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Am J Physiol Lung Cell Mol Physiol 294: L358-L367, 2008. First published December 14, 2007; doi:10.1152/ajplung.00323.2007
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Carbon nanoparticle-induced lung epithelial cell proliferation is mediated by receptor-dependent Akt activation

Klaus Unfried, Ulrich Sydlik, Katrin Bierhals, Alexander Weissenberg, and Josef Abel

Toxicology Group, Institut für Umweltmedizinische Forschung, Düsseldorf, Germany

Submitted 14 August 2007 ; accepted in final form 12 December 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Treatment of lung epithelial cells with different kinds of nano-sized particles leads to cell proliferation. Because bigger particles fail to induce this reaction, it is suggested that the special surface properties, due to the extremely small size of these kinds of materials, is the common principle responsible for this specific cell reaction. Here the activation of the protein kinase B (Akt) signaling cascade by carbon nanoparticles was investigated with regard to its relevance for proliferation. Kinetics and dose-response experiments demonstrated that Akt is specifically activated by nanoparticulate carbon particles in rat alveolar type II epithelial cells as well as in human bronchial epithelial cells. This pathway appeared to be dependent on epidermal growth factor receptor and β1-integrins. The activation of Akt by these receptors is known to be a feature of adhesion-dependent signaling. However, intracellular proteins described in this context (focal adhesion kinase pp125FAK and integrin-linked kinase) were not activated, indicating a specific signaling mechanism. Inhibitor studies demonstrate that nanoparticle-induced proliferation is mediated by phosphoinositide 3-kinases and Akt. Moreover, overexpression of mutant Akt, as well as pretreatment with an Akt inhibitor, reduced nanoparticle-specific ERK1/2 phosphorylation, which is decisive for nanoparticle-induced proliferation. With this report, we describe the activation of a pathway by carbon nanoparticles that was so far known to be triggered by ligand receptor binding or on cell adhesion to extracellular matrix proteins.

nanoparticles; carbon black; protein kinase B; integrins; epidermal growth factor receptor

THE SPECIFIC CHEMICAL AND physical properties of nanoparticulate materials are currently used for technological development in modern nanotechnology. However, apart from potential beneficial effects for humans (e.g., in nanomedicine), it is not clear whether intended or unintended exposure of humans to these nanoparticles induces adverse health effects (23). The first indications of such effects came from studies investigating ultrafine particles, such as combustion-derived nanoparticles, which are constituents of ambient airborne particulate matter (24). Here epidemiological as well as experimental studies give evidence that lung inflammation, fibrosis, lung cancer, and systemic cardiovascular diseases are correlated with exposure to these particles (12). It is generally accepted that the special size and surface properties are the driving features leading to these effects. One crucial prerequisite for the estimation of the possible hazard coming from these materials is the understanding of the molecular mechanisms by which nanoparticles induce cellular reactions (32). Within this approach, the activation of nanoparticle-specific signaling cascades leading to diverse cellular outcomes are of particular interest.

Previously, we (28) observed that treatment with two types of nanoparticles, carbon particles [nanoparticulate carbon black, (NPCB; Printex 90)] as well as silica particles (amorphous SiO2), both with a median diameter of 14 nm, induced proliferation in a rat lung epithelial cell line. Investigating signaling events relevant for this specific outcome, we were able to demonstrate that the membrane receptors epidermal growth factor receptor (EGF-R) and β1-integrin are involved in particle-induced activation of the MAPKs ERK1/2 in response to nanoparticle treatment. Respective inhibitor studies demonstrated the specificity of this signaling cascade for nanoparticle-induced proliferation.

Protein kinase B (Akt) is a serine/threonine protein kinase that has been identified as a relevant regulator of essential cell functions, such as cell survival, proliferation, motility, and expression of proinflammatory cytokines (1). It is a key element of a signaling pathway that is strictly dependent on phosphoinositide 3-kinases (PI3Ks), a group of enzymes that are located intracellularly at the cell membrane (34). On activation by receptor tyrosine kinases or G protein-coupled receptors, PI3K generates the lipid mediator phosphatidylinositol 3-phosphate (PIP3). Subsequent binding of PIP3 to Akt and to additional activating proteins results in the activation of Akt by sequential phosphorylation of two specific amino acid sites (21). Whereas phosphorylation of Thr308 by PIP3-binding phosphoinositide-dependent kinase 1 (PDK1) leads to a partial activation of Akt, a second phosphorylation at Ser473 that can be accomplished by several proteins is required for full kinase activity. Akt recognizes a variety of substrates for phosphorylation. Glucose synthase kinase-3 (GSK-3) is the best-known downstream signaling protein that is active in resting cells and is inactivated by Akt (11). In addition, proteins with an impact on cell-cycle regulation as well as on protection from apoptosis, like the proapoptotic mediator Bad or forkhead box family members, are also accepted as a substrate for Akt kinase activity (7, 34). Thus, most actions mediated by these pathways result in the downregulation of proapoptotic cell reactions and the G1/S transition of the cell cycle.

