Regulation of Skeletal Homeostasis

Abstract

Landmark advances in skeletal biology have arisen mainly from the identification of disease-causing mutations and the advent of rapid and selective gene-targeting technologies to phenocopy human disease in mice. Here, we discuss work on newly identified mechanisms controlling the remodeling of bone, communication of bone cells with cells of other lineages, and crosstalk between bone and vital organs as these relate to the therapeutic targeting of the skeleton.

Figure 1.

Molecular coupling of bone resorption with bone formation. The resorption of bone matrix by proteolytic enzymes secreted by the osteoclast, such as cathepsin K (CTSK), liberates growth factors, notably TGF-β (Tgfb) and insulin-like growth factor 1 (Igf1), from bone. Hydroxyapatite dissolution by secreted acid releases Ca²⁺ locally, which triggers intracellular Ca²⁺ release to inhibit further enzyme secretion and bone resorption. Osteoclast precursors (or preosteoclasts), derived from hematopoietic stem cells (HSCs), become differentiated into mature resorbing cells through the actions of macrophage colony–stimulating factor (MCSF) and receptor activator for nuclear factor (NF)-κB ligand (Rankl). The latter acts on the stimulatory receptor Rank to trigger TNF receptor (TNFR)–associated factor 6 (Traf6)–mediated Nfkb and NF for activated T cells 2 (Nfat2) activation. Rankl also acts on the newly discovered inhibitory GPCR, leucine-rich repeat-containing GPCR 4 (Lgr4), and Nfat2 increases Lgr4 expression to ensure feedback control of Rankl action. In contrast, osteoblasts are derived from mesenchymal stem cells (MSCs) and are recruited to the site of bone resorption by released Tgfb. MSCs differentiate into mature, mineralizing osteoblasts under the influence of Igf1 and osteoblast-derived bone morphogenetic proteins (Bmp2/4), notch, and wingless-ints (Wnt). Osteoblasts also secrete semaphorin 3a (SEMA3A), which further stimulates osteoblast precursor proliferation and differentiation through Wnt but repels osteoclast precursors. Shown also are BMP, notch, and canonical Wnt signaling pathways in osteoblast-lineage cells. Also shown is epigenetic regulation by miRNAs, where known. ALK, activin receptor–like kinase; APC, adenomatous polyposis coli; ATF4, activating transcription factor 4; CBP/p300, CREB-binding protein/E1A binding protein p300; CK1, casein kinase 1; CN, calcineurin; Co-R, co-repressor; CSL, CBF1/Su(H)/Lag-1 transcription factor complex; DKK1, Dickkopf 1; DLL1-3, delta-like canonical Notch ligands 1-3; FZD, Frizzled; GSK3β, glycogen synthase kinase-3β; HDAC1, histone deacetylase 1; HES1-5, hairy and enhancer of split 1-5; LRP5/6, LDL receptor–related protein 5/6; MAML, Mastermind-like 1; mi, miRNA; NICD, NOTCH intracellular domain; NOG, noggin; OSX, osterix; PDGF-BB, platelet-derived growth factor BB; RUNX2; runt-related transcription factor 2; S1P, sphingosine-1-phosphate; SEMA3A, semaphorin 3a; SHN2, schnurri 2; SHN3, schnurri 3; SMAD, mothers against decapentaplegic homolog; SFRP2, secreted Frizzled-related protein 2; SOST, sclerostin; TCF/LEF1, transcription factor 7/lymphoid enhancer–binding factor 1; TNFα, tumor necrosis factor-α.

Figure 2.

