The inhibitory effects of vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide on osteoclast formation are associated with upregulation of osteoprotegerin and downregulation of RANKL and RANK

The presence of a network of peptidergic nerve fibers in the skeleton, expressing several neuropeptides including vasoactive intestinal peptide (VIP), has been demonstrated. This observation, together with our findings in vitro showing that VIP can regulate the activities of osteoblasts and osteoclasts as well as the recruitment of osteoclasts, has suggested the existence of a neuro-osteogenic interplay in bone metabolism. In the present study, the effects of VIP and pituitary adenylate cyclase-activating polypeptide (PACAP), two members of the VIP/secretin/glucagon superfamily, on osteoclast formation and mRNA expression of three key regulatory proteins involved in osteoclast formation have been investigated. VIP, PACAP-27, and PACAP-38, at concentrations of 10(-6) M, all significantly inhibited formation of tartrate-resistant acid phosphatase-positive multinuclear cells (TRAP + MNC) in mouse bone marrow cultures stimulated by 1, 25(OH)(2)-vitamin D3 (D3; 10(-8) M). By using semiquantitative RT-PCR, it was found that D3 upregulated the mRNA expressions of receptor activator of NF-kappaB ligand (RANKL) and receptor activator of NF-kappaB (RANK), whereas the expression of osteoprotegerin (OPG) was downregulated in mouse bone marrow cultures stimulated by D3 for 7 days. Both VIP and PACAP-38 decreased the stimulatory effects of D3 on RANKL and RANK expression, whereas the inhibitory effect of D3 on OPG expression was reversed by VIP and PACAP-38. These observations indicate that the inhibitory effects of VIP and PACAP on osteoclast recruitment are due to regulation of the expression of key proteins involved in later stages of osteoclast differentiation.     

NMDA glutamate receptors are expressed by osteoclast precursors and involved in the regulation of osteoclastogenesis

We previously identified functional N-methyl-D-aspartate (NMDA) glutamate receptors in mature osteoclasts and demonstrated that they are involved in bone resorption in vitro. In the present work, we studied the expression of NMDA receptors (NMDAR) by osteoclast precursors and their role in osteoclastogenesis using two in vitro models, the murine myelomonocytic RAW 264.7 cell line and mouse bone marrow cells, both of which differentiate into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF) and Rank ligand (RankL). Using RT-PCR analysis with specific probes, we showed that RAW 264.7 cells and mouse bone marrow cells express mRNA of NMDAR subunits NMDA receptor 1 (NR1) and NMDA receptor 2 (NR2) A, B, and D. These subunits are expressed all along the differentiation sequence from undifferentiated precursors to mature resorbing osteoclasts. Semi-quantitative PCR analysis showed no regulation of the expression of these subunits during the differentiation process. Two specific non competitive antagonists of NMDAR, MK801 and DEP, dose-dependently inhibited osteoclast formation in both models, indicating that osteoclastogenesis requires the activation of NMDAR expressed by osteoclast precursors. MK801 had no effect when added only during the first 2 days of culture, suggesting that NMDAR are rather involved in the late stages of osteoclast formation. Finally, we demonstrated using Western-blotting and immunofluorescence that activation of NMDAR in RAW 264.7 cells by specific agonists induces nuclear translocation of NF-kappa B, a factor required for osteoclast formation. Altogether, our results indicate that osteoclast precursors express NMDAR that are involved in the osteoclast differentiation process through activation of the NF-kappa B pathway.     

