Notch signalling via rbp-j promotes myeloid differentiation

Abstract

Abstract Notch receptors are involved in the regulation of cell fate decisions, differentiation and proliferation in many developmental systems. To date, four different mammalian Notch receptors have been described. The ligands are also transmembrane proteins and include Jagged-1 and -2, and Delta-like -1, -2, -3. After ligand binding and activation, the Notch intracellular domain (N IC ) is released from the cytoplasmic membrane. N IC then translocates to the nucleus where it binds to the transcriptional repressor RBP-J, thereby converting RBP-J into a transcriptional activator. The expression of Notch on hematopoietic cells and of cognate Notch ligands on bone marrow stromal cells suggest a role for Notch signalling in the regulation of hematopoiesis. To investigate the consequences of mNotch 1 signalling in myelopoiesis, myeloid progenitor 32D and multipotent progenitor FDCP-mix cell lines were engineered to permit the conditional induction of the constitutively active intracellular domain of mNotch 1 (mN1 IC ) by the 4-hydroxytamoxifen-inducible system. In the presence of IL-3, the induction of mN1 IC activity resulted in a decrease in self renewal, a reduction of proliferation and increased myeloid differentiation of 32D and FDCP-mix cells. In the presence of hematopoietic cytokines such as GM-CSF and erythropoietin that support differentiation of FDCP-mix cells, the induction of mN1 IC led to an accelerated and increased differentiation along the granulocyte, macrophage, and erythrocyte lineage. Expression of a transcriptionally active derivative of RBP-J (RBP-J-VP16) also increased myeloid differentiation. To further test the role of Notch signalling in a physiological context, 32D or FDCP-mix cells were cultured on fibroblast layers that expressed or did not express the Notch ligand Jagged1. Similar to the induction of mN1 IC , Jagged1 accelerated myeloid differentiation of 32D and FDCP-mix cells. Taken together, our results suggest that activation of mNotch1 promotes myeloid differentiation via RBP-J transactivation.