Protease proforms as hematopoietic regulators

A common feature in myeloid leukemia is suppression of normal hematopoiesis, leading to infections, bleeding and anemia. It has been suggested for a long time that leukemic cells possess a growth-advantage by overproducing an inhibitor - a leukamia associated inhibitor. Such an inhibitor has been characterized in leukemic cells as well as subpopulations of normal bone marrow and blood cells, suggesting a role in regulation of normal hematopoiesis. In this thesis the leukemia associated inhibitor was identified as a secreted proform of proteinase 3 (PR3), normally found as active enzyme in the azurophil granules of the mature neutrophil. We could also identify other closely related serine protease proforms exhibiting the same inhibitory activity, namely azurocidin (from neutrophils), and granzymes A, B, H, K and M (from cytotoxic T/NK-cells). Our data demonstrate that hematopoietic serine proteases that leak out from cells as enzymatically inactive proforms are able to downregulate S-phase of granulopoietic progenitors. The S-phase downregulation is executed by N-terminal sequences only exposed properly with a preceding N-terminal di-propeptide. We suggest that the central event in S-phase downregulation of granulopoietic progenitors is a nitric oxide (NO)-mediated inhibition of ribonucleotide reductase, a rate-limiting enzyme in DNA-synthesis. We also present evidence for involvement of phosphatidylinositol 3-kinase, Akt/PKB and inducible NOS in the signal transduction. This thesis presents a new principle for regulation of neutrophil production, whereby a proform of an enzyme leaks out from the cell, exhibiting negative feedback on earlier developmental stages of granulopoiesis.