Endochondral ossification, an important process in vertebrate bone formation, is highly dependent on correct functioning of growth plate chondrocytes
. Proliferation of these cells determines ...longitudinal bone growth and the matrix deposited provides a scaffold for future bone formation. However, these two energy-dependent anabolic processes occur in an avascular environment
. In addition, the centre of the expanding growth plate becomes hypoxic, and local activation of the hypoxia-inducible transcription factor HIF-1α is necessary for chondrocyte survival by unidentified cell-intrinsic mechanisms
. It is unknown whether there is a requirement for restriction of HIF-1α signalling in the other regions of the growth plate and whether chondrocyte metabolism controls cell function. Here we show that prolonged HIF-1α signalling in chondrocytes leads to skeletal dysplasia by interfering with cellular bioenergetics and biosynthesis. Decreased glucose oxidation results in an energy deficit, which limits proliferation, activates the unfolded protein response and reduces collagen synthesis. However, enhanced glutamine flux increases α-ketoglutarate levels, which in turn increases proline and lysine hydroxylation on collagen. This metabolically regulated collagen modification renders the cartilaginous matrix more resistant to protease-mediated degradation and thereby increases bone mass. Thus, inappropriate HIF-1α signalling results in skeletal dysplasia caused by collagen overmodification, an effect that may also contribute to other diseases involving the extracellular matrix such as cancer and fibrosis.
Genomic actions induced by 1alpha25-dihydroxyvitamin D(3) 1,25(OH)(2)D(3) are crucial for normal bone metabolism, mainly because they regulate active intestinal calcium transport. To evaluate whether ...the vitamin D receptor (VDR) has a specific role in growth-plate development and endochondral bone formation, we investigated mice with conditional inactivation of VDR in chondrocytes. Growth-plate chondrocyte development was not affected by the lack of VDR. Yet vascular invasion was impaired, and osteoclast number was reduced in juvenile mice, resulting in increased trabecular bone mass. In vitro experiments confirmed that VDR signaling in chondrocytes directly regulated osteoclastogenesis by inducing receptor activator of NF-kappaB ligand (RANKL) expression. Remarkably, mineral homeostasis was also affected in chondrocyte-specific VDR-null mice, as serum phosphate and 1,25(OH)(2)D levels were increased in young mice, in whom growth-plate activity is important. Both in vivo and in vitro analysis indicated that VDR inactivation in chondrocytes reduced the expression of FGF23 by osteoblasts and consequently led to increased renal expression of 1alpha-hydroxylase and of sodium phosphate cotransporter type IIa. Taken together, our findings provide evidence that VDR signaling in chondrocytes is required for timely osteoclast formation during bone development and for the endocrine action of bone in phosphate homeostasis.
Cell-based therapy is a promising strategy in regenerative medicine, but the poor survival rate of the implanted cells remains a major challenge and limits clinical translation. We preconditioned ...periosteal cells to the hypoxic and ischemic environment of the bone defect site by deleting prolyl hydroxylase domain-containing protein 2 (PHD2), resulting in hypoxia-inducible factor 1 alpha (HIF-1α) stabilization. This strategy increased postimplantation cell survival and improved bone regeneration. The enhanced cell viability was angiogenesis independent but relied on combined changes in glutamine and glycogen metabolism. HIF-1α stabilization stimulated glutaminase-mediated glutathione synthesis, maintaining redox homeostasis at baseline and during oxidative or nutrient stress. Simultaneously, HIF-1α signaling increased glycogen storage, preventing an energy deficit during nutrient or oxygen deprivation. Pharmacological inhibition of PHD2 recapitulated the adaptations in glutamine and glycogen metabolism and, consequently, the beneficial effects on cell survival. Thus, targeting cellular metabolism is an appealing strategy for bone regeneration and cell-based therapy in general.
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•HIF-1α action induces survival and regenerative potential of implanted skeletal cells•Cell survival is angiogenesis independent but relies on metabolic changes•HIF-1α-mediated conversion of glutamine to glutathione preserves redox balance•HIF-1α-promoted glycogen stores inhibit energetic crisis in stress conditions
Bone regenerative strategies are hindered by the low survival rate of implanted cells. Stegen et al. show that genetic or pharmacological HIF-1α stabilization in skeletal cells prior to implantation improves cell survival by adapting glutamine and glucose metabolism to preserve redox and energy balance, resulting in enhanced bone repair.
