沉默 Vangl2 通过 MAPK 和 NF-κB 通路抑制软骨细胞中 Wnt5a 诱导的炎症反应。
Silencing of Vangl2 attenuates the inflammation promoted by Wnt5a via MAPK and NF-κB pathway in chondrocytes.
机构信息
Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guanghua School of Stomatology, Sun Yat-sen University, No.56 Lingyuan West Road, Guangzhou, 510055, Guangdong, People's Republic of China.
Department of Stomatology, Binhaiwan Central Hospital of Dongguan (also called The Fifth People's Hospital of Dongguan), The Dongguan Affiliated Hospital of Medical College of Jinan University, No.111 Humen Road, Humen Town, Dongguan City, 523905, Guangdong Province, People's Republic of China.
出版信息
J Orthop Surg Res. 2021 Feb 15;16(1):136. doi: 10.1186/s13018-021-02268-x.
BACKGROUND
The Wnt planar cell polarity (PCP) pathway is implicated in osteoarthritis (OA) both in animals and in humans. Van Gogh-like 2 (Vangl2) is a key PCP protein that is required for the orientation and alignment of chondrocytes in the growth plate. However, its functional roles in OA still remain undefined. Here, we explored the effects of Vangl2 on OA chondrocyte in vitro and further elucidated the molecular mechanism of silencing Vangl2 in Wnt5a-overexpressing OA chondrocytes.
METHODS
Chondrocytes were treated with IL-1β (10 ng/mL) to simulate the inflammatory microenvironment of OA. The expression levels of Vangl2, Wnt5a, MMPs, and related proinflammatory cytokines were measured by RT-qPCR. Small interfering RNA (siRNA) of Vangl2 and the plasmid targeting Wnt5a were constructed and transfected into ATDC5 cells. Then, the functional roles of silencing Vangl2 in the OA chondrocytes were investigated by Western blotting, RT-qPCR, and immunocytochemistry (ICC). Transfected OA chondrocytes were subjected to Western blotting to analyze the relationship between Vangl2 and related signaling pathways.
RESULTS
IL-1β induced the production of Vangl2, Wnt5a, and MMPs in a time-dependent manner and the significantly increased expression of Vangl2. Vangl2 silencing effectively suppressed the expression of MMP3, MMP9, MMP13, and IL-6 at both gene and protein levels and upregulated the expression of type II collagen and aggrecan. Moreover, knockdown of Vangl2 inhibited the phosphorylation of MAPK signaling molecules (P38, ERK, and JNK) and P65 in Wnt5a-overexpressing OA chondrocytes.
CONCLUSIONS
For the first time, we demonstrate that Vangl2 is involved in the OA process. Vangl2 silencing can notably alleviate OA progression in vitro by inhibiting the expression of MMPs and increasing the formation of the cartilage matrix and can inhibit the proinflammatory effects of Wnt5a via MAPK and NF-κB pathway. This study provides new insight into the mechanism of cartilage inflammation.
背景
Wnt 平面细胞极性（PCP）通路在动物和人类的骨关节炎（OA）中都有涉及。梵高样蛋白 2（Vangl2）是 PCP 的关键蛋白，对于生长板中软骨细胞的定向和排列是必需的。然而，其在 OA 中的功能作用仍未被定义。在这里，我们探讨了 Vangl2 对 OA 软骨细胞的影响，并进一步阐明了沉默 Wnt5a 过表达 OA 软骨细胞中 Vangl2 的分子机制。
方法
用白细胞介素-1β（10ng/ml）处理软骨细胞，模拟 OA 的炎症微环境。通过 RT-qPCR 测量 Vangl2、Wnt5a、MMPs 和相关促炎细胞因子的表达水平。构建 Vangl2 的小干扰 RNA（siRNA）和靶向 Wnt5a 的质粒，并转染 ATDC5 细胞。然后，通过 Western blot、RT-qPCR 和免疫细胞化学（ICC）研究沉默 Vangl2 在 OA 软骨细胞中的功能作用。转染 OA 软骨细胞进行 Western blot 分析，以研究 Vangl2 与相关信号通路之间的关系。
结果
IL-1β 呈时间依赖性诱导 Vangl2、Wnt5a 和 MMPs 的产生，且 Vangl2 的表达显著增加。Vangl2 沉默有效抑制了 MMP3、MMP9、MMP13 和 IL-6 的基因和蛋白水平表达，并上调了 II 型胶原和聚集蛋白聚糖的表达。此外，Vangl2 敲低抑制了 Wnt5a 过表达 OA 软骨细胞中 MAPK 信号分子（P38、ERK 和 JNK）和 P65 的磷酸化。
结论
我们首次证明 Vangl2 参与了 OA 过程。Vangl2 沉默可以通过抑制 MMPs 的表达和增加软骨基质的形成显著缓解体外 OA 进展，并通过 MAPK 和 NF-κB 通路抑制 Wnt5a 的促炎作用。本研究为软骨炎症的机制提供了新的见解。
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