Synovial Cell Line Research Articles

Low-Intensity Pulsed Ultrasound Reduces the Inflammatory Activity of Synovitis

The purpose of this study was to examine the effect of low-intensity pulsed ultrasound (LIPUS) on the cell proliferation and growth of synovial membrane cells stimulated with inflammatory cytokines, and to evaluate the effectiveness of LIPUS treatment of synovitis in the knee joints of animal models for rheumatoid arthritis. The rabbit knee synovial membrane cell line, HIG-82, was cultured in medium with or without IL-1β or TNF-α. Four hours after stimulation with the cytokines, the cells received LIPUS or sham exposure. Cell proliferation and growth were then analyzed. Using MRL/lpr mice, the anti-inflammatory effects of LIPUS were also evaluated in vivo. Stimulation with proinflammatory cytokines significantly up-regulated cell proliferation which was significantly down-regulated by LIPUS exposure. In MRL/lpr mice, exposure of knee joints to LIPUS caused a significant reduction of histological damage compared to the control. Histological lesions were significantly reduced in the joints treated with LIPUS for 3 weeks. Cox-2-positive cells in the knee joints treated with LIPUS were markedly decreased compared to the control joints. Therefore, LIPUS stimulation may be a medical treatment for joint inflammatory diseases, such as synovitis.

Delphinidin, a specific inhibitor of histone acetyltransferase, suppresses inflammatory signaling via prevention of NF-κB acetylation in fibroblast-like synoviocyte MH7A cells

Histone acetyltransferase (HAT) inhibitors (HATi) isolated from dietary compounds have been shown to suppress inflammatory signaling, which contributes to rheumatoid arthritis. Here, we identified a novel HATi in Punica granatum L. known as delphinidin (DP). DP did not affect the activity of other epigenetic enzymes (histone deacetylase, histone methyltransferase, or sirtuin1). DP specifically inhibited the HAT activities of p300/CBP. It also inhibited p65 acetylation in MH7A cells, a human rheumatoid arthritis synovial cell line. DP-induced hypoacetylation was accompanied by cytosolic accumulation of p65 and nuclear localization of IKBα. Accordingly, DP treatment inhibited TNFα-stimulated increases in NF-κB function and expression of NF-κB target genes in these cells. Importantly, DP suppressed lipopolysaccharide-induced pro-inflammatory cytokine expression in Jurkat T lymphocytes, demonstrating that HATi efficiently suppresses cytokine-mediated immune responses. Together, these results show that the HATi activity of DP counters anti-inflammatory signaling by blocking p65 acetylation and that this compound may be useful in preventing inflammatory arthritis.

Alleviating Effects of AS1892802, a Rho Kinase Inhibitor, on Osteoarthritic Disorders in Rodents

The effects of AS1892802, a selective Rho-associated coiled coil kinase (ROCK) inhibitor, on knee cartilage damage and pain behavior were examined in a rat model of osteoarthritis (OA). Monoiodoacetate (MIA) was intraarticularly injected into the right knee joints of rats. ROCK I and II mRNA levels increased in knee joints of MIA-injected rats. Our newly synthesized ROCK inhibitor, AS1892802, was injected into the ipsilateral knee or administered p.o. for 3 weeks. The compound dose-dependently and significantly inhibited of cartilage damage in the tibial plateau in a dose-dependent manner and decreased the weight distribution deficit associated with MIA injection. In addition, the compound also inhibited bradykinin induced pain responses in normal rats. In vitro, the compound could induce chondrocyte differentiation in a chondrogenic cell line and significantly inhibited IL-1β– or bradykinin-induced prostaglandin E2 production in a synovial cell line. AS1892802 prevents cartilage damage induced by MIA and has analgesic effects in rat pain models, suggesting that AS1892802 may be clinically useful for the treatment of OA.[Supplementary Figure: available only at http://dx.doi.org/10.1254/jphs.10319FP]

Dihydrotestosterone Inhibits Interleukin-1.ALPHA. or Tumor Necrosis Factor .ALPHA.-Induced Proinflammatory Cytokine Production via Androgen Receptor-Dependent Inhibition of Nuclear Factor-.KAPPA.B Activation in Rheumatoid Fibroblast-Like Synovial Cell Line

