Protease Activity Increases in Plasma, Peritoneal Fluid, and Vital Organs after Hemorrhagic Shock in Rats

Hemorrhagic shock (HS) is associated with high mortality. A severe decrease in blood pressure causes the intestine, a major site of digestive enzymes, to become permeable – possibly releasing those enzymes into the circulation and peritoneal space, where they may in turn activate other enzymes, e.g. matrix metalloproteinases (MMPs). If uncontrolled, these enzymes may result in pathophysiologic cleavage of receptors or plasma proteins. Our first objective was to determine, in compartments outside of the intestine (plasma, peritoneal fluid, brain, heart, liver, and lung) protease activities and select protease concentrations after hemorrhagic shock (2 hours ischemia, 2 hours reperfusion). Our second objective was to determine whether inhibition of proteases in the intestinal lumen with a serine protease inhibitor (ANGD), a process that improves survival after shock in rats, reduces the protease activities distant from the intestine. To determine the protease activity, plasma and peritoneal fluid were incubated with small peptide substrates for trypsin-, chymotrypsin-, and elastase-like activities or with casein, a substrate cleaved by multiple proteases. Gelatinase activities were determined by gelatin gel zymography and a specific MMP-9 substrate. Immunoblotting was used to confirm elevated pancreatic trypsin in plasma, peritoneal fluid, and lung and MMP-9 concentrations in all samples after hemorrhagic shock. Caseinolytic, trypsin-, chymotrypsin-, elastase-like, and MMP-9 activities were all significantly (p<0.05) upregulated after hemorrhagic shock regardless of enteral pretreatment with ANGD. Pancreatic trypsin was detected by immunoblot in the plasma, peritoneal space, and lungs after hemorrhagic shock. MMP-9 concentrations and activities were significantly upregulated after hemorrhagic shock in plasma, peritoneal fluid, heart, liver, and lung. These results indicate that protease activities, including that of trypsin, increase in sites distant from the intestine after hemorrhagic shock. Proteases, including pancreatic proteases, may be shock mediators and potential targets for therapy in shock

Bmcc1s, a Novel Brain-Isoform of Bmcc1, Affects Cell Morphology by Regulating MAP6/STOP Functions

The BCH (BNIP2 and Cdc42GAP Homology) domain-containing protein Bmcc1/Prune2 is highly enriched in the brain and is involved in the regulation of cytoskeleton dynamics and cell survival. However, the molecular mechanisms accounting for these functions are poorly defined. Here, we have identified Bmcc1s, a novel isoform of Bmcc1 predominantly expressed in the mouse brain. In primary cultures of astrocytes and neurons, Bmcc1s localized on intermediate filaments and microtubules and interacted directly with MAP6/STOP, a microtubule-binding protein responsible for microtubule cold stability. Bmcc1s overexpression inhibited MAP6-induced microtubule cold stability by displacing MAP6 away from microtubules. It also resulted in the formation of membrane protrusions for which MAP6 was a necessary cofactor of Bmcc1s. This study identifies Bmcc1s as a new MAP6 interacting protein able to modulate MAP6-induced microtubule cold stability. Moreover, it illustrates a novel mechanism by which Bmcc1 regulates cell morphology

SmgGDS antagonizes BPGAP1-induced Ras/ERK activation and neuritogenesis in PC12 cell differentiation

10.1091/mbc.E12-04-0300Molecular Biology of the Cell242145-156MBCE

Mast cells and mast cell tryptase enhance migration of human lung fibroblasts through protease-activated receptor 2

Background: Mast cells may activate fibroblasts and contribute to remodeling processes in the lung. However, the mechanism behind these actions needs to be further investigated. Fibroblasts are major regulators of on-going remodeling processes. Protease activated receptor 2 (PAR2) expressed by fibroblasts may be activated by serine proteases, such as the mast cell mediator tryptase. The objective in this study was to investigate the effects of mast cells and specifically mast cell tryptase on fibroblast migration and the role of PAR2 activation. Methods: Human lung fibroblasts (HFL-1) were cultured together with human peripheral blood-derived mast cells or LAD2 mast cells and stimulated with either conditioned medium from LAD2 cells or tryptase. Analyses of immunological stimulation of mast cells by IgE/anti IgE in the co-culture system were also performed. The importance of PAR2 activation by mast cells and mast cell tryptase for the migratory effects of fibroblasts was investigated by pre-treatment with the PAR2 antagonist P2pal-18S. The expression of PAR2 was analyzed on fibroblasts and mast cells. Results: The migratory capacity of HFL-1 cells was enhanced by blood-derived mast cells (p < 0.02), LAD2 cells (p < 0.001), conditioned medium (p < 0.05) and tryptase (p < 0.006). P2pal-18S decreased the induced migration caused by mast cells (p < 0.001) and tryptase (p < 0.001) and the expression of PAR2 was verified in HFL-1 cells. Mast cells immunologically stimulated with IgE/Anti IgE had no further effects on fibroblast migration. Conclusions: Mast cells and the mast cell mediator tryptase may have crucial roles in inducing lung fibroblast migration via PAR-2 activation, which may contribute to remodeling processes in chronic lung diseases