CREB Is Involved in the Regulation of C/EBPβ During 'emergency' Granulopoiesis.

Abstract 1457

Poster Board I-480

Mobilization of sufficient numbers of granulocytes to the front line of infection is prerequisite for host defense. As granulocytes have a short half-life, the production of granulocytes in the bone marrow must be tightly regulated to meet emergency demands. Our previous findings suggested that granulopoiesis at steady state is largely dependent on CCAAT enhancer binding protein α (C/EBPα) transcription factor (Zhang D.E. et al., PNAS, 1996 and Zhang P. et al., Immunity, 2004), whereas the granulopoiesis during emergency such as infections is dependent upon C/EBPβ (Hirai H. et al., Nat Immunology, 2006). Indeed the transcripts of C/EBPβ in granulocyte precursors were upregulated in response to cytokine stimulation or infection. In order to elucidate the molecular switch between C/EBPα- and C/EBPβ-dependent granulopoiesis, we developed a novel lentivirus-based reporter system. The vector carries two independent expression units, a green fluorescent protein (GFP) driven by a promoter of interest and a mouse Thy1.1 gene under the control of a constitutively active phosphoglycerate kinase (PGK) promoter. The activity of a promoter can be monitored by the intensity of GFP in Thy1.1 positive cells. Using this system, the activity of the C/EBPβpromoter was evaluated in primary bone marrow cells. A series of deletion mutants of the promoter revealed the existence of two cyclic AMP responsive elements (CRE) in the positive responsive elements during GM-CSF induced differentiation. The transcripts of CRE binding (CREB) protein were detected at higher level in hematopoietic stem cells and common myeloid progenitors than other mature cells. When a dominant negative mutant of CREB (S133A), in which the serine residue at 133aa was mutated to alanine, was retrovirally transduced into bone marrow cells, the mRNA of C/EBPβ was reduced and the proliferation/differentiation of granulocyte precursors were significantly impaired. In contrast, a constitutively active form of CREB, CREB^DIEDML, facilitated the transcription of C/EBPβ. In addition, CREB is phosphorylated and bound to the CRE in response to GM-CSF stimulation. These data suggest that CREB is involved in the regulation of granulopoiesis through upregulation of C/EBPβ.