Rheumatoid arthritis (RA) is a disease with significant gender differences in its prevalence and clinical features. Interleukin (IL)-1 and tumor necrosis factor (TNF) α produced by synoviocytes are principle inflammatory and destructive mediators of RA. We found that a potent androgen, 5α-dihydrotestosterone (DHT) inhibits IL-1α-induced production and mRNA expression of IL-8, IL-6 and IL-1β from RA patient-derived fibroblast-like synovial cell line MH7A. Promoter analysis of the IL-8 gene revealed that nuclear factor (NF)-κB activation is critical for its transcriptional activation by IL-1α, and DHT inhibited the IL-1α-induced NF-κB activation in a manner dependent on the androgen receptor (AR). DHT also inhibited the effects of TNFα on the cells overexpressed with AR, indicating that sufficient expression level of functional AR was necessary for the inhibitory effect of DHT on TNFα. These results suggest that androgen contributes to the prevention against RA and its gender difference by inhibiting IL-1α or TNFα-induced proinflammatory cytokine production from synovial fibroblast-like cells by inhibiting NF-κB activation in a manner depending on AR.

Intercellular adhesion molecule-1 on synovial cells attenuated interleukin-6-induced inhibition of osteoclastogenesis induced by receptor activator for nuclear factor κB ligand

In a co-culture of osteoclast precursor cells and synovial cells, interleukin-6 (IL-6) induces osteoclast formation. In contrast, in a monoculture of osteoclast precursor cells, IL-6 directly suppresses receptor activator for nuclear factor κB ligand (RANKL)-induced differentiation of osteoclast precursor cells into osteoclasts. In the present study, we explored why the effect of IL-6 differed between the monoculture and the co-culture systems. In the monoculture, mouse osteoclast precursor cell line, RAW 264·7 (RAW) cells were cultured with soluble RANKL (sRANKL) for 24 h or 3 days. sRANKL increased both expression of osteoclastogenesis marker, tartrate-resistant acid phosphatase isoform 5b (TRAP5b) and nuclear factor of activated T cells cytoplasmic 1 (NFATc1), whereas the co-addition of IL-6 decreased them both in a dose-dependent manner. In the co-culture, RAW cells and human synovial cell line, SW982 cells were cultured with IL-6+soluble IL-6 receptor (sIL-6R) for 3 days. TRAP5b and NFATc1 expression reduced by IL-6 was increased by the addition of SW982 cells in a manner dependent upon the number of added cells. IL-6+sIL-6R treatment significantly induced RANKL production in SW982 cells, and anti-RANKL antibody inhibited IL-6+sIL-6R-induced osteoclastogenesis. SW982 cells expressed high levels of ICAM-1 originally, and ICAM-1 expression was increased significantly by IL-6+sIL-6R. Anti-ICAM-1 antibody suppressed IL-6-induced osteoclastogenesis. Finally, in the monoculture system, addition of sICAM-1 dose-dependently restored the expression of TRAP5b reduced by IL-6. Similar results were obtained when the formation of TRAP-positive multi-nuclear cells were examined using mouse bone marrow cells. In conclusion, IL-6 gave different results in the co-culture and monoculture systems because in the co-culture, ICAM-1 from the synovial cells restored osteoclastogenesis suppressed by IL-6.

Resveratrol induces apoptosis MH7A human rheumatoid arthritis synovial cells in a sirtuin 1-dependent manner

Resveratrol, a phytoalexin, reduced the viability of MH7A cells, a human rheumatoid arthritis synovial cell line. In the apoptosis assay, resveratrol increased TUNEL-positive cells and stimulated H2A.X phosphorylation. Resveratrol disrupted mitochondrial membrane potentials in MH7A cells and stimulated cytochrome c release from the mitochondria to the cytosol. Resveratrol activated caspase-3 and caspase-9 but not caspase-8 in MH7A cells. Resveratrol upregulated the expression of the NAD-dependent deacetylase sirtuin 1 mRNA and downregulated the expression of the Bcl-XL mRNA, and resveratrol-induced MH7A cell death, mitochondrial damage, and caspase-3/-9 activation were prevented by sirtinol, an inhibitor of sirtuin 1. The results of the present study show that resveratrol induces MH7A cell apoptosis by activating caspase-9 and the effector caspase-3 along mitochondrial disruption as a result of reduced Bcl-XL expression, allowing cytochrome c release from the mitochondria into the cytosol, in a sirtuin 1-dependent manner. This suggests that resveratrol could suppress hyperplasia of synovial cells, a critical factor of rheumatoid arthritis.

