H3-cerebroside sulfate re-distribution induced by cation, opiate or phosphatidyl serine

Rabbit articular cartilage synthesizes type II collagen, comprised of alpha1(II) chains, in vivo or in vitro. Chondrocytes from the same tissue have the ability to produce type I chollagen with the chain composition 2alpha(I)-alpha2 or type II collagen, depending upon the culture systems in which they are maintained. Type I collagen and its precursors have been identified in the medium of monolayer cultures. Upon transfer from monolayer to suspension cultures, the cells synthesize type II collagen in the medium devoid of CaCl2 and type I collagen and its precursors in the complete medium.     

Association of proteoglycan degradation with catabolic cytokine and stromelysin release from cartilage cultured with fibronectin fragments

Addition of fibronectin fragments to bovine articular cartilage explant cultures results in enhanced release of metalloproteinases and rapid cartilage proteoglycan (PG) degradation and loss. The chondrolysis begins with rapid PG degradation which markedly slows after 1 week. Preliminary observations suggest that catabolic cytokines mediate chondrolytic activities of the fibronectin fragments. The objectives of this work were to investigate the correlations between: (a) release of specific cytokines; (b) release of the metalloproteinase (MMP), stromelysin-1 (MMP-3); (c) release of the tissue inhibitor of MMPs, TIMP-1, and; (d) degradation and release of PG from cultured cartilage. We report that human articular cartilage cultured with an amino-terminal 29-kDa fragment (Fn-f) at 0.1 microM, released enhanced levels of TNF-alpha, IL-1beta, and IL-1alpha with peaks at Days 2, 3, and 9, respectively. MMP-3 release was elevated with a peak at Day 6 and a profile similar to that for the Fn-f-induced cartilage PG depletion. IL-6 release was enhanced within 2 days and continued at the same level throughout the culture period but this did not lead to enhanced release of TIMP-1, a known activity of IL-6. These data suggest that in the early chondrolytic events induced in cultured cartilage by Fn-f, enhanced MMP-3 release and maximal degradation and release of PG from cultured cartilage are kinetically associated with elevated release of the catabolic cytokines, TNF-alpha, IL-1beta, and IL-1alpha. Further, a later period of slowing PG loss and slowing MMP-3 release is associated with greatly slowed release of these cytokines, but prolonged release of IL-6. This model of cartilage damage may be useful for studies of the interplay between cytokines and the effects of combinations of cytokines on cartilage homeostasis.     

The role of oncostatin M in animal and human connective tissue collagen turnover and its localization within the rheumatoid joint

OBJECTIVE: To study the interaction of interleukin-1alpha (IL-1alpha) and oncostatin M (OSM) in promoting cartilage collagen destruction. METHODS: Bovine, porcine, and human cartilage and human chondrocytes were studied in culture. The levels of collagenase (matrix metalloproteinase 1 [MMP-1]) and tissue inhibitor of metalloproteinases 1 (TIMP-1) were measured by bioassay and enzyme-linked immunosorbent assay (ELISA). The levels of OSM in rheumatoid synovial fluid were measured by ELISA. RESULTS: When combined with OSM, IL-1alpha, IL-1beta, and tumor necrosis factor alpha released proteoglycan and collagen from cartilage. OSM was the only member of the IL-6 family to have this effect. Human tendon also responded to IL-1alpha and OSM. OSM increased the production of MMP-1 and TIMP-1 but when combined with IL-1alpha, synergistically promoted MMP-1 production in human chondrocytes and synovial fibroblasts. High levels of OSM were found in human rheumatoid synovial fluids, and confocal microscopy showed that OSM was produced by macrophages in rheumatoid synovial tissue. CONCLUSION: These results highlight an important new mechanism by which there is irreversible loss of collagen from cartilage.     

