One of the least understood aspects of hematopoiesis is the process of megakaryocyte development. The recent cloning of thrombopoietin (TPO), an essential cytokine regulator of this process, allows investigation of the molecular basis for maintenance of physiologically appropriate platelet levels to proceed. Northern blots have revealed several disparate tissues to express TPO, but the cells which are responsible for TPO expression in vivo have not been identified. Two main models of TPO serum level regulation have been proposed. One asserts that TPO expression is constitutive in liver and kidney, and that serum levels are mediated via protein metabolism by an expanding or contracting platelet mass. A second suggests that in states of significant platelet variability, TPO mRNA levels may vary inversely to platelet mass. We provide experimental evidence which supports constitutive TPO expression in the liver and kidney, and mRNA-based regulation in the marrow and spleen. We plan to study the molecular basis of TPO gene regulation with an eye to understanding how the basal and inducible tissue-specific expression of the TPO gene translates into physiologically appropriate serum protein levels. To achieve these ends, we propose a research plan of three specific aims: 1. To identify the cellular and histologic sites of basal and inducible TPO production in mouse models of thrombocytopenia by in situ hybridization, RNA analysis of primary cell fractions and lines and RT-PCR; 2. To refine in vitro models of constitutive and inducible tissue-specific TPO gene expression and identify the relative contribution of transcriptional enhancement and mRNA accumulation in cells which increase TPO mRNA levels in response to thrombocytopenic sera; 3. To compare the functional organization of the TPO gene by DNAseI hypersensitive site mapping, by RNAse protection assays and 5' RACE analysis to characterize hTPO 5' mRNA isoforms during perturbations in platelet and megakarcyocyte mass, by identification of functionally relevant cis-acting elements of the TPO promoter by reporter gene analysis, refining these sequences by DNAse I footprint and mobility shift assays, and confirming the functional contribution of these sequences to constitutive and inducible tissue-specific TPO expression by site directed mutagenesis and determining their functional role by gain of function/loss of function analysis in reporter gene assays. We provide data to suggest these aims are feasible and will result in useful data as a basis for future studies. Understanding the mechanisms by which this regulator of megakaryocyte maturation is controlled will provide insight into normal and dysregulated megakaryocytopoiesis.
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