Transepithelial channels for leukocytes in the junctional epithelium.

BACKGROUND AND OBJECTIVES: The junctional epithelium (JE) has been recognized as a defensive organ rich in polymorphonuclear leukocytes (PMNs). However, the migration of PMNs through the JE has not been clearly documented. For mucosal defense, PMNs migrate outwards over the epithelium to defend the intestinal or respiratory tract on the epithelial surface. With this background, the present study investigated whether there is any structural evidence showing the transepithelial migration of PMNs through the JE in gingival mucosa. METHODS: Three-dimensional modeling of gingiva surrounding mouse molars at varying ages was performed by array tomography. Images of the serial sections for array tomography at the 800 nm thickness were obtained using back scattered electron (BSE) detector equipped in the field-emission scanning electron microscopy (FESEM). Expressions of neutrophil marker or CD47 were immunohistochemically examined on the frozen sections. RESULTS: Array tomography using FESEM and 3-dimensional modeling clearly showed that a system of epithelial channels developed between keratinocytes and generally ran along the long axis of the JE. Most PMNs were found inside the channels, rather than being scattered throughout the JE. The channels could be traced from the base of the JE to the bottom of the gingival sulcus, although some channels were fragmented and interrupted with short intercellular gaps. CONCLUSIONS: These findings suggest that the JE may be an organ for transepithelial migration of PMNs to the bottom of the gingival sulcus through epithelial channels, as occurs in the epithelial lining of other organs such as the intestinal or respiratory tract.

Characterization of junctional structures in the gingival epithelium as barriers against bacterial invasion.

BACKGROUND AND OBJECTIVE: Adherens junctions (AJs) and tight junctions (TJs) are known to play a crucial role in maintaining the physical barrier function of the epithelium. Here, we aimed to characterize the distribution of AJs and TJs throughout the gingival epithelium and to obtain insights into the physiological importance of these junctional structures. METHODS: Sections of mouse gingival tissue were examined using transmission electron microscopy (TEM) and bio-high voltage electron microscopy tomography. The gingival sections were stained for E-cadherin and JAM-A as markers of AJs and TJs, respectively, and examined using confocal microscopy and lattice structured illumination microscopy. Bacteria within the gingival epithelium were examined using in situ hybridization. RESULTS: Junctional structures, including desmosomes, AJs, and TJs, were observed throughout the gingival epithelium. The expression levels of E-cadherin were particularly low in the granular/keratinized layers of the oral epithelium (OE), while extremely low JAM-A levels were detected in the granular/keratinized layers of the sulcular epithelium (SE). The three-dimensional rendering of the junctional structures revealed that both AJs and TJs in the gingival epithelium formed discontinuous short bands or patches. Interestingly, strong bacterial signals were observed at the granular/keratinized layers of both SE and OE, but a few bacteria were detected within the junctional epithelium (JE) and the basal/spinous layers of the SE and OE. CONCLUSIONS: AJs and TJs form a discontinuous barrier throughout paracellular passage in the gingival epithelium; nevertheless, they seem to play an important role in defending against invading bacteria.

Vitamin D maintains E-cadherin intercellular junctions by downregulating MMP-9 production in human gingival keratinocytes treated by TNF-α.

PURPOSE: Despite the well-known anti-inflammatory effects of vitamin D in periodontal health, its mechanism has not been fully elucidated. In the present study, the effect of vitamin D on strengthening E-cadherin junctions (ECJs) was explored in human gingival keratinocytes (HGKs). ECJs are the major type of intercellular junction within the junctional epithelium, where loose intercellular junctions develop and microbial invasion primarily occurs. METHODS: HOK-16B cells, an immortalized normal human gingival cell line, were used for the study. To mimic the inflammatory environment, cells were treated with tumor necrosis factor-alpha (TNF-α). Matrix metalloproteinases (MMPs) in the culture medium were assessed by an MMP antibody microarray and gelatin zymography. The expression of various molecules was investigated using western blotting. The extent of ECJ development was evaluated by comparing the average relative extent of the ECJs around the periphery of each cell after immunocytochemical E-cadherin staining. Vitamin D receptor (VDR) expression was examined via immunohistochemical analysis. RESULTS: TNF-α downregulated the development of the ECJs of the HGKs. Dissociation of the ECJs by TNF-α was accompanied by the upregulation of MMP-9 production and suppressed by a specific MMP-9 inhibitor, Bay 11-7082. Exogenous MMP-9 decreased the development of ECJs. Vitamin D reduced the production of MMP-9 and attenuated the breakdown of ECJs in the HGKs treated with TNF-α. In addition, vitamin D downregulated TNF-α-induced nuclear factor kappa B (NF-κB) signaling in the HGKs. VDR was expressed in the gingival epithelium, including the junctional epithelium. CONCLUSIONS: These results suggest that vitamin D may avert TNF-α-induced downregulation of the development of ECJs in HGKs by decreasing the production of MMP-9, which was upregulated by TNF-α. Vitamin D may reinforce ECJs by downregulating NF-κB signaling, which is upregulated by TNF-α. Strengthening the epithelial barrier may be a way for vitamin D to protect the periodontium from bacterial invasion.

