Nickel nanoparticles enhance platelet-derived growth factor-induced chemokine expression by mesothelial cells via prolonged mitogen-activated protein kinase activation

Abstract

Pleural diseases (fibrosis and mesothelioma) are a major concern for individuals exposed by inhalation to certain types of particles, metals, and fibers. Increasing attention has focused on the possibility that certain types of engineered nanoparticles (NPs), especially those containing nickel, might also pose a risk for pleural diseases. Platelet-derived growth factor (PDGF) is an important mediator of fibrosis and cancer that has been implicated in the pathogenesis of pleural diseases. In this study, we discovered that PDGF synergistically enhanced nickel NP (NiNP)-induced increases in mRNA and protein levels of the profibrogenic chemokine monocyte chemoattractant protein-1 (MCP-1 or CCL2), and the antifibrogenic IFN-inducible CXC chemokine (CXCL10) in normal rat pleural mesothelial 2 (NRM2) cells in vitro. Carbon black NPs (CBNPs), used as a negative control NP, did not cause a significant increase in CCL2 or CXCL10 in the absence or presence of PDGF. NiNPs prolonged PDGF-induced phosphorylation of the mitogen-activated protein kinase family termed extracellular signal-regulated kinases (ERK)-1 and -2 for up to 24 hours, and NiNPs also synergistically increased PDGF-induced hypoxia-inducible factor (HIF)-1α protein levels in NRM2 cells. Inhibition of ERK-1,2 phosphorylation with the mitogen-activated protein kinase kinase (MEK) inhibitor, PD98059, blocked the synergistic increase in CCL2, CXCL10, and HIF-1α levels induced by PDGF and NiNPs. Moreover, the antioxidant, N-acetyl-L-cysteine (NAC), significantly reduced HIF-1α, ERK-1,2 phosphorylation, and CCL2 protein levels that were synergistically increased by the combination of PDGF and NiNPs. These data indicate that NiNPs enhance the activity of PDGF in regulating chemokine production in NRM2 cells through a mechanism involving reactive oxygen species generation and prolonged activation of ERK-1,2.

Figure 1.

Nickel nanoparticles (NiNPs) behave as NP agglomerates in cell culture medium, and are taken up by pleural mesothelial cells in vitro. NiNPs were dispersed in a bath sonicator for 2 hours in a 0.1% pluronic F-68 in phosphate buffer solution before dosing normal rat pleural mesothelial (NRM) 2 cells as indicated in Materials and Methods. NiNP-exposed NRM2 cells were collected at 24 hours and characterized using transmission electron microscopy (TEM) imaging. (A) NiNP agglomerate in control medium. (B) Size distribution showing that the majority of NiNPs had a diameter between 20 and 50 nm. (C) NiNP agglomerates (black arrows) were observed in contact with the cell membrane and within the cytoplasm of a cultured NRM2 cell. (D) Higher magnification of the black box in (C) showing NiNP agglomerates enclosed within a membrane-bound vesicle in the NRM2 cell. (E) Carbon black NP (CBNP) agglomerates (black arrows) were also observed in contact with the cell membrane and within the cytoplasm. (F) Higher magnification of the black box in (E) showing CBNP agglomerate enclosed within a membrane-bound vesicle. Subcellular structures indicated are nucleus (Nu), microvilli (Mv), mitochondria (Mi), and lysosome (Ly).

Figure 2.

NiNPs synergistically increase PDGF-induced CCL2 and CXCL10 mRNA and protein expression in NRM2 cells. NRM2 cells were grown to confluency, serum starved for 24 hours, and treated with PDGF-BB (50 ng/ml) alone or immediately prior to adding CBNPs or NiNPs (10 μg/cm²). Either mRNA or cell culture supernatants were collected for analysis by Taqman quantitative real-time RT-PCR or ELISA, respectively. (A) CCL2 mRNA analyzed at 24 hours. ***P < 0.001 compared with control determined by one-way ANOVA with post hoc Tukey test; ^##P < 0.01, between NiNPs and PDGF + NiNPs or ⁺⁺⁺P < 0.001 between PDGF and PDGF + NiNPs, as determined by two-way ANOVA with Bonferroni post test. (B) Secreted CCL2 protein at 24 hours. ***P < 0.001 compared with control, determined by one-way ANOVA; ^##P < 0.01, between NiNPs and PDGF + NiNPs or ⁺⁺P < 0.01 between PDGF and PDGF + NiNPs, as determined by two-way ANOVA. (C) CXCL10 mRNA levels at 24 hours. *P < 0.05 compared with control determined by one-way ANOVA; ^#P < 0.05, between NiNPs and PDGF + NiNPs or ⁺P < 0.05 between PDGF and PDGF + NiNPs as determined by two-way ANOVA. (D) Secreted CXCL10 protein at 48 hours. *P < 0.01 or ***P < 0.001 compared with control; ^##P < 0.01, between NiNPs and PDGF + NiNPs or ⁺⁺⁺P < 0.001 between PDGF and PDGF + NiNPs, as determined by two-way ANOVA. All data represent mean values (±SEM) of three experiments run in duplicate.

Figure 3.

