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Little is known about the molecular mechanisms of enthesis formation in mature animals. Here, we report that annexin A5 (Anxa5) plays a critical role in the regulation of bone ridge outgrowth at the entheses. We found that Anxa5 is highly expressed in the entheses of postnatal and adult mice. In Anxa5-deficient (Anxa5 -/- ) mice, the sizes of bone ridge outgrowths at the entheses of the tibias and femur were increased after age 7 weeks. Bone overgrowth was not observed at the fibrous enthesis where the fibrocartilage layer does not exist. More ALP-expressing cells were observed in the fibrocartilage layer in Anxa5 -/- mice than in wild-type (WT) mice. Calcein and Alizarin Red double labeling revealed more mineralized areas in Anxa5 -/- mice than WT mice. To examine the effects of mechanical forces, we performed tenotomy in which transmission of contractile forces by the tibial muscle was impaired by surgical muscle release. In tenotomized mice, bone overgrowth at the enthesis in Anxa5 -/- mice was decreased to a level comparable to that in WT mice at 8 weeks after the operation. The tail-suspended mice also showed a decrease in bone overgrowth to similar levels in Anxa5 -/- and WT mice at 8 weeks after hindlimb unloading. These results suggest that bone overgrowth at the enthesis requires mechanical forces. We further examined effects of Anxa5 gene knockdown (KD) in primary cultures of osteoblasts, chondrocytes, and tenocytes in vitro. Anxa5 KD increased ALP expression in tenocytes and chondrocytes but not in osteoblasts, suggesting that increased ALP activity in the fibrocartilaginous tissue in Anxa5 -/- mice is directly caused by Anxa5 deletion in tenocytes or fibrocartilage cells. These data indicate that Anxa5 prevents bone overgrowth at the enthesis, whose formation is mediated through mechanical forces and modulating expression of mineralization regulators. © 2018 American Society for Bone and Mineral Research., (© 2018 American Society for Bone and Mineral Research.)

Migration of the cells in osteoblastic lineage, including preosteoblasts and osteoblasts, has been postulated to influence bone formation. However, the molecular bases that link preosteoblastic/osteoblastic cell migration and bone formation are incompletely understood. Nck (noncatalytic region of tyrosine kinase; collectively referred to Nck1 and Nck2) is a member of the signaling adaptors that regulate cell migration and cytoskeletal structures, but its function in cells in the osteoblastic lineage is not known. Therefore, we examined the role of Nck in migration of these cells. Nck is expressed in preosteoblasts/osteoblasts, and its knockdown suppresses migration as well as cell spreading and attachment to substrates. In contrast, Nck1 overexpression enhances spreading and increases migration and attachment. As for signaling, Nck double knockdown suppresses migration toward IGF1 (insulin-like growth factor 1). In these cells, Nck1 binds to IRS-1 (insulin receptor substrate 1) based on immunoprecipitation experiments using anti-Nck and anti-IRS-1 antibodies. In vivo, Nck knockdown suppresses enlargement of the pellet of DiI-labeled preosteoblasts/osteoblasts placed in the calvarial defects. Genetic experiments indicate that conditional double deletion of both Nck1 and Nck2 specifically in osteoblasts causes osteopenia. In these mice, Nck double deficiency suppresses the levels of bone-formation parameters such as bone formation rate in vivo. Interestingly, bone-resorption parameters are not affected. Finally, Nck deficiency suppresses repair of bone injury after bone marrow ablation. These results reveal that Nck regulates preosteoblastic/osteoblastic migration and bone mass.

PTH actions on bone are exerted through multiple pathways from G-proteins coupled to a receptor PTH1R that is expressed on osteoblastic lineage cells. Physiological action of PTH may be through increased expression of RANKL, resulting in increased bone resorption and Ca( + + ) release from bone matrix by osteoclasts. Bone anabolic action of PTH by intermittent treatment is explained by ( I ) proliferation of osteogenic precursor cells, ( II ) differentiation of osteoblasts, ( III ) inhibition of apoptosis in osteoblastic cells, and ( IV ) inhibition of canonical Wnt inhibitor secretion from osteocytes. Two of these mechanisms ( II and III ) could be secondary to increased osteoclastogenesis after induction of RANKL in osteoblastic cells. Mechanisms involved in ( I ) might be controversial depending on cellular status including differentiation stages and cell density, and thus need to be clarified by further studies.

