Immune Netw.  2010 Jun;10(3):99-103. 10.4110/in.2010.10.3.99.

GSK3beta Inhibitor Peptide Protects Mice from LPS-induced Endotoxin Shock

• Ko R
• Jang HD
• Lee SY

• KMID: 2150666
• DOI: http://doi.org/10.4110/in.2010.10.3.99

Abstract

BACKGROUND
Glycogen synthase kinase 3beta (GSK3beta) is a ubiquitous serine/threonine kinase that is regulated by serine phosphorylation at 9. Recent studies have reported the beneficial effects of a number of the pharmacological GSK3beta inhibitors in rodent models of septic shock. Since most of the GSK3beta inhibitors are targeted at the ATP-binding site, which is highly conserved among diverse protein kinases, the development of novel non-ATP competitive GSK3beta inhibitors is needed.
METHODS
Based on the unique phosphorylation motif of GSK3beta, we designed and generated a novel class of GSK3beta inhibitor (GSK3i) peptides. In addition, we investigated the effects of a GSK3i peptide on lipopolysaccharide (LPS)-stimulated cytokine production and septic shock. Mice were intraperitoneally injected with GSK3i peptide and monitored over a 7-day period for survival.
RESULTS
We first demonstrate its effects on LPS-stimulated pro-inflammatory cytokine production including interleukin (IL)-6 and IL-12p40. LPS-induced IL-6 and IL-12p40 production in macrophages was suppressed when macrophages were treated with the GSKi peptide. Administration of the GSK3i peptide potently suppressed LPS-mediated endotoxin shock.
CONCLUSION
Collectively, we present a rational strategy for the development of a therapeutic GSK3i peptide. This peptide may serve as a novel template for the design of non-ATP competitive GSK3 inhibitors.

Keyword

GSK3beta; Peptide inhibitor; LPS; Cytokines

MeSH Terms

Animals
Cytokines
Glycogen Synthase Kinase 3
Glycogen Synthase Kinases
Interleukin-12 Subunit p40
Interleukin-6
Interleukins
Macrophages
Mice
Peptides
Phosphorylation
Phosphotransferases
Protein Kinases
Rodentia
Serine
Shock
Shock, Septic
Cytokines
Glycogen Synthase Kinase 3
Glycogen Synthase Kinases
Interleukin-12 Subunit p40
Interleukin-6
Interleukins
Peptides
Phosphotransferases
Protein Kinases
Serine

Figure

• 

  Figure 1 Structure of the GSKi peptide conjugated with the Hph-1 protein transduction domain. Amino acid sequences corresponding to residues 3~12 of GSK3β were chosen for the design of a GSKi peptide. The serine 9 residue, which can be phosphorylated by PKB/Akt, is highlighted in bold. The control peptide contains 11-mer of the protein transduction domain.

• 

  Figure 2 The GSKi peptide decreased pro-inflammatory cytokines production after LPS stimulation. BMDMs were pre-incubated for 2 hours with medium only and either 10 µM SB216763 or 5 µM GSK3i peptide, and then stimulated with 1 µg/ml LPS for 20 hours. Cell-free supernatants were analyzed by ELISA for production of pro-inflammatory cytokines; IL-6 (A) or IL-12p40 (B). Data represent mean±s.d. and are representative of at least three experiments.

• 

  Figure 3 The GSK3i peptide protected mice from LPS-induced endotoxin shock. 25 mg/kg of the GSK3 inhibitor SB216763 (n=15) or 30 mg/kg of the GSK3i peptide (n=15) were administered intraperitoneally to mice before injection of 15 mg/kg of the E.coli K235 LPS insult, which is the lethal dose (LD100). Sham-immunized mice were given only PBS containing 0.1% DMSO (n=4). LPS control (n=15). Administration of the GSK3 inhibitor SB216763 or GSK3i peptide protected mice from an LD100 of LPS given therapeutically. Survival of mice following LPS challenge was monitored for 7 days.

• 

  Figure 4 In vivo IL-6 in plasma, measured by ELISA 12 hours after mice were given an LD100 of LPS. Sham-immunized mice were given only PBS containing 0.1% DMSO (n=3). n=12 for LPS only, SB216763, and GSKi peptide, respectively.

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