Amyloid Peptide-Induced Cytokine and Chemokine Expression in THP-1 Monocytes Is Blocked by Small Inhibitory RNA Duplexes for Early Growth Response-1 Messenger RNA

Abstract

In Alzheimer’s disease (AD) one finds increased deposition of Aβ and also an increased presence of monocytes/macrophages in the vessel wall and activated microglial cells in the brain. AD patients show increased levels of proinflammatory cytokines by activated microglia. Here we used a human monocytic THP-1 cell line as a model for microglia to delineate the cellular signaling mechanism involved in amyloid peptides (Aβ1–40 and Aβ1–42)-induced expression of inflammatory cytokines and chemokines. We observed that Aβ peptides at physiological concentrations (125 nM) increased mRNA expression of cytokines (TNF-α, and IL-1β) and chemokines (monocyte chemoattractant protein-1 (MCP-1), IL-8, and macrophage inflammatory protein-1β (MIP-1β)). The cellular signaling involved activation of c-Raf, extracellular signal-regulated kinase-1 (ERK-1)/ERK-2, and c-Jun N-terminal kinase, but not p38 mitogen-activated protein kinase. This is further supported by the data showing that Aβ causes phosphorylation of ERK-1/ERK-2, which, in turn, activates Elk-1. Furthermore, Aβ mediated a time-dependent increase in DNA binding activity of early growth response-1 (Egr-1) and AP-1, but not of NF-κB and CREB. Moreover, Aβ-induced Egr-1 DNA binding activity was reduced >60% in THP-1 cells transfected with small interfering RNA duplexes for Egr-1 mRNA. We show that Aβ-induced expression of TNF-α, IL-1β, MCP-1, IL-8, and MIP-1β was abrogated in Egr-1 small inhibitory RNA-transfected cells. Our results indicate that Aβ-induced expression of cytokines (TNF-α and IL-1β) and chemokines (MCP-1, IL-8, and MIP-1β) in THP-1 monocytes involves activation of ERK-1/ERK-2 and downstream activation of Egr-1. The inhibition of Egr-1 by Egr-1 small inhibitory RNA may represent a potential therapeutic target to ameliorate the inflammation and progression of AD.

NLRP2, an Inhibitor of the NF-κB Pathway, Is Transcriptionally Activated by NF-κB and Exhibits a Nonfunctional Allelic Variant

“…In contrast, the constitutive promoter activity seems to rely on other transcription factors. Among the potential regulators of the NLRP2 gene, we showed that Stat3 and Gfi1 did not modify the promoter activity, but other factors including Egr-1, which promotes the expression of inflammatory cytokines (29), or Sp1, a proinflammatory protein involved in the expression of mediators of inflammation (30), should be considered in future studies. Because NLRP2 inhibits NF-B, upregulation of NLRP2 may be part of a negative regulatory loop induced via inflammatory stimuli.…”

Egr-1 binds the GnRH promoter to mediate the increase in gene expression by insulin

“…A small interfering RNA (siRNA) oligonucleotide that successfully targeted Egr-1 mRNA for degradation was published for human microglial cells (Giri et al, 2003). Using software provided by Ambion (Austin, TX), oligonucleotides for the homologous mouse sequence were designed.…”

“…Small interfering RNA specific to the human Egr-1 gene has been used by others to effectively knockdown Egr-1 mRNA levels (Giri et al, 2003). As shown Figure 4A, insulin treatment results in a 2.6 fold increase in Egr-1 protein that is not affected by the presence of sequence absent in the mouse genome (scrambled siRNA; 3.0 fold induction).…”

Overexpression of Notch1 Ectodomain in Myeloid Cells Induces Vascular Malformations through a Paracrine Pathway

“…In particular, MCP-5 was increased ϳ40-fold (range, 10 to 150) ( Figure 8). Because MCP-1 30 and FIII [31][32][33] genes have been described as targets of the transcription factor Egr-1, expression of the latter was measured and found to be increased ϳ4-fold in Tg macrophages relative to non-Tg ones (Figure 8). Together, these results indicate that N1 EC -expressing macrophages produce enhanced levels of pro-angiogenic molecules.…”

“…This activation is also supported by the increased expression of the Egr-1 transcription factor in Tg macrophages. Egr-1 seems to be a stress-responsive gene, 55 and its activation regulates the expression of several proangiogenic genes, 56 -58 including MCP-1 30 and FIII [31][32][33]59 (see below). The reprogramming of macrophages by N1 EC must be unique, because no other signaling molecule has yet been described, to our knowledge, as being able to confer to macrophages the angiogenic properties observed in these Tg mice.…”