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The bone is the main storage site for Ca2+ and Mg2+ ions in the mammalian body. Although investigations into Ca2+ signaling have progressed rapidly and led to better understanding of bone biology, the Mg2+ signaling pathway and associated molecules remain to be elucidated. Here, we investigated the role of a potential Mg2+ signaling-related lysosomal molecule, two-pore channel subtype 2 (TPC2), in osteoclast differentiation and bone remodeling. Previously, we found that under normal Mg2+ conditions, TPC2 promotes osteoclastogenesis. We observed that under low-Mg2+ conditions, TPC2 inhibited, rather than promoted, the osteoclast differentiation and that the phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) signaling pathway played a role in the TPC2 activation under low-Mg2+ conditions. Furthermore, PI(3,5)P2 depolarized the membrane potential by increasing the intracellular Na+ levels. To investigate how membrane depolarization affects osteoclast differentiation, we generated a light-sensitive cell line and developed a system for the light-stimulated depolarization of the membrane potential. The light-induced depolarization inhibited the osteoclast differentiation. We then tested the effect of myo-inositol supplementation, which increased the PI(3,5)P2 levels in mice fed a low-Mg2+ diet. The myo-inositol supplementation rescued the low-Mg2+ diet–induced trabecular bone loss, which was accompanied by the inhibition of osteoclastogenesis. These results indicate that low-Mg2+–induced osteoclastogenesis involves changes in the role of TPC2, which are mediated through the PI(3,5)P2 pathway. Our findings also suggest that myo-inositol consumption might provide beneficial effects in Mg2+ deficiency–induced skeletal diseases.

Parathyroid hormone (PTH) and 1α,25-dihydroxyvitamin D3 (VD3) are important factors in Ca(2+) homeostasis, and promote osteoclastogenesis by modulating receptor activator of nuclear factor kappa-B ligand (RANKL) mRNA expression. However, their contribution to RANKL intracellular transport (RANKLiT), including the trigger for RANKL lysosomal vesicle (RANKL-lv) fusion to the cell membrane, is unclear. In neurons, depolarization of membrane potential increases the intracellular Ca(2+) level ([Ca(2+)]i) and promotes neurotransmitter release via fusion of the synaptic vesicles to the cell membrane. To determine whether membrane depolarization also regulates cellular processes such as RANKLiT in MC3T3-E1 osteoblasts (OBs), we generated a light-sensitive OB cell line and developed a system for altering their membrane potential via delivery of a blue light stimulus. In the membrane fraction of RANKL-overexpressing OBs, PTH and VD3 increased the membrane-bound RANKL (mbRANKL) level at 10 min after application without affecting the mRNA expression level, and depolarized the cell membrane while transiently increasing [Ca(2+)]i. In our novel OB line stably expressing the channelrhodopsin-wide receiver, blue light-induced depolarization increased the mbRANKL level, which was reversed by treatment of blockers for L-type voltage-gated Ca(2+) channels and Ca(2+) release from the endoplasmic reticulum. In co-cultures of osteoclast precursor-like RAW264.7 cells and light-sensitive OBs overexpressing RANKL, light stimulation induced an increase in tartrate-resistant acid phosphatase activity and promoted osteoclast differentiation. These results indicate that depolarization of the cell membrane is a trigger for RANKL-lv fusion to the membrane and that membrane potential contributes to the function of OBs. In addition, the non-genomic action of VD3-induced RANKL-lv fusion included the membrane-bound VD3 receptor (1,25D3-MARRS receptor). Elucidating the mechanism of RANKLiT regulation by PTH and VD3 will be useful for the development of drugs to prevent bone loss in osteoporosis and other bone diseases.

Ift88 is an intraflagella transport protein, critical for the cilium, and has been shown to be required for the maintenance of chondrocytes and cartilage. However, how Ift88 is controlled by cytokines that play a role in osteoarthritis is not well understood. Therefore, we examined the effects of TGF-β on the expression of Ift88. We used ATDC5 cells as chondrocytes and analyzed the effects of TGF-β on gene expression. TGF-β treatment suppresses the levels of Ift88 mRNA in a dose-dependent manner starting from as low as 0.5 ng/mL and reaching the nadir at around 2 ng/mL. TGF-β treatment also suppresses the protein levels of Ift88. TGF-β suppression of Ift88 is still observed when the cells are cultured in the presence of a transcriptional inhibitor while the TGF-β suppression is weakened in the presence of a protein synthesis inhibitor, cycloheximide. TGF-β treatment suppresses the levels of Ift88 mRNA stability suggesting the presence of posttranscriptional regulation. TGF-β treatment reduces the number of cilia positive cells and suppresses average length of cilia. Knockdown of Ift88 by siRNA enhances TGF-β-induced increase in type II collagen mRNA expression in ATDC5 cells revealing the suppressive role of Ift88 on TGF-β-induced regulation of extracellular matrix protein expression. TGF-β also suppresses Ift88 mRNA expression in primary culture of rib chondrocytes. These data indicate that TGF-β regulates Ift88 gene expression at least in part via posttrascriptional manner. J. Cell. Physiol. 9999: 2788–2795, 2015. © 2015 Wiley Periodicals, Inc.

