The formyl peptide receptor agonist FPRa14 induces differentiation of Neuro2a mouse neuroblastoma cells into multiple distinct morphologies which can be specifically inhibited with FPR antagonists and FPR knockdown using siRNA

Abstract

The N-formyl peptide receptors (FPRs) have been identified within neuronal tissues and may serve as yet undetermined functions within the nervous system. The FPRs have been implicated in the progression and invasiveness of neuroblastoma and other cancers. In this study the effects of the synthetic FPR agonist FPRa14, FPR antagonists and FPR knockdown using siRNA on mouse neuroblastoma neuro2a (N2a) cell differentiation plus toxicity were examined. The FPRa14 (1-10μM) was found to induce a significant dose-dependent differentiation response in mouse neuroblastoma N2a cells. Interestingly, three distinct differentiated morphologies were observed, with two non-archetypal forms observed at the higher FPRa14 concentrations. These three forms were also observed in the human neuroblastoma cell-lines IMR-32 and SH-SY5Y when exposed to 100μM FPRa14. In N2a cells combined knockdown of FPR1 and FPR2 using siRNA inhibited the differentiation response to FPRa14, suggesting involvement of both receptor subtypes. Pre-incubating N2a cultures with the FPR1 antagonists Boc-MLF and cyclosporin H significantly reduced FPRa14-induced differentiation to near baseline levels. Meanwhile, the FPR2 antagonist WRW4 had no significant effect on FPRa14-induced N2a differentiation. These results suggest that the N2a differentiation response observed has an FPR1-dependent component. Toxicity of FPRa14 was only observed at higher concentrations. All three antagonists used blocked FPRa14-induced toxicity, whilst only siRNA knockdown of FPR2 reduced toxicity. This suggests that the toxicity and differentiation involve different mechanisms. The demonstration of neuronal differentiation mediated via FPRs in this study represents a significant finding and suggests a role for FPRs in the CNS. This finding could potentially lead to novel therapies for a range of neurological conditions including neuroblastoma, Alzheimer's disease, Parkinson's disease and neuropathic pain. Furthermore, this could represent a potential avenue for neuronal regeneration therapies.

Fig 1

Typical phase contrast photomicrographs (200x) exhibiting (A) untreated N2a, (B) N2a treated with 10μM FPRa14, (C) untreated IMR-32, (D) IMR-32 treated with 100μM FPRa14 (E) untreated SH-SY5Y and (F) SH-SY5Y treated with 100μM FPRa14. Images were taken after 48h incubation (scale bars represent 100μm).

Fig 2

(A) The effect of FPRa14 (0–10μM) on (A) the % differentiated N2a cells, (B) mean N2a cell perimeter, (C) mean N2a cell area and (D) mean cell count. Serum-free medium only was used as a control. Values represent mean ± SEM, taken following 24h and 48h incubation with FPRa14. Statistical analysis was performed via one-way ANOVA with Dunnett’s post hoc test. *Represents statistical significance (P<0.01) relative to appropriate incubation control. Mean total cell counts are expressed as a percentage of control.

Fig 3

(A) Key highlighting examples of the three classes of differentiated cell morphology observed following incubation with FPRa14 (2–10μM). Images were taken 48h incubation with FPRa14. (B) The effect of FPRa14 (0–10μM) on differentiated cell morphology distribution following 48h incubation with FPRa14. (C) Mean perimeter values for each N2a morphology type observed in cultures treated with FPRa14 (0–10μM). (D) Mean area values for each N2a morphology type observed in cultures treated with FPRa14 (0–10μM). Statistical analysis was performed via one-way ANOVA with Dunnett’s post hoc test. *Represents statistical significance (P<0.01) relative to undifferentiated control (UD).

Fig 4

(A) The effect of FPRa14 (10μM) on the % of differentiated N2a cells and differentiated cell morphology distribution at time points of 0-48h following agonist administration (n = 1580). (B) The effect of FPRa14 (10μM) on the % of differentiated N2a cells and differentiated cell morphology distribution at time points of 0-48h when the agonist-containing media was removed after 1h incubation in the presence of FPRa14 agonist (as indicated by dotted line) and replaced with SFM (n = 1293).

Fig 5

(A) The effect of FPRa14 (0–10μM) on the percentage differentiation of N2a cells following control siRNA, Fpr1, Fpr2, and simultaneous Fpr1 & Fpr2 siRNA treatment. (B) The effect of FPRa14 (10μM) on the proportion of the differentiated cell morphology types following Fpr1, Fpr2, and simultaneous Fpr1 & Fpr2 siRNA treatment. N2a cells were also transfected with a negative control siRNA duplex as a control. Values represent mean ±SEM, following 24h incubation with FPRa14. Statistical analysis was performed via one-way ANOVA with Dunnett’s post hoc test. *Represents statistical significance (P<0.01) relative to appropriate negative control siRNA (n = 1120). (C) The effect of FPRa14 (8μM) on the % of differentiated N2a cells after 30min incubation with Boc-MLF (0–40μM), cyclosporin H (0–40μM) or WRW4 (0–40μM). Serum-free medium only (SFM) was used as a negative control (n = 1121). (D) The effect of Boc-MLF only (40μM), cyclosporin H only (40μM), WRW4 only (40μM) and SFM on the % of differentiated N2a cells (n = 355). Values are mean ±SEM, taken after 48h of incubation with FPRa14. Statistical analysis was performed via one-way ANOVA with Dunnett’s post-hoc test. *Represents statistical significance (P<0.01) relative to serum-free medium control.

Fig 6

(A) The effect of FPRa14 (0-10mM) on % control MTT reduction in N2a cells following control siRNA, Fpr1, Fpr2, and simultaneous Fpr1 & Fpr2 siRNA treatment. Values represent mean ±SEM, following 24h incubation with FPRa14. Statistical analysis was performed via one-way ANOVA with Dunnett’s post-hoc test. *Represents statistical significance (P<0.01) relative to negative control siRNA plus FPRa14. (B) The effect of FPRa14 (0-10mM) alone, FPRa14 (0-10mM) following 30min pre-incubation with Boc-MLF (40μM) or cyclosporin H (40μM) on N2a % control MTT reduction. (C) The effect of FPRa14 (0-10mM) alone and FPRa14 (0-10mM) following 30min pre-incubation with WRW4 (40μM) on N2a % control MTT reduction. Values are mean ±SEM from six repeats. Statistical analysis was performed via one-way ANOVA with Dunnett’s post-hoc test. *Represents statistical significance (P<0.01) relative to positive FPRa14 control. (D) The effect of FPRa14 (10μM) alone or serum free media and FPRa14 (10μM) or serum free media following 30min pre-incubation with Boc-MLF (40μM), cyclosporin H (40μM) or WRW4 (40μM) on N2a % control MTT reduction. Values are mean ±SEM from six repeats. Statistical analysis was performed via one-way ANOVA with Dunnett’s post hoc test. *Represents statistical significance (P<0.01) relative to serum free media, # represents statistical significance (P<0.01) relative to FPRa14 (10μM).