Figure 1.
IGF-1 treatment up-regulates PRNP expression at both the protein and mRNA levels.
(A) and (B) Western blotting analysis for the IGF-1-induced up-regulation of PrPC both in conditions of 10% FBS and serum starvation. Compared with the control group, 100 ng/ml IGF-1 treatment for 24 hours increased the PrPC protein levels in the HeLa and SH-SY5Y cell lines. (C) Quantitative analysis of the IGF-1-induced increase of the PRNP mRNA levels in the SH-SY5Y (black columns) and HeLa (white columns) cell lines. The level of PRNP mRNA was normalized to that of GAPDH mRNA and expressed as a fold change compared to the mRNA level in the untreated group. All experiments were performed three to five times, and the data were analyzed as the mean ±S.E.M. (P<0.05).
Figure 2.
IGF-1-induced enhancement of PRNP expression depends on the activation of PI3K-Akt signaling pathway.
(A) Variation in the level of PRNP expression in HeLa and SH-SY5Y cells treated with different kinase inhibitors. Compared with the control DMSO group (lanes 1 and 5), the PI3K-Akt inhibitor LY294002 treatment group (lanes 2 and 6) demonstrated a notable reduction in the PrPC protein level. In contrast, the MEK1/2 inhibitor U0126 (lanes 3 and 7) and the JNK inhibitor SP600125 (lanes 4 and 8) had little effect on the PRNP expression. (B) Similar results were obtained when the HeLa and SH-SY5Y cell lines were co-treated with IGF-1 and inhibitors. The LY294002-treated cells (lanes 2 and 6) showed a remarkable decrease in the PrPC protein level compared with the DMSO-treated group (lanes 1 and 5). In contrast, the U0126-treated groups (lanes 3 and 7) and the SP600125-(lanes 4 and 8) showed no obvious variation. All experiments were performed three times.
Figure 3.
FOXO3a negatively regulates PRNP expression at both the mRNA and protein levels.
(A) Over-expression of FOXO1 (lane 2) and FOXO3a (lane 3), as detected by western blotting, induced a reduction of PrP protein levels in HeLa cells. (B) Western blotting showing that IGF-1 and LY294002 have an opposite function on the decrease of PrP protein levels induced by FOXO proteins over-expression. LY294002 treatment enhanced the reduction of PrP protein levels triggered by FOXO proteins, especially FOXO3a (lanes 1, 2 and 3). However, 100 ng/ml IGF-1 treatment impaired the negative regulation of FOXO proteins on PRNP (lanes 4, 5 and 6). (C) and (D) Western blotting and real-time PCR showing the efficiency of siFOXO1 and siFOXO3a in HeLa cells. Compared with the negative control groups, FOXO1 and FOXO3a were efficiently knocked-down. All experiments were performed at least three times, and the data were analyzed as the mean ± S.E.M. (P<0.05). (E) and (F) Western blotting and real-time quantitative PCR showing that the knock-down of FOXO1 and FOXO3a by targeted siRNAs affects PRNP expression at both the protein and mRNA levels. Compared with the negative control group, the knock-down of FOXO3a increased the PrPC protein level remarkably, after normalization to actin, whereas the knock-down of FOXO1 had only a slight effect on the PrPC protein level. A similar result was obtained at the mRNA level. All experiments were performed at least three times, and the data were analyzed as the mean ± S.E.M. (P<0.05).
Figure 4.
FOXO3a down-regulates PRNP expression by binding to its promoter.
(A) Two potential FOXO3a binding sites in the human PRNP promoter (dark background) at −1299 to −1292 and −1177 to −1170. (B) Potential FOXO3a binding sites in the Ovis aries and Bos taurus PRNP promoters (dark background). (C) and (D) EMSA showing that FOXO3a can bind to the human PRNP promoter. Compared with the control (lanes 1 and 2), FOXO3a bound to the PRNP promoter in a dose-dependent manner (lanes 3, 4 and 5), and this binding was abolished by a 50-fold excess of unlabeled probes (lanes 6 and 7) and mutation of the FOXO binding site (lanes 8 and 9) results in low affinity for FOXO3a. (E) Luciferase assay confirming that the human PRNP promoter recombinant has promoter-like activity. The pGL3-Basic and pGL3-Control plasmids were used as negative and positive controls, respectively. (F) Luciferase assay showing that FOXO1 and FOXO3a reduce the promoter activity of the PRNP promoter in the HeLa cell line. Compared with the control, the co-transfection with FOXO1 and FOXO3a led to a 10–30% decrease in luciferase units in 10% FBS medium, whereas a 30–45% decrease was detected when the cells were serum starved. The ‘relative luciferase unites’ stands for the ratio of firefly/renilla. All experiments were performed three to five times, and the data were analyzed as the mean ± S.E.M. (P<0.05).
Figure 5.
IGF-1 induces an increase in PRNP expression through the PI3K-Akt-FOXO3a pathway.
(A) and (B) Translocation of FOXO3a induced by different treatments. Before the treatments, GFP-FOXO3a was located predominantly in the cytoplasm. In contrast, serum starvation or treatment with LY294002 induced the nuclear translocation of GFP-FOXO3a, whereas treatment with SP600125 or U0126 had no effect on the GFP-FOXO3a translocation. However, IGF-1 was able to induce the cytoplasmic retention of GFP-FOXO3a even under serum-free conditions, whereas treatment with LY294002 led to the nuclear retention of GFP-FOXO3a even in the presence of IGF-1. (C) and (D) Western blotting showing the relationship between the phosphorylation of FOXO3a and PRNP expression. Treatment with 50 and 100 ng/ml IGF-1 remarkably increased the phosphorylation of FOXO3a in both the SH-SY5Y and HeLa cell lines but caused little variation in the total level of FOXO3a. In accordance with this increase in the phosphorylation of FOXO3a, the PrPC protein level was also increased. All protein levels were normalized to actin. (E) Western blotting showing the relationship between the dephosphorylation of FOXO3a and PRNP expression. The phosphorylation of FOXO3a was maximally inhibited after treatment with LY294002 for 1 hour, and this inhibition was gradually reduced after 4 hours. In addition, the PrPc protein levels began to decrease after treatment with LY294002 for 2 hours, with the maximal decrease appearing after 24 hours. (F) Quantitative PCR showing the variation in the PRNP mRNA levels after treatment with LY294002 for different times. No notable changes were detected after 1 hour of treatment, but the PRNP mRNA level gradually decreased over time, and an approximately 50% decrease was observed after 24 hours of treatment. All experiments were performed three times, and the data were analyzed as the mean ± S.E.M. (P<0.05).
Figure 6.
Model for the regulation of PRNP in response to IGF-1.
After cells are exposed to IGF-1, IGF-1R is activated, which then activates the PI3K-Akt pathway. The activated Akt then phosphorylates FOXO3a, leading to its translocation from the nucleus to the cytoplasm and thereby preventing FOXO3a from binding to the PRNP promoter (black arrows and streaks). With no or low levels of IGF-1 or in the presence of LY294002, the negative regulation of PI3K-Akt on FOXO3a is impaired. Then, FOXO3a is able to relocate to the nucleus, where it binds to the PRNP promoter and negatively regulates PRNP expression (red arrows and streaks).