Force-Loaded Cementocytes Regulate Osteoclastogenesis via S1P/S1PR1/Rac1 Axis.

Orthodontically induced inflammatory root resorption (OIIRR) is the major iatrogenic complication of orthodontic treatment, seriously endangering tooth longevity and impairing masticatory function. Osteoclasts are thought to be the primary effector cells that initiate the pathological process of OIIRR; however, the cellular and molecular mechanisms responsible for OIIRR remain unclear. Our previous studies revealed that cementocytes, the major mechanically responsive cells in cementum, respond to compressive stress to activate and influence osteoclasts locally. For this study, we hypothesized that the sphingosine-1-phosphate (S1P) signaling pathway, a key mechanotransduction pathway in cementocytes, may regulate osteoclasts under the different magnitudes of either physiologic compressive stress that causes tooth movement or pathologic stress that causes OIIRR. Here, we show a biphasic effect of higher compression force stimulating the synthesis and secretion of S1P, whereas lower compression force reduced signaling in IDG-CM6 cementocytes. Using conditioned media from force-loaded cementocytes, we verified the cell-to-cell communication between cementocytes and osteoclasts and show that selective knockdown of S1PR1 and Rac1 plays a role in cementocyte-driven osteoclastogenesis via the S1P/S1PR1/Rac1 axis. Most importantly, the use of inhibitors of this axis reduced or prevented the pathological process of OIIRR. The intercellular communication mechanisms between cementocytes and osteoclasts may serve as a promising therapeutic target for OIIRR.

Muscle secreted factors enhance activation of the PI3K/Akt and ß-catenin pathways in murine osteocytes.

Skeletal muscle and bone interact at the level of mechanical loading through the application of force by muscles to the skeleton and more recently focus has been placed on molecular/biochemical coupling of these two tissues. We sought to determine if muscle and muscle-derived factors were essential to the osteocyte response to loading. Botox® induced muscle paralysis was used to investigate the role of muscle contraction during in vivo tibia compression loading. 5-6 month-old female TOPGAL mice had their right hindlimb muscles surrounding the tibia injected with either BOTOX® or saline. At four days post injections when muscle paralysis peaked, the right tibia was subjected to a single session of in vivo compression loading at ∼2600 µÎµ. At 24 h post-load we observed a 2.5-fold increase in ß-catenin signaling in osteocytes in the tibias of the saline injected mice, whereas loading of tibias from Botox® injected mice failed to active ß-catenin signaling in osteocytes. This suggests that active muscle contraction produces a factor(s) that is necessary for or conditions the osteocyte's ability to respond to load. To further investigate the role of muscle derived factors, MLO-Y4 osteocyte-like cells and a luciferase based ß-catenin reporter (TOPflash-MLO-Y4) cell line we developed were treated with conditioned media (CM) from C2C12 myoblasts (MB) and myotubes (MT) and ex vivo contracted Extensor Digitorum Longus (EDL) and Soleus (Sol) muscles under static or loading conditions using fluid flow shear stress (FFSS). 10 % C2C12 myotube CM, but not myoblast or NIH3T3 fibroblast cells CM, induced a rapid activation of the Akt signaling pathway, peaking at 15 min and returning to baseline by 1-2 h under static conditions. FFSS applied to MLO-Y4 cells for 2 h in the presence of 10 % MT-CM resulted in a 6-8 fold increase in pAkt compared to a 3-4 fold increase under control or when exposed to 10 % MB-CM. A similar response was observed in the presence of 10 % EDL-CM, but not in the presence of 10 % Sol-CM. TOPflash-MLO-Y4 cells were treated with 10 ng/ml Wnt3a in the presence or absence of MT-CM. While MT-CM resulted in a 2-fold activation and Wnt3a produced a 10-fold activation, the combination of MT-CM + Wnt3a resulted in a 25-fold activation of ß-catenin signaling, implying a synergistic effect of factors in MT-CM with Wnt3a. These data provide clear evidence that specific muscles and myotubes produce factors that alter important signaling pathways involved in the response of osteocytes to mechanical load. These data strongly suggest that beyond mechanical loading there is a molecular coupling of muscle and bone.

Taurine, an osteocyte metabolite, protects against oxidative stress-induced cell death and decreases inhibitors of the Wnt/ß-catenin signaling pathway.

