Ishida Yamato, Hoshi Haruka, Kawano Kenichi, Shin Hye-Won, Katoh Yohei, Nakayama Kazuhisa
Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
Department of Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
J Cell Sci. 2025 Oct 15;138(20). doi: 10.1242/jcs.263737. Epub 2025 Jun 20.
Skeletal ciliopathies result from defects in primary cilia, which are crucial for embryonic development because they transduce extracellular signals, including Hedgehog. Selective transport of ciliary proteins is mediated by the intraflagellar transport (IFT) machinery, containing the IFT-A and IFT-B complexes and the kinesin-2 and dynein-2 motors. Biallelic loss-of-function variants in genes encoding dynein-2-specific subunits, including DYNC2LI1, cause skeletal ciliopathies. As mesenchymal stem cells (MSCs) differentiate into osteoblasts, we investigated the effects of pathogenic variants of DYNC2LI1 on osteogenic differentiation of the MSC-like line C3H10T1/2. Dync2li1-knockout cells expressing disease-causing DYNC2LI1 variants demonstrated defects in the retrograde ciliary protein trafficking, including Hedgehog pathway GPCRs, Smoothened and GPR161. Furthermore, Dync2li1-knockout cells expressing the pathogenic variants demonstrated impaired Hedgehog signaling, in particular, a reduced ratio of the GLI3 repressor form to total GLI3, resulting in impaired osteogenic differentiation of MSCs. By contrast, osteogenic differentiation via BMP signaling was derepressed in Dync2li1-knockout cells. This suggests that skeletal ciliopathies caused by DYNC2LI1 variants could be attributable in part to impaired osteogenic differentiation due to defects in Hedgehog signaling, resulting from defects in retrograde ciliary protein trafficking.
骨骼纤毛病是由初级纤毛缺陷引起的，初级纤毛对胚胎发育至关重要，因为它们可转导包括刺猬信号通路（Hedgehog）在内的细胞外信号。纤毛蛋白的选择性运输由鞭毛内运输（IFT）机制介导，该机制包含IFT-A和IFT-B复合物以及驱动蛋白-2和动力蛋白-2马达。编码动力蛋白-2特异性亚基（包括DYNC2LI1）的基因中的双等位基因功能丧失变体可导致骨骼纤毛病。由于间充质干细胞（MSC）可分化为成骨细胞，我们研究了DYNC2LI1致病变体对类MSC细胞系C3H10T1/2成骨分化的影响。表达致病DYNC2LI1变体的Dync2li1基因敲除细胞在纤毛蛋白逆行运输方面存在缺陷，包括刺猬信号通路GPCR、平滑受体（Smoothened）和GPR161。此外，表达致病变体的Dync2li1基因敲除细胞显示刺猬信号传导受损，特别是GLI3阻遏物形式与总GLI3的比例降低，导致MSC的成骨分化受损。相比之下，Dync2li1基因敲除细胞中通过骨形态发生蛋白（BMP）信号传导的成骨分化则被解除抑制。这表明由DYNC2LI1变体引起的骨骼纤毛病可能部分归因于由于逆行纤毛蛋白运输缺陷导致的刺猬信号传导缺陷而引起的成骨分化受损。