In studies investigating cell adhesion-dependent growth, Akt has been described to be relevant in the suppression of anoikis, a special form of apoptosis induced by cell detachment from extracellular matrix (ECM) proteins (15). In this context, integrin receptors play a central role in outside-in signaling leading to Akt activation (20). There is some evidence that EGF-R can be activated by integrins in fibroblasts on adhesion to ECM proteins (22). Besides integrin-dependent phosphorylation of EGF-R, a physical interaction between EGF-R and integrins was demonstrated. Further studies of epithelial cells showed that the interaction of these two receptors has a modulatory influence on the cell cycle via ERK1/2 and Akt pathways (6).

As possible intracellular mediators of this transmembrane signaling, kinases located in the focal contact are discussed. Focal adhesion kinase pp125FAK (FAK), together with members of the Src family kinases, seems to trigger Ser473 phosphorylation of Akt (27). An additional possible mediator of Akt activation is integrin-linked kinase (ILK), which has been shown to be able to interact with intracellular domains of β-integrin subunits and which has the ability to facilitate the Ser473 phosphorylation (10, 30).

In the current study, we aimed to investigate the signaling cascade linking the membrane receptors β1-integrin and EGF-R with the proliferative ERK1/2 activation in response to nano-sized particles. As described, the PI3K/Akt cascade is known to be activated cooperatively by these receptors on adhesion to ECM proteins. Our aim here was to study whether this adhesion-specific cascade is also triggered by nanoparticles via comparable mechanisms and, furthermore, leads to the activation of the proliferative ERK1/2 activation. Reactions were induced by NPCB, produced industrially in controlled combustion processes. Because these particles consist of pure carbon with very low amounts of contaminants, they are accepted as model particles to study effects of the carbonaceous core of combustion-derived nanoparticles (12). To evaluate the relevance of these signaling cascades for lung epithelial cells in different species, in addition to the well-established rat alveolar type II epithelial cell line, a human bronchial epithelial cell line was used to study time- and dose-dependent induction of nanoparticle-specific signaling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Particles and particle preparations. NPCB (14 nm in diameter; Printex 90) was obtained from Degussa (Frankfurt, Germany). Stock suspensions (1 mg/ml) of particles were prepared in PBS (RLE-6TN cells) or in HEPES-buffered saline, pH 7.4 (HBS, 16HBE14o– cells) by sonication for 60 min at 50–60 Hz, 120 W. Particles were used at end concentrations from 1 to 10 µg/cm2.

Cell culture. The RLE-6TN rat lung epithelial cell line was purchased from ATCC (Manassas, VA; Ref. 13). Cells were grown at 37°C, 5% CO2 in Ham's F-12 medium supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), amphotericin B (2.5 µg/ml), L-glutamine (2 mM), and 5% heat-inactivated FCS (Sigma, St. Louis, MO). For experiments, cells were seeded with a density of 3 x 104/cm2 and were grown to 80% confluence. After the cells were washed twice with PBS, the medium was replaced by Ham's F-12 with 0.5% FCS and cells were incubated for 20–24 h. The human bronchial epithelial cells 16HBE14o– (17) were kindly provided by Dieter C. Gruenert (California Pacific Medical Research Center Research Institute, San Francisco, CA). Cells were cultured as described in MEM supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), and 10% heat-inactivated FCS. For experiments, these cells were seeded at a density of 6 x 104/cm2 and were grown to confluence. After the cells were washed twice with HBS, cells were starved for 24 h by using MEM containing 0.5% FCS.