The neuropeptide VIP regulates the expression of osteoclastogenic factors in osteoblasts

Osteoclast formation is controlled by stromal cells/osteoblasts expressing macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), crucial for osteoclast progenitor cell proliferation, survival and differentiation, and osteoprotegerin (OPG) that inhibits the interaction between RANKL and its receptor RANK. Recent data have strongly indicated that the nervous system plays an important role in bone biology. In the present study, the effects of the neuropeptide vasoactive intestinal peptide (VIP), present in peptidergic skeletal nerve fibers, on the expression of RANKL, OPG, and M-CSF in osteoblasts and stromal cells have been investigated. VIP and pituitary adenylate cyclase-activating polypeptide 38 (PACAP-38), but not secretin, stimulated rankl mRNA expression in mouse calvarial osteoblasts. In contrast, VIP inhibited the mRNA expressions of opg and m-csf, effects shared by PACAP-38, but not by secretin. VIP did not affect rankl, opg, or m-csf mRNA expression in mouse bone marrow stromal cells (BMSCs). The effects by VIP on the mRNA expression of rankl, opg, and m-csf were all potentiated by the cyclic AMP phosphodiesterase inhibitor rolipram. In addition, VIP robustly enhanced the phosphorylation of ERK and the stimulatory effect by VIP on rankl mRNA was inhibited by the MEK1/2 inhibitor PD98059. These observations demonstrate that activation of VPAC(2) receptors in osteoblasts enhances the RANKL/OPG ratio by mechanisms mediated by cyclic AMP and ERK pathways suggesting an important role for VIP in bone remodeling.     

R848, a toll-like receptor 7 agonist, inhibits osteoclast differentiation but not survival or bone-resorbing function of mature osteoclasts

R848, also known as resiquimod, acts as a ligand for toll-like receptor 7 (TLR7) and activates immune cells. In this study, we examined the effects of R848 on differentiation, survival, and bone-resorbing function of osteoclasts. R848 inhibited osteoclast differentiation of mouse bone marrow-derived macrophages (BMMs) and human peripheral blood-derived monocytes induced by receptor activator of NF-κB ligand in a dose-dependent manner. In addition, it inhibited mouse osteoclast differentiation induced in cocultures of bone marrow cells and osteoblasts in the presence of dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. However, R848 did not affect the survival or bone-resorbing activity of mouse mature osteoclasts. R848 also upregulated the mRNA expression levels of interleukin (IL)-6, IL-12, interferon (IFN)-γ, and inducible nitric oxide synthase in mouse BMMs expressing TLR7. IFN-β was consistently expressed in the BMMs and addition of neutralizing antibodies against IFN-β to the cultures partially recovered osteoclast differentiation inhibited by R848. These results suggest that R848 targets osteoclast precursors and inhibits their differentiation into osteoclasts via TLR7.     

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide stimulate the induction of Th2 responses by up-regulating B7.2 expression.

Vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP), two structurally related neuropeptides produced within the lymphoid microenvironment, modulate several immunologic functions. We have recently demonstrated that VIP and PACAP enhance the macrophage costimulatory activity for naive CD4+ T cells exposed to allogeneic or anti-CD3 stimuli through the differential regulation of the B7 costimulatory molecules. In this study, we report on the role of VIP and PACAP on macrophage B7 expression and costimulatory function for Ag-primed CD4+ T cells, and on the macrophage-induced regulation of Th1/Th2 differentiation in vitro and in vivo. VIP and PACAP up-regulate the costimulatory activity of macrophages for Ag-primed CD4+ T cells. VIP-/PACAP-treated macrophages gain the ability to induce Th2-type cytokines such as IL-4 and IL-5 and reduce Th1-type cytokines such as IFN-gamma and IL-2. In vivo administration of VIP or PACAP in Ag-immunized mice reduce the numbers of IFN-gamma-secreting cells and enhance the numbers of IL-4-secreting cells. One of the consequences of the VIP-/PACAP-induced shift in cytokine profile is a change in the Ag-specific Ig isotype, increasing IgG1 and decreasing IgG2a levels. Finally, the preferential differentiation into Th2 effector cells after Ag stimulation induced by VIP-/PACAP-treated macrophages is mediated through the up-regulation of B7.2 expression.     

