SP600125 Enhances Temperature-Controlled Repeated Thermal Stimulation-Induced Neurite Outgrowth in PC12-P1F1 Cells

Abstract

This study evaluated the mechanism of temperature-controlled repeated thermal stimulation (TRTS)-mediated neuronal differentiation. We assessed the effect of SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, on neuronal differentiation of rat PC12-P1F1 cells, which can differentiate into neuron-like cells by exposure to TRTS or neurotrophic factors, including bone morphogenetic protein (BMP) 4. We evaluated neuritogenesis by incubating the cells under conditions of TRTS and/or SP600125. Cotreatment with SP600125 significantly enhanced TRTS-mediated neuritogenesis, whereas that with other selective mitogen-activated protein kinase (MAPK) inhibitors did not—e.g., extracellular signal-regulated kinase (ERK)1/2 inhibitor U0126, and p38 MAPK inhibitor SB203580. We tried to clarify the mechanism of SP600125 action by testing the effect of U0126 and the BMP receptor inhibitor LDN193189 on the SP600125-mediated enhancement of intracellular signaling. SP600125-enhanced TRTS-induced neuritogenesis was significantly inhibited by U0126 or LDN193189. Gene expression analysis revealed that TRTS significantly increased β3-Tubulin, MKK3, and Smad7 gene expressions. Additionally, Smad6 and Smad7 gene expressions were substantially attenuated through SP600125 co-treatment during TRTS. Therefore, SP600125 may partly enhance TRTS-induced neuritogenesis by attenuating the negative feedback loop of BMP signaling. Further investigation of the mechanisms underlying the effect of SP600125 during TRTS-mediated neuritogenesis may contribute to the future development of regenerative neuromedicine.

Physical Stimulation Methods Developed for In Vitro Neuronal Differentiation Studies of PC12 Cells: A Comprehensive Review

“…The two subclones were derived from PC12 parental cells based on their TRTS sensitivity during TRTS-mediated neuronal differentiation. Based on the new evidence acquired from the experiments using these novel subclones, the authors suggested that the BMP pathway may be required for TRTS-induced neurite outgrowth [4,7].…”

“…It can form synapses with primary cultured neurons isolated from the rat cerebral cortex [3]. PC12 cells differentiate into neuron-like cells when exposed to neurotrophic factors, including nerve growth factor (NGF) [2,4]. NGF binds to its membrane receptor, tropomyosin-related kinase A (TrkA), which is expressed in these cells.…”

“…The activation of ERK1/2 results in the elongation of neurites and the development of neuron-like characteristics in PC12 cells. NGF-induced differentiation also requires p38 MAPK, another member of the MAPK family, which may cooperate with ERK1/2 for the promotion of differentiation [4,7] (Figure 1).…”

“…PC12 cells have also been used as a suitable in vitro model to examine the effects of various types of physical stimuli on neuronal cell differentiation since the cell line was established in 1976 [1,4]. However, the development of effective methods for inducing neuronal differentiation in PC12 cells through physical means and the understanding of the underlying molecular mechanisms is ongoing.…”