PPARγ ligands inhibit nitrotyrosine formation and inflammatory mediator expressions in adjuvant-induced rheumatoid arthritis mice

Abstract

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor, whose activation has been linked to several physiologic pathways including those related to the regulation of insulin sensitivity. Here, we investigate effects of PPARγ specific ligands, rosiglitazone and pioglitazone, on formation of nitrotyrosine and increased expression of inflammatory mediators such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 and intercellular adhesion molecule-1 (ICAM-1) in adjuvant-induced murine arthritis. Administration of rosiglitazone or pioglitazone (30 mg/kg, p.o.) significantly inhibited the adjuvant-induced increase in formation of nitrotyrosine and expression of iNOS on both ankle and temporomandibular joints. Rosiglitazone also inhibited the adjuvant-induced expression of M30 positive cells, as a marker of apoptosis, in the joint tissues. In addition, treatment with rosiglitazone or pioglitazone (30 μM) inhibited lipopolysaccharide plus tumor necrosis factor (TNF)-α-induced protein expression of iNOS, cyclooxygenase-2, ICAM-1 and nitrotyrosine formation in RAW 264 cells, a murine macrophage-like cell line. Rosiglitazone or pioglitazone inhibited increase in phosphorylated I-κB (pI-κB) expression, as an index of activation of nuclear factor (NF)-κB, in both joint tissues and RAW264 cells. Furthermore, in PPARγ-transfected HEK293 cells, rosiglitazone inhibited the TNF-α-stimulated response using NF-κB-mediated transcription reporter assay. These results indicate that PPARγ ligands may possess anti-inflammatory activity against adjuvant-induced arthritis via the inhibition of NF-κB pathway.

Introduction

Many reports indicated formation of nitrotyrosine in proteins during inflammation such as arthritis, nephritis, sepsis and ischemia–reperfusion injury, because nitrotyrosine is formed by a reaction of tyrosine residue with peroxynitrite generated from superoxide and nitric oxide (NO) radicals Wada et al., 1998, Mapp et al., 2001, Noiri et al., 2001, Pfeiffer et al., 2001. Pathological roles of nitrotyrosine are still unknown. However, it is required for normal physiological conditions to reduce or metabolize the nitrated proteins, since the nitrated proteins may affect physiological regulation systems by phosphorylation of tyrosine. Few reports indicated inhibitors on the formation of nitrotyrosine, although there might be specific enzymes that recover function of the nitrated proteins (Kamisaki et al., 1998).

Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of nuclear receptor superfamily of ligand-activated transcriptional factors including steroid hormone, thyroid hormone, vitamin D, and retinoic acid Michalik and Wahli, 1999, Kersten et al., 2000. PPARγ is highly expressed in adipose tissue and plays a key role in adipocyte differentiation and insulin sensitivity. Synthesized ligands, thiazolidinedione derivatives, such as rosiglitazone, pioglitazone and troglitazone, are used as oral antihyperglycemic agents in the therapy of non-insulin-dependent diabetes mellitus Vamecq and Latruffe, 1999, Kadowaki, 2000. In addition, recent studies have shown that PPARγ may participate in control of inflammation, especially, in modulating the production of inflammatory mediators Jiang et al., 1998, Ricote et al., 1998. Actually, we have reported that the stimulation of endogenous PPARγ pathway causes anti-inflammatory responses in experimental bowel disease inflammation, such as dextran sodium sulfate and ischemia-induced colitis Nakajima et al., 2001, Saubermann et al., 2001, Wada et al., 2001. According to those observations, PPARγ ligand therapy is also suggested to suppress other inflammatory diseases such as rheumatoid arthritis, sepsis, nephritis and pancreatitis.

Rheumatoid arthritis is a chronic, destructive polyarticular joint disease, being characterized massive synovial proliferation and subintimal infiltration of inflammatory cells (Feldmann, 2001). In this report, we applied PPARγ specific ligands, rosiglitazone and pioglitazone, for adjuvant-induced murine arthritis model. We investigated the effects of PPARγ ligands on the nitrotyrosine formation and inflammatory mediator expression, such as inducible nitric oxide (iNOS), cyclooxygenase-2 and intercellular adhesion molecule-1 (ICAM-1), in ankle and temporomandibular joint tissues. First, we revealed that administration of PPARγ ligands inhibits expression of inflammatory mediators in arthritis tissues, and that suppression of nuclear factor (NF)-κB pathway may be involved in the inhibitory mechanisms of PPARγ ligands on the nitrotyrosine formation and inflammatory mediator expressions in ankle and temporomandibular joints.