Siglec-15, a member of the sialic acid-binding lectin, is a novel regulator for osteoclast differentiation

Osteoclasts are tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells derived from monocyte/macrophage-lineage precursors and are critically responsible for bone resorption. In giant cell tumor of bone (GCT), numerous TRAP-positive multinucleated giant cells emerge and severe osteolytic bone destruction occurs, implying that the emerged giant cells are biologically similar to osteoclasts. To identify novel genes involved in osteoclastogenesis, we searched genes whose expression pattern was significantly different in GCT from normal and other bone tumor tissues. By screening a human gene expression database, we identified sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) as one of the genes markedly overexpressed in GCT. The mRNA expression level of Siglec-15 increased in association with osteoclast differentiation in cultures of mouse primary unfractionated bone marrow cells (UBMC), RAW264.7 cells of the mouse macrophage cell line and human osteoclast precursors (OCP). Treatment with polyclonal antibody to mouse Siglec-15 markedly inhibited osteoclast differentiation in primary mouse bone marrow monocyte/macrophage (BMM) cells stimulated with receptor activator of nuclear factor κB ligand (RANKL) or tumor necrosis factor (TNF)-α. The antibody also inhibited osteoclast differentiation in cultures of mouse UBMC and RAW264.7 cells stimulated with active vitamin D₃ and RANKL, respectively. Finally, treatment with polyclonal antibody to human Siglec-15 inhibited RANKL-induced TRAP-positive multinuclear cell formation in a human OCP culture. These results suggest that Siglec-15 plays an important role in osteoclast differentiation.     

Regulation of osteoclastogenesis by gap junction communication

Receptor activator of NF-kappaB ligand (RANKL) is crucial in osteoclastogenesis but signaling events involved in osteoclast differentiation are far from complete and other signals may play a role in osteoclastogenesis. A more direct pathway for cellular crosstalk is provided by gap junction intercellular channel, which allows adjacent cells to exchange second messengers, ions, and cellular metabolites. Here we have investigated the role of gap junction communication in osteoclastogenesis in mouse bone marrow cultures. Immunoreactive sites for the gap junction protein connexin 43 (Cx43) were detected in the marrow stromal cells and in mature osteoclasts. Carbenoxolone (CBX) functionally blocked gap junction communication as demonstrated by a scrape loading Lucifer Yellow dye transfer technique. CBX caused a dose-dependent inhibition (significant > or = 90 microM) of the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells formed in 7- to 8-day marrow cultures stimulated by parathyroid hormone (PTH; 10 nM) or forskolin (FSK; 1 microM). Furthermore, CBX (100 microM) significantly inhibited prostaglandin E2 (PGE2; 10 microM) and 1,25(OH)2-vitamin D3 stimulated osteoclast differentiation in the mouse bone marrow cultures. Consequently, quantitative real-time polymerase chain reaction (PCR) analysis demonstrated that CBX downregulated the expression of osteoclast phenotypic markers, but without having any significant effects on RANK, RANKL, and osteoprotegerin (OPG) mRNA expression. However, the results demonstrated that CBX significantly inhibits RANKL-stimulated (100 ng/ml) osteoclastogenesis in the mouse bone marrow cultures. Taken together, our results suggests that gap junctional diffusion of messenger molecules interacts with signaling pathways downstream RANKL in osteoclast differentiation. Further studies are required to define the precise mechanisms and molecular targets involved.     

