Prg4 prevents osteoarthritis induced by dominant-negative interference of TGF-ß signaling in mice

Objective
Prg4, also known as Lubricin, acts as a joint/boundary lubricant. Prg4 has been used to prevent surgically induced osteoarthritis (OA) in mice. Surgically induced OA serves as a good model for post-traumatic OA but is not ideal for recapitulating age-related OA. Reduced expression of the TGF-β type II receptor (TGFβR2) is associated with age-related OA in clinical samples, so we previously characterized a mouse model that exhibits OA due to expression of a mutated dominant-negative form of TGFβR2 (DNIIR). Prg4 expression was significantly reduced in DNIIR mice. Furthermore, we showed that Prg4 was a transcriptional target of TGF-ß via activation of Smad3, the main signal transducing protein for TGF-ß. The objective of the present study was to determine whether maintenance of Prg4, a down-stream transcriptional target of TGF-ß, prevents OA associated with attenuated TGF-ß signaling in mice.
Design
Wild-type, DNIIR, and bitransgenic mice that express both DNIIR and Prg4, were compared. Mice were assessed with a foot misplacement behavioral test, μCT, histology, and Western blot.
Results
Compared to DNIIR mice, bitransgenic DNIIR+Prg4 mice missed 1.3 (0.4, 2.1) fewer steps while walking (mean difference (95% confidence interval)), exhibited a cartilage fibrillation score that was 1.8 (0.4, 3.1) points lower, exhibited cartilage that was 28.2 (0.5, 55.9) μm thicker, and exhibited an OARSI score that was 6.8 (-0.9, 14.5) points lower. However, maintenance of Prg4 expression did not restore levels of phosphorylated Smad3 in DNIIR mice, indicating Prg4 does not simply stimulate TGF-ß signaling.
Conclusions
Our results indicate that maintenance of Prg4 expression prevents OA progression associated with reduced TGF-β signaling in mice. Since there was no evidence that Prg4 acts by stimulating the TGF-ß signaling cascade, we propose that Prg4, a transcriptional target of TGF-ß, attenuates OA progression through its joint lubrication function.

### 研究目的
Prg4，又称润滑素（Lubricin），可作为关节边界润滑剂。Prg4曾被用于预防小鼠手术诱导的骨关节炎（osteoarthritis, OA）。手术诱导的骨关节炎是创伤后骨关节炎的良好模型，但并不适合复现年龄相关性骨关节炎。临床样本显示，转化生长因子βⅡ型受体（transforming growth factor β type II receptor, TGFβR2）表达降低与年龄相关性骨关节炎相关，因此我们此前构建了一种因表达突变的显性负性TGFβR2（dominant-negative form of TGFβR2, DNIIR）而出现骨关节炎的小鼠模型。在DNIIR小鼠体内，Prg4的表达水平显著降低。此外，我们证实Prg4是转化生长因子β（TGF-β）的转录靶标，其表达通过激活Smad3——TGF-β主要的信号转导蛋白——实现调控。本研究旨在探究维持TGF-β的下游转录靶标Prg4的表达，是否能够预防小鼠中因TGF-β信号减弱引发的骨关节炎。

### 实验设计
本研究对野生型小鼠、DNIIR小鼠以及同时表达DNIIR和Prg4的双转基因小鼠进行了比较。通过足部错位行为学测试、显微计算机断层扫描（μCT）、组织学检测以及蛋白质印迹（Western blot）对各组小鼠进行评估。

### 实验结果
与DNIIR小鼠相比，双转基因DNIIR+Prg4小鼠在行走时的错步次数减少了1.3（0.4, 2.1）步（均值差（95%置信区间）），软骨纤维化评分降低1.8（0.4, 3.1）分，软骨厚度增加28.2（0.5, 55.9）μm，OARSI评分降低6.8（-0.9, 14.5）分。然而，维持Prg4的表达并未恢复DNIIR小鼠体内磷酸化Smad3的水平，这表明Prg4并非仅通过激活TGF-β信号通路发挥作用。

### 研究结论
我们的研究结果表明，维持Prg4的表达能够预防小鼠中因TGF-β信号减弱引发的骨关节炎进展。由于未发现Prg4通过激活TGF-β信号级联发挥作用的证据，我们提出：作为TGF-β的转录靶标，Prg4可通过其关节润滑功能延缓骨关节炎的进展。