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.

Effects of In Utero Thyroxine Exposure on Murine Cranial Suture Growth

“…Previous research by our group suggests thyroxine targets calvarial cells increasing proliferation and osteogenesis when treated in vitro [51]. Western blot analysis of total perisutural tissue PCNA showed increased presence in control samples versus treated, but was not statistically significant, p = 0.1920 (Fig 3E).…”

“…When investigating cellular processes, we concentrated on known effects of thyroid hormone on calvarial cells where increased proliferation and osteogenesis have been observed in vitro [51]. Our immunohistochemistry data suggest increased PCNA, a marker of cell proliferation in the sagittal suture, and increased ALP in the coronal suture specifically, consistent with increased bony infiltration and osteoblastic activity at the suture in the high dose exposed animals.…”

Inkjet-based biopatterning of SDF-1β augments BMP-2-induced repair of critical size calvarial bone defects in mice

“…Differences were considered statistically significant if p<0.05. For quantitative qRT-PCR following standardized methodology [57, 58] we set expression differences compared to control and between groups as likely biologically significant and thus statistically significant if a 2-fold mean change increase or decrease was observed.…”

Effects of thyroxine exposure on the Twist 1 +/− phenotype: A test of gene–environment interaction modeling for craniosynostosis

“…Error bars reflect standard error of the fold change. WT control samples measure of dispersion are included in Supplemental Table S1. precursors are particularly susceptible to this axis, showing great increases in both products after in vitro administration of thyroxine (Bassett and Williams, 2003;Cray et al, 2013). It is likely that the presence of more mature cells, specifically osteoblasts in these cultures might lead to a lack of responsiveness of Twist 1 1/2 cells in this culture system.…”

“…Here, we see little modulation of Htra1 in vitro and an overall decrease in Igf signal in our Twist 1 +/− cells compared with WT controls. It is known that osteoblast precursors are particularly susceptible to this axis, showing great increases in both products after in vitro administration of thyroxine (Bassett and Williams, ; Cray et al, ). It is likely that the presence of more mature cells, specifically osteoblasts in these cultures might lead to a lack of responsiveness of Twist 1 +/− cells in this culture system.…”