DiBattista J A, Martel-Pelletier J, Wosu L O, Sandor T, Antakly T, Pelletier J P
Rheumatic Disease Unit, Notre-Dame Hospital, Montréal, Québec, Canada.
J Clin Endocrinol Metab. 1991 Feb;72(2):316-26. doi: 10.1210/jcem-72-2-316.
Glucocorticoids play an important role in the therapy of arthritic diseases. We sought, firstly, to identify, characterize and localize glucocorticoid receptors (GR) in normal human chondrocytes and, secondly, to determine whether glucocorticoid suppression of human recombinant interleukin-1 beta (rhIL-1 beta)-stimulated metalloproteases (MPs) synthesis by chondrocytes requires GR occupancy. Radioligand binding studies with cultured chondrocytes revealed the presence of high affinity-low capacity [3H]dexamethasone (DEX) binding sites with the following kinetic parameters: Kd = 12.5 +/- 1.4 nmol/L, Nmax = 57,560 +/- 3,960 sites per cell. Competition studies indicated that the DEX binding site was glucocorticoid specific and the competitive hierarchy established was: DEX greater than RU-26988 greater than RU-486 greater than cortisol greater than progesterone much greater than testosterone greater than estradiol-17 beta. Immunocytochemical studies using a specific anti-human GR antiserum identified immunoreactive material primarily in the cytoplasm with cells cultured in the absence of glucocorticoids. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis-Western immunoblotting analysis of chondrocyte cytosol detected the presence of a macromolecular species comigrating with a standard protein possessing a molecular weight of 94 kilodalton. rhIL-1 beta provoked the synthesis and secretion of the MPs stromelysin and collagenase from human chondrocytes in a saturable, coordinate, and dose-dependent fashion. DEX and cortisol inhibited the cytokine-stimulated MP synthesis in similar dose-dependent fashions: DEX, IC50 for stromelysin and collagenase suppression was 1.12 X 10(-8) mol/L and 1.26 X 10(-9) mol/L, respectively and the IC50 for cortisol was 6.3 X 10(-7) mol/L and 4.9 X 10(-8) mol/L, respectively. rhIL-1 beta failed to stimulate metalloprotease synthesis and release from chondrocytes pretreated with 10 nmol/L DEX, even after 20 days of incubation. The antiglucocorticoid, RU-486 completely reversed the DEX induced suppression of MP synthesis at 10(-7) mol/L. RU-486 alone had no effect on MP synthesis. We believe there is a biochemical rationale for the therapeutic efficacy of glucocorticoid administration in the management of arthritic diseases such as osteoarthritis and rheumatoid arthritis, and cytokines such as IL-1 are likely to be involved in the increase in MP synthesis.
糖皮质激素在关节炎疾病的治疗中发挥着重要作用。首先，我们试图鉴定、表征和定位正常人软骨细胞中的糖皮质激素受体（GR），其次，确定软骨细胞中糖皮质激素对重组人白细胞介素-1β（rhIL-1β）刺激的金属蛋白酶（MPs）合成的抑制作用是否需要GR的占据。对培养的软骨细胞进行放射性配体结合研究，发现存在高亲和力-低容量的[3H]地塞米松（DEX）结合位点，其动力学参数如下：Kd = 12.5±1.4 nmol/L，Nmax = 57,560±3,960个位点/细胞。竞争研究表明，DEX结合位点具有糖皮质激素特异性，建立的竞争等级为：DEX＞RU-26988＞RU-486＞皮质醇＞孕酮＞＞睾酮＞雌二醇-17β。使用特异性抗人GR抗血清进行的免疫细胞化学研究确定，在无糖皮质激素培养的细胞中，免疫反应性物质主要存在于细胞质中。对软骨细胞胞质溶胶进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳- Western免疫印迹分析，检测到一种与分子量为94千道尔顿的标准蛋白共迁移的大分子物质。rhIL-1β以饱和、协同和剂量依赖性方式刺激人软骨细胞合成和分泌MPs基质溶解素和胶原酶。DEX和皮质醇以类似的剂量依赖性方式抑制细胞因子刺激的MP合成：DEX对基质溶解素和胶原酶抑制的IC50分别为1.12×10-8 mol/L和1.26×10-9 mol/L，皮质醇的IC50分别为6.3×10-7 mol/L和4.9×10-8 mol/L。即使在孵育20天后，rhIL-1β也未能刺激用10 nmol/L DEX预处理的软骨细胞合成和释放金属蛋白酶。抗糖皮质激素RU-486在10-7 mol/L时完全逆转了DEX诱导的MP合成抑制。单独使用RU-486对MP合成没有影响。我们认为，糖皮质激素给药在骨关节炎和类风湿关节炎等关节炎疾病管理中的治疗效果存在生化依据，并且细胞因子如IL-1可能参与MP合成的增加。
