Faculty of Medicine and Health Sciences, Department of Anatomy and Cell Biology, McGill University, 3640 University Street, Montreal, QC, Canada.
Centre Hospitalier Universitaire Sainte-Justine Research Center, Montreal, QC, Canada.
Cell Mol Life Sci. 2024 Oct 5;81(1):419. doi: 10.1007/s00018-024-05444-4.
Fibronectin (FN) is an extracellular matrix glycoprotein essential for the development and function of major vertebrate organ systems. Mutations in FN result in an autosomal dominant skeletal dysplasia termed corner fracture-type spondylometaphyseal dysplasia (SMDCF). The precise pathomechanisms through which mutant FN induces impaired skeletal development remain elusive. Here, we have generated patient-derived induced pluripotent stem cells as a cell culture model for SMDCF to investigate the consequences of FN mutations on mesenchymal stem cells (MSCs) and their differentiation into cartilage-producing chondrocytes. In line with our previous data, FN mutations disrupted protein secretion from MSCs, causing a notable increase in intracellular FN and a significant decrease in extracellular FN levels. Analyses of plasma samples from SMDCF patients also showed reduced FN in circulation. FN and endoplasmic reticulum (ER) protein folding chaperones (BIP, HSP47) accumulated in MSCs within ribosome-covered cytosolic vesicles that emerged from the ER. Massive amounts of these vesicles were not cleared from the cytosol, and a smaller subset showed the presence of lysosomal markers. The accumulation of intracellular FN and ER proteins elevated cellular stress markers and altered mitochondrial structure. Bulk RNA sequencing revealed a specific transcriptomic dysregulation of the patient-derived cells relative to controls. Analysis of MSC differentiation into chondrocytes showed impaired mesenchymal condensation, reduced chondrogenic markers, and compromised cell proliferation in mutant cells. Moreover, FN mutant cells exhibited significantly lower transforming growth factor beta-1 (TGFβ1) expression, crucial for mesenchymal condensation. Exogenous FN or TGFβ1 supplementation effectively improved the MSC condensation and promoted chondrogenesis in FN mutant cells. These findings demonstrate the cellular consequences of FN mutations in SMDCF and explain the molecular pathways involved in the associated altered chondrogenesis.
纤连蛋白（FN）是一种细胞外基质糖蛋白，对脊椎动物主要器官系统的发育和功能至关重要。FN 突变导致常染色体显性遗传骨骼发育不良，称为角骨折型脊椎干骺端发育不良（SMDCF）。突变 FN 诱导骨骼发育受损的确切病理机制仍不清楚。在这里，我们生成了患者来源的诱导多能干细胞作为 SMDCF 的细胞培养模型，以研究 FN 突变对间充质干细胞（MSCs）及其分化为产生软骨的软骨细胞的影响。与我们之前的数据一致，FN 突变破坏了 MSC 的蛋白分泌，导致细胞内 FN 明显增加，细胞外 FN 水平显著降低。对 SMDCF 患者的血浆样本的分析也显示循环中 FN 减少。FN 和内质网（ER）蛋白折叠伴侣（BIP、HSP47）在内质网衍生的覆盖核糖体的胞质小泡中在 MSC 中积累。这些小泡的大量物质未从胞质溶胶中清除，并且较小的子集显示出溶酶体标记物的存在。细胞内 FN 和 ER 蛋白的积累增加了细胞应激标志物并改变了线粒体结构。批量 RNA 测序显示患者来源的细胞相对于对照存在特定的转录组失调。分析 MSC 分化为软骨细胞表明，突变细胞中的间充质凝聚受损，软骨形成标志物减少，细胞增殖受损。此外，FN 突变细胞表现出显著降低的转化生长因子β-1（TGFβ1）表达，这对于间充质凝聚至关重要。外源性 FN 或 TGFβ1 补充有效地改善了 FN 突变细胞的 MSC 凝聚并促进了软骨生成。这些发现表明了 SMDCF 中 FN 突变的细胞后果，并解释了相关改变的软骨生成所涉及的分子途径。
