Neutrophil proPR3 Induces S-Phase Arrest in Myeloid Progenitors by Inhibition of Ribonucleotide Reductase Mediated by Nitric Oxide.

We have shown that secreted enzymatically inactive dipeptide proforms of the hematopoietic serine proteases proteinase 3 (PR3), azurocidin and granzymes A, B, H, K, and M, are able to reduce the fraction of granulopoietic progenitors (CFU-GM) in S-phase, whereas human leukocyte elastase (HLE) and cathepsin G lack this ability. The objective of the present study was to map the specific sequence(s) of proPR3 responsible for the downmodulation of S-phase, and to elucidate the signaling pathways involved. The S-phase downmodulatory effect has a fast onset within 90 min, is reversible, non-cytotoxic and counteracted by G-CSF. The fast onset suggests a direct interruption of DNA-synthesis, i.e. an S-phase arrest. Synthetic peptides corresponding to N-terminal sequences of PR3, purified recombinant PR3 and HLE, as well as hybrid proteins constructed by an interchange of the di-propeptide and the first fourteen amino acids of the mature form of PR3 and HLE, thus creating PR3/HLE and HLE/PR3, respectively, were tested for their ability to reduce the S-phase fraction of granulopoeitic progenitors (CFU-GM). Results from synthetic peptides showed that the common N-terminal motif of PR3 and other serine proteases, i.e. IVGG or IIGG, downmodulate the S-phase of CFU-GM at 40–80 nM concentration. The dipeptide proform of HLE does not inhibit CFU-GM in S-phase, despite carrying the motif IVGG. However, if the N-terminal of HLE is presented by the HLE/PR3 hybrid protein, it is fully active, as well as the reciprocal hybrid PR3/HLE, whereas a mutant form PR3(G4R)/HLE and the tetrapeptide IVGR both are inactive. These findings demonstrate that the downmodulatory effect on CFU-GM in S-phase is mediated by the first four N-terminal amino acids IVGG of PR3, and also demonstrate that the activity is dependent on the configuration of the proform providing the correct presentation of this N-terminal motif. The S-phase arrest induced by proPR3 is mediated by a signal transduction pathway engaging phosphatidylinositol 3-kinase (PI3K), Akt/PKB, nitric oxide synthase (NOS), NO and ribonucleotide reductase. Inhibition of PI3K by wortmannin or LY294002, or Akt/PKB by 1L-6-hydroxymethyl-chiro-inositol 2-[(R)-2-O-methyl-3-O-octadecylcarbonate] abrogate the S-phase arrest induced by proPR3. The non-specific NOS-inhibitor N-monomethyl-L-arginine, the two iNOS-specific inhibitors 1400W or GW274150, or the nitric oxide scavenger carboxy-PTIO all abrogate the S-phase downmodulatory effect of proPR3. Furthermore, the effect of proPR3 is abolished by providing 2′-deoxyadenosine and 2′-deoxyguanosine to the cells, suggesting inhibition of ribonucleotide reductase, probably through direct inhibition by NO. The S-phase arrest induced by proPR3 is reproduced by SNAP, a cell-permeable NO-donor, an effect that is readily reversed by carboxy-PTIO or 2′-deoxyadenosine/2′-deoxyguanosine supplementation. The S-phase arrest induced by proPR3 is also reproduced by hydroxyurea (50 uM), a known inhibitor of ribonucleotide reductase, and is readily reversed by 2′-deoxyadenosine/2′-deoxyguanosine supplementation. The S-phase arrest induced by proPR3 or tetrapeptide IVGG has been verified by demonstration of a reduced incorporation of BrdU (bromo-deoxy-uridine) in purified myeloid progenitor cells isolated by fluorescence activated cell sorting. In conclusion, we provide evidence that the S-phase downmodulatory activity of proPR3 towards granulopoietic progenitors is an S-phase arrest caused by inhibition of ribonucleotide reductase by nitric oxide, mediated through PI3K, Akt/PKB, and NOS.