Menstrual cycle-specific inhibition of endometrial stromal cell proliferation by oncostatin M.
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We have investigated the possible roles of oncostatin M (OSM), which is a member of the interleukin-6 family of cytokines, in endometrial and endometriotic stromal cell growth. Endometrial and endometriotic stromal cells were collected from the uterus or ovarian chocolate cysts. We observed the expression of mRNA transcripts for OSM, OSM receptor subunit beta, leukaemia inhibitory factor receptor subunit (LIFR), and glycoprotein 130 in endometrial and endometriotic stromal cells. We also examined the effects of OSM (0-50 ng/ml) and LIF (0-10 ng/ml) on endometrial and endometriotic stromal cell proliferation and evaluated the effects of OSM on endometrial stromal cell differentiation. The presence of 10-50 ng/ml OSM significantly suppressed endometrial stromal cell growth in secretory phase tissue but not in proliferative phase tissue. In contrast, stromal cells in endometriotic tissues were resistant to the inhibitory effects of OSM. Addition of LIF did not influence the growth of endometrial stromal cells. We also showed that 10 ng/ml OSM stimulated markers of differentiation causing increased prolactin secretion and cyclooxygenase-2 gene expression in endometrial stromal cells from the secretory phase. These results suggest that OSM may play a pivotal role in regulating the growth and differentiation of endometrial cells. Endometriotic cells may behave differently from normal endometrial cells in terms of the inhibitory response to OSM. (+info)
Synergistic effects of glycoprotein 130 binding cytokines in combination with interleukin-1 on cartilage collagen breakdown.
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OBJECTIVE: To determine whether other glycoprotein 130 (gp130) binding cytokines can mimic the effects of oncostatin M (OSM) in acting synergistically with interleukin-1alpha (IL-1alpha) to induce cartilage collagen breakdown and collagenase expression, and to determine which receptors mediate these effects. METHODS: The release of collagen and proteoglycan was assessed in bovine and human cartilage explant cultures. Messenger RNA (mRNA) and protein production from immortalized human chondrocytes (T/C28a4) was analyzed by Northern blotting and specific enzyme-linked immunosorbent assays. Collagenase activity was measured by bioassay. Cell surface receptors were detected by flow cytometry. RESULTS: OSM in combination with IL-1alpha caused a rapid synergistic induction of matrix metalloproteinase 1 mRNA, which was sustained over a 72-hour period. Flow cytometric analyses detected both the OSM-specific receptor and the gp130 receptor at the chondrocyte cell surface, but failed to detect the leukemia inhibitory factor receptor (LIFR). Cartilage degradation assays revealed that, of the gp130 binding cytokines, only OSM and IL-6, in the presence of its soluble receptor (sIL-6R), were able to act synergistically with IL-1alpha to promote collagen release. CONCLUSION: This study demonstrates that IL-6 can mimic OSM in synergizing with IL-1alpha to induce chondrocyte-mediated cartilage collagen breakdown and collagenase production. In order to have this effect, IL-6 requires the presence of its soluble receptor. The apparent absence of LIFR explains why other gp130 binding cytokines do not act in synergy with IL-1alpha. Since OSM, IL-6, and sIL-6R levels have all been shown to be elevated in the rheumatoid joint, our findings suggest that these cytokines may be key mediators of cartilage collagen catabolism in the inflammatory arthritides. (+info)
Exclusive neutrophil recruitment with oncostatin M in a human system.
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Oncostatin M (OSM), a member of the IL-6 family has been postulated to be a potent recruiter of leukocytes, however information regarding the molecular mechanism(s) underlying this event is extremely limited. Therefore, the aim of this study was to investigate the role of OSM-mediated leukocyte recruitment in a human system in vitro under flow conditions. A parallel-plate flow chamber assay was used to examine leukocyte recruitment from whole blood by human umbilical vein endothelium treated for 24 hours with OSM. OSM in a dose-response manner revealed very significant leukocyte rolling and adhesion reaching optimal levels at a very low concentration of OSM (10 ng/ml). The OSM-induced leukocyte rolling and adhesion was comparable to levels seen with tumor necrosis factor. OSM was extremely selective for neutrophil recruitment (96%) with <3% lymphocyte recruitment. By contrast, tumor necrosis factor-alpha revealed no such selectivity, recruiting 70% neutrophils and at least 25% lymphocytes and detectable levels of eosinophils at 24 hours. The molecular mechanism underlying the leukocyte recruitment seemed to be entirely dependent on P-selectin as leukocyte recruitment could be completely blocked by the addition of a P-selectin-blocking antibody. An elevation in both P-selectin message and protein was observed with 24 hours of OSM stimulation of endothelium. By contrast, E-selectin and VCAM-1 were not detectable after OSM stimulation. Similar results were seen with passaged dermal microvascular endothelium that does not have a prestored pool of P-selectin. Based on these results, we conclude that OSM may be a very selective potent recruiter of neutrophils in more prolonged inflammatory conditions, an event exclusively dependent on P-selectin. (+info)
Activation of procollagenases is a key control point in cartilage collagen degradation: interaction of serine and metalloproteinase pathways.
