PKCη directs induction of IRF-4 expression and Ig κ gene rearrangement in pre-BCR signaling pathway

Abstract

Pre-B cell receptor (pre-BCR) signals promote pre-B cell differentiation, in which the adaptor protein B-cell linker (BLNK) plays a crucial role. However, the molecular pathways downstream of BLNK are currently unclear. Utilizing pre-B leukemia cell lines (BKO84 and others) derived from BLNK-deficient mice as in vitro models of the pre-B cell differentiation, we have demonstrated that reconstitution of BLNK as well as an active form of protein kinase C (PKC)eta induces the differentiation events, such as pre-BCR down-regulation and kappa gene rearrangement. Here we show that the same events are induced by cross-linking of pre-BCR with anti-mu antibody in these pre-B cell lines, as well as in ex vivo pre-B cells from BLNK-deficient mice, suggesting a function of BLNK as an internal cross-linker of pre-BCR. Anti-mu treatment of BKO84 cells up-regulated membrane recruitment of PKC eta and the expression of IRF-4, a transcription factor known to promote light chain gene rearrangements. Anti-mu induction of surface kappa chain on BKO84 cells was blocked by reagents that inhibit phospholipase C or PKC. Enforced expression of the active PKC eta in BKO84 cells resulted in up-regulation of IRF-4 expression. Conversely, siRNA-mediated silencing of PKC eta expression strikingly attenuated the anti-mu-induced IRF-4 expression and kappa gene rearrangement, which were restored by PKC eta reconstitution. Finally, enforced expression of IRF-4, but not of BLNK, in the PKC eta-silenced BKO84 cells resulted in kappa gene rearrangement. These results indicate that PKC eta directs the induction of IRF-4 expression downstream of BLNK in the pre-BCR signaling pathway promoting kappa gene rearrangement.