As the aged population is soaring, prevalence of osteoporosis is increasing. However, the molecular basis underlying the regulation of bone mass is still incompletely understood. Sympathetic tone acts via beta2 adrenergic receptors in bone and regulates the mass of bone which is the target organ of parathyroid hormone (PTH). However, whether beta2 adrenergic receptor is regulated by PTH in bone cells is not known. We therefore investigated the effects of PTH on beta2 adrenergic receptor gene expression in osteoblast-like MC3T3-E1 cells. PTH treatment immediately suppressed the expression levels of beta2 adrenergic receptor mRNA. This PTH effect was dose-dependent starting as low as 1 nM. PTH action on beta2 adrenergic receptor gene expression was inhibited by a transcriptional inhibitor, DRB, but not by a protein synthesis inhibitor, cycloheximide suggesting direct transcription control. Knockdown of beta2 adrenergic receptor promoted PTH-induced expression of c-fos, an immediate early response gene. With respect to molecular basis for this phenomenon, knockdown of beta2 adrenergic receptor enhanced PTH-induced transcriptional activity of cyclic AMP response element-luciferase construct in osteoblasts. Knockdown of beta2 adrenergic receptors also enhanced forskolin-induced luciferase expression, revealing that adenylate cyclase activity is influenced by beta2 adrenergic receptor. As for phosphorylation of transcription factor, knockdown of beta2 adrenergic receptor enhanced PTH-induced phosphorylation of cyclic AMP response element binding protein (CREB). These data reveal that beta2 adrenergic receptor is one of the targets of PTH and acts as a suppressor of PTH action in osteoblasts.

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.

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.

Mechanical stress is an important signal to determine the levels of bone mass. Unloading-induced osteoporosis is a critical issue in bed-ridden patients and astronauts. Many molecules have been suggested to be involved in sensing mechanical stress in bone, though the mechanisms involved in this phenomenon are not fully understood. Nck1 is an adaptor protein known to mediate signaling from plasma membrane-activated receptors to cytosolic effectors regulating actin cytoskeleton remodeling. Nck1 has also been implicated in cellular responses to endoplasmic reticulum stress. In vitro, in case of cell stress the actin cytoskeleton is disrupted and in such cases Nck1 has been reported to enter the nucleus of the cells to mediate the nuclear actin polymerization. However, the role of Nck1 in vivo during the bone response to mechanical stimuli is unknown. The purpose of this study is to examine the role of Nck1 in unloading-induced bone loss in vivo. Sciatic and femoral nerve resection was conducted. Neurectomy-based unloading enhanced Nck1 gene expression in bone about twofold. Using the Nck1 deficient mice and control Nck1+/+, effects of neurectomy-based unloading on bone structure were examined. Unloading reduced bone volume in wild type mice by 30% whereas the levels in bone loss were exacerbated to 50% in Nck1 deficient mice due to neurectomy after 4 weeks. These data demonstrate that Nck1 gene deficiency accelerates the mechanical unloading-induced bone loss suggesting Nck1 to be a crucial molecule in mechanical stress mediated regulation in bone metabolism.

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 Ca(2+) 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.

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.

Parathyroid hormone/parathyroid hormone-related protein receptor (PPR) signaling is known to be involved in tooth development. In bone, extracellular matrix protein osteopontin (OPN) is a negative regulator of PPR signaling in bone formation. However, the role of OPN in modulation of PPR action in tooth development is not understood. Therefore, we examined the tooth in double mutant mice. Constitutively active PPR was expressed specifically in the odontoblasts and osteoblasts (caPPR-tg) in the presence or absence of OPN. Radiographic analysis indicated that the length of the third molar (M3) and the incisor was decreased in the caPPR-tg mice compared to wild type, and such reduction in molar and incisor length was further enhanced in the absence of OPN (caPPR-tg OPN-KO). With respect to histology of incisors, caPPR-tg induced high cellularity and irregularity in odontoblastic shape and this was enhanced by the absence of OPN. These morphological observations suggest that OPN modulates PPR signaling that are involved in tooth formation.

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.

Schnurri (Shn)-2 is a transcriptional modulator of bone formation and bone resorption and its deficiency causes low turnover state with higher cancellous bone mass due to the defects in osteoclasts that exceeds the defects in osteoblasts in mice. We addressed whether such low turnover of bone remodeling in Shn2 deficiency may be modulated in the absence of estrogen that induces high turnover state in vivo. Ovariectomy reduced bone mass in wild type compared to sham operated control mice and such reduction in bone mass was also observed in Shn2 deficient mice. However, due to the high levels of basal bone mass in Shn2 deficient mice, the bone mass levels after ovariectomy were still comparable to sham operated wild-type mice. Analysis indicated that estrogen depletion increased bone resorption at similar levels in wild type and Shn2 deficient mice though the basal levels of osteoclast number was slightly lower in Shn2-deficient mice. In contrast, basal levels of bone marrow cell mineralization in cultures were low in Shn2-deficeint mice while estrogen depletion increased the mineralization levels to those that were comparable to sham wild type. This indicates that Shn2-deficient mice maintain bone mass at the levels comparable to wild-type sham mice even after ovariectomy-induced bone loss and this correlates with the high levels of mineralization activity in bone marrow cells after ovariectomy. J. Cell. Physiol. 226: 573–578, 2011. © 2010 Wiley-Liss, Inc.

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.