Stimulus specificity of matrix metalloproteinase dependence of human T cell migration through a model basement membrane

Chemotaxis of human T lymphoblastoma cells of the Tsup-1 line, which migrate similarly to blood T cells, through a layer of basement membrane-like Matrigel on a polycarbonate micropore filter was evoked by vasoactive intestinal peptide (VIP; concentration for a maximal response, 10(-7)M), IL-2 (10(-9)M), and the chemokines RANTES (10(-10)M) and macrophage inflammatory protein-1 alpha (10(-10)M). Chemotactic concentrations of each factor increased Tsup-1 cell secretion of matrix metalloproteinase-9 (MMP-9), with significant responses by 4 h for VIP, IL-2, and IL-4, but only after 24 h for macrophage inflammatory protein-1 alpha and RANTES, as quantified by Western blots and zymography. 3H-Labeled type IV human collagen incorporated in the Matrigel layer was degraded by migrating Tsup-1 cells, as assessed by release of radioactive fragments of the collagen. The in situ degradation of type IV collagen in Matrigel by migrating Tsup-1 cells was enhanced most significantly by VIP, IL-2, and IL-4 after 4 h at concentrations that increased the secretion of MMP-9 optimally, but only after 24 h by macrophage inflammatory protein-1 alpha and RANTES. The specific MMP inhibitor GM6001 suppressed Tsup-1 cell MMP activity evoked by all stimuli, as determined by zymography and in situ degradation of 3H-Labeled type IV human collagen. The chemotactic migration of Tsup-1 cells through Matrigel, but not through a filter alone, in response to optimal concentrations of VIP, IL-2, and IL-4, but not the chemokines, was inhibited by GM6001, with a concentration dependence similar to that for suppression of MMP activity. Thus elicitation of T cell chemotactic migration through a model basement membrane by stimuli that increase MMP activity early in the response depends on degradation of matrix proteins by MMP, whereas stimuli that recruit MMP late may rely on early activation of other proteases.     

A chemically modified tetracycline inhibits streptozotocin-induced diabetic depression of skin collagen synthesis and steady-state type I procollagen mRNA

Wasting of connective tissues including skin, bone, and cartilage have been closely associated with elevated matrix metalloproteinase (MMP) activity and depressed collagen content in the streptozotocin (STZ)-induced diabetic rat, while tetracyclines have been reported to normalize total body weight, skin hydroxyproline and collagen content in this model, in part through inhibition of MMPs. In the present study, we report the effect of CMT-1, a chemically modified tetracycline that lacks antimicrobial properties but retains divalent cation binding and MMP inhibitory activity, on diabetic skin collagen synthesis and steady-state levels of procollagen alpha 1(I) mRNA. Male, 4-month old Sprague-Dawley rats received a single injection of 75 mg/kg STZ or citrate vehicle alone and diabetic status was confirmed by positive glucosuria. Some diabetic animals received 10 mg/day of CMT-1 by oral gavage and, 28 days after STZ treatment, body weight, blood glucose values and the in vivo rates of skin collagen production were measured using the pool-expansion technique. Steady-state levels of procollagen alpha 1(I) mRNA were analyzed 21 days after STZ treatment by hybridization of total RNA with a 32P labelled cDNA to rat type I procollagen alpha 1(I) mRNA in a dot-blot assay. STZ treatment was found to significantly depress body weight, skin collagen hydroxyproline content, the in vivo rate of collagen production, and hybridizable levels of type I procollagen alpha 1(I) mRNA. CMT-1 administered daily to STZ-treated rats inhibited the diabetic depression of these parameters but had little or no effect on non-diabetic controls or on STZ-induced hyperglycemia. Thus, in addition to the inhibition of MMP mediated extracellular collagen degradation, these results suggest CMT-1 also acts to inhibit diabetic connective tissue breakdown in STZ-induced diabetes by increasing both steady-state levels of type I procollagen mRNA and collagen synthesis through mechanism(s) that are independent of the antibacterial properties of tetracyclines.     

