Colony-stimulating factor-1-induced oscillations in phosphatidylinositol-3 kinase/AKT are required for caspase activation in monocytes undergoing differentiation into macrophages.

The differentiation of human peripheral blood monocytes into resident macrophages is driven by colony-stimulating factor-1 (CSF-1), which upon interaction with CSF-1 receptor (CSF-1R) induces within minutes the phosphorylation of its cytoplasmic tyrosine residues and the activation of multiple signaling complexes. Caspase-8 and -3 are activated at day 2 to 3 and contribute to macrophage differentiation, for example, through cleavage of nucleophosmin. Here, we show that the phosphatidylinositol-3 kinase and the downstream serine/threonine kinase AKT connect CSF-1R activation to caspase-8 cleavage. Most importantly, we demonstrate that successive waves of AKT activation with increasing amplitude and duration are required to provoke the formation of the caspase-8-activating molecular platform. CSF-1 and its receptor are both required for oscillations in AKT activation to occur, and expression of a constitutively active AKT mutant prevents the macrophage differentiation process. The extracellular receptor kinase 1/2 pathway is activated with a coordinated oscillatory kinetics in a CSF-1R-dependent manner but plays an accessory role in caspase activation and nucleophosmin cleavage. Altogether, CSF-1 stimulation activates a molecular clock that involves phosphatidylinositol-3 kinase and AKT to promote caspase activation. This oscillatory signaling pathway, which is coordinated with extracellular receptor kinase 1/2 oscillatory activation, involves CSF-1 and CSF-1R and controls the terminal differentiation of macrophages.

Stability of mutant serpin/furin complexes: dependence on pH and regulation at the deacylation step.

Furin proteolytically cleaves a wide variety of proprotein substrates mainly within the trans-Golgi network (TGN) but also at the cell membrane and in endosomal compartments where pH is more acidic. Incorporation of furin recognition sequences within the reactive site loop (RSL) of alpha(1)-antitrypsin (AT) leads to the production of furin inhibitors. In an attempt to design more stable, potent, and specific serpin-based inhibitors, we constructed a series of AT and alpha(1)-antichymotrypsin (ACT) mutants by modifying the P(7)-P(1) region of their RSLs. The biochemical properties of these variants were assessed by evaluating their propensity to establish SDS-resistant complexes with furin in a variety of conditions (pH 6.0-9.0) and by measuring their association rate constants. The effect of pH during the initial steps of complex formation was minimal, suggesting that the acylation step is not rate-limiting. The decrease in stoichiometry of inhibition (SI) values observed in AT variants at high pHs was a result of the reduced pH-dependent deacylation rate, which is rate-limiting in this mechanism and which suggests increased complex stability. Conversely, the SI values for ACT mutants had a tendency to be lower at acidic pH. Transiently transfecting HEK293 cells with these mutants abolished processing of the pro-von Willebrand factor precursor but, interestingly, only the ACT variants were secreted in the media as uncleaved forms. Our results suggest that reengineering the reactive site loops of serpins to accommodate and target furin or other serine proteases must take into account the intrinsic physicochemical properties of the serpin.

Leukocyte elastase inhibition therapy in cystic fibrosis: role of glycosylation on the distribution of alpha-1-proteinase inhibitor in blood versus lung.

Cystic fibrosis patients demonstrate an increased susceptibility to bacterial lung infections. Airway infiltration by neutrophils will then lead to an increase in human leukocyte elastase (HLE) within the extracellular compartment, thereby producing deleterious effects. Here, we investigated the properties and tissue distribution of an unglycosylated, recombinant form of the HLE inhibitor alpha-1-proteinase inhibitor (alpha(1)-antitrypsin rhalpha1PI) when it is administered to the airway surface. We produced rhalpha1PI using a bacterial expression system and found the purified protein to be indistinguishable from blood-purified, glycosylated alpha1PI at inhibiting elastase in vitro. In contrast to intravenous administration, direct delivery of either alpha1PI or rhalpha1PI to the airway surface of CD-1 mice by nasal instillation produced similar highly detectable levels of protein in bronchoalveolar lavage at all time points, suggesting that glycosylation of alpha1PI does not play the same critical role in determining protein stability at the respiratory surface as it does in the vascular compartment. Interestingly, this unglycosylated rhalpha1PI was also highly protective against elastase-mediated injury 24 h after rhalpha1PI instillation and was consistently found to be significantly more protective than glycosylated blood-derived alpha1PI. Thus, these results provide evidence that aerosol delivery of rhalpha1PI could be an effective strategy for controlling HLE-dependent pathophysiology associated with cystic fibrosis lung disease.

Polyethylene glycol conjugation at Cys232 prolongs the half-life of alpha1 proteinase inhibitor.

alpha1 Proteinase inhibitor (alpha1PI), a natural inhibitor of the serine proteinase leukocyte elastase, is also an intravenous therapeutic agent used to treat hereditary emphysema and may be useful in other respiratory disorders. However, to achieve sustained suppression of leukocyte elastase, alpha1PI must be given frequently and in large amounts, thus limiting its clinical use. We hypothesized that conjugating alpha1PI with polyethylene glycol (PEG) at Cys(232) could extend the in vivo half-life of alpha1PI in blood and lung. We present evidence that site-specific conjugation with either 20 or 40 kD PEG at Cys(232) of nonglycosylated recombinant human alpha1PI (rhalpha1PI) results in an active inhibitor with prolonged in vivo stability. In addition, 72 h after airway instillation PEG-rhalpha1PI was found to be significantly better than glycosylated alpha1PI in protecting the lung against leukocyte elastase-mediated lung hemorrhage. We conclude that thiol-specific PEGylation markedly improves the in vivo pharmacokinetic profile of rhalpha1PI and represents a simple, novel strategy to address the therapeutic goal of human leukocyte elastase inhibition.