Yoshihisa Koike

Heat shock induces neurite outgrowth in PC12m3 cells via the p38 mitogen-activated protein kinase pathway

We investigated the role of the p38 mitogen-activated protein kinase (MAPK) pathway in heat-shock-induced neurite outgrowth of PC12 mutant cells in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of the PC12 mutant (PC12m3) cells were exposed to heat stress at 44 degrees C for 10 min, activity of p38 MAPK increased and neurite outgrowth was greatly enhanced. The neurite extension was inhibited by the p38 MAPK inhibitor BS203580. Longer heat treatment of PC12m3 cells provoked cell death, which was enhanced by SB203580. These findings suggest that heat-induced activation of p38 MAPK is responsible for the neurite outgrowth and survival of PC12m3 cells.

Osmotic shock-induced neurite extension via activation of p38 mitogen-activated protein kinase and CREB.

Although it is known that sustained activation of classical mitogen-induced protein kinase (MAPK, also known as ERK) induced by nerve growth factor (NGF) plays an important role in the induction of neurite outgrowth, the role of p38 MAPK in neural cell function is still not clear. We developed two neuronal cell lines from PC12 cells, PC12m3 and PC12m32, in which NGF-induced neurite outgrowth is impaired and that show neurite outgrowth in response to hyperosmotic shock. The frequencies of neurite outgrowth of PC12m3 and PC12m32 cells induced by osmotic shock were approximately 10- and 12-fold greater, respectively, than that in PC12 parental cells. The p38 MAPK pathway inhibitor SB203580 but not the ERK pathway blocker U0126 inhibited the ability of PC12m3 and PC12m32 cells to induce neurite outgrowth in response to osmotic shock. Furthermore, expression of a nonactivable form of p38 but not that of wild-type p38 significantly blocked neurite outgrowth induced by osmotic shock. The extent of phosphorylation of p38 MAPK induced by osmotic shock in PC12m32 cells was much greater than that in PC12 parental cells. The upstream kinases MKK3 and MKK6, which phosphorylate and activate p38 MAPK, also showed higher levels in PC12m32 cells than in PC12 parental cells when treated with osmotic shock. Inhibition of p38 MAPK by SB203580 resulted in inhibition of the activity of the transcription factor CREB, which is activated by osmotic shock. These findings indicate that activation of CREB mediated by a p38 pathway distinct from the NGF signaling pathway may be required for neurite outgrowth.

Microwave irradiation induces neurite outgrowth in PC12m3 cells via the p38 mitogen-activated protein kinase pathway

The increasing use of mobile phone communication has raised concerns about possible health hazard effects of microwave irradiation. We investigated damage and differentiation caused by microwave irradiation on drug-hypersensitive PC12 cell line (PC12m3). These cells showed enhancement of neurite outgrowth to various stimulants. The frequency of neurite outgrowth induced by 2.45 GHz (200 W) of microwave irradiation was approximately 10-fold greater than that of non-irradiated control cells. Incubation of PC12m3 cells with SB203580, a specific inhibitor of p38 MAPK, resulted in marked inhibition of the microwave radiation-induced neurite outgrowth. Also, activation of the transcription factor CREB induced by microwave irradiation was inhibited by SB203580. Heat shock treatment at 45 degrees C had a strong toxic effect on PC12m3 cells, whereas microwave treatment had no toxic effect on PC12m3 cells. These findings indicate that p38 MAPK is responsible for the survival of PC12m3 cells and might induce neurite outgrowth via a CREB signaling pathway when subjected to microwave irradiation.

Low-Frequency, Whole Body Vibration Induced Neurite Outgrowth by Pc12m3Cells with Impaired Nerve Growth Factor-Induced Neurite Outgrowth

Purpose: To investigate the effects of very low-frequency whole body vibration (WBV) stimulation using PC12 m3 cells. Methods: Mutant, drug-hypersensitive, PC12 m3 cells were obtained by means of continuous culture of neural PC12 cells and then stimulated by low-frequency vibration at frequencies of 7–13 Hz for 5–20 min with simultaneous exposure to nerve growth factor (NGF). Results: The frequency of neurite outgrowth by PC12 m3 cells induced by 7-Hz vibratory stimulation was approximately 3-fold greater than that induced by NGF alone. Moreover, activated cyclic-AMP responsive element (CRE)-binding protein (CREB) expression was induced in PC12 m3 cells stimulated by 7-Hz vibration. Conclusion: Low-frequency vibratory stimulation induces neurite outgrowth via a p38 mitogen-activated protein kinase/CREB signaling pathway in PC12 m3 cells. Together, these results suggest that WBV is an efficient method to stimulate neurite growth.

Heat-shock-induced three-dimensional-like proliferation of mouse fibroblasts mediated by hydroxyapatite.

The aim of the present study was to determine both the minimal and the optimal conditions under which heat treatment is effective for enhancing 3D (three‐dimensional)‐like cell proliferation. C3H10T1/2 mouse fibroblasts were cultured with hydroxyapatite granules for 10 weeks after heat treatment at 40, 41.5, 43, 44 or 45°C for 2, 10, 15, 20, 30, 45, 60, 90, 180 and 360 min. Then minimal and optimal conditions of temperature and duration of heat treatment for induction of 3D‐like proliferation of cells were determined. The minimal conditions of heat treatment to induce 3D‐like proliferation were 43°C for 2 min and the optimal conditions were 43°C for 10 min. The mean rates of formation of 3D‐like proliferation patterns by cells heat‐treated at 43°C for 2 and 10 min were significantly higher (1.7‐ and 3.7‐fold respectively) than that by untreated cells (P<0.05). We also observed significantly greater effects of heat treatment on 3D‐like proliferation at 40°C for 90 or 180 min and at 41.5°C for 15 min and 44°C for 10 min. We found that apoptosis had occurred in 7.5 and 87.0% of the cells at 1 week after heat treatment at 43°C for 10 min and 30 min respectively. Western‐blot analysis demonstrated that phosphorylation of p38 MAPK (mitogen‐activated protein kinase) was markedly increased by heat treatment at 43°C for 10 min. These findings suggest that activation of p38 MAPK by heat shock is associated with 3D‐like cell proliferation. The results of the present study should be useful for further studies aimed at elucidation of the physiological mechanisms underlying thermotherapy and hyperthermia.