Bone Marrow Stromal ST2 Cells Research Articles

Erythropoietin mediated bone formation is regulated by mTOR signaling

The role of erythropoietin (Epo) and Epo/Epo receptor (EpoR) signaling pathways for production of red blood cells are well established. However, little is known about Epo/EpoR signaling in non-hematopoietic cells. Recently, we demonstrated that Epo activates JAK/STAT signaling in hematopoietic stem cells (HSCs), leading to the production of bone morphogenetic protein 2 (BMP2) and bone formation and that Epo also directly activates mesenchymal cells to form osteoblasts in vitro. In this study, we investigated the effects of mTOR signaling on Epo-mediated osteoblastogenesis and osteoclastogenesis. We found that mTOR inhibition by rapamycin blocks Epo-dependent and -independent osteoblastic phenotypes in human bone marrow stromal cells (hBMSCs) and ST2 cells, respectively. Furthermore, we found that rapamycin inhibits Epo-dependent and -independent osteoclastogenesis in mouse bone marrow mononuclear cells and Raw264.7 cells. Finally, we demonstrated that Epo increases NFATc1 expression and decreases cathepsin K expression in an mTOR-independent manner, resulting in an increase of osteoclast numbers and a decrease in resorption activity. Taken together, these results strongly indicate that mTOR signaling plays an important role in Epo-mediated bone homeostasis.

Spleen tyrosine kinase suppresses osteoblastic differentiation through MAPK and PKCα

Spleen tyrosine kinase (Syk) is a non-receptor protein kinase present in abundance in a wide range of hematopoietic cells. Syk reportedly plays a crucial role in immune signaling in B cells and cells bearing Fcγ-activation receptors. The role of syk in osteoblastic differentiation has not been well elucidated. We report herein the role of syk in osteoblastic differentiation. We investigated the effects of two syk inhibitors on osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells and bone marrow stromal ST2 cells. Expression of syk was detected in these two cell lines. Two syk inhibitors stimulated mRNA expression of osteoblastic markers (ALP, Runx2, Osterix). Mineralization of extracellular matrix was also promoted by treatment with syk inhibitors. Knockdown of Syk caused increased mRNA expression of osteoblastic markers. In addition, syk inhibitor and knockdown of Syk suppressed phosphorylation of mitogen-activated protein kinase (MAPK) and protein kinase Cα (PKCα). Our results indicate that syk might regulate osteoblastic differentiation through MAPK and PKCα.

Static and dynamic cultivation of bone marrow stromal cells on biphasic calcium phosphate scaffolds derived from an indirect rapid prototyping technique

The adequate regeneration of large bone defects is still a major problem in orthopaedic surgery. Synthetic bone substitute materials have to be biocompatible, biodegradable, osteoconductive and processable into macroporous scaffolds tailored to the patient specific defect. Hydroxyapatite (HA) and tricalcium phosphate (TCP) as well as mixtures of both phases, biphasic calcium phosphate ceramics (BCP), meet all these requirements and are considered to be optimal synthetic bone substitute materials. Rapid prototyping (RP) can be applied to manufacture scaffolds, meeting the criteria required to ensure bone ingrowth such as high porosity and defined pore characteristics. Such scaffolds can be used for bone tissue engineering (BTE), a concept based on the cultivation of osteogenic cells on osteoconductive scaffolds. In this study, scaffolds with interconnecting macroporosity were manufactured from HA, TCP and BCP (60 wt% HA) using an indirect rapid prototyping technique involving wax ink-jet printing. ST-2 bone marrow stromal cells (BMSCs) were seeded onto the scaffolds and cultivated for 17 days under either static or dynamic culture conditions and osteogenic stimulation. While cell number within the scaffold pore system decreased in case of static conditions, dynamic cultivation allowed homogeneous cell growth even within deep pores of large (1,440 mm(3)) scaffolds. Osteogenic cell differentiation was most advanced on BCP scaffolds in both culture systems, while cells cultured under perfusion conditions were generally more differentiated after 17 days. Therefore, scaffolds manufactured from BCP ceramic and seeded with BMSCs using a dynamic culture system are the method of choice for bone tissue engineering.

