Cyclin A Is a Target Gene of Activated CREB Downstream of GM-CSF Signaling That Regulates Normal and Malignant Myelopoiesis.

Granulocyte-Macrophage Colony-Stimulating Factor stimulates the proliferation and survival of myeloid progenitor cells in vitro and in vivo. Primary AML blast cells express high affinity GM-CSF receptors. We previously reported that the basic leucine zipper transcription factor, CREB (cAMP Responsive Element Binding Protein), is phosphorylated at serine 133 in response to GM-CSF stimulation through a MEKK- and pp90RSK-dependent pathway. CREB protein and mRNA levels are elevated in the bone marrow of over 60% of AML patients at diagnosis. Furthermore, CREB is activated in primary AML blast cells that overexpress CREB. CREB overexpression in myeloid leukemia cell lines results in increased cell proliferation and survival, and decreased myeloid cell differentiation. Cells overexpressing CREB have increased numbers of cells in S phase in Brdu incorporation experiments in comparison to control cells. In addition, myeloid progenitor cells from CREB transgenic mice proliferate more and are growth-factor independent compared to cells from littermate controls. In colony assays, progenitor cells from CREB overexpressing mice are immortalized and acquire a blast-like phenotype. To understand the downstream pathways that regulate CREB-induced proliferation and survival in normal myeloid and leukemic cells, we examined the expression of two known CREB target genes, BCL-2 and Cyclin A, in CREB overexpressing myeloid leukemia cell lines. Cyclin A, but not BCL-2, was upregulated in cells overexpressing CREB compared to control cells. Cyclin A regulates G1 to S transition through activation of the cyclin-dependent kinase, CDK2. Myeloid progenitor cells from transgenic mice that overexpress CREB also overexpressed Cyclin A. We next investigated Cyclin A expression in primary blast cells from AML patients. Cyclin A protein levels were elevated in bone marrow from AML patients in which CREB was overexpressed but not in normal bone marrow. CREB has been shown to directly bind the CRE in the cyclin A promoter and increase transcription of cyclin A upon activation in response to growth factor stimulation. To study the possibility of cyclin A upregulation downstream of CREB, we examined transcriptional activation of Cyclin A in CREB overexpressing cells. A luciferase reporter construct containing the cyclin A promoter with the CRE was transfected into NFS60 cells that overexpressed CREB. A 4-fold increase in relative luciferase activity was observed in CREB overexpressing cells compared to control cells (p<0.05). Microarray analysis was performed with RNA from CREB overexpressing and non-overexpressing cells. In addition to cyclin A, several other target genes such as the pim-1 oncogene were found to be upregulated in CREB overexpressing cells. Our results suggest that aberrant cell cycle progression and increased proliferation from overexpression and activation of CREB downstream of GM-CSF signaling is a consequence of critical target genes that control myelopoiesis.