Effects Of IL-1beta Research Articles

Study the Effect of IL-1beta on Thyroid Hormones Levels in Women with Hypo- and Hyperthyroidism in the City of Ramadi

Thyroiditis is a medical condition of particular concern due to its diverse causes, including autoimmune factors, drug-induced inflammatory causes or fibrotic process, the most common of which is thyroiditis and includes hypothyroidism and hyperthyroidism. The study aimed to measure interleukin 1 beta (IL-1β) and compare it with the studied groups and the hormones TSH, T3, T4 and their relationship with IL-1β. The study included investigating the relationship between IL-1β and thyroid patients in females aged between (18-60) in the city of Ramadi as well as knowing the relationship between thyroid hormones (TSH, T4, T3) and IL-1β. 45 samples were collected from Ramadi Teaching Hospital. The current study showed that there was a significant increase in the level of IL-1β compared to the control group in hypothyroid patients at a significant level (P≤0.05), which was (2.3497ng/dl). There were no significant differences in the level of IL-1β in patients with hyperthyroidism at a significant level (P≤0.05), which was (ng/dl 2.0690) compared to the control group. The study showed that there is an inverse relationship between TSH and IL-1β (0.858) and a positive relationship between T4 and IL-1β (0.809) .We conclude from the study that interleukin 1 beta (IL-1β) has an effect on the thyroid gland, especially hypothyroidism, as it showed a highly significant increase in IL-1β in patients with hypothyroidism and no significant differences in IL-1β, as IL-1β affects thyroid hormones

O35 THE SYNERGISM BETWEEN HIGH GLUCOSE AND INTERLEUKIN-1beta PROMOTES INFLAMMASOME ACTIVATION AND THE RELEASE OF SMALL EXTRACELLULAR VESICLES IN HUMAN AORTIC SMOOTH MUSCLE CELLS

Background and objective: Hyperglycemia and inflammatory cytokines like interleukin (IL)-1beta play a key role in the vascular complications associated to diabetes melllitus (DM), such as atherosclerosis and hypertension. Understanding the pathophysiological mechanisms involved, including altered intercellular communication, is essential to identify potential therapeutical targets. We investigated the synergism of IL-1beta and high glucose on: (1) the activation of nucleotide-binding-oligomerization-domain, leucine-rich-repeat, and pyrin-domain-containing-3 (NLRP3)-inflammasome, an innate immunity component linked to vascular disease, and (2) the production and cargo of small extracellular vesicles (sEVs) as key players in vascular intercellular communication. Methods: Human aortic smooth muscle cells (HASMC) were challenged for 18 h with IL-1beta (10 ng/ml) in the presence of normal (5,5 mmol/L) or high glucose (22 mmol/L). Protein levels were quantified by Western blot, while the formation of ASC-like specks, reflecting NLRP3 activation, was assessed by indirect immunofluorescence. sEVs were determined by nanotracking analysis and endothelial senescence by the beta-galactosidase activity assay. Results: In HASMC cultures, IL-1beta favored NLRP3-inflammasome priming, as shown by NF-kappaB (phospho-p65) activation while NLRP3 and pro-IL-1beta levels were increased. The cytokine equally promoted NLRP3-inflammasome complex assembly, caspase-1 activation, and a subsequent release of mature IL-1beta in an auto-inflammatory loop. High glucose alone did not promote these effects, but it exacerbated some actions of IL-1beta and potentiated the release of HASMC-derived sEVs induced by IL-1beta, increasing their cargo content in NLRP3-inflammasome components. Moreover, these sEVs induce autocrine NLRP3-inflammasome activation in naïve HASMC and paracrine senescence in human endothelial cells. The IL-1 receptor blocker anakinra (1 mg/ml) blunted the IL-1beta effect, as well as their exacerbation by high glucose. Conclusion: High glucose exacerbates the activation of NLRP3-inflammasome by IL-1beta and alters intercellular communication by modifying the number and content of sEVs, which can expand inflammation in neighboring tissues. By preventing such synergy, biological drugs blocking IL-1beta receptors arise as potential pharmacological tools to attenuate DM associated vascular abnormalities.

