Titanium dioxide nanoparticles induced activation of extracellular signal-regulated kinases signaling pathway in human colonic epithelial Caco-2 cells is mediated by epidermal growth factor receptor

Titanium dioxide (TiO2) is one of the most applied nanomaterial and widely used in different food and non-food products as additive or coating material. In vitro and in vivo studies have shown that TiO2 nanoparticles (NPs) induce cytotoxicity and inflammation in Caco-2 cells, but the detailed mechanisms how TiO2 NPs interact with the cell and mediate their inflammatory potential remains unclear. Our recent studies on Caco-2 cells have shown that exposure to TiO2 NPs induce an inflammatory response via an epidermal growth factor receptor (EGFR)-mediated pathway. In the current study we demonstrate that 10 nm TiO2 NPs induce the activation of extracellular signal-regulated kinases (ERK) and the expression of ERK target genes Chemokine (C-C motif) ligand 2 (CCL2) and Chemokine (C-X-C motif) ligand 3 (CXCL3). This activation is dependent on EGFR, as treatment of EGFR kinase inhibitor BIBX 1382 attenuates phosphorylation of ERK1/2 and the mRNA expression of CCL2 and CXCL3. Moreover, TiO2 NPs induce the activation of the transcription factor ELK1. We thus suggest TiO2 NPs induce an EGFR/ ERK/ELK1-mediated pathway.


Introduction
Titanium dioxide (TiO 2 ) is one of the most applied nanomaterial and widely used in the food and non-food industry as additive or coating material [1]. TiO 2 nanoparticles (NPs) are characterize by its high refractive index, photo catalytic activity and stability and thus used as white pigment in cosmetics, personal care products, paints, coatings, or in food and pharmaceuticals as additive E171. Additionally, TiO 2 NPs are used in water and air purification or medical applications because of its photo catalytic and antibacterial attributes [2,3]. The growing production and wide range of applications of TiO 2 NPs will cause an increasing release into the environment and is assumed to raise the exposure especially to the gastrointestinal tract [4]. Based on current contents in food and personal care products Weir and colleagues could estimate an exposure to TiO 2 NPs of approximately 1 mg/kg*day in adults and up to 2−3 mg/kg*day in children [2].
Because of their ability to induce inflammation, cytotoxicity and genotoxicity in different cell types in vitro and in vivo [5] the effect of TiO 2 NPs on gut homeostasis is a major concern [6] . Studies using the human colon carcinoma cell line Caco-2 have shown that TiO 2 NPs induced oxidative damage [7] influenced metabolic activity and cytotoxicity [8,9] produced reactive oxygen species (ROS) [10,11] and finally induced the expression of Interleukin 8 (IL8) [12] in part, through the activations of nuclear factor (NF)-κB and p38 mitogen activated protein kinase (MAPK) pathways [13] . Little is known about the detailed mechanisms how TiO 2 NPs interact with the cell and mediate their inflammatory potential. Several studies discuss a possible activation of toll-like receptors (TLR) [14] or suggest an endocytic uptake [15][16][17][18] since NPs have been detected intracellular [19][20][21].
Moreover, our recent findings have shown that cellular response to TiO 2 NPs in Caco-2 cells are dependent on the activation of epidermal growth factor receptor (EGFR), mediated by endocytosis-related structures [12]. The underlying precise signaling cascade between EGFR activation and expression of different inflammatory markers in intestinal epithelial cells remains unknown. Studies on various cell lines declare effects to TiO 2 NPs on signaling pathways such as p38 MAPK and extracellular signal-regulated kinases 1/2 (Erk 1/2) in neutrophils [22]. c-Jun N-terminal kinases (JNK)/p38-caspase-8-Bid pathway in lymphocytes [23] . NF-κB pathway in kidney cells of mice [24] apoptotic pathways in bronchial epithelial cells via activation of caspase-8 and -3, -7 [25] and mitochondrial-mediated pathways in mouse splenocytes [26]. In macrophage-like cells TiO 2 NPs response is depended on active cathepsin B [27] and in breast, lung and liver cells by Fas upregulation and Bax activation [28] . Induction of these pathways and expression of downstream genes such as IL8 or IL-1β, are initiated by the generation of ROS or via binding of TiO 2 NPs to TLR4 at the cell membrane and further activation of NF-κB [14,29]. Besides these findings, studies using different types of NPs have shown an activation of the ERK signaling pathway [30,31] with a previous activation of EGFR in epithelial cells [32][33][34][35][36].
So far, there has been no evidence for TiO 2 NP-induced inflammatory response through EGFR activation and further induction of ERK signaling pathway and downstream mediators in Caco-2 cells. In the current study, Caco-2 cells were exposed to 10 nm TiO 2 NPs which induce a transient activation of ERK1/2 and the ERK-related transcription factor ELK1. This activation was accompanied with an increased expression of Chemokine (C-C motif) ligand 2 (CCL2) and Chemokine (C-X-C motif) ligand 3 (CXCL3) genes. We could further demonstrate that TiO 2 NP-induced ERK1/2 signaling is mediated through EGFR activation and increased after IκB-α inhibition.

