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.