PI3K/Akt signaling pathway triggers P2X7 receptor expression as a pro-survival factor of neuroblastoma cells under limiting growth conditions

Abstract

The expression of purinergic P2X7 receptor (P2X7R) in neuroblastoma cells is associated to accelerated growth rate, angiogenesis, metastasis and poor prognosis. Noticeably, P2X7R allows the survival of neuroblastoma cells under restrictive conditions, including serum and glucose deprivation. Previously we identified specificity protein 1 (Sp1) as the main factor involved in the transcriptional regulation of P2rx7 gene, reporting that serum withdrawal triggers the expression of P2X7R in Neuro-2a (N2a) neuroblastoma cell line. Here we demonstrate that PI3K/Akt pathway is crucial for the upregulation of P2X7R expression in serum-deprived neuroblastoma cells, circumstance that facilitates cell proliferation in the absence of trophic support. The effect exerted by PI3K/Akt is independent of both mTOR and GSK3, but requires the activation of EGF receptor (EGFR). Nuclear levels of Sp1 are strongly reduced by inhibition of PI3K/Akt pathway, and blockade of Sp1-dependent transcription with mithramycin A prevents upregulation of P2rx7 gene expression following serum withdrawal. Furthermore, atypical PKCζ plays a key role in the regulation of P2X7R expression by preventing phosphorylation and, consequently, activation of Akt. Altogether, these data indicate that activation of EGFR enhanced the expression of P2X7R in neuroblastoma cells lacking trophic support, being PI3K/Akt/PKCζ signaling pathway and Sp1 mediating this pro-survival outcome.

Figure 1. P2X7R expression is upregulated in serum-deprived neuroblastoma cells.

(A) Changes in P2X7 transcript levels in N2a cell line cultured either in standard culture medium (FBS) for 24 h or in serum free medium (SF) for the indicated time periods. Total RNA was extracted and P2X7 mRNA was quantified by Q-PCR as described in Methods. GAPDH was used as a control for differences in cDNA input. Results are mean ± s.e.m. of three independent experiments in triplicate; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 vs FBS (ANOVA with the Dunnett’s post hoc test). (B) Immunoblotting depicting the presence of endogenous P2X7R in whole-cell lysates from N2a cells cultured either in FBS for 24 or in SF at the indicated time points. Cell lysates were analyzed by western blotting with anti-P2X7R (intracellular epitope) antibody. GAPDH was used as internal loading control. Histogram represents P2X7 protein levels at the indicated time periods obtained by densitometry and normalization to GAPDH. The values represent mean ± s.e.m. of three independent experiments in duplicate. *P ≤ 0.05, ***P ≤ 0.001 vs FBS (ANOVA with the Dunnett’s post hoc test). (C) Double immunofluorescence for P2X7R (green) and α-tubulin (red) in N2a cells cultured either in FBS for 24 h or in SF medium for 72 h. Nuclei were counterstained with DAPI (blue). Insets depict enlarged views (2.5X magnification) of delimited area. Scale bar = 15 μm. (D) Proliferation of serum-starved N2a cells treated with LY294002 (50 μM), BBG (5 or 10 μM), A740003 (10 or 50 μM) and/or ARL67156 (100 μM) for 48 h. Values were normalized to those obtained from untreated control cells, set as 100% proliferation rate. Results are mean ± s.e.m. of three independent experiments in triplicate. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 vs control (ANOVA with the Dunnett’s post hoc test); ^###P < 0.001 (ANOVA with the Sidak’s post hoc test).

Figure 2. Involvement of PI3K and PKC in the regulation of P2X7R expression in neuroblastoma cells following serum withdrawal.

(A) N2a cells were incubated in FBS medium for 24 h or in SF medium in absence (control) or presence of LY294002 (50 μM, PI3K inhibitor), U0126 (10 μM, MEK/ERK1/2 inhibitor), H89 (1 μM, PKA inhibitor), GF109203X (5 μM, pan PKC inhibitor) or KN93 (1 μM, CaMKII inhibitor) in SF medium for 24 h. Total RNA was extracted and P2X7 mRNA was quantified by Q-PCR, using GADPH as housekeeping gene. Normalized P2X7 transcript levels in cells cultured in FBS was set as 100%. Results are mean ± s.e.m. of three independent experiments in triplicate. **P ≤ 0.01 vs control (ANOVA with the Dunnett’s post hoc test). (B) Immunoblotting showing the presence of endogenous P2X7R in N2a cells cultured in SF medium for 24 h in the absence (control) or presence of either LY294002 or GF109203X. Whole-cell lysates were analyzed by western blotting with anti-P2X7R antibody. GAPDH was used as internal loading control. Histogram represents levels of P2X7 protein in control cells compared to treated cells and were obtained by densitometry and normalization to GAPDH. Values are mean ± s.e.m. of three independent experiments in duplicate; *P ≤ 0.05, **P ≤ 0.01 vs control (ANOVA with the Dunnett’s post hoc test). (C) Immunofluorescence for P2X7R (green) in N2a cells cultured in SF medium for 24 h in the absence (control) or presence of either LY294002 or GF109203X. Scale bar = 15 μm.