NLRP3 Inflammasome Products IL‐1beta and IL‐18 Have A Direct Effect on Cardiomyocytes

ObjectiveWe previously demonstrated a significant role for NLRP3 inflammasome activation in cardiomyocytes in cardiac inflammation, remodeling and contractile dysfunction in response to stressors such as Angiotensin II infusion and pressure overload. There is limited data regarding responses of cardiomyocytes to interleukin‐1 beta (IL‐1beta) and interleukin 18 (IL‐18), which are generated as a result of NLRP3 activation. The goal of this study was to determine the effects of IL‐1beta and IL‐18 on cardiomyocytes.MethodsIsolated Neonatal Rat Ventricular Myocytes (NRVMs) were treated for various times with a range of doses of recombinant IL‐1beta or IL‐18. Western Blotting was used to measure the activation state of protein kinases and downstream signaling pathways. Quantitative Polymerase Chain Reaction (q‐PCR) was used to assess the expression of pro‐inflammatory cytokines and chemokines in cardiomyocytes.ResultsWestern blotting showed a significant increase in phosphorylation of multiple protein kinases and molecules that regulate apoptosis and other inflammatory functions after IL‐1beta and IL‐18 treatment, with maximal responses seen at 1ng/ml IL‐1beta and 10ng/ml IL‐18. Phosphorylated mitogen‐activated protein kinases (MAPKs) including JNK and P38 as well as p‐STAT3 were increased several fold at 10 and 30 mins of treatment with either cytokine. q‐PCR analysis of NRVM mRNA revealed increases in gene expression of multiple pro‐inflammatory cytokines and chemokines in cells treated with either IL‐1beta and IL‐18. CXCL‐10, TGF‐beta, IL‐18, and NLRP3 were all upregulated by both IL‐1beta or IL‐18. Interestingly, upregulation of CCL2, IL‐6, CXCL1, and TNF‐alpha resulted from treatment with IL‐1beta but not with IL‐18. We are currently analyzing the effects of IL‐1beta and IL‐18 on cell viability to further determine the direct effect of these products of the NLRP3 inflammasome in regulating cardiomyocyte apoptosis basally or in response to stressors.ConclusionsWe have previously shown that the cardiomyocyte is a source of proinflammatory cytokines and chemokines, including IL‐1beta and IL‐18. Our results suggest the possibility that IL‐1beta and IL‐18 activate a pro‐inflammatory positive feedback loop in cardiomyocytes which enhances and sustains cardiac inflammation, contributing to cardiac remodeling under stress conditions.

Novel pendrin inhibitor attenuates airway hyperresponsiveness and mucin expression in experimental murine asthma

Novel pendrin inhibitor attenuates airway hyperresponsiveness and mucin expression in experimental murine asthma

Low-Intensity Pulsed Ultrasound (LIPUS) Promotes BMP9-Induced Osteogenesis and Suppresses Inflammatory Responses in Human Periodontal Ligament-Derived Stem Cells.

We previously reported that low intensity pulsed ultrasound (LIPUS) promotes marrow stromal cell (MSC) osteogenesis and suppresses the LPS-induced inflammatory response in osteoblasts. Here, we examined the effects of LIPUS on human periodontal ligament-derived stem cells (hPDLSCs) in chronic inflammatory bone disease, such as periodontitis. hPDLSCs were collected from 3 healthy third molars. hPDLSCs were induced to differentiate by either recombinant BMP2 or BMP9 with or without daily LIPUS treatment (20 min/d). hPDLSCs were also stimulated by Porphyromonas gingivalis-derived LPS (LPS-PG), IL-1beta, and TNF-alpha with or without LIPUS. Matrix mineralization was evaluated by alizarin red S staining. The expression of genes for osteogenic makers and for inflammatory cytokines were analyzed by real time RT-PCR. LIPUS promoted BMP9-induced osteogenesis of hPDLSCs based on increases in both cell calcification and osteogenic marker expression. In contrast, LIPUS did not affect BMP2-induced osteogenic differentiation. LIPUS-induced Noggin expression was potentially involved in the differential response of the cells. Either LPS-PG, IL-1beta, or TNF-alpha-induced ERK phosphorylation and IL-8, CCL2, and RANKL expression were decreased in LIPUS-treated hPDLSCs. Moreover, the inhibitory effects of LPS-PG and IL-1beta on osteogenesis of hPDLSCs were significantly blocked by LIPUS. LIPUS is an effective tool to promote osteogenic differentiation under inflammatory conditions.

