Studer R K, Georgescu H I, Miller L A, Evans C H
Ferguson Laboratory for Orthopaedic Research and the University of Pittsburgh School of Medicine, Pennsylvania 15213, USA.
Arthritis Rheum. 1999 Feb;42(2):248-57. doi: 10.1002/1529-0131(199902)42:2<248::AID-ANR6>3.0.CO;2-S.
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).
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.
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.
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.
一氧化氮（NO）由白细胞介素 - 1β（IL - 1β）激活的关节软骨细胞大量产生。如果NO生成受阻，IL - 1β对蛋白聚糖合成的大部分抑制作用将被阻止。我们检验了这样一个假设，即NO对蛋白聚糖合成的这种抑制作用继发于软骨细胞转化生长因子β（TGFβ）的变化。
研究了兔关节软骨细胞和软骨切片的单层原代培养物。NO生成通过培养基中亚硝酸盐积累来测定。用貂肺上皮细胞生物测定法测量软骨细胞和软骨条件培养基（CM）中的TGFβ生物活性。蛋白聚糖合成通过将35S - 硫酸钠掺入通过PD - 10柱上的凝胶过滤从未掺入标记中分离的大分子中来测量。
IL - 1β在12小时内使软骨细胞CM中的活性TGFβ增加；到24小时时，活性和潜伏性TGFβ均有显著增加。NG - 单甲基 - L - 精氨酸（L - NMA）增强了总TGFβ的增加，而不影响早期TGFβ的激活。IL - 1β在24小时时刺激了TGFβ3的非NO依赖性短暂增加；然而，TGFβ1没有变化。当用L - NMA抑制NO合成时，IL - 1β在培养24 - 72小时内增加了CM中TGFβ1的浓度。L - 精氨酸（10 mM）逆转了L - NMA对NO生成的抑制作用，并阻止了TGFβ1的增加。抗TGFβ1抗体阻止了暴露于IL - 1β + L - NMA的软骨细胞中蛋白聚糖合成的恢复，证实了在IL - 1β处理的软骨细胞中NO对TGFβ1的抑制部分影响了蛋白聚糖合成减少。此外，NO供体S - 亚硝基 - N - 乙酰青霉胺逆转了L - NMA引起的TGFβ和蛋白聚糖合成的增加。器官培养中的软骨切片也得到了类似结果。预先暴露于TGFβ1后CM中TGFβ1水平的自分泌增加也被NO阻断。
NO可通过减少暴露于IL - 1β的软骨细胞中TGFβ1的产生间接调节蛋白聚糖合成。它阻止了自分泌刺激的TGFβ1增加，从而可能减少这种细胞因子在软骨细胞中的合成代谢作用。