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OBJECTIVE: Bovine and human cartilages in explant culture respond to proinflammatory cytokines with the up-regulation of procollagenases. In stimulated bovine nasal cartilage (BNC), >90% of collagen is released by day 14 of culture, but collagen release is rarely seen before day 7. The aim of this study was to investigate if activation of procollagenases is a rate-limiting step in cartilage collagen breakdown. METHODS: BNC and human articular cartilage explants were cultured with interleukin-1alpha (IL-1alpha) and/or oncostatin M (OSM) with or without test reagents. Collagen levels were determined by assay of hydroxyproline. Collagenase activity was measured using the diffuse fibril assay. RESULTS: The addition of procollagenase activators, matrix metalloproteinase 3 (MMP-3), and APMA to IL-1alpha/OSM-stimulated BNC resulted in early release of collagen. The release with APMA was completely blocked by the addition of tissue inhibitor of metalloproteinases 1. This shows that procollagenases are present early in the culture period, but cartilage collagen breakdown does not happen until activation occurs. The addition of plasminogen to IL-1alpha/OSM-stimulated cartilage produced early collagen release in bovine and a significant increase in human cartilage. Thus, plasminogen activators (PAs) are present and convert plasminogen to plasmin, a known activator of several MMPs, including collagenases. Addition of alpha1-proteinase inhibitor or a urokinase-type PA inhibitor, 7-amino-4-chloro-3-(3-isothiureidopropoxy) isocoumarin, partially blocked the breakdown of collagen from IL-1alpha/OSM-treated bovine cartilage. This suggests that serine proteinases are involved in the activation cascades of procollagenases that result in cartilage collagen breakdown. CONCLUSION: The activation of procollagenases is a key control point in cartilage collagen breakdown, and serine proteinase pathways activate MMPs. (+info)
Oncostatin M regulates the synthesis and turnover of gp130, leukemia inhibitory factor receptor alpha, and oncostatin M receptor beta by distinct mechanisms.
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The cytokine receptor subunits gp130, leukemia inhibitory factor receptor alpha (LIFRalpha), and oncostatin M receptor beta (OSMRbeta) transduce OSM signals that regulate gene expression and cell proliferation. After ligand binding and activation of the Janus protein-tyrosine kinase/STAT and mitogen-activated protein kinase signal transduction pathways, negative feedback processes are recruited. These processes attenuate receptor action by suppression of cytokine signaling and by down-regulation of receptor protein expression. This study demonstrates that in human fibroblasts or epithelial cells, OSM first decreases the level of gp130, LIFRalpha, and OSMRbeta by ligand-induced receptor degradation and then increases the level of the receptors by enhanced synthesis. The transcriptional induction of gp130 gene by OSM involves STAT3. Various cell lines expressing receptor subunits to the different interleukin-6 class cytokines revealed that only LIFRalpha degradation is promoted by activated ERK and that degradation of gp130, OSMRbeta, and a fraction of LIFRalpha involves mechanisms that are separate from signal transduction. These mechanisms include ligand-mediated dimerization, internalization, and endosomal/lysosomal degradation. Proteosomal degradation appears to involve a fraction of receptor subunit proteins that are ubiquitinated independently of ligand binding. (+info)
Interstitial collagens I, III, and VI sequester and modulate the multifunctional cytokine oncostatin M.
