Histamine contributes to increased RANKL to osteoprotegerin ratio through altered nuclear receptor 4A activity in human chondrocytes.

OBJECTIVE: To elucidate histamine receptor-mediated signaling pathways, transcriptional events, and target gene expression in human cartilage. METHODS: Histamine modulation of cartilage destruction was assessed by Safranin O staining and proteoglycan release. H(1) , H(2) , H(3) , and H(4) histamine receptor-dependent regulation of transcription factors (nuclear receptor 4A1 [NR4A1], NR4A2, and NR4A3), RANKL, and osteoprotegerin (OPG) messenger RNA (mRNA) levels were measured in primary and SW-1353 chondrocyte cells using quantitative polymerase chain reaction and selective histamine receptor antagonists. Soluble RANKL and OPG protein levels were determined using enzyme-linked immunosorbent assays. NR4A protein levels and transactivity were evaluated by Western blot analysis, immunocytochemistry, and luciferase reporter assays. Stable depletion of NR4A1-3 was achieved by lentiviral transduction of NR4A short hairpin RNA. RESULTS: Primary human chondrocyte cells expressed differential steady-state levels of H(1) -H(4) histamine receptor mRNA. In combination with tumor necrosis factor α, histamine significantly promoted cartilage proteoglycan depletion and release. Histamine modulated the expression of NR4A1-3 orphan receptors in primary and immortalized human chondrocyte cells in a time- and concentration-dependent manner. Histamine selectively signaled through H(1) and H(2) histamine receptors in chondrocytes to modulate RANKL and NR4A2 expression. The temporal effects of histamine on NR4A2 gene transcription were reduced in cells pretreated with inhibitors directed against protein kinase A, MAPK, and NF-κB signaling pathways. Histamine modulated the expression of RANKL with modest effects on OPG levels, leading to increased RANKL:OPG mRNA and protein ratios. Stable knockdown of NR4A1-3 expression resulted in reduced endogenous OPG levels and the loss of histamine-dependent regulation of RANKL expression. CONCLUSION: Our findings indicate that histamine, via H(1) and H(2) histamine receptors, contributes to joint disease by enhancing the ratio of RANKL to OPG expression through altered NR4A activity in human chondrocyte cells.

Increased expression of the orphan nuclear receptor NURR1 in psoriasis and modulation following TNF-alpha inhibition.

The orphan nuclear receptor NURR1 belongs to the NR4A subfamily of transcription factors which are emerging as important mediators of cytokine and growth factor signaling. The transcriptional function of these ligand-independent and constitutively active receptors is controlled at the level of expression and nuclear localization. This study examines the expression of NURR1 in psoriasis and biological effects on this receptor following inhibition of tumor necrosis factor-alpha (TNF-alpha) signaling. We report increased expression of NURR1 mRNA and protein in involved psoriasis skin compared with uninvolved and normal skin, which correlates significantly (P=0.0055) with clinical measures of the psoriasis area and severity index. Enhanced NURR1 expression localizes to both nucleus and cytoplasm of cells of involved epidermis, blood vessels, and inflammatory infiltrates, in contrast to predominant cytoplasmic distribution in uninvolved and normal skin. Endogenous NURR1 levels are rapidly and selectively increased in response to proinflammatory agonists and growth factors in normal dermal endothelial cells. Following TNF-alpha inhibition with infliximab or etanercept, NURR1 mRNA and protein levels in involved skin are significantly decreased and cytoplasmic distribution is restored. These findings establish the aberrant expression and distribution of NURR1 in psoriasis and suggest that clinical benefits of TNF-alpha inhibition may be mediated through altered NURR1 activity.

A role for type 1alpha corticotropin-releasing hormone receptors in mediating local changes in chronically inflamed tissue.

