Influence of macrophage products on the release of plasminogen activator, collagenase, beta-glucuronidase and prostaglandin E2 by articular chondrocytes.
ABSTRACT: We describe the effects of products of mononuclear phagocytes on the secretory activity of chondrocytes. The primary confluent cultures of rabbit articular chondrocytes were exposed to standard medium alone or enriched with conditioned medium obtained from cultures of rabbit peritoneal macrophages, the mouse macrophage cell line P388D1 or human blood mononuclear cells. Four markers of release were assessed, the neutral proteinases plasminogen activator and collagenase, the acid hydrolase beta-glucuronidase and prostaglandin E2, and the kinetics of their changes were monitored. Chondrocytes that were cultured in standard medium secreted large amounts of plasminogen activator, some beta-glucuronidase, but no collagenase, and released only minor amounts of prostaglandin E2. The addition of conditioned medium from rabbit macrophages induced a rapid release of large quantities of prostaglandin E2 and an abundant secretion of collagenase, while abolishing or strongly decreasing plasminogen activator secretion. In addition, beta-glucuronidase secretion was markedly enhanced. The decrease in secretion of plasminogen activator appeared to reflect a diminished production, since no evidence was found for the generation of inhibitors or for an accelerated extracellular breakdown of the enzyme. Conditioned media of the mouse and human mononuclear cells influenced the secretory activities of rabbit articular chondrocytes in a similar way, suggesting that the factor (or factors) acting on chondrocytes is produced by a variety of macrophages, and that its action is not species-restricted. The time course and concentration-dependence of the effects observed indicate that the secretion of plasminogen activator and collagenase are influenced in a strictly reciprocal fashion by the macrophage products. The release of prostaglandin E2 paralleled that of collagenase.
Project description:Homogeneous catabolin from pig leucocytes induced proteoglycan breakdown, but not collagen breakdown, in explants of articular cartilage. It augmented lectin-induced proliferation of mouse thymocytes, stimulated production of prostaglandin E2 and collagenase by fibroblasts and chondrocytes, and increased Ca2+ release from mouse calvarial explants, all at concentrations down to 50 pM. In view of these effects it was concluded that pig catabolin is a form of interleukin 1.
Project description:Although an inflammatory microenvironment is required for successful implantation, an inflammatory overreaction is one of the causes of unexplained recurrent pregnancy losses (uRPL). Prostaglandin E2 (PGE2) plays a pivotal role in regulating immune balance during early pregnancy, and it can stimulate inflammatory reactions via prostaglandin E2 receptor 3 (EP3). However, the role of PGE2 receptor signaling in the uRPL remains unknown. We aimed to investigate whether EP3 signaling is involved in the mechanism of uRPL. Via immunohistochemistry we could show that the expression of cyclooxygenase-2, EP3 and G protein alpha inhibitor 1 (Gi1) was enhanced in the decidua of the uRPL group in comparison to the control group in first-trimester placentas. In vitro, we demonstrated that sulprostone (an EP1/EP3 agonist) inhibited the secretion of beta-hCG and progesterone in JEG-3 cells and the secretion of beta-hCG in HTR-8/SVneo cells while it induced the expression of plasminogen activator inhibitor type 1 in JEG-3 cells. In addition, PGE2/sulprostone was able to stimulate the expression of Gi1, phosphorylated-extracellular signal-regulated kinases 1/2 (p-ERK1/2) and p53. L-798,106 (an EP3-specific antagonist) suppressed the expression of EP3 and p-ERK1/2 without affecting the secretion of beta-hCG. Elevated activation of EP3 signaling in first-trimester placentas plays an important role in regulating the inflammatory microenvironment, the hormone secretion of extravillous trophoblasts and the remodeling of extracellular matrix in the fetal-maternal interface. L-798,106 might be a 'potential therapeutic candidate' for the treatment of uRPL.
Project description:Androgen dependent induction of the ETS related gene (ERG) expression in more than half of all prostate cancers results from gene fusions involving regulatory sequence of androgen regulated genes (i.e. TMPRSS2, SLC45A3 and NDRG1) and protein coding sequence of the ERG. Emerging studies in experimental models underscore the functions of ERG in prostate tumorigenesis. However, biological and biochemical functions of ERG in prostate cancer (CaP) remain to be elucidated. This study suggests that ERG activation plays a role in prostaglandin signaling because knockdown of ERG expression in TMPRSS2-ERG fusion containing CaP cells leads to altered levels of the 15-hydroxy-prostaglandin dehydrogenase (HPGD), a tumor suppressor and prostaglandin catabolizing enzyme, and prostaglandin E2 (PGE2) . We demonstrate that HPGD expression is regulated by the binding of the ERG protein to the core promoter of this gene. Moreover, prostaglandin E2 dependent cell growth and urokinase-type plasminogen activator (uPA) expression are also affected by ERG knockdown. Together, these data imply that the ERG oncoprotein in CaP cells positively influence prostaglandin mediated signaling, which may contribute to tumor progression.