Clinical and experimental studies of JPYS in reducing side-effects of chemotherapy in late-stage gastric cancer

Matrix metalloproteinases (MMPs) are a group of enzymes thought to be responsible for both normal connective-tissue-matrix remodelling and the accelerated breakdown associated with tumor development. These MMPs and tissue inhibitor of MMPs (TIMP1) could be expressed by either the cancer or the stromal cells. Expression of mRNAs encoding interstitial collagenase (MMP1), 72-kD type IV collagenase (MMP2) and stromelysin (MMP3), which are probably involved in tumor invasion and metastasis, and of TIMP1 were studied in human mammary pathology by in situ hybridization and Northern blot analysis. Out of 6 benign lesions, 2 expressed MMP2 mRNAs. mRNAs encoding MMP1 and MMP3 were detectable in occasional stromal and tumor cells in 2 out of 17 carcinomas. Thirteen out of 17 cancers expressed MMP2 mRNA throughout the tumor in stromal cells close to noninvasive tumor clusters and well-differentiated invasive cancer cells. TIMP1 mRNA expression was detected in noninvasive and well-differentiated invasive tumor cells. These data suggest that there is a cooperation between tumor and stromal cells, in particular for the production of 72-kD type IV collagenase, involved in the disruption of basement membranes. A lack of TIMP1 expression from invasive cancer cells would also contribute to matrix destruction.     

Batimastat (BB-94) inhibits matrix metalloproteinases of equine laminitis

A method for culturing explants of lamellar hoof was developed to investigate the process of lamellar separation that occurs in laminitis. Explants, consisting of hoof wall, dermal and epidermal lamellae and the adjacent sub-lamellar connective tissue remained intact when cultured in tissue culture medium for 2 days. However, when cultured in the presence of the matrix metalloproteinase (MMP) activator aminophenylmercuric acetate (APMA), the lamellae separated when tension was applied by pulling the hoof wall in an opposite direction to the connective tissue. The separation occurred between the epidermal basal cells and the basement membrane therefore mimicking the lesion of laminitis. Electrophoresis of culture medium from control hoof explants into gradient polyacrylamide gels co-polymerised with gelatin revealed that the explants had produced 2 gelatinases of molecular weight 92 and 72 kDa corresponding to EqMMP-9 and EqMMP-2 respectively. Minor bands of lower molecular weight were the active forms of these enzymes. The zymograms of culture medium from APMA treated explants revealed an increase in the amount of active MMPs. Equine polymorphs cultured for 2 days produced only EqMMP-9. Lamellar explant medium from horses with acute laminitis contained increased amounts of zymogen and active EqMMP-2 and EqMMP-9 particularly in explants from the fore hooves. Zymography of homogenates of normal lamellar hoof tissue revealed only EqMMP-2 and a minor active band. However, homogenates of lamellar tissue from horses with laminitis showed that EqMMP-9 was present as well as increased EqMMP-2 in both zymogen and active forms. Addition of the MMP inhibitor batimastat (BB-94) to the culture medium of APMA treated explants prevented lamellar separation. BB-94 incubated with polyacrylamide strips containing the MMPs from laminitis affected lamellar explants inhibited enzymatic activity at a concentration of 1 mmol/l. It is concluded that activation of MMPs may be responsible for the lamellar separation seen in laminitis and that MMP inhibitors may be useful clinically for preventing this process.     

Different collagenase gene products have different roles in degradation of type I collagen

Vertebrate collagenases, matrix metalloproteinases (MMPs), cleave type I collagen at a single helical locus. We show here that rodent interstitial collagenases (MMP-13), but not human fibroblast collagenase (MMP-1), cleave type I collagen at an additional aminotelopeptide locus. Collagenase cDNAs and chimeric constructs in pET-3d, juxtaposing MMP-13 sequences amino-terminal to the active site in the catalytic domain and MMP-1 sequences carboxyl-terminal and vice versa, were expressed in Escherichia coli. Assays utilized collagen from wild type (+/+) mice or mice that carry a targeted mutation (r/r) that encodes substitutions in alpha1(I) chains that prevent collagenase cleavage at the helical locus. MMP-13 and chimeric molecules that contained the MMP-13 sequences amino-terminal to the active site cleaved (+/+) collagen at the helical locus and cleaved cross-linked (r/r) collagen in the aminotelopeptide (beta components converted to alpha chains). Human MMP-1 and chimeric MMP-1/MMP-13 with MMP-1 sequences amino-terminal to the active site cleaved collagen at the helical locus but not in the aminotelopeptide. All activities were inhibited by TIMP-1, 1,10-phenanthroline, and EDTA. Sequences in the distal two-thirds of the catalytic domain determine the aminotelopeptide-degrading capacity of MMP-13.     

