Takuya Notomi

Sympathetic control of bone mass regulated by osteopontin

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.

Anabolic action of parathyroid hormone regulated by the β2-adrenergic receptor

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 β2-adrenergic receptor (β2AR), respectively. These receptors activate a common cAMP/PKA signal transduction pathway mediated through the stimulatory heterotrimeric G protein. Activation of β2AR 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 β2AR. β2AR deficiency suppressed both iPTH-induced increase in bone formation and resorption. We showed that the lack of β2AR 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 β2AR, two G protein-coupled receptors from distinct families, which control bone formation and PTH anabolism.

Nanogel‐based scaffold delivery of prostaglandin E2 receptor–specific agonist in combination with a low dose of growth factor heals critical‐size bone defects in mice

OBJECTIVE Regeneration of bone requires the combination of appropriate drugs and an appropriate delivery system to control cell behavior. However, the delivery of multiple drugs to heal bone is complicated by the availability of carriers. The aim of this study was to explore a new system for delivery of a selective EP4 receptor agonist (EP4A) in combination with low-dose bone morphogenetic protein 2 (BMP-2). METHODS Combined delivery of EP4A and BMP-2 was carried out with a nanogel-based scaffold in the shape of a disc, to repair critical-size circle-shaped bone defects in calvariae that otherwise did not heal spontaneously. RESULTS Combination treatment with EP4A and low-dose BMP-2 in nanogel efficiently activated bone cells to regenerate calvarial bone by forming both outer and inner cortical plates as well as bone marrow tissue to regenerate a structure similar to that of intact calvaria. EP4A enhanced low-dose BMP-2-induced cell differentiation and activation of transcription events in osteoblasts. CONCLUSION These data indicate that combined delivery of EP4A and low-dose BMP-2 via nanogel-based hydrogel provides a new system for bone repair.

Phospholipase C-dependent Ca2+-sensing pathways leading to endocytosis and inhibition of the plasma membrane vacuolar H+-ATPase in osteoclasts.

In osteoclasts, elevation of extracellular Ca2+ is an endogenous signal that inhibits bone resorption. We recently found that an elevation of extracellular Ca2+ decreased proton extrusion through the plasma membrane vacuolar H+-ATPase (V-ATPase) rapidly. In this study we investigated mechanisms underlying this early Ca2+-sensing response, particularly in reference to the activity of the plasma membrane V-ATPase and to membrane retrieval. Whole cell clamp recordings allowed us to measure the V-ATPase currents and the cell capacitance (C(m)) simultaneously. C(m) is a measure of cell surface. Extracellular Ca2+ (2.5-40 mM) decreased C(m) and the V-ATPase current simultaneously. The decreased C(m), together with the enhanced uptake of a lipophilic dye (FM1-43), indicated that Ca2+ facilitated endocytosis. The endocytosis was blocked by dynamin inhibitors (dynasore and dynamin-inhibitory peptide), by small interfering RNA (siRNA) targeting for dynanmin-2 and also by bafilomycin A(1), a blocker of V-ATPases. The extracellular Ca2+-induced endocytosis and inhibition of the V-ATPase current were diminished by a phospholipase C inhibitor (U73122) and siRNA targeting for phospholipase C gamma2 subunit. Holding the cytosolic Ca2+ at either high (0.5-5 microM) or low levels or inhibiting calmodulin by an inhibitor (W7) or an antibody (anti-CaM) decreased the stimulated endocytosis and the inhibition of the V-ATPase current. These data suggest that extracellular Ca2+ facilitated dynamin- and V-ATPase-dependent endocytosis in association with an inhibition of the plasma membrane V-ATPase. Phospholipase C, cytosolic Ca2+, and calmodulin were involved in the signaling pathways. Membrane retrieval and the plasma membrane V-ATPase activity may cooperate during the early phase of Ca2+-sensing response in osteoclasts.

