Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
Histamine Contributes to Inflammatory Joint Disease by Regulating the Expression of RANKL and OPG through Altered NR4A Activity in Human Chondrocyte Cells.
Marzaioli2, Viviana, McMorrow2, Jason P., McEvoy1, Alice, Murphy2, Evelyn P.
Mast cells, a major source of histamine in vivo, are found in increased numbers in rheumatoid arthritis synovial tissue. In the K/BxN arthritis model, mast cells are required to promote autoantigen-induced inflammatory arthritis. It has recently been shown that IL-33 enhances autoantibody-mediated articular inflammation via IgG mast cell degranulation and pro-inflammatory mediator release. Furthermore, histamine stimulates chondrocyte production of matrix metalloproteinases and osteoclast differentiation directly, and indirectly, through altered RANKL and OPG expression in osteoblasts. The aim of this study was to elucidate the receptor mediated signalling pathways and transcriptional events regulated by histamine in human chondrocytes.
Histamine receptors (HR1,-2, 3,-4), nuclear transcription factors (NR4A1,-2,-3), RANKL and OPG mRNA levels were measured in human chondrocytes using real-time PCR (qPCR). HR subtype involvement was monitored using selective HR antagonists. NR4A protein levels and transactivity were evaluated by western blot, immunocytochemistry and luciferase reporter assays. ShRNA for control and NR4A1, 2, 3 were generated using lentivirus transduction.
Differential expression of histamine receptor subtypes (HR14) was detected. HR1 and HR2 receptors are highly expressed in chondrocyte cells with minimal expression of HR3 and HR4. Histamine robustly modulates RANKL and OPG levels, in a time-and concentration-dependent fashion, leading to significantly increased RANKL/OPG expression ratio (p<0.005). Histamine rapidly and differentially modulates expression of all three NR4A transcription factors. Within 2-hours, histamine maximally modulates NR4A3 (300-fold), NR4A2 (100-fold) and NR4A1 (10-fold) transcript levels. The study of HR receptor antagonists reveals that histamine selectively signals through HR1 and HR2 in chondrocyte cells to modulate RANKL, OPG and NR4A13 expression. Our data further demonstrates histamine-dependent activation of NF-kB and CREB signalling pathways through HR1 and HR2 receptors, respectively. Consistent with mRNA analysis, histamine promotes NR4A nuclear localization and significantly enhances the capacity of NR4A proteins to mediate target gene expression. Specific knockdown of NR4A-1,-2,-3 mRNA and protein levels (>50%) results in significantly reduced endogenous production of OPG (>60%) by chondrocytes. Finally, in NR4A-depleted cells, histamine regulation of RANKL and OPG expression is completely lost.
These data reveal that histamine, through HR1 and HR2, contributes to the development of inflammatory joint disease by regulating the expression of RANKL and OPG through altered NR4A activity in human chondrocyte cells.
To cite this abstract, please use the following information:
Marzaioli, Viviana, McMorrow, Jason P., McEvoy, Alice, Murphy, Evelyn P.; Histamine Contributes to Inflammatory Joint Disease by Regulating the Expression of RANKL and OPG through Altered NR4A Activity in Human Chondrocyte Cells. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :1482