Cell proliferation. The incorporation of 5-bromo-2'-deoxyuridine (BrdU) was monitored as a parameter for DNA synthesis by using the BrdU Labeling and Detection kit III (Roche Applied Science, Mannheim, Germany). BrdU was added to the culture medium 4 h before cell fixation. The assay was performed according to the manufacturer's protocol as follows: cells were fixed, DNA was partially digested with nuclease, and a peroxidase-labeled antibody to BrdU was added. Finally, the peroxidase substrate was added, resulting in a color change that was measured at 405 nm by using an ELISA reader.

Adhesion assays. Adhesion assays were carried out as described previously (31). Briefly, cells were serum starved for 20 h, washed twice with PBS, and harvested by being scraped in PBS containing 5 mM EDTA. Cells were spun at 760 rpm for 6 min, resuspended in Ham's F-12 medium without FCS, seeded on plates coated with 5 µg/cm2 fibronectin (10 µg/ml PBS; BD Biosciences, Bedford, MA), and maintained in Ham's F-12 for 30–90 min.

Transfection. The dominant-negative (kinase-dead) Akt construct was a kind gift from T. Franke (Columbia University, New York, NY) (14). The Akt mutant contains a point mutation K179M in the ATP-binding domain. The construct was generated as Hind III-BamH I inserts in pCMV6 vectors generated by the laboratory of T. Franke. The RLE-6TN cells were transfected at 40–50% confluence by using Lipofectamine Plus reagent (Invitrogen, Paisley, UK) as described (18). The transfections were carried out by incubating cells for 3 h with 0.25 µg DNA, 6 µl Plus reagent, and 4 µl Lipofectamine reagent per milliliter of culture medium (Ham's F-12 supplemented with 0.5% FCS and no antibiotics). The culture medium was replaced by fresh supplemented Ham's F-12 with 5% FCS. Sixteen hours later, cells were starved for 20 h with Ham's F-12 with 0.5% FCS before particle exposure.

Kinase activity assays. The Akt assay (Cell Signaling Technology, Danvers, MA) is a nonradioactive assay kit based on the ability of immunoprecipitated Akt to phosphorylate GSK-3 {alpha}/β at Ser21 and was performed following the manufacturer's guidelines. Briefly, 200–300 µg of protein was immunoprecipitated by using an immobilized Akt antibody, and immunoprecipitates were incubated with a GSK-3 {alpha}/β fusion protein as a substrate. Phosphorylation of Ser21 of GSK-3 {alpha}/β as a measure of Akt kinase activity was then detected by Western blotting with an antibody that recognizes phospho-GSK-3 {alpha}/β. The ILK assay also used the GSK-3 {alpha}/β fusion protein as the substrate. Protein (300 µg) from each sample was immunoprecipitated with 4 µg ILK antibody (Upstate, Dundee, UK). The following steps were performed as described for the Akt assay.

Treatment with particles, antibodies, peptides, and inhibitors. Cells were treated with particles for up to 24 h in the absence or presence of inhibitors. Lactate dehydrogenase activity was determined in supernatants to ensure that particle treatment, as well as pretreatment with inhibitors or transient transfection, did not induce cytotoxicity and cell death. Inhibitors specific for the signaling steps were added to the cells at 30 min (PI3K) or 60 min (EGF-R, integrins, Akt) before particle treatment. Concentrations of the respective inhibitors are indicated in the figure legends. Peptides were dissolved in sterile H2O or 5% acetic acid according to the instructions of the manufacturer. The integrin-blocking antibody and the control antibody were diluted in PBS. Pharmacological inhibitors were dissolved in DMSO, methanol, ethanol, or sterile H2O according to the instructions of the manufacturer. In detail, the following inhibitors and control substances were used: β1-integrin-blocking antibody Ha2/5 (Anti-CD29) and mouse-anti-hamster IgM (both BD Pharmingen, San Diego, CA); integrin-blocking peptide H-Arg-Gly-Asp-Ser-OH (RGDS) and control peptide H-Gly-Arg-Ala-Asp-Ser-Pro-OH (GRADSP; both Calbiochem); N-[3-chlorophenyl]-6,7-dimethoxy-4-quinazolinamine (tyrphostin AG 1478; Sigma); {D-3-deoxy-2-O-methyl-myoinositol 1-[(R)-2-methoxy-3-(octadecyloxy)propyl hydrogenphosphate]} (SH-5; Alexis Biochemicals, Lausen, Switzerland); and 4-[(3-bromophenyl)amino]-6,7-dimethoxyquinazoline (compound 32, AG 1517; Calbiochem).