Identification of two pore channel 2 as a novel regulator of osteoclastogenesis

Background: Two-pore channel 2 (TPC2) is a calcium channel, but its function in osteoclasts is unknown. Results: TPC2 expression levels in osteoclast precursor cells were increased upon osteoclast differentiation induced by RANKL. Down-regulation of TPC2 expression suppressed RANKL-induced intracellular Ca2+ signaling as well as nuclear localization of NFATc1 and inhibited osteoclastogenesis. Conclusion: TPC2 regulates osteoclastogenesis. Significance: TPC2 plays a critical role in osteoclast differentiation. Osteoclast differentiation is one of the critical steps that control bone mass levels in osteoporosis, but the molecules involved in osteoclastogenesis are still incompletely understood. Here, we show that two-pore channel 2 (TPC2) is expressed in osteoclast precursor cells, and its knockdown (TPC2-KD) in these cells suppressed RANKL-induced key events including multinucleation, enhancement of tartrate-resistant acid phosphatase (TRAP) activities, and TRAP mRNA expression levels. With respect to intracellular signaling, TPC2-KD reduced the levels of the RANKL-induced dynamic waving of Ca2+ in RAW cells. The search for the target of TPC2 identified that nuclear localization of NFATc1 is retarded in TPC2-KD cells. Finally, TPC2-KD suppressed osteoclastic pit formation in cultures. We conclude that TPC2 is a novel critical molecule for osteoclastogenesis.

MURF1 deficiency suppresses unloading-induced effects on osteoblasts and osteoclasts to lead to bone loss

Loss of mechanical stress or unloading causes disuse osteoporosis that leads to fractures and deteriorates body function and affects mortality rate in aged population. This bone loss is due to reduction in osteoblastic bone formation and increase in osteoclastic bone resorption. MuRF1 is a muscle RING finger protein which is involved in muscle wasting and its expression is enhanced in the muscle of mice subjected to disuse condition such as hind limb unloading (HU). However, whether MuRF1 is involved in bone loss due to unloading is not known. We therefore examined the effects of MuRF1 deficiency on unloading‐induced bone loss. We conducted hind limb unloading of MuRF1 KO mice and wild‐type control mice. Unloading induced about 60% reduction in cancellous bone volume (BV/TV) in WT mice. In contrast, MuRF1 deficiency suppressed unloading‐induced cancellous bone loss. The cortical bone mass was also reduced by unloading in WT mice. In contrast, MuRF1 deficiency suppressed this reduction in cortical bone mass. To understand whether the effects of MuRF1 deficiency suppress bone loss is on the side of bone formation or bone resorption, histomorphometry was conducted. Unloading reduced bone osteoblastic formation rate (BFR) in WT. In contrast, MuRF1 deficiency suppressed this reduction. Regarding bone resorption, unloading increased osteoclast number in WT. In contrast, MURF1 deficiency suppressed this osteoclast increase. These data indicated that the ring finger protein, MURF1 is involved in disuse‐induced bone loss in both of the two major bone remodeling activities, osteoblastic bone formation and osteoclastic bone resorption. J. Cell. Biochem. 112: 3525–3530, 2011.

Osteoblastic differentiation enhances expression of TRPV4 that is required for calcium oscillation induced by mechanical force

Mechanical stress is known to alter bone mass and the loss of force stimuli leads to reduction of bone mass. However, molecules involved in this phenomenon are incompletely understood. As mechanical force would affect signaling events in cells, we focused on a calcium channel, TRPV4 regarding its role in the effects of force stimuli on calcium in osteoblasts. TRPV4 expression levels were enhanced upon differentiation of osteoblasts in culture. We found that BMP-2 treatment enhanced TRPV4 gene expression in a dose dependent manner. BMP-2 effects on TRPV4 expression were suppressed by inhibitors for transcription and new protein synthesis. In these osteoblasts, a TRPV4-selective agonist, 4α-PDD, enhanced calcium signaling and the effects of 4α-PDD were enhanced in differentiated osteoblasts compared to the control cells. Fluid flow, as a mechanical stimulation, induced intracellular calcium oscillation in wild type osteoblasts. In contrast, TRPV4 deficiency suppressed calcium oscillation significantly even when the cells were subjected to fluid flow. These data suggest that TRPV4 is involved in the flow-induced calcium signaling in osteoblasts.

Per-1 is a specific clock gene regulated by parathyroid hormone (PTH) signaling in osteoblasts and is functional for the transcriptional events induced by PTH

Per‐1 is one of the clock genes and is known to regulate various biological events including bone mass determination. Parathyroid hormone is anabolic to bone while the mechanism of its action is not fully understood. Here, we examined the role of PTH on Per‐1 gene expression under osteoblast specific PTH signaling. Constitutively active PTH receptor (caPPR) expressed specifically in osteoblasts in transgenic mice activates Per‐1 gene expression in bone. This is specific as expression of other clock gene Bmal‐1 is not affected by caPPR over‐expression. Per‐1 is also expressed in osteoblastic cell line. Interestingly, Per‐1 expression is required for PTH signaling‐induced CRE dependent transcription. This is forming a positive feed back loop in the anabolic action of PTH signaling and Per‐1 in bone. These data indicate that PTH singling in osteoblasts activates Per‐1 gene expression in vivo in association with its anabolic action in bone at least in part through the regulation of transcriptional events. J. Cell. Biochem. 112: 433–438, 2011.

