Wiweger Malgorzata I, de Andrea Carlos E, Scheepstra Karel W F, Zhao Zhe, Hogendoorn Pancras C W
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.
Orphanet J Rare Dis. 2014 Mar 14;9:35. doi: 10.1186/1750-1172-9-35.
Mutations in the EXT genes disrupt polymerisation of heparan sulphates (HS) and lead to the development of osteochondroma, an isolated/sporadic- or a multifocal/hereditary cartilaginous bone tumour. Zebrafish (Danio rerio) is a very powerful animal model which has shown to present the same cartilage phenotype that is commonly seen in mice model and patients with the rare hereditary syndrome, Multiple Osteochondroma (MO).
Zebrafish dackel (dak) mutant that carries a nonsense mutation in the ext2 gene was used in this study. A panel of molecular, morphological and biochemical analyses was used to assess at what step bone formation is affected and what mechanisms underlie changes in the bone formation in the ext2 mutant.
During bone development in the ext2-/- zebrafish, chondrocytes fail to undergo terminal differentiation; and pre-osteoblasts do not differentiate toward osteoblasts. This inadequate osteogenesis coincides with increased deposition of lipids/fats along/in the vessels and premature adipocyte differentiation as shown by biochemical and molecular markers. Also, the ext2-null fish have a muscle phenotype, i.e. muscles are shorter and thicker. These changes coexist with misshapen bones. Normal expression of runx2 together with impaired expression of osterix and its master regulator--xbp1 suggest that unfolded protein responses might play a role in MO pathogenesis.
Heparan sulphates are required for terminal differentiation of the cartilaginous template and consecutive formation of a scaffold that is needed for further bone development. HS are also needed for mesenchymal cell differentiation. At least one copy of ext2 is needed to maintain the balance between bone and fat lineages, but homozygous loss of the ext2 function leads to an imbalance between cartilage, bone and fat lineages. Normal expression of runx2 and impaired expression of osterix in the ext2-/- fish indicate that HS are required by osteoblast precursors for their further differentiation towards osteoblastic lineage. Lower expression of xbp1, a master regulator of osterix, suggests that HS affect the 'unfolded protein response', a pathway that is known to control bone formation and lipid metabolism. Our observations in the ext2-null fish might explain the musculoskeletal defects that are often observed in MO patients.
EXT基因的突变会破坏硫酸乙酰肝素（HS）的聚合，并导致骨软骨瘤的发生，骨软骨瘤是一种孤立性/散发性或多灶性/遗传性软骨性骨肿瘤。斑马鱼（Danio rerio）是一种非常强大的动物模型，已显示出与小鼠模型以及患有罕见遗传性综合征——多发性骨软骨瘤（MO）的患者中常见的相同软骨表型。
本研究使用了在ext2基因中携带无义突变的斑马鱼达克（dak）突变体。采用了一系列分子、形态学和生化分析方法，以评估骨形成在哪个步骤受到影响，以及ext2突变体中骨形成变化的潜在机制。
在ext2基因敲除的斑马鱼骨发育过程中，软骨细胞无法进行终末分化；前成骨细胞也不会向成骨细胞分化。这种成骨不足与血管周围脂质/脂肪沉积增加以及脂肪细胞过早分化同时出现，这通过生化和分子标记得以证实。此外，ext2基因缺失的鱼具有肌肉表型，即肌肉更短更厚。这些变化与畸形的骨骼同时存在。runx2的正常表达以及osterix及其主要调节因子xbp1的表达受损表明，未折叠蛋白反应可能在MO发病机制中起作用。
硫酸乙酰肝素是软骨模板终末分化以及进一步骨发育所需支架连续形成所必需的。HS对于间充质细胞分化也很必要。维持骨和脂肪谱系之间的平衡至少需要一个ext2拷贝，但ext2功能的纯合缺失会导致软骨、骨和脂肪谱系之间的失衡。ext2基因敲除鱼中runx2的正常表达和osterix的表达受损表明，HS是成骨细胞前体向成骨谱系进一步分化所必需的。osterix的主要调节因子xbp1的较低表达表明，HS影响“未折叠蛋白反应”，这是一种已知可控制骨形成和脂质代谢的途径。我们在ext2基因缺失鱼中的观察结果可能解释了MO患者中经常观察到的肌肉骨骼缺陷。
