Gene Expression In Synovial Fibroblasts Research Articles

The long non-coding RNA HOTAIR contributes to joint-specific gene expression in rheumatoid arthritis

Although patients with rheumatoid arthritis (RA) typically exhibit symmetrical joint involvement, some patients develop alternative disease patterns in response to treatment, suggesting that different molecular mechanism may underlie disease progression depending on joint location. Here, we identify joint-specific changes in RA synovium and synovial fibroblasts (SF) between knee and hand joints. We show that the long non-coding RNA HOTAIR, which is only expressed in knee SF, regulates more than 50% of this site-specific gene expression in SF. HOTAIR is downregulated after stimulation with pro-inflammatory cytokines and is expressed at lower levels in knee samples from patients with RA, compared with osteoarthritis. Knockdown of HOTAIR in knee SF increases PI-Akt signalling and IL-6 production, but reduces Wnt signalling. Silencing HOTAIR inhibits the migratory function of SF, decreases SF-mediated osteoclastogenesis, and increases the recruitment of B cells by SF. We propose that HOTAIR is an important epigenetic factor in joint-specific gene expression in RA.

A novel diindolylmethane analog, 1,1-bis(3’-indolyl)-1-(p-chlorophenyl) methane, inhibits the tumor necrosis factor-induced inflammatory response in primary murine synovial fibroblasts through a Nurr1-dependent mechanism

The progression of rheumatoid arthritis involves the thickening of the synovial lining due to the proliferation of fibroblast-like synoviocytes (FLS) and infiltration by inflammatory cells. Tumor necrosis factor alpha (TNFα) is a pro-inflammatory cytokine involved in progression of the disease. Under rheumatoid conditions, FLS express the tumor necrosis factor (TNF)-recognition complex (TNFR1, TNFR2, VCAM-1 and ICAM-1), which induces local macrophage activation and leads to downstream nuclear factor κB (NF-κB) signaling. The NF-κB-regulated inflammatory gene, cyclooxygenase (COX), increases synthesis of prostaglandins that contribute to the propagation of inflammatory damage within the joint. Because the nuclear orphan receptor, NR4A2 (Nurr1), can negatively regulate NF-κB-dependent inflammatory gene expression in macrophages, we postulated that activation of this receptor by the Nurr1 ligand 1,1-bis(3’-indolyl)-1-(p-chlorophenyl) methane (C-DIM12) would modulate inflammatory gene expression in synovial fibroblasts by inhibiting NF-κB. Treatment with C-DIM12 suppressed TNFα-induced expression of adhesion molecules and NF-κB regulated genes in primary synovial fibroblasts including vascular adhesion molecule 1 (VCAM-1), PGE2 and COX-2. Immunofluorescence studies indicated that C-DIM12 did not prevent translocation of p65 and stabilized nuclear localization of Nurr1 in synovial fibroblasts. Knockdown of Nurr1 expression by RNA interference prevented the inhibitory effects of C-DIM12 on inflammatory gene expression, indicating that the anti-inflammatory effects of this compound are Nurr1-dependent. Collectively, these data suggest that this receptor may be a viable therapeutic target in RA.

Clock gene expression in different synovial cells of patients with rheumatoid arthritis and osteoarthritis