CIZ/NMP4 (Cas interacting zinc finger protein, Nmp4, Zfp384) is a transcription factor that is known to regulate matrix related-proteins. To explore the possible pathophysiological role of CIZ/NMP4 in arthritis, we examined CIZ/NMP4 expression in articular cartilage in arthritis model. CIZ/NMP4 was expressed in the articular chondrocytes of mice at low levels while its expression was enhanced when arthritis was induced. Arthritis induction increased clinical score in wild type mice. In contrast, CIZ/NMP4 deficiency suppressed such rise in the levels of arthritis score and swelling of soft tissue. CIZ/NMP4 deficiency also reduced invasion of inflammatory cells in joint tissue. Quantitative PCR analyses of mRNA from joints revealed that arthritis-induced increase in expressions of IL-1β was suppressed by CIZ/NMP4 deficiency. CIZ/NMP4 bound to IL-1β promoter and activated its transcription. The increase in CIZ/NMP4 in arthritis was also associated with enhancement in bone resorption and cartilage matrix degradation. In fact, RANKL, a signaling molecule prerequisite for osteoclastogenesis and, MMP-3, a clinical marker for arthritis were increased in joints upon arthritis induction. In contrast, CIZ/NMP4 deficiency suppressed the arthritis-induced increase in bone resorption, expression of RANKL and MMP-3 mRNA. Thus, CIZ/NMP4 plays a role in the development of arthritis at least in part through regulation of key molecules related to the arthritis.

Objective: Apoptosis of chondrocytes in articular cartilage has been observed in rheumatoid arthritis patients. However, molecules involved in such chondrocyte apoptosis in arthritic joints have not been fully understood. We previously observed that apoptosis of chondrocytes is enhanced in a murine arthritis model induced by injection with anti–type II collagen antibodies and lipopolysaccharide (mAbs/LPS), and osteopontin (OPN) deficiency suppresses chondrocyte apoptosis in this arthritis model in vivo. To understand how OPN deficiency renders resistance against chondrocyte apoptosis, we examined the cellular basis for this protection. Design: Chondrocytes were prepared from wild-type and OPN-deficient mouse ribs, and tumor necrosis factor (TNF)–α–induced cell death was examined based on lactate dehydrogenase (LDH) release assay and TUNEL assay. Results: TNF-α treatment induced LDH release in wild-type chondrocytes, while OPN deficiency suppressed such LDH release in the cultures of these cells. TNF-α–induced increase in the number of TUNEL-positive cells was observed in wild-type chondrocytes, while OPN deficiency in chondrocytes suppressed the TNF-α induction of TUNEL-positive cells. OPN deficiency suppressed TNF-α–induced increase in caspase-3 activity in chondrocytes in culture. Furthermore, OPN overexpression in chondrocytes enhanced TNF-α–induced apoptosis. Conclusion: These results indicated that the presence of OPN in chondrocytes is involved in the susceptibility of these cells to TNF-α–induced apoptosis.

Ift88 is an intraflagella transport protein, critical for the cilium, and has been shown to be required for the maintenance of chondrocytes and cartilage. However, how Ift88 is controlled by cytokines that play a role in osteoarthritis is not well understood. Therefore, we examined the effects of TGF-β on the expression of Ift88. We used ATDC5 cells as chondrocytes and analyzed the effects of TGF-β on gene expression. TGF-β treatment suppresses the levels of Ift88 mRNA in a dose-dependent manner starting from as low as 0.5 ng/mL and reaching the nadir at around 2 ng/mL. TGF-β treatment also suppresses the protein levels of Ift88. TGF-β suppression of Ift88 is still observed when the cells are cultured in the presence of a transcriptional inhibitor while the TGF-β suppression is weakened in the presence of a protein synthesis inhibitor, cycloheximide. TGF-β treatment suppresses the levels of Ift88 mRNA stability suggesting the presence of posttranscriptional regulation. TGF-β treatment reduces the number of cilia positive cells and suppresses average length of cilia. Knockdown of Ift88 by siRNA enhances TGF-β-induced increase in type II collagen mRNA expression in ATDC5 cells revealing the suppressive role of Ift88 on TGF-β-induced regulation of extracellular matrix protein expression. TGF-β also suppresses Ift88 mRNA expression in primary culture of rib chondrocytes. These data indicate that TGF-β regulates Ift88 gene expression at least in part via posttrascriptional manner.

The Na+–K+pump current (Ip) and the h-current (Ih) flowing through hyperpolarization-activated channels (h-channels) participate in generating the resting potential. These two currents are thought to be produced independently. We show here bidirectional interactions between Na+–K+pumps and h-channels in mesencephalic trigeminal neurons. Activation ofIhleads to the generation of two types of ouabain-sensitiveIpwith temporal profiles similar to those of instantaneous and slow components ofIh, presumably reflecting Na+transients in a restricted cellular space. Moreover, theIpactivated by instantaneousIhcan facilitate the subsequent activation of slowIh. Such counteractive and cooperative interactions were also disclosed by replacing extracellular Na+with Li+, which is permeant through h-channels but does not stimulate the Na+–K+pump as strongly as Na+ions. These observations indicate that the interactions are bidirectional and mediated by Na+ions. Also after substitution of extracellular Na+with Li+, the tailIhwas reduced markedly despite an enhancement ofIhitself, attributable to a negative shift of the reversal potential forIhpresumably caused by intracellular accumulation of Li+ions. This suggests the presence of a microdomain where the interactions can take place. Thus, the bidirectional interactions between Na+–K+pumps and h-channels are likely to be mediated by Na+microdomain. Consistent with these findings, hyperpolarization-activated and cyclic nucleotide-modulated subunits (HCN1/2) and the Na+–K+pumpα3 isoform were colocalized in plasma membrane of mesencephalic trigeminal neurons having numerous spines.

