LILRA3 enhances the proliferation of U937 cells through a combination of Akt and MEK/Erk signaling pathways
The proliferation capacity of the two established U937 cell lines was evaluated using CCK-8 assay by detecting OD values at 450 nm at 12, 24, 48 and 72 hours. After 24 hours of culturing, enhanced proliferation was observed for the LILRA3-overexpressing U937 cells compared with the cells expressing the null vector, and the difference was quite significant at 48 and 72 hours (p=0.016 for 48 hours; p=0.003 for 72 hours) (Figure 5A). Western blotting was applied to investigate the potential signaling pathway, and we found that overexpression of LILRA3 significantly increased the levels of phosphorylated Akt , MEK and P38 MAPK(Figure 5B-5D). Given that the MEK and Akt pathways are closely related to proliferation, our ensuing research mainly focused on exploring the upstream and downstream factors of these two kinases. The levels of phosphorylated Erk (the accepted downstream protein of MEK),PDK1 and c-Raf (the classical kinases for Akt and MEK, respectively), PI3K (the conventional upstream protein of both Akt and MEK), and Foxo3a (the shared downstream protein of MEK and Akt) were further examined. Intriguingly, we found that phosphorylation of Erk, PDK1, c-Raf and PI3K was significantly increased but that of Foxo3a decreased in the presence of LILRA3 (Figure 5C, 5E, 5F and 5G). Phosphorylation of P65 , SAPK/JNK andmTOR was also investigated, but no difference was found (see Supplementary Figure 2). Because both Akt and MEK can be activated by PI3K, we speculated that the two pathways might function synergistically to regulate U937 cell proliferation. To confirm our hypothesis, LILRA3-overexpressing cells were seeded into 6-well plates and pretreated with specific inhibitors of MEK (GSK1120212, 1 μmol/L), Akt (MK-2206 2HCL, 5 μmol/L) or PI3K (LY294002, 20 mmol/L); the three inhibitors were purchased from Selleckchem (Houston, USA). Western blotting was used to evaluate the inhibitory efficiency, and the CCK-8 assay was carried out at 24, 48 and 72 hours to detect cell viability. We found that the three inhibitors could dramatically decrease phosphorylation of their targeted proteins (Figure 6A-6C) and could sharply reduce cell viability after 24 hours of culture compared with cells treated with dimethyl sulfoxide (DMSO) (Figure 6D). Most importantly, as shown in Figure 6C and 6D, LY294002 (a specific inhibitor of PI3K) also markedly decreased phosphorylation of Akt and MEK, with the highest inhibitory efficiency on cell proliferation compared with the other inhibitors. These results were consistent with our speculation. Foxo3a is a well-known critical transcription factor involved in cell growth. Although we detected a noteworthy decline in phosphorylation of this protein in cells pretreated with the three inhibitors, this result was not in accordance with our finding when the level of phosphorylated Foxo3a was detected between LILRA3-overexpressing and null vector cells. Indeed, Foxo3a phosphorylation should be enhanced by activated Akt and MEK; in contrast, we detected a decreased level when LILRA3 was overexpressed. A possible hypothesis to explain this phenomenon is that LILRA3 might have a direct/indirect inhibitory effect on Foxo3 phosphorylation; further investigations are needed to verify this speculation. In conclusion, our data indicate that LILRA3 regulates cell proliferation, most likely through PI3K/Akt and PI3K/MEK/Erk signaling pathways.