Western blotting. After exposure, cells were washed twice with ice-cold PBS or HBS and were lysed in modified RIPA buffer [25 mM Tris·HCl, pH 7.4, 150 mM NaCl, 0.1 mM EDTA, 1% Nonidet P-40, 0.1% SDS, 1% deoxycholate, 0.025% NaN3, 1% protease inhibitor cocktail, and 1% phosphatase inhibitor cocktail (both inhibitor cocktails from Sigma)]. Aliquots (5 µg) of total cell protein were separated by SDS-PAGE (10%) and were transferred onto PVDF membranes (Hybond-P, Amersham Biosciences, Little Chalfont, UK). The activation of Akt, ERK1/2, and FAK were determined by their phosphorylation status by using phospho-Akt (Ser473) antibodies, phospho-p44/42 MAPK (Thr202/Tyr204) antibodies (Cell Signaling Technology), and phospho-FAK (Tyr397) antibodies (Upstate), respectively, according to the manufacturer's instructions. Total Akt, ERK1/2, ILK, and FAK were monitored with Akt antibodies, p44/42 MAPK antibodies, ILK1 antibodies (Cell Signaling Technology), and FAK antibodies (clone 4.47; Upstate), respectively. Signal strength was detected by using the ECL Plus Western Blotting Detection System (Amersham Biosciences) as recommended. All Western blot results were analyzed densitometrically. Only differences that proved to be statistically significant were considered.

Statistical analysis. All experiments were repeated independently at least three times. Mean values were compared statistically by using Student's t-test. Differences were assessed as significant when P ≤ 0.05. Although some results are presented as relative increases, statistics were performed with the use of absolute data.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Activation of Akt by NPCB. Nanoparticles have been shown to act as an extracellular stimulus that induces signaling dependent on the membrane receptors EGF-R and β1-integrin (28). Because Akt activation was known to be initiated by these receptors on cell attachment, we investigated whether this signaling cascade is also triggered by nanoparticles.

First, the Akt phosphorylation of Ser473 was investigated by using a phospho-specific antibody in time-course experiments (Fig. 1A). Although the total amount of Akt protein present in the cell was not influenced by the particle treatment, phosphorylation increased significantly after 8 h of treatment with 5 µg/cm2 NPCB. Because of an increase in background signals, the particle-induced response after 16 h proved not to be statistically significant. The elevated background after 16 h has been described earlier (5) as induced by the starving conditions under which the cells have to be kept to identify treatment-specific reactions. Interestingly, this time course in phosphorylation has striking similarities to the time course in ERK1/2 activation that we described in our previous studies (28).


Figure 1
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  		Fig. 1. Activation of protein kinase B (Akt) by nanoparticulate carbon black (NPCB) in RLE-6TN cells. A: time-course experiments showing quantification of Ser473 phosphorylation and representative Western blots of Ser473 phosphorylation and total Akt protein after treatment with 5 µg/cm2 NPCB. B: phosphorylation of glucose synthase kinase (GSK) fusion protein by immunoprecipitated Akt from cells treated for 8 h with 5 µg/cm2 NPCB (M, mock experiment without Akt-specific antibody). C: quantification and representative Western blot of Ser473 phosphorylation of a dose response after 8 h of treatment with indicated dosages of NPCB. Total Akt served as loading control. Gray bars, NPCB-treated cells; open bars, PBS-treated cells. *Significantly different from PBS-treated controls (P < 0.05).

 
To verify the significance of the phosphorylation signal for kinase activity, we performed specific kinase activity assays with immunoprecipitated Akt from cells treated for 8 h with 5 µg/cm2 NPCB (Fig. 1B). A clear increase in enzyme activity was observed in the assay detecting the phosphorylation of the GSK-3 fusion protein. We therefore conclude that phosphorylation of Ser473 in this cell system is representative of Akt activation.

The specificity of the nanoparticle-induced reaction was demonstrated in a second set of experiments. A clear dose response was observed when cells were treated for 8 h with increasing dosages of NPCB (Fig. 1C).

Nanoparticle-induced signaling in human bronchial epithelial cells. To investigate whether the parallel activation of ERK1/2 and Akt by NPCB represents a cell reaction of general relevance in lung epithelial cells in humans as well as in rodents, 16HBE14o– human bronchial epithelial cells were tested for the activation of ERK1/2 and Akt signaling. Comparable with the results obtained with rat lung epithelial cells, in this experimental system, both signaling pathways show similar activation patterns for ERK1/2 (Fig. 2A) and Akt (Fig. 2C), with a maximal activation after 12 h of treatment. Again, the specificity was demonstrated by dose-response experiments (Fig. 2B and D). This parallel activation of signaling pathways by the same stimulus in two different species indicates that the observed events are part of one mutual cascade. This hypothesis was investigated in detail in RLE-6TN cells in the following experiments.