Abstract 289: VEGFR/PDGFR blockade impairs tumor-stroma interactions through multiple mechanisms that inhibit bone metastatic homing

Abstract Bone microenvironment and tumor-stromal interactions are critical for the initiation and development of metastasis. The aim of this study was to delineate the role of VEGFR/PDGFR axes in bone metastatic homing and colonization using a multi-tyrosine kinase inhibitor (SU11248). SU blocks 7 different receptors, including VEGFRs and PDGFRs, resulting in the blockade of many biological functions. Here we show that SU blocks lung cancer metastasis to the bone through different mechanisms. Incubation of bone metastatic lung A549M1 cells with SU in vitro had no effect on cell proliferation whereas a potent effect on bone marrow (BM) stromal ST2 cells, and BM-derived endothelial cells (BMEC) was seen. The inhibitory effects of SU over ST2 cells were mediated through PDGFRbeta, which was highly expressed by ST2 cells. Direct heterotypic adhesion of tumor cells to a ST2 monolayer was significantly decreased by SU, whereas no effects were found in BMEC or HUVEC monolayers. SU markedly impaired invasion in vitro. Furthermore, SU inhibited the adhesion of A549M1 cells to collagen I. To evaluate these effects in an in vivo model of bone homing, we pretreated mice with SU two days before intracardiac (i.c.) inoculation of luciferase expressing A549M1 cells. Dramatic inhibition of bioluminescence was observed in SU treated mice 4 days post-inoculation. Quantification of A549M1 colony forming units after BM flushing revealed identical results suggesting an effect of SU in bone homing. To test SU effects in bone metastasis colonization, we used SU alone or in combination with the zoledronic acid (ZA) daily, starting from 6 days after i.c. of A549 M1 cells. As expected, ZA treatment slightly increased survival, whereas SU or combination led to a double lifespan. Bioluminescence imaging showed a moderate decrease in skeletal tumor burden in single ZA and SU treated mice, whereas marked reduction in osteolytic lesions assessed by X-Ray imaging and microCT scans was only evident in ZA treated mice. However, combination abrogated tumor burden and severely prevented the development of osteolytic lesions as compared to SU treated mice, which correlated with the number of TRAP positive cells at tumor-bone interphase. Treatment of tumor-free mice with SU revealed a potent proapoptotic effect on BM stromal PDGFRbeta+ compartment, as seen by extensive active Caspase-3 immunoreactiviy 24h after treatment. This effect over stroma leads to severe haemorrhage after 4 days of treatment, indicating a severe disruption of the BM microenvironment by SU. These data suggest that disruption of VEGFR/PDGFR axes alters heterotypic tumor-stromal cell and tumor-matrix interactions during bone homing preventing osseous colonization. We suggest that SU in combination with ZA could be suitable for the treatment of osteolytic bone metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 289.

The Effect of Microstructure of Octacalcium Phosphate on the Bone Regenerative Property

The present study was designed to investigate whether the microstructure of synthetic octacalcium phosphate (OCP) affects its intrinsic bone regenerative properties as a scaffold and its conversion process into hydroxyapatite (HA). Our previous studies indicated that an agregate of OCP crystals, consisting of randomly oriented plate-like crystals, are capable of enhancing both osteoblastic cell differentiation in vitro and bone regeneration. While the transformation of OCP into HA has been considered in relation to the stimulatory capacity of OCP in bone regeneration, little is known about the effect of the microstructure of OCP granules on these capabilities. Two types of OCP granules, with identical diameters (300-500 microm) but composed of crystals with distinct crystal dimensions (4.0 and 26.6 microm length), were prepared (hereafter referred to as fine OCP granules [F-OCP] and coarse OCP granules [C-OCP], respectively). The intergranule distances and the porosity, including the intergranule spaces, were 108.5 microm and 93.7% for F-OCP, and 67.5 microm and 95.7% for C-OCP, as estimated by mercury intrusion. The OCP granules were implanted in mouse critical-sized calvarial defects for up to 14 days. Histological examination demonstrated that osteoblastic cells aligned on the surface of F-OCP at day 7 and formed new bone around the granules up to day 14. On the other hand, cells around C-OCP were sparse at day 7, and resulted in only slight bone formation around the granules at day 14. X-ray diffraction showed that both OCP granules tended to be converted to an apatite structure with similar conversion velocity by the implantation. Adhesion of mouse bone marrow stromal ST-2 cells was markedly inhibited on C-OCP compared to F-OCP in vitro. These results suggested that the microstructure consisting of plate-like crystals of OCP controls cell adhesion on the crystal surfaces and their resultant bone regenerative properties as well as the physicochemical effect associated with the transitory nature of OCP previously reported.

