CREB Overexpression In Vivo Results in Increased Proliferation, Blast Transformation, and Earlier Engraftment of Myeloid Progenitor Cells.

Abstract

CREB or cAMP responsive element binding protein is a 43-kDa-basic/leucine zipper (bZip) transcription factor that regulates gene expression through the activation of cAMP-dependent or -independent signal transduction pathways. CREB promotes growth and survival in a variety of cell types and is overexpressed in the bone marrow of greater than 60% of AML patients. To understand the role of CREB in myelopoiesis, we characterized the effects of CREB overexpression in transgenic mice. We created mice in which CREB expression was targeted to the myeloid lineage using the hMRP8 promoter. CREB transgenic mice showed evidence of monocytosis, compared to age-matched littermate controls. We performed colony assays with methylcellulose containing SCF, IL-6, and IL-3. Bone marrow cells from CREB transgenic mice formed robust colonies earlier and had increased numbers of colony forming units (CFU-GM) when compared to control mice. Cytospin analysis of these cells showed the presence of more immature myeloid cells compared to controls. At day 12, cells from colonies were 50% c-Kit positive, 83% Gr-1 positive, and 67% Mac-1 positive by FACS analysis. To assess self-renewal of progenitors from CREB transgenic mice, serial replating experiments were performed. Bone marrow cells from transgenic mice were highly successful in repopulating the methylcellulose containing SCF, IL-6, and IL-3, in contrast to the control cells, which were unable to grow after serial replating. Following tertiary replating of the CREB transgenic mouse bone marrow, we observed that the colonies (96+3.5) appeared more homogeneous with immature cells that were >99% c-Kit positive and <1% GR-1, Mac-1 positive. These results suggest that persistent expression of CREB leads to a blast-like phenotype in the absence of differentiation. To determine whether increased CREB expression confers growth factor-independence, we cultured bone marrow cells in methylcellulose that did not contain cytokines. We observed a 10-fold increase in the numbers of cells from CREB transgenic mice (two different founder lines) compared to normal bone marrow. When cultured in methylcellulose containing M-CSF, the bone marrow cells from CREB transgenic mice formed larger and significantly greater numbers of colonies. However, these cells did not grow in the presence of G-CSF or EPO alone. To determine if the myeloproliferative (monocytosis) phenotype was transplantable into wild type recipient mice we injected 4x106 bone marrow cells from CREB transgenic mice into wild type C57/BL6 recipient mice. Serial analysis of the peripheral blood counts and cell surface markers by FACS analysis showed earlier myeloid engraftment at 6 weeks following transplantation compared to normal control mice. The transgenic recipients showed increased monocytes and neutrophils in the peripheral blood with a corresponding increase in Mac-1 positive, Gr-1 positive cell populations at 8 weeks after transplantation. At the same time, the lymphocyte count was significantly lower in CREB transgenic recipient mice than controls. Our results suggest that CREB plays a critical role in the regulation of normal hematopoiesis and stem cell self-renewal.