Synergistic action of transforming growth factor-beta and insulin-like growth factor-I induces expression of type II collagen and aggrecan genes in adult human articular chondrocytes

Reexpression of aggrecan and type II collagen genes in dedifferentiated adult human articular chondrocytes (AHAC) in suspension culture varied widely depending on the specific lot of bovine serum used to supplement the culture medium. Some lots of serum provided strong induction of aggrecan and type II collagen expression by AHAC while others did not stimulate significant production of these hyaline cartilage extracellular matrix molecules even following several weeks in culture. Addition of 50 ng/ml insulin-like growth factor-I (IGF-I) to a deficient serum lot significantly enhanced its ability to induce aggrecan and type II collagen mRNA. Given this observation, IGF-I and other growth factors were tested in defined serum-free media for their effects on the expression of these genes. Neither IGF-I nor insulin nor transforming growth factor beta (TGF-beta) alone stimulated induction of aggrecan or type II collagen production by dedifferentiated AHAC. However, TGF-beta 1 or TGF-beta 2 combined with IGF-I or insulin provided a strong induction as demonstrated by RNase protection and immunohistochemical assays. Interestingly, type I collagen, previously shown to be downregulated in serum supplemented suspension cultures of articular chondrocytes, persisted for up to 12 weeks in AHAC cultured in defined medium supplemented with TGF-beta and IGF-I.     

Effect of the tripeptide glycyl-L-histidyl-L-lysine on the proliferation and synthetic activity of chick embryo chondrocytes

Under certain conditions chondrocytes form lattices with cartilage collagens, which may serve as cartilage implants. It is necessary to find the optimal conditions for culturing chondrocytes. Three different supports are compared: (a) plastic; (b) cartilage collagens; and (c) insoluble skin collagen solubilized under denaturing conditions (ISC-40). The effect of culture medium supplementation with the tripeptide (Gly-His-Lys)2.Cu.2H2O.2NaCl (GHK) on chondrocyte proliferation and synthetic activity is studied, with particular attention paid to collagen types I, II and III. The collagen supports stimulated chondrocyte proliferation, but on the ISC-40 support they started to dedifferentiate rather early. In the primary culture, chondrocytes on all three supports synthesized mainly collagen type II, and only small amounts of types I and III. In the first passage the synthesis of these two collagen types increased, relative to collagen type II, at least on the cartilage collagen support. Supplementation of culture medium with GHK stimulated chondrocyte proliferation in the primary structure mostly on the ISC-40 support. On the other two types of supports the stimulatory effect of GHK was expressed mostly in the first passages. The collagen synthetic rate was increased by GHK on both of the collagen supports; on the cartilage collagen support collagen type II was synthesized predominantly and on the ISC-40 support types I and III were mostly formed. It is suggested that supplementation of culture medium with GHK may be useful in the preparation of cartilage implants.     

Cytogenetic analysis of sister chromosome sets in the second polar body and in pronuclei of unicellular mouse embryos. I. Frequency and origin of aneuploidy in embryos heterozygous for the reciprocal chromosome translocation T[14;15]6Ca]

