Elk-3 Is a Transcriptional Repressor of Nitric-oxide Synthase 2

Abstract

The inducible isoform of nitric-oxide synthase (NOS2), a key enzyme catalyzing the dramatic increase in nitric oxide by lipopolysaccharide (LPS), plays an important role in the pathophysiology of endotoxemia and sepsis. Recent evidence suggests that Ets transcription factors may contribute to NOS2 induction by inflammatory stimuli. In this study, we investigated the role of Ets transcription factors in the regulation of NOS2 by LPS and transforming growth factor (TGF)-␤1. Transient transfection assays in macrophages showed that Ets-2 produced an increase in NOS2 promoter activity, whereas the induction by Ets-1 was modest and NERF2 had no effect. Elk-3 (Net/Erp/Sap-2a) markedly repressed NOS2 promoter activity in a dose-dependent fashion, and overexpression of Elk-3 blunted the induction of endogenous NOS2 message. Mutation of the Net inhibitory domain of Elk-3, but not the C-terminal-binding protein interaction domain, partially alleviated this repressive effect. We also found that deletion of the Ets domain of Elk-3 completely abolished its repressive effect on the NOS2 promoter. LPS administration to macrophages led to a dose-dependent decrease in endogenous Elk-3 mRNA levels, and this decrease in Elk-3 preceded the induction of NOS2 mRNA. In a mouse model of endotoxemia, the expression of Elk-3 in kidney, lung, and heart was significantly down-regulated after systemic administration of LPS, and this down-regulation also preceded NOS2 induction. Moreover, TGF-␤1 significantly increased endogenous Elk-3 mRNA levels that had been down-regulated by LPS in macrophages. This increase in Elk-3 correlated with a TGF-␤1-induced down-regulation of NOS2. Taken together, our data suggest that Elk-3 is a strong repressor of NOS2 promoter activity and mRNA levels and that endogenous expression of Elk-3 inversely correlates with NOS2. Thus, Elk-3 may serve as an important mediator of NOS2 gene expression.Sepsis is a systemic inflammatory response to severe infection that frequently progresses to refractory hypotension, multiple organ system failure, and death (1). In a subset of patients, the release of bacterial cell wall-derived lipopolysaccharide (LPS 1 or endotoxin) initiates a cascade of inflammatory events (2). This response to infection is mediated, in part, by macrophage activation and the release of proinflammatory cytokines (3). Among the mediators induced by these signal cascades, nitric oxide (NO) acts as a key regulator of hypotension and end organ damage (4, 5). The production of nitric oxide from L-arginine is catalyzed by NO synthases (NOS) (6). Of the three isoforms of NOS identified in mammalian cells, the expression of NOS2 is inducible by many proinflammatory stimuli, such as interleukin-1␤, tumor necrosis factor-␣, interferon-␥, and bacterial LPS, depending on the target cell. Thus, the study of NOS2 gene regulation is of particular importance in our understanding and management of sepsis and other inflammatory disorders. Ets proteins are a family of transcription factors that share a unique and highly...

Activation of a TGF-β-Specific Multistep Gene Expression Program in Mature Macrophages Requires Glucocorticoid-Mediated Surface Expression of TGF-β Receptor II

“…Besides ID3, early TGF-␤ response genes in M2 macrophages include well established targets of TGF-␤, such as the AP-1 family member JUNB (27), the ETS-domain transcription factor ELK3 involved in TGF-␤-mediated suppression of LPS-induced NO synthase 2(NOS2) (46), and the runt-domain containing transcription factor RUNX3, necessary for TGF-␤ induced suppression of DC maturation (47). Induced expression of the vitamin D receptor provides an additional mechanism for the known synergistic antiproliferative effect of TGF-␤ and vitamin D as observed in myeloid leukemia cell lines (48).…”

Endotoxin-Induced Down-Regulation of Elk-3 Facilitates Heme Oxygenase-1 Induction in Macrophages

“…We have previously noted that Elk-3 is down-regulated during an inflammatory stimulus in cultured macrophages (34). Thus, to investigate the temporal relationship between Elk-3 and HO-1 regulation during endotoxin exposure, we assessed mRNA levels for both Elk-3 and HO-1 in mouse tissue and cells.…”

“…TCF expression plasmids were gifts from Dr. P. Oettgen (Beth Israel Deaconess Medical Center, Boston, MA; pCI-Elk-3) and Dr. F. M. Stanley (New York University School of Medicine, New York, NY; pcDNA3-Elk-1 and pcDNA3-Sap-1a). ELK-3⌬A (Elk-3 without the A domain) in pCMV/Myc/nuc expression vector (Invitrogen Life Technologies) was generated as previously described (34). All constructs were verified by sequencing.…”

“…Elk-3, more than Elk-1, has been shown to be a repressor of gene transcription in the absence of MAPK activation (28,33). Recently, we have demonstrated that Elk-3 repressed NO synthetase-2 expression during LPS exposure in macrophages (34). Thus, the goal of this study was to determine new pathways for HO-1 gene repression during an inflammatory stimulus, particularly involving the Ets family member Elk-3.…”

Transcription of the Caveolin-1 Gene Is Differentially Regulated in Lung Type I Epithelial and Endothelial Cell Lines

“…Nitric-oxide synthase 2 that catalyzes nitric oxide production, for instance, can be regulated by several ETS factors. ESE-1 and ETS-2 increase nitric-oxide synthase 2 promoter activity, whereas ELK-3 represses nitricoxide synthase 2 via an inhibitory domain (36). Other ETS domain proteins including ETS-1, ETS-2, PEA3, and ERM are known to have inhibitory regions flanking the Ets domain that affects their transcriptional and DNA binding activities (27,28,31).…”