Effect of Serum IL-1beta of PCSO-524 and Firocoxib in Dogs Undergoing Medial Patellar Luxation Repair

Effect of Serum IL-1beta of PCSO-524 and Firocoxib in Dogs Undergoing Medial Patellar Luxation Repair

Distinct expression of SREBP-2 in normal and osteoarthritic chondrocytes

Purpose: Recent reports have elucidated that osteoarthritis (OA) is a metabolic disease with the epigenetic and proteomic analysis studies for various lipid metabolism-related genes and proteins comparing osteoarthritic cartilage with normal. Sterol regulatory element-binding protein 2 (SREBP2) is a ubiquitously expressed transcription factor that controls cholesterol homeostasis by stimulating transcription of sterol-regulated genes, such as 3-hydroxy-3-methyl glutaryl CoA reductase (HMGCR) [1]. Apart from regulating cholesterol homeostasis, several studies have provided evidence that SREBP2 and its various single nucleotide polymorphisms (SNPs) also play an central role in the pathogenesis of osteoarthritis, however, the association of SREBP2 with articular chondrocytes arthritogenic effects of IL-1beta (IL-1β) has been poorly understood [2, 3]. In this study, we investigated the association between SREBP2 expression and IL-1β-induced articular chondrocytes arthritogenic effects. Methods: Human cartilage were obtained from total knee arthroplasty for osteoarthritis or total hip arthroplasty for fracture of neck of femur (normal control group). Meanwhile, the normal and osteoarthritic cartilage isolated from of C57BL/6 mice and surgically induced osteoarthritis models. Then, immunohistochemistry staining and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to detect the expression of SREBP2, HMGCR, SOX9, type II collagen and type X collagen. Moreover, we treated normal chondrocytes with different concentration of IL-1β, and examined the expression of the above genes. Results: We found that the expression of SREBP-2, HMGCR, SOX9, type II collagen and type X collagen in OA patients and normal controls were significantly different, and similar results obtained in the two C57BL/6 mice groups. After induced by IL-1β, normal chondrocytes turned hypertrophy-like chondrocytes, and notably, the expression of SREBP-2, type X collagen and HMGCR were upregulated, and SOX9 and type II collagen were suppressed in vitro. Conclusions: These data suggest that SREBP-2 has a distinct expression in normal and osteoarthritic chondrocytes. Proinflammatory cytokine IL-1β may influence the hypertrophy and function of chondrocyte in part through SREBP-2.

Cytokine signaling-1 suppressor is inducible by IL-1beta and inhibits the catabolic effects of IL-1beta in chondrocytes: its implication in the paradoxical joint-protective role of IL-1beta

IntroductionAlthough IL-1β is believed to be crucial in the pathogenesis of osteoarthritis (OA), the IL-1β blockade brings no therapeutic benefit in human OA and results in OA aggravation in several animal models. We explored the role of a cytokine signaling 1 (SOCS1) suppressor as a regulatory modulator of IL-1β signaling in chondrocytes.MethodsCartilage samples were obtained from patients with knee OA and those without OA who underwent surgery for femur-neck fracture. SOCS1 expression in cartilage was assessed with immunohistochemistry. IL-1β-induced SOCS1 expression in chondrocytes was analyzed with quantitative polymerase chain reaction and immunoblot. The effect of SOCS1 on IL-1β signaling pathways and the synthesis of matrix metalloproteinases (MMPs) and aggrecanase-1 was investigated in SOCS1-overexpressing or -knockdown chondrocytes.ResultsSOCS1 expression was significantly increased in OA cartilage, especially in areas of severe damage (P < 0.01). IL-1β stimulated SOCS1 mRNA expression in a dose-dependent pattern (P < 0.01). The IL-1β-induced production of MMP-1, MMP-3, MMP-13, and ADAMTS-4 (aggrecanase-1, a disintegrin and metalloproteinase with thrombospondin motifs 4) was affected by SOCS1 overexpression or knockdown in both SW1353 cells and primary human articular chondrocytes (all P values < 0.05). The inhibitory effects of SOCS1 were mediated by blocking p38, c-Jun N-terminal kinase (JNK), and nuclear factor κB (NF-κB) activation, and by downregulating transforming growth factor-β-activated kinase 1 (TAK1) expression.ConclusionsOur results show that SOCS1 is induced by IL1-β in OA chondrocytes and suppresses the IL-1β-induced synthesis of matrix-degrading enzymes by inhibiting IL-1β signaling at multiple levels. It suggests that the IL-1β-inducible SOCS1 acts as a negative regulator of the IL-1β response in OA cartilage.

