| Glial cell line-derived neurotrophic factor (GDNF) was first isolated by virtue of its ability to induce dopamine uptake and cell survival in cultures of embryonic ventral midbrain dopaminergic neurons. Its amino acid sequence indicated that it was a member of the transforming growth factor-( supergene family. Its ability to rescue dopaminergic neurons supported the idea that GDNF might ameliorate degeneration of dopaminergic neurons in patients with Parkinson's disease (PD). In addition, GDNF also has neurotrophic effects on motoneurons (MNs). GDNF was found to promote survival, neurite outgrowth, and choline acetyltransferase activity in MNs. These investigators suggest that GDNF may be useful in the treatment of MNs disorders like amyotrophic lateral sclerosis. All these findings strengthened the point that GDNF is functionally important in nervous system. GDNF signal via multicomponent receptors that consist of the Ret receptor tyrosine kinase plus a glycosylphosphatidylinositol (GPI)-linked coreceptor named GDNF receptor ( (GFR(), which gives binding specificity. Binding of GDNF to Ret and GFR( induces Ret phosphorylation. After phosphorylation, Ret induces the activation of several intracellular pathways, among which the mitogen-activated protein (MAP) kinase and the phosphatidylinositol 3-kinase (PI 3-kinase) are of particular interest. Rat pheochromocytoma PC12 cells, which are dependent on neurotrophic factors for survival in serum-free media and express low levels of Ret, were grown without serum in the presence of GDNF, soluble GFR(1 or both, and examined 7 days later. With GDNF or soluble GFR(1 alone, only a few neurite-bearing, phase-bright, live cells were found. In contrast, when PC12 cells were cultured with both GDNF and GFR(1, an increase in the number of living cells with neurites was observed. Therefore, we transfected GFR(1 expression vector into wild-type PC12 cells to establish a PC12 cell line that could coexpress Ret and GFR(1. The gene engineered PC12 cells could provide an unlimited source of clonal cells for studies on the mechanism of action of GDNF. It was reported that PI 3-kinase has been implicated in the survival-promoting mechanisms and MAP kinase seems to be involved in differentiation processes in nerve growth factor signal transduction pathway. To examine the contribution of these pathways to the survival and differentiation effect triggered by GDNF, we used PD98059, a specific inhibitor of MAP kinase kinase (MEK), and LY294002, a selective inhibitor of PI 3-kinase. GDNF had produced a clear increase in numbers of differentiated PC12-GFR(1 cells after 7 days of culture. Similarly, GDNF could promote the survival of PC12-GFR(1 cells cultured in serum-free medium. Using neurite outgrowth in PC12 cells as a measure of differentiation, GDNF could promote PC12-GFR(1 cells differentiation. PD98059 specifically blocks the activation of MAP kinase, via inhibition of MAP kinase kinase (MEK). PC12-GFR(1 cells were treated with various concentrations of PD98059 for 15 min before the addition of GDNF. The compound blocked GDNF-induced neurite formation in a dose-dependent manner, without significantly altering cell viability. These data indicate that the MAP kinase pathway is absolutely required for GDNF-induced neuronal differentiation in PC12-GFR(1 cells and the downstream elements activated by MEK-MAP kinases are not necessary for GDNF-mediated survival. In this report we describe the use of a specific inhibitor of MAP kinase to block the differentiation effect of GDNF on PC12-GFR(1 cells, providing pharmacological evidence that the MAP kinase pathway is required for the differentiation effect of GDNF.LY294002 at doses that inhibited the phosphorylation of Akt, a down-stream element of the PI 3-kinase, functionally block PI 3-kinase activity. Cells were cultured in medium supplemented with GDNF in the presence or absence of LY294002. The effects of LY294002 on the survival and differentiation of PC12-GFR(1 cells were evaluated. The re... |
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