Citations

Abstract Osteogenesis imperfecta (OI) is a heterogeneous group of inherited connective tissue disorders primarily caused by dominant mutations in COL1A1 or COL1A2 that impair type I procollagen folding and secretion. Misfolded collagen accumulates in the endoplasmic reticulum (ER), triggering ER stress and osteoblast dysfunction and bone fragility. Current pharmacologic therapy focuses on inhibiting bone resorption but has limited efficacy and does not address the underlying biology of the disease. The epigenetic regulator Polycomb-repressive complex 2 (PRC2) has emerged as an important regulator of bone formation. Genetic and pharmacologic disruption of PRC2 enhanced osteogenic differentiation in wild-type cells. Here we demonstrate that inhibition of the PRC2 through targeting its essential component embryonic ectoderm development (EED) enhances osteogenic differentiation, improves bone architecture in male Col1a2+/G610C OI mouse models, modulates the integrated stress response (ISR), and improves ER morphology in OI cells. These findings identify EED inhibition as a novel epigenetic strategy to restore collagen homeostasis and improve skeletal integrity in OI.

Abstract Germline and osteoblast-directed deletion of G protein-gated inwardly rectifying K+ channel 3 (Girk3) was recently shown to increase bone mass after 18 wk of age in male mice. Here, we show that germline Girk3 deletion also increases trabecular and cortical bone mass and increases the mechanical strength of the femur in female mice after 18 wk of age. Unlike male mice, however, osteoblast-directed Girk3 deletion using 2.3 kb-Col1a1-Cre does not increase bone mass in adult female mice. To discover mechanisms underlying high bone mass in female Girk3−/− mice, bulk RNA-sequencing was performed on 2-d-old calvarial bone, revealing lower expression of proinflammatory cytokines such as IL-1β and IL-6 in Girk3−/− mice. Accordingly, cytokines and chemokines are largely suppressed in the circulation of adult Girk3−/− mice compared to WT littermates. The cytokines GM-CSF, IL-1β, IL-2, and IL-9 are reduced in the serum of both male and female Girk3−/− mice, while eotaxin, IFNγ, MIP-1α, and others are sexually dimorphic. Histomorphometry reveals that osteoclast activity is modestly reduced in Girk3−/− bone, which is supported by in vitro osteoclast resorption assays. However, deletion of Girk3 in myeloid-lineage cells with LysM-Cre is not sufficient to recapitulate high bone mass in either male or female mice. Moreover, female Girk3−/− mice are not protected from ovariectomy-induced bone loss. Finally, single-cell screening using cytometry by time-of-flight in the BM revealed no differences in immune cell abundances due to global Girk3 deletion. Taken together, while Girk3 regulates inflammatory cytokine expression in the bone and serum, deletion of Girk3 in myeloid-lineage cells does not affect bone mass.

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.

ABSTRACT Endochondral ossification is tightly controlled by a coordinated network of signaling cascades including parathyroid hormone (PTH). Pleckstrin homology (PH) domain and leucine rich repeat phosphatase 1 (Phlpp1) affects endochondral ossification by suppressing chondrocyte proliferation in the growth plate, longitudinal bone growth, and bone mineralization. As such, Phlpp1−/− mice have shorter long bones, thicker growth plates, and proportionally larger growth plate proliferative zones. The goal of this study was to determine how Phlpp1 deficiency affects PTH signaling during bone growth. Transcriptomic analysis revealed greater PTH receptor 1 (Pth1r) expression and enrichment of histone 3 lysine 27 acetylation (H3K27ac) at the Pth1r promoter in Phlpp1-deficient chondrocytes. PTH (1-34) enhanced and PTH (7-34) attenuated cell proliferation, cAMP signaling, cAMP response element-binding protein (CREB) phosphorylation, and cell metabolic activity in Phlpp1-inhibited chondrocytes. To understand the role of Pth1r action in the endochondral phenotypes of Phlpp1-deficient mice, Phlpp1−/− mice were injected with Pth1r ligand PTH (7-34) daily for the first 4 weeks of life. PTH (7-34) reversed the abnormal growth plate and long-bone growth phenotypes of Phlpp1−/− mice but did not rescue deficits in bone mineral density or trabecular number. These results show that elevated Pth1r expression and signaling contributes to increased proliferation in Phlpp1−/− chondrocytes and shorter bones in Phlpp1-deficient mice. Our data reveal a novel molecular relationship between Phlpp1 and Pth1r in chondrocytes during growth plate development and longitudinal bone growth. © 2021 American Society for Bone and Mineral Research (ASBMR).