IRCCS Istituto Ortopedico Galeazzi, 20161 Milan, Italy.
Department of Biomedical, Surgical and Dental Sciences, University of Milan, 20157 Milan, Italy.
Int J Mol Sci. 2022 May 16;23(10):5575. doi: 10.3390/ijms23105575.
Connexin 43 (Cx43) exerts pivotal functions in articular chondrocytes (CH). It is involved in the communication among cells and between cells and the extracellular environment, and it contributes to the maintenance of the correct cell phenotype. The pro-inflammatory cytokine TNFα induces a reduction in Cx43 expression in CH. Here, we studied the dynamics of this decrease in expression. We evaluated Cx43 protein and gene expression and the involvement of C-terminal domain (CTD) cleavage and proteasomal degradation. Treatments able to counteract TNFα action were also examined, together with Gap Junction (GJ) functionality and Cx43 localization. TNFα induced a significant reduction in Cx43 expression already at day 1, and the down modulation reached a peak at day 3 (-46%). The decrease was linked to neither gene expression modulation nor CTD cleavage. Differently, the proteasome inhibitor MG132 reverted TNFα effect, indicating the involvement of proteasomal degradation in Cx43 reduction. In addition, the co-treatment with the anabolic factor TGF-β1 restored Cx43 levels. Cx43 decrease occurred both at the membrane level, where it partially influenced GJ communication, and in the nucleus. In conclusion, TNFα induced a rapid and lasting reduction in Cx43 expression mostly via the proteasome. The down modulation could be reverted by cartilage-protective factors such as MG132 and TGF-β1. These findings suggest a possible involvement of Cx43 perturbation during joint inflammation.
间隙连接蛋白 43（Cx43）在关节软骨细胞（CH）中发挥关键作用。它参与细胞间以及细胞与细胞外环境间的通讯，有助于维持正确的细胞表型。促炎细胞因子 TNFα可诱导 CH 中 Cx43 表达减少。在此，我们研究了这种表达减少的动态变化。我们评估了 Cx43 蛋白和基因表达以及 C 末端结构域（CTD）切割和蛋白酶体降解的参与情况。还研究了能够对抗 TNFα 作用的治疗方法，以及缝隙连接（GJ）功能和 Cx43 定位。TNFα 在第 1 天就可诱导 Cx43 表达显著减少，并且下调在第 3 天达到峰值（-46%）。这种减少与基因表达调节或 CTD 切割无关。相反，蛋白酶体抑制剂 MG132 逆转了 TNFα 的作用，表明 Cx43 减少与蛋白酶体降解有关。此外，与合成代谢因子 TGF-β1 的共同处理恢复了 Cx43 水平。Cx43 的减少发生在细胞膜水平，部分影响 GJ 通讯，以及细胞核内。总之，TNFα 诱导 Cx43 表达迅速且持久减少，主要通过蛋白酶体。下调可以通过软骨保护因子如 MG132 和 TGF-β1 逆转。这些发现提示在关节炎症期间 Cx43 失调可能涉及其中。