FGF2 stimulates SDF‐1 expression through the Erm transcription factor in Sertoli cells

Ets-related molecule (Erm) is a member of the Ets transcription factor family. Erm is known to be an important factor for the self-renewal of Spermatogonial stem cells (SSCs) and the maintenance of spermatogenesis. We investigated the molecular mechanism of Erm regulation on SDF-1 in TM4 Sertoli cells. Erm and Sdf-1 levels were up-regulated after FGF2 treatment in TM4 cells, whereas these levels were significantly decreased by FGF2 in ST2 bone marrow stromal cells. Knockdown of Erm by siRNA in the presence of FGF2 decreased the Sdf-1 levels in TM4 cells. The expression levels of Erm were similar and Erm overexpression increased the Sdf-1 in both TM4 and ST2 cells. FGFR subtype analysis revealed that FGFR4 was expressed in TM4 cells but not in ST2 cells. A blocking experiment also confirmed that FGFR4 is partly responsible for the up-regulation of Erm and SDF-1 induced by FGF2 stimulation in TM4 cells. FGF2 and ERM increased the activity of Sdf-1 gene promoter region in a dose-dependent manner. EMSA revealed that ERM strongly binds to the -846 to -851 nucleotide region of the potential Ets binding site (EBS) in the Sdf-1 promoter. In addition, CXCR4, the SDF-1 receptor, was expressed in spermatogonia and Sertoli cells in the seminiferous tubules of the mouse testis. Our results indicate that ERM directly regulates Sdf-1 gene expression by interacting with its cis-acting element in response to FGF2 stimulation in TM4 cells.

Dose-Dependent Osteogenic Effect of Octacalcium Phosphate on Mouse Bone Marrow Stromal Cells

Octacalcium phosphate (OCP) has been advocated to be a precursor of biological apatite crystals in bones and teeth. Our previous studies showed that synthetic OCP stimulates bone regeneration, followed by the progressive conversion of OCP into hydroxyapatite (HA), when implanted in bone defects. However, the precise mechanism to induce the osteogenic phenotype in osteoblasts by OCP has not been identified. The present study was designed to investigate whether the physicochemical aspect, specific to and derived from the structural properties of OCP, influences the function of an osteoblastic cell line, mouse bone marrow stromal ST-2 cells. Different amounts of synthetic OCP and synthetic sintered ceramic HA were coated onto 48-well tissue culture plates. The amounts of OCP and HA were controlled to strengthen their intrinsic physicochemical properties, in which the milieu around the crystals will be modified during the culture. The roughness of the OCP coatings was independent of the amount of coating. Chemical analyses of the supernatants of the OCP coatings revealed that the concentration of Ca2+ decreased with increasing amounts of OCP, while the concentration of inorganic phosphate increased markedly, most probably through OCP--apatite conversion. ST-2 cells were cultured on the OCP or HA coatings up to day 21. The OCP coating caused a significant decrease in cell attachment and in the initial stage of proliferation, dependent upon the amount of coating. On the other hand, OCP enhanced the expression of osteogenic markers, including type I collagen, alkaline phosphatase, and osterix. However, HA did not alter the expression of these markers in ST-2 cells cultured on different amounts of HA coating. These results demonstrated that OCP is capable of inducing the differentiation of stromal cells into osteoblastic cells, especially differentiation into early stage osteoblastic cells, prior to reaching the stage of mature osteoblastic cell lineage.