JNK-associated scattered growth of YD-10B oral squamous carcinoma cells while maintaining the epithelial phenotype.

Cell scattering of epithelial carcinoma cancer cells is one of the critical event in tumorigenesis. Cells losing epithelial cohesion detach from aggregated epithelial cell masses and may migrate to fatal organs through metastasis. The present study investigated the molecular mechanism by which squamous cell carcinoma cells grow scattered at the early phase of transformation while maintaining the epithelial phenotype. We studied YD-10B cells, which are established from human oral squamous cell carcinoma, because the cells grow scattered without the development of E-cadherin junctions (ECJs) under routine culture conditions despite the high expression of functional E-cadherin. The functionality of their E-cadherin was demonstrated in that YD-10B cells developed ECJs, transiently or persistently, when they were cultured on substrates coated with a low amount of fibronectin or to confluence. The phosphorylation of JNK was up-regulated in YD-10B cells compared with that in human normal oral keratinocyte cells or human squamous cell carcinoma cells, which grew aggregated along with well-organized ECJs. The suppression of JNK activity induced the aggregated growth of YD-10B cells concomitant with the development of ECJs. These results indicate for the first time that inherently up-regulated JNK activity induces the scattered growth of the oral squamous cell carcinoma cells through down-regulating the development of ECJ despite the expression of functional E-cadherin, a hallmark of the epithelial phenotype.

Substrate roughness induces the development of defective E-cadherin junctions in human gingival keratinocytes.

PURPOSE: The entry of bacteria or harmful substances through the epithelial seal of human gingival keratinocytes (HGKs) in the junctional epithelium (JE) is blocked by specialized intercellular junctions such as E-cadherin junctions (ECJs). However, the influence of roughened substrates, which may occur due to apical migration of the JE, root planing, or peri-implantitis, on the development of the ECJs of HGKs remains largely unknown. METHODS: HGKs were cultured on substrates with varying levels of roughness, which were prepared by rubbing hydrophobic polystyrene dishes with silicon carbide papers. The activity of c-Jun N-terminal kinase (JNK) was inhibited with SP600125 or by transfection with JNK short hairpin RNA. The development of intercellular junctions was analyzed using scanning electron microscopy or confocal laser scanning microscopy after immunohistochemical staining of the cells for E-cadherin. The expression level of phospho-JNK was assessed by immunoblotting. RESULTS: HGKs developed tight intercellular junctions devoid of wide intercellular gaps on smooth substrates and on rough substrates with low-nanometer dimensions (average roughness [Ra]=121.3±13.4 nm), although the ECJs of HGKs on rough substrates with low-nanometer dimensions developed later than those of HGKs on smooth substrates. In contrast, HGKs developed short intercellular junctions with wide intercellular gaps on rough substrates with mid- or high-nanometer dimensions (Ra=505.3±115.3 nm, 867.0±168.6 nm). Notably, the stability of the ECJs was low on the rough substrates, as demonstrated by the rapid destruction of the cell junction following calcium depletion. Inhibition of JNK activity promoted ECJ development in HGKs. JNK was closely associated with cortical actin in the regulation of ECJs in HGKs. CONCLUSIONS: These results indicate that on rough substrates with nanometer dimensions, the ECJs of HGKs develop slowly or defectively, and that this effect can be reversed by inhibiting JNK.

ROCK inhibition activates MCF-7 cells.

