Calreticulin Promotes Proliferation and Migration But Inhibits Apoptosis in Schwann Cells

Abstract

BACKGROUND Previous studies indicated that calreticulin (CRT) regulated various biological processes. This study was aimed to investigate the function of CRT in Schwann cells (SCs). MATERIAL AND METHODS SCs were separated from sciatic nerves of mice and were transfected with pcDNA3.1-CRT (pc-CRT), small interfering RNA targets CRT (siCRT), or their corresponding negative controls. The expression of CRT was determined by quantitative reverse transcription PCR (qRT-PCR) and Western blot analysis. Then, cell proliferation, migration, and apoptosis were measured by 3-(4, 5-dimethylhiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, modified 2-chamber migration assay, and flow cytometry, respectively. Finally, the phosphorylation levels of key kinases in the phosphatidylinositol-3-kinase (PI3K)/AKT and the extracellular signal-regulated kinase/ribosomal S6 kinase 2 (ERK/S6) pathways were detected by Western blot analysis. RESULTS Overexpression of CRT remarkably increased viability (P<0.05, P<0.01 or P<0.001) and migration (P<0.001), but inhibited apoptosis (P<0.05). The CRT-knockdown showed the inverse impacts on viability (P<0.05 or P<0.001), migration (P<0.001), and apoptosis (P<0.001). Additionally, the phosphorylation levels of AKT (Thr308 and Ser473), ERK, and S6 were all up-regulated in CRT-overexpressed cells (P<0.001), and were down-regulated in CRT-knockdown cells (P<0.05, P<0.01 or P<0.001). CONCLUSIONS Overexpression of CRT in SCs promoted cell proliferation and migration but suppressed cell apoptosis. The PI3K/AKT and ERK/S6 pathways might be involved in the functional effects of CRT on SCs.

Figure 1

The difference in calreticulin (CRT) expression in transfected Schwann cells (SCs). SCs were transfected with pcDNA3.1 (pcNC), pcDNA3.1-CRT (pc-CRT), non-silencing small interfering RNA (siNC), or specific small interfering RNA against CRT (siCRT). After transfection, cells were harvested for quantitative real-time (qRT)-PCR (A) and Western blot analysis (B). Data presented are the mean of at least 3 independent experiments. Error bars indicate SD. ** P<0.01; *** P<0.001.

Figure 2

Effect of CRT on proliferation in SCs. SCs were transfected with pcNC, pc-CRT, siNC, or siCRT. Cells transfected with pcNC and siNC served as negative control for pc-CRT and siCRT transfected cells, respectively. Proliferation was measured by MTT assay. Data presented are the mean of 3 independent experiments. Error bars indicate SD. * P<0.05; ** P<0.01; *** P<0.001.

Figure 3

Influence of CRT on migration in SCs. SCs were transfected with pcNC, pc-CRT, siNC, or siCRT. Cells with pcNC and siNC served as negative control for pc-CRT and siCRT transfected cells, respectively. Cell migration was measured by a modified 2-chamber assay. Data presented are the mean of 3 independent experiments. Error bars indicate SD. *** P<0.001.

Figure 4

Promotion of apoptosis in CRT-knockdown SCs. SCs were transfected with pcNC, pc-CRT, siNC, or siCRT. Cells with pcNC and siNC served as negative control for pc-CRT and siCRT transfected cells, respectively. Cell apoptosis was measured by flow cytometry. Data presented are the mean of 3 independent experiments. Error bars indicate SD. *** P<0.001.

Figure 5

Overexpression of CRT in SCs activated the PI3K/AKT and ERK/S6 signaling pathways. SCs were transfected with pcNC, pc-CRT, siNC, or siCRT. Cells with pcNC and siNC served as negative control for pc-CRT and siCRT transfected cells, respectively. (A) expression levels of kinases in transfected cells. After transfection, total proteins extracted from transfected cells underwent Western blot analysis. (B) phosphorylation rates of different kinases in transfected cells. The band intensity was estimated by Image Lab™ software. The phosphorylation rate is expressed as the relative intensity of phosphorylated kinases/kinases and the final results were normalized by GAPDH. Data presented are the mean of 3 independent experiments. Error bars indicate SD. *** P<0.001.