Preparation of TiO 2 NPs
The preparation and characterization of the used TiO 2 NPs were described in our previous study [13]. Briefly, titanium (IV) dioxide (anatase) particles were obtained from Alfa Aesar (Alfa Aesar GmbH & Co KG, Germany). Particles were used with a nominal size of 10 nm (Stock Number 44690, Lot B19T020; specific surface area 120 m 2 g -1 ) and a measured size of 14.4 nm [13]. The particles form agglomerates in a size of 673 × 455 nm [13]. Stock solution was prepared with deionized water to a final concentration of 2 mg ml -1 . The dispersion was sonicated at 23 KHZ and 150 W (MSE Ltd, United Kingdom) for two minutes and finally autoclaved. Immediately before treatment of the cells, the dispersion was diluted in cell culture medium as indicated below.

Western blot analyses
Caco-2 cells were seeded in 6-well plates (8.87 cm -2 per well) (Sarstedt, Germany) at a density of 1-2×10E5 cells cm -2 . At confluency, prepared medium with 10 nm TiO 2 NPs were added to the cells at a final concentration of 40 μg cm -2 cell growth surface or remain untreated. After indicated time cells were washed twice with cold PBS and lysed 20 min on ice in RIPA buffer (Sigma-Aldrich, U.S.) added with inhibitors (1 mM PMSF, 1 mM Na 3 VO 4 , 1x complete protease inhibitor cocktail (Santa Cruz Biotechnology, U.S.), 1x phosphatase inhibitor cocktail 3 (Sigma-Aldrich, U.S.)). Lysates were clarified by centrifugation (12.000 g for 6 min) and protein concentration was measured using bicinchoninic acid assay (BCA) reagent. 40 μg protein from each preparation were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Carl Roth GmbH + Co.KG, Germany). Membranes were blocked in 5% (w/v) BSA/TBST (20 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% (v/v) Tween 20) for 1 h at room temperature. Primary antibody incubation p-ERK1/2 1:200 (sc-7383, Santa Cruz Biotechnology, U.S.) in 5% BSA/TBST took place at 4°C overnight, followed by secondary antibody incubation 1:500 in TBST (anti-mouse HRP (PAB10782, Abnova, Taiwan)) for 2 h at room temperature. The antibody was detected with enhanced chemiluminescence (ECL) reagent (MBL International Corporation, U.S.) and visualized with Fusion solo S (Vilber Lourmat Deutschland GmbH, Germany). Densitometry of the bands was analyzed using FusionCapt Advance Solo 4s software and the ratio of p-ERK to total protein, detected by ponceau staining, determined. Expressions of the treated samples were set in relation to the untreated control sample. Experiments were performed at least three times.

Electrophoretic mobility shift assay (Emsa)
Binding of ELK1 was investigated by non-radioactive EMSA using LightShift EMSA Optimization and Control Kit (Thermo Fisher Scientific Inc., U.S.) according to manufacture's protocol. In detail, Caco-2 cells were seeded in 6-well plates (Sarstedt AG & Co, Germany) at a density of 1-2×10E5 cells cm -2 . At confluency, cells were stimulated with 10 nm TiO 2 NPs at a final concentration of 40 μg cm -2 cell growth surface or remain unstimulated. After indicated time cells were washed twice with ice cold PBS and lysed 20 min on ice in lysis buffer (10 mM HEPES, 1.5 mM MgCl 2, 10 mM KCl, 1 mM DTT, 1 mM PMSF, 1x complete protease inhibitor cocktail (Santa Cruz Biotechnology, U.S.)) adapted from [37]. Cells were scraped, placed in a microlitre tube and centrifuged 20 min at 11.000 g at 4°C to separate cytosolic and nuclear fractions. Supernatant was decanted and pellet was suspended in extraction buffer to lyse nuclei (20 mM HEPES, 1.5 mM MgCl 2 , 0.42 M NaCl, 0.2 mM EDTA, 25 % (v/v) glycerol, 1 mM DTT, 1 mM PMSF, 1x complete protease inhibitor cocktail, [37]. 2 µg of nucleus extraction were used for binding reaction with biotin-labeled DNA for ELK1: 5'-TTTGCAAAATGCAGGAATTGTTTTCACAGT-3' [38]. Samples were separated by 6 % native polyacrylamide gel and transferred to a nylon membrane (GE Healthcare, U.K.). Biotin were detected with peroxidase-coupled streptavidin and ECL (Thermo Fisher Scientific Inc., U.S.) and visualized and quantified with Fusion solo S (Vilber Lourmat Deutschland GmbH, Germany). Densitometry of the binding complex was analyzed using FusionCapt Advance Solo 4s software and expressions of the treated samples were set in relation to the untreated control sample. Experiments were performed at least three times.

