Abstract
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.
Highlights
The thyroid hormones, triiodothyronine (T3) and thyroxine (T4) are essential for the growth and maintenance of all cells and organ systems in the body
As craniosynostosis is primarily a bone problem, bone is found where a patent joint should be, we focused our attention to osteogenic factors, as well as biochemical markers that have been previously associated with craniosynostosis
Cells were cultured in alpha minimum Eagles medium supplemented with penicillin/ streptomycin and 1% fetal bovine serum (FBS)
Summary
The thyroid hormones, triiodothyronine (T3) and thyroxine (T4) are essential for the growth and maintenance of all cells and organ systems in the body. There is significant morbidity due to craniosynostosis resulting in various secondary effects including secondary fusion of other sutures, altered cranial base growth, and midface growth discrepancies [10], elevated intracranial pressure [6], altered intracranial volume [6,11], and dilation of the subarachnoid spaces [12]. Such events may result in optic nerve compression, papilledema, and if left uncorrected, may lead to optic atrophy, blindness [13], cognitive disabilities, and mental retardation [14], which pose extensive, costly, and recurrent clinical and surgical management problems [15,16]
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