G2A Protects Mice against Sepsis by Modulating Kupffer Cell Activation: Cooperativity with Adenosine Receptor 2b.

G2A is a GPCR abundantly expressed in immune cells. G2A-/- mice showed higher lethality, higher plasma cytokines, and an impaired bacterial clearance in response to a murine model of sepsis (cecal ligation and puncture), which were blocked by GdCl3, an inhibitor of Kupffer cells. Anti-IL-10 Ab reversed the impaired bacterial clearance in G2A-/- mice. Indomethacin effectively blocked both the increased i.p. IL-10 levels and the impaired bacterial clearance, indicating that disturbed PG system is the proximal cause of these phenomena. Stimulation with LPS/C5a induced an increase in Escherichia coli phagocytosis and intracellular cAMP levels in G2A+/+ peritoneal macrophages but not G2A-/- cells, which showed more PGE2/nitrite release and intracellular reactive oxygen species levels. Heterologous coexpression of G2A and adenosine receptor type 2b (A2bAR) induced a synergistic increase in cAMP signaling in a ligand-independent manner, with the evidence of physical interaction of G2A with A2bAR. BAY 60-6583, a specific agonist for A2bAR, increased intracellular cAMP levels in Kupffer cells from G2A+/+ but not from G2A-/- mice. Both G2A and A2bAR were required for antiseptic action of lysophosphatidylcholine. These results show inappropriate activation of G2A-/- Kupffer cells to septic insults due to an impaired cAMP signaling possibly by lack of interaction with A2bAR.

Isoflavone-enriched whole soy milk powder stimulates osteoblast differentiation.

Functional foods with high nutritive values and potential therapeutic potential is a prerequisite for today's ailing world. Soybeans exert beneficial effects on human health. It contains plentiful polyunsaturated fatty acids and dietary fibers along with several isoflavonoids having bioactivity for improving health. Recent studies have shown that soybean isoflavones can have a positive effect on bone growth. The current study was designed to observe any impact of isoflavone-enriched soy milk powder (I-WSM) on inducing osteogenic properties at cellular and molecular levels. Precisely, we have evaluated the effect of I-WSM on the bone differentiation process. Our results show that I-WSM has the ability to stimulate osteogenic properties in osteoblasts both at the initial and terminal stages of differentiation. Treatment of I-WSM on osteoblasts demonstrates the inductive effect on the expression of osteogenic transcriptional factors like Runx2 and Osterix. Moreover, I-WSM increased the expression of the extracellular matrix protein osteocalcin, required for the formation of scaffold for bone mineralization. The estrogen signaling pathway was utilized by I-WSM to induce osteogenic activity. Taken together, here we report the cellular and molecular events mediated by I-WSM to exert an osteogenic effect in osteoblasts, which will help to understand its mechanism of action and project it as a remedy for the bone-related disease. Taken together, I-WSM has the ability to exert the osteogenic effect in osteoblasts via the estrogen signaling pathway and thus might be projected as a remedy for a bone-related disease like osteoporosis.

Antimicrobial Potential of Silver Nanoparticles Synthesized Using Medicinal Herb Coptidis rhizome.

Coptidis rhizome contains several alkaloids that are bioactive agents of therapeutic value. We propose an eco-friendly method to synthesize biocompatible silver nanoparticles (AgNPs) using the aqueous extract of Coptidis rhizome. Silver ions were reduced to AgNPs using the aqueous extract of Coptidis rhizome, indicating that Coptidis rhizome can be used for the biosynthesis of AgNPs. The time and the concentration required for conversion of silver ions into AgNPs was optimized using UV-absorbance spectroscopy and inductively coupled plasma spectroscopy (ICP). Biosynthesized AgNPs showed a distinct UV-Visible absorption peak at 420 nm. ICP analysis showed that the time required for the completion of biosynthesis was around 20 min. Microscopic images showed that nanoparticles synthesized were of spherical shape and the average diameter of biosynthesized AgNPs was less than 30 nm. XRD analysis also confirmed the size of AgNps and revealed their crystalline nature. The interaction of AgNPs with phytochemicals present in Coptidis rhizome extract was observed in FTIR analysis. The antimicrobial property of AgNPs was evaluated using turbidity measurements. Coptidis rhizome-mediated biosynthesized AgNPs showed significant anti-bacterial activities against Escherichia coli and Staphylococcus aureus that are commonly involved in various types of infections, indicating their potential as an effective anti-bacterial agent.

