Eased basal Erk phosphorylation and blunted the response to FGF2 treatment (Figure 5A). To investigate the contribution of FGF signaling pathways to TRIII/CDK2 list FGF2-induced neuronal differentiation, we blocked FGF receptor kinase activity with VEGFR1/Flt-1 Storage & Stability pharmacologic inhibitors (PD-173074, SU-5402) or a dominant-negative FGFR1 construct (ref. 42; Figure 5, B and C; and Supplemental Figure five, B and D). In all situations, inhibition of FGF receptor tyrosine kinase function attenuated the differentiating effects of TRIII expression in the presence and absence of exogenous FGF2. Similarly, pharmacologic inhibition of downstream MEK/Erk MAPK signaling with U0126 and CI-1030 attenuated the differentiating effects of TRIII expression in the presence and absence of ligand (Figure 5B and Supplemental Figure 5, C and D). These benefits demonstrate that TRIII and its GAG chains market neuronal differentiation and enhance FGF2-induced differentiation in NB cells by way of FGF receptors and downstream Erk MAPK signaling. T RIII and FGF2 cooperate to induce Id1 expression. Related to previous function demonstrating that FGF2 promotes differentiation of neural crest erived cells through Erk MAPK and also the transcription issue inhibitor of DNA binding 1 (Id1) (30), we located that FGF2 induced Id1 protein expression in NB cells inside 1 hour of remedy, followed by a gradual lower in expression (Figure 6A). Interestingly, TRIII knockdown fully abrogated FGF2induced Id1 expression. We also observed increases in Id1 protein levels in response to FGF2 more than the longer time course of neuronal differentiation; this raise was inhibited by TRIII knockdown and might be rescued by restoring TRIII expression with GAG modifications (Figure 6B). Likewise, basal Id1 expression and FGF2-induced increases in Id1 expression have been enhanced by TRIII overexpression within a GAG-dependent manner (Supplemental Figure 5E). TRIII- and FGF2-induced Id1 expression changes have been abroVolume 123 Quantity 11 November 2013http://jci.orgresearch articleFigureTRIII promotes neuronal differentiation of NB cells. Transient transductions with TRIII-GFP, GFP handle, nontargeted control shRNA (shNTC), or shRNA to TRIII (shTRIII). (A) Phase microscopy of 5Y cells 96 hours immediately after plating. Original magnification, 0; scale bar: 100 M. (B) Time course of 5Y cell neurite length (mean of 3 fields SEM). Adenoviral transduction at 24 hours. P 0.0001 for key effects of time and receptor expression (2-way ANOVA); interaction P 0.05; P 0.05, P 0.01, P 0.001 (Bonferroni post-hoc comparisons shown for TRIII-GFP compared to GFP and handle). (C) 5Y cell neurite length (imply of 3 fields SEM) following 96 hours of TRIII knockdown. P 0.0001 (2-tailed Student’s t test). (D) Western blot for neurofilament 160 kDa (NF160), tyrosine hydroxylase (TH), neuron-specific enolase (NSE), 3-tubulin, and GAP43 just after 96-hour transduction. Densitometry for NF160 normalized to -actin is shown as % handle. (E) Quantification of differentiation markers from three independent experiments in 5Y cells normalized to -actin (mean improve above manage SEM). P 0.05 for all markers (1-sample Student’s t test). (F) Differentiation markers following 72-hour TRIII knockdown and rescue with knockdown-resistant rat TRIII (rTRIII). Densitometry for NF160 normalized to -actin is shown as % control. (G) Quantification of NF160 from three independent experiments (imply SEM) in SHEP cells normalized to -actin. P 0.05 (1-sample t test and 2-tailed Student’s t test). (H.