Enhanced osteoclastogenesis in patients with tophaceous gout: Urate crystals promote osteoclast development through interactions with stromal cells

To analyze cellular mechanisms of bone erosion in gout. Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) from patients with gout were analyzed for the presence of osteoclast precursors. Fixed tophus and bone samples were analyzed by immunohistochemistry. Mechanisms of osteoclastogenesis were studied by culturing murine preosteoclast RAW 264.7 cells, bone marrow stromal ST2 cells, and human synovial fibroblasts with monosodium urate monohydrate (MSU) crystals. PBMCs from patients with severe erosive gout had the preferential ability to form osteoclast-like cells in culture with RANKL and monocyte colony-stimulating factor (M-CSF). The number of PBMC-derived tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells strongly correlated with the number of tophi (r = 0.6296, P = 0.630). Patients with severe erosive and tophaceous gout also had higher circulating concentrations of RANKL and M-CSF. Furthermore, greater numbers of TRAP-positive multinucleated cells were cultured from SFMCs derived from gouty knee effusions than from paired PBMCs (P = 0.004). Immunohistochemical analysis demonstrated numerous multinucleated cells expressing osteoclast markers within tophi and at the interface between soft tissue and bone. MSU crystals did not directly promote osteoclast formation from RAW 264.7 cells in vitro. However, MSU crystals inhibited osteoprotegerin gene and protein expression in ST2 cells and human synovial fibroblasts, without significantly altering RANKL gene expression. Conditioned medium from ST2 cells cultured with MSU crystals promoted osteoclast formation from RAW 264.7 cells in the presence of RANKL. Chronic tophaceous and erosive gout is characterized by enhanced osteoclast development. These data provide a rationale for the study of osteoclast-targeted therapies for the prevention of bone damage in chronic gout.

Dose-Dependent Osteogenic Effect of Octacalcium Phosphate on Mouse Bone Marrow Stromal Cells

Octacalcium phosphate (OCP) has been advocated to be a precursor of biological apatite crystals in bones and teeth. Our previous studies showed that synthetic OCP stimulates bone regeneration, followed by the progressive conversion of OCP into hydroxyapatite (HA), when implanted in bone defects. However, the precise mechanism to induce the osteogenic phenotype in osteoblasts by OCP has not been identified. The present study was designed to investigate whether the physicochemical aspect, specific to and derived from the structural properties of OCP, influences the function of an osteoblastic cell line, mouse bone marrow stromal ST-2 cells. Different amounts of synthetic OCP and synthetic sintered ceramic HA were coated onto 48-well tissue culture plates. The amounts of OCP and HA were controlled to strengthen their intrinsic physicochemical properties, in which the milieu around the crystals will be modified during the culture. The roughness of the OCP coatings was independent of the amount of coating. Chemical analyses of the supernatants of the OCP coatings revealed that the concentration of Ca2+ decreased with increasing amounts of OCP, while the concentration of inorganic phosphate increased markedly, most probably through OCP--apatite conversion. ST-2 cells were cultured on the OCP or HA coatings up to day 21. The OCP coating caused a significant decrease in cell attachment and in the initial stage of proliferation, dependent upon the amount of coating. On the other hand, OCP enhanced the expression of osteogenic markers, including type I collagen, alkaline phosphatase, and osterix. However, HA did not alter the expression of these markers in ST-2 cells cultured on different amounts of HA coating. These results demonstrated that OCP is capable of inducing the differentiation of stromal cells into osteoblastic cells, especially differentiation into early stage osteoblastic cells, prior to reaching the stage of mature osteoblastic cell lineage.

Effect of Octacalcium Phosphate Ionic Dissolution Products on Osteoblastic Cell Differentiation

Our previous studies suggested that synthetic octacalcium phosphate (OCP) enhances bone regeneration more than hydroxyapatite (HA). However, the molecular mechanisms to induce osteogenic phenotype in osteoblast by OCP have not been identified. OCP tended to convert into an apatite structure in vivo and in vitro, and its process was accompanied by calcium consumption from the surrounding solution and the release of phosphate ions into the solution at a physiological condition. The present study was designed to investigate whether the dissolution of ionic products of OCP affects on proliferation and differentiation of mouse bone marrow stromal ST-2 cells in vitro. The number of cells treated with OCP-conditioned medium was slightly decreased in comparison to that of control at day 7. On the other hand, the level of alkaline phosphatase activity increased in OCP-conditioned medium. These results demonstrated that OCP is capable of inducing osteoblastic cell differentiation in ST-2 cells.