Figure 2
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  		Fig. 2. Time- and dose-dependent activation of ERK1/2 and Akt by NPCB in human bronchial epithelial cells (16HBE14o–), quantifications, and representative Western blots. A: ERK1/2 activation over time induced by 10 µg/cm2 NPCB. B: ERK1/2 dose response after 12 h treatment with NPCB. C: Akt activation over time induced by 10 µg/cm2 NPCB. D: Akt dose response after 12 h treatment with NPCB. Gray bars, samples treated with NPCB; open bars, cells treated with HEPES-buffered saline (HBS). *Significantly different from HBS-treated controls (P < 0.05).

 
Influence of membrane receptors. Because it had been shown that the membrane receptors EGF-R and β1-integrin are relevant for ERK1/2 activation by nanoparticles (28), it was investigated by inhibitor assays whether these receptors are also involved in Akt activation. The NPCB-dependent phosphorylation of EGF-R has been demonstrated in assays detecting phosphotyrosine in immunoprecipitated EGF-R (28). In these studies, the use of tyrphostin AG 1478 (an inhibitor of the receptor kinase activity) resulted in a significant decrease in NPCB-induced ERK1/2 activation. We therefore tested this inhibitor as well as the more specific EGF-R inhibitor AG 1517 (compound 32) for their effects on Akt phosphorylation (Fig. 3A). Pretreatment of the cells with these substances led to a significant decrease in nanoparticle-induced Akt activation. Under the chosen cell-starving conditions, the inhibitors had no effect on background signals. The solvent DMSO also induced a slight increase in phosphorylation, which proved not to be significant and was not within the same effect range as NPCB.


Figure 3
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  		Fig. 3. Influence of membrane-receptor inhibitors on nanoparticle-induced Akt phosphorylation on Ser473 in RLE-6TN cells. A: quantification and representative Western blot investigating role of epidermal growth factor receptor (EGF-R). B: quantification and representative Western blot investigating role of integrins. Gray bars, samples treated with 5 µg/cm2 NPCB; open bars, cells treated with PBS or respective solvent controls. AG 1478, N-[3-chlorophenyl]-6,7-dimethoxy-4-quinazolinamine; AG 1517, 4-[(3-bromophenyl)amino]-6,7-dimethoxyquinazoline; RGDS, integrin-blocking peptide H-Arg-Gly-Asp-Ser-OH; GRADSP, control peptide H-Gly-Arg-Ala-Asp-Ser-Pro-OH. *Significantly different from PBS-treated controls (P < 0.05). {dagger}Significantly different from samples treated with NPCB and respective solvent controls (P < 0.05).

 
The influence of integrins was also tested with two types of inhibitors. Integrin activation can be blocked either with specific peptides representing repetitive protein sequences of ECM proteins or with integrin-inactivating antibodies. The results of both types of inhibition experiments are shown in Fig. 3B. The peptide RGD occurs in some types of ECM proteins and is recognized by several combinations of the heterodimeric integrins. The decrease of NPCB-induced Akt phosphorylation in cells pretreated with RGD demonstrates that this process is dependent on integrins. The control peptide GRADSP, as well as the respective solvents (acetic acid; H2O) had no effect. To specify the type of integrin receptor responsible for these effects, β1-integrin was blocked by incubating the cells with the β1-integrin-inactivating antibody (anti-CD29). Again, a significant reduction of the nanoparticle-induced phosphorylation that did not occur with the control antibody was observed. These data give a clear indication that Akt activation induced by NPCB is strongly dependent on the membrane receptors EGF-R and β1-integrin.

Linkage to intracellular signaling. Integrin-dependent signaling in the context of cell adhesion and detachment is frequently mediated by kinases located in the focal contacts (8). FAK is a protein tyrosine kinase that is activated by autophosphorylation on Tyr397 when focal contacts are formed during cell adhesion (25). The activation can be investigated with the respective phospho-specific antibody. In earlier studies, we described that FAK is not activated in mesothelial cells treated with fibrous particles (5). In the current study, again, FAK appears not to play a role in the particle-specific reaction. As shown in Fig. 4A, during the time course of 24 h in cells treated with NPCB, no autophosphorylation of FAK was observed, whereas treatment of the cells with 0.1 µm hydrogen peroxide for 1 h significantly increased FAK phosphorylation.


