FGF Stimulated PC-1 Expression is regulated by Msx2 and Twist

Mutations in fibroblast growth factor receptors (FGFR's), msx2 and twist are associated with the aberrant mineralization phenotype of the craniosynostosis syndromes. Pyrophosphate is a critical regulator of bone mineralization and the calcification of normally non-mineralized tissues, therefore we hypothesized that FGF signaling would regulate expression of the pyrophosphate generating enzyme, PC-1. We previously showed that FGF2 significantly and specifically induces expression of this enzyme in calvarial pre-osteoblastic cells, and that this is mediated by the transcription factor Runx2. Objectives: To expand upon these findings, here we investigate the regulation of PC-1 gene expression by craniosynostosis syndrome associated transcription factors, msx2 and twist. Methods: mRNA expression was analyzed by real time PCR. Protein expression was analyzed by western. PC-1 promoter activity was monitored by luciferase assay. Transcription factor binding was analyzed by electrophoretic mobility shift assay and chromatin immunoprecipitation. Results: Here we show that calvarial pre-osteoblastic PC-1 gene expression is up-regulated by msx2 and down-regulated by twist. Sequence analysis of the PC-1 gene promoter reveals one putative msx2 and five putative twist binding sites. Experiments utilizing PC-1 gene promoter/ luciferase reporter constructs confirm that msx2 increases PC-1 gene promoter activity and expression, while twist inhibits this. Conclusions: These results suggest that the transcription of PC-1 is mediated by msx2 and twist. As craniosynostosis associated mutations in FGF receptors and msx2 are known to be activating, while those in twist are known to be inactivating, these findings are highly consistent with the hypothesis that calvarial pre-osteoblastic PC-1 expression plays a critical role in the molecular pathogenesis of craniosynostosis. (NIH/NIDCR DE007057 & DE11723)