Dormant carcinoma cancer cells showing epithelial characteristics can be activated to dissipate into the surrounding tissue or organs through epithelial-mesenchymal transition (EMT). However, the molecular details underlying the activation of dormant cancer cells have been less explored. In this study, we examined the molecular pathway to activate dormant breast cancer cells. Rho-associated kinase (ROCK) inhibition disrupted cell junction, promoted cell proliferation and migration / invasion in both two-dimensional and three-dimensional substrates. The disintegration of cell junction upon ROCK inhibition, coupled with the loss of E-cadherin and b-catenin from the cell membrane, was associated with the activation of Rac1 upon ROCK inhibition. Migration / invasion also increased upon ROCK inhibition. However, the activation of MCF-7 cells upon ROCK inhibition was not associated with the up-regulation of typical EMT markers, such as snail and slug. Based on these results, we suggest the potential risk for dormant cancer cells to dissipate through non-typical EMT when ROCK activity is down-regulated.

Enhanced regeneration of rabbit mandibular defects through a combined treatment of electrical stimulation and rhBMP-2 application.

We evaluated the new bone regeneration of a rabbit mandibular defect using hBMSCs under electrical stimulation combined with rhBMP-2 in this study. An inner scaffold prepared by setting a collagen sponge with hBMSCs and hydrogel was placed into a polycaprolactone (PCL) outer box, and an electrical stimulation device was installed between the inner scaffold and the outer box. There were three experimental groups depending on electrical stimulation and application of rhBMP-2. The experimental group was divided into the following three groups. Group 1, in which rhBMP-2 (5 µg/defect) was added to hydrogel and electrical stimulation was not applied; Group 2, in which rhBMP-2 (5 µg/defect) was added as in Group 1 and electrical stimulation was applied; and Group 3, in which electrical stimulation was applied and rhBMP-2 (5 µg/defect) was injected directly into defect site. The delivered electrical stimulation was charge-balanced bi-phasic electric current pulses, and electrical stimulation was conducted for 7 days. The stimulation parameters of the bi-phasic electrical current set at an amplitude of 20 µA, a duration of 100 µs and a frequency of 100 Hz. Four weeks after surgery, new bone formation in each group was evaluated using radiography, histology, and micro-computed tomography (µCT). Groups 2 and 3 exhibited a significant increase in new bone formation compared to Group 1, while Group 3 showed the highest level of new bone regeneration. In a comparison between two groups, Group 2 showed a higher bone volume (BV) by 260 % (p < 0.01) compared with Group 1, and Group 3 showed a higher BV by 442 % (p < 0.01) compared with Group 1. The trend of the bone surface density (ratio of new bone to the real defect volume, BS/TV), trabecular number, and connectivity was identical to that of the BV. The total bone mineral density (BMD) of Groups 2 and 3 showed values higher by the ratios of 103 % (p < 0.01) and 107.5 % (p < 0.01) compared with Group 1, respectively. Part BMD for Groups 2 and 3 showed higher values by the ratios of 104.9 % (p < 0.01) and 122.4 % (p < 0.01) compared with Group 1, respectively. These results suggest that the combined treatment of electrical stimulation, hBMSCs, a collagen sponge, hydrogel, and rhBMP-2 was effective for bone regeneration of large-size mandibular defects. The application of rhBMP-2 with an injection following electrical stimulation demonstrated better efficiency as regards bone regeneration.

Low-Rac1 activity downregulates MC3T3-E1 osteoblastic cell motility on a nanoscale topography prepared on polystyrene substrates in vitro.

Surface roughness affects various cell activities, including osteoblast motility, which may have an effect on bone regeneration. Defective cell signaling, which is associated with the slow motility of osteoblasts on a substrate with rough topology at nanometer dimensions (Ra = 123.8 ± 29.1 nm), was studied. Osteoblasts grown on the rough surface at nanometer dimensions showed the high activities of small GTPase RhoA and Rho-associated kinase (ROCK) on the rough surface at nanometer dimensions and downregulated Rac1 activity compared to the smooth surface. The inhibition of ROCK in the cells with Y-27632, a specific ROCK inhibitor, reversed the low-cell motility. In addition, the transfection of constitutively active Rac1 reversed the low-cell motility. However, Rac1 inhibition abolished the reversal of low-cell motility induced by ROCK inhibition. These results indicate that upregulated RhoA/ROCK activity suppresses Rac1 activity to decrease the motility of osteoblasts on a rough surface at nanometer dimensions, and the low motility of osteoblasts on a rough surface at nanometer dimensions can be reversed by ROCK inhibition.

The RhoA-ROCK-PTEN pathway as a molecular switch for anchorage dependent cell behavior.