Interleukin-1 Beta Promotes the Expression of Prox-1 and Vascular Endothelial Growth Factor Receptor Type-3 in Adult Human Dermal Fibroblasts

Abstract 4738 Background and Aims:We recently reported that human interleukin 1 beta (IL-1 beta) stimulates bone-marrow stromal myofibroblasts to express a hematopoietic stem cell marker, CD34 (16th EHA, and 53rdASH meeting). To characterize precisely the effects of IL-1 beta to the stromal cells, adult human dermal fibroblasts (HDF) were cultured with IL-1 beta, and the morphological changes and molecular expressions were observed. Materials and Methods:HDF were purchased, and cultured in knockout DMEM medium with 15% KSR with or without recombinant human IL-1 beta for 3 weeks. The morphological changes and the expression of specific hematopoiesis-related genes were analyzed time-dependently. The concentration of hematopoietic growth factors in the culturing supernatants was also measured. Results:When HDF were cultured with human IL-1 beta for 3 weeks, the cellular morphology changed to the filamentous appearance. RT-PCR analyses revealed that cells expressed Prox-1, a master molecule for lymphatic duct neogenesis, and also vascular endothelial growth factor receptor (VEGFR) type-3. Smad7 and PAI1 were also expressed; however, LYVE1, a marker for lymphatic vascular endothelial cell, was not induced. VEGF-C production was also demonstrated at RNA and protein levels, and VEGF-C and VEGFR type-3 system played an important role in morphological changes and cell-proliferation. Discussion:We previously reported that when HDF were cultured in DMEM/F12 medium with IL-1 beta and EPO, hematopoietic markers were induced. When HDF were cultured in knockout DMEM, a condition for immature cell-growth at ES cell levels, and IL-1 beta, lymphatic duct neogenesis-related genes were activated, and HDF converted their morphology without an introduction of any genes externally. We are now making clear the precise action of IL-1 beta to HDF using microarray analysis. Disclosures:No relevant conflicts of interest to declare.

Abstract B25: The ETS transcription factor ESE1 promotes activation of the NFKB pathway in prostate tumors

Abstract ETS transcription factors are important elements in the pathogenesis of prostate cancer. Here, we report that the ETS factor ESE1, which is expressed in normal prostate epithelial cells, is frequently upregulated in prostate tumors and functionally linked to activation of the NFkB pathway and inflammation. Immunohistochemistry and QRT-PCR in normal prostate and primary prostate tumor samples showed increased expression of ESE1 in about 35% of tumors. Over-expression of ESE1 was observed both in the presence and absence of ERG over-expression, indicating that activation of ESE1 could have an independent role in a subset of prostate tumors. Furthermore, elevated levels of ESE1 were significantly associated with higher rates of biochemical relapses in patients with primary tumors treated with radical prostatectomy (Log Rank (Mantel-Cox)=0.018). Overexpression of ESE1 in prostate cancer cells, like LNCaP and 22RV1, promoted a more aggressive phenotype with increased growth in soft-agar, resistance to anoikis and cell migration. When injected in nude mice, ESE1 over-expressing prostate cancer cells exhibited greater ability to form lung metastasis compared to control cells. ESE1 affected transcription of many genes and ChIP showed binding of ESE1 to ETS binding sites in the promoter of key genes involved in cell adhesion, metastasis and inflammation. Relevantly, we observed that mRNA and protein level of the NFkB subunit NFKB1 (p50) was increased in stable ESE1 expressing cells. Analysis of NFKB1 promoter revealed the presence of a previously unknown ETS binding site and ChIP demonstrated binding of ESE1 to this site, consistent with transcriptional activation of the gene by ESE1. Immunofluorescence microscopy showed a marked intranuclear localization of the NFkB p50/p65 complex, indicating that ESE1 promoted also NFkB activation. Consistently, the activity of the NFkB-responsive luciferase reporter was increased in ESE1 expressing cells compared to control cells. Furthermore, we found that the pro-inflammatory cytokine IL-1beta increased ESE1 mRNA and protein level in LNCaP cells while promoting intranuclear localization of the NFkB complex. This was associated with the induction of anoikis resistance and cell migration. All these effects were rescued by siRNA mediated knockdown of ESE1 indicating that ESE1 had a key role in mediating the effects of IL-1beta in these cells. We propose that the production of pro-inflammatory cytokines like IL-1beta within the tumor microenvironment results in the upregulation of ESE1 and establishment of a feed-forward loop leading to the constitutive activation of the NFkB linked to the ability of ESE1 to induce p50 transcription and NFkB nuclear translocation. These data show an important oncogenic role of ESE1 in prostate tumors and reveal a previously unidentified link between this ETS factor and the NFkB pathway. Citation Format: Nicole Longoni, Maurizia Mello-Grand, Manuela Sarti, Sandra Pinton, Cecilia Dallavalle, Giovanna Chiorino, Carlo V. Catapano, Giuseppina M. Carbone. The ETS transcription factor ESE1 promotes activation of the NFKB pathway in prostate tumors [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B25.