NiNPs enhance and prolong PDGF-induced extracellular signal–regulated kinase (ERK) phosphorylation, but not PDGF receptor (PDGF-R) β in rat pleural mesothelial cells. NRM2 cells were grown to confluency, serum starved, and treated with PDGF-BB (50 ng/ml) or PDGF-BB plus NiNPs (10 μg/cm²), and cell lysates were collected at 2 and 24 hours after exposure. (A) Western blot analysis showing strong PDGF-induced phosphorylated ERK (p-ERK) at 2 hours that is almost completely diminished by 24 hours. NiNP coexposure did not alter PDGF-induced ERK phosphorylation at 2 hours, but enhanced PDGF-induced activation at 24 hours. Total ERK protein levels were not changed by any of the treatments. (B) Densitometry of the blots in (A) showed the relative ratio of p-ERK to total ERK protein for each time point. Densitometric analysis was performed using Image J analysis software, as described in Materials and Methods, and represent mean values (±SEM) of three experiments. (C) Western blot analysis showed PDGF-BB–induced phosphorylation of PDGF-Rβ at 15 minutes that is not affected by NiNPs. PDGF-BB–induced PDGF-Rβ phosphorylation was diminished by 24 hours and not affected by NiNPs. **P < 0.01 compared with untreated (control) and NiNP-exposed cells; ***P < 0.001 compared with cells treated with control or NiNPs alone, as determined by one-way ANOVA with post hoc Tukey’s test and ^P < 0.05 compared with cells treated with PDGF alone, as determined by two-way ANOVA with Bonferroni’s post test. Both Western blots shown are representative of at least three independent experiments that produced similar results.

Figure 4.

CXCL10 and CCL2 expression in rat pleural mesothelial cells is regulated by the MEK-ERK signaling pathway. NRM2 cells were grown to confluency, serum starved, and then pretreated with 20 μM PD98059 (black bars) for 1 hour before adding PDGF (50 ng/ml) or NiNPs (10 μg/cm²), alone or in combination, for 24 or 48 hours. (A) Western blot analysis showing p-ERK and total ERK protein levels in cell lysates from NRM2 cells. (B) CXCL10 mRNA levels at 24 hours and secreted protein levels at 48 hours from NRM2 cells were measured using TaqMan quantitative real-time RT-PCR or ELISA, respectively. (C) CCL2 mRNA and secreted protein levels were measured at 24 hours after exposure. Data represent mean values (±SEM) of three experiments run in duplicate. **P < 0.01 or ***P < 0.001 compared with cells in the same exposure group treated with mitogen-activated protein kinase kinase (MEK) inhibitor, PD98059, and determined by two-way ANOVA with Bonferroni’s post test.

Figure 5.

PDGF enhancement of NiNP-induced hypoxia-inducible factor (HIF)-1α is mediated by MAPK signaling. NRM2 cells were grown to confluency, serum starved, and then pretreated with 20 μM PD98059 (black bars) for 1 hour before adding PDGF (50 ng/ml) or NiNPs (10 μg/cm²), alone or in combination, for 24 or 48 hours. (A) Western blot analysis of HIF-1α protein levels in cell lysates from NRM2 cells showing that PDGF enhancement of NiNP-induced HIF-1α is reduced by PD98059. (B) Densitometry of Western blots in (A) showing the relative ratio of HIF-1α to β-actin protein for each treatment. Densitometric analysis was performed using Image J analysis software, as described in Materials and Methods. All data represent mean values (±SEM) of three independent experiments. ***P < 0.001 compared with control or PDGF treatment with or without PD98059, as determined by one-way ANOVA with post hoc Tukey’s test; ^###P < 0.001 comparing NiNP to PDGF + NiNP treatment without PD98059; or ^P < 0.05 compared with cells in the same exposure group treated without MEK inhibitor, PD98059, as determined by two-way ANOVA with Bonferroni’s post test. Western blot shown is a representative of at least three independent experiments that produced similar results.

Figure 6.

HIF-1α and CCL2 expression induced by NiNPs and PDGF is reduced by the antioxidant, N-acetyl-L-cysteine (NAC). NRM2 cells were grown to confluency, serum starved, and then pretreated with 5 mM NAC for 1 hour before adding PDGF (50 ng/ml) or NiNPs (10 μg/cm²), alone or in combination, for 24 hours. (A) Representative Western blot of HIF-1α protein levels in cell lysates from NRM2 cells showing that induction of HIF-1α levels after treatment with NiNPs or NiNPs and PDGF-BB were both reduced by NAC. (B) Densitometry of three independent Western blots, including the representative blot displayed in (A) showing the relative ratio of HIF-1α to β-actin protein for each treatment. Densitometric analysis was performed using Image J analysis software, as described in Materials and Methods. (C) Western blot showing increased levels of p-ERK-1,2 after 24-hour treatment with the combination of PDGF and NiNPs, which were reduced by 1-hour pretreatment with NAC. Note that ERK-1,2 phosphorylation is transiently induced by PDGF alone at 2 hours, and returns to basal levels by 24 hours (see Figure 3). (D) ELISA results showing that CCL2 protein induced by the combination of NiNPs and PDGF is significantly reduced by pretreatment with NAC. The ELISA data are from a single experiment with samples run in triplicate. *P < 0.05 or **P < 0.01 compared with cells in the same exposure group treated without NAC, as determined by two-way ANOVA with Bonferroni’s post test.

Figure 7.

Schematic illustration showing the hypothetical cell signaling mechanism involved in the synergistic induction of HIF-1α and chemokines by NiNPs and PDGF in rat pleural mesothelial cells. NiNPs are taken up by pleural mesothelial cells to generate reactive oxygen species (ROS), increase HIF-1α, and stimulate chemokine production. PDGF-BB, a growth factor produced by activated alveolar macrophages or other cellular sources, binds to PDGF-Rβ on mesothelial cells to stimulate ERK phosphorylation and marginally increase chemokine production. NiNPs are postulated to increase the duration of PDGF-induced ERK phosphorylation, and thereby synergistically increase the production of CCL2 and CXCL10, which play important roles in the progression and resolution of pleural fibrogenesis, respectively.