Expression of estrogen receptor-alpha in cells of the osteoclastic lineage

 Estrogen deficiency at the menopause is associated with an increased rate of bone loss and subsequent risk of skeletal fracture. Whilst cells of the osteoblastic lineage are known to express estrogen receptors, the presence of estrogen receptors in osteoclasts remains controversial. We have examined expression of the classic estrogen receptor, estrogen receptor-alpha (ERα), during osteoclast differentiation. In situ mRNA hybridisation with a digoxygenin-labelled riboprobe to ERα mRNA, together with immunocytochemical analysis using a human ERα-specific monoclonal antibody demonstrated similar findings and confirmed the expression of ERα in chondroblasts and osteoblasts from human fetal bone and mineralising human bone marrow cultures. ERα expression was detected in human bone marrow cultures treated with 1,25(OH)₂D₃ and macrophage colony-stimulating factor and in macrophage cultures treated with 1,25(OH)₂D₃. However, in an in vitro model of human osteoclast formation, no ERα expression was observed in the osteoclasts that developed. The human preosteoclast TCG 51 cell line showed strong expression of ERα in contrast to the low levels observed in the more mature bone resorptive TCG 23 cell line. No expression was detectable in osteoclasts cultured from giant cell tumour of bone (GCTB) tissue or in osteoclasts in Pagetic, GCTB, or hyperparathyroid bone tissues. In conclusion, preosteoclasts express detectable levels of ERα, but osteoclast maturation and bone resorption is associated with loss of ERα expression. This indicates that ERα expression and regulation may play a role in osteoclast formation. Accepted: 4 November 1998     

Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide Potentiate c-fos Expression Induced by Glutamate in Cultured Cortical Neurons

Abstract: Previous reports have demonstrated that glutamate stimulates c- fos mRNA expression in primary cultures of mouse cerebral cortical neurons. We show here that vasoactive intestinal peptide (VIP) induces c- fos mRNA expression; however, this effect of VIP is completely inhibited by the noncompetitive NMDA receptor antagonist MK-801, therefore indicating that VIP stimulates c- fos expression in a glutamate-dependent manner. A similar effect was observed with pituitary adenylate cyclase-activating polypeptide27 (PACAP27). At the intracellular level, coactivation of protein kinases A and C mediates the glutamate-dependent stimulation of c- fos expression evoked by VIP, because either H-89 or staurosporin inhibits the effect of VIP as well as that of glutamate. These results point to a "biochemical AND gate" mechanism, which implies the obligatory activation of both protein kinases A and C in the transduction of c- fos expression. In summary, this article provides evidence that VIP and PACAP27 potentiate the effect of glutamate, the principal effector on c- fos expression, suggesting that both peptides can increase the "throughput" or "strength" of glutamate-containing circuits in the cerebral cortex.     

Statins modulate the levels of osteoprotegerin/receptor activator of NFkappaB ligand mRNA in mouse bone-cell cultures.

Statins stimulate bone formation partly by inducing osteoblast differentiation, although there is controversy about the effects of statins on bone mineral density and fracture risk. Several studies have revealed that statins suppress bone resorption. However, the mechanism by which statins inhibit bone resorption is still unclear. The present study was performed to clarify the effects of statins on osteoclast formation as well as the levels of osteoprotegerin (OPG) and receptor activator of NFkappaB ligand (RANKL) mRNA in mouse bone-cell cultures by semiquantitative RT-PCR. 10(-8) M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] significantly stimulated osteoclast formation and 10(-6) M statins (mevastatin and simvastatin) significantly antagonized osteoclast formation stimulated by 1,25(OH)2D3 in mouse bone-cell cultures, including both osteoblasts and osteoclasts. 10(-6) M mevastatin and simvastatin increased the level of OPG mRNA in mouse bone-cell cultures. On the other hand, 10(-6) M mevastatin and simvastatin inhibited the level of RANKL mRNA in these cultures. In conclusion, the present study demonstrates that statins inhibit osteoclast formation in mouse bone-cell cultures. Moreover, statins also increased and decreased the levels of OPG and RANKL mRNA expression in these cultures, respectively. The modulation of OPG/RANKL may be involved in the inhibition of osteoclast formation by statins.     