Oncostatin M synergistically induces CXCL10 and ICAM‐1 expression in IL‐1β‐stimulated‐human gingival fibroblasts

Periodontitis is a chronic bacterial infection of tooth-supporting structures. T-helper type 1 (Th1) cells are related to the exacerbation of periodontal disease. Human gingival fibroblasts (HGFs), the major cell type in periodontal connective tissues, are involved in immunological response in periodontal tissues. However, it is uncertain whether HGFs are related to Th1 response. Chemokine (C-X-C motif) ligand 10 (CXCL10) is a cytokine, that is related to Th1 cells migration. Intercellular adhesion molecule (ICAM)-1 is involved in Th1 cells retention and activation in inflamed tissue. The aim of this study is to examine the effect of oncostatin M (OSM) on CXCL10 and ICAM-1 expression in HGFs. OSM stimulation induced CXCL10 and ICAM-1 expression in HGFs. Moreover, the synergistic effects of CXCL10 release and ICAM-1 expression in HGFs were observed with combined stimulation of interleukin (IL)-1beta and OSM. OSM increased type 1 IL-1 receptor (IL-1R1) expression, and IL-1beta enhanced OSMRbeta expression on HGFs. IL-1beta + OSM stimulation enhanced the phosphorylation of inhibitor of nuclear factor kappaB (IkappaB)-alpha, signal transducer and activator of transcription (STAT)3, c-Jun N terminal kinase (JNK), and protein kinase B (Akt) compared to OSM or IL-1beta stimulation. CXCL10 production from OSM + IL-1beta stimulated HGFs was suppressed by nuclear factor (NF)-kappaB, STAT3, JNK, and phosphoinositide-3-kinase (PI3K) inhibitors. On the other hand, only NF-kappaB and STAT3 inhibitors suppressed ICAM-1 expression enhanced by OSM + IL-1beta treatment. These effects of OSM and IL-1beta may promote Th1 cells infiltration and retention in periodontally diseased tissues and be related to exacerbation of periodontal disease.

Differential effects of p38MAP kinase inhibitors on the expression of inflammation‐associated genes in primary, interleukin‐1β‐stimulated human chondrocytes

A main challenge in the therapy of osteoarthritis (OA) is the development of drugs that will modify the disease. Reliable test systems are necessary to enable an efficient screening of therapeutic substances. We therefore established a chondrocyte-based in vitro cell culture model in order to characterize different p38MAPK inhibitors. Interleukin-1beta (IL-1beta)-stimulated human OA chondrocytes were treated with the p38MAPK inhibitors Birb 796, pamapimod, SB203580 and the new substance CBS-3868. Birb 796- and SB203580-treated cells were analysed in a genome-wide microarray analysis. The efficacy of all inhibitors was characterized by quantitative gene expression analysis and the quantification of PGE(2) and NO release. Microarray analysis revealed inhibitor-specific differences in gene expression. Whereas SB203580 had a broad effect on chondrocytes, Birb 796 counteracted the IL-1beta effect more specifically. All p38MAPK inhibitors significantly inhibited the IL-1beta-induced gene expression of COX-2, mPGES1, iNOS, matrix metalloproteinase 13 (MMP13) and TNFRSF11B, as well as PGE(2) release. Birb 796 and CBS-3868 showed a higher efficacy than SB203580 and pamapimod at inhibiting the expression of COX-2 and MMP13 genes, as well as PGE(2) release. In the case of mPGES1 and TNFRSF11B gene expression, CBS-3868 exceeded the efficacy of Birb 796. Our test system could differentially characterize inhibitors of the same primary pharmaceutical target. It reflects processes relevant in OA and is based on chondrocytes that are mainly responsible for cartilage degradation. It therefore represents a valuable tool for drug screening in between functional in vitro testing and in vivo models.

