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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

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Abstract

Microglia are the immune cells of the CNS. Brain injury triggers phenotypic changes in microglia including regulation of surface antigens. The serine proteinase α-thrombin can induce profound changes in neural cell physiology via cleavage of proteinase-activated receptors (PARs). We recently demonstrated that pharmaceutical-grade recombinant human α-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 PAR1 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.

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Abbreviations

CNS:

Central nervous system

PAR:

Proteinase-activated receptor

rh-thr:

Pharmaceutical-grade recombinant human thrombin

ERK 1/2:

Extracellular signal-regulated kinase 1/2

JNK:

c-Jun activated kinase

TNF:

Tumor necrosis factor

LPS:

Lipopolysaccharide/endotoxin

PMX:

Polymyxin B sulfate

DMEM:

Dulbecco’s modified Eagle’s Medium

FBS:

Fetal bovine serum

MSFM:

Macrophage serum-free medium

PBS:

Phosphate buffered saline

EDTA:

Ethylenediaminetetraacetic acid

Ig:

Immunoglobulin

MAPK:

Mitogen-activated protein kinase

HPRT:

Hypoxanthine phosphoribosyltransferase

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Acknowledgements

This work was supported by NIH/NINDS grants NS44337 (TM) and NS047309 (JRW). The authors thank ZymoGenetics, Inc., Seattle, WA, USA for the generous gift of pharmaceutical-grade recombinant human α-thrombin.

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Authors and Affiliations

  1. Department of Neurology, School of Medicine, University of Washington, Box 356465, 1959 NE Pacific Street, Seattle, WA, 98195-6465, USA

    Jonathan R. Weinstein, Matthew Zhang, Mansur Kutlubaev, Richard Lee, Caroline Bishop & Thomas Möller

  2. ZymoGenetics, Inc, Seattle, WA, 98102, USA

    Henrik Andersen

  3. Institute for Neuropathology, University of Göttingen, Gottingen, 37075, Germany

    Uwe-Karsten Hanisch

  4. Center for Neurogenetics and Neurotherapeutics, School of Medicine, University of Washington, Seattle, WA, 98195-7110, USA

    Thomas Möller

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  1. Jonathan R. Weinstein
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Correspondence to Jonathan R. Weinstein.

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Weinstein, J.R., Zhang, M., Kutlubaev, M. et al. 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. Neurochem Res 34, 445–452 (2009). https://doi.org/10.1007/s11064-008-9803-9

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