The proliferation of anchorage-dependent cells of mesenchymal origin requires the attachment of the cells to substrates. Thus, cells that are poorly attached to substrates exhibit retarded cell cycle progression or apoptotic death. A major disadvantage of most polymers used in tissue engineering is their hydrophobicity; hydrophobic surfaces do not allow cells to attach firmly and, therefore, do not allow normal proliferation rates. In this study, we investigated the molecular mechanism underlying the reduced proliferation rate of cells that are poorly attached to substrates. There was an inverse relationship between the activity of the small GTPase RhoA (RhoA) and the cell proliferation rate. RhoA activity correlated inversely with the strength of cell adhesion to the substrates. The high RhoA activity in the cells poorly attached to substrates caused an increase in the activity of Rho-associated kinase (ROCK), a well-known effector of RhoA that upregulated the activity of phosphatase and tensin homolog (PTEN). The resulting activated PTEN downregulated Akt activity, which is essential for cell proliferation. Thus, the cells that were poorly attached to substrates showed low levels of cell proliferation because the RhoA-ROCK-PTEN pathway was hyperactive. In addition, RhoA activity seemed to be related to focal adhesion kinase (FAK) activity. Weak FAK activity in these poorly attached cells failed to downregulate the high RhoA activity that restrained cell proliferation. Interestingly, reducing the expression of any component of the RhoA-ROCK-PTEN pathway rescued the proliferation rate without physico-chemical surface modifications. Based on these results, we suggest that the RhoA-ROCK-PTEN pathway acts as a molecular switch to control cell proliferation and determine anchorage dependence. In cells that are poorly attached to substrates, its inhibition is sufficient to restore cell proliferation without the need for physico-chemical modification of the material surface.

Mechanisms by which the inhibition of specific intracellular signaling pathways increase osteoblast proliferation on apatite surfaces.

Osteoblasts proliferate slowly on the surface of calcium phosphate apatite which is widely used as a substrate biomaterial in bone regeneration. Owing to poor adhesion signaling in the cells grown on the calcium phosphate surface, inadequate growth factor signaling is generated to trigger cell cycle progression. The present study investigated an intracellular signal transduction pathway involved in the slow cell proliferation in osteoblasts grown on the calcium phosphate surface. Small GTPase RhoA and phosphatase and tensin homolog (PTEN) were more activated in cells grown on the surface of calcium phosphate apatite than on tissue culture plate. Specific inhibition of RhoA and PTEN induced the cells on calcium phosphate apatite surface to proliferate at a similar rate as cells on tissue culture plate surface. Specific inhibition of ROCK, which is a downstream effector of RhoA and an upstream activator of PTEN also increased proliferation of these osteoblasts. Present results indicate that physical property of calcium phosphate crystals that impede cell proliferation may be surmounted by the inhibition of the RhoA/ROCK/PTEN pathway to rescue delayed proliferation of osteoblasts on the calcium phosphate apatite surface. In addition, specific inhibition of ROCK promoted cell migration and osteoblast differentiation. Inhibition of the RhoA/ROCK/PTEN intracellular signaling pathway is expected to enhance cell activity to promote and accelerate bone regeneration on the calcium phosphate apatite surface.

Array of amorphous calcium phosphate particles improves cellular activity on a hydrophobic surface.

Poor interaction between cells and surfaces, especially hydrophobic surfaces, results in delayed proliferation and increased apoptosis due to low cell adhesion signaling. To improve cell adhesion, hydrophilic array of amorphous calcium phosphate (ACP) was fabricated on a surface. A phosphate-buffered solution containing calcium ions was prepared at low temperature to prevent spontaneous precipitation. Then, the ion solution was heated to generate nuclei of ACP nanoparticles. The ACP nanoparticles adhered to the hydrophobic polystyrene surface forming an array composed of ACP particles. Multiple treatments of these nuclei with fresh CaP ion solutions increased the diameter and decreased the solubility of ACP particles enough to mediate cellular adhesion. The particle density in the array was dependent on the ion concentration of the CaP ion solutions. The ACP array improved a wide variety of activities when osteoblastic MC3T3-E1 cells were cultured on the ACP array fabricated on a hydrophobic bacteriological dish surface, compared to those cultured without the ACP array in vitro. The use of ACP array resulted in a lower apoptosis and also increased the spreading of cells to form stress fibers and focal contacts. Cells cultured on the ACP array proliferated more than cells cultured on a hydrophobic surface without the ACP array. The ACP array increased the expression of markers of differentiation in osteoblast. These results indicate that an array of ACP can be used as a coating material for enhancing biocompatibility in tissue engineering or biomaterials rather than modifying the surface with organic molecules.