Genome-wide Profiling of Interleukin-4 and STAT6 Transcription Factor Regulation of Human Th2 Cell Programming

Dissecting the molecular mechanisms by which T helper (Th) cells differentiate to effector Th2 cells is important for understanding the pathogenesis of immune-mediated diseases, such as asthma and allergy. Because the STAT6 transcription factor is an upstream mediator required for interleukin-4 (IL-4)-induced Th2 cell differentiation, its targets include genes important for this process. Using primary human CD4(+) T cells, and by blocking STAT6 with RNAi, we identified a number of direct and indirect targets of STAT6 with ChIP sequencing. The integration of these data sets with detailed kinetics of IL-4-driven transcriptional changes showed that STAT6 was predominantly needed for the activation of transcription leading to the Th2 cell phenotype. This integrated genome-wide data on IL-4- and STAT6-mediated transcription provide a unique resource for studies on Th cell differentiation and, in particular, for designing interventions of human Th2 cell responses.

Inhibition Effect of IL-1β on Calcium Channels Currents in Cultured Cortical Neurons of Rat

Interleukin-1beta (IL-1beta) is an important proinflammatory cytokine that exert wide biological functions in the central nervous system (CNS). Voltage-gated Ca2+ channels in plasma membrane are essential components of Ca2+ signal whose change on pathological conditions is closely related to processes of diseases. Although both IL-1beta and Ca2+ channels have key roles in injury and disease of the brain, so far their relations have been reported in few studies. The effects of IL-1beta on Ca2+ channels in cultured cortical neurons of rats were investigated using patch-clamp recording in present research. Our results showed that both 10 and 50 ng/mL of IL-1beta treatment inhibit the Ca2+ currents in time and dose dependent manner, without the change of activation properties of voltage-gated Ca2+ channels (VGCC)s.

The Inorganic Pyrophosphate Transporter ANK Preserves the Differentiated Phenotype of Articular Chondrocyte

The differentiated phenotype of chondrocyte is lost in pathological situations and after interleukin (IL)-1beta challenge. Wnt proteins and the inorganic pyrophosphate (PP(i)) transporter Ank regulate the differentiation process in many cell types. We investigated the possible contribution of Ank and/or PP(i) to the maintenance of the differentiated chondrocyte phenotype with special care to Wnt signaling. Primary articular chondrocytes lost their phenotype upon IL-1beta challenge, with cessation of type II collagen and Sox-9 expression. Ank expression and PP(i) transport were strongly reduced by IL-1beta, whereas Wnt-5a was the only Wnt protein increased. Transient overexpression of Ank counteracted most of IL-1beta effects on Type II collagen, Sox-9, and Wnt-5a expression. When resting chondrocytes were transfected with a siRNA against Ank, this reproduced the phenotype induced by IL-1beta. In both cases, no markers for hypertrophic chondrocytes were detected. The conditioned supernatant from chondrocytes knocked-down for Ank contained Wnt-5a, which activated Tcf/Lef reporter plasmids and promoted translocation of beta-catenin into the nucleus without activating the c-Jun N-terminal kinase (JNK) pathway. Supplementation with PP(i) compensated for most effects of Ank deficiency on Type II collagen, Sox-9, and Wnt-5 expression, both in IL-1beta and Ank knock-down conditions. Phenotype changes induced by IL-1beta were also supported by activation of the JNK pathway, but this latter was not sensitive to PP(i) supplementation. Altogether our data demonstrate that the transport of PP(i) by ANK contributed to the maintenance of the differentiated phenotype of chondrocyte by controlling the canonical Wnt pathway in a Wnt-5a-dependent manner.