Figure 3. Akt activation induced by serum withdrawal triggers upregulation of P2X7R receptor expression in neuroblastoma cells.

(A) Changes in Akt phosphorylation in N2a cells cultured in SF medium for the indicated time points. Whole-cell lysates were analyzed by western blotting with antibodies against phospho-Akt (Thr³⁰⁸) or total Akt. Histogram represents relative levels of phospho-Akt (pAkt) during the whole detection period, obtained by densitometry and normalization to total Akt. The values represent mean ± s.e.m. of four independent experiments in duplicate. *P ≤ 0.05, **P ≤ 0.01 vs time = 0 (ANOVA with the Dunnett’s post hoc test). (B) N2a cells were incubated in absence or presence of API-1 (10 μM, Akt inhibitor) in SF medium for 24 h. Total RNA was extracted and P2X7 mRNA was quantified by Q-PCR, using GADPH as housekeeping gene. Results are mean ± s.e.m. of three independent experiments in triplicate. ***P ≤ 0.001 vs control (t-test). (C) Immunoblotting depicting the presence of endogenous P2X7R in whole-cell lysates from N2a cells cultured in SF medium for 24 h in the absence (control) or presence of API-1. Whole-cell lysates were analyzed by western blotting with anti-P2X7R antibody. GAPDH was used as internal loading control. Histogram represents levels of P2X7 protein in control cells compared to treated cells, obtained by densitometry and normalization to GAPDH. Values are mean ± s.e.m. of three independent experiments in duplicate; **P ≤ 0.01 vs control (t-test). (D) Immunofluorescence for P2X7R (green) in N2a cells cultured in SF medium for 24 h in the absence (control) or presence of API-1. Scale bar = 15 μm.

Figure 4. Sp1 transcription factor is upregulated in serum-deprived neuroblastoma cells.

(A) Changes in Sp1 transcript levels in N2a cells cultured either in FBS medium for 24 h or in SF medium for the indicated time periods. Total RNA was extracted and Sp1 mRNA was quantified by Q-PCR. GAPDH was used as a control for differences in cDNA input. Results are mean ± s.e.m. of three independent experiments in triplicate. *P ≤ 0.05, ***P ≤ 0.001 vs FBS (ANOVA with the Dunnett’s post hoc test). (B) Immunoblotting depicting the presence of endogenous Sp1 in whole-cell lysates from N2a cells cultured in FBS for 24 or in SF at different time points. Cell lysates were analyzed by western blotting with anti-Sp1 antibody. GAPDH was used as an internal loading control. Histogram represents total Sp1 levels during the whole detection period, obtained by densitometry and normalization to GAPDH. The values represent mean ± s.e.m. of three independent experiments in duplicate. *P ≤ 0.05, **P ≤ 0.01 vs FBS (ANOVA with the Dunnett’s post hoc test). (C) Double immunofluorescence for Sp1 (green) and α-tubulin (red) in N2a cells cultured either in FBS for 24 h or in SF medium for 72 h. Nuclei were counterstained with DAPI (blue). Scale bar = 15 μm.

Figure 5. PI3K/Akt-dependent upregulation of P2X7 expression in serum-deprived neuroblastoma cells requires Sp1 factor.

(A) Immunofluorescence for Sp1 (green) in N2a cells cultured in SF medium for 24 h in the absence (control) or presence of either LY294002 or API-1. Scale bar = 15 μm. (B) Immunoblotting depicting the presence of endogenous Sp1 in nuclear fractions of control and either LY294002- or API-1-treated N2a cells cultured in SF medium for 24 h. Histone H2B was used as nuclear fraction marker. Histogram represents levels of Sp1 protein in control cells compared to treated cells, obtained by densitometry and normalization to H2B. The values represent mean ± s.e.m. of four independent experiments in duplicate. **P ≤ 0.01, ***P ≤ 0.001 vs control (ANOVA with the Dunnett’s post hoc test). (C) Serum-deprived N2a cells were incubated in absence or presence of either LY294002 or API-1 for 24 h. Total RNA was extracted and Sp1 transcript was quantified by Q-PCR, using GADPH as housekeeping gene. Results are mean ± s.e.m. of three independent experiments in triplicate. **P ≤ 0.01, ***P ≤ 0.001 vs control (ANOVA with the Dunnett’s post hoc test). (D) Changes in P2X7 transcript levels in N2a cells cultured either in FBS for 24 h or in SF medium for 48 h in absence (control) or presence of mithramycin A (300 nM, Sp1 inhibitor). Total RNA was extracted and P2X7 mRNA was quantified by Q-PCR, using GADPH as a housekeeping gene. Normalized P2X7 transcript levels in cells cultured in FBS was set as 100%. Results are mean ± s.e.m. of three independent experiments in triplicate; ***P ≤ 0.001 vs FBS (ANOVA with the Dunnett’s post hoc test); ^###P < 0.001 (ANOVA with the Sidak’s post hoc test).

