Role of the MAPK and PI3-Kinase/Akt Pathways in the Pre-B Cell Receptor (pre-BCR)-Induced NF-κb Activation and Rag1 and Rag2 Down Regulation.

Abstract 2667

Poster Board II-643

The pre-BCR acts as a critical checkpoint in pre-B cell development and might be also involved in leukemogenesis. Using the 697 and Nalm6 human pre-B cell lines, we have previously shown that pre-BCR stimulation resulted in cell cycle progression associated with activation of number of adaptors and signaling pathways including the PI3-Kinase/Akt, Ras/MAPK, AP1 and the canonical NFkB pathway. We have also demonstrated that Src kinases together with Syk played a crucial role in controlling the pre-BCR-associated functions, acting upstream the above-mentioned signaling pathways. Pre-BCR crosslinking also induced down regulation of Rag1 and Rag2 transcription. In this study we aimed to evaluate the role of MAPK and Akt in the pre-BCR-induced NF-kB activation and Rag1/2 down modulation. For this purpose the 697 pre-B cells and normal bone marrow primary pre-B cells were treated with the U0126 and LY294002, and with MEK1/2 and Akt inhibitors, respectively. A dominant negative form of Akt fused to the HIV1 Tat peptide was also used to inhibit the PI3-Kinase/Akt pathway. We bring evidence that LY294002 could alter the pre-BCR-induced NF-kB activation by inhibiting : i) p105 degradation, ii) p50 NF-kB1 nuclear translocation and, iii) the binding of p50 to an oligonucleotide containing a specific consensus sequence. On the contrary, U0126 significantly enhanced p105 degradation, indicating that MAPK and Akt exerted antagonistic effects on the pre-BCR-induced NF-kB activation. Strikingly the baseline levels of Rag1 and Rag2 transcripts were increased in the LY294002 but not the U0126-treated pre-B cells. Futhermore, both inhibitors were shown to induce a strong increase in the expression of Rag1 and Rag2 transcripts upon pre-BCR crosslinking, suggesting that this receptor exerts dual effects on Rag1/2 expression with a predominant negative regulatory component mediated by both PI3-K and MAPK. No changes in the levels of Pax5, E2A, EBF, IFR4, IRF8, FOXO1, FOXO3, Myb, MAZ, LEF1 and SP1 (transcription factors implied in the regulation of Rag1 and Rag2 transcription) were observed in the pre-BCR stimulated or unstimulated-697 cells, treated or not with the MAPK and Akt inhibitors. Our results suggest that the pre-BCR signaling is a complex and tightly self-controlled process, which deregulation might alter cell growth and survival pathways via NF-kB as well as genomic stability trough Rag1/2 expression.