Antimicrobial Potential of Silver Nanoparticles Synthesized Using Medicinal Herb Coptidis rhizome.

Coptidisrhizome contains several alkaloids that are bioactive agents of therapeutic value. We propose an eco-friendly method to synthesize biocompatible silver nanoparticles (AgNPs) using the aqueous extract of Coptidisrhizome. Silver ions were reduced to AgNPs using the aqueous extract of Coptidisrhizome, indicating that Coptidisrhizome can be used for the biosynthesis of AgNPs. The time and the concentration required for conversion of silver ions into AgNPs was optimized using UV-absorbance spectroscopy and inductively coupled plasma spectroscopy (ICP). Biosynthesized AgNPs showed a distinct UV-Visible absorption peak at 420 nm. ICP analysis showed that the time required for the completion of biosynthesis was around 20 min. Microscopic images showed that nanoparticles synthesized were of spherical shape and the average diameter of biosynthesized AgNPs was less than 30 nm. XRD analysis also confirmed the size of AgNps and revealed their crystalline nature. The interaction of AgNPs with phytochemicals present in Coptidisrhizome extract was observed in FTIR analysis. The antimicrobial property of AgNPs was evaluated using turbidity measurements. Coptidisrhizome-mediated biosynthesized AgNPs showed significant anti-bacterial activities against Escherichia coli and Staphylococcus aureus that are commonly involved in various types of infections, indicating their potential as an effective anti-bacterial agent.

Lysophosphatidic acid enhances breast cancer cells-mediated osteoclastogenesis.

Lysophosphatidic acid (LPA) is known to play a critical role in breast cancer metastasis to bone. In this study, we tried to investigate any role of LPA in the regulation of osteoclastogenic cytokines from breast cancer cells and the possibility of these secretory factors in affecting osteoclastogenesis. Effect of secreted cytokines on osteoclastogenesis was analyzed by treating conditioned media from LPA-stimulated breast cancer cells to differentiating osteoclasts. Result demonstrated that IL-8 and IL-11 expression were upregulated in LPA-treated MDA-MB-231 cells. IL-8 was induced in both MDA-MB-231 and MDA-MB-468, however, IL-11 was induced only in MDA-MB-231, suggesting differential LPARs participation in the expression of these cytokines. Expression of IL-8 but not IL-11 was suppressed by inhibitors of PI3K, NFkB, ROCK and PKC pathways. In the case of PKC activation, it was observed that PKCδ and PKCµ might regulate LPA-induced expression of IL-11 and IL-8, respectively, by using specific PKC subtype inhibitors. Finally, conditioned Medium from LPA-stimulated breast cancer cells induced osteoclastogenesis. In conclusion, LPA induced the expression of osteolytic cytokines (IL-8 and IL-11) in breast cancer cells by involving different LPA receptors. Enhanced expression of IL-8 by LPA may be via ROCK, PKCu, PI3K, and NFkB signaling pathways, while enhanced expression of IL-11 might involve PKCδ signaling pathway. LPA has the ability to enhance breast cancer cells-mediated osteoclastogenesis by inducing the secretion of cytokines such as IL-8 and IL-11.

Quercetin induces apoptosis and cell cycle arrest in triple-negative breast cancer cells through modulation of Foxo3a activity.

