Bone marrow-derived mesenchymal stem cells up-regulate acetylcholine receptor delta subunit through NRG/ErbB3-mediated mitogen-activated protein kinase pathway

Abstract

To investigate the effect of bone marrow-derived mesenchymal stem cells (BMSCs) on the expression of acetylcholine receptor delta subunit (AChRd), the murine skeletal muscle cell line Sol8 were grown in DMEM with 20% fetal bovine serum added with (conditional medium group) or without (control group) conditional medium of BMSC cells for 48 hours. RT-PCR and Western blot were performed to access the mRNA and protein levels of AChRd in Sol8 cells, respectively. Western blot was used to detect total and phosphorylated protein levels of Ras, Raf-1, Mek1/2, and Erk1/2, respectively. NRG-1 antibody added in conditional medium of BMSCs, si-ErbB3, and four Ras/Raf/MEK/ERK pathway inhibitors (FTS, Sulindac, U0126, and PD98059) were using to investigate the effect of AChRd levels. Our studies indicated that expression of AChRd was significantly enhanced in the conditional medium group when compared with those in control group and phosphorylation of Ras, Raf, Erk1/2 in Sol8 cells was also increased. Although gene silencing for ErbB3 gene, adding of NRG-1 antibody in conditional medium of BMSCs or treatment of Ras/Raf/MEK/ERK pathway inhibitors can down-regulate expression of AChRd and phosphorylation, which suggesting that the Ras/Raf/MEK/ERK pathway may be involved in BMSCs-induced expression of AChRd.

Figure 1

The level of AChRd mRNA and protein in Sol8 cells is up‐regulated when added with conditional medium of BMSCs. Sol8 cells added with (Conditional Medium group) or without (Control group) conditional medium of BMSC cells were cultured in 35 mm dish for 48 hours. The mRNA level of AChRd was detected using RT‐PCR (A). The protein level of AChRd was detected using Western Blot (B). GAPDH is shown as an internal standard.

Figure 2

The level of AChRd mRNA and protein in Sol8 cells is down‐regulated after neutralized with NRG‐1 antibody in conditional medium of BMSCs. The mRNA and protein levels of NRG‐1 in BMSCs and Sol8 cells were detected by RT‐PCR and Western Blot, respectively (A). The levels of NRG‐1 in conditional medium of BMSC and Sol8 cells were detected by ELISA, respectively (B). The mRNA levels of AChRd in Sol8 cells were detected by RT‐PCR after neutralized with 0, 5, 10, and 15 μg/mL NRG‐1 antibodies in conditional medium of BMSCs, respectively (C). The protein levels of AChRd in Sol8 cells were detected by Western Blot after neutralized with 0, 5, 10, and 15 μg/mL NRG‐1 antibodies in conditional medium of BMSCs, respectively (D).

Figure 3

The level of AChRd mRNA and protein in Sol8 cells is down‐regulated after ErbB3 RNAi. The efficiency of ErbB3 RNAi in Sol8 cells was evaluated using RT‐PCR (A) and Western blot analysis (B). The mRNA levels of AChRd in Sol8 cells were detected by RT‐PCR after ErbB3 RNAi (C). The protein levels of AChRd in Sol8 cells were detected by Western Blot after ErbB3 RNAi (D).

Figure 4

Effect of BMSCs conditional medium on the Ras/Raf/MEK/ERK pathway of Sol8 cells. Western blotting analysis was used to detect the total and phosphorylated form of Ras, Raf‐1,Mek1/2, and ERK1/2, respectively. GAPDH is shown as an internal standard.

Figure 5

Effect of the Ras/Raf/MEK/ERK pathway inhibitor on AChRd protein in Sol8 cells. Western blotting analysis was used to detect AChRd protein in Sol8 cells after treated by FTS (A), Sulindac sulfide (B), U0126 (C), and PD98059 (D), respectively. GAPDH is shown as an internal standard.