Taurine has been shown to have positive effects on bone mass, which are thought to be due in part to its cytoprotective effects on osteoblasts and here we show that taurine also protects osteocytes against cell death due to reactive oxygen species. Using the IDG-SW3 cell line, the expression of the taurine uptake transporter Taut/Slc6a6 is increased during osteoblast to osteocyte differentiation. Taurine had no effect on genes associated with osteoblast to osteocyte differentiation such as Dmp1, Phex or osteocalcin, even at high doses, but a slight yet significant inhibition of alkaline phosphatase was observed at the highest dose (50 mM). No effect was seen on the osteoclast regulatory genes Rankl and Opg, however the wnt antagonist Sost/sclerostin was potently and dose-dependently downregulated in response to taurine supplementation. Taurine also significantly inhibited Dkk1 mRNA expression, but only at 50 mM. Interestingly, osteocytes were found to also be able to synthesize taurine intracellularly, potentially as a self-protective mechanism, but do not secrete the metabolite. A highly significant increase in the expression of cysteine dioxygenase (Cdo), a key enzyme necessary for the production of taurine, was observed with osteoblast to osteocyte differentiation along with a decrease in methionine, the precursor of taurine. For the first time, we describe the synthesis of taurine by osteocytes, potentially to preserve viability and to regulate bone formation through inhibition of sclerostin.

Mechanical regulation of bone homeostasis through p130Cas-mediated alleviation of NF-κB activity.

Mechanical loading plays an important role in bone homeostasis. However, molecular mechanisms behind the mechanical regulation of bone homeostasis are poorly understood. We previously reported p130Cas (Cas) as a key molecule in cellular mechanosensing at focal adhesions. Here, we demonstrate that Cas is distributed in the nucleus and supports mechanical loading-mediated bone homeostasis by alleviating NF-κB activity, which would otherwise prompt inflammatory processes. Mechanical unloading modulates Cas distribution and NF-κB activity in osteocytes, the mechanosensory cells in bones. Cas deficiency in osteocytes increases osteoclastic bone resorption associated with NF-κB-mediated RANKL expression, leading to osteopenia. Upon shear stress application on cultured osteocytes, Cas translocates into the nucleus and down-regulates NF-κB activity. Collectively, fluid shear stress-dependent Cas-mediated alleviation of NF-κB activity supports bone homeostasis. Given the ubiquitous expression of Cas and NF-κB together with systemic distribution of interstitial fluid, the Cas-NF-κB interplay may also underpin regulatory mechanisms in other tissues and organs.

Case report: Electron microscopic evaluation of bone from a patient treated with cinacalcet hydrochloride, maxacalcitol, and alfacalcidol for hyperparathyroid bone disease with secondary hyperparathyroidism.

Evaluation of bone is of great importance in chronic kidney disease patients, as these patients are at an increased risk for fractures. We treated a hemodialysis patient suffering from hyperparathyroid bone disease with cinacalcet hydrochloride and concurrent administration of maxacalcitol and alfacalcidol for a year. Hyperparathyroid bone disease is characterized by cortical thinning, increased cortical porosity, reduced trabecular bone volume, and increased hypomineralized matrix volume, and there is little information to date about the effects of treatment with cinacalcet hydrochloride on the bone fragility in patients with hyperparathyroid bone disease. In the present study, histological and backscattered electron microscopic evaluation of this combination treatment revealed an excellent improvement of both bone volume and bone morphology. This treatment improved cortical thinning, cortical porosity, and trabecular thinning. Furthermore, the treatment also reduced hypomineralized matrix volume, indicative of improved mineralization by osteocytes. We speculate that the intermittent maxacalcitol administration may have effectively stimulated the vitamin D receptors expressed on osteocytes and osteoblasts, resulting in increased mineralization. Our approach for evaluating the bone in patients with chronic kidney disease by backscattered electron microscopy is novel.

The role of the wnt/ß-catenin signaling pathway in formation and maintenance of bone and teeth.

