CCR2 depletion triggers the activation of STAT1 to enhance BCR
signaling
CCR2 is associated with the activity of the JAK/STAT pathway and its
downstream transcriptional
factors.21 Furthermore,
the inhibition on the CCL2-CCR2 axis was shown to affect the activity of
NF-κB.30 To ascertain
the effect of CCR2 KO on the transcriptional factors downstream of
JAK/STAT pathway, we determined the activation of STAT1, NF-κB, and
STAT5 in B cells using CFm and western blotting. Upon sAg stimulation,
the colocalization between pSTAT1 and BCR in CCR2 deficient B cells
significantly increased within 10 min after stimulation (Fig.
6A-C ), which was in contrast to the observations for WT B cells. For
pNF-κB, its colocalization with BCR was notably higher in CCR2 deficient
B cells at 5 and 30 min post-stimulation (Fig. 6D-F ). Lastly,
we observed increased colocalization of pSTAT5 and BCR at 5 min after
stimulation in CCR2 depleted B cells (Fig. 6G-I ). While the
pSTAT1, pNF-κB, and pIKKB protein expression levels increased in CCR2 KO
B cells upon sAg stimulation, there was no difference in pSTAT5 protein
expression (Fig. 6J ).
Previous studies showed that Mst1 plays a role in cell proliferation by
influencing the AKT/mTOR signaling
pathway,31 and the
JAK/STAT downstream transcriptional factor
activation.32 To
identify the possible sequential effect of Mst1-mTORC1-STAT1 on BCR
signaling, we pretreated CCR2 KO B cells with the STAT1 inhibitor,
fludarabine, prior to sAg stimulation. Following the fludarabine
treatment, the levels of pCD19, pSHIP, pAkt, pS6, Dock8, pNF-kB, pIKKB,
and pMst1 were corrected towards the expression levels observed in WT B
cells (Fig. 6K ). Similarly, following pretreatment with
XMU-MP-1 (Fig. 6L) or rapamycin (Fig. 6M) , the levels
of pSTAT1 and pNF-kB in CCR2 KO B cells were rescued to levels
comparable to WT B cells, with the exception of pSTAT5 levels, which
displayed little to no changes before and after the rapamycin treatment.
This exception may reflect that STAT5 is not particularly affected by
Mst1 and mTORC1 suppression. Altogether, by connecting the upstream and
downstream signaling pathways, CCR2 might regulate BCR signaling via the
Mst1-mTORC1-STAT1 pathway.