Generation and Analysis of Transgenic Mice Reveal a Role for CRE Binding Proteins in Multiple Stages of B Cell Development, Functional Maturation, Proliferation and Apoptosis.

Cyclic AMP response element binding protein-1 (CREB-1) is expressed in various stages of B cell development in the bone marrow. Antigen receptor induced DNA binding activity and activation dependent phosphorylation suggests a role for CREB-1 in early B cell development and functional maturation in-vivo. This was directly tested in transgenic mice over expressing a dominant negative Ser^119-ala phosphomutant CREB-1 in bone marrow (BM). Northern blot analysis of total RNA and Western blot analysis of the protein isolated from 4 independent transgenic lines revealed expression of the transgene in the BM and peripheral B cells. Flow cytometric analysis of BM cells from transgenic (Tg) and non-transgenic (NTg) littermates revealed ~65% reduction in B220⁺/IgM⁻ pro-B and pre-B population (7.56±0.89% NTg vs. 3.5±1.1% Tg mice) and B220⁺/IgM⁺ immature and mature B cells (4.4±0.98 in NTg vs 1.4± 0.19% in Tg mice). Detailed analysis of BM cells from Tg and NTg littermates by multiparameter flow cytometry revealed ~70% increase in B220⁺CD43⁺CD24^+(int) pre-BI cells (39% in NTg vs 67% in Tg mice), and ~60% decrease in B220⁺CD43⁺CD24^++(high) pre-BII B cells (46% in Ntg vs 20% in Tg mice), indicating a developmental block in pre-BI to pre-BII transition in mCREB-1 mice. Adoptive transfer of BM cells from Tg or NTg mice into sub-lethally irradiated RAG-2^−/− mice, revealed cell intrinsic defect in Tg bone marrow B cells. RT-PCR analysis of RNA from Tg pre-BI B cells revealed increased c-jun and decreased jun-B transcripts with minimal changes in c-fos, PCNA, mb-1 and vpreB. In contrast to pre-BI cells, increased c-jun and junB transcripts were observed in pre-BII B cells. Cell cycle analysis exhibited a consistent decrease in S phase entry in pre-BII B cells from Tg compared to NTg mice at 48 hours without stimulation (41% in NTg vs 11% in Tg) or in response to IL-7 (48% in NTg vs 27% in Tg). Consistent with the BM defect, mCREB-1 Tg mice revealed ~ 40% decrease in the IgM⁺B220⁺ cells (29.3±0.96% in NTg vs. 17.7±2.9% Tg) in the spleen. This is further reflected in significant reduction in the absolute number of mature B cells in the spleen [(36±3.6 in NTg vs. 22 ±1.9% in Tg mice, p<0.01]. Interestingly when compared to NTg mice, mCREB-1 Tg mice revealed about 45% decrease in CD21^dimCD23^high follicular B cells with minimal change in the CD21^highCD23^dim marginal zone B cell (16.5±1.4x10⁶ in NTg vs 9.5±0.92 x10⁶ cells in Tg mice P<0.01). In contrast to the bone marrow and spleen, analysis of peritoneal cells revealed significant increase in the total cell number and B220⁺IgM⁺ population in Tg mice compared to NTg littermates [22±3% in NTg vs 33±2.6% in Tg mice (p<0.01)]. The increased cell number and the abnormal distribution reflected a 3–4 fold increase in IgM^highIgD^lowMac-1^Int B1 B cells. This increase is reflected in both CD5⁺ B-1a and CD5⁻ B-1b B cells in the Tg mice and is attributed to resistance to apoptosis as evidenced by Annexin V/PI staining. These studies provide the first evidence for a role for CRE binding proteins in multiple stages of B cell development, functional maturation, proliferation and apoptosis. Ongoing studies are aimed to define the role of CREB-1 transcription factor in human B cell malignancies including chronic lymphocytic leukemia and acute lymphocytic leukemia.