MIC-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF-beta superfamily.
ABSTRACT: Macrophages play a key role in both normal and pathological processes involving immune and inflammatory responses, to a large extent through their capacity to secrete a wide range of biologically active molecules. To identify some of these as yet not characterized molecules, we have used a subtraction cloning approach designed to identify genes expressed in association with macrophage activation. One of these genes, designated macrophage inhibitory cytokine 1 (MIC-1), encodes a protein that bears the structural characteristics of a transforming growth factor beta (TGF-beta) superfamily cytokine. Although it belongs to this superfamily, it has no strong homology to existing families, indicating that it is a divergent member that may represent the first of a new family within this grouping. Expression of MIC-1 mRNA in monocytoid cells is up-regulated by a variety of stimuli associated with activation, including interleukin 1beta, tumor necrosis factor alpha (TNF-alpha), interleukin 2, and macrophage colony-stimulating factor but not interferon gamma, or lipopolysaccharide (LPS). Its expression is also increased by TGF-beta. Expression of MIC-1 in CHO cells results in the proteolytic cleavage of the propeptide and secretion of a cysteine-rich dimeric protein of Mr 25 kDa. Purified recombinant MIC-1 is able to inhibit lipopolysaccharide -induced macrophage TNF-alpha production, suggesting that MIC-1 acts in macrophages as an autocrine regulatory molecule. Its production in response to secreted proinflammatory cytokines and TGF-beta may serve to limit the later phases of macrophage activation.
Project description:Macrophage inhibitory cytokine-1 (MIC-1), also known as prostate-derived factor (PDF), is a molecule of the TGF-β superfamily and has been associated with the progression of various types of diseases including prostate cancer. Initially identified from activated macrophages, the MIC-1 gene may provide a potential link between inflammation and prostate cancer. In this context, we performed MIC-1 expression analysis using mouse prostate tissues to determine whether there was any correlation with age and inflammation. Reverse transcription PCR analysis on RNA samples isolated from prostate lobes from prostate-specific antigen transgenic mice of varying ages revealed that MIC-1 gene expression is extremely low to non-detectable in the prostate tissues obtained from young mice, while its expression increases in the prostate tissues harvested from elderly mice. Increased MIC-1 gene expression in the mouse prostate was found to be associated with an increased level of infiltrating lymphocytes. To confirm this observation, we showed that inflammation-associated cytokines (IL-1β and TNF-α) significantly upregulate the secretion of the MIC-1 protein in a human prostate cancer cell line (LNCaP cells), while cytokines IL-6 and granulocyte macrophage colony-stimulating factor were less effective. Taken together, these data indicated that inflammation-associated cytokines may play a critical role in the functional regulation of the MIC-1 gene in the early stages of prostate cancer development. More studies are required to understand the biological activity of MIC-1 gene regulation in the development and progression of prostate cancer.
Project description:Macrophage inhibitory cytokine-1 (MIC-1) gene is a member of transforming growth factor-beta superfamily and was reported to be highly overexpressed in human prostate cancer using microarray technology. The aim of this study was to evaluate the quantitative expression of MIC-1 in malignant and benign prostate tissues and to associate expression levels with clinicopathological parameters of prostate cancer. Matched (paired) prostatic tissue samples from the cancerous and noncancerous parts of the same prostates were obtained from 66 patients who underwent radical prostatectomy. Quantitative RT-PCR was performed using SYBR Green I on the Roche LightCycler system. Macrophage inhibitory cytokine-1 gene overexpression in cancerous tissues was observed in 88% of cases, compared to noncancerous tissues (P<0.001). The expression level of MIC-1 in cancerous tissues was significantly higher than in noncancerous tissue (P<0.001). Higher expression of MIC-1 gene was significantly associated with higher Gleason score (P=0.004). The expression of the MIC-1 gene in prostate cancer is significantly higher than in noncancerous tissues, especially in more aggressive forms of the disease (Gleason score>5). This is in contrast to prostate-specific antigen that is downregulated in higher-grade tumours. The upregulation of MIC-1 in prostate cancer and in advanced and more aggressive prostatic tumours suggests that MIC-1 protein should be evaluated as a potential diagnostic and prognostic biomarker.