Effects of Low-Intensity Pulsed Ultrasound on the Expression and Activity of Hyaluronan Synthase and Hyaluronidase in IL-1β-Stimulated Synovial Cells

The purpose of this study is to examine effects of low-intensity pulsed ultrasound (LIPUS) on metabolism of hyaluronan (HA) in synovial membrane cells stimulated by IL-1β. Rabbit knee synovial membrane cell line, HIG-82, was cultured in medium with the presence or absence of 1 ng/mL IL-1β, and after 4 h the cell was exposed to LIPUS for 15 min. The mRNA levels of HA synthase (HAS) 2,3, hyaluronidase (HYAL) 2, and cyclooxygenase (COX)-2 were examined by real-time PCR analysis. Concentrations of HA and PGE₂ were quantified by use of enzyme linked immunosorbent assay (ELISA). The COX-2 level was analyzed by western blotting. Gene levels of HAS2 and HAS3 in IL-1β-stimulated cells were up-regulated significantly (p < 0.01) by LIPUS. HYAL2 mRNA was up-regulated by the treatment with IL-1β, whereas down-regulated significantly (p < 0.01) by the following LIPUS exposure. Furthermore, IL-1β stimulation enhanced COX-2 and PGE₂ expression as compared to the untreated control, and IL-1β-induced COX-2 and PGE₂ expression was inhibited by LIPUS. These results suggest that LIPUS enhanced HA synthesis and inhibited HYAL2 expression, leading to the accumulation of high-molecular weight HA. Therefore, LIPUS stimulation may be a better candidate as medical remedy to treat inflammatory joint diseases accompanied with HA degradation in synovial fluid.

Luteolin suppresses IL-1β-induced cytokines and MMPs production via p38 MAPK, JNK, NF-kappaB and AP-1 activation in human synovial sarcoma cell line, SW982

Matrix metalloproteinases (MMPs) play an important role in tissue degradation in rheumatoid synovium and inflammatory cytokines are essential in the pathogenesis of rheumatoid arthritis (RA). This study was conducted to evaluate the efficacy of luteolin in regulating interleukin-1β (IL-1β)-induced production of MMPs (MMP-1 and -3) and cytokines (tumor necrosis factor (TNF)-α and IL-6) in human synovial cell line, SW982. Treatment with luteolin at 1 or 10μM significantly (P<0.05) inhibited IL-1β-induced MMPs (MMP-1 and -3) and cytokines (TNF-α and IL-6) production when measured by enzyme-linked immunosorbent assay (ELISA). The mitogen-activated protein kinases (MAPKs) represent an attractive target for RA because they can regulate MMP and cytokine expression. The effects of luteolin on the activation of MAPKs and transcription factors were also examined in SW982 cells by ELISA. IL-1β-induced JNK and p38 activation were inhibited by luteolin. Moreover, IL-1β-induced activator protein-1 (AP-1) and nuclear factor-kappaB (NF-κB) activation were inhibited by luteolin. These results suggest that luteolin reduces the production of MMPs and cytokines in SW982 cells by inhibiting MAPKs (JNK and p38) and transcription factors (AP-1 and NF-κB).