Parathyroid hormone (PTH), the major calcium-regulating hormone, and norepinephrine (NE), the principal neurotransmitter of sympathetic nerves, regulate bone remodeling by activating distinct cell-surface G protein-coupled receptors in osteoblasts: the parathyroid hormone type 1 receptor (PTHR) and the β(2)-adrenergic receptor (β(2)AR), respectively. These receptors activate a common cAMP/PKA signal transduction pathway mediated through the stimulatory heterotrimeric G protein. Activation of β(2)AR via the sympathetic nervous system decreases bone formation and increases bone resorption. Conversely, daily injection of PTH (1-34), a regimen known as intermittent (i)PTH treatment, increases bone mass through the stimulation of trabecular and cortical bone formation and decreases fracture incidences in severe cases of osteoporosis. Here, we show that iPTH has no osteoanabolic activity in mice lacking the β(2)AR. β(2)AR deficiency suppressed both iPTH-induced increase in bone formation and resorption. We showed that the lack of β(2)AR blocks expression of iPTH-target genes involved in bone formation and resorption that are regulated by the cAMP/PKA pathway. These data implicate an unexpected functional interaction between PTHR and β(2)AR, two G protein-coupled receptors from distinct families, which control bone formation and PTH anabolism.

The sympathetic nervous system suppresses bone mass by mechanisms that remain incompletely elucidated. Using cell-based and murine genetics approaches, we show that this activity of the sympathetic nervous system requires osteopontin (OPN), a cytokine and one of the major members of the noncollagenous extracellular matrix proteins of bone. In this work, we found that the stimulation of the sympathetic tone by isoproterenol increased the level of OPN expression in the plasma and bone and that mice lacking OPN (OPN-KO) suppressed the isoproterenol-induced bone loss by preventing reduced osteoblastic and enhanced osteoclastic activities. In addition, we found that OPN is necessary for changes in the expression of genes related to bone resorption and bone formation that are induced by activation of the sympathetic tone. At the cellular level, we showed that intracellular OPN modulated the capacity of the β2-adrenergic receptor to generate cAMP with a corresponding modulation of cAMP-response element binding (CREB) phosphorylation and associated transcriptional events inside the cell. Our results indicate that OPN plays a critical role in sympathetic tone regulation of bone mass and that this OPN regulation is taking place through modulation of the β2-adrenergic receptor/cAMP signaling system.

The relationship between bone and nervous system has been considered based on clinical observations such as Reflex Sympathetic Dystrophy (RSD) or ectopic bone formation associated with spinal cord injury. Sympathtic nervous tone has been reported to control both bone formation and bone resorption. Unloading induces bone loss due to an increase in bone resorption and decrease in bone formation. Both of these two arms are shown to be influenced by sympathetic tone. In addition, cannabinoid receptor has been observed to be involved in regulation of bone mass. Psychiatric diseases such as depression has also been suggested to linked to the alteration in the levels of bone mass. These observations together point to importance of the relationship between bone mass and nervous system.

Male sex hormones play a critical role in regulation of bone metabolism. In male mice lacking androgen receptor (AR), osteopenia and high turnover state in bone remodeling have been reported. However, androgen receptor's role in disuse-induced osteopenia is not known. Therefore, we examined the effects of AR deficiency on unloading-induced bone loss. Wild type or androgen receptor deficient mice (ARKO) were subjected to hind limb unloading (HU) or normal housing (Control). The groups of mice were as follows; wild type control mice (Group WT-Cont), ARKO control mice (Group ARKO-Cont), wild type HU mice (Group WT-HU), and ARKO-HU mice (Group ARKO-HU). HU reduced cancellous bone mass in ARKO (ARKO-HU) by about 70% compared to ARKO-Cont and this reduction rate was over two-fold more than that of wild type (WT-HU) (reduction by less than 30% compared to WT-Cont). Combination of ARKO and HU (ARKO-HU) resulted in the least levels of cortical bone mass and bone mineral density among the four groups. ARKO-HU group indicated the highest levels of systemic bone resorption marker, deoxypyridinoline. Osteoclast development levels in the cultures in ARKO-HU derived bone marrow cells were the highest among the four groups. These data suggest that combination of androgen receptor deficiency and hind limb unloading results in exacerbation of disuse-induced osteopenia due to the enhanced levels of bone resorption.

JunD is an activator protein-1 (AP-1) component though its function in skeletal system is still not fully understood. To elucidate the role of JunD in the regulation of bone metabolism, we analyzed JunD-deficient mice. JunD deficiency significantly increased bone mass and trabecular number. This bone mass enhancement was due to JunD deficiency-induced increase in bone formation activities in vivo. Such augmentation of bone formation was associated with simultaneous increase in bone resorption while the former was dominant over the latter as accumulation of bone mass occurred in JunD-deficient mice. In a pathological condition relevant to postmenopausal osteoporosis, ovariectomy reduced bone mass in wild type (WT) mice as known before. Interestingly, JunD deficiency suppressed ovariectomy-induced increase in bone resorption and kept high bone mass. In addition, JunD deficiency also enhanced new bone formation after bone marrow ablation. Examination of molecular bases for these observations revealed that JunD deficiency enhanced expression levels of c-jun, fra-1, and fra-2 in bone in conjunction with elevated expression levels of runx2, type I collagen, and osteocalcin. Thus, JunD is involved in estrogen depletion-induced osteopenia via its action to suppress bone formation and to enhance bone resorption.

