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Effects of cyclofenil diphenol, an agent which disrupts Golgi structure, on proteoglycan synthesis in chondrocytes.

ABSTRACT: 1. Cyclofenil diphenol (F6060), a weak non-steroidal oestrogen, was shown previously to inhibit [35S]proteoglycan synthesis [Mason, Lineham, Phillipson & Black (1984) Biochem. J. 223, 401-412] and to induce fragmentation of the Golgi apparatus into small vesicles [Lancaster, Fryer, Griffiths & Mason (1989) J. Cell Sci. 92, 271-280] in cultures of Swarm chondrosarcoma chondrocytes. Two structurally related compounds, F6204 and F6091, show a similar concentration-related effect, with complete inhibition of [35S]proteoglycan synthesis at 90 micrograms/ml. The apparent [3H]protein synthesis is only approx. 40% inhibited with [3H]lysine as precursor. Stilboestrol, clomiphene and tamoxiphen are also potent inhibitors of [35S]proteoglycan synthesis. 2. Syntheses of chondroitin 4-[35S]sulphate and chondroitin 6-[35S]sulphate, which are Golgi-mediated events, are inhibited 40-68% and 3-48% respectively by concentrations of cyclofenil between 50 and 70 micrograms/ml. [3H]Hyaluronan synthesis, which occurs by a different mechanism at the plasma membrane, is inhibited by 47-66%. These results suggest that cyclofenil may act via more than one inhibitory mechanism. Cyclofenil diphenol inhibits polymerization of chondroitin sulphate on to p-nitrophenyl beta-xyloside even when the chondrocytes are loaded with the initiator prior to treatment. 3. Cyclofenil diphenol interferes with the cellular uptake of amino acids via the system A carrier, as shown by inhibition of uptake of methylaminoisobutyric acid, a specific substrate for this system. The drug had no effect on the uptake of 2-deoxyglucose by the cells. 4. Cyclofenil diphenol (90 micrograms/ml) caused a decrease in the pool size of UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and UDP-hexoses, but this was insufficient to account for the accompanying profound inhibition of [35S]proteoglycan synthesis. Entry of [3H]glucosamine into the cell and into the UDP-N-acetylhexosamine pool did not appear to be affected. 5. Cyclofenil diphenol inhibited the substitution of 3H-labelled proteoglycan core protein with chondroitin sulphate chains. Core protein was identified in treated cultures on the basis of immunoprecipitation with an antiserum against the hyaluronate-binding region and distinguished from precipitated proteoglycan on SDS/PAGE.

SUBMITTER: Mason RM

PROVIDER: S-EPMC1130717 | biostudies-other | 1992 Jan

REPOSITORIES: biostudies-other

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Project description:BackgroundSphingosine-1-phosphate (S1P), a downstream metabolite of ceramide, induces various bioactivities via two distinct pathways: as an intracellular second messenger or through receptor activation. The receptor for S1P (S1PR) is the family of Endothelial differentiation, sphingolipid G-protein-coupled receptor (EDG). We have here attempted to reveal the expression of EDG/S1PR in human articular chondrocytes (HAC), exploring the implications of S1P in cartilage degradation.MethodsArticular cartilage specimens were obtained from patients with rheumatoid arthritis (RA), osteoarthritis (OA) or traumatic fracture (representing normal chondrocytes) who underwent joint surgery. Isolated HAC were cultured in vitro by monolayer and stimulated with S1P in the presence or absence of inhibitors of signaling molecules. Stimulated cells and culture supernatants were collected and subjected to analyses using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA).ResultsAll of the tested HAC samples showed positive results in terms of EDG/S1PR expression in basal condition. When HAC was stimulated with S1P, a significant increase in prostaglandin (PG) E2 production was observed together with enhanced expression of cyclooxygenase (COX)-2. S1P stimulated extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) in HAC, and the PGE2 induction was abrogated by PD98059 and SB203580. Pertussis toxin inhibited the PGE2 induction from HAC by S1P, suggesting an essential role for Gi protein. S1P also attenuated the expression of proteoglycan aggrecan, a component of cartilage matrix, in HAC at transcriptional level.ConclusionIt was suggested that the S1P-induced PGE2 was at least in part involved in the aggrecan-suppressing effect of S1P, seeing as COX inhibitors attenuated the effect. Accordingly, S1P might play an important role in cartilage degradation in arthritides.

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Effects of cyclofenil diphenol, an agent which disrupts Golgi structure, on proteoglycan synthesis in chondrocytes.

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