Figure 4

A conditionally active form of Raf-1 (ΔRaf-1:ER) protects HT22 neuronal cells against serum deprivation-induced cell death via activation of ERK. (A, B) Activation of Raf-1 provides protection against serum deprivation-induced apoptosis. HT22-ΔRaf-1:ER cells were serum-starved in the presence or absence of 4OHT (200 nM) for twenty-four hours. The reduction of colorless 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide by mitochondrial NAD(P)H-dependent dehydrogenases to formazan dye crystals was used as an indicator for cell survival (MTT assay). (A); ⋆, values statistically significantly different (p < 0.005) from those of cells incubated in the absence of serum and PD98059, but in the presence of 4OHT. (B) HT22ΔRaf-1:ER cells or HT22pac cells cells were serum-starved in the presence or absence of 4OHT for twenty-four hours. The cytotoxic effect of serum withdrawal was determined by phase contrast microscopy. The neuroprotection was abrogated by incubation with the MEK inhibitor PD98059, indicating that the ERK signaling pathway is essential for ΔRaf-1:ER mediated neuroprotection. (C) Kinetics of ERK2 and Elk-1 phosphorylation after stimulation with BDNF or 4OHT. BDNF stimulation triggers a rapid, but transient activation of ERK in TrkB receptor expressing HT22 cells (HT22-TrkB.TK cells, upper panel), whereas activation of the ΔRaf-1:ER fusion protein leads to a sustained activation of ERK, lasting for hours (HT22-ΔRaf-1:ER cells, lower panel). The kinetics of ERK phosphorylation and activation correlate very well with phosphorylation and activity of the ternary complex factor Elk-1, a nuclear substrate of ERK that functions as a key regulator of serum response element-driven gene transcription. (D) Prolonged activation of ERK is essential for neuroprotection. HT22 cells expressing ΔRaf-1:ER were serum-starved and incubated with 4OHT for twenty-four hours. PD98059 was added simultaneously (t = 0), or eight (t = 8) or sixteen hours (t = 16) after stimulation of the cells with 4OHT. Cells were analyzed twenty-four hours after stimulation by either the MTT assay (left) or by phase contrast microscopy (right) (reproduced from [31] with copyright permission from Blackwell Publishing, Oxford).