Quercetin, a plant-derived flavonoid found in fruits, vegetables and tea, has been known to possess bioactive properties such as anti-oxidant, anti-inflammatory and anti-cancer. In this study, anti-cancer effect of quercetin and its underlying mechanisms in triple-negative breast cancer cells was investigated. MTT assay showed that quercetin reduced breast cancer cell viability in a time and dose dependent manner. For this, quercetin not only increased cell apoptosis but also inhibited cell cycle progression. Moreover, quercetin increased FasL mRNA expression and p51, p21 and GADD45 signaling activities. We also observed that quercetin induced protein level, transcriptional activity and nuclear translocation of Foxo3a. Knockdown of Foxo3a caused significant reduction in the effect of quercetin on cell apoptosis and cell cycle arrest. In addition, treatment of JNK inhibitor (SP 600125) abolished quercetin-stimulated Foxo3a activity, suggesting JNK as a possible upstream signaling in regulation of Foxo3a activity. Knockdown of Foxo3a and inhibition of JNK activity reduced the signaling activities of p53, p21 and GADD45, triggered by quercetin. Taken together, our study suggests that quercetin induces apoptosis and cell cycle arrest via modification of Foxo3a signaling in triple-negative breast cancer cells.

Application of Bioactive Quercetin in Oncotherapy: From Nutrition to Nanomedicine.

Phytochemicals as dietary constituents are being explored for their cancer preventive properties. Quercetin is a major constituent of various dietary products and recently its anti-cancer potential has been extensively explored, revealing its anti-proliferative effect on different cancer cell lines, both in vitro and in vivo. Quercetin is known to have modulatory effects on cell apoptosis, migration and growth via various signaling pathways. Though, quercetin possesses great medicinal value, its applications as a therapeutic drug are limited. Problems like low oral bioavailability and poor aqueous solubility make quercetin an unreliable candidate for therapeutic purposes. Additionally, the rapid gastrointestinal digestion of quercetin is also a major barrier for its clinical translation. Hence, to overcome these disadvantages quercetin-based nanoformulations are being considered in recent times. Nanoformulations of quercetin have shown promising results in its uptake by the epithelial system as well as enhanced delivery to the target site. Herein we have tried to summarize various methods utilized for nanofabrication of quercetin formulations and for stable and sustained delivery of quercetin. We have also highlighted the various desirable measures for its use as a promising onco-therapeutic agent.

Methoxy poly(ethylene glycol)-poly(lactide) nanoparticles encapsulating quercetin act as an effective anticancer agent by inducing apoptosis in breast cancer.

PURPOSE: To overcome the therapeutic restrictions offered by hydrophobic quercetin (Qu), this study aims to synthesize MPEG-PLA encapsulated Qu nanoparticle and to evaluate their anticancer efficacy. MATERIALS AND METHODS: In vitro anticancer potential and apoptotic studies were done by cell cytotoxicity assay and flow cytometry, respectively. MPEG-PLA-Qu nanoparticles were evaluated for anticancer efficacy in vivo using xenograft mice model. TUNEL assay was performed to observe the frequency of apoptotic cells in vivo. RESULTS: The hydrodynamic particle size, polydispersity index, zeta potential and drug loading % of MPEG-PLA-Qu nanoparticle was 155.3 ± 3.2 nm, 0.2 ± 0.05, -3.14 mV and 5.3 ± 1.1%, respectively. Also, MPEG-PLA-Qu showed sustained drug release for 10 days. In vitro results showed that MPEG-PLA-Qu could efficiently induce apoptosis in triple negative breast cancer cell line (MDA-MB-231) with higher amount of quercetin in cell lysate treated with MPEG-PLA-Qu in comparison to free quercetin. In xenograft model for breast cancer, peritumorally injected MPEG-PLA-Qu significantly inhibited the tumor growth. Moreover, TUNEL assay showed more occurrence of apoptotic cells in MPEG-PLA-Qu treated tumors compared to free quercetin at similar dose. CONCLUSION: Our data suggest that MPEG-PLA-Qu nanoparticle can have a promising clinical potential for the treatment of breast cancer.

Nanoparticle based insulin delivery system: the next generation efficient therapy for Type 1 diabetes.

