Mesotrypsin, a brain trypsin, activates selectively proteinase‐activated receptor‐1, but not proteinase‐activated receptor‐2, in rat astrocytes

Abstract

Proteinase-activated receptors (PARs), a subfamily of G protein-coupled receptors, which are activated by serine proteases, such as trypsin, play pivotal roles in the CNS. Mesotrypsin (trypsin IV) has been identified as a brain-specific trypsin isoform. However, its potential physiological role concerning PAR activation in the brain is largely unknown. Here, we show for the first time that mesotrypsin, encoded by the PRSS3 (proteinase, serine) gene, evokes a transient and pronounced Ca 2+ mobilization in both primary rat astrocytes and retinal ganglion RGC-5 cells, suggesting a physiological role of mesotrypsin in brain cells. Mesotrypsin mediates Ca 2+ responses in rat astrocytes in a concentration-dependent manner, with a 50% effective concentration (EC 50 ) value of 25 nM. The maximal effect of mesotrypsin on Ca 2+ mobilization in rat astrocytes is much higher than that observed in 1321N1 human astrocytoma cells, indicating that the activity of mesotrypsin is species-specific. The pre-treatment of cells with thrombin or the PAR-1-specific peptide TRag (AlapFluoro-Phe-Arg-Cha-HomoArg-Tyr-NH 2 , synthetic thrombin receptor agonist peptide), but not the PAR-2-specific peptide, reduces significantly the mesotrypsin-induced Ca 2+ response.Treatment with the PAR-1 antagonist SCH79797 confirms that mesotrypsin selectively activates PAR-1 in rat astrocytes. Unlike mesotrypsin, the two other trypsin isoforms, cationic and anionic trypsin, activate multiple PARs in rat astrocytes. Therefore, our data suggest that brain-specific mesotrypsin, via the regulation of PAR-1, is likely to be involved in multiple physiological/pathological processes in the brain. Keywords: anionic trypsin, calcium signaling, cationic trypsin, mesotrypsin, proteinase-activated receptors. The proteinase-activated receptors (PARs) are a four-member subfamily of G protein-coupled receptors. They are widely expressed in the CNS, including neurons, astrocytes, microglial cells, oligodendrocytes and rat brain capillary endothelial cells, and play a pivotal role in multiple physiological and pathological processes in the brain, such as ischemic stroke and astrogliosis (Striggow et al. 2000(Striggow et al. , 2001Dömötör et al. 2002;Suo et al. 2002;Wang et al. 2002a;Xi et al. 2003;Wang et al. 2004;Nicole et al. 2005). PAR is activated by proteolytic cleavage of its N-terminus by certain serine proteinases, such as thrombin and trypsin. Thus, a new N-terminus is unmasked, acting as a tethered ligand. This ligand can interact with the extracellular loop-2, initiating multiple signal transductions (Wang and Reiser 2003). Trypsin, which is primarily isolated from human pancreas, can activate not only PAR-2 and PAR-4, but also PAR-1 (Nystedt et al. 1995;Böhm et al. 1996;Xu et al. 1998;Wang et al. 2002a;Wang et al. 2004). It is well known that the human pancreas secretes three isoforms of trypsinogen, which are encoded by the PRSS (proteinase, serine) genes PRSS1 (cationic trypsinogen), PRSS2 (anionic trypsinogen) and PRSS3 (mesotrypsinogen). Unlike c...

Trypsin IV or Mesotrypsin and p23 Cleave Protease-activated Receptors 1 and 2 to Induce Inflammation and Hyperalgesia

“…Our results showing that trypsin IV can activate PAR 2 agree with our previous observation that impure preparations of trypsin IV can activate this receptor but contradict other reports that mesotrypsin does not activate PAR 2 in human epithelial cells (30) or rat astrocytes (31). These discrepancies could be attributable to differences in protease preparations, species, levels of receptor expression, or cell type, and further experimentation is necessary to address these issues.…”

“…However, whereas trypsin I and II signal to human epithelial cell lines expressing PAR 1 and PAR 2 , mesotrypsin is inactive (30). In contrast, mesotrypsin signals to rat brain astrocytes by cleaving PAR 1 but not PAR 2 (31). Thus, the capacity of trypsin IV/mesotrypsin to activate PARs depends on the preparation of proteases, the cell type, and the species.…”

Expression of plasminogen activator inhibitor-1 by olfactory ensheathing glia promotes axonal regeneration

“…PAR-1 is a G-protein-coupled receptor mainly activated by thrombin and other serine proteinases-proteolytic cleavage (Vu et al, 1991;Wang et al, 2006). PAR-1 may couple to different G proteins (Hung et al, 1992;Offermanns et al, 1994), mediating diverse activities.…”

Determinants of Affinity and Proteolytic Stability in Interactions of Kunitz Family Protease Inhibitors with Mesotrypsin

“…Mesotrypsin is highly resistant to inhibition by many polypeptide trypsin inhibitors (39 -41), although it is readily inhibited by serpin-type inhibitors with Arg in the reactive loop (42,43). Although it cleaves peptide anilide model substrates with kinetic constants similar to other trypsins (44 -46), it displays reduced or no activity toward several specific protein substrates of other trypsins (44,(47)(48)(49) and, unlike other trypsins, is incapable of autoactivation (44). By contrast, mesotrypsin is far more active than other trypsins in cleavage of canonical protease inhibitors at the reactive site peptide bond (44,46,50).…”