Department of Medical Sciences, Section of Clinical Pharmacology, Uppsala University, UAS, Ing 70 3tr Foa2 Lab22, 75185 Uppsala, Sweden.
Calcif Tissue Int. 2012 Mar;90(3):219-29. doi: 10.1007/s00223-011-9561-6. Epub 2012 Jan 4.
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 male rats high doses of vitamin A and performed microarray analysis of diaphyseal bone with and without marrow after 1 week, i.e., just before the first fractures appeared. Of the differentially expressed genes in cortical bone, including marrow, 98% were upregulated. 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 with a GO term, which principally represented extracellular matrix. This is consistent with the histological findings of increased endosteal/marrow osteoblast number. Fourteen genes, including Cyp26b1, which is known to be upregulated 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 was upregulated. Further analysis of the major gene-expression changes revealed apparent augmented Wnt signaling in the sample containing bone marrow but reduced Wnt signaling in cortical bone. Moreover, induced expression of hypoxia-associated genes was found only in samples containing bone marrow. Together, these results highlight the importance of compartment-specific analysis of bone and corroborate previous observations of compartment-specific effects of vitamin A, with reduced activity in cortical bone but increased activity in the endosteal/marrow compartment. We specifically identify potential key osteoblast-, Wnt signaling-, and hypoxia-associated genes in the processes leading to spontaneous fractures.
维生素 A 是唯一已知的能在大鼠体内引发自发性骨折的化合物。为了阐明这种作用的分子机制，我们给雄性幼鼠喂食大剂量的维生素 A ，并在第 1 周（即在首次出现骨折之前）对骨干进行微阵列分析，无论是否有骨髓。在包括骨髓在内的皮质骨中差异表达的基因中，有 98%是上调的。相比之下，无骨髓的高维生素 A 皮质骨中，有 37%的差异表达基因表达下调。基因本体论（GO）分析表明，只有包含骨髓的样本与 GO 术语相关，该术语主要代表细胞外基质。这与骨内膜/骨髓成骨细胞数量增加的组织学发现一致。我们选择了包括 Cyp26b1 在内的 14 个基因进行实时 PCR 验证，该基因已知受维生素 A 上调。此外，对骨切片的免疫组织化学染色证实了骨特异性分子骨粘连蛋白的上调。对主要基因表达变化的进一步分析表明，在含有骨髓的样本中，Wnt 信号明显增强，但在皮质骨中，Wnt 信号减弱。此外，仅在含有骨髓的样本中发现了与缺氧相关基因的诱导表达。总之，这些结果强调了对骨进行特定部位分析的重要性，并证实了维生素 A 的特定部位效应的先前观察结果，即在皮质骨中活性降低，但在内骨膜/骨髓部位活性增加。我们特别确定了在自发性骨折发生过程中潜在的关键成骨细胞、Wnt 信号和缺氧相关基因。