The Wnt signaling pathway is known as one of the important molecular cascades that regulate cell fate throughout lifespan. The Wnt signaling pathway is further separated into the canonical signaling pathway that depends on the function of ß-catenin (Wnt/ß-catenin pathway) and the noncanonical pathways that operate independently of ß-catenin (planar cell polarity pathway and Wnt/Ca(2+) pathway). The Wnt/ß-catenin signaling pathway is complex and consists of numerous receptors, inhibitors, activators, modulators, phosphatases, kinases and other components. However, there is one central, critical molecule to this pathway, ß-catenin. While there are at least 3 receptors, LRP 4, 5 and 6, and over twenty activators known as the wnts, and several inhibitors such as sclerostin, dickkopf and secreted frizzled-related protein, these all target ß-catenin. These regulators/modulators function to target ß-catenin either to the proteasome for degradation or to the nucleus to regulate gene expression. Therefore, the interaction of ß-catenin with different factors and Wnt/ß-catenin signaling pathway will be the subject of this review with a focus on how this pathway relates to and functions in the formation and maintenance of bone and teeth based on mainly basic and pre-clinical research. Also in this review, the role of this pathway in osteocytes, bone cells embedded in the mineralized matrix, is covered in depth. This pathway is not only important in mineralized tissue growth and development, but for modulation of the skeleton in response to loading and unloading and the viability and health of the adult and aging skeleton.

The Cementocyte-An Osteocyte Relative?

Cementum is a mineralized tissue covering the tooth root that functions in tooth attachment and posteruptive adjustment of tooth position. During formation of the apically located cellular cementum, some cementoblasts become embedded in the cementoid matrix and become cementocytes. As apparently terminally differentiated cells embedded in a mineralized extracellular matrix, cementocytes are part of a select group of specialized cells, also including osteocytes, hypertrophic chondrocytes, and odontoblasts. The differentiation and potential function(s) of cementocytes are virtually unknown, and the question may be posed whether the cementocyte is a dynamic actor in cementum in comparable fashion with the osteocyte in the skeleton, responding to changing tooth functions and endocrine signals and actively directing local cementum metabolism. This review summarizes the literature with regard to cementocytes, comparing them to their closest "cousins," the osteocytes, where insights gained from osteocyte studies serve to inform the critical examination of cementocytes. The review identifies important unanswered questions about these cells regarding their origins, differentiation, morphology and lacuno-canalicular system, selective markers, and potential functions.

CCL7 is a protective factor secreted by mechanically loaded osteocytes.

In a search for factors up-regulated by mechanical strain in osteocytes, we discovered that chemokine (C-C motif) ligand 7 (CCL7), a chemotactic myokine, was highly expressed in MLO-Y4 osteocyte-like cells. Although MLO-Y4 cells secrete potent chemotactic factors for osteoclast precursors, CCL7 was not responsible for this activity. CCL7 was increased in osteocytes in response to tooth movement in vivo. Since mechanical loading plays a crucial role in maintaining osteocyte viability, CCL7 was tested for protective activity and found to be protective against cell death induced by dexamethasone and etoposide. CCL7 specific antibody partially, but in combination with indomethacin, completely abrogated the protective effects of fluid flow shear stress against dexamethasone-induced cell death. CCL7 activated the ß-catenin pathway through phosphorylation of glycogen synthase kinase 3 (GSK-3), suggesting that this pathway is responsible for the observed protective effects. A related cytokine, CCL2, also produced by MLO-Y4 cells but not regulated by mechanical loading, proved to be more potent and protected against cell death induced by not only dexamethasone, but also by Tumor Necrosis Factor α (TNFα). Whereas osteocytes may produce CCL2 in constitutively low levels, a major function of mechanically induced CCL7 may be to selectively protect osteocytes in an autocrine manner against glucocorticoid-induced cell death.

Skeletal muscle secreted factors prevent glucocorticoid-induced osteocyte apoptosis through activation of ß-catenin.

