Blood platelets mediate hemostasis as well as diverse pathologic processes, including thrombosis and inflammation, and thrombocytopenia is a frequent, dose-limiting complication of cancer chemotherapy. The importance of platelets in diseases that are common in the United States is thus well recognized. This makes it necessary to understand how terminally differentiated megakaryocytes (MKs) fragment and release blood platelets. Despite the discovery of thrombopoietin (Tpo) and its signal transduction pathways in MKs, the cellular and molecular basis of terminal MK maturation and platelet release remain unknown. Mice lacking the erythro-megakaryocytic transcription factor NF-E2 have severe, usually lethal, thrombocytopenia resulting from a late arrest in MK cytoplasmic differentiation. Hence, they constitute a powerful and unique model to study thrombocytopoiesis, and are an important tool in the P.I.'s long-term goal of defining molecular aspects of platelet production. MKs deficient in NF-E2 have a cell-autonomous defect and fail to produce proplatelets, the precursors of blood platelets, in culture. However, the transcriptional targets of NF-E2 and the relevant biochemical pathways of terminal MK differentiation are not known. Initial efforts to identify genes that fail to be expressed in the absence of NF-E2 have led to a MK-and platelet-specific beta tubulin isoform (beta1) that normally appears late in MK differentiation, localizes to proplatelets and platelet marginal bands, and possibly mediates the cytoplasmic reorganization that accompanies platelet release. Beta1 tubulin expression is completely lost in the absence of NF-E2 function. Preliminary data thus strongly suggest that loss of beta1 tubulin expression in MKs might explain in part why blood platelets are not produced in the absence of NF-E2.
The Specific Aims of this proposal include determining whether the absence of beta1 tubulin in NF-E2-deficient cells reflects direct regulation by this transcription factor, and whether restoring beta1 tubulin expression in the defective MKs is sufficient to rescue some or all aspects of thrombocytopoiesis. Further, we propose to use gene targeting in mice to test the essential roles of beta1 tubulin in vivo, independent of its putative relation to NF-E2. Finally, we propose to use a previously successful mRNA subtraction strategy to identify and characterize additional molecular targets of NF-E2 that may participate in platelet biogenesis. The results of these studies should substantially improve the current understanding of how MKs produce blood platelets.
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