Neural and neuroendocrine control of bone remodeling. Bone formation is regulated by signals from the SNS, controlled centrally by leptinergic neurons via serotonin action on Htr2c in the ventromedial hypothalamus (VMH). Enhanced sympathetic tone is initiated through Foxhead box protein O1 (FoxO1) via its control of dopamine β-hydroxylase (Dbh) expression. Secretion of norepinephrine at the nerve terminals innervating osteoblasts is regulated by the endocannabinoid receptor, Cb1, and its ligand 2-arachidonoylglycerol (2AG). Norepinephrine activates the Adrb2 receptor to activate activating transcription factor 4 (ATF4) signaling, ultimately modulating expression of circadian clock genes, notably period (Per) and cryptochrome (Cry), to inhibit bone formation. Adrb2 also signals via the neuropeptide cocaine and amphetamin-regulated transcript (Cart) to modulate the Rankl section and, thus, osteoclast activation. Signals arising from IL-1 (Il1)-, cannabinoid receptor 2 (Cb2)–, and neuropeptide Y (Npy)–expressing neurons regulate bone remodeling, as do peripheral nicotinic acetylcholine (ACH) receptors (nAchr). Neurohypophyseal and pituitary hormones, namely growth hormone (Gh), adrenocorticotrophic hormone (Acth), follicle-stimulating hormone (Fsh), thyroid-stimulating hormone (Tsh), prolactin (Prl), oxytocin (Oxt), and arginine vasopressin (Avp), also regulate both osteoclasts and osteoblasts directly through GPCRs. Certain ligands—namely Oxt, TSHβ variant (TSHβv), and Acth—are also produced in bone marrow by macrophages and/or osteoblasts. ADRB2, β-adrenergic receptor 2; CART, cocaine and amphetamine–regulated transcript; HTR, 5-hydroxytryptamine receptor; Lep, leptin; MAPK, mitogen-activated protein kinase; Ser, serotonin; VEGF, vascular endothelial growth factor.

Figure 3.

Crosstalk of bone cells within bone marrow regulates bone remodeling, adipogenesis, hematopoiesis, and angiogenesis. Bone cells interact with multiple bone marrow cells, including HSCs, MSCs, adipocytes, T cells, erythroid precursors, and CD31^hiendomucin (Emcn)^hi endothelial cells of H-type vessels. Osteocytes and osteoblasts communicate with osteoclasts by secreting Rankl, which acts on the Rank receptor that interacts with two ITAM-containing immune receptors, DNAX-activating protein of 12 kD (Dap12) and Fc receptor γ subunit (Fcrγ), as well as with Lgr4, a negative regulator. Osteoclasts are also controlled both directly and via the osteoblast by multiple pro-osteoclastic and antiosteoclastic cytokines, prominently Tnfα, IL-1, IL-4, IL-6, IL-17, and Ifnγ. White adipocytes, prominently seen in hypogonadal states, are MSC derived and secrete adipokines, notably adiponectin, which interacts with osteoblastic receptors. Adipocytes also secrete leptin (Lep), which not only interacts with adipocyte and osteoblast leptin receptors (Leprs) but also crosses the blood-brain barrier to regulate the hypothalamic relay of sympathetic signaling to bone. In addition to osteoblastic suppression exerted through Adrb2, sympathetic nerves innervating MSCs regulate the HSC niché and hematopoiesis via C-X-C motif chemokine ligand 12 (Cxcl2) release that primarily causes HSC egress. Through the activation of a hypoxia-inducible factor 1a (Hif1a) pathway in response to hypoxia, MSCs also produce erythropoietin (Epo) to cause erythrocytosis but also, through notch and NOG, to regulate angiogenesis. OPG, osteoprotegerin; Th17, T helper 17 cell.

Figure 4.