Immunocytochemistry
Caco-2 cells were seeded in removable 12-well chambers (ibidi GmbH, Germany) at a density of 1-2×10E5 cells cm -2 . At confluency prepared medium with 10 nm TiO 2 NPs were added to the cells at a final concentration of 40 μg cm -2 cell growth surface or remain untreated. After indicated incubation time cells were washed with PBS and fixed with cold 4 % paraformaldehyde in PBS for 30 min. After washing, cells were permeabilized with 0.1% Trion X-100 in PBS for 10 min and blocked with 10% donkey serum in PBS for 1 h. Primary antibody p-ERK1/2 (sc-7383, Santa Cruz Biotechnology, U.S.) was diluted 1:100 in 10% donkey serum in PBS and cells were incubated overnight at 4°C. Following PBS wash, cells were incubated 1 h at RT with secondary antibody Alexa Fluor ® 546 (A10036, Molecular Probes ® , U.S.) 1:200 in 10% donkey serum in PBS. After washing with PBS cells were mounted with Dapi-Fluoromount-G™ clear mounting media (Southern Biotech™, U.S.). Pictures were acquired with a confocal laser scanning microscope TCS SP8 (Leica Microsystems GmbH, Germany).

m RNA expression analysis
For mRNA expression analysis cells were seeded in 24-well or 6-well culture plates (Sarstedt AG & Co., Germany) at a density of 1-2×10E5 cells cm -2 . After reaching confluency, 10 nm TiO 2 particles were added at a final concentration of 40 μg cm -2 cell growth surface or remain untreated. At indicated time points cells were washed twice with PBS and proceed to RNA extraction. RNA was extracted using the GeneJET RNA Purification Kit (Thermo Fisher Scientific GmbH, Germany) and first strand cDNA synthesis was prepared using RevertAid™ H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific GmbH, Germany) as described by the manufacturer. Real-time PCR was performed on a 7500 Real-Time PCR Systems (Life Technologies Inc., U.S.) using HOT FIREPol ® EvaGreen ® qPCR Mix Plus ROX (Solis BioDyne, Estonia). Sequences of primers (used at 0.2 µM) were as follows: B2M (Beta-2 microglobulin) forward: GCAAGGACTGGTCTTTCTATCT, reverse: TAACTATCTTGGGCTGTGACAAA; CCL2 (Chemokine (C-C motif) ligand 2) forward: CCCAAAGAAGCTGTGATCTTCA; reverse: TCTGGGGAAAGCTAGGGGAA, CXCL3 (chemokine (C-X-C motif) ligand 3) forward: CCCAAACCGAAGTCATAGCCA, reverse: ACCCTGCAGGAAGTGTCAA; GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) forward: AGAGCACAAGAGGAAGAGAGAG, reverse: GGTTGAGCACAGGGTACTTTATT. Parameters for qPCR were: 95°C for 15 min, 40 cycles of 10 s 95°C, 30 s 60°C and 30 s 72°C. After cycling, melting curve analysis was performed. Expressions of the different genes was normalized to the expressions of GAPDH and B2M and compared to control using delta delta C t method [39]. Experiments were performed four times.

Cells treatment
Inhibitor of IκB-a phosphorylation Bay11-7082 (InvivoGene, USA), inhibitor of p38α and p38β MAPK SB202190 (InvivoGene, USA) and inhibitor of EGFR kinases BIBX 1382 (Santa Cruz Biotechnology, Inc., U.S.) were dissolved in DMSO to obtain a stock solution at 50 mM. Epidermal growth factor (EGF) at a concentration of 100 ng ml -1 was used as positive control in the EGFR/ERK signaling pathway as described [40]. Confluent monolayer of Caco-2 cells were incubated for 1 h with 50 µM BIBX 1382, 5 µM SB202190 or 30 min with 50 µM Bay11-7082 and subsequently stimulated with NPs, EGF or left unstimulated. After treatment for indicated time, cells were washed twice with cold PBS and processed as described above. Results of the treated cells are presented as fold change to untreated cells.