We aimed to estimate the number of AMPA receptors (AMPARs) bound by the quantal transmitter packet, their single-channel conductance and their density in the postsynaptic membrane at cerebellar Purkinje cell synapses. The synaptic and extrasynaptic AMPARs were examined in Purkinje cells in 2- to 4-day-old rats, when they receive synaptic inputs solely from climbing fibres (CFs). Evoked CF EPSCs and whole-cell AMPA currents displayed roughly linear current-voltage relationships, consistent with the presence of GluR2 subunits in synaptic and extrasynaptic AMPARs. The mean quantal size, estimated from the miniature EPSCs (MEPSCs), was approximately 300 pS. Peak-scaled non-stationary fluctuation analysis of spontaneous EPSCs and MEPSCs gave a weighted-mean synaptic channel conductance of approximately 5 pS (approximately 7 pS when corrected for filtering). By applying non-stationary fluctuation analysis to extrasynaptic currents activated by brief glutamate pulses (5 mM), we also obtained a small single-channel conductance estimate for extrasynaptic AMPARs (approximately 11 pS). This approach allowed us to obtain a maximum open probability (Po,max) value for the extrasynaptic receptors (Po,max = 0.72). Directly resolved extrasynaptic channel openings in the continued presence of glutamate exhibited clear multiple-conductance levels. The mean area of the postsynaptic density (PSD) of these synapses was 0.074 microm2, measured by reconstructing electron-microscopic (EM) serial sections. Postembedding immunogold labelling by anti-GluR2/3 antibody revealed that AMPARs are localised in PSDs. From these data and by simulating error factors, we estimate that at least 66 AMPARs are bound by a quantal transmitter packet at CF-Purkinje cell synapses, and the receptors are packed at a minimum density of approximately 900 microm-2 in the postsynaptic membrane.

To determine both the preventive and recovery effects of tower climbing exercise on mass, strength, and local turnover of bone in ovariectomized (OVX) rats, we carried out two experiments. In experiment I, 60 Sprague-Dawley rats, 12 months of age, were assigned to four groups: a Baseline Control, Sham-Operated Sedentary, OVX-Sedentary and OVX-Exercise rats. Rats voluntarily climbed a 200-cm tower to drink water from a bottle set at the top. At 3 months, OVX elevated both the femoral cortex and lumbar trabecular turnover, leading to a reduction in bone mass and strength. However, in OVX-Exercise rats, those values were maintained at the same level as in the Sham-Sedentary rats. Thus, the climbing exercise, started after 3 days of OVX, prevented OVX-induced cortical and trabecular bone loss by depressing turnover elevation. After confirming the preventive effect, we evaluated the recovery effect of exercise. In experiment II, 90 Sprague-Dawley rats, 12 months of age, were assigned to six groups: a Baseline control, two groups of Sham-Operated Sedentary and OVX-Sedentary, and OVX-Exercise rats. The exercise started 3 months after the OVX operation. At 3 months, OVX increased the trabecular bone formation rate and osteoclast surface, leading to a decrease in compressive strength. In the midfemur, the cross-sectional area, moment of inertia, and bending load values decreased. At 6 months, in the OVX-Exercise rats, the parameters of breaking load in both the lumbar and midfemur, lumbar bone mass, and the total cross-sectional area recovered to the same levels as those in the Sham-Sedentary rats. However, the cortical bone area did not recover. Periosteal bone formation increased, while endosteal bone formation decreased. These results showed that the climbing exercise had both a preventive and recovery effect on bone strength in OVX rats. In the mid-femur, effects on bone formation were site-specific, and the cross-sectional morphology was improved without an increase in cortical bone area, supporting cortical drift by mechanical stimulation.

An ion channel's function depends largely on its location and density on neurons. Here we used high-resolution immunolocalization to determine the subcellular distribution of the hyperpolarization-activated and cyclic-nucleotide-gated channel subunit 1 (HCN1) in rat brain. Light microscopy revealed graded HCN1 immunoreactivity in apical dendrites of hippocampal, subicular and neocortical layer-5 pyramidal cells. Quantitative comparison of immunogold densities showed a 60-fold increase from somatic to distal apical dendritic membranes. Distal dendritic shafts had 16 times more HCN1 labeling than proximal dendrites of similar diameters. At the same distance from the soma, the density of HCN1 was significantly higher in dendritic shafts than in spines. Our results reveal the complex cell surface distribution of voltage-gated ion-channels, and predict its role in increasing the computational power of single neurons via subcellular domain and input-specific mechanisms.

Metabotropic gamma-aminobutyric acid receptors (GABA(B)Rs) are involved in modulation of synaptic transmission and activity of cerebellar and thalamic neurons. We used subtype-specific antibodies in pre- and postembedding immunohistochemistry combined with three-dimensional reconstruction of labelled profiles and quantification of immunoparticles to reveal the subcellular distribution of pre- and postsynaptic GABA(B)R1a/b and GABA(B)R2 in the rat cerebellum and ventrobasal thalamus. GABA(B)R1a/b and R2 were extensively colocalized in most brain regions including the cerebellum and thalamus. In the cerebellum, immunoreactivity for both subtypes was prevalent in the molecular layer. The most intense immunoreactivity was found in Purkinje cell spines with a high density of immunoparticles at extrasynaptic sites peaking at around 240 nm from glutamatergic synapses between spines and parallel fibre varicosities. This is in contrast to dendrites at sites around GABAergic synapses where sparse and random distribution was found for both subtypes. In addition, more than one-tenth of the synaptic membrane specialization of spine-parallel fibre synapses were labelled at pre- or postsynaptic sites. Weak immunolabelling for both subtypes was also seen in parallel fibres but only rarely in GABAergic axons. In the ventrobasal thalamus, immunolabelling for both receptor subtypes was intense over the dendritic field of thalamocortical cells. Electron microscopy demonstrated an extrasynaptic localization of GABA(B)R1a/b and R2 exclusively in postsynaptic elements. Quantitative analysis further revealed the density of GABA(B)R1a/b around GABAergic synapses was higher than glutamatergic synapses on thalamocortical cell dendrites. The distinct localization of GABA(B)Rs relative to synaptic sites in the cerebellum and ventrobasal thalamus suggests that GABA(B)Rs differentially regulate activity of different neuronal populations.

