Expression data from hypervitaminosis A rat diaphyseal bone

Vitamin A is the only known compound that produces spontaneous fractures in rats. In an effort to resolve the molecular mechanism behind this effect, we fed young rats high doses of vitamin A and performed a global transcriptional analysis of diaphyseal bone after one week, i.e. just before the first fractures appeared. Microarray gene expression analysis revealed that 68 transcripts were differentially expressed in hypervitaminotic cortical bone and 118 transcripts were found when the bone marrow was also included. 98% of the differentially expressed genes in the bone marrow sample were up-regulated. In contrast, hypervitaminotic cortical bone without marrow showed reduced expression of 37% of differentially expressed genes. Gene Ontology (GO) analysis revealed that only samples containing bone marrow were associated to a GO term, which principally represented extracellular matrix (ECM). This is consistent with the histological findings of increased endosteal bone formation. Four of the genes in this ECM cluster and four other genes, including Cyp26b1 which is known to be up-regulated by vitamin A, were selected and verified by real-time PCR. In addition, immunohistochemical staining of bone sections confirmed that the bone-specific molecule, osteoadherin (Omd) was up-regulated. Further analysis of the major gene expression changes revealed distinct differences between cortical bone and bone marrow, e.g. there appeared to be augmented Wnt signaling in the bone marrow but reduced Wnt signaling in cortical bone. Moreover, induced expression of hypoxia-associated genes was only found in samples containing bone marrow. Together, these results corroborate our previous observations of compartment-specific effects of vitamin A, with reduced periosteal but increased endosteal bone formation, and suggest important roles for Wnt signaling and hypoxia in the processes leading to spontaneous fractures.维生素A是目前已知唯一可在大鼠体内诱发自发性骨折的化合物。为阐明该效应背后的分子机制，我们给幼年大鼠投喂高剂量维生素A，并在给药一周后（即首批自发性骨折出现前夕）对其骨干骨（diaphyseal bone）开展全局转录组分析。微阵列基因表达分析（Microarray gene expression analysis）显示，高维生素A血症大鼠的皮质骨（cortical bone）中存在68个差异表达转录本；若同时纳入骨髓（bone marrow）样本，则可检测到118个差异表达转录本。骨髓样本中98%的差异表达基因呈上调趋势；与之相反，剔除骨髓的高维生素A血症皮质骨中，37%的差异表达基因呈现表达下调。基因本体（Gene Ontology，GO）分析显示，仅包含骨髓的样本可富集到与细胞外基质（extracellular matrix，ECM）相关的GO条目，且该条目为主要富集项。这与骨内膜骨形成增强的组织学观测结果一致。我们选取了该细胞外基质簇中的4个基因，以及另外4个基因（包括已知可被维生素A上调的Cyp26b1），通过实时荧光定量聚合酶链式反应（real-time PCR）验证了其表达变化。此外，对骨切片进行免疫组织化学染色（immunohistochemical staining）证实，骨特异性分子骨黏附蛋白（osteoadherin，Omd）呈上调表达。对主要基因表达变化的进一步分析揭示了皮质骨与骨髓之间的显著差异：例如，骨髓中Wnt信号通路（Wnt signaling）呈现激活增强，而皮质骨中该通路则被抑制。此外，缺氧相关基因的诱导表达仅在包含骨髓的样本中被检测到。综上，本研究结果验证了我们此前的观测结论：维生素A具有组织分区特异性效应——骨膜骨形成受到抑制，而骨内膜骨形成增强；同时提示Wnt信号通路与缺氧过程在自发性骨折的发生发展中发挥关键作用。