Abstract
BackgroundThe differentiation of hematopoietic stem cells into platelet-forming megakaryocytes is of fundamental importance to hemostasis. Constitutive apoptosis is an integral, yet poorly understood, facet of megakaryocytic (Mk) differentiation. Understanding Mk apoptosis could lead to advances in the treatment of Mk and platelet disorders.ResultsWe used a Gene-ontology-driven microarray-based transcriptional analysis coupled with protein-level and activity assays to identify genes and pathways involved in Mk apoptosis. Peripheral blood CD34+ hematopoietic progenitor cells were induced to either Mk differentiation or, as a negative control without observable apoptosis, granulocytic differentiation. Temporal gene-expression data were analyzed by a combination of intra- and inter-culture comparisons in order to identify Mk-associated genes. This novel approach was first applied to a curated set of general Mk-related genes in order to assess their dynamic transcriptional regulation. When applied to all apoptosis associated genes, it revealed a decrease in NF-κB signaling, which was explored using phosphorylation assays for IκBα and p65 (RELA). Up-regulation was noted among several pro-apoptotic genes not previously associated with Mk apoptosis such as components of the p53 regulon and TNF signaling. Protein-level analyses probed the involvement of the p53-regulated GADD45A, and the apoptosis signal-regulating kinase 1 (ASK1). Down-regulation of anti-apoptotic genes, including several of the Bcl-2 family, was also detected.ConclusionOur comparative approach to analyzing dynamic large-scale transcriptional data, which was validated using a known set of Mk genes, robustly identified candidate Mk apoptosis genes. This led to novel insights into the molecular mechanisms regulating apoptosis in Mk cells.
Highlights
The differentiation of hematopoietic stem cells into platelet-forming megakaryocytes is of fundamental importance to hemostasis
A CD41a+AnnexinV+Propidium-iodide— apoptotic Mk cell population was detected by flow cytometry as early as day 5 and a significant increase in apoptosis was observed between days 9 and 12 (Figure 1D)
We show only the last three analyses (Figure 3A-D) because the comparison to day 0 CD34+ cells is uninformative for apoptosis-related genes since initial cytokine treatment is likely to cause expression changes in apoptosis-related genes that are unrelated to Mk differentiation and terminal Mk apoptosis is not detected until day 5 (Figure 1D)
Summary
The differentiation of hematopoietic stem cells into platelet-forming megakaryocytes is of fundamental importance to hemostasis. Constitutive apoptosis is an integral, yet poorly understood, facet of megakaryocytic (Mk) differentiation. Committed Mk progenitors undergo endomitosis and become polyploid with multilobated nuclei. At this stage, Mk cells undergo morphological changes including the development of a demarcation membrane system and dramatic increase in cell size [4]. Polyploidization and platelet release are linked to a program of constitutive apoptosis. Expression of Bcl-2, another anti-apoptotic gene, was decreased in a megakaryoblastic cell line and was low and unchanged during thrombopoietin (Tpo)-driven maturation of cord-blood CD34+ cell derived Mk cells [6]. Bcl-2 over-expression throughout the murine hematopoietic compartment led to a 50% reduction in platelet levels with no change in Mk cell numbers [13]. Irregular patterns of Mk apoptosis have been associated with Mk-cell-related diseases including immune thrombocytopenic purpura [14]
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