Patients with rheumatoid arthritis (RA) show modulated circadian rhythms of inflammatory cytokines and cortisol, which may be associated with a modified expression of clock genes. The expression of major clock genes was previously studied in synovial tissues and fibroblasts of patients with RA and osteoarthritis (OA). We therefore especially aimed to examine the localization of clock genes at the cellular level in synovial tissue. Furthermore we were interested in studying the expression of the D site of albumin promoter (albumin D-box) binding protein (DBP) at the immunohistochemical level in human samples. Methods used include the in situ expression of the clock genes Brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 (Bmal 1), Circadian Locomotor Output Cycles Kaput (Clock), Period 1 and 2 (Per 1 and Per 2), and DBP was examined by immunohistochemistry in synovial tissues of patients with RA or OA. Additionally, expression profiles of different clock genes were determined over 24h by real time PCR in synovial fibroblasts (SFs) after a 2h serum shock or TNF-α. Results show that all clock genes investigated were found to be expressed both in RA and OA synovial tissues. Double staining against cell specific markers revealed that clock proteins were especially seen in macrophages, SFs and B-lymphocytes. Cell counting showed that clock proteins were found in approximately 5–20% of cells. Additionally, preliminary cell culture experiments showed that TNF-α treatment resulted in differential 24h expression profiles between RA and OA samples and also compared to the results obtained from the serum shock experiments. From our study we conclude that the major clock genes, including DBP, are expressed in samples from RA and OA patients, especially in macrophages and synovial fibroblasts, but also in B-lymphocytes. Preliminary experiments suggest that TNF-α seems to be able to modify clock gene expression in synovial fibroblasts.

Influence of CYB5A gene variants on risk of rheumatoid arthritis and local endocrine function in the joint

Rheumatoid arthritis (RA) is a multifactorial disease with a complex genetic background. An autoimmune etiology is involved in RA onset and progression but other pathways like neuroendocrine effects are potentially important. Androgens such as dehydroepiandrosterone, androstenedione and testosterone act anti-inflammatory. Endogenous de novo synthesis of androgens depends mainly on two enzyme activities of cytochrome P450c17: 17-alpha-hydroxylase and 17,20 lyase. A co-factor, particularly for 17,20-lyase activity, is cytochrome b5 (type A), encoded by the CYB5A gene on chromosome 18. We hypothesized that gene variants in CYB5A trigger the local amount of hormones, especially androgens and side products in this pathway, in the joint and therefore affect the susceptibility to RA. We screened two available genome wide association study (GWAS) data sets for association between single nucleotide polymorphisms (SNPs) in or near the CYB5A gene and RA. Both GWAS revealed RA-associated polymorphisms in the CYB5A gene: rs1790834 (p = 0.0073, OR = 0.83) and rs1790858 (p = 0.0095, OR = 0.44), respectively. We sought to replicate these findings in our Slovac RA sample with n = 521 RA cases and n = 321 healthy controls. For rs1790834 localized in intron 1 of the CYB5A gene, association was found with an OR = 0.69, i.e., a protective effect of the rare allele (p = 0.008). Genotype dependent gene expression in synovial fibroblasts, a cell type potentially responsible for androgen biosynthesis in the joint, showed an association between the rare allele of rs1790834 and increased CYB5A expression (p In conclusion, this study demonstrates for the first time that cofactors of important androgen-generating enzymes such as cytochrome b5A plays an important role for RA susceptibility. The interaction with neuroendocrine factors will be discussed.

Circadian Timekeeping Is Disturbed in Rheumatoid Arthritis at Molecular Level

IntroductionPatients with rheumatoid arthritis (RA) have disturbances in the hypothalamic-pituitary-adrenal (HPA) axis. These are reflected in altered circadian rhythm of circulating serum cortisol, melatonin and IL-6 levels and in chronic fatigue. We hypothesized that the molecular machinery responsible for the circadian timekeeping is perturbed in RA. The aim of this study was to investigate the expression of circadian clock in RA.MethodsGene expression of thirteen clock genes was analyzed in the synovial membrane of RA and control osteoarthritis (OA) patients. BMAL1 protein was detected using immunohistochemistry. Cell autonomous clock oscillation was started in RA and OA synovial fibroblasts using serum shock. The effect of pro-inflammatory stimulus on clock gene expression in synovial fibroblasts was studied using IL-6 and TNF-α.ResultsGene expression analysis disclosed disconcerted circadian timekeeping and immunohistochemistry revealed strong cytoplasmic localization of BMAL1 in RA patients. Perturbed circadian timekeeping is at least in part inflammation independent and cell autonomous, because RA synovial fibroblasts display altered circadian expression of several clock components, and perturbed circadian production of IL-6 and IL-1β after clock resetting. However, inflammatory stimulus disturbs the rhythm in cultured fibroblasts. Throughout the experiments ARNTL2 and NPAS2 appeared to be the most affected clock genes in human immune-inflammatory conditions.ConclusionWe conclude that the molecular machinery controlling the circadian rhythm is disturbed in RA patients.

