Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-Cho, Kita-ku, Okayama-City, Okayama 700-8525, Japan.
J Cell Sci. 2012 Jun 15;125(Pt 12):2965-72. doi: 10.1242/jcs.101956. Epub 2012 Mar 27.
Low-density lipoprotein receptor-related protein 1 (LRP1) is known to be a receptor for signal transmission and endocytosis. We have previously reported that LRP1 regulates WNT-β-catenin and protein kinase C signaling in chondrocytes, represses the hypertrophy of chondrocytes during endochondral ossification and that LRP1 is colocalized with a ligand, CCN family member 2 (CCN2; also known as connective tissue growth factor, CTGF), which conducts endochondral ossification, in chondrocytes. However, the role of LRP1 in the endocytic transport of CCN2 in chondrocytes is not yet understood. In the present study, we investigated the interaction between LRP1 and CCN2 during endocytic trafficking. Small interfering RNA (siRNA)-mediated knockdown of LRP1 in chondrocytic HCS-2/8 cells showed that the amount of exogenous CCN2 binding and/or incorporation was decreased in the LRP1 downregulated cells. Importantly, we observed that CCN2 internalization in chondrocytes was dependent on clathrin, and internalizated CCN2 was colocalized with an early or recycling endosome marker. Transcytosis of CCN2 through HCS-2/8 cells was confirmed by performing experiments with a trans-well apparatus, and the amount of transcytosed CCN2 was decreased by an LRP1 antagonist. These findings rule out possible leakage and confirm the crucial involvement of LRP1 during experimental transcytosis. Moreover, under hypoxic conditions that mimic the cartilaginous microenvironment, the level of LRP1 and the amount of transcytosed CCN2 increased, and these increases were neutralized by treatment with the LRP1 antagonist. The distribution of LRP1 and its antagonist in the growth plate in vivo was consistent with that of CCN2 in this tissue, which is produced by and transported by LRP1 from the chondrocytes in the prehypertrophic layer. These findings suggest that LRP1 mediates the transcytosis of CCN2, which might be a crucial event that determines the distribution of CCN2 in cartilage.
低密度脂蛋白受体相关蛋白 1（LRP1）是一种已知的信号转导和内吞作用受体。我们之前曾报道过，LRP1 调节软骨细胞中的 WNT-β-连环蛋白和蛋白激酶 C 信号通路，抑制软骨细胞在软骨内骨化过程中的肥大，并且 LRP1 与配体 CCN 家族成员 2（CCN2；也称为结缔组织生长因子，CTGF）共定位，CCN2 介导软骨内骨化。然而，LRP1 在软骨细胞中 CCN2 的内吞转运中的作用尚不清楚。在本研究中，我们研究了 LRP1 和 CCN2 在内吞运输过程中的相互作用。在软骨细胞 HCS-2/8 中用小干扰 RNA（siRNA）介导的 LRP1 敲低表明，下调 LRP1 的细胞中外源 CCN2 结合和/或掺入减少。重要的是，我们观察到 CCN2 在软骨细胞中的内化依赖于网格蛋白，内化的 CCN2 与早期或再循环内体标志物共定位。通过使用 Transwell 装置进行实验证实了 CCN2 通过 HCS-2/8 细胞的转胞吞作用，并且 LRP1 拮抗剂减少了转胞吞作用的 CCN2 量。这些发现排除了可能的渗漏，并证实了 LRP1 在实验性转胞吞作用中的关键作用。此外，在模拟软骨微环境的缺氧条件下，LRP1 水平和转胞吞作用的 CCN2 量增加，用 LRP1 拮抗剂处理可中和这些增加。LRP1 及其拮抗剂在体内生长板中的分布与该组织中 CCN2 的分布一致，CCN2 由 LRP1 从肥大前层的软骨细胞中产生并转运。这些发现表明 LRP1 介导了 CCN2 的转胞吞作用，这可能是决定 CCN2 在软骨中分布的关键事件。
