Human chondrocytes respond discordantly to the protein encoded by the osteoarthritis susceptibility gene GDF5.
Human chondrocytes respond discordantly to the protein encoded by the osteoarthritis susceptibility gene GDF5.
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DOI：
10.1371/journal.pone.0086590
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Loughlin J
中科院分区：
文献类型：
--
作者：
Ratnayake M;Plöger F;Santibanez-Koref M;Loughlin J
由SNP rs143383介导的导致GDF5表达减少的遗传缺陷与大关节骨关节炎密切相关。我们推测，应用外源性GDF5蛋白可以减轻这种缺陷，作为第一步，我们评估了这种应用对原发骨关节炎软骨细胞基因表达的影响。用野生型重组小鼠和人GDF5蛋白对关节置换术后骨关节炎患者软骨细胞进行培养。我们还研究了GDF5的变体，一个与受体BMPR-IA有更高的亲和力，另一个对GDF5拮抗剂noggin不敏感。以转化生长因子-β-1处理软骨细胞作为阳性对照。单层和微团培养软骨细胞，定量聚合酶链式反应检测软骨分解代谢和合成蛋白基因的表达。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结构证明了GDF5信号转位到细胞核的能力。细胞内Smads的磷酸化证明了GDF5在软骨细胞中诱导细胞内信号的能力。软骨细胞经转化生长因子-β-1培养后，MMP1、MMP13表达下调，TIMP1、COL2A1表达上调。相比之下，无论采用何种培养技术，用野生型GDF5或其变异体培养的软骨细胞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外源性GDF5的培养没有可预测的反应。这可能是骨关节炎疾病过程的原因或结果，如果外源性GDF5治疗要作为克服该基因导致骨关节炎易感性的遗传缺陷的潜在手段，则需要克服。
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.
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影响因子：
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作者：
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通讯作者：
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