Peripheral corticotropin-releasing hormone (CRH) is an important regulator of localized inflammatory responses. The aim of this study is to define the pathological signaling pathways in which peripheral CRH receptor-mediated responses reside. We report that PECAM-1-expressing synovial membrane endothelial cells are the principal source of CRH receptor subtype 1alpha in chronically inflamed synovial tissue (ST). Analysis of ST from an early arthritis patient cohort (n = 9) established that expression of CRH-R1alpha significantly (P < 0.03) colocalized with PECAM-1 and E-selectin expression in vivo. Freshly excised ST explants released a mediator(s) that acts to promote CRH-R1alpha mRNA to levels present in inflamed human synovium (n = 8). We tested the ability of conditioned medium and individual inflammatory mediators to modulate CRH-R1alpha expression. Histamine selectively induced the expression of CRH-R1alpha, and these effects were mediated through the histamine receptor type 1. Ectopic expression of CRH-R1alpha in normal human endothelial and synoviocyte cells resulted in the induction of the orphan receptor NR4A2 through the reconstitution of cAMP/protein kinase A/cAMP response element-binding protein signaling and identified a role for CRH in modulating nuclear factor kappaB transcriptional activity. CRH enhanced the expression of nitric-oxide synthase (NOS III) to promote NO production from CRH-R1alpha-expressing cells. These data establish a role for CRH receptor-mediated responses in regulating vascular changes associated with chronic synovitis.

Modulation of orphan nuclear receptor NURR1 expression by methotrexate in human inflammatory joint disease involves adenosine A2A receptor-mediated responses.

Modulation by proinflammatory mediators indicate that NURR1 induction represents a point of convergence of distinct signaling pathways, suggesting an important common role for this transcription factor in mediating multiple inflammatory signals. The present study identifies NURR1 as a molecular target of methotrexate (MTX) action in human inflammatory joint disease and examines the mechanism through which MTX modulates NURR1 expression. MTX significantly suppresses expression of NURR1 in vivo in patients with active psoriatic arthritis (n = 10; p < 0.002) who were prescribed low-dose MTX for management of peripheral arthritis. Importantly, reduction in NURR1 levels correlate (n = 10; r = 0.57; p = 0.009) with changes in disease activity score (both clinical and laboratory parameters). MTX selectively modulates NURR1 levels induced by inflammatory stimuli and growth factors in resident cell populations of synovial tissue. In primary human synoviocytes and microvascular endothelial cells, we observe dose-dependent differential effects of MTX on steady-state and inducible NURR1 levels. Our data confirms that adenosine, and its stable analog 5'-N-ethylcarboxamideadenosine, can mimic the differential effects of MTX on NURR1 transcription. In addition, we verify that the inhibitory effect of low-dose MTX on NURR1 activation is mediated through the adenosine receptor A2. More specifically, our data distinguishes the selective involvement of the A2A receptor subtype in these responses. In summary, these findings establish the nuclear orphan receptor NURR1 as a molecular target of MTX action in human inflammatory joint disease and demonstrate that the immunomodulatory actions of MTX on NURR1 expression are mediated through adenosine release.

Cyclooxygenase 2-derived prostaglandin E2 production by corticotropin-releasing hormone contributes to the activated cAMP response element binding protein content in rheumatoid arthritis synovial tissue.

OBJECTIVE: To determine a mechanism by which corticotropin-releasing hormone (CRH) promotes human inflammatory joint disease progression. METHODS: An ex vivo synovial tissue culture system was established to investigate the functional properties of CRH at peripheral sites of inflammation. CRH- and interleukin-1 beta (IL-1 beta)-induced prostaglandin E(2) (PGE(2)) production from 10 fresh rheumatoid arthritis (RA) synovial tissue (ST) explants was quantified using a competitive enzyme-linked immunosorbent assay. Modulation of PGE(2) levels was further examined following selective and nonselective cyclooxygenase 2 (COX-2) inhibition. Nuclear extracts were analyzed by electrophoretic mobility shift assays to determine functional cAMP response element binding protein (CREB) activity in response to CRH and PGE(2) in isolated primary synovial cell populations. Western blot analysis measured levels of total and activated (phosphospecific) CREB/activating transcription factor (ATF) family members prior to and following stimulation. RESULTS: CRH, in a time- and dose-dependent manner, significantly (P = 0.022) up-regulated PGE(2) production from 10 fresh RA ST explants. Costimulation of RA ST with CRH and IL-1 beta significantly augmented (P = 0.036) the effects on PGE(2) production additively over 24 hours. We demonstrated that selective COX-2 inhibitors prevent the induction of PGE(2) by both CRH and IL-1 beta. Further, we provided evidence that CRH and PGE(2) signal through the induction of CREB and phosphorylated CREB/ATF family members in RA ST and in isolated primary RA cell populations. CONCLUSION: Our findings underscore the pathogenic role that CRH may play in modulating inflammatory joint disease and establish the CREB/ATF family of transcription factors as principal effector molecules of proinflammatory mediator action in RA.