Figure 4
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  		Fig. 4. Focal adhesion kinase (FAK) and integrin-linked kinase (ILK) in NPCB-treated RLE-6TN cells. A: quantification and representative Western blots of Tyr397 autophosphorylation of FAK and total FAK in cells treated with 5 µg/cm2 NPCB or 1 µM H2O2 for 1 h (light gray bar). B: phosphorylation of GSK fusion protein by immunoprecipitated ILK from cells either treated with 5 µg/cm2 NPCB or cells grown on fibronectin (M, mock experiment without ILK-specific antibody). C: quantification and representative Western blot of ILK in cells treated with 5 µg/cm2 NPCB. Gray bars, NPCB-treated cells; open bars, PBS-treated cells. *Significantly different from PBS-treated controls (P < 0.05).

 
A second candidate for mediating integrin effects to intracellular signaling is ILK (4), because this protein of the focal contact has been described to suppress anoikis via activation of the Akt pathway. The influence of NPCB on ILK was tested on the level of kinase activity. Figure 4B clearly demonstrates that phosphorylation of a GSK-3 fusion protein from immunoprecipitated ILK did not occur after treatment of cells with NPCB, whereas cell adhesion on fibronectin-coated dishes revealed increasing ILK activity between 30 and 90 min. Additional measurements of ILK protein levels during a time course of 24 h ensured that the lack of activity after particle treatment was not due to changes in ILK protein content (Fig. 4C).

The role of PI3K and Akt in nanoparticle-induced proliferation. Akt signaling is described to be strictly dependent on PI3K, which is also located at the intracellular side of the plasma membrane. The influence of this protein on Akt activation was investigated in independent experiments using the pharmacological inhibitors LY-294002 and Wortmannin (Fig. 5A). As expected, both inhibitors significantly decreased NPCB-induced Akt activation, whereas the effect of Wortmannin appeared to be stronger than the effect of LY-294002.


Figure 5
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  		Fig. 5. Influence of phosphoinositide 3-kinase (PI3K) inhibitors on Akt, ERK1/2, and proliferation in RLE-6TN cells. A: quantification and representative Western blots of Ser473 phosphorylation. B: quantification and representative Western blots of ERK1/2 phosphorylation. C: proliferation determined by measurement of 5-bromo-2'-deoxyuridine (BrdU) incorporation 24 h after particle treatment. Gray bars, cells treated with either 5 µg/cm2 (Western blot) or 10 µg/cm2 (BrdU assay) NPCB; open bars, cells treated with PBS or solvent controls. *Significantly different from PBS-treated controls (P < 0.05). {dagger}Significantly different from samples treated with NPCB and respective solvent controls (P < 0.05).

 
Comparing these data with the ERK1/2 data published previously, striking similarities between ERK1/2 and Akt with regard to kinetics as well as to their dependence on EGF-R and β1-integrin are obvious. It was therefore further investigated whether these two signaling pathways are induced in parallel by a common mechanism involving the membrane receptors or whether ERK1/2 activation and subsequent proliferation are mediated by PI3K. First, the influence of PI3K inhibitors on ERK1/2 phosphorylation was tested in Western blot experiments with a phospho-ERK1/2 antibody. (Fig. 5B) Interestingly, both inhibitors revealed similar results as for Akt activation (Fig. 5A). Again, with the chosen doses, the effect of Wortmannin was stronger than that of LY-294002.

If this effect on ERK1/2 had a physiological relevance, it would be reproducible on the level of proliferation. In an earlier study (5), we were able to demonstrate that BrdU incorporation is a specific marker for nanoparticle-induced cell proliferation. We therefore investigated BrdU incorporation after 24 h of particle treatment in the presence of PI3K inhibitors (Fig. 5C). Preincubation of cells with Wortmannin led to a significant decrease of NPCB-induced proliferation. LY-294002 also showed a reduction of the proliferation rate; however, because of high standard deviations, this effect proved not to be statistically significant. This lower inhibition of proliferation by LY-294002 most likely reflects the weaker inhibitory effect of this substance on the phosphorylation of Akt and ERK1/2 compared with Wortmannin.