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit LPS-stimulated MIP-1alpha production and mRNA expression

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are neuropeptides with immunomodulatory properties, including the regulation of several proinflammatory mediators. Such mediators, for example chemokines, influence trafficking of inflammatory cells and contribute to shaping the immune response. In the present work, we studied the effect of VIP and PACAP on the CC chemokine macrophage inflammatory protein-1 alpha (MIP-1alpha) production in LPS-stimulated RAW 264.7 macrophage cell line. VIP and PACAP inhibited the production of MIP-1alpha in a dose-dependent manner and over a broad spectrum of LPS concentrations. The use of selective agonists and antagonists of VIP/PACAP receptors showed that type 1 VIP receptor (VPAC1) is the major receptor involved, but the type 2 VIP receptor (VPAC2) may be also implicated. By using selective PKA and PKC inhibitors and cAMP mimicked agents, we demonstrated a cAMP-dependent signalling pathway for the inhibitory effect of VIP/PACAP on MIP-1alpha production, although a minor non-mediated cAMP pathway was also involved. mRNA expression studies showed a down-regulation of MIP-1alpha gene expression by VIP and PACAP. Taken together, the present work strongly supports an anti-inflammatory role of VIP and PACAP by a new mechanism associated with impairment of a key component of the chemokine network.     

Expression of connexin 43 mRNA in microisolated murine osteoclasts and regulation of bone resorption in vitro by gap junction inhibitors

Several studies have demonstrated that connexin 43 (Cx43) mediates signals important for osteoblast function and osteogenesis. The role of gap junctional communication in bone resorption is less clear. We have investigated the expression of Cx43 mRNA in osteoclasts and bone resorption cultures and furthermore, the functional importance of gap junctional communication in bone resorption. RT-PCR analysis demonstrated Cx43 mRNA expression in mouse bone marrow cultures and in osteoclasts microisolated from the marrow cultures. Cx43 mRNA was also expressed in bone resorption cultures with osteoclasts and osteoblasts/stromal cells incubated for 48h on devitalized bone slices. An up-regulation of Cx43 mRNA was detected in parathyroid (PTH)-stimulated (0.1 nM) bone resorption. Two inhibitors of gap junction communication, 18alpha-glycyrrhetinic acid (30 microM) and oleamide (100 microM), significantly inhibited PTH- and 1,25-(OH)(2)D(3)-stimulated osteoclastic pit formation. In conclusion, our data indicate a functional role for gap junction communication in bone resorption.     

High levels of vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor mRNA expression in primary and tumor lymphoid cells

Neuropeptides exert a variety of putative immunomodulatory actions. Despite the molecular cloning of multiple forms of receptors for several neuropeptides with putative immunomodulatory effects, including vasoactive intestinal peptide (VIP), the related peptide pituitary adenylate cyclase-activating peptide (PACAP), the opiate peptides, tachykinins, somatostatin and corticotropin-releasing factor, it has not been reported that any of the receptor genes are expressed at significant levels in cells of the immune system. The low level of expression of these receptors and lack of knowledge concerning receptor subtype has impeded progress in understanding how neuropeptides regulate immune function. For example, it is not understood why VIP produces immunomodulatory effects at concentrations far below its receptor-binding affinity. Receptors for VIP and PACAP have recently been cloned. We show here by Northern blot analysis that the VIP/PACAP₁ receptor mRNA is present in total RNA prepared from mouse spleen B- and T-lymphocytes. The VIP/PACAP₁ receptor mRNA was also present in human peripheral blood lymphocytes, and in a B-lymphocyte and a myelocytic cell line. The mRNA for a second form of the receptor, the VIP/PACAP₂ receptor, was not expressed at detectable levels in normal cells, but was detected in several human T-cell lines and a murine mast cell line. The results indicate that VIP/PACAP₁ and perhaps VIP/PACAP₂ receptors mediate the diverse effects of VIP and PACAP on immune cells.     