Project description:Tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta(1) (TGF-beta(1)) are peptides with multiple biological activities that influence neoplastic, immunologic and fibroproliferative diseases. There are clear interrelationships and overlap between the actions of TNF-alpha and TGF-beta(1) in lung fibrosis; therefore, we postulated that TNF-alpha may play a significant role in regulating TGF-beta(1) expression in lungs. We recently reported that TNF-alpha activates the extracellular regulated kinase (ERK)-specific pathway in fibroblasts resulting in stabilization of TGF-beta(1) mRNA and increased expression of TGF-beta(1). In the current study, we further investigated the molecular mechanisms involved in TNF-alpha regulation of TGF-beta(1) expression. Nuclear run-on assays showed that treatment of Swiss 3T3 fibroblasts with TNF-alpha increased transcription of the TGF-beta(1) gene in an ERK independent manner. Pre-treatment with the activator protein-1 (AP-1) inhibitor curcumin attenuated TNF-alpha induced transcription of the TGF-beta(1) gene. TNF-alpha induced increased levels of c-Jun and C-Fos in the nucleus accompanied by phosphorylation of c-Jun. In electrophoretic mobility shift assays, AP-1 binding to an AP-1 binding site found within the TGF-beta(1) promoter was increased in nuclear extracts from Swiss 3T3 fibroblasts treated with TNF-alpha. Together, these results suggest that TNF-alpha induces expression and DNA binding of AP-1 resulting in increased transcription of the TGF-beta(1) gene. It is essential to know which transcription pathways are activated because of the wide distribution of TNF-alpha and TGF-beta(1), the general lack of effective treatments for fibroproliferative disease and the possibility that targeting the correct transcription factors could be palliative.
Project description:Alpha-melanocyte stimulating hormone (α-MSH) is involved in normal skin wound healing and also has anti-inflammatory properties. The association of α-MSH to polyelectrolyte layers with various supports has been shown to improve these anti-inflammatory properties. This study aimed to evaluate the effects of nanofibrous membrane functionalized with α-MSH linked to polyelectrolyte layers on gingival cell inflammatory response. Human oral epithelial cells (EC) and fibroblasts (FB) were cultured on plastic or electrospun Poly-#-caprolactone (PCL) membranes with α-MSH covalently coupled to Poly-L-glutamic acid (PGA-α-MSH), for 6 to 24 h. Cells were incubated with or without Porphyromonas gingivalis lipopolysaccharide (Pg-LPS). Cell proliferation and migration were determined using AlamarBlue test and scratch assay. Expression of interleukin-6 (IL-6), tumor necrosis factor (TNF-α), and transforming growth factor-beta (TGF-β) was evaluated using RT-qPCR method. Cell cultures on plastic showed that PGA-α-MSH reduced EC and FB migration and decreased IL-6 and TGF-β expression in Pg-LPS stimulated EC. PGA-α-MSH functionalized PCL membranes reduced proliferation of Pg-LPS stimulated EC and FB. A significant decrease of IL-6, TNF-α, and TGF-β expression was also observed in Pg-LPS stimulated EC and FB. This study showed that the functionalization of nanofibrous PCL membranes efficiently amplified the anti-inflammatory effect of PGA-α-MSH on gingival cells.