Axial patterning in embryonic skeletogenesis associates with coordinated programming of somitogenesis and angiogenesis. As seen in endochondral bone formation, skeletogenesis is closely related to angiogenesis during development. PlexinD1 is a member of plexin family, is expressed in central nervous system and endothelium, and plays a role in blood vessel patterning and endothelium positioning during embryonic development. Here, we examined the effects of PlexinD1 deficiency on skeletogenesis. Three-dimensional micro CT examination revealed that PlexinD1 deficiency resulted in axial skeletal patterning defects including malformation in vertebral body and rib bone shape. Histological examination of the vertebral bodies and long bones showed that PlexinD1 deficiency altered the development of cartilage. PlexinD1 deficiency did not affect the levels of von Willebrand factor staining in relatively large vessels not attached but close to the vertebral body of mice. However, PlexinD1 deficiency reduced the von Willebrand factor (vWf) staining in most of the microvasculatures attached to the vertebral bone. PlexinD1 was expressed in osteoblastic cells and bone tissues of newborn and adult mice. As most of the homozygous knockout mice did not survive, we examined the role of PlexinD1 in bone formation in heterozygous adult mice subjected to bone marrow ablation. However, PlexinD1 heterozygous knockout did not reveal defects in new bone formation. In conclusion, PlexinD1 is involved in the patterning of axial skeletogenesis.

Loss of mechanical stress causes bone loss. However, the mechanisms underlying the unloading-induced bone loss are largely unknown. Here, we examined the effects of gold-thioglucose (GTG) treatment, which destroys ventromedial hypothalamus (VMH), on unloading-induced bone loss. Unloading reduced bone volume in control (saline-treated) mice. Treatment with GTG-reduced bone mass and in these GTG-treated mice, unloading-induced reduction in bone mass levels was not observed. Unloading reduced the levels of bone formation rate (BFR) and mineral apposition rate (MAR). GTG treatment also reduced these parameters and under this condition, unloading did not further reduce the levels of BFR and MAR. Unloading increased the levels of osteoclast number (Oc.N/BS) and osteoclast surface (Oc.S/BS). GTG treatment did not alter the basal levels of these bone resorption parameters. In contrast to control, GTG treatment suppressed unloading-induced increase in the levels of Oc.N/BS and Oc.S/BS. Unloading reduced the levels of mRNA expression of the genes encoding osteocalcin, type I collagen and Cbfa1 in bone. In contrast, GTG treatment suppressed such unloading-induced reduction of mRNA expression. Unloading also enhanced the levels of fat mass in bone marrow and mRNA expression of the genes encoding PPARgamma2, C/EBPalpha, and C/EBPbeta in bone. In GTG-treated mice, unloading did not increase fat mass and the levels of fat-related mRNA expression. These results indicated that GTG treatment suppressed unloading-induced alteration in bone loss., (2006 Wiley-Liss, Inc.)

Molecular mechanisms underlying unloading-induced reduction of bone formation have not yet been fully understood. In vitro, Runx2 has been suggested to be involved in mechanical signaling in osteoblasts. However, the roles of Runx2 in vivo during the bone response to mechanical stimuli have not yet been known. The purpose of this paper was to examine the roles of Runx2 in unloading-induced bone loss in vivo. Tail suspension was conducted for 2 wk using 9- to 11-wk-old Runx2 heterozygous knockout mice (Runx2(+/-)) and wild-type (Wt) littermates. Bones were subjected to two-dimensional micro-x-ray computed tomography, bone histomorphometry and RT-PCR analyses. Loss of half Runx2 gene dosage-exacerbated unloading-induced bone loss in trabecular and cortical envelopes. Unloading-induced reduction in mineral apposition rate and bone formation rate in cortical bone as well as trabecular bone was exacerbated in Runx2(+/-) mice, compared with Wt mice. Bone resorption parameters were not significantly affected by unloading or Runx2(+/-) genotype. Basal Runx2 and osterix mRNA levels in bone were reduced by 50% in Wt, whereas unloading in Runx2(+/-) mice did not further alter Runx2 and osterix mRNA levels. In contrast, osteocalcin mRNA levels were reduced by unloading, regardless of Runx2 gene dosage. These data demonstrated that full Runx2 gene dosage is required for maintaining normal function of osteoblasts in mechanical unloading or nonphysiological condition. Finally, we propose Runx2 as a critical target gene in unloading to alter osteoblastic activity and bone formation in vivo.