Effects of the novel immunosuppressant FTY720 in a murine rheumatoid arthritis model

We investigated the effects and mechanisms by which FTY720 (FTY) inhibits arthritis development in the SKG mouse rheumatoid arthritis (RA) model. FTY (1mg/kg/day) administration suppressed the progression of laminarin-induced arthritis in SKG mice. FTY treatment decreased IL-6 and TNF-alpha expression in synovial fibroblast cells and the number of inflammatory cells overall. Bone destruction was also suppressed by treatment with FTY. The numbers of CD4(+) and CD8(+) T cells were significantly increased in the thymus and decreased in the spleen in FTY-treated SKG mice. FTY enhanced IL-4 production by CD4(+) T cells stimulated by allogeneic spleen cells and inhibited prostaglandin E(2) (PGE(2)) production by a TNF-alpha-stimulated synovial fibroblast cell line. These results indicate that FTY can inhibit arthritis in SKG mice via sequestration of autoimmune CD4(+) T cells in the thymus, enhancement of Th2 immune responses, and inhibition of PGE(2) production by synovial cells.

Modulation of Hyaluronan Fragmentation by Interleukin-1 Beta in Synovial Membrane Cells

Hyaluronan (HA) plays a crucial role in the lubricating and buffering properties of synovial fluid. The purpose of this study was to examine the effects of interleukin (IL)-1beta on HA degradation in cultured synovial membrane cells. The rabbit synovial membrane cell line HIG-82 was cultured with and without IL-1beta. The amounts of HA of varying molecular weights in the medium were analyzed using high-performance liquid chromatography, the mRNA levels of HA synthase (HAS) and hyaluronidase (HYAL) were analyzed by means of real-time PCR, and HYAL activity was analyzed by HA zymography. The amounts of HA with a molecular weight lower than 300 kDa, and between 300 and 1900 kDa, in the culture medium of HIG-82 cells were significantly higher in the presence of IL-1beta. However, the amount of HA with a molecular weight greater than 1900 kDa was significantly lower in the presence of IL-1beta. Both HAS2 and HAS3 mRNA levels were upregulated by treatment with IL-1beta. So, too, were the levels of HYAL1 and HYAL2 mRNA, which resulted in enhanced HYAL activity. However, HYAL activity was inhibited by transfection of HYAL2-siRNA. Our results suggest that IL-1beta is a crucial factor in the fragmentation of HA in inflammatory joints.

Interleukin‐1 β increases gap junctional communication among synovial fibroblasts via the extracellular‐signal‐regulated kinase pathway

The gap junction protein, Cx43 (connexin 43), has been implicated in the aetiology of osteoarthritis. Studies have revealed that the size and number of gap junctions increase in synovial biopsies from patients with osteoarthritis. Furthermore, pharmacological inhibition of Cx43 function has been shown to reduce IL-1beta (interleukin-1beta)-induced metalloproteinase production by synovial fibroblasts in vitro. In the present study, we examined the link between IL-1beta and Cx43 function. We demonstrated that treatment of a rabbit synovial fibroblast cell line with IL-1beta markedly increased the level of the Cx43 protein in a concentration- and time-dependent manner. The impact on Cx43 protein levels appeared to occur post-transcriptionally, as mRNA levels are unaffected by IL-1beta administration. Additionally, we showed by fluorescence microscopy that IL-1beta alters the cellular distribution of Cx43 to cell-cell junctions and is concomitant with a striking increase in gap junction communication. Furthermore, we demonstrated that the increase in Cx43 protein, and the associated change in protein localization and gap junction communication following IL-1beta treatment, are dependent upon activation of the ERK (extracellular-signal-regulated kinase) signalling cascade. These data show that IL-1beta acts through the ERK signalling cascade to alter the expression and function of Cx43 in synovial fibroblasts.

Effects of hesperetin on the production of inflammatory mediators in IL-1β treated human synovial cells

This study was conducted to evaluate the efficacy of hesperetin in regulating interleukin-1β (IL-1β)-induced production of the matrix metalloproteinase (MMP)-3 and IL-6 in human synovial cell line, SW982. Treatment with hesperetin at 1 or 10 μM significantly ( P < 0.05) inhibited IL-1β-induced MMP-3 and IL-6 production when measured by enzyme-linked immunosorbent assay (ELISA). The effects of hesperetin on the activation of mitogen-activated protein kinases (MAPKs) were also examined in SW982 cells by ELISA assay. IL-1β-induced JNK activation was inhibited by hesperetin. These results suggest that hesperetin reduces the production of MMP and IL-6 in SW982 synovial cells by inhibiting JNK.

