Identification of Elastase as a Secretory Protease from Cultured Rat Microglia

Abstract

In the course of studying the secretory products of microglia, we detected protease activity in the conditioned medium. Various proteins (casein, histone, myelin basic protein, and extracellular matrix) were digested. The protease activity was characterized by using purified myelin basic protein as a substrate. Maximal activity was observed at neutral pH levels (7-8), which was different from the optimum pH level of proteolytic activity observed in the cell homogenate. The activity was inhibited approximately 60 and 50% by 1 mM phenylmethylsulfonyl fluoride and 40 microM elastatinal, respectively. In gel filtration, the major activity, which was inhibited in the presence of N-methoxysuccinyl-Ala-Ala-Pro-Val-methyl chloride, eluted at a position corresponding to a molecular mass of approximately 25 kDa. These results suggest that the major protease present in microglial conditioned medium is elastase or an elastase-like protease. This suggestion was confirmed by the finding that the 25-kDa protein band was stained with anti-elastase antiserum by western blotting. De novo synthesis of elastase in microglia was supported by [35S]methionine incorporation. In the presence of lipopolysaccharide, the secretory elastase decreased. These results demonstrate that microglia secrete proteases, one of which was identified as elastase. The significance of this enzyme production in physiological and pathological conditions is discussed.

Involvement of vasodilator-stimulated phosphoprotein in UDP-induced microglial actin aggregation via PKC- and Rho-dependent pathways

“…In vivo imaging techniques revealed that the activation of the P2Y 12 receptor was required to extend the processes toward a lesion site in damaged brain tissue [2,9]. These studies indicate the prominent role of P2Y 12 receptor activation in microglial motility. We also reported that P2Y 6 receptor activation stimulated by its ligand uridine 5′-diphosphate (UDP) induced dynamic cytoskeletal arrangement and process movement in microglia, resulting in increase of microsphere phagocytosis [1].…”

“…Activation of the P2Y 12 receptor also causes integrin-dependent microglial process extension [8]. In vivo imaging techniques revealed that the activation of the P2Y 12 receptor was required to extend the processes toward a lesion site in damaged brain tissue [2,9]. These studies indicate the prominent role of P2Y 12 receptor activation in microglial motility.…”

“…These nucleotides are released from damaged cells and act on their cognate receptors, P2X and P2Y. Microglia express ionotropic P2X 4,7 and metabotropic P2Y 6,12 receptors [1]. We previously showed that extracellular adenosine 5′-triphosphate (ATP) and adenosine 5′-diphosphate (ADP) induce membrane ruffling and chemotaxis by activation of P2Y 12 receptors in microglia [1,[5][6][7].…”

“…We previously showed that extracellular adenosine 5′-triphosphate (ATP) and adenosine 5′-diphosphate (ADP) induce membrane ruffling and chemotaxis by activation of P2Y 12 receptors in microglia [1,[5][6][7]. Activation of the P2Y 12 receptor also causes integrin-dependent microglial process extension [8]. In vivo imaging techniques revealed that the activation of the P2Y 12 receptor was required to extend the processes toward a lesion site in damaged brain tissue [2,9].…”

IRF8 is a transcriptional determinant for microglial motility

“…Mouse primary cultured microglia were prepared as described previously [5,35]. Briefly, a mixed glial culture was prepared from neonatal IRF8 knockout, hetero-knockout, and wildtype mice and maintained for 10-16 days in DMEM with 10 % fetal bovine serum [5,36].…”