Project description:Substance P (SP) is found in increased concentrations in inflamed joints and is believed to play a role in joint pathology. Culture of bovine articular chondrocytes with SP or with the related mammalian tachykinins neurokinin A or B (NKA or NKB) produced no effect on prostaglandin E2 (PGE2) or collagenase production. However, the C-terminal fragment of SP, SP-(7-11), increased PGE2 and collagenase production at concentrations greater than 1 microM. The N-terminal fragments SP-(1-4) and SP-(1-6) had no effect on PGE2 or collagenase production. In addition, SP-(7-11), but not intact SP, SP-(1-4), SP-(1-6), SP-(8-11) or SP-(9-11), nor the tachykinins NKA and NKB, caused an increase in the intracellular calcium concentration as measured by the fluorescent dye Fura-2. The maximal change in intra-cellular calcium induced by 10 microM SP-(7-11) was 140 +/- 30 nM. We postulate that cleavage of SP by neutral endopeptidases which are present in the synovial fluid and which yield SP-(7-11) may be of biological importance in chondrocyte-mediated cartilage pathology.
Project description:1. Explants of rabbit skin and synovium in tissue culture secreted a specific collagenase into their culture media. Primary cultures of fibroblast-like cells, which were obtained from these tissues and maintained in culture for up to 14 subculture passages, also secreted high activities of a specific collagenase into serum-free culture medium. Secretion of enzyme activity from the cell monolayer was at constant rate for over 100h and continued for up to 8 days in serum-free culture medium. The enzymic activity released was proportional to the number of cells in the monolayer. 2. The fibroblast collagenase was maximally active between pH7 and 8. At 24 degrees C the collagenase decreased the viscosity of collagen in solution by 60%. The collagen molecule was cleaved into three-quarters and one-quarter length fragments as demonstrated by electron microscopy of segment-long-spacing crystallites (measured as native collagen molecules aligned with N-termini together along the long axis), and by polyacrylamide-gel electrophoresis of the denatured products. The collagenase hydrolysed insoluble collagen, reconstituted collagen fibrils and gelatin, but had no effect on haemoglobin or Pz-Pro-Leu-Gly-Pro-d-Arg (where Pz=4-phenylazobenzyloxycarbonyl). 3. The fibroblast collagenase was partially purified by gel filtration and the molecular weight was estimated as 38000. The activity of the partially purified enzyme was stimulated by 4-chloromercuribenzoate, inhibited by EDTA, cysteine, 1,10-phenanthroline and serum, but was unaffected by di-isopropyl phosphorofluoridate, Tos-LysCH(2)Cl and pepstatin. 4. Long-term cell cultures originating from rabbit skin or synovium from rabbits with experimentally induced arthritis also secreted specific collagenase. Human fibroblasts released only very small amounts of collagenase.
Project description:Collagenase-3 (matrix metalloprotease-13) is a recently discovered human collagenase produced in normal articular cartilage chondrocytes and thought to be involved in the pathological process of osteoarthritis. We have sequenced and characterized 1.6 kb of the human collagenase-3 gene 5'-flanking region. The transcription start site was located 22 bp upstream from the ATG start codon. Sequence analysis of the 5'-flanking region revealed the presence of the consensus recognition sites for the TATA and CCAAT DNA-binding proteins, activator protein-1 and E26 transformation specific/polyoma virus enhancer, as well as three core motifs of hormone response elements. Transient transfection assays demonstrated that a small fragment of 133 bp, containing the activator protein-1 and E26 transformation specific/polyoma virus enhancer sites promoted transcription in normal and osteoarthritic human chondrocytes with significantly higher activity than the original 1.6 kb fragment. Nucleotide sequence comparison of the promoter region of human collagenase-3 revealed a stronger similarity to the mouse collagenase-1 promoter than to the human collagenase-1 promoter.
Project description:Adenylate cyclase [ATP pyrophosphate lyase (cyclizing), EC 18.104.22.168] was shown to be present in cultured human articular chondrocytes. Optimal conditions of incubation time, protein and substrate concentrations and pH were determined in whole cell lysates. Maximal activity occurred at pH 8.5 with no decrease in activity up to pH 10.0. Adenylate cyclase activity of particulate membrane preparations was enhanced by the addition of crude cytosol preparations. The prostaglandins E1, E2, F1 alpha, F2 alpha, D2, B1, B2, A1 and A2, as well as adrenaline and isoprenaline, stimulated adenylate cyclase derived from either adult or foetal chondrocytes. No significant stimulation was observed in the presence of human calcitonin or glucagon. Bovine parathyroid hormone always significantly stimulated the adenylate cyclase derived from foetal chondrocytes, but not from adult chondrocytes. Preincubation of the chondrocytes in culture with indomethacin and with or without supernatant medium from cultured mononuclear cells increased the responsiveness of the adenylate cyclase to prostaglandin E1.
