Elsevier

Clinical Immunology

Volume 118, Issues 2–3, February–March 2006, Pages 276-283
Clinical Immunology

Inflammatory modulation of PPARγ expression and activity

https://doi.org/10.1016/j.clim.2005.09.018Get rights and content

Abstract

Nitric oxide (NO) production increases with age in the lupus-prone MRL/lpr mouse, paralleling disease activity. One mechanism for excess NO production in MRL/lpr mice may be a defect in down-regulatory mechanisms of the iNOS pathway. A potential modulator of NO is the nuclear hormone receptor peroxisome proliferation activated receptor gamma (PPARγ). We demonstrate that renal PPARγ protein expression was altered as disease progressed in MRL/lpr mice, which paralleled increased iNOS protein expression. Additionally, MRL/lpr-derived primary mesangial cells expressed less PPARγ than BALB/c mesangial cells and produced more NO in response to LPS and IFNγ. Furthermore, PPARγ activity was reduced in mesangial cells following exposure to inflammatory mediators. This activity was restored with the addition of a NOS enzyme inhibitor. These results indicate that the activation of inflammatory pathways may lead to reduced activity and expression of PPARγ, further exacerbating the disease state.

Introduction

The MRL/MpJ-Faslpr (MRL/lpr) mouse model of systemic lupus erythematosus (SLE) is characterized by SLE-like renal histopathology, auto-antibody production, arthritis, and vasculitis [1], [2], [3]. It has been demonstrated by our laboratory and others that enhanced NO production occurs in MRL/lpr mice [4], [5], [6], [7], [8]. Spontaneous NO production in these mice is reflective of renal pathology and increases with age. Additionally, stimulated NO production by MRL/lpr macrophages is significantly elevated compared to normal BALB/c mice [6].

The excess production of NO in MRL/lpr mice is likely multifactorial. We hypothesize that a potential cause of chronically enhanced NO production is a defect in the modulators of the inducible nitric oxide synthase (iNOS) inflammatory pathway. One protein involved in down-regulation of inflammatory mediators, including NO, is the nuclear hormone receptor peroxisome proliferation activated receptor gamma (PPARγ) [9], [10], [11].

Evidence for a role for PPARγ agonists in suppressing inflammation in mouse macrophage cell lines was recently demonstrated [9], [10], [11]. Activation of PPARγ with either synthetic thiazoledinediones (TZDs) or prostaglandin J2 (PGJ2) metabolites inhibited the production of inflammatory mediators such as NO, IL-6, and TNFα [4], [10]. Previous studies by our laboratory indicated that 15-deoxy-Δ12, 14PGJ2 (15ΔPGJ2) and TZDs reduced NO production and iNOS expression in mesangial cells from MRL/lpr mice [4], [5]. Though PPARγ agonists inhibit the production of some inflammatory mediators, Ponstler et al. demonstrated that PPARγ actually stimulates COX-2 through a PPARγ response element (PPRE) present in the COX-2 promoter [12]. A recently identified endogenous PPARγ agonist, lysophosphatidic acid (LPA), has not been studied for anti-inflammatory function through PPARγ [13]. However, it has recently been shown that 15ΔPGJ2 can inhibit NF-κB translocation independent of PPARγ activation [14]. Thus, the role of PPARγ as an intrinsic regulator of inflammation remains unclear.

PPARγ is a member of the nuclear hormone receptor family and is primarily expressed in adipocytes, monocytes, and macrophages and is less expressed in the heart and kidney [15], [16]. PPARγ has three isoforms. Isoforms 1 and 3 are identical and when fully translated only differ in their splice variants [17]. PPARγ2 is predominantly found in adipocytes and differs from the other isoforms at its extended N-terminus [17], [18]. PPARγ has the structural features of most nuclear hormone receptors [19], [20]. The N-terminus contains the ligand-independent transcriptional activation domain (AF1). This site contains a serine that can be phosphorylated by MAP kinases. Such alterations decrease ligand-binding affinity and PPARγ activation [21], [22], [23], [24].

Based on the enhanced NO production in MRL/lpr mice and that PPARγ functions as a repressor of iNOS, we analyzed the expression of PPARγ protein and mRNA, and PPARγ function in MRL/lpr kidneys and mesangial cells. The results indicate that PPARγ expression/function is diminished in the chronic inflammatory state present in the kidney of MRL/lpr mice and likely contribute to the enhanced inflammatory state characteristic of lupus nephritis.

Section snippets

Reagents

Lipopolysaccharide (LPS) was purchased from Sigma (St. Louis, MO), IFNγ from PharMingen (San Diego, CA), and 15ΔPGJ2 from Cayman Chemical (Ann Arbor, MI). Media and fetal calf sera (FCS) were purchased from Invitrogen (Carlsbad, CA). All other reagents were from Sigma (St. Louis, MO). NG-monomethyl-l-arginine (l-NMMA) was purchased from Paragon Biochemicals (Salt Lake City, UT). Ciglitazone was purchased from Biomol (Plymouth, PA). GW347845X (GW) was a gift from Kathleen Brown of

iNOS expression is upregulated in aging MRL/lpr mice

Kidneys were harvested from aging MRL/lpr and BALB/c mice at 9, 15, and 21 weeks, and the kidney cortices were used in protein and RNA analysis. As shown in Fig. 1, iNOS protein and mRNA expression increased with aging and disease progression in MRL/lpr kidney cortices from 3 different mice at each time point in each group. A representative blot is shown. Elevated levels of iNOS mRNA and protein were evident beginning at 15 weeks. In contrast, neither iNOS protein nor mRNA was detected in

Discussion

Our in vitro and in vivo findings provide evidence for a dysfunctional intrinsic PPARγ pathway of inflammatory suppression in MRL/lpr mice. Our data suggest that PPARγ expression is decreased in chronic inflammatory states, like lupus nephritis, while PPARγ activity is reduced in the induced acute inflammatory setting. In primary and immortalized mesangial cells from MRL/lpr mice, there is a baseline reduction in PPARγ expression as compared with BALB/c control mesangial cells. In the B6

Acknowledgments

This research was supported by the Medical Research Service, Ralph H. Johnson VAMC, and by NIH grants AR45476 and AR47451. Ms. Crosby was supported by a Dissertation Fellowship from the American Association of University Women and NIH Medical Scientist Training Program grant NGMS-GM08716.

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