Growth Differentiation Factor-15 Prevents Low Potassium-induced Cell Death of Cerebellar Granule Neurons by Differential Regulation of Akt and ERK Pathways

Abstract

Growth differentiation factor-15 (GDF-15) is a novel member of the transforming growth factor-␤ superfamily and has been shown to be induced in neurons subsequent to lesions. We have therefore begun to study its putative role in the regulation of neuron survival and apoptosis. Cultured cerebellar granule neurons (CGN) survive when maintained in high K ؉ (25 mM) but undergo apoptosis when switched to low K ؉ (5 mM). GDF-15 prevented death of CGN in low K ؉ . This effect could be blocked by phosphatidylinositol 3-kinase/Akt pathway inhibitors LY294002 or wortmannin. In contrast, mitogen-activated protein kinase (MEK)/extracellular-signal-regulated kinase (ERK) pathway inhibitors U0126 and PD98059 potentiated GDF-15 mediated survival and prevented cell death in low K ؉ even without factor treatment. Immunoblots revealed GDF-15-induced phosphorylation of Akt and glycogen synthase kinase-3␤. This activation was suppressed by phosphatidylinositol 3-kinase inhibitors. Low K ؉ induced delayed and persistent ERK activation, which was blocked by MEK inhibitors or GDF-15. ERK activation induced c-Jun, a member of the AP-1 transcription factor family. GDF-15 or U0126 prevented c-Jun activation. Furthermore, we show that GDF-15 prevented generation of reactive oxygen species, a known activator of ERK. Together, our data suggest that GDF-15 prevents apoptosis in CGN by activating Akt and inhibiting endogenously active ERK. Transforming growth factor-␤s (TGF-␤s)1 comprise a superfamily of contextually acting cytokines with a broad array of biological activities. Depending on the developmental stage and cell type TGF-␤s regulate diverse processes including development, cell proliferation, differentiation, survival, and death (1-4). Members of the TGF-␤ superfamily can be subdivided into subfamilies of structurally closer related proteins, including the TGF-␤s 1-3, bone morphogenetic proteins, the growth/differentiation factors (GDFs), activins, and the glial cell line-derived neurotrophic factor (GDNF) family (5-7).All TGF-␤ famliy members, except members of the GDNF subfamily, signal through heteromeric complexes of type I and type II serine/threonine kinases receptors. Within the type I and type II receptor families there are several subgroups, e.g. bone morphogenetic protein receptors, depending on their respective ligands (for reviews, see Refs. 7 and 8). GDNF, neurturin, persephin, and artemin signal through a heteromeric receptor complex consisting of the receptor tyrosine kinase Ret (9, 10) and glycosylphosphatidylinositol-linked ␣-receptors (11).We and others (12, 13) have recently cloned a novel member of the TGF-␤ superfamily, GDF-15/macrophage inhibitory cytokine-1 (MIC-1). The protein does not belong to one of the known TGF-␤ subfamilies and represents a divergent member of the TGF-␤ superfamily. GDF-15 is widely synthesized in the central nervous system, most strongly expressed in the choroid plexus, and secreted into the cerebrospinal fluid, from where it may penetrate into the brain parenchyma (14 -16). We have p...

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“…CGNs were isolated and cultured from 8-d-old Wistar rats as described previously (Subramaniam et al, 2003). Cells were resuspended in high K ϩ [Eagle's basal medium containing 10% fetal calf serum, 25 mM KCl, 2 mM glutamine, and 0.5% (v/v) penicillin/streptomycin], switched to low K ϩ (Eagle's basal medium, 5 mM KCl, 2 mM glutamine, and 0.5% penicillin/streptomycin) at day 4, treated with additives for the indicated time points, and processed for the specific assays described below.…”

“…Western blot analysis was performed as described previously (Subramaniam et al, 2003). Densitometric quantification of Western blots was done using NIH Image (version 1.61) software.…”