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Inhibition of transforming growth factor beta production by nitric oxide-treated chondrocytes: implications for
R K Studer1, H I Georgescu, L A Miller
1Ferguson Laboratory for Orthopaedic Research and the University of Pittsburgh School of Medicine, Pennsylvania 15213, USA.
Objective:
Nitric oxide (NO) is generated copiously by articular chondrocytes activated by interleukin-1beta (IL-1beta). If NO production is blocked, much of the IL-1beta 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 (TGFbeta).
Methods:
Monolayer, primary cultures of lapine articular chondrocytes and cartilage slices were studied. NO production was determined as nitrite accumulation in the medium. TGFbeta bioactivity in chondrocyte- and cartilage-conditioned medium (CM) was measured with the mink lung epithelial cell bioassay. Proteoglycan synthesis was measured as the incorporation of 35S-sodium sulfate into macromolecules separated from unincorporated label by gel filtration on PD-10 columns.
Results:
IL-1beta increased active TGFbeta in chondrocyte CM by 12 hours; by 24 hours, significant increases in both active and latent TGFbeta were detectable. NG-monomethyl-L-arginine (L-NMA) potentiated the increase in total TGFbeta without affecting the early TGFbeta activation. IL-1beta stimulated a NO-independent, transient increase in TGFbeta3 at 24 hours; however, TGFbeta1 was not changed. When NO synthesis was inhibited with L-NMA, IL-1beta increased CM concentrations of TGFbeta1 from 24-72 hours of culture. L-arginine (10 mM) reversed the inhibitory effect of L-NMA on NO production and blocked the increases in TGFbeta1. Anti-TGFbeta1 antibody prevented the restoration of proteoglycan synthesis by chondrocytes exposed to IL-1beta + L-NMA, confirming that NO inhibition of TGFbeta1 in IL-1beta-treated chondrocytes effected, in part, the decreased proteoglycan synthesis. Furthermore, the increase in TGFbeta and proteoglycan synthesis seen with L-NMA was reversed by the NO donor S-nitroso-N-acetylpenicillamide. Similar results were seen with cartilage slices in organ culture. The autocrine increase in CM TGFbeta1 levels following prior exposure to TGFbeta1 was also blocked by NO.
Conclusion:
NO can modulate proteoglycan synthesis indirectly by decreasing the production of TGFbeta1 by chondrocytes exposed to IL-1beta. It prevents autocrine-stimulated increases in TGFbeta1, thus potentially diminishing the anabolic effects of this cytokine in chondrocytes.
Insights
Nitric oxide (NO) decreases transforming growth factor beta 1 (TGFbeta1) production in chondrocytes treated with interleukin-1beta (IL-1beta). This NO action partially explains IL-1beta
Area of Science:
- Biochemistry
- Cell Biology
- Rheumatology
Background:
- Articular chondrocytes produce nitric oxide (NO) when stimulated by interleukin-1beta (IL-1beta).
- NO production is linked to IL-1beta's inhibition of proteoglycan synthesis.
- The role of NO in mediating this inhibition via transforming growth factor beta (TGFbeta) was investigated.
Purpose of the Study:
- To test the hypothesis that NO's inhibitory effect on proteoglycan synthesis is secondary to changes in chondrocyte TGFbeta.
- To elucidate the molecular mechanisms linking NO, TGFbeta, and proteoglycan synthesis in chondrocytes.
Main Methods:
- Primary cultures of lapine articular chondrocytes and cartilage slices were utilized.
- Nitrite accumulation measured NO production; TGFbeta bioactivity assessed using mink lung epithelial cells.
- Proteoglycan synthesis quantified by 35S-sodium sulfate incorporation.
Main Results:
- IL-1beta increased active and latent TGFbeta in chondrocyte-conditioned medium.
- Inhibition of NO synthesis with L-NMA potentiated IL-1beta-induced TGFbeta1 increases.
- Blocking NO restored proteoglycan synthesis, an effect reversed by anti-TGFbeta1 antibody, indicating NO's inhibitory role on TGFbeta1 production.
Conclusions:
- Nitric oxide modulates chondrocyte proteoglycan synthesis indirectly by reducing TGFbeta1 production.
- NO prevents autocrine increases in TGFbeta1, potentially mitigating anabolic effects in chondrocytes.
- These findings highlight a novel regulatory pathway in cartilage homeostasis and disease.