Data analysis
Results are presented as means ± standard error (SEM). Treated samples are presented as fold change to untreated controls. Statistical analysis was carried out using GraphPad Prism 4 software (GraphPad Software, Inc., U.S.) and comparisons of the means were performed by ANOVA followed by Tukey's multiple comparison test. Differences were considered to be statistically significant at p<0.05.

Results and discussion
Nanosized TiO 2 particles and their impacts on in vivo and in vitro systems are currently researched intensively in biomedical studies. There are different effects described on cellular level in various cell types. TiO 2 NPs shown an impact on oxidative stress, inflammation or apoptosis [5], but the precise cellular mechanisms are still unknown. Several studies discuss a possible activation of toll-like receptors (TLR) [14,29] or suggest an endocytic uptake [15][16][17][18] since NPs have been detected intracellular [19][20][21]. Moreover, our recent findings have shown that cellular response to TiO 2 NPs in Caco-2 cells are dependent on the activation of epidermal growth factor receptor (EGFR), mediated by endocytic-related structures [13]. Activation of EGFR initiates the induction of downstream signaling pathways, including ERK, JNK and p38 MAPK pathways [41,42]. The present study attempted to provide an insight of the signaling pathway responsible for the NP-induced inflammatory response.

TiO 2 NPs activate ERK1/2 kinases
ERK is part of the Ras/Raf/MEK/ERK signal transduction cascade, which is activated following stimulation by various extracellular factors besides EGF like insulin, other growth factors, osmotic stress, cytokines or trans membrane G-protein coupled receptors [43]. To investigate the role of ERK in the NP-induced inflammatory response, p-ERK expression was determined by western blot analyses and immunocytochemie. As shown in Figure 1 expression of p-ERK1/2 in Caco-2 cells was induced after treatment with 10 nm TiO 2 NPs compared to untreated control at any time point (Figure 1A to F). Western blot analyses revealed exposure to 10 nm TiO 2 induce a transient activation of p-ERK1/2 (Figure 2A). Levels of p-ERK1/2 consequently increased in a time dependent manner until 60 min after NPs exposure and is significantly induced at 60 min by 10.2±3.1 compared to untreated control cells ( Figure 2B). Activation of ERK1/2 faded 90 min after NPs exposure. It was shown EGF treatment increase  the levels of p-ERK1/2 peaking at 2, 5 and 10 min [44]. Therefore the observed induction of ERK1/2 by NPs is slightly slower than with EGF. If the NP-induced ERK1/2 activation resulted from an activation of EGFR or another mechanism is not known. To go more into detail in this issue we further used inhibitors which intervene the ERK and NF-κB pathways.

Activation of EKR1/2 is dependent on EGFR
To analyses signal transduction of the NP-induced inflammatory response, we used three different inhibitors after stimulation with 10 nm TiO 2 and EGF. In our recent studies we have shown that NPinduced inflammatory response was dependent on EGFR, NF-κB and p38 MAPK activation [13].
It is known activation of EGFR leads to the phosphorylation of ERK1/2 [41]. As shown by western blot analysis p-EKR1/2 was induced 30 min after NPs and EGF stimulation by factor 2.5 ± 0.4 and 4.2 ± 0.9 compared to untreated control cells ( Figure 3A). Densitometric analysis of western blots revealed that treatment with BIBX1382 (EGFR inhibitor) lead to a decreased p-ERK1/2 by 68 % in NP-treated cells and by 83 % in EGF-treated cells compared to the stimulated specimens without inhibitor treatment ( Figure 3B). These results shown that NPinduced activation of ERK1/2 is dependent on activation of EGFR. Study with ultrafine carbon black particles support our observations, which shown a transactivation of EGFR and further stimulation of ERK in human bronchial epithelial cells [45].
As published earlier, the NP-induced inflammatory response can be inhibited by BAY11-7082 [13]. Using the same inhibitor at identical concentration, which inhibited the phosphorylation of IκB-α, we shown that p-ERK1/2 is elevated in comparison to untreated cells. Inactivation of IκB-α phosphorylation by BAY11-7082 resulted in a significant increase of p-ERK1/2 in NP2-and EGF-treated specimens by factor 5.7 ± 2.3 and 5.5 ± 1.0, respectively ( Figure 3A and 3B). Thus manipulation of the NF-κB pathway confound ERK signaling through a possible feedback mechanism. Different studies already demonstrated interactions between NF-κB and ERK signaling pathway [46][47][48]. Having shown p38 MAPK is also involved in NPinduced inflammatory response [13], we analyze whether p38 MAPK is required for NP-induced ERK activation. Treatment with p38α and p38β MAPK inhibitor SB202190 did not show an influence on NP-induced activation of ERK1/2 ( Figure 3A and 3B). Activation of p38 MAPK is therefore not required for the NP-induced activation of ERK1/2, but is involved in the NP-induced expression of IL8 mRNA as shown in our previous study [13]. Thus, results demonstrate exposure to 10 nm TiO 2 NPs induce the activation of both p38 MAPK and NF-κB/ERK pathways. If this activation is responsible for the induction of ERK-related gene expression was further investigated.

