Noncanonical regulation of insulin-mediated ERK activation by phosphoinositide 3-kinase γ.

Classically Class IB phosphoinositide 3-kinase (PI3Kγ) plays a role in extracellular signal-regulated kinase (ERK) activation following G-protein coupled receptor (GPCR) activation. Knock-down of PI3Kγ unexpectedly resulted in loss of ERK activation to receptor tyrosine kinase agonists such as epidermal growth factor or insulin. Mouse embryonic fibroblasts (MEFs) or primary adult cardiac fibroblasts isolated from PI3Kγ knock-out mice (PI3KγKO) showed decreased insulin-stimulated ERK activation. However, expression of kinase-dead PI3Kγ resulted in rescue of insulin-stimulated ERK activation. Mechanistically, PI3Kγ sequesters protein phosphatase 2A (PP2A), disrupting ERK-PP2A interaction, as evidenced by increased ERK-PP2A interaction and associated PP2A activity in PI3KγKO MEFs, resulting in decreased ERK activation. Furthermore, ß-blocker carvedilol-mediated ß-arrestin-dependent ERK activation is significantly reduced in PI3KγKO MEF, suggesting accelerated dephosphorylation. Thus, instead of classically mediating the kinase arm, PI3Kγ inhibits PP2A by scaffolding and sequestering, playing a key parallel synergistic step in sustaining the function of ERK, a nodal enzyme in multiple cellular processes.

Plasminogen promotes macrophage phagocytosis in mice.

The phagocytic function of macrophages plays a pivotal role in eliminating apoptotic cells and invading pathogens. Evidence implicating plasminogen (Plg), the zymogen of plasmin, in phagocytosis is extremely limited with the most recent in vitro study showing that plasmin acts on prey cells rather than on macrophages. Here, we use apoptotic thymocytes and immunoglobulin opsonized bodies to show that Plg exerts a profound effect on macrophage-mediated phagocytosis in vitro and in vivo. Plg enhanced the uptake of these prey by J774A.1 macrophage-like cells by 3.5- to fivefold Plg receptors and plasmin proteolytic activity were required for phagocytosis of both preys. Compared with Plg(+/+) mice, Plg(-/-) mice exhibited a 60% delay in clearance of apoptotic thymocytes by spleen and an 85% reduction in uptake by peritoneal macrophages. Phagocytosis of antibody-mediated erythrocyte clearance by liver Kupffer cells was reduced by 90% in Plg(-/-) mice compared with Plg(+/+) mice. A gene array of splenic and hepatic tissues from Plg(-/-) and Plg(+/+) mice showed downregulation of numerous genes in Plg(-/-) mice involved in phagocytosis and regulation of phagocytic gene expression was confirmed in macrophage-like cells. Thus, Plg may play an important role in innate immunity by changing expression of genes that contribute to phagocytosis.

Macrophage gene expression and foam cell formation are regulated by plasminogen.

BACKGROUND: Deciphering the molecular and cellular processes that govern macrophage foam cell formation is critical to understanding the basic mechanisms underlying atherosclerosis and other vascular pathologies. METHODS AND RESULTS: Here, we identify a pivotal role of plasminogen (Plg) in regulating foam cell formation. Deficiency of Plg inhibited macrophage cholesterol accumulation on exposure to hyperlipidemic conditions in vitro, ex vivo, and in vivo. Gene expression analysis identified CD36 as a regulated target of Plg, and macrophages from Plg(-/-) mice had decreased CD36 expression and diminished foam cell formation. The Plg-dependent CD36 expression and foam cell formation depended on conversion of Plg to plasmin, binding to the macrophage surface, and the consequent intracellular signaling that leads to production of leukotriene B4. Leukotriene B4 rescued the suppression of CD36 expression and foam cell formation arising from Plg deficiency. CONCLUSIONS: Our findings demonstrate an unanticipated role of Plg in the regulation of gene expression and cholesterol metabolism by macrophages and identify Plg-mediated regulation of leukotriene B4 as an underlying mechanism.

Phenylalanine-rich peptides potently bind ESAT6, a virulence determinant of Mycobacterium tuberculosis, and concurrently affect the pathogen's growth.

BACKGROUND: The secretory proteins of Mycobacterium tuberculosis (M. tuberculosis) have been known to be involved in the virulence, pathogenesis as well as proliferation of the pathogen. Among this set, many proteins have been hypothesized to play a critical role at the genesis of the onset of infection, the primary site of which is invariably the human lung. METHODOLOGY/PRINCIPAL FINDINGS: During our efforts to isolate potential binding partners of key secretory proteins of M. tuberculosis from a human lung protein library, we isolated peptides that strongly bound the virulence determinant protein Esat6. All peptides were less than fifty amino acids in length and the binding was confirmed by in vivo as well as in vitro studies. Curiously, we found all three binders to be unusually rich in phenylalanine, with one of the three peptides a short fragment of the human cytochrome c oxidase-3 (Cox-3). The most accessible of the three binders, named Hcl1, was shown also to bind to the Mycobacterium smegmatis (M. smegmatis) Esat6 homologue. Expression of hcl1 in M. tuberculosis H37Rv led to considerable reduction in growth. Microarray analysis showed that Hcl1 affects a host of key cellular pathways in M. tuberculosis. In a macrophage infection model, the sets expressing hcl1 were shown to clear off M. tuberculosis in much greater numbers than those infected macrophages wherein the M. tuberculosis was not expressing the peptide. Transmission electron microscopy studies of hcl1 expressing M. tuberculosis showed prominent expulsion of cellular material into the matrix, hinting at cell wall damage. CONCLUSIONS/SIGNIFICANCE: While the debilitating effects of Hcl1 on M. tuberculosis are unrelated and not because of the peptide's binding to Esat6-as the latter is not an essential protein of M. tuberculosis-nonetheless, further studies with this peptide, as well as a closer inspection of the microarray data may shed important light on the suitability of such small phenylalanine-rich peptides as potential drug-like molecules against this pathogen.