Serum calcium levels are tightly controlled by an integrated hormone-controlled system that involves active vitamin D 1,25(OH)(2)D, which can elicit calcium mobilization from bone when intestinal ...calcium absorption is decreased. The skeletal adaptations, however, are still poorly characterized. To gain insight into these issues, we analyzed the consequences of specific vitamin D receptor (Vdr) inactivation in the intestine and in mature osteoblasts on calcium and bone homeostasis. We report here that decreased intestinal calcium absorption in intestine-specific Vdr knockout mice resulted in severely reduced skeletal calcium levels so as to ensure normal levels of calcium in the serum. Furthermore, increased 1,25(OH)(2)D levels not only stimulated bone turnover, leading to osteopenia, but also suppressed bone matrix mineralization. This resulted in extensive hyperosteoidosis, also surrounding the osteocytes, and hypomineralization of the entire bone cortex, which may have contributed to the increase in bone fractures. Mechanistically, osteoblastic VDR signaling suppressed calcium incorporation in bone by directly stimulating the transcription of genes encoding mineralization inhibitors. Ablation of skeletal Vdr signaling precluded this calcium transfer from bone to serum, leading to better preservation of bone mass and mineralization. These findings indicate that in mice, maintaining normocalcemia has priority over skeletal integrity, and that to minimize skeletal calcium storage, 1,25(OH)(2)D not only increases calcium release from bone, but also inhibits calcium incorporation in bone.
VEGF is crucial for metaphyseal bone vascularization. In contrast, the angiogenic factors required for vascularization of epiphyseal cartilage are unknown, although this represents a developmentally ...and clinically important aspect of bone growth. The VEGF gene is alternatively transcribed into VEGF(120), VEGF(164), and VEGF(188) isoforms that differ in matrix association and receptor binding. Their role in bone development was studied in mice expressing single isoforms. Here we report that expression of only VEGF(164) or only VEGF(188) (in VEGF(188/188) mice) was sufficient for metaphyseal development. VEGF(188/188) mice, however, showed dwarfism, disrupted development of growth plates and secondary ossification centers, and knee joint dysplasia. This phenotype was at least partly due to impaired vascularization surrounding the epiphysis, resulting in ectopically increased hypoxia and massive chondrocyte apoptosis in the interior of the epiphyseal cartilage. In addition to the vascular defect, we provide in vitro evidence that the VEGF(188) isoform alone is also insufficient to regulate chondrocyte proliferation and survival responses to hypoxia. Consistent herewith, chondrocytes in or close to the hypoxic zone in VEGF(188/188) mice showed increased proliferation and decreased differentiation. These findings indicate that the insoluble VEGF(188) isoform is insufficient for establishing epiphyseal vascularization and regulating cartilage development during endochondral bone formation.
Treatment of bone metastases is largely symptomatic and is still an unmet medical need. Current therapies mainly target the late phase of tumor-induced osteoclast activation and hereby inhibit ...further metastatic growth. This treatment method is, however, less effective in preventing initial tumor engraftment, a process that is supposed to depend on the bone microenvironment. We explored whether bone-derived placental growth factor (PlGF), a homologue of vascular endothelial growth factor-A, regulates osteolytic metastasis. Osteogenic cells secrete PlGF, the expression of which is enhanced by bone-metastasizing breast tumor cells. Selective neutralization of host-derived PlGF by anti-mouse PlGF (alphaPlGF) reduced the incidence, number, and size of bone metastases, and preserved bone mass. alphaPlGF did not affect metastatic tumor angiogenesis but inhibited osteoclast formation by preventing the upregulation of the osteoclastogenic cytokine receptor activator of NF-kappaB ligand in osteogenic cells, as well as by blocking the autocrine osteoclastogenic activity of PlGF. alphaPlGF also reduced the engraftment of tumor cells in the bone and inhibited their interaction with matrix components in the metastatic niche. alphaPlGF therefore inhibits not only the progression of metastasis but also the settlement of tumor in the bone. These findings identify novel properties of PlGF and suggest that alphaPlGF might offer opportunities for adjuvant therapy of bone metastasis.
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