Abstract
To promote the functional restoration of the nervous system following injury, it is necessary to provide optimal extracellular signals that can induce neuronal regenerative activities, particularly neurite formation. This study aimed to examine the regulation of neuritogenesis by temperature-controlled repeated thermal stimulation (TRTS) in rat PC12 pheochromocytoma cells, which can be induced by neurotrophic factors to differentiate into neuron-like cells with elongated neurites. A heating plate was used to apply thermal stimulation, and the correlation of culture medium temperature with varying surface temperature of the heating plate was monitored. Plated PC12 cells were exposed to TRTS at two different temperatures via heating plate (preset surface temperature of the heating plate, 39.5°C or 42°C) in growth or differentiating medium for up to 18 h per day. We then measured the extent of growth, neuritogenesis, or acetylcholine esterase (AChE) activity (a neuronal marker). To analyze the mechanisms underlying the effects of TRTS on these cells, we examined changes in intracellular signaling using the following: tropomyosin-related kinase A inhibitor GW441756; p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580; and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126 with its inactive analog, U0124, as a control. While a TRTS of 39.5°C did not decrease the growth rate of cells in the cell growth assay, it did increase the number of neurite-bearing PC12 cells and AChE activity without the addition of other neuritogenesis inducers. Furthermore, U0126, and SB203580, but not U0124 and GW441756, considerably inhibited TRTS-induced neuritogenesis. These results suggest that TRTS can induce neuritogenesis and that participation of both the ERK1/2 and p38 MAPK signaling pathways is required for TRTS-dependent neuritogenesis in PC12 cells. Thus, TRTS may be an effective technique for regenerative neuromedicine.
Highlights
Neurite outgrowth is a key process in the development of functional neuronal circuits and the regeneration of the nervous system following injury
To examine the effects of temperature-controlled repeated thermal stimulation (TRTS) on the viability of PC12 cells, we investigated the effect of the two TRTS treatments on cell proliferation in a growth medium
PC12 cells were incubated in growth medium for 7 days and exposed to TRTS for a total of 18 h per day, or left untreated for 7 days, and the number of cells attached to the bottom of the well in each case was evaluated on the indicated days
Summary
Neurite outgrowth is a key process in the development of functional neuronal circuits and the regeneration of the nervous system following injury. To improve the outcomes of individuals with neurodegenerative diseases and injury, it is necessary to understand and develop optimal extracellular signals that can induce neuronal regenerative activities, those that enhance cellular neurogenesis [1,2,3]. Treatment of PC12 cells with NGF induces activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), which are part of the mitogen-activated protein kinase (MAPK) family, via activation of the NGF receptor tropomyosin-related kinase A (TrkA). Activation of ERK1/2 leads to neurite elongation and development of neuron-like phenotypic characteristics in PC12 cells [15,16]. Differentiation via NGF requires the participation of p38 MAPK, another MAPK family member, which is mediated by ERK1/2 [17,18]
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