Ceramide Kinase Inhibition Blocks IGF-1-Mediated Survival of Otic Neurosensory Progenitors by Impairing AKT Phosphorylation

Abstract

Sphingolipids are bioactive lipid components of cell membranes with important signal transduction functions in health and disease. Ceramide is the central building block for sphingolipid biosynthesis and is processed to form structurally and functionally distinct sphingolipids. Ceramide can be phosphorylated by ceramide kinase (CERK) to generate ceramide-1-phosphate, a cytoprotective signaling molecule that has been widely studied in multiple tissues and organs, including the developing otocyst. However, little is known about ceramide kinase regulation during inner ear development. Using chicken otocysts, we show that genes for CERK and other enzymes of ceramide metabolism are expressed during the early stages of inner ear development and that CERK is developmentally regulated at the otic vesicle stage. To explore its role in inner ear morphogenesis, we blocked CERK activity in organotypic cultures of otic vesicles with a specific inhibitor. Inhibition of CERK activity impaired proliferation and promoted apoptosis of epithelial otic progenitors. CERK inhibition also compromised neurogenesis of the acoustic-vestibular ganglion. Insulin-like growth factor-1 (IGF-1) is a key factor for proliferation, survival and differentiation in the chicken otocyst. CERK inhibition decreased IGF-1-induced AKT phosphorylation and blocked IGF-1-induced cell survival. Overall, our data suggest that CERK is activated as a central element in the network of anti-apoptotic pro-survival pathways elicited by IGF-1 during early inner ear development.

Keywords: NVP-231; PF-543; ceramide metabolism enzymes; development; otic progenitors; sphingolipids.

FIGURE 1

Gene expression of enzymes involved in ceramide processing and IGF1 expression in early inner ear development in chicken. (A) Scheme of the main ceramide metabolism enzymes (upper part) and their gene expression at HH18 related to CERK. Data are represented as the mean ± SEM of technical triplicates of five independent experiments. (B) Expression of CERK and (C) IGF1 from otic vesicle pools obtained from HH17 to HH21 chicken embryos normalized to HH17. Data are represented as mean ± SEM of three independent experiments performed in technical triplicates. (D) Schematic cartoon showing the aspect of the ex vivo organotypic culture model of otic vesicles dissected from HH18 embryos. The culture mimics in vivo otic vesicle (OV) morphogenesis and the development of the acoustic-vestibular ganglion (AVG) in the ventromedial region. (A), anterior; (D), dorsal. (E) CERK otic vesicle expression levels after 4 h ex ovo culture in serum-free medium, 1 μM CKi, 10 nM IGF-1, or IGF-1 plus CKi. Data are represented as mean ± SEM of a technical triplicate from otic vesicle pools (n = 15). Statistical significance was calculated by one-way ANOVA followed by Dunnett’s T3 (A,B) or Bonferroni’s (E) post hoc test. (A) *p < 0.05, ***p < 0.001 vs CERK; (B) *p < 0.05 vs HH17; (E) **p < 0.01, ***p < 0.001 vs control and ^#p < 0.05 vs IGF-1.

FIGURE 2

The ceramide rheostat supports ex ovo inner ear morphogenesis. (A) Otic vesicles were dissected from HH18 embryos and cultured for 20 h in serum-free medium (SFM), 1 μM CKi, 10 nM IGF-1 alone or with CKi. Morphological otic vesicle aspect and immunofluorescence staining for TUNEL (cyan) and PH3 (red) of at least 4 otic vesicle per condition from three independent experiments are shown. Scale bar: 150 μm. Quantification of (B) otic vesicle areas, (C) TUNEL-positive and (D) PH3-labeled cells with CKi (0.1 and 1 μM) with or without IGF-1. Results are shown as the mean ± SEM relative to the SFM condition. (E) Effect of CKi (1 μM) on PCNA protein levels from otic vesicles cultured and processed for western blotting. The results are presented as the mean ± SEM of three independent experiments with n = 1 otic vesicle per condition relative to the IGF-1 data. A representative blot is shown above. (F) Quantification of EdU-positive cells obtained from confocal images and normalized to data from SFM (the first bar). Data are represented as mean ± SEM of n = 7 otic vesicles per condition. (G) Effect of CKi on the activation of AKT (Ser-473, pAKT) determined by western blotting. The results are presented as the mean ± SEM of four independent experiments with n = 15 otic vesicles per condition relative to the IGF-1 data. Representative blots are shown above. Statistical significance was calculated by one-way ANOVA followed by Dunnett’s T3 post hoc test (B–D,G) or Bonferroni’s post hoc test (E,F). *p < 0.05, **p < 0.01, ***p < 0.001 vs control; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs IGF-1.

FIGURE 3

CERK inhibition disrupts early otic neurogenesis. (A) Confocal images of representatives otic vesicles immunostained with SOX2 (red) or G4 (white) after 20 h of culture in serum-free medium (SFM), 1 μM CKi, 10 nM IGF-1, or IGF-1 plus CKi. Scale bar: 150 μm. Histograms represent the quantification of the AVG size (B), SOX2 levels in otic vesicle (C) and G4 levels in AVG (D) normalized to data for SFM. Data are represented as mean ± SEM of two independent experiments, n = 8 otic vesicles per condition. (E) Magnification showing the AVG area with arrowheads pointing to neurite processes (white). Scale bar: 50 μm. (F) Histogram represents neurite length mean ± SEM from n = 8 otic vesicles per condition. Statistical significance was calculated by one-way ANOVA followed by Bonferroni’s post hoc test (B–D) or Dunnett’s T3 post hoc test (F). *p < 0.05, **p < 0.01, ***p < 0.001 vs control; ^###p < 0.001 vs IGF-1.

FIGURE 4

Inhibition of endogenous SPHK1 has no effect on early inner ear development. (A) Morphological aspect of organotypic cultures after 20 h in serum-free medium (SFM), 1 μM PF-543, 10 nM IGF-1, IGF-1 plus PF-543. Immunostaining with TUNEL (cyan) and PH3 (red) of at least 5 otic vesicle per condition. Scale bar: 150 μm. (B) Quantification of otic vesicle area, (C) TUNEL-positive or (D) PH3-labeled cells in the presence or absence of PF-543 (0.25 and 1 μM) and IGF-1 (10 nM). (E) SOX2 intensity (F) and Tuj-1 intensity as neurogenesis markers normalized to data for SFM. Data are represented as mean ± SEM from at least n = 4 otic vesicles per condition. Statistical significance was calculated by one-way ANOVA followed by Bonferroni’s (B,E) or Dunnett’s T3 post hoc test (C). *p < 0.05, **p < 0.01, ***p < 0.001 vs control.