Diabetic cases have increased rapidly in recent years throughout the world. Currently, for type-1 diabetes mellitus (T1DM), multiple daily insulin (MDI) injections is the most popular treatment throughout the world. At this juncture, researchers are trying to develop different insulin delivery systems, especially through oral and pulmonary route using nanocarrier based delivery system. This next generation efficient therapy for T1DM may help to improve the quality of life of diabetic patients who routinely employ insulin by the subcutaneous route. In this paper, we have depicted various next generation nanocarrier based insulin delivery systems such as chitosan-insulin nanoparticles, PLGA-insulin nanoparticles, dextran-insulin nanoparticles, polyalkylcyanoacrylated-insulin nanoparticles and solid lipid-insulin nanoparticles. Modulation of these insulin nanocarriers may lead to successful oral or pulmonary insulin nanoformulations in future clinical settings. Therefore, applications and limitations of these nanoparticles in delivering insulin to the targeted site have been thoroughly discussed.

Lys1110 of TRPM2 is critical for channel activation.

TRPM2 (transient receptor potential melastatin 2) is a non-selective Ca2+-permeable cation channel activated by ADPR (adenosine diphosphoribose) and H2O2. It is widely expressed in mammalian cells and plays an important role in the regulation of various cell functions. However, the mechanisms of TRPM2 channel activation are not fully understood. Previously, we reported that TRPM2 channel activation is induced by high intracellular Cl- concentration. In the present study, we investigated the functional role of Lys1110 in the membrane-proximal C-terminal region by site-directed mutagenesis. Replacement of the positively charged amino acid lysine (Lys1110) with the neutrally charged amino acid asparagine (K1110N) or the negatively charged amino acid glutamic acid (K1110E) generated mutants that failed to induce an increase in free cytosolic calcium concentration ([Ca2+]i) not only by intracellular injection of Cl-, but also by H2O2 or ADPR. However, a mutant generated by replacing the lysine residue with a positively charged amino acid arginine (K1110R) displayed channel activity similar to wild-type TRPM2. Interestingly, in the K1107N/K1110N double-point mutant, the impaired function of the K1110N mutant in response to ADPR and H2O2, but not to Cl-, was recovered. There were no changes in protein expression, membrane trafficking and oligomerization of the mutant channels. The extent of [Ca2+]i increase by H2O2 in HEK (human embryonic kidney)-293 cells expressing TRPM2 mutants was well correlated with the degree of susceptibility to H2O2-induced cell death. These results display the crucial role of a positively charged amino acid residue at position 1110 for TRPM2 channel activity.

Effect of Wnt3a on keratinocytes utilizing in vitro and bioinformatics analysis.

Wingless-type (Wnt) signaling proteins participate in various cell developmental processes. A suppressive role of Wnt5a on keratinocyte growth has already been observed. However, the role of other Wnt proteins in proliferation and differentiation of keratinocytes remains unknown. Here, we investigated the effects of the Wnt ligand, Wnt3a, on proliferation and differentiation of keratinocytes. Keratinocytes from normal human skin were cultured and treated with recombinant Wnt3a alone or in combination with the inflammatory cytokine, tumor necrosis factor α (TNFα). Furthermore, using bioinformatics, we analyzed the biochemical parameters, molecular evolution, and protein-protein interaction network for the Wnt family. Application of recombinant Wnt3a showed an anti-proliferative effect on keratinocytes in a dose-dependent manner. After treatment with TNFα, Wnt3a still demonstrated an anti-proliferative effect on human keratinocytes. Exogenous treatment of Wnt3a was unable to alter mRNA expression of differentiation markers of keratinocytes, whereas an altered expression was observed in TNFα-stimulated keratinocytes. In silico phylogenetic, biochemical, and protein-protein interaction analysis showed several close relationships among the family members of the Wnt family. Moreover, a close phylogenetic and biochemical similarity was observed between Wnt3a and Wnt5a. Finally, we proposed a hypothetical mechanism to illustrate how the Wnt3a protein may inhibit the process of proliferation in keratinocytes, which would be useful for future researchers.

Biomolecule-mediated synthesis of selenium nanoparticles using dried Vitis vinifera (raisin) extract.