Project description:1. The immunological cross-reactivity between rabbit collagenases from a variety of normal and pathological sources was examined. The specific antibody raised against collagenase secreted from normal rabbit synovial fibroblasts gave reactions of complete identity with collagenases secreted from fibroblasts derived from rabbit skin, and from synovium from experimentally arthritic rabbits. 2. The rabbit fibroblast collagenase was immunologically identical with collagenases obtained from the organ culture medium of normal rabbit skin, synovium, ear fibrocartilage and subchondral bone. 3. Collagenases from the culture media of normal rabbit synovium and from hyperplastic synovium of rabbits made experimentally arthritic were identical. 4. The collagenase secreted from rabbit fibroblasts gave a reaction completely identical with that of a collagenase extracted directly from a rabbit carcinoma. 5. IgG (immunoglobulin G) from a specific antiserum to rabbit fibroblast collagenase was a potent inhibitor of the collagenases obtained from the culture media of the various rabbit cells and tissues. 6. Collagenases from human synovium and from mouse macrophages and bone were neither precipitated nor inhibited by antibodies to rabbit collagenase. 7. No immunoreactive material was found in lysates of rabbit polymorphonuclear leucocyte granules with the specific antisera to rabbit fibroblast collagenase. No evidence for inactive forms of rabbit collagenase in lysates of the rabbit synovial fibroblasts could be found, either by double immunodiffusion against the specific collagenase, or by displacement of active enzyme from inhibition by the IgG.
Project description:Mesenchymal stem cells (MSCs) administered for therapeutic purposes can be activated by interferon-? (IFN-?) secreted from natural killer cells in injured tissues and exert anti-inflammatory effects. These processes require a substantial period of time, leading to a delayed onset of MSCs' therapeutic effects. In this study, we investigated whether pretreatment with IFN-? could potentiate the anti-fibrotic ability of MSCs in rats with ischemia-reperfusion injury (IRI) and unilateral ureter obstruction. Administration of MSCs treated with IFN-? strongly reduced infiltration of inflammatory cells and ameliorated interstitial fibrosis compared with control MSCs without IFN-? treatment. In addition, conditioned medium obtained from IFN-?-treated MSCs decreased fibrotic changes in cultured cells induced by transforming growth factor-?1 more efficiently than that from control MSCs. Most notably, secretion of prostaglandin E2 from MSCs was significantly increased by treatment with IFN-?. Increased prostaglandin E2 in conditioned medium obtained from IFN-?-treated MSCs induced polarization of immunosuppressive CD163 and CD206-positive macrophages. In addition, knockdown of prostaglandin E synthase weakened the anti-fibrotic effects of MSCs treated with IFN-? in IRI rats, suggesting the involvement of prostaglandin E2 in the beneficial effects of IFN-?. Administration of MSCs treated with IFN-? might represent a promising therapy to prevent the progression of renal fibrosis.
Project description:OBJECTIVE: To determine whether 17beta-oestradiol (E2) modulates interleukin (IL) 1beta-induced proteoglycan degradation in chondrocytes, and to analyse the part played by metalloproteinases (MMPs) in this process. METHODS: Primary cultured rabbit articular chondrocytes were prepared and treated with 10 ng/ml IL1beta combined or not with 0.1-10 nM E2. Neosynthesised proteoglycans (PGs) were evaluated after incorporation of [(35)SO(4)]sulphate and further analysed after chromatography on a Sepharose 2B column. Chondrocyte mRNA levels of aggrecan, MMP-1, -3, -13, and tissue inhibitor of metalloproteinase-1 (TIMP-1) were studied by northern blot. MMP-1 activity was measured by zymography. MMP-1 gene transcription was studied by transient transfection of chondrocytes with an MMP-1-luciferase construct. RESULTS: E2 modulated the IL1beta-induced total sulphated PGs in rabbit articular chondrocytes, which decreased as the E2 concentration was increased. At a low concentration (0.1 nmol/l) E2 counteracts the IL1beta-induced decrease in sulphated PG, while at high concentration (10 nmol/l) E2 enhances the IL1beta effects. A biphasic E2 effect was also observed on IL1beta-induced disaggregation of PG, 53-58 kDa gelatinolytic activity, and MMP-1, -3, and -13 mRNA levels. In contrast, E2 did not modify the level of aggrecan mRNA and had no effect on TIMP-1 mRNA expression. Finally, simultaneous addition of IL1beta and E2 (0.1-10 nmol/l) did not modify IL1beta-induced MMP-1-luciferase activity, suggesting that E2 effects probably occur at the post-transcriptional level of MMP gene expression. CONCLUSION: Oestrogen concentration may have an inverse effect on IL1beta stimulated proteoglycan degradation and MMP production by chondrocytes.