REDD2 Gene Is Upregulated by Modified LDL or Hypoxia and Mediates Human Macrophage Cell Death

Abstract

Objective-Cholesterol accumulation in macrophages is known to alter macrophage biology. In this article we studied the impact of macrophage cholesterol loading on gene expression and identified a novel gene implicated in cell death. Methods and Results-The regulated in development and DNA damage response 2 (REDD2) gene was strongly upregulated as THP-1 macrophages are converted to foam cells. These results were confirmed by Northern blot of RNA from human monocyte-derived macrophages (HMDM) treated with oxidized LDL (oxLDL). Human REDD2 shares 86% amino acid sequence identity with murine RTP801-like protein, which is 33% identical to RTP801, a hypoxia-inducible factor 1-responsive gene involved in apoptosis. Treatment of HMDM with desferrioxamine, a molecule that mimics the effect of hypoxia, increased expression of REDD2 in a concentration-dependent fashion. Transfection of U-937 and HMEC cells with a REDD2 expression vector increased the sensitivity of the cells for oxLDL-induced cytotoxicity, by inducing a shift from apoptosis toward necrosis. In contrast, suppression of mRNA expression using siRNA approach resulted in increased resistance to oxLDL treatment. Key Words: REDD2 Ⅲ macrophages Ⅲ atherosclerosis Ⅲ hypoxia Ⅲ oxLDL Ⅲ necrosis Ⅲ apoptosis I t is well-established that elevation of plasma low-density lipoproteins (LDL) is a risk factor for atherosclerosis, and that lesion-prone areas show increased intramural retention of lipoproteins by the extracellular matrix. 1 Many of these lipoproteins are modified mainly through glycation, aggregation, protease degradation, and oxidation and as such become increasingly atherogenic. 2,3 Several studies have indicated that a diversity of effects on macrophage function can be attributed to modified LDL. These include growth stimulation, 4 -6 proinflammatory effects such as expression of inflammatory cytokines, 7 increase in cytotoxicity 8 and in expression of metalloproteinases, inhibition of expression of inducible nitric oxide synthase, 9,10 and effects on lipid metabolism and accumulation. [11][12][13] The human leukemia cell line THP-1 has been used as a model for the monocyte-macrophage lineage. 14 On treatment with phorbol esters, these cells take on the characteristics of activated macrophages, including decreased LDL receptor expression and increased expression of scavenger receptors. These changes in gene expression enable the cells to take-up modified lipoproteins and subsequently to develop a foam cell-like morphology. Using a powerful DNA microarray approach, Shiffman et al 15 have identified several upregulated genes in THP-1 cells loaded with oxidized LDL (oxLDL). In addition, Yuchang et al have also reported several differentially expressed genes in oxLDL-treated compared with nontreated THP-1 cells using the subtractive approach. 16 In this study, we have combined the subtractive and the DNA microarray approaches and identified additional genes induced in cholesterol loaded THP-1 macrophages. Among the most significant upregulated genes, one express...

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Emerging role for regulated in development and DNA damage 1 (REDD1) in the regulation of skeletal muscle metabolism

“…In silico analysis revealed a new mouse gene product (designated RTP801L for RTP801-like; DDIT4L; REDD2) that displayed 57% sequence identity to both rat and human RTP801. Subsequently, Northern blot analysis showed REDD2 mRNA was induced in a dose-dependent manner in macrophages incubated with oxidized LDL or the iron chelator desferrioxamine, the latter being a chemical mimetic of hypoxia (19). REDD2 mRNA was increased in various nonmuscle cell lines by different stressors, including osmotic stress (92), ROS (59), and radiation (37) as well as immunomodulatory agents such as endotoxin (TLR4 agonists) and zymosan (TLR2 agonist) (58).…”