Project description:Transcriptional profiling of human umbilical vein endothelial cells following stimulation with tumour necrosis factor alpha and transforming growth factor beta singly or combined for 8 hr All stimulations were for 8 hr - TNF-α vs no cytokine; TGF-β1 vs no cytokine; TNF-α & TGF-β1 vs TNF-α alone; TNF-α & TGF-β1 vs TGF-β1 alone
Project description:Using circulating inflammatory markers and magnetic resonance imaging (MRI), recent studies have associated inflammation with brain volumetric measures. Macrophage Inhibitory Cytokine-1 (MIC-1/GDF15) is a divergent transforming growth factor - beta (TGF-?) superfamily cytokine. To uncover the underlying mechanisms of the previous finding of a negative association between MIC-1/GDF15 serum levels and cognition, the present study aimed to examine the relationship of circulating MIC-1/GDF15 levels with human brain gray matter (GM) volumes, in a community-dwelling sample aged 70-90 years over two years (Wave 1: n = 506, Wave 2: n = 327), of which the age-related brain atrophy had been previously well defined. T1-weighted MRI scans were obtained at both waves and analyzed using the FMRIB Software Library and FreeSurfer. The results showed significantly negative associations between MIC-1/GDF15 serum levels and both subcortical and cortical GM volumes. GM volumes of the whole brain, cortex, temporal lobe, thalamus and accumbens showed significant mediating effects on the associations between MIC-1/GDF15 serum levels and global cognition scores. Increases in MIC-1/GDF15 serum levels were associated with decreases in cortical and subcortical GM volume over two years. In conclusion, MIC-1/GDF15 serum levels were inversely associated with GM volumes both cross-sectionally and longitudinally.
Project description:Tumor necrosis factor-alpha (TNF) is an inflammatory cytokine that induces context-dependent proliferation, survival, and apoptosis responses in hepatocytes. TNF stimulates and enhances growth factor-mediated hepatocyte proliferation and survival following partial hepatectomy, but also acts in concert with other inflammatory cytokines of the innate immune response during viral infection to induce apoptosis in hepatocytes. In other epithelial cell types, TNF has recently been shown to stimulate autocrine release of transforming growth factor-alpha (TGF-alpha) and interleukin-1 (IL-1) family ligands. Here, we examine the role of these autocrine ligands in modulating TNF-induced proliferation and apoptosis in primary hepatocytes. We show that TNF-induced hepatocyte proliferation is regulated by an inducible, coupled, and self-antagonizing autocrine cascade involving the pro-proliferative TGF-alpha and IL-1 receptor antagonist (IL-1ra) ligands and antiproliferative IL-1alpha/beta ligands. Moreover, cooperative stimulation of hepatocyte proliferation by combined TNF and TGF-alpha treatment is self-limited through antiproliferative autocrine IL-1alpha/beta feedback. We show that TNF potently induces apoptosis of adenovirus-infected hepatocytes in a manner similarly determined through the integrated activity of a coupled TGF-alpha-IL-1alpha/beta-IL-1ra autocrine cascade. Exogenous TGF-alpha can either enhance or diminish apoptosis in adenoviral vector-treated and TNF-treated hepatocytes, in a biphasic relationship also mediated by autocrine IL-1alpha/beta feedback.We demonstrate that TNF-induced hepatocyte proliferation and apoptosis are both governed by a self-antagonizing TGF-alpha-IL-1alpha/beta-IL-1ra autocrine cascade in vitro, and thus identify multiple molecular targets for control of TNF-regulated hepatocyte phenotypic responses related to liver regeneration and adenoviral gene therapy.