CXCL12 gene expression is upregulated by hypoxia and growth arrest but not by inflammatory cytokines in rheumatoid synovial fibroblasts

Objectives CXCL12 is a constitutively expressed chemokine with important homeostatic functions. Increased CXCL12 expression has been observed in several inflammatory conditions, including rheumatoid arthritis (RA). This study was undertaken to identify potential mechanisms of regulation of CXCL12 gene expression by human fibroblasts under normal or inflammatory conditions. Methods Synovial fibroblasts (SF) were cultured from RA and osteoarthritis (OA) synovial tissues. CXCL12 mRNA expression was analysed by real time quantitative RT-PCR in RA-SF under different growth conditions, and exposed to hypoxia or to different pro-inflammatory factors. A 5′ CXCL12 −1.4 kb promoter region fragment was cloned in a luciferase reporter plasmid and its activity analysed in human fibroblasts. Results CXCL12 mRNA expression was not constitutively increased in RA- compared to OA-SF. LPS, pro-inflammatory cytokines or growth factors did not induce CXCL12 mRNA expression in SF. Hypoxia and growth arrest by either serum starvation or confluent growth induced CXCL12 mRNA and protein expression in SF. Constitutive and induced expression of CXCL12 in fibroblasts was regulated at the transcriptional level by specific regions of the −1.4 kb promoter. Conclusions Pro-inflammatory factors and cytokines do not up-regulate CXCL12 gene expression in SF. Growth arrest and hypoxia are potentially important inducers of CXCL12 expression in human fibroblasts and operate by regulating transcriptional activity of the promoter.

Distinct roles of Smad pathways and p38 pathways in cartilage-specific gene expression in synovial fibroblasts

The role of TGF-β/bone morphogenetic protein signaling in the chondrogenic differentiation of human synovial fibroblasts (SFs) was examined with the adenovirus vector–mediated gene transduction system. Expression of constitutively active activin receptor–like kinase 3 (ALK3CA) induced chondrocyte-specific gene expression in SFs cultured in pellets or in SF pellets transplanted into nude mice, in which both the Smad and p38 pathways are essential. To analyze downstream cascades of ALK3 signaling, we utilized adenovirus vectors carrying either Smad1 to stimulate Smad pathways or constitutively active MKK6 (MKK6CA) to activate p38 pathways. Smad1 expression had a synergistic effect on ALK3CA, while activation of p38 MAP kinase pathways alone by transduction of MKK6CA accelerated terminal chondrocytic differentiation, leading to type X collagen expression and enhanced mineralization. Overexpression of Smad1 prevented MKK6CA-induced type X collagen expression and maintained type II collagen expression. In a mouse model of osteoarthritis, activated p38 expression as well as type X collagen staining was detected in osteochondrophytes and marginal synovial cells. These results suggest that SFs can be differentiated into chondrocytes via ALK3 activation and that stimulating Smad pathways and controlling p38 activation at the proper level can be a good therapeutic strategy for maintaining the healthy joint homeostasis and treating degenerative joint disorders.

Distinct roles of Smad pathways and p38 pathways in cartilage-specific gene expression in synovial fibroblasts.

