Expression of TGF-βs and their receptors is differentially modulated by reactive oxygen species and nitric oxide in human articular chondrocytes

Objectives: To study the effects exerted by two antioxidants, N-monomethyl-L-arginine (L-NMMA), as an inhibitor of nitric oxide (NO) synthesis, and N-acetylcysteine (NAC), a reactive oxygen species (ROS) scavenger, on the expression of the major growth factor involved in cartilage repair, TGF-beta, under the three isoforms beta1, beta2 and beta3, and the receptors I and II of this factor, using lipopolysaccharide (LPS)-treated human chondrocytes in culture. Methods: Suspension cultures of human chondrocytes derived from the knee of osteoarthritic patients were treated for 48 h with lipopolysaccharide (LPS) (10 mug/ml), L-NMMA (0.5 mM) or NAC (1 mM). Nitrite levels were assayed on the culture media using the Griess spectrophotometric method. After total RNA extraction, the expression of inducible NO synthase (iNOS), TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta receptors I and II, was determined by semi-quantitative polymerase chain-reaction (RT-PCR). Results: LPS induced a dramatic increase of both NO production and iNOS mRNA level. The addition of L-NMMA (0.5 mM) abolished NO production without affecting iNOS mRNA levels. In contrast NAC (1 mM) strongly synergized with LPS to stimulate NO synthesis. LPS treatment did not significantly alter TGF-beta1 expression whereas L-NMMA inhibited its production. TGF-beta2 mRNA level was decreased by LPS and was not changed in the presence of L-NMMA. On the other hand, NAC was capable of counteracting the LPS-induced inhibition of TGF-beta2 expression. TGFbeta3 mRNA level was markedly reduced by LPS alone, or with both L-NMMA and NAC. Finally, the expression of TGF-betaRI was slightly increased in the presence of combined LPS and L-NMMA or NAC whereas that of TGFbeta-RII was reduced in the same conditions. Conclusions: The modulation of TGF-beta system was found to be differentially controlled by NO and ROS productions. Indeed, the control exerted on TGF-beta expression varied according to the isoform: TGF-beta1 mRNA level depends on NO whereas that of TGF-beta2 is regulated by ROS and TGF-beta3 seems to be unaffected by both of them. The expression of TGF-beta receptors appeared to be modulated by NO and ROS levels. The relevance of the present findings to osteoarthritis (OA) physiopathology and the potential use of antioxidant therapy to treat this disease are discussed. (C) 2001 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved.