Abstract
Bone marrow stromal cells (BMSCs) are considered as candidates for regenerative therapy and a useful model for studying neuronal differentiation. The role of basic fibroblast growth factor (bFGF) in neuronal differentiation has been previously studied; however, the signaling pathway involved in this process remains poorly understood. In this study, we investigated the signaling pathway in the bFGF-induced neuronal differentiation of canine BMSCs. bFGF induced the mRNA expression of the neuron marker, microtubule associated protein-2 (MAP2) and the neuron-like morphological change in canine BMSCs. In the presence of inhibitors of fibroblast growth factor receptors (FGFR), phosphatidylinositol 3-kinase (PI3K) and Akt, i.e., SU5402, LY294002, and MK2206, respectively, bFGF failed to induce the MAP2 mRNA expression and the neuron-like morphological change. bFGF induced Akt phosphorylation, but it was attenuated by the FGFR inhibitor SU5402 and the PI3K inhibitor LY294002. In canine BMSCs, expression of FGFR-1 and FGFR-2 was confirmed, but only FGFR-2 activation was detected by cross-linking and immunoprecipitation analysis. Small interfering RNA-mediated knockdown of FGFR-2 in canine BMSCs resulted in the attenuation of bFGF-induced Akt phosphorylation. These results suggest that the FGFR-2/PI3K/Akt signaling pathway is involved in the bFGF-induced neuronal differentiation of canine BMSCs.
Highlights
Neuronal differentiation is a complex process coordinated by the down-regulation of pluripotent genes and concomitant up-regulation of neuron-specific lineage genes
We have previously reported that mRNA expression of the neuron marker microtubule associated protein-2 (MAP2) and neuronlike morphology were observed in basic fibroblast growth factor (bFGF)-induced neuronal cell differentiation of canine Bone marrow stromal cells (BMSCs) by bFGF [28]
To elucidate the signaling pathway involved in bFGF-induced neuronal differentiation of canine BMSCs, we first examined the effect of the fibroblast growth factor receptors (FGFR) inhibitor SU5402 (20 μM) on bFGF-induced MAP2 mRNA expression. bFGF significantly induced MAP2 mRNA expression in the absence of this inhibitor, but its effect was attenuated in the presence of the inhibitor (Fig 1A)
Summary
Neuronal differentiation is a complex process coordinated by the down-regulation of pluripotent genes and concomitant up-regulation of neuron-specific lineage genes. Established cell culture models are frequently used to study in vitro neuronal differentiation. These models exhibit neuron-like morphology and express neuronal marker mRNAs and proteins in response to several neurotrophins, growth factors, and chemical compounds. Rat adrenal pheochromocytoma cells (PC12) differentiate into sympathetic neuron-like cells, which are characterized by neurite outgrowth, electrical excitability, and expression of neuronal markers in response to nerve growth factor (NGF) [1,2,3]. Mouse neuroblastoma cells (Neuro-2A) exhibited neuron-like morphology in the presence of the cannabinoid receptor agonist HU210 [4, 5]. In human neuroblastoma cell lines (SK-N-SH, BE(2)-C, and NB1643), all-trans retinoic acid induced neurite outgrowth and expression of neuronal markers [6, 7]. Adult tissue stem cells have been reported to possess neuronal differentiation potency, and considered as a useful tool for neuronal differentiation research [8, 9]
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