Rho-associated kinase (ROCK) inhibition reverses low cell activity on hydrophobic surfaces.

Hydrophobic polymers do not offer an adequate scaffold surface for cells to attach, migrate, proliferate, and differentiate. Thus, hydrophobic scaffolds for tissue engineering have traditionally been physicochemically modified to enhance cellular activity. However, modifying the surface by chemical or physical treatment requires supplementary engineering procedures. In the present study, regulation of a cell signal transduction pathway reversed the low cellular activity on a hydrophobic surface without surface modification. Inhibition of Rho-associated kinase (ROCK) by Y-27632 markedly enhanced adhesion, migration, and proliferation of osteoblastic cells cultured on a hydrophobic polystyrene surface. ROCK inhibition regulated cell-cycle-related molecules on the hydrophobic surface. This inhibition also decreased expression of the inhibitors of cyclin-dependent kinases such as p21(cip1) and p27(kip1) and increased expression of cyclin A and D. These results indicate that defective cellular activity on the hydrophobic surface can be reversed by the control of a cell signal transduction pathway without physicochemical surface modification.

Nuclear factor I-C is essential for odontogenic cell proliferation and odontoblast differentiation during tooth root development.

Our previous studies have demonstrated that nuclear factor I-C (NFI-C) null mice developed short molar roots that contain aberrant odontoblasts and abnormal dentin formation. Based on these findings, we performed studies to elucidate the function of NFI-C in odontoblasts. Initial studies demonstrated that aberrant odontoblasts become dissociated and trapped in an osteodentin-like mineralized tissue. Abnormal odontoblasts exhibit strong bone sialoprotein expression but a decreased level of dentin sialophosphoprotein expression when compared with wild type odontoblasts. Loss of Nfic results in an increase in p-Smad2/3 expression in aberrant odontoblasts and pulp cells in the subodontoblastic layer in vivo and primary pulp cells from Nfic-deficient mice in vitro. Cell proliferation analysis of both cervical loop and ectomesenchymal cells of the Nfic-deficient mice revealed significantly decreased proliferative activity compared with wild type mice. In addition, Nfic-deficient primary pulp cells showed increased expression of p21 and p16 but decreased expression of cyclin D1 and cyclin B1, strongly suggesting cell growth arrest caused by a lack of Nfic activity. Analysis of the pulp and abnormal dentin in Nfic-deficient mice revealed an increase in apoptotic activity. Further, Nfic-deficient primary pulp cells exhibited an increase in caspase-8 and -3 activation, whereas the cleaved form of Bid was hardly detected. These results indicate that the loss of Nfic leads to the suppression of odontogenic cell proliferation and differentiation and induces apoptosis of aberrant odontoblasts during root formation, thereby contributing to the formation of short roots.

Drosophila Atlastin regulates the stability of muscle microtubules and is required for synapse development.

Hereditary spastic paraplegia (HSP) is an inherited neurological disorder characterized by progressive spasticity and weakness of the lower extremities. The most common early-onset form of HSP is caused by mutations in the human gene that encodes the dynamin-family GTPase Atlastin-1 (Atl-1). Recently, loss of the Drosophila ortholog of Atl-1 (Atl) has been found to induce locomotor impairments from the earliest adult stages, suggesting the developmental role of atlastin-subfamily GTPases. Here, we provide evidence that Atl is required for normal growth of muscles and synapses at the neuromuscular junction (NMJ). Atl protein is highly expressed in larval body-wall muscles. Loss-of-function mutations in the atl gene reduce the size of muscles and increase the number of synaptic boutons. Rescue of these defects is accomplished by muscular, but not neuronal expression of Atl. Loss of Atl also disrupts ER and Golgi morphogenesis in muscles and reduces the synaptic levels of the scaffold proteins Dlg and alpha-spectrin. We also provide evidence that Atl functions with the microtubule-severing protein Spastin to disassemble microtubules in muscles. Finally, we demonstrate that the microtubule-destabilizing drug vinblastine alleviates synapse and muscle defects in atl mutants. Together, our results suggest that Atl controls synapse development and ER and Golgi morphogenesis by regulating microtubule stability.

A histomorphometric study on collagen-apatite composite as a graft material: the influence of gap size at the titanium-bone interface in animal model.