Osteocytes as mechanosensors in the inhibition of bone resorption due to mechanical loading

Bone has the ability to adjust its structure to meet its mechanical environment. The prevailing view of bone mechanobiology is that osteocytes are responsible for detecting and responding to mechanical loading and initiating the bone adaptation process. However, how osteocytes signal effector cells and initiate bone turnover is not well understood. Recent in vitro studies have shown that osteocytes support osteoclast formation and activation when co-cultured with osteoclast precursors. In this study, we examined the osteocytes' role in the mechanical regulation of osteoclast formation and activation. We demonstrated here that (1) mechanical stimulation of MLO-Y4 osteocyte-like cells decreases their osteoclastogenic-support potential when co-cultured with RAW264.7 monocyte osteoclast precursors; (2) soluble factors released by these mechanically stimulated MLO-Y4 cells inhibit osteoclastogenesis induced by ST2 bone marrow stromal cells or MLO-Y4 cells; and (3) soluble RANKL and OPG were released by MLO-Y4 cells, and the expressions of both were found to be mechanically regulated. Our data suggest that mechanical loading decreases the osteocyte's potential to induce osteoclast formation by direct cell–cell contact. However, it is not clear that osteocytes in vivo are able to form contacts with osteoclast precursors. Our data also demonstrate that mechanically stimulated osteocytes release soluble factors that can inhibit osteoclastogenesis induced by other supporting cells including bone marrow stromal cells. In summary, we conclude that osteocytes may function as mechanotransducers by regulating local osteoclastogenesis via soluble signals.

A Novel Regulatory Role for Stromal-derived Factor-1 Signaling in Bone Morphogenic Protein-2 Osteogenic Differentiation of Mesenchymal C2C12 Cells

Stromal-derived factor 1 (SDF-1) is a chemokine with important functions in development and postnatal tissue homeostasis. SDF-1 signaling via the G-protein-coupled receptor CXCR4 regulates the recruitment of stem and precursor cells to support tissue-specific repair or regeneration. In this study we examined the contribution of SDF-1 signaling to osteogenic differentiation of mesenchymal C2C12 cells induced by bone morphogenic protein 2 (BMP2). Blocking SDF-1 signaling before BMP2 stimulation by treatment with siRNA, antibodies against SDF-1 or CXCR4, or the G-protein-coupled receptor inhibitor pertussis toxin strongly suppressed BMP2 induction of osteogenic differentiation in C2C12 cells, as evidenced by an early decrease in the expression of the myogenesis inhibitor Id1, the osteogenic master regulators Runx2 and Osx, the osteoblast-associated transcription factors JunB, Plzf, Msx2, and Dlx5, and later of the bone marker proteins osteocalcin and alkaline phosphatase. Similarly, blocking SDF-1/CXCR4 signaling strongly inhibited BMP2-induced osteogenic differentiation of ST2 bone marrow stromal cells. Moreover, we found that the interaction between SDF-1 and BMP2 signaling was mediated via intracellular Smads and MAPK activation. Our data provide the first evidence for a co-requirement of the SDF-1/CXCR4 signaling axis in BMP2-induced osteogenic differentiation of C2C12 and ST2 cells and, thus, uncover a new potential target for modulation of osteogenesis.

Osteoconductivity of Porous Titanium Having Young’s Modulus Similar to Bone and Surface Modification by OCP

The present study was designed to investigate whether porous titanium (Ti) having Young’s modulus similar to bone has osteoconductive characteristics in rat critical-sized calvarial bone defect. The effect of coating by octacalcium phosphate (OCP) was also examined. OCP is known as a precursor of initial mineral crystals of biological apatite in bones and teeth. Ti powder was prepared by plasma rotating electrode process in an Ar atmosphere. Then, porous Ti disks, 8 mm in diameter with 1 mm thick, were obtained using the particles ranging from 300 to 500 +m, by sintering at 1573 K without applied pressure. The disks had about 35 vol% in porosity and about 10 GPa in Young’s modulus which corresponds to that of human cortical bone. Newly formed bone was observed so as to fill the pore up at 12 weeks, confirming the ability to conduct the ingrowths of the bone tissue. Although in vitro study showed that proliferation of mouse bone marrow stromal ST-2 cells was inhibited on the dishes coated by OCP rather than the control dish, OCP coating on porous Ti seemed to stimulate the bone formation in vivo. Taken together, it seems likely that porous Ti having Young’s modulus similar to bone shows osteoconductive characteristics to conduct bone ingrowths. OCP could be a potential coating agent to assist bone regeneration on porous Ti.

