IP3 3-kinase opposes NGF driven neurite outgrowth

Abstract

The inositol (1,4,5) trisphosphate 3-kinases comprise a family of enzymes (A, B, and C) that phosphorylate the calcium mobilising molecule inositol (1,4,5) trisphosphate (IP(3)) to generate inositol (1,3,4,5) tetrakisphosphate. This molecule can function as a second messenger, but its roles are not completely understood. The A isoform of inositol (1,4,5) trisphosphate 3-kinase localises to filamentous actin within dendritic spines in the hippocampus and is implicated in the regulation of spine morphology and long term potentiation, however the mechanisms through which it signals in neuronal cells are not completely understood. We have used NGF driven neurite outgrowth from PC12 cells as a platform to examine the impact of signaling via inositol (1,4,5) trisphosphate 3-kinase activity in a neuronal cell. We have found that the catalytic activity of the enzyme opposes neurite outgrowth, whilst pharmacological inhibition of inositol (1,4,5) trisphosphate 3-kinase leads to a significant increase in neurite outgrowth, and we show that the reduction in neurite outgrowth in response to inositol (1,4,5) trisphosphate 3-kinase activity correlates with reduced ERK activity as determined by western blotting using phosphorylation-specific antibodies. Our findings suggest a novel neuronal signaling pathway linking metabolism of IP(3) to signaling via ERK.

Figure 1. IP₃ 3-KA co-localises with F-actin at the PC12 cell growth cone, and inhibits neurite outgrowth.

(A) Growth cone of PC12 cell stably expressing high levels of IP₃ 3-KA-GFP, fixed ten days after culture in the presence of 100 ng/ml NGF, and labeled with phalloidin-594. IP₃ 3-KA-GFP co-localises with F-actin. Scale bar is 10 µm. (B) Quantitative analysis of the effects of IP₃ 3-KA-GFP expression on neurite outgrowth. Neurite outgrowth was quantified using the Cellomics Arrayscan Neurite Outgrowth algorithm to measure the percentage of cells with neurites, and average neurite length (described in full in materials and methods). IP₃ 3-KA expression results in an inhibition of neurite outgrowth. Data are presented as mean +/− SEM. ***p<0.001, by ANOVA with post-hoc analysis. (C) Arrayscan images of PC12 cells expressing varying levels of IP₃ 3-KA-GFP, fixed four days after culture in the presence of 100 ng/ml NGF, and labeled using Hoescht 33342 and the Cellomics Neurite Outgrowth HitKit to identify neuritis. Scale bar is 100 µm.

Figure 2. The catalytic activity of IP₃ 3-KA opposes NGF-driven neurite outgrowth from PC12 cells.

(A) Localisation of mutant IP₃ 3-KA constructs in PC12 cell neurites. IP₃ 3-KA D260A/K262A-GFP is enriched at the growth cone, whilst IP₃ 3-KA 66-459-GFP is not. Scale bar is 10 µm. (B) Quantitative analysis of the effects of mutated IP₃ 3-KA expression on neurite outgrowth. D260A/K262A-GFP overexpression results in an increase in the number of cells with neurites and decreases neurite length. IP₃ 3-KA 66-459-GFP expression results in a decrease in both indices of neurite outgrowth. Data are presented as mean +/− SEM. *p<0.05, ***p<0.001, by ANOVA with post-hoc analysis. (C) Arrayscan images of PC12 cells expressing mutant IP₃ 3-KA-GFP, fixed four days after culture in the presence of 100 ng/ml NGF, and labeled using Hoescht 33342 and the Cellomics Neurite Outgrowth HitKit to identify neuritis. Scale bar is 100 µm.

Figure 3. Inhibition of IP₃ 3-kinase increases neurite outgrowth from PC12 cells, and IP₃ 3-KA-PC12 cells.

(A) Analysis of the effects of C5 on wild type PC12 cells and cells expressing high levels of IP₃ 3-KA. Differentiating PC12 cells in the presence of C5 results in an increase in two indices of neurite outgrowth. Data are presented as mean +/− SEM. ***p<0.001, by ANOVA with post-hoc analysis. (B) Arrayscan images of PC12 cells expressing high levels of IP₃ 3-KA, fixed four days after culture in the presence of 100 ng/ml NGF, and labeled using Hoescht 33342 and the Cellomics Neurite Outgrowth HitKit to identify neurites (using a 10× objective). (C) Arrayscan images of PC12 cells, fixed four days after culture in the presence of 100 ng/ml NGF, and labeled as for (B). Scale bar is 100 µm.

Figure 4. IP₃ 3-KA activity results in attenuation of ERK phosphorylation.

IP₃ does not appear to be required for NGF driven neurite outgrowth. (A) Analysis of the effects of XeC blockade of the IP₃ receptor, on PC12 cells expressing high levels of IP₃ 3-KA and on wild type PC12 cells. XeC has no effect on control PC12 cells, but partially overcomes the inhibition of neurite outgrowth caused by IP₃ 3-KA expression. Data are presented as mean +/− SEM. **p<0.01, by ANOVA with post-hoc analysis. (B) Determination of ERK activation in control PC12 cells and IP₃ 3-KA expressing cells using a suboptimal dose of NGF, in the presence of either 40 µM C5 or vehicle. Lower blot represents ERK activation as detected by phospho-specific antibodies against ERK1 and ERK2. Upper blot represents total ERK, as detected using an antibody that detects both ERK1 and ERK2. All antibodies were visualised with an HRP conjugated secondary antibody. The blot shown is representative of three independent experiments. (C) Densitometric quantification of ERK activation, as depicted in (B). **p<0.01, Data are presented as mean +/− SEM. ***p<0.001, by ANOVA with post-hoc analysis. IP₃ 3-KA expression attenuates ERK activation, whilst C5 inhibition of IP₃ 3-kinase results in increased ERK activity.