The role of TGF-beta/bone morphogenetic protein signaling in the chondrogenic differentiation of human synovial fibroblasts (SFs) was examined with the adenovirus vector-mediated gene transduction system. Expression of constitutively active activin receptor-like kinase 3 (ALK3CA) induced chondrocyte-specific gene expression in SFs cultured in pellets or in SF pellets transplanted into nude mice, in which both the Smad and p38 pathways are essential. To analyze downstream cascades of ALK3 signaling, we utilized adenovirus vectors carrying either Smad1 to stimulate Smad pathways or constitutively active MKK6 (MKK6CA) to activate p38 pathways. Smad1 expression had a synergistic effect on ALK3CA, while activation of p38 MAP kinase pathways alone by transduction of MKK6CA accelerated terminal chondrocytic differentiation, leading to type X collagen expression and enhanced mineralization. Overexpression of Smad1 prevented MKK6CA-induced type X collagen expression and maintained type II collagen expression. In a mouse model of osteoarthritis, activated p38 expression as well as type X collagen staining was detected in osteochondrophytes and marginal synovial cells. These results suggest that SFs can be differentiated into chondrocytes via ALK3 activation and that stimulating Smad pathways and controlling p38 activation at the proper level can be a good therapeutic strategy for maintaining the healthy joint homeostasis and treating degenerative joint disorders.

Synergistic effect of interleukin‐1β and tumor necrosis factor α on interleukin‐8 gene expression in synovial fibroblasts. evidence that interleukin‐8 is the major neutrophil‐activating chemokine released in response to monokine activation

To investigate both the involvement of chemokines in general and the relative importance of specific chemokines in rheumatoid arthritis (RA), we characterized the effect of the monokines tumor necrosis factor alpha (TNF alpha) and interleukin-1 beta (IL-1 beta) on the synthesis of neutrophil-activating factors by synovial fibroblasts isolated from the joints of patients with RA. Neutrophil-stimulating activity was assessed by determining intracellular calcium mobilization. IL-8 synthesis and secretion was assessed by specific enzyme-linked immunosorbent assay, and IL-8 messenger RNA (mRNA) levels were determined by Northern blot. Treatment of synovial fibroblasts with IL-1 beta and TNF alpha resulted in the production of an activity which induced intracellular calcium mobilization in peripheral blood neutrophils. The 2 monokines combined had a synergistic effect on the release of the neutrophil-stimulating activity. The effect of the 2 monokines required gene transcription and translation, and closely mimicked the pattern of IL-8 secretion induced in these cells by the monokines. We confirmed that the majority of the neutrophil-stimulating activity was IL-8 by 3 different approaches: cross-desensitization experiments with IL-8, melanoma growth-stimulatory activity, and neutrophil-activating peptide 2, stimulation of calcium mobilization in cells transfected with the IL-8 receptor complementary DNA, and inhibition of the activity following pretreatment of the supernatants with an anti-IL-8 antibody. TNF alpha and IL-1 beta induced a time- and dose-dependent release of immunoreactive IL-8. A synergistic effect of TNF alpha and IL-1 beta was also observed for both IL-8 production and accumulation of IL-8 mRNA. These results indicate that the monokines TNF alpha and IL-1 beta synergistically activate IL-8 expression and protein secretion by synovial fibroblasts, and that under these conditions, IL-8 appears to be the major neutrophil-activating factor released.

Regulation of Collagenase Gene Expression in Synovial Fibroblastsa

Regulation of Collagenase Gene Expression in Synovial Fibroblasts^a

Interleukin‐1 induces interleukin‐1α and interleukin‐1β gene expression in synovial fibroblasts and peripheral blood monocytes

Cellular interactions involved in the chronic inflammatory response, characteristic of those found in the joints of rheumatoid arthritis patients, were investigated by examining the effect of interleukin-1 (IL-1), tumor necrosis factor alpha, and gamma-interferon on the regulation of IL-1 gene expression and production by synovial fibroblasts. Biologically active IL-1 was detected in lysates of IL-1-treated rat and human fibroblasts that had been isolated from synovial tissue by collagenase digestion. Northern blot analysis of RNA isolated from these cells revealed the expression of IL-1 alpha and IL-1 beta transcripts. Neither the IL-1 transcripts nor the biologic activity of IL-1 was found in untreated synovial fibroblasts. The messenger RNA induction in synovial cells was followed by a time- and dose-dependent expression of intracellular IL-1 activity. Human monocytes and human skin fibroblasts also responded to IL-1 treatment by producing IL-1-specific transcripts. These observations suggest that IL-1 plays a key role in stimulating immune and inflammatory responses and in sustaining those responses through continued production at sites of inflammation.