Stability of mRNA influences osteoporotic bone mass via CNOT3

Significance Osteoporosis is a highly prevalent disease affecting nearly 20 million people in the United States and is life-threatening in elderly patients. However, underlying pathophysiology regarding the posttranscriptional control of bone resorption is incompletely understood. CNOT3 is a molecule involved in mRNA stability in yeast to mammalian cells, but its role in bone regulation is not known. We discovered that Cnot3 deficiency specifically enhances receptor activator of NF-κB (RANK) mRNA stability and leads to osteopenia in healthy young adult animals. Moreover, Cnot3 levels are reduced in ageing-induced osteoporosis, and Cnot3 deficiency further exacerbates such osteoporosis significantly. As a mechanism, Cnot3 binds to RANK mRNA and its 3′-UTR renders Cnot3-dependent instability to the reporter gene. Our results reveal Cnot3 regulation in aging-induced osteoporosis. Osteoclastogenesis is under the control of posttranscriptional and transcriptional events. However, posttranscriptional regulation of osteoclastogenesis is incompletely understood. CNOT3 is a component of the CCR4 family that regulates mRNA stability, but its function in bone is not known. Here, we show that Cnot3 deficiency by deletion of a single allele induces osteoporosis. Cnot3 deficiency causes an enhancement in bone resorption in association with an elevation in bone formation, resulting in high-turnover type bone loss. At the cellular level, Cnot3 deficiency enhances receptor activator of NF-κB ligand (RANKL) effects on osteoclastogenesis in a cell-autonomous manner. Conversely, Cnot3 deficiency does not affect osteoblasts directly. Cnot3 deficiency does not alter RANKL expression but enhances receptor activator of NF-κB (RANK) mRNA expression in bone in vivo. Cnot3 deficiency promotes RANK mRNA stability about twofold in bone marrow cells of mice. Cnot3 knockdown also increases RANK mRNA expression in the precursor cell line for osteoclasts. Anti-CNOT3 antibody immunoprecipitates RANK mRNA. Cnot3 deficiency stabilizes luciferase reporter expression linked to the 3′-UTR fragment of RANK mRNA. In contrast, Cnot3 overexpression destabilizes the luciferase reporter linked to RANK 3′-UTR. In aged mice that exhibit severe osteoporosis, Cnot3 expression levels in bone are reduced about threefold in vivo. Surprisingly, Cnot3 deficiency in these aged mice further exacerbates osteoporosis, which also occurs via enhancement of osteoclastic activity. Our results reveal that CNOT3 is a critical regulator of bone mass acting on bone resorption through posttranscriptional down-regulation of RANK mRNA stability, at least in part, even in aging-induced osteoporosis.

Migration Linked to FUCCI-Indicated Cell Cycle Is Controlled by PTH and Mechanical Stress

Bone metabolism is maintained via balanced repetition of bone resorption by osteoclasts and bone formation by osteoblasts. Osteoblastic cells are capable of conducting self‐renewal and differentiation that are basically associated with cell‐cycle transition to enable cell specification and bone formation. Osteoblasts are also migrating to fill the resorption cavity curved by osteoclasts during bone remodeling to maintain homeostasis of bone mass whose imbalance leads to osteoporosis. However, technical difficulties have hampered the research on the dynamic relationship between cell cycle and migration in osteoblasts. In this report, we overcome these problems by introducing fluorescent ubiquitination‐based cell cycle indicator (FUCCI) reporter system in calvarial osteoblastic cells and reveal that the cells in G1 as well as S/G2/M phase are migrating. Furthermore, the osteoblastic cells in S/G2/M phase migrate faster than those in G1 phase. Interestingly, parathyroid hormone (PTH) as an anabolic agent enhances migration velocity of the cells. Mechanical stress, another anabolic signal, also enhances migration velocity. In contrast, in the presence of both PTH and mechanical stress, the migration velocity returns to the base line levels revealing the interaction between the two anabolic stimuli in the regulation of cell migration. Importantly, PTH and mechanical stress also interact when they regulate the transition of cell cycle. These data demonstrate that osteoblastic migration is linked to cell cycle and it is under the control of mechanical and chemical stimuli that coordinate to regulate bone mass. J. Cell. Physiol. 229: 1353–1358, 2014.