Glutamatergic regulation of bone remodeling

L-glutamate (Glu) is the predominant neuromediator in the mammalian central nervous system (CNS). Bone is highly innervated and there is growing evidence of a neural control of bone cell metabolism. The recent discovery of Glu-containing nerve fibers in bone and Glu receptors (GluR) and transporters in bone cells suggest that this neuromediator may also act as a signaling molecule in bone and regulate bone cell function. Our previous studies have demonstrated that ionotropic N-Methyl-D-Aspartate (NMDA) GluR are highly expressed by mammalian osteoclasts. NMDA receptors (NMDAR) are heteromers associating the NR1 subunit and one of the four types of NR2 subunits (NR2A to D). We showed that osteoclasts express NR1, NR2B and NR2D subunits, suggesting a molecular diversity of NMDAR in these cells. Electrophysiological studies have confirmed that NMDAR are functional in mature osteoclasts, and features of Glu-induced current recorded in these cells indicate a major NR2D subunit composition. Using an in vitro assay of bone resorption, we showed that several antagonists of NMDAR binding to different sites of the receptor inhibit bone resorption. In particular, the specific NMDAR channel blocker MK801 had no effect on osteoclast attachment to bone and survival while it rapidly decreased the percentage of osteoclasts with actin ring structures that are associated with actively resorbing osteoclasts. NMDAR may thus be involved in adhesion-induced formation of the sealing zone required for bone resorption. NMDAR are also expressed by osteoclast precursors isolated from mouse bone marrow. We recently confirmed the presence of NR1, NR2B and NR2D in these cells and demonstrated their expression at all differentiation stages from osteoclast precursors to mature resorbing osteoclasts. No regulation of these subunits mRNA expression levels was observed throughout the osteoclastic differentiation sequence. Activation of NMDAR may therefore represent a new mechanism for regulating osteoclast formation and activity. While the origin of Glu in bone is still unknown, the possibility of a glutamatergic neurotransmission in this tissue is suggested by the detection of Glu in nerve fibers in close contact to bone cells. Furthermore, we recently demonstrated that sciatic neurectomy in growing rats induces a bone loss associated with a reduction of nerve profiles immunostained for Glu. These results suggest that Glu may be released from glutamatergic nerve profiles present in bone and therefore contribute to the local regulation of bone cell function.     

Extracellular ADP is a powerful osteolytic agent: evidence for signaling through the P2Y(1) receptor on bone cells.

There is increasing evidence that extracellular nucleotides act on bone cells via P2 receptors. This study investigated the action of ADP and 2-methylthioADP, a potent ADP analog with selectivity for the P2Y(1) receptor, on osteoclasts, the bone-resorbing multinuclear cells. Using three different assays, we show that ADP and 2-methylthioADP at nanomolar to submicromolar levels caused up to fourfold to sixfold increases in osteoclastic bone resorption. On mature rat osteoclasts, cultured for 1 day on polished dentine disks, peak effects on resorption pit formation were observed between 20 nM and 2 microM of ADP. The same concentrations of ADP also stimulated osteoclast and resorption pit formation in 10-day mouse marrow cultures on dentine disks. In 3-day explant cultures of mouse calvarial bones, the stimulatory effect of ADP on osteoclast-mediated Ca(2+) release was greatest at 5-50 microM and equivalent to the maximal effects of prostaglandin E(2). The ADP effects were blocked in a nontoxic manner by MRS 2179, a P2Y(1) receptor antagonist. Using in situ hybridization and immunocytochemistry, we found evidence for P2Y(1) receptor expression on both osteoclasts and osteoblasts; thus, ADP could exert its actions both directly on osteoclasts and indirectly via P2Y(1) receptors on osteoblasts. As a major ATP degradation product, ADP is a novel stimulator of bone resorption that could help mediate inflammatory bone loss in vivo.     

Differential regulation of activity-dependent neuroprotective protein in rat astrocytes by VIP and PACAP

Activity-dependent neuroprotective protein (ADNP) was shown to be a vasoactive intestinal peptide (VIP) responsive gene in astrocytes derived from the cerebral cortex of newborn rats. The present study was set out to identify VIP receptors that are associated with increases in ADNP expression in developing astrocytes. Using VIP analogues specific for the VPAC1 and the VPAC2 receptors, it was discovered that VIP induced changes in ADNP expression in astrocytes via the VPAC2 receptor. The constitutive synthesis of ADNP and VPAC2 was shown to be age-dependent and increased as the astrocyte culture developed. Pituitary adenylate cyclase-activating polypeptide (PACAP) also induced changes in ADNP expression. The apparent changes induced by VIP and PACAP on ADNP expression were developmentally dependent, and while stimulating expression in young astrocytes, an inhibition was demonstrated in older cultures. In conclusion, VIP, PACAP and the VPAC2 receptor may all contribute to the regulation of ADNP gene expression in the developing astrocyte.     

