Critical role of protein L-isoaspartyl methyltransferase in basic fibroblast growth factor-mediated neuronal cell differentiation

Abstract

We aimed to study the role of protein L-isoaspartyl methyltransferase (PIMT) in neuronal differentiation using basic fibroblast growth factor (bFGF)-induced neuronal differentiation, characterized by cell-body shrinkage, long neurite outgrowth, and expression of neuronal differentiation markers light and medium neurofilaments (NF). The bFGF-mediated neuronal differentiation of PC12 cells was induced through activation of mitogen-activated protein kinase (MAPK) signaling molecules [MAPK kinase 1/2 (MEK1/2), extracellular signal-regulated kinase 1/2 (ERK1/2), and p90RSK], and phosphatidylinositide 3-kinase (PI3K)/Akt signaling molecules PI3Kp110β, PI3Kp110γ, Akt, and mTOR. Inhibitors (adenosine dialdehyde and S-adenosylhomocysteine) of protein methylation suppressed bFGF-mediated neuronal differentiation of PC12 cells. PIMTeficiency caused by PIMT-specific siRNA inhibited neuronal differentiation of PC12 cells by suppressing phosphorylation of MEK1/2 and ERK1/2 in the MAPK signaling pathway and Akt and mTOR in the PI3K/Akt signaling pathway. Therefore, these results suggested that PIMT was critical for bFGF-mediated neuronal differentiation of PC12 cells and regulated the MAPK and Akt signaling pathways. [BMB Reports 2016; 49(8): 437-442].

Fig. 1.. bFGF induces neuronal differentiation of PC12 cells. (A) Neuronal differentiation of PC12 cells was induced by treatment with 50 ng/ml bFGF. (B, C) The mRNA and protein levels of NF-M and NF-L from PC12 cells treated with 50 ng/ml bFGF for indicated times were analyzed by RT-PCR and Western blot.

Fig. 2.. bFGF-mediated neuronal differentiation of PC12 cells activates MAPK and PI3K/Akt signaling pathways. (A, B, D, E, G) Levels of phosphorylated and total MEK1/2, ERK1/2, p90RSK, PI3Kp110β, PI3Kp110γ, Akt, NF-M, and mTOR from whole lysates of PC12 cell treated with 50 ng/ml bFGF in the absence or presence of U0126 (D left), LY294002 (D right) or rapamycin (G) for 3 days were analyzed by Western blotting. (C, F) Neuronal differentiation of PC12 cells was induced by treatment with 50 ng/ml bFGF in the absence or presence of U0126 (10 μM), LY294002 (50 μM) or rapamycin (100 nM) for 6 days.

Fig. 3.. PIMT induces bFGF-mediated neuronal differentiation of PC12 cells. (A, D) Neuronal differentiation of PC12 cells was triggered by bFGF (50 ng/ml) treatment in the absence or presence of AdOx (1 and 5 μM), SAH (1 and 10 μM) or under transfection with control SCR or PIMT siRNA for 6 days. (B, C, E) NF-M, Tuj-1, and PIMT from whole lysates of PC12 cells treated with bFGF in the absence or presence of AdOx (upper) and SAH (lower) for 3 days or under transfection with 20 nM control SCR siRNA or PIMT siRNA (1-3) for 2 (C) or 3 (E) days were detected by Western blot.

Fig. 4.. PIMT induces bFGF-mediated neuronal differentiation of PC12 cells by activating MAPK and Akt signaling pathways. (A-C) Phosphorylated and total forms of c-Raf, MEK1/2, ERK1/2, p90RSK, PI3Kp110b, PI3Kp110g, PDK1, Akt, and mTOR from PC12 cells, transfected with control SCR or PIMT siRNA for 48 h, treated with 50 ng/ml bFGF for 5 or 30 min were detected by Western blot. (D) Binding level of MEK1 with PIMT was evaluated by immunoprecipitation with anti-PIMT from whole lysates of PC12 cells treated with vehicle or 50 ng/ml bFGF for 5 min. (E) Proposed model for the role of PIMT in bFGF-mediated neuronal differentiation of PC12 cells.