The c‐Raf inhibitor GW5074 provides neuroprotection in vitro and in an animal model of neurodegeneration through a MEK‐ERK and Akt‐independent mechanism

Abstract

Cerebellar granule neurons undergo apoptosis when switched from a medium containing high potassium (HK) to one that has low potassium (LK). LK-induced cell death is blocked by GW5074 {5-Iodo-3-[(3,5-dibromo-4-hydroxyphenyl) methylene]-2-indolinone}, a synthetic drug that inhibits c-Raf activity in vitro. GW5074 has no direct effect on the activities of several apoptosis-associated kinases when assayed in vitro. In contrast to its effect in vitro, treatment of neurons with GW5074 causes c-Raf activation (when measured in vitro in the absence of the drug) and stimulates the Raf-MEK-ERK pathway. Treatment of neurons with GW5074 also leads to an increase in the activity of B-Raf, which is not inhibited by GW5074 in vitro at concentrations at which the drug exerts its neuroprotective effect. PD98059 and U0126, two distinct inhibitors of MEK, block the activation of ERK by GW5074 but have no effect on its ability to prevent cell death. Overexpression of a dominant-negative form of Akt does not reduce the efficacy of GW5074, demonstrating an Akt-independent mechanism of action. Neuroprotection is inhibited by SN-50, a specific inhibitor of nuclear factor-kappa B (NF-jB) and by the Ras inhibitor S-trans, trans-farnesylthiosalicylic acid (FTS) implicating NF-jB and Ras in the neuroprotective signaling pathway activated by GW5074. In addition to preventing LK-induced apoptosis, treatment with GW5074 protects against the neurotoxic effects of MPP+ and methylmercury in cerebellar granule neurons, and glutathione depletion-induced oxidative stress in cortical neurons. Furthermore, GW5074 prevents neurodegeneration and improves behavioral outcome in an animal model of Huntington's disease. Given its neuroprotective effect on distinct types of cultured neurons, in response to different neurotoxic stimuli, and in an animal model of neurodegeneration, GW5074 could have therapeutic value against neurodegenerative pathologies in humans.

Secondhand smoke exposure induces Raf/ERK/MAPK-mediated upregulation of cerebrovascular endothelin ETA receptors

“…We chose the Raf-1 inhibitor GW5074 to further demonstrate the involvement of ERK in the ET receptor upregulation after SHS. The specificity and efficacy of GW5074 for inhibiting Raf-1 in-vivo has been established in previous studies [32,33]. Lakey et al and Chin et al reported that GW5074 is a potent Raf-1 inhibitor and examined the effect of GW5074 on purified Raf-1 and confirmed that GW5074 selectively inhibits Raf-1 in vivo .…”

“…Raf phosphorylates MEK1/2, which in turn phosphorylates and activates ERK1/2 and then leads to activation of transcription factors [32]. The ERK1/2 pathway is a major effector of Raf.…”

“…For the treatment group, GW5074 was administrated to the animals once every day for 8 weeks in addition to the same condition of SHS exposure. The dosage of GW5074 was based on a previous study [32,34]. This exposure type may resemble SHS exposure and that the level of nicotine in the animals compare well with at seen in plasma of human smokers.…”

Cardiovascular risk factors regulate the expression of vascular endothelin receptors

“…Both U0126 and SB386023 decreased organ culture-induced ET B receptor upregulation (Henriksson et al, 2004). Raf-1 (c-Raf) is 20 a MAPK required for Ras/MEK/ERK signal transduction, which is the first enzyme in the MAPK cascade consisting of the three protein kinases Raf-1, MEK and ERK (Chin et al, 2004). Inhibition of Raf-1 attenuated cigarette smoke-induced expression of endothelin receptors in mice (Lei et al, 2008).…”

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“…Control animals received vehicle injections. Doses were chosen according to our preliminary studies and literature [8,9].…”