Corticotropin-releasing hormone signaling in synovial tissue vascular endothelium is mediated through the cAMP/CREB pathway.

Modulation of locally produced corticotropin-releasing hormone (CRH) is a component of the cytokine network in human inflammatory arthritis. CRH signaling, through the CRH-receptor subtype R1alpha, may play a role in both vascular changes and pathologic mechanisms associated with joint inflammation. Furthermore, the peripheral actions of CRH may be mediated in part through the NURR subfamily of nuclear orphan receptors. The aim of this study was to establish the signaling mechanisms through which CRH receptor-mediated responses contribute to gene regulation in inflamed synovial vasculature. Immunohistochemical analysis of serial rheumatoid arthritis (RA) tissue sections demonstrates CRH and NURR1 expression in the synovial lining layer, subsynovial lining layer, and the vascular endothelium. The identical pattern of immunolocalization confirms that NURR1 is produced at the same synovial sites shown to produce CRH. The distribution of specific NURR1 staining on the synovial vasculature parallels that observed for CRH-R1 expression. Using primary synovial tissue endothelial cells, we demonstrate that CRH induces specific CREB-1 and ATF-2 binding to the NURR1 promoter. We further provide evidence that CRH signaling can be mimicked by activation of cAMP/PKA/CREB using forskolin in primary human microvascular endothelial cells. These data indicate that the CRH receptor-dependent inflammatory response in synovial tissue endothelium is mediated through the cAMP/CREB signaling pathway.

Activation of nuclear orphan receptor NURR1 transcription by NF-kappa B and cyclic adenosine 5'-monophosphate response element-binding protein in rheumatoid arthritis synovial tissue.

Modulation of the NURR subfamily of nuclear receptors may be an important mechanism regulating pathways associated with inflammatory joint disease. We examined the signaling mechanisms through which inflammatory mediators, produced by rheumatoid arthritis (RA) synovial tissue, contribute to the regulation of the NURR subfamily. Markedly enhanced expression of NURR1 is observed in synovial tissue of patients with RA compared with normal subjects. Modulation by proinflammatory mediators in primary RA and normal synoviocytes shows that PGE(2), IL-1beta, and TNF-alpha markedly enhance NURR1 mRNA and protein levels in contrast to other subfamily members, NUR77 and NOR-1. We have established that transcriptional activation of the NURR1 gene by IL-1beta and TNF-alpha requires a proximal promoter region that contains a consensus NF-kappaB DNA-binding motif. IL-1beta- and TNF-alpha-induced NF-kappaB binding to this site is due predominantly to p65-p50 heterodimer and p50 homodimer subunit protein complexes. We further demonstrate a direct CREB-1-dependent regulation by PGE(2) situated at promoter region -171/-163. Moreover, analyses confirm the presence of CREB-1 and NF-kappaB p50 and p65 subunit binding to the NURR1 promoter under basal conditions in freshly explanted RA synovial tissue. In summary, enhanced NF-kappaB- and CREB-1-binding activity on the NURR1 promoter by inflammatory mediators delineates novel mechanisms in the regulation of NURR1 transcription. PGE(2)-, TNF-alpha-, and IL-1beta-dependent stimulation of the NURR1 gene implies that NURR1 induction represents a point of convergence of at least two distinct signaling pathways, suggesting an important common role for this transcription factor in mediating multiple inflammatory signals.