A novel PPAR response element in the murine iNOS promoter

Abstract

The nuclear hormone receptor peroxisome proliferation activated receptor gamma (PPARγ) is a modulator of inflammation including down-regulation of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production. PPARγ agonists reduce iNOS expression and NO production in a dose-dependent manner in macrophages, mesangial cells and other inflammatory cells. However, the mechanisms involved in the inhibition of iNOS expression by PPARγ and its agonists are not fully understood. Here we show that the PPARγ agonist ciglitazone dose-dependently inhibited a murine iNOS-luciferase reporter construct by up to 50% in transfected mesangial cells. Blocking de novo protein synthesis in mesangial cells had no effect on PPARγ agonist activity, indicating that ciglitazone acts directly to inhibit iNOS transcription. We identified a novel PPAR response element (PPRE) in the murine iNOS promoter that is homologous to the PPRE consensus sequence. In binding assays PPARγ directly binds to this response element in vitro and can function as a positive element in response to PPARγ agonists when placed in front of a reporter gene. Site-directed mutagenesis of this PPRE in a murine iNOS promoter/reporter construct did not block the inhibitory activity of a synthetic PPARγ agonist on the iNOS promoter/reporter construct in transfected mesangial cells. However, the mutated construct exhibited lower basal expression, and higher expression in response to inflammatory stimuli compared to the intact construct. These data suggest that the iNOS PPRE contributes to positive basal expression and negative expression of iNOS in response to inflammatory stimuli. The PPRE is not necessary, however, for synthetic PPARγ agonists to inhibit iNOS expression.

Introduction

Inducible nitric oxide synthase (iNOS) is an enzyme that is up-regulated in inflammatory states and converts arginine into citrulline and NO (MacMicking et al., 1997). When produced in nanomolar amounts by endothelial NOS (eNOS) or neuronal NOS (nNOS), NO has vital vasodilatory and neurotransmitter effects (MacMicking et al., 1997). In contrast, micromolar concentrations of NO are produced by iNOS when induced by an infectious agent (MacMicking et al., 1997). However, when NO production is unchecked, as occurs in some autoimmune diseases, cellular damage occurs (Liew, 1995). These deleterious effects of NO are mediated by its metabolites including peroxynitrite (Liew, 1995). In addition to direct tissue damage, this free radical is also capable of altering molecular and protein targets in the cell, changing basic cellular functions (Oates et al., 1999). For example, NO can affect catalase function and hinder the ability of the cell to clear potentially harmful oxygen radicals (Keng et al., 2000, Mohan and Das, 1997). These damaging effects occur in inflammatory diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis and inflammatory bowel disease (Oates et al., 2002). In lupus nephritis, mesangial cells mediate the hyper-inflammatory state found in glomeruli. Mesangial cells are part of the supporting smooth muscle structure of the individual glomeruli. Given proper stimuli, they are capable of acquiring macrophage-like qualities and may secrete inflammatory mediators that can contribute to a chronic inflammatory response (Beck et al., 1998). These mediators include NO, super oxide radicals and TNFα. Thus, mesangial cells are a critical executor of chronic renal inflammation found in lupus nephritis. Drugs that inhibit iNOS expression or NO production improve the disease state in mouse models of SLE (Gilkeson et al., 1997, Weinberg et al., 1994). One intrinsic mediator involved in the down-regulation of iNOS is PPARγ.

Once activated by agonists, PPARγ heterodimerizes with RXR and binds to a peroxisome proliferator response element (PPRE) in the promoters of target genes (Hsu et al., 1998). The PPRE consists of a direct repeat (DR1) with a consensus sequence of 5′-AGGTCANAGGTCA-3′. It has been demonstrated that RXR binds to the 5′ repeat of the response element, while PPAR binds to the 3′ repeat (IJpenberg et al., 1997). A 5′ flanking sequence of five to six nucleotides is also important for proper binding to this response element (Hsu et al., 1998, IJpenberg et al., 1997). PPARγ activates genes with a PPRE in their promoter regions, such as the fatty acid transport protein (FATP) gene (Frohnert et al., 1999).

The role of PPARγ in suppressing inflammation in mouse macrophage cell-lines has been clearly demonstrated (Alleva et al., 2002, Jiang et al., 1998, Ricote et al., 1998). Activation of PPARγ with either thiazoledinediones (TZDs) or PGJ₂ metabolites inhibits inflammatory mediator production, including NO (Colville-Nash et al., 1998, Reilly et al., 2000). However, the exact mechanism by which PPARγ agonists and activated PPARγ interferes with the transcriptional activation of iNOS is unclear. Previous studies show that ligand-bound PPARγ utilizes co-activators necessary for NF-κB activity and inhibit iNOS expression through competition for co-activators (Li et al., 2000). This model is referred to as transrepression. However, very high concentrations of TZDs are necessary to demonstrate such repression as well as activation in several cell lines and disease-states (Kawahito et al., 2000, Lewis et al., 2001, Su et al., 1999). Additionally, 15-deoxy-Δ^12,14-PGJ₂ inhibits NF-κB translocation independent of PPARγ activation (Straus et al., 2000) and we have shown that synthetic PPARγ agonists can inhibit iNOS independent of PPARγ (Crosby et al., 2005). Despite this independent action of PPARγ agonists, we have also shown that endogenous PPARγ can modulate iNOS expression and NO production in the absence of synthetic agonists. Macrophages that express PPARγ produce less iNOS and NO in response to inflammatory mediators compared to cells not expressing PPARγ (Crosby et al., 2005). Thus PPARγ may be acting as an inflammatory modulator even in the absence of a synthetic agonist via action of the yet unidentified intrinsic agonist.

To understand more fully the mechanism involved in the down-regulation of iNOS expression by synthetic PPARγ agonists, we investigated whether the TZD ciglitazone exerts a direct effect on the murine iNOS promoter and the role of a novel PPRE in the murine iNOS promoter. Our results indicate that the PPARγ agonist, ciglitazone inhibits iNOS expression and NO production by a direct mechanism that does not involve the up-regulation of an intermediate protein. Furthermore, we identified a PPRE in the murine iNOS promoter that binds PPARγ in vitro and found that the PPRE is not necessary for the inhibition of iNOS expression by a synthetic agonist, but likely is involved in regulating basal expression and intrinsic down-regulation, possibly through an endogenous agonist.