Matrix metalloproteinases have been implicated to play a vital role in glioma invasion as they degrade extracellular matrix to facilitate the subsequent migration of tumor cells into the surrounding brain tissue. The cytokine Interleukin-10 (IL-10) was detected recently in glial tumors in vivo. Expression of specific IL-10 mRNA as well as blood serum levels of IL-10 in glioma patients increased with malignancy suggesting a functional role of IL-10 in glioma progression. Moreover, glioma cell migration in vitro was enhanced in the presence of IL-10. We therefore investigated the expression of the matrix metalloproteinases (MMPs) stromelysin-1 (MMP-3), 72-kDa collagenase (MMP-2), 92-kDa collagenase (MMP-9), matrilysin (MMP-7) and the human macrophage metalloelastase (MMP-12). In addition, a possible relation between exposure of glioma cells to IL-10 and invasiveness of these cells due to MMP expression was analyzed. Experiments with Matrigel coated Boyden chambers revealed a pronounced dose dependent effect of IL-10 on glioma invasiveness. The synthetic MMP-inhibitor Marimastat markedly reduced cell invasion in the Boyden chambers confirming the significance of MMPs in the process of invasion. Subsequently, the expression level of MMPs and the serine protease uPA was investigated in 7 glioma cell lines (U373, GaMG, U251, GHE, SNB19, U138 and D54) by RT-PCR. In all but one cell line no enhancement of MMP expression by IL-10 was detected. Matrilysin in U373 cells was the only protease found to be upregulated in the presence of IL-10 dependent on cell density. The present data suggest that IL-10 related effects on the invasive properties of the cell lines are not directly mediated by an upregulation of matrix metalloproteinase expression.     

Type II collagen pro-alpha-chains containing a Gly574Ser mutation are not incorporated into the cartilage matrix of transgenic mice

The biochemical consequences of a type II procollagen mutation that contained a Gly574Ser amino acid substitution were analyzed in a transgenic mouse strain. The mutation correlated with one previously characterized in a patient with the lethal human chondrodysplasia, hypochondrogenesis (Horton et al., 1992), and resulted in a similar shortlimbed phenotype. There were fewer collagen fibrils present in the transgenic cartilage and reduced immunofluorescence of cartilage matrix using a type II collagen antibody. Pepsin-extracted collagen from transgenic mouse embryo cartilage was analyzed electrophoretically and indicated less type II as well as type XI collagen compared to their wild-type littermates. A pulse-chase experiment was performed to evaluate the biosynthesis and fate of type II collagen. Chondrocytes isolated from transgenic tissue synthesized fewer stable molecules, resulting in decreased secretion of the procollagen chains. By amino acid sequence analysis of the type II collagen peptides from cartilage of transgenic mouse embryos, serine was not detected at residue 574, the site mutated in the transgene. Based on sequence data, we believe that the molecules incorporated into collagen fibrils of the extracellular matrix, while fewer in number, were composed of normal alpha 1(II) chains.     

Expression of CD54 (intercellular adhesion molecule-1) and the beta 1 integrin CD29 is modulated by a cyclic AMP dependent pathway in activated primary rat microglial cell cultures

Integrins mediate cell attachment to a variety of extracellular matrix proteins. These interactions play an important role in morphogenesis and differentiation. The mediating functions of integrins during chondrogenesis in vitro were investigated by using mesenchymal cells from limb buds of day 12 mouse embryos. The cells were treated with anti-beta 1, -alpha 1, and -alpha 5 integrin antibodies (a) from day 1 to day 3 and (b) from day 3 to day 7 of cultivation. The total culture period was 7 days. The presence of exogenous anti-beta 1, but not -alpha 1 and -alpha 5 integrin antibodies, from day 1 to 3 completely inhibited the differentiation of blastemal cells to chondroblasts and the formation of cartilage matrix. On the other hand, the presence of exogenous anti-beta 1, -alpha 1, and -alpha 5 integrin antibodies from day 3 of cultivation onwards had no effect. Immunoblotting and immunomorphological findings in the cultures treated with anti-beta 1 antibody from day 1 to day 3 revealed a pattern of integrins and collagen composed of beta 1, alpha 1, alpha 5 beta 1 integrins and collagen type I. The cartilage-specific chondroitin sulfate proteoglycan (CSPG) could not be demonstrated in these cultures. The cultures treated later (day 3 to day 7) showed a pattern of beta 1, alpha 3, alpha 5 beta 1, and alpha v beta 3 integrins, collagen types I and II, and CSPG identical to that of the untreated controls. These findings indicate that beta 1-integrins play a crucial role in early cartilage differentiation and point to a possible important cell-matrix interaction in the induction of chondrogenesis.     