Figure 6. Akt phosphorylation induced by serum withdrawal in neuroblastoma cells is dependent on EGFR activation.

(A) Serum-starved N2a cells were challenged with either vehicle (PBS) or EGF (100 ng/mL) for the indicated time periods. The expression of phospho-Akt (Thr³⁰⁸) and total Akt were detected by immunoblotting of whole-cell extracts. Histogram represents relative levels of phospho-Akt (pAkt) during the whole detection period, obtained by densitometry and normalization to total Akt. The values represent mean ± s.e.m. of four independent experiments in triplicate. *P ≤ 0.05, ***P ≤ 0.001 vs time = 0 (ANOVA with the Dunnett’s post hoc test); ^#P < 0.05 (ANOVA with the Sidak’s post hoc test). (B) Serum-starved N2a cells were incubated either in FBS for 24 h or in SF for 24 h or 48 h in absence (control) or presence of AG147801 (1 μM, EGFR antagonist). Then, total RNA was extracted and P2X7 mRNA was quantified by Q-PCR, using GADPH as a housekeeping gene. Normalized P2X7 transcript levels in cells cultured in FBS medium for 24 h was set as 100%. Results are mean ± s.e.m. of three independent experiments in triplicate; *P ≤ 0.05, **P ≤ 0.01 vs FBS (ANOVA with the Dunnett’s post hoc test); ^#P < 0.05 (ANOVA with the Sidak’s post hoc test). (C) Changes in Akt phosphorylation in N2a cells cultured in either FBS or in SF medium for 30 min in absence (control) or presence of AG147801. Whole-cell lysates were analyzed by western blotting with antibodies against phospho-Akt (Thr³⁰⁸) or total Akt. Histogram represents relative levels of phospho-Akt (pAkt) obtained by densitometry and normalization to total Akt. The values represent mean ± s.e.m. of three independent experiments in duplicate. **P ≤ 0.01 vs FBS (ANOVA with the Dunnett’s post hoc test); ^#P < 0.05 (ANOVA with the Sidak’s post hoc test).

Figure 7. PKCζ decreased P2rx7 gene expression by preventing Akt activation in serum deprived neuroblastoma cells.

(A) Changes in P2X7 transcript levels in serum-deprived N2a cells cultured for 48 h in absence or presence of 10 μM GF109203X and/or 300 nM mithramycin A (Sp1 inhibitor). GADPH was used as housekeeping gene. Results are mean ± s.e.m. of three independent experiments in triplicate; **P ≤ 0.01 vs control (ANOVA with the Dunnett’s post hoc test); ^###P ≤ 0.001 (ANOVA with the Sidak’s post hoc test). (B) Serum-deprived N2a cells were incubated for 24 h in absence (control) or presence of LY294002 (50 μM), API-1 (10 μM) and/or GF109203X (10 μM). P2X7 mRNA was quantified by Q-PCR, using GADPH as housekeeping gene. Results are mean ± s.e.m. of three independent experiments in triplicate. **P ≤ 0.01, ***P ≤ 0.001 vs control (ANOVA with the Dunnett’s post hoc test); ^###P ≤ 0.001 (ANOVA with the Sidak’s post hoc test). (C) Immunoblotting depicting the presence of Sp1 in nuclear fractions of N2a cells cultured for 24 h in SF medium in absence (control) or presence of LY294002 (50 μM) and/or GF109203X (10 μM). Histone H2B was used as nuclear fraction marker. Histogram represents levels of Sp1 protein obtained by densitometry and normalization to H2B expression. The values represent mean ± s.e.m. of three independent experiments in duplicate. *P ≤ 0.05, ***P ≤ 0.001 vs control (ANOVA with the Dunnett’s post hoc test); ^###P ≤ 0.001 (ANOVA with the Sidak’s post hoc test). (D) Immunofluorescence for Sp1 (green) in N2a cells cultured in SF medium for 24 h in the absence (control) or presence of LY294002 and/or GF109203X. Scale bar = 15 μm. (E) Serum-starved N2a cells were challenged with either vehicle (PBS) or GF109203X for the indicated time periods. The expression of phospho-Akt (Thr³⁰⁸) and total Akt were detected by immunoblotting of whole-cell extracts. Histogram represents relative levels of phospho-Akt (pAkt) obtained by densitometry and normalization to total Akt levels. The values represent mean ± s.e.m. of four independent experiments in duplicate. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 vs t = 0 (ANOVA with the Dunnett’s post hoc test); ^#P ≤ 0.05, ^##P ≤ 0.01, ^###P ≤ 0.001 (ANOVA with the Sidak’s post hoc test).

Figure 8. Serum withdrawal upregulates pro-survival P2X7R in neuroblastoma cells.

Serum starvation triggers EGF-independent activation of EGFR and, consequently, activation of PI3K/Akt pathway, resulting in Sp1 phosphorylation and induction of P2rx7 gene expression. Moreover, PKCζ activation reduces Akt phosphorylation, thereby providing a negative feedback loop to regulate Akt activity. Higher levels of P2×7R allow cell proliferation of N2a cells even in the absence of trophic support.