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The binding of certain growth factors and cytokines to components of the extracellular matrix can regulate their local availability and modulate their biological activities. We show that oncostatin M (OSM), a profibrogenic cytokine and modulator of cancer cell proliferation, specifically binds to collagen types I, III, IV, and VI, immobilized on polystyrene or nitrocellulose. Single collagen chains inhibit these interactions in a dose-dependent manner. Cross-inhibition experiments of collagen-derived peptides point to a limited set of OSM-binding collagenous consensus sequences. Furthermore, this interaction is found for OSM but not for other interleukin-6 type cytokines. OSM binding to collagens is saturable, with dissociation constants around 10(-8) m and estimated molar ratios of 1-3 molecules of OSM bound to one molecule of triple helical collagen. Furthermore, collagen-bound OSM is biologically active and able to inhibit proliferation of A375 melanoma cells. We conclude that abundant interstitial collagens dictate the spatial pattern of bioavailable OSM. This interaction could be exploited for devising collagenous peptide-antagonists that modulate OSM bioactivity in tumor growth and fibrotic disorders like rheumatoid arthritis and hepatic fibrosis. (+info)
Activation of signal transducer and activator of transcription 1 (STAT1) is not sufficient for the induction of STAT1-dependent genes in endothelial cells. Comparison of interferon-gamma and oncostatin M.
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We compared human endothelial cell (EC) responses to interferon-gamma (IFN gamma) and oncostatin M (OnM), cytokines that utilize Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling. Both cytokines cause phosphorylation of Tyr residue 701 and Ser residue 727 of STAT1, as shown by immunoblotting. Both activate DNA binding of STAT1 homodimers, shown by electrophoretic mobility shift assay. However, only IFN gamma increases expression of three STAT1-dependent gene products examined, namely transporter associated with antigen processing-1 (TAP1), interferon regulatory factor-1 (IRF1), and class I major histocompatibility complex (MHC) protein, as demonstrated by immunoblotting. Only IFN gamma increases TAP1 transcription assessed by reporter gene assay. OnM pretreatment or co-treatment does not inhibit IFN gamma responses. Interestingly, IFN gamma activation of STAT1 is considerably more long-lived than that produced by OnM. To determine whether duration is functionally significant, we transduced EC with a chimeric receptor containing extracellular domains of platelet-derived growth factor receptor beta and intracellular regions of gp130, the signaling subunit of the OnM receptor, mutated to prevent binding of the tyrosine phosphatase SHP-2. Addition of platelet-derived growth factor to such transduced cells produces STAT1 activation that is comparable in magnitude and duration to that caused by IFN gamma, but still fails to induce TAP1, IRF1, or class I MHC molecules. OnM also activates STAT1 but not transcription of STAT1-dependent genes in HepG2 cells. Transient transfection of HepG2 cells with a STAT-defective mouse IFN gamma receptor failed to complement the OnM STAT signal. We conclude that STAT1 activation is necessary but not sufficient for induction of transcription of IFN gamma-responsive genes. However, signals provided by IFN gamma other than STAT1 activation cannot be provided in trans to complement the response to OnM. (+info)
The critical role of the PE21 element in oncostatin M-mediated transcriptional repression of the p53 tumor suppressor gene in breast cancer cells.
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Cytokine oncostatin M (OM) exerts growth-inhibitory and differentiative effects on breast cancer cells. Previously we showed that the transcription from the p53 gene in breast cancer cells was down regulated by OM. To elucidate the molecular mechanisms underlying the OM effect on p53 transcription, in this study, we dissected the p53 promoter region and analysed the p53 promoter activity in breast tumor cells. We showed that treatment of MCF-7 cells with OM induced a dose- and time-dependent suppression of p53 promoter activity. The p53 promoter activity was decreased to 35% of control at 24 h and further decreased to 20% at 48 h by OM at concentrations of 5 ng/ml and higher. Deletion of the 5'-flanking region of the p53 promoter from -426 to -97 did not affect the OM effect. However, further deletion to -40 completely abolished the repressive effect of OM. The p53 promoter region -96 to -41 contains NF-kappaB and c-myc binding sites, and a newly identified UV-inducible element PE21. Mutations to disrupt NF-kappaB binding or c-myc binding to the p53 promoter decreased the basal promoter activity without affecting the OM-mediated suppression, whereas mutation at the PE21 motif totally abolished the OM effect. We further demonstrated that insertion of PE21 element upstream of the thymidine kinase minimal promoter generated an OM response analogous to that of the p53 promoter. Finally, we detected the specific binding of a nuclear protein with a molecular mass of 87 kDa to the PE21 motif. Taken together, we demonstrate that OM inhibits the transcription of the p53 gene through the PE21 element. Thus, the PE21 element is functionally involved in p53 transcription regulated by UV-induction and OM suppression. (+info)