The purpose of this study was to evaluate the healing process of collagen-apatite composite (CAC) at the titanium-bone interface in animal model. Small gaps (0.5 or 1.0 mm-sized wells) were prepared in the epoxy-resin block implants coated with pure titanium. The gaps were filled with CAC or demineralized freeze-dried bone (DFDB). The titanium-coated epoxy-resin block implants were inserted in the tibia of rabbit for 4 weeks or 8 weeks. The microscopic features of bony healing process in the grafted gaps were examined and analyzed. In the histomorphometric analysis, CAC group showed higher fraction of newly-formed bone than DFDB group in both 0.5 and 1.0 mm gap subgroup at 4-week specimen (P < 0.05). In the transmission electron microscopic examinations, osteoblasts of the newly-formed bone of CAC group showed more cellular activity than that of DFDB group. From the results, it was expected that CAC had more beneficial property on early bony healing process than DFDB at the titanium-bone interface.

Trolox prevents osteoclastogenesis by suppressing RANKL expression and signaling.

Excessive receptor activator of NF-kappaB ligand (RANKL) signaling causes enhanced osteoclast formation and bone resorption. Thus, down-regulation of RANKL expression or its downstream signals may be a therapeutic approach to the treatment of pathological bone loss. In this study, we investigated the effects of Trolox, a water-soluble vitamin E analogue, on osteoclastogenesis and RANKL signaling. Trolox potently inhibited interleukin-1-induced osteoclast formation in bone marrow cell-osteoblast coculture by abrogating RANKL induction in osteoblasts. This RANKL reduction was attributed to the reduced production of prostaglandin E(2) via a down-regulation of cyclooxygenase-2 activity. We also found that Trolox inhibited osteoclast formation from bone marrow macrophages induced by macrophage colony-stimulating factor plus RANKL in a reversible manner. Trolox was effective only when present during the early stage of culture, which implies that it targets early osteoclast precursors. Pretreatment with Trolox did not affect RANKL-induced early signaling pathways, including MAPKs, NF-kappaB, and Akt. We found that Trolox down-regulated the induction by RANKL of c-Fos protein by suppressing its translation. Ectopic overexpression of c-Fos rescued the inhibition of osteoclastogenesis by Trolox in bone marrow macrophages. Trolox also suppressed interleukin-1-induced osteoclast formation and bone loss in mouse calvarial bone. Taken together, our findings indicate that Trolox prevents osteoclast formation and bone loss by inhibiting both RANKL induction in osteoblasts and c-Fos expression in osteoclast precursors.

Disruption of Nfic causes dissociation of odontoblasts by interfering with the formation of intercellular junctions and aberrant odontoblast differentiation.

We reported previously that Nfic-deficient mice exhibit short and abnormal molar roots and severely deformed incisors. The objective of this study is to address the mechanisms responsible for these changes using morphological, IHC, and RT-PCR analysis. Nfic-deficient mice exhibited aberrant odontoblasts and abnormal dentin formation in molar roots and the labial crown analog of incisors. The most striking changes observed in these aberrant odontoblasts were the loss of intercellular junctions and the decreased expression of ZO-1 and occludin. As a result, they became dissociated, had a round shape, and lost their cellular polarity and arrangement as a sheet of cells. Furthermore, the dissociated odontoblasts became trapped in dentin-like mineralized tissue, resembling osteodentin in the overall morphology. These findings suggest that loss of the Nfic gene interferes with the formation of intercellular junctions that causes aberrant odontoblast differentiation and abnormal dentin formation. Collectively, these changes in odontoblasts contributed to development of molars with short and abnormal roots in Nfic-deficient mice.

Brain-type creatine kinase has a crucial role in osteoclast-mediated bone resorption.

Osteoclasts differentiate from precursor cells of the monocyte-macrophage lineage and subsequently become activated to be competent for bone resorption through programs primarily governed by receptor activator of nuclear factor-kappaB ligand in cooperation with macrophage colony-stimulating factor. Proteins prominently expressed at late phases of osteoclastogenesis and with a supportive role in osteoclast function are potential therapeutic targets for bone-remodeling disorders. In this study, we used a proteomics approach to show that abundance of the brain-type cytoplasmic creatine kinase (Ckb) is greatly increased during osteoclastogenesis. Decreasing Ckb abundance by RNA interference or blocking its enzymatic activity with a pharmacological inhibitor, cyclocreatine, suppressed the bone-resorbing activity of osteoclasts grown in vitro via combined effects on actin ring formation, RhoA GTPase activity and vacuolar ATPase function. Activities of osteoclasts derived from Ckb-/- mice were similarly affected. In vivo studies showed that Ckb-/- mice were better protected against bone loss induced by ovariectomy, lipopolysaccharide challenge or interleukin-1 treatment than wild-type controls. Furthermore, administration of cyclocreatine or adenoviruses harboring Ckb small hairpin RNA attenuated bone loss in rat and mouse models. Our findings establish an important role for Ckb in the bone-resorbing function of osteoclasts and underscore its potential as a new molecular target for antiresorptive drug development.