Akt and nanoparticle-induced ERK1/2 activation. Because the differences in PI3K inhibitor efficiency were comparable for Akt, ERK1/2, and proliferation, it seems possible that activation of ERK1/2 is mediated by Akt itself. This hypothesis was tested with two independent assays. First, increasing dosages of the Akt inhibitor SH-5 were used to preincubate cells before nanoparticle treatment. As expected, the inhibitor had a dose-dependent, significant inhibitory effect on nanoparticle-induced phosphorylation of Akt (Fig. 6A, left). A comparable dose response of the Akt inhibitor was observed on the level of ERK1/2, which proved to be statistically significant for the highest concentration (1 µM; Fig. 6A, right). In a second independent assay, Akt activity was reduced by transfection of the cells with the well-known system of the Akt dominant-negative mutant established by Franke et al. (14). Figure 6B demonstrates that the diminished Akt activity in dominant-negative transfected cells resulted in a reduction of ERK1/2 activation in cells treated for 8 h with 5 µg/cm2 NPCB.


Figure 6
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  		Fig. 6. Influence of Akt activity on ERK1/2 phosphorylation in RLE-6TN cells induced by NPCB. A: quantification and representative Western blots of Ser473 phosphorylation (left) and ERK1/2 phosphorylation (right). B: Western blot analysis of Ser473 phosphorylation and ERK1/2 phosphorylation in cells transfected with dominant-negative Akt, [pCMV6 HA Akt (K179M)], or with vector pCMV6 HA. C: proliferation determined by measurement of BrdU incorporation 24 h after treatment. Gray bars, cells treated with either 5 µg/cm2 (Western blot) or 10 µg/cm2 (BrdU assay) NPCB; open bars, cells treated with PBS or solvent controls. *Significantly different from PBS-treated controls (P < 0.05). {dagger}Significantly different from samples treated with NPCB and respective solvent controls (P < 0.05). §Significantly lower than PBS-treated controls (P < 0.05).

 
Furthermore, the effect of Akt inhibition was tested on the level of proliferation (Fig. 6C). Because cells transiently transfected with the dominant-negative Akt mutant lose the Akt block during cell division, this experiment was performed with SH-5. As a phosphatidylinositol analog, this substance specifically blocks the phosphorylation of Akt at Ser473. The pretreatment with SH-5 resulted in a significant reduction of the particle-induced proliferation. These data demonstrate a causal connection between the activity of Akt and the ERK1/2-dependent nanoparticle-specific proliferation.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
NPCB activates the Akt cascade. The signaling cascade via PI3K and Akt is mostly considered to be a pathway induced by ligand binding to growth factor receptors, which helps the cell to survive and which induces cell division (1). Besides this well-known mode of activation, the pathway can be triggered via integrins and is therefore able to respond to extracellular stress such as cell detachment, mechanical stimuli, and cell-shape changes (2, 4). So far, there are only few reports indicating a role of integrin-dependent Akt activation on xenobiotic stress. Cell survival after genotoxic stress induced by ionizing radiation appears to be mediated by a β1-integrin-mediated Akt activation (9). Treatment of mesothelial cells with asbestos fibers resulted in Ser473 phosphorylation of Akt, which also appeared to be integrin dependent (5).

In the current study, we were able to show that the Akt pathway is specifically induced by combustion-derived nanoparticles. Akt is activated on the level of the Ser473 phosphorylation by NPCB in a dose- and time-dependent manner. This molecular reaction was demonstrated for lung epithelial cells from humans and rats, indicating a specific role of this pathway in nanoparticle cell interaction. Some studies indicate that nanoparticles may enter the body via several pathways and may be responsible for diverse pathogenic endpoints in a variety of target tissues (24). Further studies with other cell types will show whether Akt activation is a general process of interaction between nanoparticles and cells triggering specific signaling.

Akt activation is dependent on integrins and EGF-R. As already demonstrated (28), nanoparticle-induced proliferation as well as the ERK1/2 pathway is dependent on EGF-R and on β1-integrin. Using the respective inhibitor assays, we show here that the activation of Akt is also dependent on these membrane receptors. In our earlier studies, we demonstrated that, parallel to proliferation, apoptosis can be induced by the same nanoparticles (28). This cell reaction, which is mediated by the MAPKs JNK1/2, is also dependent on EGF-R but not on integrins. Thus EGF-R appears to trigger two signaling pathways leading to opposing endpoints. Because β1-integrins appear to be relevant for nanoparticle-specific proliferation, they may be considered the switch decisive for the cell fate by triggering the respective pathways. In systems investigating cell adhesion, a cooperative effect of these two membrane receptors has been observed (6, 22). These studies suggest EGF-R as target for integrin-mediated outside-in signaling resulting in the activation of the classical Ras-Raf-MEK cascade, leading to the phosphorylation of the ERK1/2 MAPKs. However, the mechanism of integrin-dependent activation of EGF-R is not understood so far.