Distant interactions between bone and vital organs. In addition to the central nervous system and pituitary, a number of vital organs have connections with bone cells, mainly osteoblasts. Uncarboxylated osteocalcin is thought to interact via a G protein–coupled receptor, family C, group 6, member A (GPCR6A) to enhance insulin secretion from pancreatic β-cells, improve peripheral glucose tolerance by acing on white adipocytes, increase muscle mass, elevate testosterone secretion, stimulate brain development, and improve cognition. White adipocytes produce adiponectin and leptin (Lep); the latter not only acts on osteoblast LEPRs but also crosses the blood-brain barrier to regulate the SNS, which in turn, causes lipolysis through Adrb1/2/3 activation. Osteocytes produce fibroblast growth factor (Fgf)23, a hormone that interacts with the Fgf receptor (Fgfr) and Klotho in the renal tubule to increase phosphate secretion; the kidney, in turn, generates active 1,25-dihydroxyvitamin D₃ (1,25-D₃)to stimulate bone resorption and increase Ca and P absorption from the gut. Less is known about muscle–bone connectivity, except that irisin, produced during exercise, stimulates cortical bone mass, and osteoblastic osteocalcin is thought to maintain muscle mass. Adrb, β-adrenergic receptor; Dlk1, delta-like noncanonical Notch ligand 1; Insr, insulin receptor; VMH, ventromedial hypothalamus.

Figure 5.

Current and near-future therapies to prevent and treat bone loss in osteoporosis. Therapies to prevent and/or treat osteoporosis either act to suppress bone resorption (termed antiresorptive or anticatabolic agents) or stimulate new bone formation (anabolic agents). Either action allows bone formation to exceed bone resorption either in absolute or relative terms and therefore, has a net positive effect on bone mass. Estrogen (E₂) and the SERM raloxifene (and other similar drugs in the pipeline) act on the estrogen receptor α (ERα) in a tissue-specific manner. Thus, raloxifene is proestrogenic in reducing osteoclastogenesis and hence, reduces the risk of vertebral fractures, as noted in the Multiple Outcomes of Raloxifene Evaluation Trial. In contrast, it is antiestrogenic in reducing breast epithelial cell proliferation with potent effects in reducing ER⁺ breast cancers in the Study of Tamoxifen and Raloxifene and Continuing Outcomes Relevant to Evista (raloxifene) Trials. It is therefore used for osteoprotection during early menopause when osteoclastogenesis is high, particularly in patients with a high risk of breast cancer. The currently approved bisphosphonates (alendronate, risedronate, ibandronate, and zoledronic acid) display a high avidity to bone hydroxyapatite with which their N-atoms form H-bonds. When an osteoclast resorbs bone, the drug is released, enters the osteoclast by pinocytosis, and inhibits the enzyme farnesyl pyrophosphate synthase (FPPS). The blocking of farnesylation of small GTP-binding proteins is the basis of their potent antiresorptive actions in osteoporosis and skeletal metastasis. Besides inhibiting FPPS, bisphosphonates also interact with other targets, such as EGFRs, and may therefore exert anticancer actions directly. They also have known antiangiogenic actions, likely arising from a weak action on vascular EGFRs. Denosumab is a human monoclonal antibody that binds to and prevents the interaction of osteoblast- and osteocyte-derived RANKL to the osteoclast receptor RANK. In doing so, denosumab reduces osteoclastogenesis, inhibits bone resorption by mature cells, and induces osteoclast apoptosis. It reduces the risk of fracture at all sites (Fracture Reduction Evaluation of Denosumab in Osteoporosis Every 6 Months Trial) and is used for high-risk osteoporosis. The CTSK antagonist, odanacatib, was recently withdrawn from active development as a result of potential off-target effects; nonetheless, the phase III clinical studies do prove that CTSK is a valuable therapeutic target for osteoporosis. The only approved anabolic agent teriparatide or recombinant N-terminal fragment 1-34 of PTH [PTH(1-34)] acts directly on the PTH/PTH-related protein (PTHrP) receptor on the osteoblast to stimulate its bone-forming action with a substantial reduction of fracture risk when given intermittently. The new, likely-to-be-approved anabolic PTHrP(1-34) (abaloparatide) mimics the action of PTH mechanistically but displays a more rapid effect in reducing vertebral fracture risk in clinical trials that compare either abaloparatide or teriparatide against placebo (Abaloparatide Comparator Trial In Vertebral Endpoints Trial). Romosozumab, currently in clinical development, is a humanized monoclonal antibody to the WNT inhibitor SOST. By removing SOST from the LRP5/6 receptor, romosozumab triggers WNT signaling to a therapeutic advantage in osteoporosis.