Faculty of Medicine and Health Sciences, Department of Anatomy and Cell Biology, McGill University, Montreal, QC, Canada.
CHU Sainte-Justine Research Center, Montreal, QC, Canada.
Matrix Biol. 2024 Nov;133:86-102. doi: 10.1016/j.matbio.2024.08.002. Epub 2024 Aug 17.
Fibronectin (FN) is a ubiquitous extracellular matrix glycoprotein essential for the development of various tissues. Mutations in FN cause a unique form of spondylometaphyseal dysplasia, emphasizing its importance in cartilage and bone development. However, the relevance and functional role of FN during skeletal development has remained elusive. To address these aspects, we have generated conditional knockout mouse models targeting the cellular FN isoform in cartilage (cFNKO), the plasma FN isoform in hepatocytes (pFNKO), and both isoforms together in a double knockout (FNdKO). We used these mice to determine the relevance of the two principal FN isoforms in skeletal development from postnatal day one to the adult stage at two months. We identified a distinct topological FN deposition pattern in the mouse limb during different gestational and postnatal skeletal development phases, with prominent levels at the resting and hypertrophic chondrocyte zones and in the trabecular bone. Cartilage-specific cFN emerged as the predominant isoform in the growth plate, whereas circulating pFN remained excluded from the growth plate and confined to the primary and secondary ossification centers. Deleting either isoform independently (cFNKO or pFNKO) yielded only relatively subtle changes in the analyzed skeletal parameters. However, the double knockout of cFN in the growth plate and pFN in the circulation of the FNdKO mice significantly reduced postnatal body weight, body length, and bone length. Micro-CT analysis of the adult bone microarchitecture in FNdKO mice exposed substantial reductions in trabecular bone parameters and bone mineral density. The mice also showed elevated bone marrow adiposity. Analysis of chondrogenesis in FNdKO mice demonstrated changes in the resting, proliferating and hypertrophic growth plate zones, consistent alterations in chondrogenic markers such as collagen type II and X, decreased apoptosis of hypertrophic chondrocytes, and downregulation of bone formation markers. Transforming growth factor-β1 and downstream phospho-AKT levels were significantly lower in the FNdKO than in the control mice, revealing a crucial FN-mediated regulatory pathway in chondrogenesis and bone formation. In conclusion, the data demonstrate that FN is essential for chondrogenesis and bone development. Even though cFN and pFN act in different regions of the bone, both FN isoforms are required for the regulation of chondrogenesis, cartilage maturation, trabecular bone formation, and overall skeletal growth.
纤连蛋白（FN）是一种普遍存在的细胞外基质糖蛋白，对各种组织的发育至关重要。FN 突变导致一种独特的脊椎-干骺端发育不良形式，强调其在软骨和骨骼发育中的重要性。然而，FN 在骨骼发育中的相关性和功能作用仍然难以捉摸。为了解决这些问题，我们生成了针对软骨细胞中细胞纤连蛋白（cFN）同种型（cFNKO）、肝细胞中血浆纤连蛋白（pFN）同种型（pFNKO）以及两种同种型的条件性敲除（FNdKO）的小鼠模型。我们使用这些小鼠来确定在从出生后第一天到两个月的成年阶段的骨骼发育过程中，两种主要 FN 同种型的相关性。我们在不同的胚胎和出生后骨骼发育阶段发现了小鼠肢体中独特的 FN 沉积模式，在静止和肥大软骨细胞区和小梁骨中存在明显的水平。在生长板中，特异性的 cFN 成为主要同种型，而循环的 pFN 仍然被排除在生长板之外，并局限于初级和次级骨化中心。单独删除任何一种同种型（cFNKO 或 pFNKO）仅导致分析的骨骼参数发生相对较小的变化。然而，FNdKO 小鼠中生长板中的 cFN 和循环中的 pFN 的双重敲除显著降低了出生后的体重、体长和骨长。FNdKO 小鼠的成年骨微结构的 micro-CT 分析显示，小梁骨参数和骨矿物质密度有显著降低。这些小鼠还表现出骨髓脂肪增多。对 FNdKO 小鼠的软骨发生分析显示，静止、增殖和肥大生长板区发生变化，软骨形成标志物如 II 型和 X 型胶原的一致性改变，肥大软骨细胞凋亡减少，以及骨形成标志物的下调。FNdKO 小鼠中的转化生长因子-β1 和下游磷酸化 AKT 水平明显低于对照小鼠，揭示了 FN 介导的软骨发生和骨形成中的关键调节途径。总之，数据表明 FN 对软骨发生和骨骼发育至关重要。尽管 cFN 和 pFN 在骨骼的不同区域起作用，但两种 FN 同种型都需要调节软骨发生、软骨成熟、小梁骨形成和整体骨骼生长。