Pigment epithelium derived factor as an anti-inflammatory factor against decrease of glutamine synthetase expression in retinal Müller cells under high glucose conditions

The pathogenesis of diabetic retinopathy (DR) is similar to that of a chronic inflammatory disease. A predominant function of Müller cells is to regulate glutamate levels, but in DR the function is compromised. The present study was performed to investigate the role of pigment epithelial derived factor (PEDF) on the expression of glutamine synthetase (GS) in rat retinal Müller cells under high glucose conditions, and to study the possible mechanism for PEDF against decrease of GS expression in retinal Müller cells under high glucose conditions. The role of PEDF on the expressions of GS and interleukin-1beta (IL-1beta) in retinal Müller cells under normal and high glucose conditions was measured by western blotting, real-time RT-PCR, or immunocytochemistry. In order to confirm the effect of PEDF on GS against the role of IL-1beta, the PEDF siRNA method was used. High glucose increased the expression of IL-1beta, but decreased the expressions of GS and PEDF in retinal Müller cells. PEDF increased the expression of GS and decreased the expression of IL-1beta in retinal Müller cells under high glucose conditions. The effect of IL-1beta on expression of GS was inhibited by PEDF. Moreover, down-regulation of PEDF expression by siRNA resulted in significantly increasing the expression of IL-1beta, but decreasing the expression of GS in retinal Müller cells. PEDF increases expression of GS against the effect of IL-1beta in retinal Müller cells under high glucose conditions. These findings suggested that PEDF may act as an anti-inflammatory factor against decrease of GS expression in retinal Müller cells in diabetic retinopathy.

Cytokine‐dependent balance of mitogenic effects in primary human lung fibroblasts related to cyclic AMP signaling and phosphodiesterase 4 inhibition

Interleukin-1beta (IL-1beta) and basic fibroblast growth factor (bFGF) are important regulators of proliferation, and their expression is increased in lungs of patients with asthma, idiopathic pulmonary fibrosis (IPF), or chronic obstructive pulmonary disease (COPD). We investigated the effect of IL-1beta and bFGF on proliferation of human lung fibroblasts and the role of COX-2, PGE(2), and cAMP in this process. Furthermore, the effect of phosphodiesterase (PDE) 3 and 4 inhibition was analyzed. In primary human lung fibroblasts low concentrations of IL-1beta (<10 pg/ml) potentiated the bFGF-induced DNA synthesis, whereas higher concentrations revealed antiproliferative effects. Higher concentrations of IL-1beta-induced COX-2 mRNA and protein associated with an increase in PGE(2) and cAMP, and all of these parameters were potentiated by bFGF. The PDE4 inhibitor piclamilast concentration-dependently reduced proliferation by a partial G1 arrest. The PDE3 inhibitor motapizone was inactive by itself but enhanced the effect of the PDE4 inhibitor. This study demonstrates that bFGF and IL-1beta act in concert to fine-tune lung fibroblast proliferation resulting in amplification or reduction. The antiproliferative effect of IL-1beta is likely attributed to the induction of COX-2, which is further potentiated by bFGF, and the subsequent generation of PGE(2) and cAMP. Inhibition of PDE4 inhibition (rather than PDE3) may diminish proliferation of human lung fibroblasts and therefore could be useful in the therapy of pathological remodeling in lung diseases.

Molecular Mechanism of Proinflammatory Cytokine-Mediated Squamous Metaplasia in Human Corneal Epithelial Cells

The cornified envelope protein small proline-rich protein 1B (SPRR1B) is a biomarker for squamous metaplasia. Proinflammatory cytokines IL-1beta and IFN-gamma are potent inducers of ocular surface keratinization and SPRR1B expression. Here the molecular mechanisms controlling SPRR1B gene expression in response to IL-1beta and IFN-gamma are elucidated. A 3-kb fragment of the SPRR1B gene 5'-flanking region was amplified from human chromosome 1, sequentially deleted, and cloned into a luciferase vector. Constructs were transiently transfected into human corneal epithelial cells, and activity was assessed in response to IL-1beta, IFN-gamma, or basal medium. Functional cis-elements responding to IL-1beta and IFN-gamma were characterized by site-directed mutagenesis and gel mobility shift assay. Effects of mitogen-activated protein kinases p38, ERK, and JNK were assessed using inhibitors and dominant-negative mutants. Results were validated by real-time RT-PCR. The first 620 bp of the SPRR1B 5'-flanking region regulated constitutive expression and increased promoter activity in response to IL-1beta and IFN-gamma. Corresponding cis-elements for IL-1beta and IFN-gamma were bound by cAMP response element binding protein (CREB) and zinc-finger E-box binding homeobox 1 (ZEB1), respectively. Inhibition of p38 abolished the stimulatory effects of IL-1beta and IFN-gamma on SPRR1B, whereas inhibition of JNK and ERK had no effect. Dominant-negative mutants targeting p38alpha and p38beta2 blocked cytokine-induced SPRR1B promoter activity and mRNA expression. SPRR1B is upregulated by the proinflammatory cytokines IL-1beta and IFN-gamma via p38 MAPK-mediated signaling pathways that lead to the activation of transcription factors CREB and ZEB1, respectively. These results identify key intracellular signaling intermediates involved in the pathogenesis of immune-mediated ocular surface squamous metaplasia.

