title: 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.affiliation: Department of Pharmacology, School of Medicine, Nantong University, Nantong, Jiangsu, People's Republic of China.
authors.forename: Chao
authors.initials: C
authors.lastname: Huang
authors.affiliation: Department of Pharmacology, School of Medicine, Nantong University, Nantong, Jiangsu, People's Republic of China.
authors.forename: Xu
authors.initials: X
authors.lastname: Lu
authors.affiliation: Department of Pharmacology, School of Medicine, Nantong University, Nantong, Jiangsu, People's Republic of China.
authors.forename: Jia
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Pharmacology, School of Medicine, Nantong University, Nantong, Jiangsu, People's Republic of China.
authors.forename: Lijuan
authors.initials: L
authors.lastname: Tong
authors.affiliation: Department of Pharmacology, School of Medicine, Nantong University, Nantong, Jiangsu, People's Republic of China.
authors.forename: Bo
authors.initials: B
authors.lastname: Jiang
authors.affiliation: Department of Pharmacology, School of Medicine, Nantong University, Nantong, Jiangsu, People's Republic of China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
mesh_terms: D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism*; D002460:Cell Line; D004790:Enzyme Induction / Q000187:drug effects / Q000502:physiology; D055785:Gene Knockdown Techniques / Q000379:methods; D018840:HSP70 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism*; D051379:Mice; D017628:Microglia / Q000187:drug effects / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D000099151:MAP Kinase Kinase Kinase 7
chemical_list: D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D018840:HSP70 Heat-Shock Proteins; D020930:MAP Kinase Kinase Kinases; D016328:NF-kappa B; D052247:Nitric Oxide Synthase Type II; D016376:Oligonucleotides, Antisense; D017923:Sodium-Hydrogen Exchangers; D000099151:MAP Kinase Kinase Kinase 7; C069139:growth factor-activatable Na-H exchanger NHE-1; C496317:Nos2 protein, mouse
keywords: Ca2+; inhibitor of κB kinase; intracellular pH value; lipopolysaccharide