FAK and ILK are not activated by NPCB. FAK and ILK are classical signaling proteins dependent on integrin transmembrane signaling, playing an important role in PI3K and Akt activation in adhesion-dependent cell reactions (15, 17). Both enzymes appear not to be activated with doses relevant for nanoparticle effects and within the time frame of the induced signaling. It cannot be excluded that at higher doses these signaling proteins can be activated. However, because of cytotoxic effects of high nanoparticle concentrations, it is doubtful whether such an activation would be a specific response on nanoparticle stress. From these data, we conclude that although nanoparticle-induced signaling depends on membrane receptors known to be relevant for adhesion-dependent signaling, the intracellular events differ with regard to the respective stimulus. There are already a few reports indicating integrin-dependent Akt activation that is not mediated by the well-known focal adhesion proteins (19, 33). The relevance of these events for nanoparticle-induced signaling remains to be demonstrated.

NPCB-induced Akt activation is responsible for proliferative ERK1/2 signaling. The striking similarities in activation of ERK1/2 and Akt with respect to time and dose, as well as to receptor dependence, opens the question of whether these pathways are triggered in parallel by a common upstream event or whether they are parts of one cascade leading to one endpoint. The PI3K-Akt pathway is mostly considered to be a pathway independent from the ERK1/2 pathway. Whereas ERK1/2 induce proliferation via the transcription factor Elk-1 and the activation of cyclin-dependent kinases (3, 26), Akt has an impact on the cell cycle via the forkhead box, as described (7). Our results, however, show that PI3K as well as Akt are responsible for nanoparticle-induced ERK1/2 activation and the resulting proliferation. Because PI3K has an upstream position within this cascade, it must be considered that Akt mediates the link to the MAPK cascade. It is, however, not clear by which molecular mechanism ERK1/2 is activated by Akt. One could speculate that proteins of the ERK1/2 pathway are activated either by GSK-3, as a protein downstream of Akt that is able to phosphorylate a broad range of proteins, or by Akt itself, because some proteins of this cascade show structural similarities to GSK-3 (1, 11).

Because nanoparticles may be considered relevant for the pathogenesis of several diseases, including cancer, the initial effects triggering the described pathways are of special interest. The involvement of integrins suggests a potential of outside-in signaling following an interaction of the particles with extracellular structures of the membrane. However, nanoparticles have been described to enter the cell easily without using specific uptake mechanisms (16). It therefore may be speculated that the activation of the described pathway is triggered by intracellular processes comparable with the phosphorylation of EGF-R by intracellularly generated reactive oxygen species (32).

The present data, summarized in Fig. 7, demonstrate that the signaling pathway involving PI3K and Akt is activated by nano-sized carbon particles. The activation of this cascade is dependent on the membrane receptors EGF-R and β1-integrin and has a direct impact on nanoparticle-induced proliferation. This endpoint is triggered by an Akt-dependent phosphorylation of ERK1/2. Thus this study describes a specific role of Akt in the activation of proliferative MAPK signaling in lung epithelial cells.


Figure 7
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  		Fig. 7. Signaling cascade induced by NPCB in RLE-6TN cells. Both membrane receptors, EGF-R and β1-integrin, probably cooperatively induce proliferative signaling via ERK1/2. Data from this study demonstrate that PI3K and Akt are main mediators of this signaling between membrane receptors and MAPKs. Furthermore, no evidence for an involvement of focal contact proteins FAK and ILK could be obtained (modified after Ref. 32).

 


   ACKNOWLEDGMENTS
 
The excellent technical assistance of Ragnhild Wirth and Winfried Brock is gratefully acknowledged.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Unfried, Institut für umweltmedizinische Forschung, Auf’m Hennekamp 50, 40225 Düsseldorf, Germany (e-mail: klaus.unfried{at}uni-duesseldorf.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 RESULTS
 DISCUSSION
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