KDM6A-Mediated Regulation of Cranial Frontal Bone Suture Fusion in Mice Is Sex Dependent.

The five flat bones of developing cranial plates are bounded by fibrous sutures, which remain open during development to accommodate for the growing brain. Kdm6A is a demethylase that removes the epigenetic repressive mark, trimethylated lysine 27 on histone 3 (H3K27me3), from the promoters of osteogenic genes, and has previously been reported to promote osteogenesis in cranial bone cells. This study generated a mesenchyme-specific deletion of a histone demethylase, Kdm6a, to assess the effects of Kdm6a loss, in cranial plate development and suture fusion. The results showed that the loss of Kdm6a in Prx1+ cranial cells caused increased anterior width and length in the calvaria of both male and female mice. However, the posterior length was further decreased in female mice. Moreover, loss of Kdm6a resulted in suppression of late suture development and calvarial frontal bone formation predominantly in female mice. In vitro assessment of calvaria cultures isolated from female Kdm6a knockout mice found significantly suppressed calvarial osteogenic differentiation potential, associated with decreased gene expression levels of Runx2 and Alkaline Phosphatase and increased levels of the suppressive mark, H3K27me3, on the respective gene promoters. Conversely, cultured calvaria bone cultures isolated from male Kdm6a knockout mice exhibited an increased osteogenic differentiation potential. Interestingly, the milder effects on cranial suture development in Kdm6a knockout male mice, were associated with an overcompensation of the Kdm6a Y-homolog, Kdm6c, and increased expression levels of Kdm6b in calvarial bone cultures. Taken together, these data demonstrate a role for Kdm6a during calvarial development and patterning, predominantly in female mice, and highlight the potential role of Kdm6 family members in patients with unexplained craniofacial deformities.

Pharmacological targeting of KDM6A and KDM6B, as a novel therapeutic strategy for treating craniosynostosis in Saethre-Chotzen syndrome.

BACKGROUND: 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-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. RESULTS: This 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. 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. Pharmacological 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.

miRNA-376c-3p Mediates TWIST-1 Inhibition of Bone Marrow-Derived Stromal Cell Osteogenesis and Can Reduce Aberrant Bone Formation of TWIST-1 Haploinsufficient Calvarial Cells.

Key transcription factors, which activate or repress master gene regulators and signaling pathways, tightly regulate self-renewal and cell lineage differentiation of bone marrow-derived stromal cells (BMSC). Among these factors is the basic helix-loop-helix transcription factor Twist-related protein 1 (TWIST-1), which is important in BMSC self-renewal, life span, and differentiation. Another layer of gene regulation comes from microRNAs (miRNAs). miRNAs are short noncoding RNAs that interfere with translation of specific target mRNAs and thereby regulate diverse biological processes, including BMSC lineage commitment. However, little is known of how TWIST-1-regulated miRNAs control osteogenic commitment, and influence the fate of bone precursor cells. In this study, we have discovered a novel TWIST-1-regulated miRNA, miR-376c-3p. Reduced miR-376c-3p expression by a miR-376c-3p inhibitor or due to TWIST-1 haploinsufficiency promotes alkaline phosphatase (ALP) activity, mineral deposition, and expression of osteoblast-associated genes in BMSC and calvarial cells. Conversely, overexpression of miR-376c-3p using a miR-376c-3p mimic inhibited BMSC proliferation and the osteogenic potential of BMSC and TWIST-1 haploinsufficient calvarial cells. This was demonstrated by a decrease in insulin growth factor 1 receptor (IGF1R) levels, Akt signaling, ALP activity, mineral deposition, and expression of osteoblast-associated genes. Thus, miR-376c-3p reduces IGF1R/Akt signaling in BMSC and is one mechanism by which osteogenesis may be inhibited. Overall, we have identified miR-376c-3p as a TWIST-1-regulated miRNA, which plays an important role in the osteogenesis of bone precursor cells and can mediate TWIST-1 inhibition of osteogenesis. Furthermore, overexpression of miRNA-376c-3p in TWIST-1 haploinsufficient calvarial cells can decrease the aberrant osteogenesis of these cells, which contributes to increased calvarial bone volume and premature fusion of the coronal sutures.