β adrenergic stimulation suppresses bone formation in vivo while its actions in osteoblastic differentiation are still incompletely understood. We therefore examined the effects of β2 adrenergic stimulation on osteoblast-like MC3T3-E1 cells focusing on BMP-induced alkaline phosphatase expression. Morphologically, isoproterenol treatment suppresses BMP-induced increase in the numbers of alkaline phosphatase-positive small foci in the cultures of MC3T3-E1 cells. Biochemically, isoproterenol treatment suppresses BMP-induced enzymatic activity of alkaline phosphatase in a dose-dependent manner. Isoproterenol suppression of alkaline phosphatase activity is observed even when the cells are treated with high concentrations of BMP. With respect to cell density, isoproterenol treatment tends to suppress BMP-induced increase in alkaline phosphatase expression more in osteoblasts cultured at higher cell density. In terms of treatment protocol, continuous isoproterenol treatment is compared to cyclic treatment. Continuous isoproterenol treatment is more suppressive against BMP-induced increase in alkaline phosphatase expression than cyclic regimen. At molecular level, isoproterenol treatment suppresses BMP-induced enhancement of alkaline phosphatase mRNA expression. Regarding the mode of isoproterenol action, isoproterenol suppresses BMP-induced BRE-luciferase activity. These data indicate that isoproterenol regulates BMP-induced alkaline phosphatase expression in osteoblast-like MC3T3E1 cells.

Zinc is a trace element in the mammalian body, and increasing evidence shows its critical role in bone development and osteoclastogenesis. The relationships between zinc and voltage-gated ion channels have been reported; however, the effects of zinc on membrane potential and the related ion channels remain unknown. In this study, we found that zinc-induced hyperpolarization in RAW264.7 cells (RAW) was promoted by inhibition of hyperpolarization-activated cyclic nucleotide modulated channels (HCNs). In electrophysiological experiments with RAW-derived osteoclasts, HCNs were functional and generated hyperpolarization-activated inward currents (Ih) with properties similar to the Ih recorded in excitable cells such as neurons and cardiomyocytes. Quantitative PCR of HCN subunits HCN1 and HCN4 in RAW cells showed detectable levels of HCN1 mRNA and HCN4 expression was the highest of all four subunits. HCN4 knockdown decreased osteoclastic Ih and promoted osteoclastogenesis in the presence of zinc, but not in the absence of zinc. To determine the effect of membrane hyperpolarization on osteoclastogenesis, we developed a light-controllable membrane potential system in RAW cells by stably expressing the light-driven outward proton pump, Archaerhodopsin3 (Arch). Arch activation by yellow-green light hyperpolarizes the cell membrane. Light-induced hyperpolarization accelerated osteoclast differentiation in the presence of receptor activator of nuclear factor kappa-B ligand (RANKL). Thus, HCN activation reduced the hyperpolarization-related promotion of osteoclast differentiation in the presence of zinc. This study revealed the novel role of HCN and membrane potential in non-excitable osteoclasts.

As the aged population is soaring, prevalence of osteoporosis is increasing. However, the molecular basis underlying the regulation of bone mass is still incompletely understood. Sympathetic tone acts via beta2 adrenergic receptors in bone and regulates the mass of bone which is the target organ of parathyroid hormone (PTH). However, whether beta2 adrenergic receptor is regulated by PTH in bone cells is not known. We therefore investigated the effects of PTH on beta2 adrenergic receptor gene expression in osteoblast-like MC3T3-E1 cells. PTH treatment immediately suppressed the expression levels of beta2 adrenergic receptor mRNA. This PTH effect was dose-dependent starting as low as 1 nM. PTH action on beta2 adrenergic receptor gene expression was inhibited by a transcriptional inhibitor, DRB, but not by a protein synthesis inhibitor, cycloheximide suggesting direct transcription control. Knockdown of beta2 adrenergic receptor promoted PTH-induced expression of c-fos, an immediate early response gene. With respect to molecular basis for this phenomenon, knockdown of beta2 adrenergic receptor enhanced PTH-induced transcriptional activity of cyclic AMP response element-luciferase construct in osteoblasts. Knockdown of beta2 adrenergic receptors also enhanced forskolin-induced luciferase expression, revealing that adenylate cyclase activity is influenced by beta2 adrenergic receptor. As for phosphorylation of transcription factor, knockdown of beta2 adrenergic receptor enhanced PTH-induced phosphorylation of cyclic AMP response element binding protein (CREB). These data reveal that beta2 adrenergic receptor is one of the targets of PTH and acts as a suppressor of PTH action in osteoblasts.

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.

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.