Bone formation enhanced by implanted octacalcium phosphate involving conversion into Ca-deficient hydroxyapatite

The present study was designed to investigate whether hydrolysis of synthetic octacalcium phosphate (OCP) into hydroxyapatite affects bone formation. Mouse bone marrow stromal ST-2 cells and primary calvarial osteoblastic cells were cultured on the dishes pre-coated with OCP or its hydrolyzed Ca-deficient hydroxyapatite (OCP hydrolyzate; HL). The capacity of proliferation and differentiation was determined up to day 20. Granules of OCP and HL were implanted into critical-size rat calvaria defects for 4 and 12 weeks, and then bone formation was measured by histomorphometry. Structural changes of incubated and implanted OCP were determined by X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). The proliferation of both ST-2 and primary osteoblasts cultured on OCP or HL was initially inhibited, whereas their differentiation to osteoblasts was promoted at last. Implantation of OCP in bone defect more significantly enhanced bone formation than that of HL until 12 weeks. OCP tended to convert to apatite in vitro and in vivo. The conversion of the implanted OCP was ascertained to advance gradually with implantation periods. Taken together, these results suggest that OCP supports appositional bone formation and OCP-apatite conversion may be involved in this stimulatory capacity of OCP.

Dominant Negative N-Cadherin Inhibits Osteoclast Differentiation by Interfering With β-Catenin Regulation of RANKL, Independent of Cell-Cell Adhesion

We studied the effects of dominant negative N-cadherin (NCadDeltaC) expression in ST2 cells on their ability to support osteoclastogenesis. Expression of NCadDeltaC in ST2 cells did not decrease cell-to-cell adhesion but significantly reduced osteoclast formation when co-cultured with BMMs. NCadDeltaC inhibited beta-catenin/TCF signaling, resulting in decreased RANKL expression, which could contribute to the reduced osteoclast formation. Cadherin is a calcium-dependent cell adhesion molecule that plays major roles during embryonic development and morphogenesis. Classic cadherins interact with beta-catenin, which is also involved in the Wnt signaling pathway. We tested whether disruption of N-cadherin function in stromal cells by dominant negative N-cadherin affects their ability to support osteoclastogenesis by altering heterotypic interaction with osteoclast precursors. ST2 cells were transduced with retrovirus encoding extracellular domain-truncated, dominant negative N-cadherin (NCadDeltaC) and co-cultured with bone marrow macrophages (BMMs) to study the ability to support osteoclastogenesis. As a downstream target of NCadDeltaC, beta-catenin/T-cell factor (TCF) transcriptional activity was analyzed using TOPflash reporter construct. Real-time RT-PCR analysis and RANKL-luciferase reporter assays were performed to study the effects of NCadDeltaC on the osteoprotegerin (OPG)/RANKL system. Immunoblotting analysis showed that primary bone marrow stromal cells, ST2 cells, and BMMs expressed N-cadherin. Retroviral expression of NCadDeltaC in ST2 cells did not significantly inhibit cell adhesion but markedly impaired the formation of TRACP(+) osteoclasts (>40%) when co-cultured with BMMs. However, the inhibition of osteoclastogenesis was not reproduced by neutralizing antibody against N-cadherin. Expression of NCadDeltaC, however, strongly suppressed beta-catenin/TCF transcriptional activity in ST2 cells, which was rescued by constitutively active beta-catenin adenovirus (Ad DeltaN46 beta-catenin) or constitutively active TCF mutant (pCS2-VP16DeltabetaXTCF-3). As a potential downstream target of Wnt signaling, we found that the expression of RANKL was reduced in ST2 cells expressing NCadDeltaC. Moreover, Wnt-3A, Ad DeltaN46 beta-catenin, and VP16DeltabetaXTCF-3 increased the expression of RANKL and enhanced the transcriptional activity of mouse RANKL promoter in ST2 cells. Our data suggest that expression of dominant negative N-cadherin in ST2 cells suppressed osteoclastogenesis by interfering with beta-catenin regulation of RANKL independent of cell-cell adhesion.

