Orphan Nuclear Receptor NR4A2 Is Constitutively Expressed in Cartilage and Upregulated in Inflamed Synovium From hTNF-Alpha Transgenic Mice

Abstract

Orphan nuclear receptor 4A2 (NR4A2/Nurr1) is a constitutively active transcription factor with potential roles in the onset and progression of inflammatory arthropathies. NR4A2 is overexpressed in synovium and cartilage from individuals with rheumatoid arthritis (RA), psoriatic arthritis, and osteoarthritis. This study documents the expression and tissue localization of NR4A2 and upstream regulator nuclear factor kappa B (NF-κB) in the human tumor necrosis factor-alpha (hTNF-α) transgenic mouse model of RA. Since TNF-α is a potent inducer of NR4A2 in vitro, we hypothesized that NR4A2 would also be upregulated and active during disease progression in this model. Expression levels of NR4A2, related receptors NR4A1 (Nur77) and 3 (NOR1), and NF-κB₁ transcripts were quantified by RT-qPCR in hTNF-α and wild-type joints at three stages of disease. The protein distribution of NR4A2 and NF-κB subunit RelA (p65) was analyzed by quantitative immunohistochemistry. Global gene expression of 88 RA-related genes was also screened and compared between groups. Consistent with previous reports on the hTNF-α model, transgenic mice exhibited significant weight loss and severely swollen paws by 19 weeks of age compared to age-matched wild-type controls. NR4A1-3 and NF-κB₁ were constitutively expressed at disease onset and in healthy joints. NF-κB₁ transcript levels increased 2-fold in hTNF-α paws with established disease (12 weeks), followed by a 2-fold increase in NR4A2 at the late disease stage (19 weeks). NR4A2 and RelA proteins were overexpressed in inflamed synovium prior to symptoms of arthritis, suggesting that gene expression changes documented in whole paws were largely driven by elevated expression in diseased synovium. Broader screening of RA-related genes by RT-qPCR identified several differentially expressed genes in hTNF-α joints including those encoding inflammatory cytokines and chemokines, matrix-degrading enzymes and inhibitors, cell surface receptors, intracellular signaling proteins and transcription factors. Consensus binding sites for NR4A receptors and NF-κB₁ were enriched in the promoters of differentially expressed genes suggesting central roles for these transcription factors in this model. This study is the first comprehensive analysis of NR4A2 in an animal model of RA and validates the hTNF-α model for testing of small molecules and genetic strategies targeting this transcription factor.

Keywords: NFkB (RelA); NR4A2 gene; TNF-α; cartilage; inflammation; nuclear receptors; rheumatoid arthritis; synovium.

FIGURE 1

Progression of arthritis in hTNF-α transgenic mice. Wild-type and hTNF-α mice were studied at three stages of disease: early (8 weeks), established (12 weeks), and late (19 weeks). (A) Mean clinical score based on total paw swelling ±standard deviation. (B) Mean body mass (g) ± standard deviation. Unpaired t-test, *p < 0.05.

FIGURE 2

Gene expression analysis of target transcription factors. RNA was extracted from the paws of wild-type and hTNF-α transgenic mice at early, established, and late timepoints. Relative levels of NF-κB₁ (A), NR4A1 (B), NR4A2 (C), NR4A3 mRNA (D) were measured by RT-qPCR and normalized to TBP. Horizontal bars represent mean expression levels for each group. Mann Whitney test, *p < 0.05, **p < 0.005.

FIGURE 3

Localization of RelA in cartilage and synovium. RelA was detected in paws of wild-type and hTNF-α mice at early (A,D), established (B,E), and late (C,F) stages of disease by immunohistochemistry. Isotype control was applied to a transgenic section from the late stage of disease (G). Representative images at 400× magnification are shown. Scoring of RelA positive cells in synovium (H) and cartilage (I). Unpaired t-test, **p < 0.005, ***p < 0.0005.

FIGURE 4

Localization of NR4A2 in cartilage and synovium. NR4A2 protein was detected in paws of wild-type and hTNF-α mice at early (A,D), established (B,E), and late (C,F) stages of disease by immunohistochemistry. Isotype control was applied to a transgenic section from the late stage of disease (G). Representative images at 400× magnification are shown. Scoring of NR4A2 positive cells in synovium (H) and cartilage (I). Unpaired t-test, *p < 0.05, ***p < 0.0005.

FIGURE 5

Global gene expression profiles in hTNF-α joints. RT-qPCR panels were used to measure the expression of 88 genes associated with RA in wild-type and hTNF-α paws at early and late stages of disease. (A) Genes induced by greater than 2-fold at early stage. (B) Genes repressed by greater than 0.5-fold at early stage. (C) Genes induced by greater than 2-fold at late stage. (D) Genes repressed by greater than 0.5-fold at late stage. (E) Comparison of induced genes at early and late stages.