Growth suppression and apoptosis induction in synovial sarcoma cell lines by a novel NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ)

Synovial sarcoma is a relatively common soft tissue sarcoma with an aggressive clinical course. Although surgery is currently the first treatment modality, improvement of adjuvant chemotherapy is deemed essential to improve the clinical outcome. Nuclear factor-κB (NF-κB) is constitutively activated in various cancer cells and has emerged as a potential therapeutic molecular target; however, the possible involvement of NF-κB in the pathology of sarcomas remains to be clarified. Herein we examined the effects of a novel NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) on two synovial sarcoma-derived cell lines, HS-SY-II and SYO-1. The growth of both cell lines was completely inhibited by DHMEQ and apoptosis was induced at 10 μg/ml. Additionally, we found that DHMEQ showed additive effects when used in combination with other cytotoxic agents. These observations indicate that inhibition of NF-κB activity may serve as a potential therapeutic target for synovial sarcoma.

Inhibition of cyclooxygenase 2 expression by diallyl sulfide on joint inflammation induced by urate crystal and IL-1β

Investigation of the effects of diallyl sulfide (DAS), a garlic sulfur compound, on joint tissue inflammatory responses induced by monosodium urate (MSU) crystals and interleukin-1beta (IL-1beta). The HIG-82 synovial cell line was used to establish the experimental model and DAS regime. Primary cultures of articular chondrocytes and synovial fibroblasts obtained from patients undergoing joint replacement for osteoarthritis were used in experimental studies. Cyclooxygenase (COX) expression following MSU crystal and IL-1beta stimulation with/without DAS co-incubation was assessed by reverse transcription-polymerase chain reaction (RT-PCR), western blotting, and immunocytochemistry and nuclear factor-kappa B (NF-kappaB) activation determined by electrophoretic mobility shift assay. Prostaglandin E2 (PGE(2)) production was measured by enzyme-linked immunosorbent assay (ELISA). DAS effects on COX gene expression in an MSU crystal-induced acute arthritis in rats were assessed by RT-PCR. MSU crystals upregulated COX-2 expression in HIG-82 cells and this was inhibited by co-incubation with DAS. DAS inhibited MSU crystal and IL-1beta induced elevation of COX-2 expression in primary synovial cells and chondrocytes. Production of PGE(2) induced by crystals was suppressed by DAS and celecoxib. MSU crystals had no effect on expression of COX-1 in synovial cells. NF-kappaB was activated by MSU crystals and this was blocked by DAS. Increased expression of COX-2 in synovium following intraarticular injection of MSU crystals in a rat model was inhibited by co-administration of DAS. DAS prevents IL-1beta and MSU crystal induced COX-2 upregulation in synovial cells and chondrocytes and ameliorates crystal induced synovitis potentially through a mechanism involving NF-kappaB. Anti-inflammatory actions of DAS may be of value in treatment of joint inflammation.

Effect of local viral transfer of interleukin 10 gene on a rabbit arthritis model induced by interleukin 1β

Interleukin 1beta (IL-1beta) is the principal mediator in the pathogenesis of rheumatoid arthritis. Continuous injection of interleukin 1beta (IL-1beta) into the knee articular cavities of animals can induce models that resemble rheumatoid arthritis. The objective of this study was to evaluate the feasibility of local recombinant retrovirus viral interleukin 10 (rRV-vIL-10) gene transfer treatment of a rabbit model of arthritis induced by IL-1beta. An hIL-1beta-induced rabbit rheumatoid arthritis model was established using the MFG-hIL-1beta-neo-HIG-82 cell line, which is capable of continuous secretion of hIL-1beta. After transfecting the rabbit synovial fibroblast cell line (MFG-hIL-1beta-neo-HIG-82) with rRV-vIL-10, G418 was then added to identify the positive clone. The rRV-vIL-10 positive clone was injected into the established rabbit rheumatoid arthritis model through intra-articular injection. Successful gene transfer was determined by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. The levels of IL-1beta before and after treatment were determined by enzyme- linked immunosorbent assay. Retrovirus vector was an effective vector both to synoviocytes in vitro and synovium tissue in vivo as confirmed by RT-PCR and immunohistochemistry. The rabbit arthritis model treated with rRV-vIL-10 showed a dramatic remission of arthritis and a decline in the level of cytokines such as IL-1beta. Retrovirus-mediated transfection of vIL-10 successfully transferred the gene into rabbit synovium ex vivo and was able to suppress intra-articular inflammation response to IL-1beta.

