Regulation of inflammatory responses by neuregulin-1 in brain ischemia and microglial cells in vitro involves the NF-kappa B pathway.
We previously demonstrated that neuregulin-1 (NRG-1) was neuroprotective in rats following ischemic stroke. Neuroprotection by NRG-1 was associated with the suppression of pro-inflammatory gene expression in brain tissues. Over-activation of brain microglia can induce pro-inflammatory gene expression by activation of transcriptional regulators following stroke. Here, we examined how NRG-1 transcriptionally regulates inflammatory gene expression by computational bioinformatics and in vitro using microglial cells.



To identify transcriptional regulators involved in ischemia-induced inflammatory gene expression, rats were sacrificed 24 h after middle cerebral artery occlusion (MCAO) and NRG-1 treatment. Gene expression profiles of brain tissues following ischemia and NRG-1 treatment were examined by microarray technology. The Conserved Transcription Factor-Binding Site Finder (CONFAC) bioinformatics software package was used to predict transcription factors associated with inflammatory genes induced following stroke and suppressed by NRG-1 treatment. NF-kappa B (NF-kB) was identified as a potential transcriptional regulator of NRG-1-suppressed genes following ischemia. The involvement of specific NF-kB subunits in NRG-1-mediated inflammatory responses was examined using N9 microglial cells pre-treated with NRG-1 (100 ng/ml) followed by lipopolysaccharide (LPS; 10 μg/ml) stimulation. The effects of NRG-1 on cytokine production were investigated using Luminex technology. The levels of the p65, p52, and RelB subunits of NF-kB and IkB-α were determined by western blot analysis and ELISA. Phosphorylation of IkB-α was investigated by ELISA.



CONFAC identified 12 statistically over-represented transcription factor-binding sites (TFBS) in our dataset, including NF-kBP65. Using N9 microglial cells, we observed that NRG-1 significantly inhibited LPS-induced TNFα and IL-6 release. LPS increased the phosphorylation and degradation of IkB-α which was blocked by NRG-1. NRG-1 also prevented the nuclear translocation of the NF-kB p65 subunit following LPS administration. However, NRG-1 increased production of the neuroprotective cytokine granulocyte colony-stimulating factor (G-CSF) and the nuclear translocation of the NF-kB p52 subunit, which is associated with the induction of anti-apoptotic and suppression of pro-inflammatory gene expression.



Neuroprotective and anti-inflammatory effects of NRG-1 are associated with the differential regulation of NF-kB signaling pathways in microglia. Taken together, these findings suggest that NRG-1 may be a potential therapeutic treatment for treating stroke and other neuroinflammatory disorders.
Morehouse School of Medicine, 720 Westview Drive, Atlanta, 30310, Georgia.
Lauren J
LJ
Simmons
Morehouse School of Medicine, 720 Westview Drive, Atlanta, 30310, Georgia.
Monique C
MC
Surles-Zeigler
Division of Biomedical Sciences, University of California-Riverside School of Medicine, 900 University Ave, Riverside, CA, 92521, USA.
Yonggang
Y
Li
Georgia Highlands College, 5441 GA-20, Cartersville, GA, 30121, USA.
Gregory D
GD
Ford
Morehouse School of Medicine, 720 Westview Drive, Atlanta, 30310, Georgia.
Gale D
GD
Newman
Division of Biomedical Sciences, University of California-Riverside School of Medicine, 900 University Ave, Riverside, CA, 92521, USA. byron.ford@medsch.ucr.edu.
Byron D
BD
Ford
D000818:Animals; D002461:Cell Line, Transformed; D019295:Computational Biology; D016207:Cytokines / Q000378:metabolism; D004660:Encephalitis / Q000188:drug therapy* / Q000209:etiology*; D004797:Enzyme-Linked Immunosorbent Assay; D016179:Granulocyte Colony-Stimulating Factor / Q000378:metabolism; D020672:I-kappa B Proteins / Q000378:metabolism; D020244:Infarction, Middle Cerebral Artery / Q000150:complications* / Q000473:pathology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D046228:Microarray Analysis; D017628:Microglia / Q000187:drug effects*; D016328:NF-kappa B / Q000378:metabolism; D020890:Neuregulin-1 / Q000627:therapeutic use*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D016207:Cytokines; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B; D020890:Neuregulin-1; D012333:RNA, Messenger; D016179:Granulocyte Colony-Stimulating Factor
Bioinformatics; Gene expression; Inflammation; Ischemia; Microarray; Neuregulin; Stroke; Transcription factor-binding site (TFBS)