It is a widely held belief that the sole effect of muscle on bone is through mechanical loading. However, as the two tissues are intimately associated, we hypothesized that muscle myokines may have positive effects on bone. We found that factors produced by muscle will protect osteocytes from undergoing cell death induced by dexamethasone (dex), a glucocorticoid known to induce osteocyte apoptosis thereby compromising their capacity to regulate bone remodeling. Both the trypan blue exclusion assay for cell death and nuclear fragmentation assay for apoptosis were used. MLO-Y4 osteocytes, primary osteocytes, and MC3T3 osteoblastic cells were protected against dex-induced apoptosis by C2C12 myotube conditioned media (MT-CM) or by CM from ex vivo electrically stimulated, intact extensor digitorum longus (EDL) or soleus muscle derived from 4 month-old mice. C2C12 MT-CM, but not undifferentiated myoblast CM prevented dex-induced cell apoptosis and was potent down to 0.1 % CM. The CM from EDL muscle electrically stimulated tetanically at 80 Hz was more potent (10 fold) in prevention of dex-induced osteocyte death than CM from soleus muscle stimulated at the same frequency or CM from EDL stimulated at 1 Hz. This suggests that electrical stimulation increases production of factors that preserve osteocyte viability and that type II fibers are greater producers than type I fibers. The muscle factor(s) appears to protect osteocytes from cell death through activation of the Wnt/ß-catenin pathway, as MT-CM induces ß-catenin nuclear translocation and ß-catenin siRNA abrogated the positive effects of MT-CM on dex-induced apoptosis. We conclude that muscle cells naturally secrete factor(s) that preserve osteocyte viability.

Bisphosphonate-related osteonecrosis of the jaw: model and diagnosis with cone beam computerized tomography.

Intravenous bisphosphonate (BP) therapy has become the standard of care for the treatment of cancers that metastasize to bone. BPs are associated with osteonecrosis of alveolar bones, a condition known as osteonecrosis of the jaw (ONJ). The incidence or pathogenesis of ONJ is largely unknown. The lesions are characterized by areas of exposed necrotic bone that do not heal after 8 weeks in the absence of radiation to the head and neck. ONJ lesions have been recalcitrant to conventional therapies. Lesions in cancer patients treated with BPs develop in association with periodontal disease, tooth extraction and/or in association with increased mechanical force due to partial/complete dentures. We hypothesized that intravenous BPs in cancer patients impair normal bone remodeling, thereby increasing the incidence of osteonecrotic lesions and that these lesions can be detected using cone beam computerized tomography (CBCT). From CBCTs taken at the University of Missouri at Kansas City School of Dentistry, 26 subjects had a cancer diagnosis and were on BP therapy. From these 26 subjects, 18 presented visible, exposed necrotic bone. We observed both sclerotic and radiolucent lesions. Lesions could be detected and measured in reconstructed images where most were found to expand to large areas of the bone. We were able to identify necrotic bodies or 'involucrums' within the ONJ lesions, suggesting that this could be the mechanism for the formation of a clinically visible sequestrum. We propose that CBCT can potentially identify and follow the progression of both pre- and postclinical lesions in ONJ patients, allowing better diagnosis and assessment of disease status.

Periostin is essential for the integrity and function of the periodontal ligament during occlusal loading in mice.

BACKGROUND: The ability of the periodontal ligament (PDL) to absorb and distribute forces is necessary for periodontal homeostasis. This adaptive response may be determined, in part, by a key molecule, periostin, which maintains the integrity of the PDL during occlusal function and inflammation. Periostin is primarily expressed in the PDL and is highly homologous to betaig-H3 (transforming growth factor-beta [TGF-beta] inducible gene). Cementum, alveolar bone, and the PDL of periostin-null mice dramatically deteriorate following tooth eruption. The purpose of this study was to determine the role of periostin in maintaining the functional integrity of the periodontium. METHODS: The periodontia from periostin-null mice were characterized followed by unloading the incisors. The effect of substrate stretching on periostin expression was evaluated using a murine PDL cell line. Real-time reverse transcription-polymerase chain reaction was used to quantify mRNA levels of periostin and TGF-beta. TGF-beta1 neutralizing antibodies were used to determine whether the effects of substrate stretching on periostin expression are mediated through TGF-beta. RESULTS: Severe periodontal defects were observed in the periostin-null mice after tooth eruption. The removal of masticatory forces in periostin-null mice rescue the periodontal defects. Periostin expression was increased in strained PDL cells by 9.2-fold at 48 hours and was preceded by a transient increase in TGF-beta mRNA in vitro. Elevation of periostin in response to mechanical stress was blocked by the addition of 2.5 ng/ml neutralizing antibody to TGF-beta1, suggesting that mechanical strain activates TGF-beta to have potential autocrine effects and to increase periostin expression. CONCLUSION: Mechanical loading maintains sufficient periostin expression to ensure the integrity of the periodontium in response to occlusal load.

