Abstract
The effects of hymenialdisine (SK&F 108752) were evaluated on interleukin-1 (IL-1)-induced proteoglycan (PG) degradation, PG synthesis, nitric oxide (NO) production, and inducible nitric oxide synthase (iNOS) gene expression in bovine articular cartilage (BAC) and/or cartilage-derived chondrocytes. Cartilage disks from 0- to 3-month-old calves were treated with IL-1α or retinoic acid. PG release was determined by measuring glycosaminoglycan release, and nitrite production was measured as a readout for NO. Inhibition of iNOS gene expression was measured by Northern blot analysis in IL-1α-stimulated, cartilage-derived chondrocytes. To measure PG synthesis, chondrocytes were established in alginate beads and treated with hymenialdisine, and then [35S]sulfate incorporation into PGs was determined. Hymenialdisine inhibited IL-1α-stimulated PG breakdown in BAC in a dose-related manner with an IC50 of approximately 0.6 μM. Herbimycin, a protein tyrosine kinase inhibitor, also inhibited PG breakdown, whereas RO 32-0432, a protein kinase C inhibitor, had no effect. Both hymenialdisine and herbimycin also were able to inhibit retinoic acid-stimulated PG release. IL-1α-stimulated NO production in BAC was inhibited by hymenialdisine and herbimycin at similar concentrations. The effect on iNOS gene expression was determined by Northern blot analysis in chondrocytes grown in monolayer, and inhibition by hymenialdisine was observed with an IC50 of approximately 0.8 μM. In chondrocytes cultured in alginate beads, IL-1α inhibited PG synthesis, whereas hymenialdisine stimulated synthesis at low concentrations (0.6 and 1.25 μM), and higher doses (2.5 μM) were not stimulatory. Compounds with this profile may have utility in the treatment of osteoarthritis.
Footnotes
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Send reprint requests to: Alison M. Badger, Department of Bone and Cartilage Biology, SmithKline Beecham Pharmaceuticals, 709 Swedeland Rd., King of Prussia, PA 19406. E-mail:Alison_M_Badger{at}SBPHRD.com
- Abbreviations:
- IL-1
- interleukin 1
- TNF
- tumor necrosis factor
- BAC
- bovine articular cartilage
- DMMB
- dimethylmethylene blue
- GAG
- glycosaminoglycans
- iNOS
- inducible nitric oxide
- MMP
- matrix metalloproteinase
- NMMA
- NG-monomethyl-l-arginine
- NO
- nitric oxide
- PG
- proteoglycan
- PTK
- protein tyrosine kinase
- PKC
- protein kinase C
- NF-κB
- nuclear factor-κB
- DMEM
- Dulbecco’s modified Eagle’s medium
- FBS
- fetal bovine serum
- FCS
- fetal calf serum
- CPC
- cetylpyridiniumchloride-precipitable
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- LPS
- lipopolysaccharide
- Received December 9, 1998.
- Accepted April 2, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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