IL-1β modulate the Ca2+-activated big-conductance K channels (BK) via reactive oxygen species in cultured rat aorta smooth muscle cells

The large conductance Ca(2+)-activated K(+) (BK) channel, abundantly expressed in vascular smooth muscle cells, plays a critical role in controlling vascular tone. Activation of BK channels leads to membrane hyperpolarization and promotes vasorelaxation. BK channels are activated either by elevation of the intracellular Ca(2+) concentration or by membrane depolarization. It is also regulated by a diversity of vasodilators and vasoconstrictors. Interleukin-1beta (IL-1beta) is one of the cytokines that play important roles in the development and progression of a variety of cardiovascular diseases. The effects of IL-1beta on vascular reactivity are controversial, and little is known about the modulation of BK channel function by IL-1beta. In this study, we investigated how IL-1beta modulates BK channel function in cultured arterial smooth muscle cells (ASMCs), and examined the role of H(2)O(2) in the process. We demonstrated that IL-1beta had biphasic effects on BK channel function and membrane potential of ASMCs, that is both concentration and time dependent. IL-1beta increased BK channel-dependent K(+) current and hyperpolarized ASMCs when applied for 30 min. While long-term (24-48 h) treatment of IL-1beta resulted in decreased expression of alpha-subunit of BK channel, suppressed BK channel activity, decreased BK channel-dependent K(+) current and depolarization of the cells. H(2)O(2) scavenger catalase completely abolished the early effect of IL-1beta, while it only partly diminished the long-term effect of IL-1beta. These results may provide important molecular mechanisms for therapeutic strategies targeting BK channel in inflammation-related diseases.

ERK1/2 Activation Induced by Inflammatory Cytokines Compromises Effective Host Tissue Integration of Engineered Cartilage

Proinflammatory cytokines are known to provoke degradative signaling cascades that promote extracellular matrix disintegration in articular cartilage. Because integration of the repair tissue into the surrounding native cartilage to produce a mechanically stable interface has a profound impact on the viability and functionality of the restored joint surface, this study examined the effects of proinflammatory cytokines on the properties of tissue-engineered cartilage in the context of integration. Using an established in vitro cartilage defect model, we examined the integration of chondrocyte-laden agarose constructs into native articular cartilage and the biochemical and biomechanical alterations of these implants upon treatment with interleukin 1-beta (IL1-beta) and tumor necrosis factor-alpha (TNF-alpha). Additionally, we probed extracellular regulated kinase (ERK) signaling involvement in response to proinflammatory cytokines. The time-dependent accumulation of extracellular matrix and concomitant increase in Young's modulus observed in the absence of cytokines was significantly decreased upon IL1-beta and TNF-alpha treatment. Push-out test showed the highest interface strength in hybrid cultures maintained without cytokines, which was significantly lowered with IL1-beta and TNF-alpha treatment. Histological characteristics of the interface region are consistent with the biochemical findings. Treatment with an inhibitor of ERK pathway antagonized the deleterious effects caused by both cytokines. This study is the first to show the functional catastrophic effects of IL1-beta and TNF-alpha on the biochemical, structural, and integrative properties of tissue-engineered cartilage and their significant counteraction by the blockade of ERK signaling pathway. With the discovery of new potential chemical entities, ERK inhibitor may emerge as a new therapeutic approach for functional integration and mechanical integrity of an engineered cartilage to the host tissue and, therefore, enhance long-term viability and functionality of the restored joint surface.