Evaluation of the suppressive actions of glucosamine on the interleukin-1β-mediated activation of synoviocytes

Recently, we found that administration of glucosamine to adjuvant arthritis, a model for rheumatoid arthritis, suppressed the progression of arthritis in rats. To clarify its anti-inflammatory mechanism, we evaluated the actions of glucosamine on the activation of synoviocytes in vitro. Synoviocytes isolated from human synovial tissues were stimulated with interleukin (IL)-1beta in the presence of 0.01-1 mM glucosamine. IL-8 and prostaglandin (PG) E(2) were measured by ELISA, and nitric oxide was quantitated by Griess assay. IL-8 mRNA was detected by RT-PCR. Furthermore, the effect of glucosamine on the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and the binding of [(125)I] IL-1beta to its receptors were examined using a primary human synovial cell line (CSABI- 479). Glucosamine significantly suppressed the IL-1beta-induced IL-8 production as well as its mRNA expression (p < 0.05) at 1 mM. Furthermore, glucosamine (1 mM) inhibited the IL-1beta-induced nitric oxide and PGE(2) production (p < 0.05). Moreover, glucosamine suppressed the IL-1beta-induced phosphorylation of p38 MAPK (p < 0.05 at >0.1 mM) and the IL-1beta-binding to its receptors (p < 0.05 at 1 mM). These observations suggest that glucosamine can suppress the IL-1beta-mediated activation of synoviocytes (such as IL-8-, nitric oxide- and PGE(2)-production, and phosphorylation of p38 MAPK), thereby possibly exhibiting antiinflammatory actions in arthritis.

Identification and Characterization of an RTX Toxin in the Emerging Pathogen Kingella kingae

Kingella kingae is an emerging bacterial pathogen that is increasingly recognized as the causative agent of a variety of pediatric diseases, including septic arthritis and osteomyelitis. The pathogenesis of K. kingae disease is believed to begin with colonization of the upper respiratory tract. In the present study, we examined interactions between K. kingae and cultured respiratory epithelial cells and observed potent cytotoxicity, detected by both microscopy and lactic acid dehydrogenase (LDH) release assays. Experiments with synovial and macrophage cell lines revealed cytotoxicity for these cell types as well. Using mariner mutagenesis and a screen for loss of cytotoxicity, a genetic locus encoding an RTX toxin system was identified. Disruption of the K. kingae RTX locus resulted in a loss of cytotoxicity for respiratory epithelial, synovial, and macrophage cell lines. DNA sequence analysis demonstrated that the RTX locus is flanked by insertion elements and has a reduced G+C content compared to that of the whole genome. Two relatively less invasive Kingella species, K. oralis and K. denitrificans, were found to be noncytotoxic and to lack the RTX region, as determined by LDH release assays and Southern blotting. We concluded that K. kingae expresses an RTX toxin that has wide cellular specificity and was likely acquired horizontally. The possible roles for this toxin in the pathogenesis of K. kingae disease include breaching of the epithelial barrier and destruction of target tissues, such as synovium (joint lining).

Estrogen receptor alpha regulates matrix metalloproteinase-13 promoter activity primarily through the AP-1 transcriptional regulatory site

