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Neurochemical Research

, Volume 34, Issue 3, pp 445–452 | Cite as

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

  • Jonathan R. Weinstein
  • Matthew Zhang
  • Mansur Kutlubaev
  • Richard Lee
  • Caroline Bishop
  • Henrik Andersen
  • Uwe-Karsten Hanisch
  • Thomas Möller
Original Paper

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.

Keywords

Thrombin Microglia CD95(Fas) Proteinase-activated receptor (PAR) Extracellular signal-regulated kinase 1/2 (ERK 1/2) 

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

Notes

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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Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Jonathan R. Weinstein
    • 1
  • Matthew Zhang
    • 1
  • Mansur Kutlubaev
    • 1
  • Richard Lee
    • 1
  • Caroline Bishop
    • 1
  • Henrik Andersen
    • 2
  • Uwe-Karsten Hanisch
    • 3
  • Thomas Möller
    • 1
    • 4
  1. 1.Department of Neurology, School of MedicineUniversity of WashingtonSeattleUSA
  2. 2.ZymoGenetics, IncSeattleUSA
  3. 3.Institute for NeuropathologyUniversity of GöttingenGottingenGermany
  4. 4.Center for Neurogenetics and Neurotherapeutics, School of MedicineUniversity of WashingtonSeattleUSA

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