Smad7 induces G0/G1 cell cycle arrest in mesenchymal cells by inhibiting the expression of G1 cyclins

The major Smad pathways serve in regulating the expression of genes downstream of TGFbeta signals. In this study, we examined the effects of sustained Smad7 expression in cultured cells. Interestingly, Smad7 caused various mesenchymal cells, including NIH3T3 fibroblast and ST2 bone-marrow stromal cells, to undergo a marked morphological alteration into a flattened cell shape, but kept them alive for as long as 60 days. Furthermore, Smad7 arrested the proliferation of the cells even before they reached confluence. These cells became quiescent in G0/G1 phase and accumulated a hypophosphorylated form of retinoblastoma. The cytostatic effect of Smad7 was closely associated with a preceding decrease in the levels of G1 cyclins, such as cyclin D1 and cyclin E. Accordingly, ectopic cyclin E was able to overcome the Smad7-induced arrest of proliferation. These results indicate that Smad7 functions upstream of G1 cyclins and suggest a novel role for Smad7 as an antiproliferative factor. In contrast to the growth of mesenchymal cells, that of epithelial cells was little susceptible to Smad7. The present findings raise the possibility that a link between Smad7 and the G1 to S phase transition may also contribute to the cell cycle control by certain Smad7-inducing stimuli in a cell-type-dependent fashion.

Cytokines link osteoblasts and inflammation: microarray analysis of interleukin-17- and TNF-α-induced genes in bone cells

The novel cytokine interleukin (IL)-17 has been implicated in many infectious and autoimmune settings, especially rheumatoid arthritis. Consistent with its proinflammatory effects on bone, osteoblast cells are highly responsive to IL-17, particularly in combination with other inflammatory cytokines. To better understand the spectrum of activities controlled by IL-17, we globally profiled genes regulated by IL-17 and tumor necrosis factor alpha (TNF-alpha) in the preosteoblast cell line MC3T3-E1. Using Affymetrix microarrays, 80-90 genes were up-regulated, and 19-50 genes were down-regulated with IL-17 and TNF-alpha as compared with TNF-alpha alone. These included proinflammatory chemokines and cytokines, inflammatory genes, transcriptional regulators, bone-remodeling genes, signal transducers, cytoskeletal genes, genes involved in apoptosis, and several unknown or unclassified genes. The CXC family chemokines were most dramatically induced by IL-17 and TNF-alpha, confirming the role of IL-17 as a potent mediator of inflammation and neutrophil recruitment. Several transcription factor-related genes involved in inflammatory gene expression were also enhanced, including molecule possessing ankyrin repeats induced by lipopolysaccharide/inhibitor of kappaBzeta (MAIL/kappaBzeta), CCAAT/enhancer-binding protein delta (C/EBPdelta), and C/EBPbeta. We also identified the acute-phase gene lipocalin-2 (LCN2/24p3) as a novel IL-17 target, which is regulated synergistically by TNF-alpha and IL-17 at the level of its promoter. A similar but not identical pattern of genes was induced by IL-17 and TNF-alpha in ST2 bone marrow stromal cells and murine embryonic fibroblasts. This study provides a profile of genes regulated by IL-17 and TNF-alpha in osteoblasts and suggests that in bone, the major function of IL-17 is to cooperate and/or synergize with other cytokines to amplify inflammation.

