, Alam S & Lee JW (2004): Cytotoxicity of oxidized low-density lipoprotein in cultured RPE cells is dependent on the formation of 7-ketocholesterol. Invest Ophthalmol Vis Sci 45: 28302837.
  • Roebuck KA (1999): Regulation of interleukin-8 gene expression. J Interferon Cytokine Res 19: 429438.
  • Roh MI, Kim HS, Song JH, Lim JB, Koh HJ & Kwon OW (2009a): Concentration of cytokines in the aqueous humor of patients with naive, recurrent and regressed CNV associated with amd after bevacizumab treatment. Retina 29: 523529.
  • Roh MI, Kim HS, Song JH, Lim JB & Kwon OW (2009b): Effect of intravitreal bevacizumab injection on aqueous humor cytokine levels in clinically significant macular edema. Ophthalmology 116: 8086.
  • Rydberg EK, Salomonsson L, Hulten LM, Noren K, Bondjers G, Wiklund O, Bjornheden T & Ohlsson BG (2003): Hypoxia increases 25-hydroxycholesterol-induced interleukin-8 protein secretion in human macrophages. Atherosclerosis 170: 245252.
  • Steck TL & Lange Y (2010): Cell cholesterol homeostasis: mediation by active cholesterol. Trends Cell Biol 20: 680687.
  • Steelman LS, Pohnert SC, Shelton JG, Franklin RA, Bertrand FE & McCubrey JA (2004): JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in cell cycle progression and leukemogenesis. Leukemia 18: 189218.
  • Steelman LS, Abrams SL, Whelan J et al. (2008): Contributions of the Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT pathways to leukemia. Leukemia 22: 686707.
  • Tomoyori H, Carvajal O, Nakayama M, Kishi T, Sato M, Ikeda I & Imaizumi K (2002): Lymphatic transport of dietary cholesterol oxidation products, cholesterol and triacylglycerols in rats. Biosci Biotechnol Biochem 66: 828834.
  • Vejux A & Lizard G (2009): Cytotoxic effects of oxysterols associated with human diseases: induction of cell death (apoptosis and/or oncosis), oxidative and inflammatory activities, and phospholipidosis. Mol Aspects Med 30: 153170.
  • Vine DF, Croft KD, Beilin LJ & Mamo JC (1997): Absorption of dietary cholesterol oxidation products and incorporation into rat lymph chylomicrons. Lipids 32: 887893.
  • Wolf JS, Chen Z, Dong G et al. (2001): IL (interleukin)-1alpha promotes nuclear factor-kappaB and AP-1-induced IL-8 expression, cell survival, and proliferation in head and neck squamous cell carcinomas. Clin Cancer Res 7: 18121820.
  • Yang XM, Wang YS, Zhang J et al. (2009): Role of PI3K/Akt and MEK/ERK in mediating hypoxia-induced expression of HIF-1alpha and VEGF in laser-induced rat choroidal neovascularization. Invest Ophthalmol Vis Sci 50: 18731879.
  • Zhu Y, Culmsee C, Klumpp S & Krieglstein J (2004): Neuroprotection by transforming growth factor-beta1 involves activation of nuclear factor-kappaB through phosphatidylinositol-3-OH kinase/Akt and mitogen-activated protein kinase-extracellular-signal regulated kinase1,2 signaling pathways. Neuroscience 123: 897906.
  • Zhuang ZY, Xu H, Clapham DE & Ji RR (2004): Phosphatidylinositol 3-kinase activates ERK in primary sensory neurons and mediates inflammatory heat hyperalgesia through TRPV1 sensitization. J Neurosci 24: 83008309.