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Abstract
We previously reported that glial cell line-derived neurotropic factor (GDNF) receptor α1 (GFRα1) is a direct target of apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1). In the present study, we further analyzed the physiological roles of Ape1/Ref-1-induced GFRα1 expression in Neuro2a mouse neuroblastoma cells. Ape1/Ref-1 expression caused the clustering of GFRα1 immunoreactivity in lipid rafts in response to GDNF. We also found that Ret, a downstream target of GFRα1, was functionally activated by GDNF in Ape1/Ref-1-expressing cells. Moreover, GDNF promoted the proliferation of Ape1/Ref-1-expressing Neuro2a cells. Furthermore, GFRα1-specific RNA experiments demonstrated that the downregulation of GFRα1 by siRNA in Ape1/Ref-1-expressing cells impaired the ability of GDNF to phosphorylate Akt and PLCγ-1 and to stimulate cellular proliferation. These results show an association between Ape1/Ref-1 and GDNF/GFRα signaling, and suggest a potential molecular mechanism for the involvement of Ape1/Ref-1 in neuronal proliferation.
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Fig. 1.
GFRα1 colocalizes with a lipid raft marker in Ape1/Ref-1-expressing Neuro2a cells. (A) Neuro2a, SN4741, and SKNSH cells were transfected with empty vector (vector) or Ape1/Ref-1 expression vector (Ape1) then harvested 48 h later. Total cell extracts were prepared for immunoblotting as indicated. (B) Neuro2a cells were transfected with empty or Ape1/Ref-1 expression vector and then harvested at the indicated times after transfection. Total cell extracts were prepared for immunoblotting as indicated. (C) Neuro2a/Ape1 and vector/Neuro2a cells were immunostained using polyclonal anti-GFRα1 antibodies tagged with Alexa fluor. (D) Neuro2a/Ape1 cells were stained with anti-GFRα1 antibodies and cholera toxin (CTX), which specifically binds the lipid raft ganglioside GM1. The images show the colocalization of GFRα1 and CTX.
Fig. 2.
The effect of Ape1/Ref-1 on GDNF/GFRα1 signaling in Neuro2a cells. (A) Vector/Neuro2a (vector) and Neuro2a/Ape1 (Ape1) cells were treated with or without 30 ng/ml GDNF for 10 min. The cells were then lysed and subjected to immunoprecipitation (IP) with anti-Ret antibodies and analyzed for GFRα1, c-Src, phospho-Ret (pRet), and Ret by immunoblotting. (B) Vector/Neuro2a and Neuro2a/Ape1 cells were incubated with or without 30 ng/ml GDNF. At the indicated times, cell extracts were prepared and examined for activated phospho-Ret by immunoblotting.
Fig. 3.
Ape1/Ref-1 increases Akt and PLCγ-1 phosphorylation in response to GDNF through GFRα1. (A) GDNF-induced Akt and PLCγ-1 phosphorylation in Ape1/Ref-1-expressing Neuro2a cells. Vector/Neuro2a and Neuro2a/Ape1 cells were incubated with or without 30 ng/ml GDNF for the indicated times and total cell extracts were prepared for immunoblotting as indicated. (B) The amounts of phospho-Akt and Phospho-PLCγ-1 were quantified by densitometry and corrected for the amount of Akt and PLCγ-1 in the corresponding lysate, rerspectively. Levels of phospho-Akt and phospho-PLCγ-1 are expressed relative to its level in non-NGEF-treated cells (0 min) transfected with control vector. The data shown are the means±S.D. from three separate experiments. (C) Neuro2a/Ape1 cells were transfected with control or GFRα1 siRNA. At 48 h after transfection, the cells were incubated with or without 30 ng/ml GDNF for the indicated times. Total cell lysates were prepared for immunoblotting as indicated. (D) The amounts of phospho-Akt and Phospho-PLCγ-1 were quantified by densitometry and corrected for the amount of Akt and PLCγ-1 in the corresponding lysate, respectively. Levels of phospho-Akt are expressed relative to its level in non-NGEF-treated cells (0 min) transfected with control siRNA. The data shown are the means±S.D. from three separate experiments.
Fig. 4.
Ape1/Ref-1 expression increases neuronal proliferation in response to GDNF. Vector/Neuro2a and Neuro2a/Ape1 cells were transfected with control (sicont) or GFRα1-siRNA (siGFRα1). At 24 h after transfection, the cells were incubated with or without 30 ng/ml GDNF for up to 48 h. The number of cells was then determined by counting every 24 h after GDNF treatment. Each value is the mean±S.D. from three separate experiments. ∗∗p<0.01.
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