Pribadi, C.Camp, E.Cakouros, D.Anderson, P.Glackin, C.Gronthos, S.2021-02-122021-02-122020Stem Cell Research and Therapy, 2020; 11(1):529-1-529-141757-65121757-6512http://hdl.handle.net/2440/129807Background: 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. 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. Methods: The present study utilized Twist-1 haploinsufficient (Twist-1(del/+)) 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. Results: This study showed that the histone methyltransferase EZH2, an osteogenesis inhibitor, is downregulated in calvarial cells derived from Twist-1(del/+) 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-1(del/+) 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-1(del/+) calvarial cells in vitro and reduced mineralized bone formation in Twist-1(del/+) 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-1(del/+) mice prevented premature suture fusion and kept the sutures open up to postnatal day 20. Conclusion: The 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.en© The Author(s). 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.Epigenetics; KDM6A; KDM6B; calvarial cells; osteogenesis; coronal sutures, TWIST-1; Twist-1(del/+) mice; Saethre-Chotzen syndrome; craniosynostosisJournal article100003164410.1186/s13287-020-02051-50006001011000052-s2.0-85097377549559417Pribadi, C. [0000-0001-9634-2678]Cakouros, D. [0000-0001-6136-0761]Anderson, P. [0000-0002-3730-4652]Gronthos, S. [0000-0002-6225-3084]