Many females develop bone diseases such as osteoporosis, and joint diseases such as osteoarthritis after menopause when estrogen levels decline. As estrogen receptors (ER) are present in such tissues, it is possible that the loss of estrogen at menopause influences the expression of enzymes such as members of the MMP family of proteinases to affect bone and connective tissue metabolism. The present study was undertaken to assess a possible relationship between ER-α and MMP-13 expression at the promoter level, and to determine how such a relationship could be modulated by ligands such as estrogen. Using a rabbit synovial cell line lacking endogenous ER, a transient transfection system with an ER-α construct, and a series of MMP-13 promoter-luciferase constructs of varying lengths and with specific mutations in transcription factor binding sites, it was found that ER-α can significantly enhance MMP-13 promoter activity via the AP-1 site, with modulatory influences by the Runx and PEA-3 sites on this ER-α dependent enhancement of the promoter activity. This enhancement by ER-α was significantly depressed in the presence of 17-ß-estradiol in a dose dependent manner. The influence of tamoxifen and raloxifen on the activity of the ER-α was consistent with their known agonist/antagonist activity. These findings indicate that loss of estrogen in vivo could potentially lead to enhanced expression of MMP-13, a proteinase that has been implicated in both osteoporosis and osteoarthritis, and thus contribute to the development and progression of these conditions.

Stromelysin-1 expression is activated in vivo by Ets-1 through palindromic head-to-head Ets binding sites present in the promoter

Regulation of the gene expression of Stromelysin-1 (matrix metalloproteinase-3), a member of the matrix metalloproteinase family, is critical for tissue homeostasis. The Stromelysin-1 promoter is known to be transactivated by Ets proteins through palindromic head-to-head Ets binding sites (EBS), an unusual configuration among metalloproteinase promoters. Patterns of increased co-expression of Stromelysin-1 and Ets-1 genes have been observed in pathological processes such as rheumatoid arthritis, glomerulonephritis and tumor invasion. In this context, we show in a synovial fibroblastic model cell line (HIG-82), which is able to co-express Stromelysin-1 and Ets-1, that the EBS palindrome is essential for the expression of Stromelysin-1. More precisely, using electrophoretic mobility shift assays, DNA affinity purification and chromatin immunoprecipitation, we demonstrate that endogenous Ets-1, but not Ets-2, is present on this palindrome. The use of a dominant-negative form of Ets-1 and the decrease of Ets-1 amount either by fumagillin, an antiangiogenic compound, or by short interfering RNA show that the activation rate of the promoter and the expression of Stromelysin-1 correlate with the level of endogenous Ets-1. Thus, it is the first demonstration, using this cellular model, that endogenously expressed Ets-1 is actually a main activator of the Stromelysin-1 promoter through its effective binding to the EBS palindrome.

Regulatory role of heme oxygenase 1 in inflammation of rheumatoid arthritis

To examine the expression and pathogenetic roles of heme oxygenase 1 (HO-1), an inducible heme-degrading enzyme with antiinflammatory properties, in rheumatoid arthritis (RA). HO-1 expression in synovial tissue from patients with RA, patients with osteoarthritis, and patients with noninflammatory joint diseases was determined by immunoblotting and immunohistochemistry. Effects of various agents, such as hemin (a chemical inducer of HO-1), small interfering RNA (siRNA) specific for HO-1, HO-1 expression vector, and antirheumatic agents, on HO-1 expression in RA synovial cell lines were analyzed by real-time reverse transcription-polymerase chain reaction (PCR) and immunoblotting. Cytokine synthesis was evaluated by real-time PCR and enzyme-linked immunosorbent assay. HO-1 was expressed more abundantly in the lesions of synovial tissue from patients with RA than in those from the other patient groups. Hemin, auranofin, and HO-1 expression vector induced HO-1 and reduced expression of tumor necrosis factor alpha (TNFalpha) messenger RNA, lipopolysaccharide (LPS)-induced secretion of interleukin-6 (IL-6) and IL-8, and expression of cyclooxygenase 2 in the synovial cell lines. Treatment with HO-1-specific siRNA augmented the synthesis of TNFalpha, IL-6, and IL-8 and canceled the suppressive effects of auranofin on TNFalpha secretion. When hemoglobin, as a scavenger of carbon monoxide, was added to auranofin-treated synovial cell lines, LPS-dependent production of IL-6 and IL-8 was increased. Our data demonstrate that HO-1 is expressed in RA synovial tissues and plays a regulatory role in the development of inflammation. The pharmacologic effects of auranofin depend, in part, on the levels of HO-1, suggesting that HO-1 induction is a novel therapeutic strategy for RA.