Role of the plasminogen activator and matrix metalloproteinase systems in epidermal growth factor- and scatter factor-stimulated invasion of carcinoma cells

Normal as well as neoplastic cells traverse extracellular matrix barriers by mobilizing proteolytic enzymes in response to epidermal growth factor (EGF)-EGF receptor (EGFR) or hepatocyte growth factor/scatter factor (SF)-c-Met interactions. The plasminogen activator-plasminogen axis has been proposed to play a key role during cell invasion, but the normal development of plasminogen activator- as well as that of plasminogen-deficient mice supports the existence of alternate proteolytic systems that permit cells to traverse extracellular matrix barriers. To characterize the role that matrix-degrading proteinases play in EGF- or SF-stimulated invasion, a human squamous carcinoma cell line (UM-SCC-1) was triggered atop the matrices of type I collagen or human dermal explants in a three-dimensional culture system. During EGF- or SF-induced invasion, UM-SCC-1 cells expressed urokinase-type plasminogen activator (uPA) and uPA receptor as well as the matrix metalloproteinases (MMPs), membrane-type MMP-1, collagenase 1, stromelysin 1, and gelatinase B. Despite the presence of a positive correlation between uPA receptor-uPA expression and growth factor-stimulated invasion, UM-SCC-1 invasion was not affected by inhibitors directed against the plasminogen activator-plasminogen axis. In contrast, both recombinant and synthetic MMP inhibitors completely suppressed invasion by either EGF- or SF-stimulated cells without affecting either proteinase expression or cell motility across collagen-coated surfaces. These data demonstrate that MMPs, but not the plasminogen activator-plasmin system, can directly regulate the ability of either EGF- or SF-stimulated tumor cells to invade interstitial matrix barriers.     

Preparation of the myristoylated and nonmyristoylated form of recombinant recoverin in E. coli cells and comparison of their functional activity]

Numerous data strongly suggest the involvement of cytokines and the matrix metalloproteinase collagenase (MMP-1) in the pathogenesis of periodontitis. Recently, we have demonstrated that, upon culturing under the influence of IL-1 alpha + EGF, a large amount of inactive procollagenase (MMP-1) is stored in the extracellular matrix of periosteal tissue. We now show that this endogenous reservoir of proenzyme can be operative after activation with plasmin and is able to induce a rapid and almost complete breakdown of the collagenous extracellular matrix. The level of collagen degradation following activation showed a strong correlation with the amount of proenzyme that was incorporated in the tissue. The highest level of degradation (70% of the total amount of collagenous proteins) was found with the IL-1 alpha + EGF-treated explants, followed by those treated with IL-1 alpha alone (35%). Explants cultured with EGF or in the absence of cytokines, containing only small amounts of procollagenase, showed little collagen breakdown following plasmin activation (7%). Inhibition of metalloproteinases by EDTA, or blockage of plasmin by PMSF, prevented the degradation in all explants irrespective of the amount of proenzyme present in the tissue. Our findings demonstrate that endogenous proenzyme stored in a native connective tissue matrix can be activated at a later time interval which results in a massive breakdown of the tissue. This study shows a possible pathway of collagenase-induced breakdown without recent de novo synthesis of the enzyme. Such a sequence may be operative in chronic inflammatory diseases, such as periodontitis, where production of procollagenase under the influence of cytokines spans a longer time period, whereas breakdown is often characterized by a cyclic behaviour.     

