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Sfrp4 Expression in Thyroxine Treated Calvarial Cells: A Novel Target

Evidence suggests alterations of thyroid hormone levels can disrupt normal craniofacial development. Most data suggest the major targets of thyroid hormones to be the Htra1/Igf1 pathway. Recent discovery by our group suggests involvement of targets of the WNT pathway, specifically overexpression of antagonist Sfrp4 in the presence of excess thyroid hormone. Here we aimed to model these interactions in vitro to determine if thyroid hormone directly drives Sfrp4 expression in cells relevant to craniofacial development. Affymetrix transcriptional profiling found Sfrp4 to be overexpressed in cranial suture derived cells stimulated with thyroxine in vitro. Bioinformatics interrogation of the SFRP4 promoter identified multiple putative binding sites for thyroid hormone receptors. Experimentation with several cell lines demonstrated that thyroxine treatment induced Sfrp4 expression. However, protein levels did not correlate with induction of gene expression, demonstrating that Sfrp4 mRNA and protein levels are not tightly coupled. Transcriptional and protein analyses demonstrate thyroid hormone receptor binding to the proximal promoter of the target gene Sfrp4. Interestingly functional analysis of stem cells, fibroblasts, or primary culture cells after thyroxine stimulation for alkaline phosphatase activity shows only pre‐osteoblasts are pushed to become positive after thyroid hormone stimulation, suggesting cell type susceptibility. Finally, we added recombinant SFRP4 to pre‐osteoblasts in combination with thyroxine treatment and observed a significant decrease in alkaline phosphatase positivity, suggesting SFRP4 may be a key regulatory molecule that prevents thyroxine driven osteogenesis. The implications for this relationship indicate SFRP4 as a potential diagnostic or therapeutic target for hyperostotic craniofacial disorders.