Torres, Haydee M

Abstract Osteoporosis and other metabolic bone diseases are prevalent in the aging population. While bone has the capacity to regenerate throughout life, bone formation rates decline with age and contribute to reduced bone density and strength. Identifying mechanisms and pathways that increase bone accrual in adults could prevent fractures and accelerate healing. G protein-gated inwardly rectifying K+ (GIRK) channels are key effectors of G protein-coupled receptor signaling. Girk3 was recently shown to regulate endochondral ossification. Here, we demonstrate that deletion of Girk3 increases bone mass after 18 weeks of age. Male 24-week-old Girk3-/- mice have greater trabecular bone mineral density and bone volume fraction than wildtype (WT) mice. Osteoblast activity is moderately increased in 24-week-old Girk3-/- mice compared to WT mice. In vitro, Girk3-/- bone marrow stromal cells (BMSCs) are more proliferative than WT BMSCs. Calvarial osteoblasts and BMSCs from Girk3-/- mice are also more osteogenic than WT cells, with altered expression of genes that regulate the wingless-related integration site (Wnt) family. Wnt inhibition via Dickkopf-1 (Dkk1) or β-catenin inhibition via XAV939 prevents enhanced mineralization, but not proliferation, in Girk3-/- BMSCs and slows these processes in WT cells. Finally, selective ablation of Girk3 from cells expressing Cre recombinase from the 2.3 kb-Col1a1 promoter, including osteoblasts and osteocytes, is sufficient to increase bone mass and bone strength in male mice at 24 weeks of age. Taken together, these data demonstrate that Girk3 regulates progenitor cell proliferation, osteoblast differentiation, and bone mass accrual in adult male mice.

AbstractBackgroundSkeletal development requires precise extrinsic and intrinsic signals to regulate processes that form and maintain bone and cartilage. Notch1 is a highly conserved signaling receptor that regulates cell fate decisions by controlling the duration of transcriptional bursts. Epigenetic molecular events reversibly modify DNA and histone tails by influencing the spatial organization of chromatin and can fine‐tune the outcome of a Notch1 transcriptional response. Histone deacetylase 1 and 2 (HDAC1 and HDAC2) are chromatin modifying enzymes that mediate osteoblast differentiation. While an HDAC1‐Notch interaction has been studied in vitro and in Drosophila, its role in mammalian skeletal development and disorders is unclear. Osteosclerosis is a bone disorder with an abnormal increase in the number of osteoblasts and excessive bone formation.MethodsHere, we tested whether Hdac1/2 contribute to the pathogenesis of osteosclerosis in a murine model of the disease owing to conditionally cre‐activated expression of the Notch1 intracellular domain in immature osteoblasts.ResultsImportantly, selective homozygous deletions of Hdac1/2 in osteoblasts partially alleviate osteosclerotic phenotypes (Col2.3kb‐Cre; TGRosaN1ICD/+; Hdac1flox/flox; Hdac2flox/flox) with a 40% decrease in bone volume and a 22% decrease in trabecular thickness in 4 weeks old when compared to male mice with heterozygous deletions of Hdac1/2 (Col2.3 kb‐Cre; TGRosaN1ICD/+; Hdac1flox/+; Hdac2flox/+). Osteoblast‐specific deletion of Hdac1/2 in male and female mice results in no overt bone phenotype in the absence of the Notch1 gain‐of‐function (GOF) allele.ConclusionsThese results provide evidence that Hdac1/2 contribute to Notch1 pathogenic signaling in the mammalian skeleton. Our study on epigenetic regulation of Notch1 GOF‐induced osteosclerosis may facilitate further mechanistic studies of skeletal birth defects caused by Notch‐related GOF mutations in human patients, such as Adams‐Oliver disease, congenital heart disease, and lateral meningocele syndrome.