Role of NFAT Transcription Factors in Megakaryopoiesis
Megakaryopoiesis is a complex process in which immature hematopoietic stem cells of the bone marrow differentiate, influenced by cytokines and cells of vascular and osteoblastic “niches”, into mature megakaryocytes, the origin of peripheral blood platelets. NFAT (Nuclear Factor of Activated T cells) transcription factors are a family of five proteins that are primarily known for their central role in the regulation of inducible gene expression in activated T cells, but also have been shown to regulate processes of differentiation and tissue adaptation in various cell types.
We have previously demonstrated that NFAT factors are expressed in human CD34+ hematopoietic progenitor cells as well as in bone marrow megakaryocytes. During the myeloid differentiation of CD34+ cells, expression of NFAT is regulated in a lineage-specific manner, in which it is maintained or upregulated during megakaryopoiesis, while it is suppressed during the differentiation of neutrophil cells. The role of NFAT in the megakaryocytic lineage, however, is unknown.
The aim of the current project is to understand the role of NFAT in megakaryocytic cells. Two complementary experimental systems will be used. First, NFAT family members are overexpressed by retroviral transduction in human CD34+ cells; a second group of CD34+ cells is transduced with VIVIT, a specific peptide inhibitor of NFAT activation. CD34+ cells overexpressing NFAT or VIVIT are then compared to control cells with respect to several aspects of megakaryopoiesis. As a variation of this method, NFATc2 knockout mice serve as a model to study the role of NFATc2 in murine megakaryopoiesis. In a second approach, megakaryocytic cells transduced with NFAT, VIVIT or vector control are used to identify the global gene expression profile (both constitutive and inducible) regulated by NFAT in megakaryocytes by microarray analysis.
In summary, we expect that the current project will help to define a new role for NFAT transcription factors in megakaryocytes and therefore will significantly add to the understanding of relevant signaling pathways regulating gene expression in these cells.