Periodontal breakdown in the Dmp1 null mouse model of hypophosphatemic rickets.

Dentin Matrix Protein 1 (DMP1) is highly expressed in alveolar bone and cementum, which are important components of the periodontium. Therefore, we hypothesized that Dmp1 is critical for the integrity of the periodontium, and that deletion may lead to increased susceptibility to disease. An early-onset periodontal defect was observed in the Dmp1 null mouse, a mouse model of hypophosphatemic rickets. The alveolar bone is porous, with increased proteoglycan expression. The cementum is also defective, as characterized by irregular, punctate fluorochrome labeling and elevated proteoglycan. The osteocyte and cementocyte lacuno-canalicular system of both alveolar bone and cementum is abnormal, with irregular lacunar walls and fewer canaliculi. As a consequence, there is significant interproximal alveolar bone loss, combined with detachment between the periodontal ligament (PDL) and cementum. We propose that defective alveolar bone and cementum may account for the periodontal breakdown and increased susceptibility to bacterial infection in Dmp1 null mice.

Photolysis of a caged peptide reveals rapid action of N-ethylmaleimide sensitive factor before neurotransmitter release.

The time at which the N-ethylmaleimide-sensitive factor (NSF) acts during synaptic vesicle (SV) trafficking was identified by time-controlled perturbation of NSF function with a photoactivatable inhibitory peptide. Photolysis of this caged peptide in the squid giant presynaptic terminal caused an abrupt (0.2 s) slowing of the kinetics of the postsynaptic current (PSC) and a more gradual (2-3 s) reduction in PSC amplitude. Based on the rapid rate of these inhibitory effects relative to the speed of SV recycling, we conclude that NSF functions in reactions that immediately precede neurotransmitter release. Our results indicate the locus of SNARE protein recycling in presynaptic terminals and reveal NSF as a potential target for rapid regulation of transmitter release.

DMP1-targeted Cre expression in odontoblasts and osteocytes.

Odontoblasts in dentin and osteocytes in bone contain dendritic processes. To test if their dendrites share a common feature, we compared their cellular morphology as visualized using scanning electron microscopy. Analysis of our data showed that both cells share an identical dendritic canalicular system and express extensive processes forming a complex network within the mineralized matrix. Because dentin matrix protein 1 (DMP1), an extracellular matrix protein, is highly expressed in both types of cells, we next tested, using a transgenic approach, whether a 9.6-kb Dmp1 promoter-4-kb 1st intron would be able to target Cre cDNA in these cells for expression/deletion of other genes in odontoblasts and osteocytes. We determined the specificity and efficiency of Cre activity by crossing Dmp1-Cre mice with ROSA26 reporter mice. Results showed that odontoblasts and osteocytes were specifically targeted, suggesting that this animal model will be useful for the preferential study of gene functions in both types of cells.

Mechanism by which MLO-A5 late osteoblasts/early osteocytes mineralize in culture: similarities with mineralization of lamellar bone.

The mechanisms whereby bone mineralizes are unclear. To study this process, we used a cell line, MLO-A5, which has highly elevated expression of markers of the late osteoblast such as alkaline phosphatase, bone sialoprotein, parathyroid hormone type 1 receptor, and osteocalcin and will mineralize in sheets, not nodules. In culture, markers of osteocytes and dendricity increase with time, features of differentiation from a late osteoblast to an early osteocyte. Mineral formation was examined using transmission electron microscopy, scanning electron microscopy with energy-dispersive X-ray analysis, and atomic force microscopy. At 3-4 days of culture, spheres of approximately 20-50 nm containing calcium and phosphorus were observed budding from and associated with developing cellular projections. By 5-6 days, these calcified spheres were associated with collagen fibrils, where over time they continued to enlarge and to engulf the collagen network. Coalescence of these mineralized spheres and collagen-mediated mineralization were responsible for the mineralization of the matrix. Similar calcified spheres were observed in cultured fetal rat calvarial cells and in murine lamellar bone. We propose that osteoid-osteocytes generate spherical structures that calcify during the budding process and are fully mineralized on their developing cellular processes. As the cellular process narrows in diameter, these mineralized structures become associated with and initiate collagen-mediated mineralization.