Department of Oral Biology, Georgia Regents University, Augusta, Georgia, United States of America.
PLoS One. 2013 Jul 23;8(7):e69067. doi: 10.1371/journal.pone.0069067. Print 2013.
The incidence of craniosynostosis is one in every 1,800-2500 births. The gene-environment model proposes that if a genetic predisposition is coupled with environmental exposures, the effects can be multiplicative resulting in severely abnormal phenotypes. At present, very little is known about the role of gene-environment interactions in modulating craniosynostosis phenotypes, but prior evidence suggests a role for endocrine factors. Here we provide a report of the effects of thyroid hormone exposure on murine calvaria cells. Murine derived calvaria cells were exposed to critical doses of pharmaceutical thyroxine and analyzed after 3 and 7 days of treatment. Endpoint assays were designed to determine the effects of the hormone exposure on markers of osteogenesis and included, proliferation assay, quantitative ALP activity assay, targeted qPCR for mRNA expression of Runx2, Alp, Ocn, and Twist1, genechip array for 28,853 targets, and targeted osteogenic microarray with qPCR confirmations. Exposure to thyroxine stimulated the cells to express ALP in a dose dependent manner. There were no patterns of difference observed for proliferation. Targeted RNA expression data confirmed expression increases for Alp and Ocn at 7 days in culture. The genechip array suggests substantive expression differences for 46 gene targets and the targeted osteogenesis microarray indicated 23 targets with substantive differences. 11 gene targets were chosen for qPCR confirmation because of their known association with bone or craniosynostosis (Col2a1, Dmp1, Fgf1, 2, Igf1, Mmp9, Phex, Tnf, Htra1, Por, and Dcn). We confirmed substantive increases in mRNA for Phex, FGF1, 2, Tnf, Dmp1, Htra1, Por, Igf1 and Mmp9, and substantive decreases for Dcn. It appears thyroid hormone may exert its effects through increasing osteogenesis. Targets isolated suggest a possible interaction for those gene products associated with calvarial suture growth and homeostasis as well as craniosynostosis.
颅缝早闭的发病率为每 1800-2500 例出生一例。基因-环境模型提出，如果遗传易感性与环境暴露相结合，其影响可能是相乘的，导致严重的异常表型。目前，人们对基因-环境相互作用在调节颅缝早闭表型中的作用知之甚少，但先前的证据表明内分泌因素起作用。在这里，我们报告了甲状腺激素暴露对鼠颅骨细胞的影响。将鼠源性颅骨细胞暴露于药物甲状腺素的临界剂量下，并在处理后 3 天和 7 天进行分析。终点测定设计用于确定激素暴露对成骨标志物的影响，包括增殖测定、定量碱性磷酸酶（ALP）活性测定、Runx2、Alp、Ocn 和 Twist1 的靶向 qPCR 测定、28853 个靶基因的基因芯片阵列和具有 qPCR 验证的靶向成骨微阵列。甲状腺素暴露以剂量依赖的方式刺激细胞表达 ALP。在培养 7 天时，增殖没有观察到差异模式。靶向 RNA 表达数据证实 Alp 和 Ocn 的表达在培养 7 天时增加。基因芯片阵列表明，46 个基因靶标存在实质性表达差异，靶向成骨微阵列表明 23 个靶标存在实质性差异。由于它们与骨骼或颅缝早闭的已知关联，选择了 11 个基因靶标进行 qPCR 确认（Col2a1、Dmp1、Fgf1、2、Igf1、Mmp9、Phex、Tnf、Htra1、Por 和 Dcn）。我们证实了 Phex、FGF1、2、Tnf、Dmp1、Htra1、Por、Igf1 和 Mmp9 的 mRNA 实质性增加，以及 Dcn 的实质性减少。似乎甲状腺激素可能通过增加成骨作用来发挥其作用。分离的靶标表明，与颅骨缝生长和动态平衡以及颅缝早闭相关的那些基因产物可能存在相互作用。
