robustly modulating RANKL with modest effects on OPG, leading to ... enhancing the expression ratio of RANKL/OPG through altered NR4A activity, in human.
Histamine contributes to joint inflammation through altered NR4A activity, in human chondrocyte cells Alyssa Gilmore1, Viviana Marzaioli1, Jason P. McMorrow1, Hannes Angerer3, Daniel Crean1, Davide Zocco1, Peadar Rooney, Doug Veale, Ursula Fearon, Martina Gogarty, Alice N. McEvoy2, Martin H. Stradner3 and Evelyn P. Murphy1*. UCD School of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland 1. Introduction Mast cells are central players in perpetuating inflammatory responses during arthritic diseases, and may contribute to the initiation of joint specific autoimmune damage. Increased histamine levels in synovial fluid, tissue and cartilage of rheumatoid- and osteo- arthritis patients support a role for histamine in regulating cartilage homeostasis.
3.3 Stable depletion of NR4A1-3 receptor levels reduces endogenous OPG expression, resulting in enhanced RANKL/OPG mRNA and protein levels. A
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IgG
NR4A1, 2 E20 Ab
shRNA shRNA NR4A1-3 scram
The NR4A subfamily of orphan nuclear receptors (NR4A1-3) are constitutively active transcriptional regulators of cytokine and growth factor responses in diseases characterised by chronic or inappropriate inflammation. As increased levels of NR4A2 have been measured in OA and RA joint tissues, we hypothesized that NR4A receptors may mediate histamine-dependent modulation of gene expression in cartilage. Here, we elucidate the histamine receptor-mediated signaling pathways, transcriptional events and target gene expression in human chondrocyte cells.
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Figure 1: Bone homeostasis depends on a fine balance between receptor activator of NF-κB-ligand (RANKL) and osteoprotegerin (OPG) expression. RANKL binds to receptors on pre-osteoclast cells, signalling their maturation into activated osteoclasts, leading to bone resorption. OPG is a natural decoy of RANKL. The ratio between RANKL and OPG production dictates whether osteoclastogenesis will occur; favouring RANKL results in absorption while OPG is an anti-resorptive agent.
2. Methods Histamine modulation of cartilage destruction was assessed by Safranin-O staining and proteoglycan release. Histamine receptor (H1-4R) dependent regulation of nuclear transcription factors NR4A1-3; receptor activator of NF-κB-ligand (RANKL); and osteoprotegerin (OPG) mRNA levels were measured in human primary (n=8) and SW-1353 chondrocyte cells using QPCR and selective HR antagonists. sRANKL and OPG protein levels were determined by ELISA. NR4A protein levels were evaluated by western and immunocytochemistry. Stable depletion of NR4A1-3 was achieved by lentiviral transduction of NR4A shRNA.
3. Results 3.1 Primary human cartilage explants show that histamine, with/without TNFα, promotes cartilage destruction and proteoglycan depletion
Figure 4 : Human SW-1353 chondrocyte cells were tranduced with a control scrambled shRNA or shRNA lentiviral construct specific for NR4A1, 2 and 3 receptors. NR4A expression levels in stable cell lines were analyzed by A) by qRT-PCR. B) immunofluorescence following incubation with NR4A2 E20 antibody or isotype-matched non-immune IgG (Red staining NR4A, blue staining DAPI, magnification 100x). C) NR4A2 and TATA binding (TBP) protein levels measured in control and shRNA NR4A1-3 cells left untreated or treated with histamine (10-5M) for 4 hours and analyzed by western analysis D) Transcript levels of RANKL and OPG mRNA levels in control and shRNA NR4A1-3 cells measured by qRT-PCR. E) RANKL/OPG mRNA expression ratio. F) RANKL/OPG protein ratio. ** p
