Musculoskeletal Research Group, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom.
Biopharm GmbH, Heidelberg, Germany.
PLoS One. 2014 Jan 21;9(1):e86590. doi: 10.1371/journal.pone.0086590. eCollection 2014.
A genetic deficit mediated by SNP rs143383 that leads to reduced expression of GDF5 is strongly associated with large-joint osteoarthritis. We speculated that this deficit could be attenuated by the application of exogenous GDF5 protein and as a first step we have assessed what effect such application has on primary osteoarthritis chondrocyte gene expression. Chondrocytes harvested from cartilage of osteoarthritic patients who had undergone joint replacement were cultured with wildtype recombinant mouse and human GDF5 protein. We also studied variants of GDF5, one that has a higher affinity for the receptor BMPR-IA and one that is insensitive to the GDF5 antagonist noggin. As a positive control, chondrocytes were treated with TGF-β1. Chondrocytes were cultured in monolayer and micromass and the expression of genes coding for catabolic and anabolic proteins of cartilage were measured by quantitative PCR. The expression of the GDF5 receptor genes and the presence of their protein products was confirmed and the ability of GDF5 signal to translocate to the nucleus was demonstrated by the activation of a luciferase reporter construct. The capacity of GDF5 to elicit an intracellular signal in chondrocytes was demonstrated by the phosphorylation of intracellular Smads. Chondrocytes cultured with TGF-β1 demonstrated a consistent down regulation of MMP1, MMP13 and a consistent upregulation of TIMP1 and COL2A1 with both culture techniques. In contrast, chondrocytes cultured with wildtype GDF5, or its variants, did not show any consistent response, irrespective of the culture technique used. Our results show that osteoarthritis chondrocytes do not respond in a predictable manner to culture with exogenous GDF5. This may be a cause or a consequence of the osteoarthritis disease process and will need to be surmounted if treatment with exogenous GDF5 is to be advanced as a potential means to overcome the genetic deficit conferring osteoarthritis susceptibility at this gene.
由 SNP rs143383 介导的遗传缺陷导致 GDF5 表达减少，与大关节骨关节炎密切相关。我们推测，这种缺陷可以通过外源性 GDF5 蛋白的应用得到缓解，作为第一步，我们评估了这种应用对原发性骨关节炎软骨细胞基因表达的影响。从接受关节置换术的骨关节炎患者的软骨中采集软骨细胞，用野生型重组小鼠和人 GDF5 蛋白培养。我们还研究了 GDF5 的变体，一种对受体 BMPR-IA 具有更高亲和力，另一种对 GDF5 拮抗剂 noggin 不敏感。作为阳性对照，软骨细胞用 TGF-β1 处理。软骨细胞在单层和微团中培养，通过定量 PCR 测量编码软骨分解代谢和合成代谢蛋白的基因表达。证实了 GDF5 受体基因的表达及其蛋白产物的存在，并通过激活荧光素酶报告基因构建体证明了 GDF5 信号向核内易位的能力。通过细胞内 Smads 的磷酸化证明了 GDF5 激活软骨细胞内信号的能力。用 TGF-β1 培养的软骨细胞表现出 MMP1、MMP13 的一致下调和 TIMP1、COL2A1 的一致上调，两种培养技术均如此。相比之下，用野生型 GDF5 或其变体培养的软骨细胞无论使用哪种培养技术，都没有表现出任何一致的反应。我们的结果表明，骨关节炎软骨细胞对用外源性 GDF5 培养不会以可预测的方式做出反应。这可能是骨关节炎疾病过程的原因或结果，如果要将外源性 GDF5 作为克服该基因赋予骨关节炎易感性的遗传缺陷的潜在手段进行推进，就需要克服这一问题。
