Maternal regulation of SATB2 in osteo‐progeniters impairs skeletal development in offspring

Abstract

This is an open access article under the terms of the Creat ive Commo ns Attri butio n-NonCo mmercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.Abbreviations: ALP, alkaline phosphatase; BMC, bone mineral content; BMD, bone mineral density; BMP4, bone morphogenetic protein 4; BMSCs, bone marrow-derived mesenchymal stem cells; ChIP-seq, chromatin immunoprecipitation with massively parallel DNA sequencing; cko, conditional knockout; E18.5, embryonic day 18.5; EOCCs, embryonic osteogenic calvarial cells; Ezh2, enhancer of zeste homolog 2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; H3K27me3, tri-methylation of lysine 27 on histone H3; HFD, high fat diet; IGF1, insulin-like growth factor 1; Osx, osterix; PND, postnatal day; pQCT, peripheral quantitative computerized tomography; SATB2, special AT-rich sequence-binding protein 2; Tcfap2a, transcription factor AP2 alpha. AbstractNutritional status during intrauterine and/or early postnatal life has substantial influence on adult offspring health. Along these lines, there is a growing body of evidence illustrating that high fat diet (HFD)-induced maternal obesity can regulate fetal bone development. Thus, we investigated the effects of maternal obesity on both fetal skeletal development and mechanisms linking maternal obesity to osteoblast differentiation in offspring. Embryonic osteogenic calvarial cells (EOCCs) were isolated from fetuses at gestational day 18.5 (E18.5) of HFD-induced obese rat dams. We observed impaired differentiation of EOCCs to mature osteoblasts from HFD obese dams. ChIP-seq-based genome-wide localization of the repressive histone mark H3K27me3 (mediated via the polycomb histone methyltransferase, enhancer of zeste homologue 2 [Ezh2]) showed that this phenotype was associated with increased enrichment of H3K27me3 on the gene of SATB2, a critical transcription factor required for osteoblast differentiation. Knockdown of Ezh2 in EOCCs and ST2 cells increased SATB2 expression; while Ezh2 overexpression in EOCCs and ST2 cells decreased SATB2 expression. These data were consistent with experimental results showing strong association between H3K27me3, Ezh2, and SATB2 in cells from rats and humans. We have further presented that SATB2 mRNA and protein expression were increased in bones, and increased trabecular bone mass from pre-osteoblast specific Ezh2 deletion (Ezh2 flox/flox Osx-Cre + cko) mice compared with those from control Cre + mice. These findings indicate that maternal HFD-induced obesity may be associated with decreasing fetal pre-osteoblastic cell differentiation, under epigenetic control of SATB2 expression via Ezh2-dependent mechanisms. K E Y W O R D Sbone, epigenetic, high fat diet, osteoblast 2512 | CHEN Et al.

Ezh2 mediates epigenetic regulation of osteoclastogenesis and bone remodeling in mice

“…Compared with all other genotypic littermates generated by in-bred breeding, Ezh2 deletion in pre-osteoclastic LysM-Cre positive cells promotes bone mass accretion. Indeed, we provide clear evidence in vivo that deletion of Ezh2 in pre-osteoclastic cells inhibits osteoclastogenesis in young adult mice (Chen et al , 2020).…”

“…These data suggest that maternal HFD-induced obesity increases Ezh2 expression in fetal osteoprogenitors, while increased Ezh2 activity suppressed expression of osteoblastic marker genes such as SATB2. Ezh2 facilities association of H3K27me3 with SATB2 in fetal cells from both obese dams in rodents and obese mothers in human (Chen et al , 2020). We described that deletion of Ezh2 gene in osteoblastic specific cell lineage increased bone density, especially trabecular bone density and bone mineral content, and increased SATB2 expression in bone (Chen et al , 2020).…”

“…Ezh2 facilities association of H3K27me3 with SATB2 in fetal cells from both obese dams in rodents and obese mothers in human (Chen et al , 2020). We described that deletion of Ezh2 gene in osteoblastic specific cell lineage increased bone density, especially trabecular bone density and bone mineral content, and increased SATB2 expression in bone (Chen et al , 2020). Together with data presented in our current report, these results clearly demonstrats that Ezh2 expression controls both osteoblast and osteoclast differentiation and activity.…”

“…Murine osteoclast cell line RAW264.7 cells (from ACCT) and non-adherent bone marrow cells were cultured in 96-well plates (2×10 4 cells/well) in the presence or absence of 30 ng/ml of RANKL, in α-MEM supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT), penicillin (100 Units/ml), streptomycin (100 µg/ml), and glutamine (4 mM). These cell cultures were previously described in our laboratory (Chen et al , 2020). After 5 days for bone marrow cell cultures, the cells were fixed with 4% paraformaldehyde and stained for TRAPase activity using a TRAPase staining kit according to the manufacturer’s protocols (Sigma-Aldrich, Acid phosphatase leukocyte, procedure No.…”

“…Despite evidence for a role of Ezh2 in bone cell differentiation, it is not known if this system is involved in maternal HFD-obesity-associated offspring programming of bone development. Depend on different Cre mouse models were used, deletion of Ezh2 in osteoblastic cell lineage resulted in unclear bone developmental patterns (Chen et al , 2020). We recently reported that, using Osterix Cre mouse model, although deletion of Ezh2 in osteoblast precursors resulted in clear osteogenic gene higher expression in bone, bone mass evaluated by peripheral quantitative CT scan showed obscure phenotype when conditional Ezh2 deletion mice compared with Osterix Cre or Ezh flox/flox mice as controls (Levaot et al , 2015).…”