Expression of and role for ovarian cancer G-protein-coupled receptor 1 (OGR1) during osteoclastogenesis

Osteoclasts differentiate from hematopoietic mononuclear precursor cells under the control of both colony stimulating factor-1 (CSF-1, or M-CSF) and receptor activator of NF-kappaB ligand (RANKL, or TRANCE, TNFSF11) to carry out bone resorption. Using high density gene microarrays, we followed gene expression changes in long bone RNA when CSF-1 injections were used to restore osteoclast populations in the CSF-1-null toothless (csf1(tl)/csf1(tl)) osteopetrotic rat. We found that ovarian cancer G-protein-coupled receptor 1 (OGR1, or GPR68) was strongly up-regulated, rising >6-fold in vivo after 2 days of CSF-1 treatments. OGR1 is a dual membrane receptor for both protons (extracellular pH) and lysolipids. Strong induction of OGR1 mRNA was also observed by microarray, real-time RT-PCR, and immunoblotting when mouse bone marrow mononuclear cells and RAW 264.7 pre-osteoclast-like cells were treated with RANKL to induce osteoclast differentiation. Anti-OGR1 immunofluorescence showed intense labeling of RANKL-treated RAW cells. The time course of OGR1 mRNA expression suggests that OGR1 induction is early but not immediate, peaking 2 days after inducing osteoclast differentiation both in vivo and in vitro. Specific inhibition of OGR1 by anti-OGR1 antibody and by small inhibitory RNA inhibited RANKL-induced differentiation of both mouse bone marrow mononuclear cells and RAW cells in vitro, as evidenced by a decrease in tartrate-resistant acid phosphatase-positive osteoclasts. Taken together, these data indicate that OGR1 is expressed early during osteoclastogenesis both in vivo and in vitro and plays a role in osteoclast differentiation.     

Expression of Pituitary Adenylate Cyclase-Activating Peptide (PACAP) and PAC1 in the Periodontal Ligament After Tooth Luxation

Pituitary adenylate cyclase-activating peptide (PACAP) is widely distributed throughout the nervous system. PACAP not only acts as a neurotransmitter but also elicits a broad spectrum of biological action via the PACAP-specific receptor, PAC1. However, no studies have investigated PACAP and PAC1 in the periodontal ligament (PDL), so we aimed to perform this investigation in rats after tooth luxation. In the PDL of an intact first molar, there are few osteoclasts and osteoblasts. However, at days 3 and 5 after luxation, large PAC1-positive cells, thought to be osteoclasts because of their expression of the osteoclast marker, tartrate-resistant acid phosphatase, were detected in appreciable numbers. Osteoblast numbers increased dramatically on day 7 after luxation, and PAC1-positive mononuclear small cells were increased at day 14, many of which expressed the osteoblast marker, alkaline phosphatase. PACAP-positive nerve fibers were rarely detected in the PDL of intact first molars, but were increasingly evident at this site on days 5 and 7 after luxation. Double-immunofluorescence analysis demonstrated the relationship between PACAP-positive nerve fibers and PAC1-positive osteoclasts/-blasts in the PDL. At 5 days after luxation, PACAP-positive nerve fibers appeared in close proximity to PAC1-positive osteoclasts. At 7 days after luxation, PACAP-positive nerve fibers appeared in close proximity to PAC1-positive osteoblasts. These results suggest that PACAP may have effects on osteoclasts and osteoblasts in the PDL after tooth luxation and thus regulate bone remodeling after these types of injury.