REGULATION OF MEMBRANE BIOSYNTHESIS BY CSF-1

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The long-term goal of this research plan is to contribute to the understanding of how key metabolic pathways required for the proliferation of hematopoietic cells are regulated by growth factors. Our investigations will center on colony-stimulating factor 1 (CSF-1), a hematopoietic growth factor required for the proliferation, differentiation and survival of mononuclear phagocytes. We have selected the BAC1.2F5 cell line as the model macrophage system. These cells exhibit an absolute CSF-1 requirement for both growth and viability and arrest in the early G1 phase of the cell cycle when transiently deprived of CSF-1, thus permitting investigations of cell cycle-specific metabolic alterations. Like normal macrophages, BAC1.2F5 cell proliferation is blocked by ligands that increase intracellular cAMP. Our research plan focuses on the regulation of membrane phospholipid biosynthesis by both CSF-1 and cAMP in BAC1.2F5 cells. While it is apparent that cells must increase their net rate of phospholipid formation during the cell cycle to produce daughter cells, little information is available on the relationship between the cell cycle and phospholipid formation. The rate-controlling enzyme in the BAC1.2F5 phospholipid biosynthetic pathway is CTP:phosphocholine cytidylyltransferase, and our working hypothesis is that modulation of cytidylyltransferase activity is the major mechanism by which membrane phospholipid biogenesis is regulated by CSF-1 and cAMP. Cytidylyltransferase has been extensively studied in nondividing cells and its enzymatic activity is enhanced by association with membranes and attenuated by phosphorylation. We have generated evidence for growth factor regulation of cytidylyltransferase mRNA levels, illustrating an additional level of control over the cellular activity of this key enzyme. Defining the role of CSF1 and cAMP in governing cytidylyltransferase catalytic activity in BAC1.2F5 cells will contribute to our understanding of the mechanisms that coordinate macrophage growth and membrane biogenesis. The project is organized around three specific aims: (1) To characterize the regulated expression of CTP:phosphocholine cytidylyltransferase mRNA by CSF-1; (2) To determine the relationship between post-translational modifications of cytidylyltransferase and the rate of membrane formation during the cell cycle; and (3) To define the role of cytidylyltransferase in the cAMP inhibition of macrophage membrane phospholipid biogenesis.