Reciprocal cross-talk between RANKL and interferon-gamma-inducible protein 10 is responsible for bone-erosive experimental arthritis.

OBJECTIVE: Interferon-gamma-inducible protein 10 (IP-10; also called CXCL10), a chemokine important in the migration and proliferation of T cells, is induced in a wide variety of cell types. However, the role of IP-10 in rheumatoid arthritis (RA) remains largely unknown. The purpose of this study was to examine the potential role of IP-10 in bone resorption and RA through examination of a mouse model of collagen-induced arthritis (CIA). METHODS: The effects of IP-10 on mouse T cells during osteoclast differentiation were examined in migration assays. The bone-erosive activity of IP-10 was determined in vivo in a mouse model of CIA by histologic and immunostaining analyses. Cytokine levels in serum and culture medium were measured with sandwich enzyme-linked immunosorbent assays. RESULTS: Serum concentrations of IP-10 were significantly higher in mice with CIA than in control mice. RANKL greatly induced IP-10 expression in osteoclast precursors, but not in mature osteoclasts. IP-10 stimulated the expression of RANKL and tumor necrosis factor alpha (TNFalpha) in CD4+ T cells and induced osteoclastogenesis in cocultures of CD4+ T cells and osteoclast precursors. However, IP-10 did not induce RANKL or TNFalpha in CD8+ T cells. Treatment with neutralizing antibody to IP-10 significantly inhibited the infiltration of CD4+ T cells and F4/80+ macrophages into the synovium and attenuated bone destruction in mice with CIA. Furthermore, levels of RANKL and TNFalpha were inhibited by antibody to IP-10. Bone erosion was observed in mice infected with an IP-10 retrovirus. CONCLUSION: Our findings suggest that IP-10 plays a critical role in the infiltration of CD4+ T cells and F4/80+ macrophages into inflamed joints and causes bone destruction. Our results provide the first evidence that IP-10 contributes to the recruitment of inflammatory cells and is involved in bone erosion in inflamed joints.

Stimulation by TLR5 modulates osteoclast differentiation through STAT1/IFN-beta.

Osteoclasts are bone-resorbing cells that are differentiated from hemopoietic precursors of the monocyte-macrophage lineage. Stimulation of TLRs has been shown to positively or negatively modulate osteoclast differentiation, depending on the experimental condition. However, the molecular mechanism by which this modulation takes place remains unclear. In the present study, we examined the effects of flagellin, a specific microbial ligand of TLR5, on the receptor activator of NF-kappaB ligand (RANKL)-stimulated osteoclastogenesis. Flagellin suppressed RANKL induction of c-Fos protein expression in bone marrow-derived macrophages without affecting c-Fos mRNA expression. Ectopic overexpression of c-Fos and a constitutively active form of NFATc1 reversed the flagellin-induced anti-osteoclastogenic effect. The inhibitory effect of flagellin was mediated by IFN-beta production. Flagellin stimulated IFN-beta expression and release in bone marrow-derived macrophages, and IFN-beta-neutralizing Ab prevented the flagellin-induced c-Fos down-regulation and the anti-osteoclastogenic effect. IFN-beta gene induction by flagellin, LPS, or RANKL was dependent on STAT1 activation. Treatment with flagellin or RANKL stimulated STAT1 activation, and STAT1 deficiency or the JAK2 inhibitor AG490 dramatically prevented IFN-beta induction in response to flagellin or RANKL. In addition, STAT1 deficiency abolished the anti-osteoclastogenic effect induced by flagellin or LPS. In contrast, flagellin stimulated osteoclast differentiation in cocultures of osteoblasts and bone marrow cells without inducing IFN-beta. Thus, IFN-beta acts as a critical modulator of osteoclastogenesis in response to TLR5 activation.