Inhibition of transforming growth factor beta production by nitric oxide-treated chondrocytes - Implications for matrix synthesis

Objective. Nitric oxide (NO) is generated copiously by articular chondrocyt es activated by interleukin-1 beta (IL-1 beta). If NO production is blocked , much of the IL-1 beta inhibition of proteoglycan synthesis is prevented. We tested the hypothesis that this inhibitory effect of NO on proteoglycan synthesis is secondary to changes in chondrocyte transforming growth factor beta (TGF beta). Methods. Monolayer, primary cultures of lapine articular chondrocytes and c artilage slices were studied. NO production nas determined as nitrite accum ulation in the medium. TGF beta bioactivity in chondrocyte- and cartilage-c onditioned medium (CM) was measured with the mink lung epithelial cell bioa ssay, Proteoglycan synthesis Was measured as the incorporation of S-35-sodi um sulfate into macromolecules separated from unincorporated label by gel f iltration on PD-10 columns. Results. IL-1 beta increased active TGF beta in chondrocyte CM by 12 hours; by 24 hours, significant increases in both active and latent TGF beta were detectable. N-G-monomethyl-L-arginine (L-NMA) potentiated the increase in total TGF beta without affecting the early TGF beta activation. IL-1 beta s timulated a NO-independent, transient increase in TGF beta 3 at 24 hours; h owever, TGF beta 1 was not changed. When NO synthesis was inhibited with L- MMA, IL-1 beta increased CM concentrations of TGF beta 1 from 24-72 hours o f culture. L-arginine (10 mM) reversed the inhibitory effect of L-NMA on NO production and blocked the increases in TGF beta 1, Anti-TGF beta 1 antibo dy prevented the restoration of proteoglycan synthesis by chondrocytes expo sed to IL-1 beta + L-NMA, confirming that NO inhibition of TGF beta 1 in IL -1 beta-treated chondrocytes effected, in part, the decreased proteoglycan synthesis. Furthermore, the increase in TGF beta and proteoglycan synthesis seen with L-NMA was reversed by the NO donor S-nitroso-N-acetylpenicillami de. Similar results were seen with cartilage slices in organ culture. The a utocrine increase in CM TGF beta 1 levels following prior exposure to TGF b eta 1 was also blocked by NO. Conclusion. NO can modulate proteoglycan synthesis indirectly by decreasing the production of TGF beta 1 by chondrocytes exposed to IL-1 beta. It prev ents autocrine-stimulated increases in TGF beta 1, thus potentially diminis hing the anabolic effects of this cytokine in chondrocytes.