Department of Biomedical Engineering, University of Alabama at Birmingham, 1530 3rd Avenue South, Birmingham, AL 35294-0005, USA.
Arthritis Res Ther. 2012 Mar 6;14(2):R49. doi: 10.1186/ar3762.
Previous studies have indicated that transforming growth factor β (TGF-β) signaling has a critical role in cartilage homeostasis and repair, yet the mechanisms of TGF-β's chondroprotective effects are not known. Our objective in this study was to identify downstream targets of TGF-β that could act to maintain biochemical and biomechanical properties of cartilage.
Tibial joints from 20-week-old mice that express a dominant-negative mutation of the TGF-β type II receptor (DNIIR) were graded histologically for osteoarthritic changes and tested by indentation to evaluate their mechanical properties. To identify gene targets of TGF-β, microarray analysis was performed using bovine articular chondrocytes grown in micromass culture that were either treated with TGF-β or left untreated. Phosphoadenosine phosphosynthetase 2 (PAPSS2) was identified as a TGF-β-responsive gene. Papss2 expression is crucial for proper sulfation of cartilage matrix, and its deficiency causes skeletal defects in mice and humans that overlap with those seen in mice with mutations in TGF-β-signaling genes. Regulation of Papss2 was verified by real time RT-PCR and Western blot analyses. Alterations in sulfation of glycosaminoglycans were analyzed by critical electrolyte concentration and Alcian blue staining and immunofluorescence for chondroitin-4-sulfate, unsulfated chondroitin and the aggrecan core protein.
DNIIR mutants showed reduced mechanical properties and osteoarthritis-like changes when compared to wild-type control mice. Microarray analysis identified a group of genes encoding matrix-modifying enzymes that were regulated by TGF-β. Papss2 was upregulated in bovine articular chondrocytes after treatment with TGF-β and downregulated in cartilage from DNIIR mice. Articular cartilage in DNIIR mice demonstrated reduced Alcian blue staining at critical electrolyte concentrations and reduced chondroitin-4-sulfate staining. Staining for unsulfated chondroitin sulfate was increased, whereas staining for the aggrecan core protein was comparable in DNIIR and wild-type mice.
TGF-β maintains biomechanical properties and regulates expression of Papss2 and sulfation of glycosaminoglycans in mouse articular cartilage.
先前的研究表明转化生长因子 β（TGF-β）信号在软骨稳态和修复中起着关键作用，但 TGF-β 的软骨保护作用的机制尚不清楚。我们在这项研究中的目的是确定 TGF-β 的下游靶标，这些靶标可以维持软骨的生化和生物力学特性。
从小鼠的胫骨关节中提取 20 周龄表达 TGF-β 型 II 受体显性负突变（DNIIR）的组织，进行组织学分级以评估骨关节炎变化，并通过压痕试验评估其机械性能。为了鉴定 TGF-β的基因靶点，使用在微团培养中生长的牛关节软骨进行微阵列分析，这些软骨要么用 TGF-β处理，要么不处理。磷酸腺苷磷酸合酶 2（PAPSS2）被鉴定为 TGF-β 反应基因。Papss2 的表达对于软骨基质的适当硫酸化至关重要，其缺乏会导致小鼠和人类出现骨骼缺陷，这些缺陷与 TGF-β 信号转导基因发生突变的小鼠相似。通过实时 RT-PCR 和 Western blot 分析验证了 Papss2 的调控。通过临界电解质浓度和阿尔辛蓝染色以及软骨素 4-硫酸盐、未硫酸化软骨素和核心蛋白聚糖的免疫荧光分析来分析糖胺聚糖的硫酸化改变。
与野生型对照小鼠相比，DNIIR 突变体的机械性能降低，并出现骨关节炎样变化。微阵列分析鉴定了一组编码基质修饰酶的基因，这些基因受 TGF-β调节。TGF-β 处理后牛关节软骨中 Papss2 上调，DNIIR 小鼠软骨中 Papss2 下调。DNIIR 小鼠的关节软骨在临界电解质浓度下的阿尔辛蓝染色减少，软骨素 4-硫酸盐染色减少。未硫酸化软骨素硫酸化染色增加，而 DNIIR 和野生型小鼠的核心蛋白聚糖染色相似。
TGF-β 维持小鼠关节软骨的生物力学特性，并调节 Papss2 的表达和糖胺聚糖的硫酸化。
