Abstract
BackgroundDuring development, excessive osteogenic differentiation of mesenchymal progenitor cells (MPC) within the cranial sutures can lead to premature suture fusion or craniosynostosis, leading to craniofacial and cognitive issues. Saethre-Chotzen syndrome (SCS) is a common form of craniosynostosis, caused by TWIST-1 gene mutations. Currently, the only treatment option for craniosynostosis involves multiple invasive cranial surgeries, which can lead to serious complications.MethodsThe present study utilized Twist-1 haploinsufficient (Twist-1del/+) mice as SCS mouse model to investigate the inhibition of Kdm6a and Kdm6b activity using the pharmacological inhibitor, GSK-J4, on calvarial cell osteogenic potential.ResultsThis study showed that the histone methyltransferase EZH2, an osteogenesis inhibitor, is downregulated in calvarial cells derived from Twist-1del/+ mice, whereas the counter histone demethylases, Kdm6a and Kdm6b, known promoters of osteogenesis, were upregulated. In vitro studies confirmed that siRNA-mediated inhibition of Kdm6a and Kdm6b expression suppressed osteogenic differentiation of Twist-1del/+ calvarial cells. Moreover, pharmacological targeting of Kdm6a and Kdm6b activity, with the inhibitor, GSK-J4, caused a dose-dependent suppression of osteogenic differentiation by Twist-1del/+ calvarial cells in vitro and reduced mineralized bone formation in Twist-1del/+ calvarial explant cultures. Chromatin immunoprecipitation and Western blot analyses found that GSK-J4 treatment elevated the levels of the Kdm6a and Kdm6b epigenetic target, the repressive mark of tri-methylated lysine 27 on histone 3, on osteogenic genes leading to repression of Runx2 and Alkaline Phosphatase expression. Pre-clinical in vivo studies showed that local administration of GSK-J4 to the calvaria of Twist-1del/+ mice prevented premature suture fusion and kept the sutures open up to postnatal day 20.ConclusionThe inhibition of Kdm6a and Kdm6b activity by GSK-J4 could be used as a potential non-invasive therapeutic strategy for preventing craniosynostosis in children with SCS.Graphical abstractPharmacological targeting of Kdm6a/b activity can alleviate craniosynostosis in Saethre-Chotzen syndrome. Aberrant osteogenesis by Twist-1 mutant cranial suture mesenchymal progenitor cells occurs via deregulation of epigenetic modifiers Ezh2 and Kdm6a/Kdm6b. Suppression of Kdm6a- and Kdm6b-mediated osteogenesis with GSK-J4 inhibitor can prevent prefusion of cranial sutures.
Highlights
During development, excessive osteogenic differentiation of mesenchymal progenitor cells (MPC) within the cranial sutures can lead to premature suture fusion or craniosynostosis, leading to craniofacial and cognitive issues
Twist-1del/+ calvarial cells exhibit increased expression and upregulated enzymatic activity of Kdm6a and Kdm6b Calvarial cells derived from 15-day-old Twist-1del/+ mice cultured under osteogenic inductive conditions were found to express reduced transcript levels of Twist-1 and Ezh2, whereas gene expression levels of Kdm6a, Kdm6b, and the early (Runx2) and late bone-associated markers were upregulated, compared to wild type calvarial cells (Fig. 1a)
Parallel studies found that the level of Alizarin redpositive mineralized deposits was significantly reduced in cultures of siRNA Kdm6a or Kdm6b knockdown Twist-1del/+ calvarial cells under osteogenic conditions, compared with scrambled siRNA-treated cells (Fig. 2d)
Summary
Excessive osteogenic differentiation of mesenchymal progenitor cells (MPC) within the cranial sutures can lead to premature suture fusion or craniosynostosis, leading to craniofacial and cognitive issues. The only treatment for craniosynostosis is invasive cranial surgery, mainly involving the removal of the affected sutures and remodeling of the skull [14, 15]. These procedures could negatively impact the quality of life of children with craniosynostosis, leading to serious complications and enforcing the need for invasive surgical procedures [16,17,18]. It has been revealed that epigenetic mechanisms play a significant role in craniosynostosis where studies of genetically identical twins reported that one twin displayed craniosynostosis, whereas the other displayed normal skull development [23, 24]. Until now, no study has thoroughly examined the role of epigenetics in SCS
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