Expression of nerve growth factor and its high-affinity receptor Trk-A in the rat pancreas during embryonic and fetal life

We recently reported that nitric oxide (NO), which is produced by chondrocytes treated with interleukin-1beta (IL-1), releases basic fibroblast growth factor (bFGF) stored in the matrix of articular chondrocytes. To clarify the mechanism of the IL-1-induced bFGF release, we investigated the production and gene expression of bFGF, matrix metalloproteinases (MMPs), syndecan 3, and inducible NO synthase (iNOS) by IL-1-treated rabbit articular chondrocytes. IL-1 stimulated not only the release of bFGF but also the production of it. Gelatin and casein zymography revealed that IL-1 stimulated the production of not only MMP-9 but also MMP-3. The increase in the production of these MMPs preceded the IL-1-stimulated bFGF release. An MMP inhibitor partially suppressed the release of bFGF, indicating that matrix degradation is at least partially involved in the IL-1-stimulated bFGF release even if increased production of bFGF is related to the release. IL-1 sequentially stimulated mRNA expression of iNOS, membrane type 1-MMP, MMP-9 and -3, and bFGF, in that order. NG-Monomethyl-L-arginine, an inhibitor of NO production, inhibited gene expression of MMP-9 and bFGF. These findings suggest that elevation of the NO level via iNOS mRNA expression stimulated by IL-1 mediates gene expression and production of MMPs and bFGF, resulting in the release of bFGF, and also reveal molecular mechanisms implicating the degradation of articular cartilage followed by angiogenesis in the synovium in arthritic joints.     

SB 203580 inhibits p38 mitogen-activated protein kinase, nitric oxide production, and inducible nitric oxide synthase in bovine cartilage-derived chondrocytes

Nitric oxide (NO) is implicated in a number of inflammatory processes and is an important mediator in animal models of rheumatoid arthritis and in in vitro models of cartilage degradation. The pyridinyl imidazole SB 203580 inhibits p38 mitogen-activated protein (MAP) kinase in vitro, blocks proinflammatory cytokine production in vitro and in vivo, and is effective in animal models of arthritis. The purpose of this study was to determine whether SB 203580 could inhibit p38 MAP kinase activity, NO production, and inducible NO synthase (iNOS) in IL-1 stimulated bovine articular cartilage/chondrocyte cultures. The results indicated that SB 203580 inhibited both IL-1 stimulated p38 MAP kinase activity in isolated chondrocytes and NO production in bovine chondrocytes and cartilage explants with an IC50 value of approximately 1 microM. To inhibit NO production, SB 203580 had to be present in cartilage explant cultures during the first 8 h of IL-1 stimulation, and activity was lost when it was added 24 h following IL-1. SB 203580 did not inhibit iNOS activity, as measured by the conversion of arginine to citrulline, when added directly to cultures where the enzyme had already been induced, but had to be present during the induction period. Using a 372-bp probe for bovine iNOS we demonstrated inhibition of IL-1-induced mRNA by SB 203580 at both 4 and 24 h following IL-1 treatment. The iNOS mRNA levels were consistent with NO levels in 24-h cell culture supernatants of the IL-1-stimulated bovine chondrocytes used to obtain the RNA.     

FGF signal transduction in PC12 cells: comparison of the responses induced by endogenous and chimeric receptors

Loss of articular cartilage, which is the most important pathological lesion occurring in osteoarthritis, has been shown to be enzymatically mediated. The matrix metalloproteinases (MMPs) are a group of enzymes which have been implicated in this degradation of articular cartilage matrix. The use of pharmacological agents to inhibit this catabolic process in the joint is a potential route for therapeutic intervention. The gelatinase MMPs, MMPs-2 and 9, were purified by affinity chromatography from equine cell cultures. The ability of phenylbutazone, flunixin, betamethasone, dexamethasone, methylprednisolone acetate (MPA), hyaluronan, pentosan polysulphate and polysulphated glycosaminoglycan (PSGAG) to inhibit equine MMPs-2 and 9 were assessed by two degradation assays. Whilst some agents did have direct effects on MMP activity, these effects were only obtained at concentrations which were unlikely to be achieved for any length of time in vivo. It is improbable that any pharmacological agent, currently used in the horse, has a significant effect on gelatinase MMP activity.