Drug-Loaded Carbon Nanohorns: Adsorption and Release of Dexamethasone in Vitro

Single-wall carbon nanohorns (SWNHs) are recently discovered nanostructured spherical aggregates of graphitic tubes. The unique physicochemical properties of SWNHs, including their large surface area, suggest their possible utility as carriers in drug delivery systems. Here we investigated the in vitro binding and release of the antiinflammatory glucocorticoid dexamethasone (DEX) by as-grown SWNHs and their oxidized form, oxSWNHs. Adsorption analyses using [3H]-DEX determined the amount of DEX adsorbed by oxSWNHs to be 200 mg for each gram of oxSWNHs in 0.5 mg/mL of DEX solution, which was approximately 6 times larger than that obtained for as-grown SWNHs. Adsorption kinetics indicated that oxSWNHs had higher affinity for DEX than as-grown SWNHs. Treatment of oxSWNHs at 1200 degrees C under H2, which removed the oxygen-containing functional groups on oxSWNHs, did not diminish the high affinity for DEX, suggesting that oxygen-containing functional groups have little contribution for the affinity. DEX-oxSWNH complexes exhibited sustained release of DEX into phosphate-buffered saline (pH 7.4) at 37 degrees C and more rapid biphasic release into culture medium. The biological integrity of the released DEX form was confirmed by activation of glucocorticoid response element-driven transcription in mouse bone marrow stromal ST2 cells and induction of alkaline phosphatase in mouse osteoblastic MC3T3-E1 cells. Notably, synthesis of SWNHs does not require a metal catalyst, the toxicity of which could become problematical in clinical use, and no cytotoxicity was observed in cells cultured in the presence of oxSWNHs under our conditions. Taken together, these observations highlight the potential utility of SWNHs in drug delivery systems.

Generation of bone-resorbing osteoclasts from B220+ cells: its role in accelerated osteoclastogenesis due to estrogen deficiency.

Estrogen deficiency stimulates both osteoclastic bone resorption and pre-B lymphopoiesis, the interrelationships between which remain unknown. To investigate the involvement of an increase in the number of B220+ cells in accelerated osteoclastogenesis after estrogen deficiency, we first examined whether ovariectomy (OVX) increased the frequency of clonogenic osteoclast precursors in bone marrow. The results were that after OVX, the frequency of clonogenic osteoclast precursors is increased in bone marrow, suggesting that accumulated osteoclast precursors contribute to accelerated osteoclastogenesis. Further, we found that cocultures of B220+ cells purified from bone marrow cells and stromal ST2 cells in the presence of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] gave rise to osteoclasts that can resorb bone and express calcitonin receptors. When the frequencies of clonogenic osteoclast precursors in the purified B220+ and B220- cell fractions were compared, it was found that the fractions gave rise to osteoclasts at similar frequencies, which rules out the possibility of cross-contamination and suggests that the two fractions contain comparable numbers of osteoclast precursors. Furthermore, we identified cells that are positive for both tartrate-resistant acid phosphatase (TRAP) and B220, not only in cocultures of B220+ and ST2 cells, but also in freshly isolated unfractionated bone cells. Therefore, it is concluded that at least a subfraction of B220+ cells are capable of generating osteoclasts and that the increase in the number of B220+ cells caused by estrogen deficiency may contribute to accelerated bone resorption by this novel osteoclastogenesis pathway.

Interleukin-18 Up-Regulates Osteoprotegerin Expression in Stromal/Osteoblastic Cells

Osteoprotegerin (OPG) and osteoclast differentiation factor (ODF) are crucial regulators of osteoclastogenesis. To determine the biological role of interleukin (IL)-18 produced by stromal/osteoblastic cells in osteoclastogenesis, we examined the effects of IL-18 on the OPG and ODF mRNA levels in these cells. When bone marrow stromal ST2 cells, osteoblastic MC3T3-E1 cells, and mouse calvarial osteoblasts were stimulated with IL-18, the expression of OPG mRNA, but not ODF mRNA, was transiently increased, its expression reaching a maximal level at 3 h after the beginning of the culture. In accordance with this observation, all these cells expressed the mRNAs of two IL-18 receptor components and MyD88, an adapter molecule involved in IL-18 signaling. Moreover, in these cells, mitogen-activated protein kinase was phosphorylated after stimulation with IL-18. These results suggest that stromal/osteoblastic cells are IL-18-responsive cells and that IL-18 may inhibit osteoclastogenesis by up-regulating OPG expression, without stimulation of ODF production, in stromal/osteoblastic cells.