Inhibition of endogenous heat shock protein 70 attenuates inducible nitric oxide synthase induction via disruption of heat shock protein 70/Na(+) /H(+) exchanger 1-Ca(2+) -calcium-calmodulin-dependent protein kinase II/transforming growth factor β-activated kinase 1-nuclear factor-κB signals in BV-2 microglia.

Abstract

Inducible nitric oxide synthase (iNOS) critically contributes to inflammation and host defense. The inhibition of heat shock protein 70 (Hsp70) prevents iNOS induction in lipopolysaccharide (LPS)-stimulated macrophages. However, the role and mechanism of endogenous Hsp70 in iNOS induction in microglia remains unclear. This study addresses this issue in BV-2 microglia, showing that Hsp70 inhibition or knockdown prevents LPS-induced iNOS protein expression and nitric oxide production. Real-time PCR experiments showed that LPS-induced iNOS mRNA transcription was blocked by Hsp70 inhibition. Further studies revealed that the inhibition of Hsp70 attenuated LPS-stimulated nuclear translocation and phosphorylation of nuclear factor (NF)-κB as well as the degradation of inhibitor of κB (IκB)-α and phosphorylation of IκB kinase β (IKKβ). This prevention effect of Hsp70 inhibition on IKKβ-NF-κB activation was found to be dependent on the Ca(2+) /calcium-calmodulin-dependent protein kinase II (CaMKII)/transforming growth factor β-activated kinase 1 (TAK1) signals based on the following observations: 1) chelation of intracellular Ca(2+) or inhibition of CaMKII reduced LPS-induced increases in TAK1 phosphorylation and 2) Hsp70 inhibition reduced LPS-induced increases in CaMKII/TAK1 phosphorylation, intracellular pH value, [Ca(2+) ]i , and CaMKII/TAK1 association. Mechanistic studies showed that Hsp70 inhibition disrupted the association between Hsp70 and Na(+) /H(+) exchanger 1 (NHE1), which is an important exchanger responsible for Ca(2+) influx in LPS-stimulated cells. These studies demonstrate that the inhibition of endogenous Hsp70 attenuates the induction of iNOS, which likely occurs through the disruption of NHE1/Hsp70-Ca(2+) -CaMKII/TAK1-NF-κB signals in BV-2 microglia, providing further insight into the functions of Hsp70 in the CNS.

• Authors: Chao Huang, Xu Lu, Jia Wang, Lijuan Tong, Bo Jiang, Wei Zhang
• Journal: Journal of neuroscience research (J Neurosci Res) Vol. 93 Issue 8 Pg. 1192-202 (Aug 2015) ISSN: 1097-4547 [Electronic] United States
• PMID: 25691123 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2015 Wiley Periodicals, Inc.

Chemical References

• HSP70 Heat-Shock Proteins
• NF-kappa B
• Oligonucleotides, Antisense
• Sodium-Hydrogen Exchangers
• growth factor-activatable Na-H exchanger NHE-1
• Nitric Oxide Synthase Type II
• Nos2 protein, mouse
• Calcium-Calmodulin-Dependent Protein Kinase Type 2
• MAP Kinase Kinase Kinases
• MAP kinase kinase kinase 7

Topics

• Animals
• Calcium-Calmodulin-Dependent Protein Kinase Type 2 (metabolism)
• Cell Line
• Enzyme Induction (drug effects, physiology)
• Gene Knockdown Techniques (methods)
• HSP70 Heat-Shock Proteins (antagonists & inhibitors, metabolism)
• MAP Kinase Kinase Kinases (metabolism)
• Mice
• Microglia (drug effects, metabolism)
• NF-kappa B (metabolism)
• Nitric Oxide Synthase Type II (antagonists & inhibitors, metabolism)
• Oligonucleotides, Antisense (pharmacology)
• Sodium-Hydrogen Exchangers (metabolism)