Maximizing peak bone mass is an important factor in osteoporosis prevention. Resistance exercise increases bone mass and strength, while nutritional supplements have beneficial effects on bone loss reduction. We have previously shown that the combined intake of sucrose and amino acids (AA), which is strongly insulinogenic, efficiently increased muscle protein synthesis. To investigate the effects of sugar and an AA solution immediately after resistance exercise, we compared insulinogenic sucrose and non-insulinogenic fructose combined with an AA solution with or without resistance exercise. Sucrose intake immediately after resistance exercise increased the trabecular bone mass and compressive maximum load compared with fructose+AA intake after exercise. Additionally, combined sucrose+AA and exercise increased trabecular bone formation and decreased bone resorption more than combined fructose and exercise. Serum insulin levels were greatly increased by sucrose+AA intake with exercise. In culture experiments, neither sugar+AA affected osteoblast and osteoclast differentiation. In a gene expression study, sucrose+AA intake after resistance exercise was shown to upregulate the Runx2 expression level and decrease RANKL/OPG ratio. These results suggest that the combined intake of sucrose and an AA solution immediately after resistance exercise exerts anabolic effects on bone by altering gene expression related to bone remodeling. Although translation of our bone remodeling findings from animal to human studies has been challenging, our findings suggest that exercise with sugar+AA intake may contribute to improved bone health.

Bone development is a dynamic process that requires cell motility and morphological adaptation under the control of actin cytoskeleton. This actin cytoskeleton system is regulated by critical modulators including actin-binding proteins. Among them, profilin1 (Pfn1) is a key player to control actin fiber structure, and it is involved in a number of cellular activities such as migration. During the early phase of body development, skeletal stem cells and osteoblastic progenitor cells migrate to form initial rudiments for future skeletons. During this migration, these cells extend their process based on actin cytoskeletal rearrangement to locate themselves in an appropriate location within microenvironment. However, the role of Pfn1 in regulation of mesenchymal progenitor cells (MPCs) during skeletal development is incompletely understood. Here we examined the role of Pfn1 in skeletal development using a genetic ablation of Pfn1 in MPCs by using Prx1-Cre recombinase. We found that Pfn1 deficiency in MPCs caused complete cleft sternum. Notably, Pfn1-deficient mice exhibited an absence of trabecular bone in the marrow space of appendicular long bone. This phenotype is location-specific, as Pfn1 deficiency did not largely affect osteoblasts in cortical bone. Pfn1 deficiency also suppressed longitudinal growth of long bone. In vitro, Pfn1 deficiency induced retardation of osteoblastic cell migration. These observations revealed that Pfn1 is a critical molecule for the skeletal development, and this could be at least in part associated with the retardation of cell migration.

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.

Tumor metastasis to bone is a serious pathological situation that causes severe pain, and deterioration in locomoter function. However, the mechanisms underlying tumor metastasis is still incompletely understood. CIZ/NMP4 is a nucleocytoplasmic shuttling protein and its roles in tumor cells have not been known. We, therefore, hypothesized the role of CIZ/NMP4 in B16 melanoma cells that metastasize to bone. CIZ/NMP4 is expressed in B16 cells. The CIZ/NMP4 expression levels are correlated to the metastatic activity in divergent types of melanoma cells. Overexpression of CIZ/NMP4 increased B16 cell migration in Trans-well assay. Conversely, siRNA-based knockdown of CIZ/NMP4 suppressed migratory activity of these cells. As RANKL promotes metastasis of tumor cells in bone, we tested its effect on CIZ in melanoma cells. RANKL treatment enhanced CIZ/NMP4 expression. This increase of CIZ by RANKL promoted migration. Conversely, we identified CIZ/NMP4 binding site in the promoter of RANKL. Furthermore, luciferase assay indicated that CIZ/NMP4 overexpression enhanced RANKL promoter activities, revealing a positive feedback loop of CIZ/NMP4 and RANKL in melanoma. These observations indicate that CIZ/NMP4 is critical regulator of metastasis of melanoma cells. J. Cell. Physiol. 227: 2807–2812, 2012. © 2012 Wiley Periodicals, Inc.

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.

Bone has been known to adapt to mechanical stress (Amin 2010; Beier and Loeser 2010; Currey 2010; Temiyasathit and Jacobs 2010). The presence of mechanical stress increases bone mass and the absence of mechanical stress reduces bone mass. Bending of weight-bearing long bone increases pressure on the concave side and decreases it on the convex side. Under such circumstances, bone is accumulated on the concave side and is reduced on the convex side. This can be seen after angular deformity due to malunion of fractures in children where minor angular deformity could be corrected during the growth of the children.

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.

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.

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 ( Cm) simultaneously. Cm is a measure of cell surface. Extracellular Ca2+ (2.5–40 mM) decreased Cm and the V-ATPase current simultaneously. The decreased Cm, 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 A1, 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 γ2 subunit. Holding the cytosolic Ca2+ at either high (0.5–5 μM) 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.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that generate Ih currents are widely distributed in the brain and have been shown to contribute to various neuronal functions. In the present study, we investigated the functions of Ih in the motion-sensitive projection neurons [wide field vertical (WFV) cells] of the superior colliculus, a pivotal visual center for detection of and orientating to salient objects. Combination of whole cell recordings and immunohistochemical investigations suggested that HCN1 channels dominantly contribute to the Ih in WFV cells among HCN isoforms expressed in the superficial superior colliculus and mainly located on their expansive dendritic trees. We found that blocking Ih suppressed the initiation of short- and fixed-latency dendritic spike responses and led instead to long- and fluctuating-latency somatic spike responses to optic fiber stimulations. These results suggest that the dendritic Ih facilitates the dendritic initiation and/or propagation of action potentials and ensures that WFV cells generate spike responses to distal synaptic inputs in a sensitive and robustly time-locked manner, probably by acting as continuous depolarizing drive and fixing dendritic membrane potentials close to the spike threshold. These functions are different from known functions of dendritic Ih revealed in hippocampal and neocortical pyramidal cells, where they spatiotemporally limit the propagations of synaptic inputs along the apical dendrites by reducing dendritic membrane resistance. Thus we have revealed new functional aspects of Ih, and these dendritic properties are likely critical for visual motion processing in these neurons.