TiO 2 NPs induced binding of ELK1
The best-studied nuclear target of phosphorylated ERK is the transcription factor ELK1 [28] Stimulation with 10 nm TiO 2 increased the binding of ELK1 to ELK1-specific biotin-labeled oligos up to 45 min after TiO 2 exposure ( Figure 4A) and declined 60 min after NPs treatment. Densitometric analyses revealed a 1.8 ± 0.5-fold increase of the binding complex compared to untreated control. This time course and amount is typical for activated ELK1 [49].

NP-induced activation of EGFR/ERK/ELK1 signaling pathway is related to the expression of CCL2 and CXCL3
It has been shown that activation of ERK1/2 induce the expression of CCL2 and CXCL3 mRNA [50]. Expression analysis revealed an increase by factor 11.0 ± 2.6 and 12.6 ± 5.3 3 h after NPs exposure of CCL2 and CXCL3 mRNA, respectively ( Figure 5A and B). Preincubation with EGFR kinase inhibitor BIBX 1382 decreased the NPinduced expression by 77 % and by 70 % of CCL2 and CXCL3 mRNA. 6 h after NPs treatment mRNA expression of CCL2 and CXCL3 was induced by 4.8 ± 0.4-fold and by 3.3 ± 0.5-fold, respectively. Treatment with BIBX 1382 resulted in a 54% and 58% reduction of NP-induced expression of these genes. The results demonstrate that NP-induced expression of ERK target genes is dependent on the activation of Caco-2 cells were pre-treated with 50 µM BIBX 1382, 5 µM SB202190 or 50 µM Bay11-7082 and exposed to 10 nm TiO 2 (co<ncentration of 40 µg cm -2 of the cell growth surface), 100 ng ml -1 EGF for 30 min or remained untreated (w/o inhibitor). A -Phosphorylated ERK1/2 was evaluated by western blot. B -Densitometric analyses of the treated samples are presented as fold change to the untreated control sample. Expression of p-ERK1/2 was normalized to total protein expression. Shown are representative western blots of three independent experiments. Data presented the mean ± SEM. Two-way ANOVA followed by Tukey's multiple comparison test (p<0.05). Asterisks (*) represent significant differences to the untreated control w/o inhibitor, plus (+) represent significant differences to NP2-or EGF-treated cells w/o inhibitor, respectively. EGFR. It was shown that the expression of CXCL3 and CCL2 mRNA was increased after different inflammatory stimuli and was part of an inflammatory response, mediated by EGFR [51,52]. EGFR-dependent ERK1/2 activation and downstream signaling shown regulatory effects on cellular transformation, intestinal cell migration and wound healing [50,[53][54][55]. Moreover, Unfried et al. demonstrated that carbon NPs induce proliferation via ERK1/2 signaling in lung epithelial cells [56]. This activation was dependent on EGFR. If the TiO 2 NP-induced activation of these pathways in Caco-2 cells promote cell migration and transformation remains unknown. Further experiments are needed to clarify this.

Conclusion
We demonstrated that exposure to 10 nm TiO 2 NPs induced the activation of ERK1/2 and the expression of ERK1/2 target genes Chemokine (C-C motif) ligand 2 (CCL2) and Chemokine (C-X-C motif) ligand 3 (CXCL3). This cellular response to TiO 2 NPs is dependent on the activation EGFR. Furthermore, TiO 2 NPs induce the activation of the transcription factor ELK1 and thus suggest an EGFR/ ERK/ELK1 signaling pathway. IκB-α was shown to interact in the NPinduced ERK1/2 signaling cascade as treatment with inhibitor BAY11-7082 increase the NP-induced phosphorylation of ERK1/2.

Declaration of interest
The authors declare that they have no competing interests.