Effects of thyroxine exposure on osteogenesis in mouse calvarial pre-osteoblasts

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.

Figure 1. Proliferation: Cell proliferation assay after thyroxine treatment.

Reference line indicates control (untreated) response and experimental group plotted as percent response compared to reference (error bars = standard error of the mean). Note the increases in proliferation after 3 and 7 days of treatment with the exception of the highest dose after 7 days.

Figure 2. (a) Quantitative Alkaline Phosphatase: Alkaline Phosphatase activity assay indicative of cell differentiation.

Reference line indicates control (untreated) response and experimental group plotted as percent response compared to reference (error bars = standard error of the mean). Note the great increases in response after 7 days of treatment for doses 10⁻⁵ through 10⁻⁷mM. (b) Alkaline Phosphatase Stain. Representative assay for Alkaline Phosphatase after 7 days in culture with thyroxine treatment. PM indicated proliferation media only. −4,−5,−6,−7,−8,−9,10 indicated thyroxine dose doses 10⁻⁴ through 10⁻¹⁰mM.

Figure 3. Gene Expression: Fold change mRNA for markers of osteoblastogenesis after thyroxine treatment for 3 or 7 days (error bars = standard error of the mean fold change).

Note greater than two-fold up-regulations (reference lines) for alkaline phosphatase and osteocalcin.

Figure 4. Array Confirmation: Confirmation of targets from gene arrays of interest.

Expressed as fold changes mRNA after thyroxine treatment for 3 or 7 days (error bars = standard error of the mean fold change). Note two-fold changes (reference lines) or greater for targets: Phex, DMP1, HTRA1,FGF1,FGF2, POR, DCN, IGF1, and MMP9.