Project description:The normal structure and function of articular cartilage are the result of a precisely balanced interaction between anabolic and catabolic processes. The transforming growth factor-beta (TGF-beta) family of growth factors generally exerts an anabolic or repair response; in contrast, proinflammatory cytokines such as interleukin 1 beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) exert a strong catabolic effect. Recent evidence has shown that IL-1beta, and TNF-alpha, and the TGF-beta signaling pathways share an antagonistic relationship. The aim of this study was to determine whether the modulation of the response of articular chondrocytes to TGF-beta by IL-1beta or TNF-alpha signaling pathways occurs through regulation of activity and availability of mothers against DPP (Drosophila) human homologue (Smad) proteins.Human articular chondrocytes isolated from knee joints from patients with osteoarthritis (OA) or normal bovine chondrocytes were cultured in suspension in poly-(2-hydroxyethyl methacrylate)-coated dishes with either 10% fetal bovine serum media or serum-deprived media 6h before treatment with IL-1beta alone, TNF-alpha alone or IL-1beta followed by TGF-beta. Nuclear extracts were examined by electrophoretic mobility-shift assays (EMSA) for nuclear factor-kappa B (NF-kappaB) and Smad3/4 deoxyribonucleic acid (DNA) binding. Nuclear extracts were also subjected to the TranSignal Protein/DNA array (Panomics, Redwood City, CA) enabling the simultaneous semiquantitative assessment of DNA-binding activity of 54 different transcription factors. Nuclear phospho-Smad2/3 and total Smad7 protein expression in whole cell lysates were studied by Western blot. Cytoplasmic Smad7, type II collagen alpha 1 (COL2A1), aggrecan and SRY-related high mobility group-Box gene 9 (SOX-9) mRNA expression were measured by real-time polymerase chain reaction (PCR).The DNA-binding activity of Smad3/4 in the TranSignal Protein/DNA array was downregulated by TNF-alpha (46%) or IL-1beta treatment (42%). EMSA analysis showed a consistent reduction in Smad3/4 DNA-binding activity in human articular chondrocytes treated with IL-1beta or TNF-alpha. TGF-beta-induced Smad3/4 DNA-binding activity and Smad2/3 phosphorylation were also reduced following pretreatment with IL-1beta in human OA and bovine chondrocytes. Real-time PCR and Western blot analysis showed that IL-1beta partially reversed the TGF-beta stimulation of Smad7 mRNA and protein levels in TGF-beta-treated human OA cells. In contrast, TGF-beta-stimulated COL2A1, aggrecan, and SOX-9 mRNA levels were abrogated by IL-1beta.IL-1beta or TNF-alpha exerted a suppressive effect on Smad3/4 DNA-binding activity in human articular chondrocytes, as well as on TGF-beta-induced stimulation of Smad3/4 DNA-binding activity and Smad2/3 phosphorylation in human OA and bovine articular chondrocytes. IL-1beta partially reversed the increase in TGF-beta-stimulated Smad7 mRNA or protein levels suggesting that Smad7 may not be involved in the suppression of TGF-beta signaling induced by IL-1beta or TNF-alpha in articular chondrocytes. The balance between the IL-1beta or TNF-alpha and the TGF-beta signaling pathways is crucial for maintenance of articular cartilage homeostasis and its disruption likely plays a substantial role in the pathogenesis of OA.
Project description:To identify the cytokines secreted by mesenchymal-like cancer cells that activate macrophages, the cytokine profiles of conditioned media from MCF7, MCF7 induced to undergo EMT by treatment of TGF-β, TNF-α and prolonged mammosphere culture, and MDA-MB-231 cells were analyzed by RayBio® Human Cytokine Antibody Array V. 5 samples. There are 5 groups: MCF7, MCF7 induced to undergo EMT by treatment of TGF-β (TGF-β-MCF7), TNF-α (TNF-α-MCF7), prolonged mammosphere culture (MCF7M), and MDA-MB-231 cells
Project description:Treatment of rat glomerular mesangial cells with transforming growth factor beta 2 (TGF beta 2) stimulates prostaglandin E2 (PGE2) synthesis. Actinomycin D, cycloheximide and diclofenac attenuate the TGF beta 2-induced PGE2 formation. As shown previously, two proinflammatory cytokines, interleukin 1 beta (IL-1 beta) and tumour necrosis factor alpha (TNF alpha), are potent stimuli for PGE2 and phospholipase A2 secretion from mesangial cells. We report here that, whereas TGF beta 2 potentiates the IL-1 beta- and TNF alpha-evoked PGE2 production, it strongly inhibits the phospholipase A2 secretion induced by both cytokines. In addition, the inhibitory effect of TGF beta 2 on phospholipase A2 secretion is not due to the augmented PGE2 formation.