Voltage-gated proton (H+) channels play a pivotal role in compensating charge and pH imbalances during respiratory bursts in phagocytes. Lactic acidosis is a clinically important metabolic condition accompanying various tissue disorders in which the extracellular pH and the intracellular pH often change in parallel. In this study, we investigated the responses of the H+ channel in microglia to lactate-induced pH disturbances using the perforated-patch recordings. Na-lactate (pH 6.8) acidified the cells and activated the H+ channel within 5 min. This early activation was correlated with increases in the pH gradient across the plasma membrane (DeltapH) and was dose-dependent over a concentration range of 10-150 mM. At 10 mM, the change in DeltapH was only slight, but the H+ currents continued to increase over an hour after the cell acidosis was stabilized. Prolonged exposure to lactate (10-20 mM,1 h) increased the amplitude by two to threefold. The late activation was not explained by increased DeltapH but by changes in the property of the channel per se. Pretreatment with staurosporine and chelerythrine, inhibitors for protein kinase C, prevented the late activation. These results suggest that the H+ channel could be activated greatly during long-lasting lactic acidosis through both DeltapH-dependent and -independent mechanisms.

Hyperpolarization-activated cation currents (I(h)) contribute to various physiological properties and functions in the brain, including neuronal pacemaker activity, setting of resting membrane potential, and dendritic integration of synaptic input. Four subunits of the Hyperpolarization-activated and Cyclic-Nucleotide-gated nonselective cation channels (HCN1-4), which generate I(h), have been cloned recently. To better understand the functional diversity of I(h) in the brain, we examined precise immunohistochemical localization of four HCNs in the rat brain. Immunoreactivity for HCN1 showed predominantly cortical distribution, being intense in the neocortex, hippocampus, superior colliculus, and cerebellum, whereas those for HCN3 and HCN4 exhibited subcortical distribution mainly concentrated in the hypothalamus and thalamus, respectively. Immunoreactivity for HCN2 had a widespread distribution throughout the brain. Double immunofluorescence revealed colocalization of immunoreactivity for HCN1 and HCN2 in distal dendrites of pyramidal cells in the hippocampus and neocortex. At the electron microscopic level, immunogold particles for HCN1 and HCN2 had similar distribution patterns along plasma membrane of dendritic shafts in layer I of the neocortex and stratum lacunosum moleculare of the hippocampal CA1 area, suggesting that these subunits could form heteromeric channels. Our results further indicate that HCNs are localized not only in somato-dendritic compartments but also in axonal compartments of neurons. Immunoreactivity for HCNs often occurred in preterminal rather than terminal portions of axons and in specific populations of myelinated axons. We also found HCN2-immunopositive oligodendrocytes including perineuronal oligodendrocytes throughout the brain. These results support previous electrophysiological findings and further suggest unexpected roles of I(h) channels in the brain.

To investigate the relationship between the effects of bone turnover and bone marrow cell development in bone cells, we developed a mouse voluntary climbing exercise model. Climbing exercise increased bone volume and transient osteogenic potential of bone marrow. This model would be suitable for investigating the mechanistic roles of mechanical loading. Introduction: The relationship between bone mass gain and local bone formation and resorption in mechanically loaded bone is not well understood. Materials and Methods: Sixty-five C57BL/6J mice, 8 weeks of age, were assigned to five groups: a baseline control and two groups each of ground control and climbing exercise mice for 2 and 4 weeks. Mice were housed in a 100-cm tower and had to climb toward a bottle placed at the top to drink water. Results: Compared with the ground control, bone mineral density of the left femur increased in the climbing mice at 4 weeks. At 2 and 4 weeks, bone formation rate (BFR/BS) of periosteal surface, the cross-sectional area, and moment of inertia were increased in the climbing mice, whereas BFR/BS and eroded surface (ES/BS) of endosteal surface did not differ. The trabecular bone volume (BV/TV) of the proximal tibia increased in climbing mice, and osteoclast surface (Oc.S/BS) and osteoclast number decreased at 2 weeks. At 4 weeks, there were increases in BV/TV and parameters of bone formation, including mineralized surface, mineral apposition rate, and bone formation rate. In marrow cell cultures from the tibia, the number of alkaline phosphatase+ colony forming units-fibroblastic and the area of mineralized nodule formation in climbing mice were increased, and the number of osteoclast-like TRACP+ multinucleated cells was lower at 2 weeks. At 4 weeks, these parameters recovered to the levels of the ground controls. Conclusion: Our results indicate that climbing increased trabecular bone volume and reduced bone resorption, with a subsequent increase in bone formation. Intermittent climbing downregulates marrow osteoclastogenic cells and upregulates osteogenic cells initially, but further exercise seemed to desensitize them. Cortical envelopes were enlarged earlier, but the response seems to differ from trabecular bone.

