Thrombin-Induced Regulation of CD95(Fas) Expression in the N9 Microglial Cell Line: Evidence for Involvement of Proteinase-Activated Receptor1 and Extracellular Signal-Regulated Kinase 1/2

Abstract

Microglia are the immune cells of the CNS. Brain injury triggers phenotypic changes in microglia including regulation of surface antigens. The serine proteinase alpha-thrombin can induce profound changes in neural cell physiology via cleavage of proteinase-activated receptors (PARs). We recently demonstrated that pharmaceutical-grade recombinant human alpha-thrombin (rh-thr) induces a restricted set of proteolysis-dependent changes in microglia. CD95(Fas) is a cell-death receptor that is up-regulated in microglia by inflammatory stimuli. Here we characterized the effect of rh-thr on CD95(Fas) expression in the N9 microglial cell line. Dose-response and time course studies demonstrated maximal effects at 100 U/ml and 24 h, respectively. Regulation of expression was seen at both the surface protein and steady-state mRNA levels. The rh-thr-induced effects were mimicked by PAR(1) agonist peptides and blocked by pharmacologic inhibitors selective for extracellular signal-regulated kinase 1/2 (ERK 1/2). Rh-thr also induced a rapid and sustained phosphorylation of ERK 1/2. Thrombin-induced regulation of CD95(Fas) could modulate the neuroinflammatory response in a variety of neurological disorders.

α-Synuclein Activates Microglia by Inducing the Expressions of Matrix Metalloproteinases and the Subsequent Activation of Protease-Activated Receptor-1

“…To our knowledge, this is the first report of PAR-1 cleavage by MMP-3, MMP-8, or MMP-9. A recent report showed that thrombin (a ligand of PAR-1) induced CD95 expression via PAR-1 activation in N9 microglial cells (32). Interestingly, the sites of PAR-1 cleavage by MMP-3, -8, and -9 were the same as those of thrombin.…”

“…During our search for cell surface receptors involved in MMP-mediated microglial activation, we noted that PAR-1 had been reported to mediate MMP-1-induced endothelial activation and tumorigenesis and thrombin-induced microglial activation (31,32). Thus, to test the possible involvement of PAR-1 in a-synuclein-induced microglial activation, FIGURE 4.…”

Protease-Activated Receptor–1 Expression in Rat Microglia after Trimethyltin Treatment

“…Although the physiological significance of this upregulation remains to be determined, it is interesting to note that over the past years, several studies have suggested the involvement of microglial PARs, especially PAR-1, in the balance of inflammatory processes in the nervous system (Weinstein et al 2008;Weinstein et al 2009). In particular, our group has observed that the administration of PAR-1 activating peptides (TRAP6 and TFLLR) inhibits the production of the proinflammatory cytokines TNF-α and IL-6 in microglial cells treated with lipopolysaccharide, while promoting the release of the anti-inflammatory cytokine IL-10.…”

IgM-Dependent Phagocytosis in Microglia Is Mediated by Complement Receptor 3, Not Fcα/μ Receptor

“…To detect the mRNA expression of Fcα/μR in mouse microglia, RNA isolation (including on-column DNase digestion), reverse transcription and quantitative Real-Time polymerase chain reaction (qRT-PCR) were done as previously described (28). In brief, qRT-PCR was performed using the 7500 Real Time PCR System (Applied Biosystems).…”