To determine the effects of a tower climbing exercise on mass, strength, and local turnover of bone, 70 9-wk-old Sprague-Dawley rats were assigned to seven groups: a baseline control and three groups of sham-operated sedentary, orchidectomized (ORX)-sedentary and ORX-exercise rats. Rats voluntarily climbed a 200-cm tower to drink water from a bottle set at the top. At 4 wk, the periosteal bone formation rate (BFR), moment of inertia, bone mineral content, bone mineral density, and bending load at the midfemur were maintained in ORX-exercise rats, whereas these parameters were reduced in ORX-sedentary rats. At 8 wk, the periosteal mineral apposition rate and BFR in ORX-exercise rats were significantly higher, whereas the parameters in ORX-sedentary rats did not differ compared with sham-sedentary rats. In ORX-exercise rats, the trabecular mineralizing surface, BFR, and bone volume of the lumbar vertebrae were maintained at the same levels as those in the sham-sedentary group, whereas the osteoclast surface decreased compared with the ORX-sedentary group. However, the climbing exercise did not affect bone mineral content, bone mineral density, or the compression load of the lumbar vertebrae. These results show that, in the midfemur, the voluntary climbing exercise maintained cortical bone mass and strength by stimulating periosteal bone formation and partially prevented ORX-induced trabecular bone loss, depressing the elevation of turnover. Interestingly, in ORX rats, the climbing exercise had the opposite effect on bone formation at the periosteal femoral cortical bone, where the exercise increased the bone formation compared with vertebral trabecular bone, where the exercise decreased it.

Metabotropic gamma-aminobutyric acid receptors (GABA(B)Rs) are involved in modulation of synaptic transmission and activity of cerebellar and thalamic neurons. We used subtype-specific antibodies in pre- and postembedding immunohistochemistry combined with three-dimensional reconstruction of labelled profiles and quantification of immunoparticles to reveal the subcellular distribution of pre- and postsynaptic GABA(B)R1a/b and GABA(B)R2 in the rat cerebellum and ventrobasal thalamus. GABA(B)R1a/b and R2 were extensively colocalized in most brain regions including the cerebellum and thalamus. In the cerebellum, immunoreactivity for both subtypes was prevalent in the molecular layer. The most intense immunoreactivity was found in Purkinje cell spines with a high density of immunoparticles at extrasynaptic sites peaking at around 240 nm from glutamatergic synapses between spines and parallel fibre varicosities. This is in contrast to dendrites at sites around GABAergic synapses where sparse and random distribution was found for both subtypes. In addition, more than one-tenth of the synaptic membrane specialization of spine-parallel fibre synapses were labelled at pre- or postsynaptic sites. Weak immunolabelling for both subtypes was also seen in parallel fibres but only rarely in GABAergic axons. In the ventrobasal thalamus, immunolabelling for both receptor subtypes was intense over the dendritic field of thalamocortical cells. Electron microscopy demonstrated an extrasynaptic localization of GABA(B)R1a/b and R2 exclusively in postsynaptic elements. Quantitative analysis further revealed the density of GABA(B)R1a/b around GABAergic synapses was higher than glutamatergic synapses on thalamocortical cell dendrites. The distinct localization of GABA(B)Rs relative to synaptic sites in the cerebellum and ventrobasal thalamus suggests that GABA(B)Rs differentially regulate activity of different neuronal populations.

To determine the effects of tower climbing exercise on mass, strength, and local turnover of bone, 50 Sprague-Dawley rats, 10 weeks of age, were assigned to five groups: a baseline control and two groups of sedentary and exercise rats. Rats voluntarily climbed the 200-cm tower to drink water from the bottle set at the top of it. In 4 weeks, the trabecular bone formation rate (BFR/bone surface [BS]), bone volume (BV/TV), and trabecular thickness (Tb.Th) of both the lumbar vertebra and tibia and the bone mineral density (BMD) of the tibia increased, while the osteoclast surface (Oc.S) decreased. The parameter values in the midfemur, such as the total cross-sectional area, the moment of inertia, the periosteal mineralizing surface (MS/BS), mineral apposition rate (MAR), BFR/BS, and bending load increased, while the endosteal MAR decreased. In 8 weeks, the increases in the bone mineral content (BMC), BMD of the femur and tibia, and the bending load values of the femur were significant, but the climbing exercise did not increase BMC, BMD, or the compression load of the lumbar vertebra. Although the periosteal MS/BS, MAR, and BFR/BS increased, the endosteal MS/BS, MAR, and BFR/BS decreased. These results show that climbing exercise has a beneficial effect on the femoral cortex and tibia trabecular, rather than the vertebral trabecular. In the midfemur, effects on bone formation are site specific, supporting accelerated cortical drift by mechanical stimulation.

To determine the effects of resistance exercise on mass, strength and local turnover of bone, 50 Sprague Dawley rats, 8 weeks of age, were assigned to five groups: a baseline control and two groups of sedentary and exercising rats. The trunk of the rats was kept upright during electrically stimulated jumping exercise for 1 h every other day. In 4 weeks, the trabecular mineralizing surface per bone surface (MS/BS), bone formation rate per bone surface (BFR/BS) and the compression load of the lumbar body increased and the number of osteoclasts decreased, but bone mineral density (BMD) and structure did not increase. In the mid femur, the cross-sectional area, the cortical bone area, the moment of inertia, the periosteal MS/BS, BFR/BS and the bending load increased in the exercise group. In 8 weeks, the increases in BMD, structure and load values were significant in both the lumbar and mid femur. At both 4 and 8 weeks, the MS/BS for the endocortical surface of mid femur were not increased and mineral apposition rate (MAR) remained reduced. These results show that jumping exercise increases the mass and strength of the lumbar vertebrae and mid femur by stimulating bone formation and accelerates cortical drift by both increasing periosteal bone formation and reducing the endocortical MAR.