Biomolecule-mediated nanoparticle synthesis has recently the gained attention of researchers due to its ecofriendly and non-toxic nature. Metabolites from plant extracts represent a better alternative to chemical methods to fulfill the growing demand for non-hazardous nanoparticle synthesis routes. Selenium and its nanoparticles have an extensive range of applications. Thus, biofabrication of selenium nanoparticles can be potentially useful in various fields. This study reports a green approach to biosynthesize selenium nanoparticles (Se-np) using dried Vitis vinifera (raisin) extracts. The biosynthesized selenium nanoparticles were characterized using transmission electron microscope (TEM), dynamic light scattering (DLS), X-ray diffraction (XRD), energy dispersive X-ray (EDX) spectroscopy and Fourier transform infrared spectroscopy (FTIR). Transmission electron microscopic images revealed the spherical shape of biosynthesized selenium nanoparticles and a size range of 3-18 nm. Dynamic light scattering also confirmed the average particle size of 8.12 ± 2.5 nm with 0.212 PDI. The crystalline nature of selenium nanoparticles was confirmed by the X-ray diffraction study. Moreover, as inferred from the FTIR spectrum, the presence of highly stable lignin biopolymer on the surface of selenium nanoballs suggests a possible role as capping agent.

Protective effects of ferulic acid in amyloid precursor protein plus presenilin-1 transgenic mouse model of Alzheimer disease.

We previously reported the protective effects of long-term administration of ferulic acid against the in vivo toxicity of ß-amyloid peptide administered intracerebroventricularly in mice. In the present study, we investigated the effects of ferulic acid in transgenic amyloid precursor protein (APP)swe/presenilin 1 (PS1)dE9 (APP/PS1) mouse model of Alzheimer disease (AD). Chronic (for 6 months from the age of 6 to 12 months) oral administration of ferulic acid at a dose of 5.3 mg/kg/day significantly enhanced the performance in novel-object recognition task, and reduced amyloid deposition and interleukin-1 beta (IL-1ß) levels in the frontal cortex. These results suggest that ferulic acid at a certain dosage could be useful for prevention and treatment of AD.

Interplay between cartilage and subchondral bone contributing to pathogenesis of osteoarthritis.

Osteoarthritis (OA) is a common debilitating joint disorder, affecting large sections of the population with significant disability and impaired quality of life. During OA, functional units of joints comprising cartilage and subchondral bone undergo uncontrolled catabolic and anabolic remodeling processes to adapt to local biochemical and biological signals. Changes in cartilage and subchondral bone are not merely secondary manifestations of OA but are active components of the disease, contributing to its severity. Increased vascularization and formation of microcracks in joints during OA have suggested the facilitation of molecules from cartilage to bone and vice versa. Observations from recent studies support the view that both cartilage and subchondral bone can communicate with each other through regulation of signaling pathways for joint homeostasis under pathological conditions. In this review we have tried to summarize the current knowledge on the major signaling pathways that could control the cartilage-bone biochemical unit in joints and participate in intercellular communication between cartilage and subchondral bone during the process of OA. An understanding of molecular communication that regulates the functional behavior of chondrocytes and osteoblasts in both physiological and pathological conditions may lead to development of more effective strategies for treating OA patients.

Rspo 1 promotes osteoblast differentiation via Wnt signaling pathway.

R-spondin (Rspo)s proteins are a new group of Wnt/beta-catenin signaling agonists. These signaling molecules are known to be involved in the developmental stages of skeletal system. Recent studies in various murine osteoblast models have proposed that Rspo 1 may interact with Wnt signaling pathway to induce differentiation in osteoblasts. Though findings in murine osteoblasts implicate a synergestic role of Rspo 1 with Wnt signaling, still no study has addressed the similar role in more clinically applicable osteoblast models i.e., human cell lines or primary cells. Therefore, in the present study, we investigated the possible role of Rspo 1 during differentiation process of human in vitro osteoblast cell models like primary osteoblasts or human osteoprogenitor cell line hFOB 1.19 along with murine preosteoblast cell line MC3T3 E-1. Our results showed increase in Rspo 1 at transcript level during differentiating phase of human primary osteoblasts and human FOB 1.19 cells. We also found that Rspo 1 (100 ng/mL) acts additively with Wnt3a to activate Wnt signaling, as confirmed by luciferase activity after transfection of TOPFLASH construct to hFOB 1.19 cells. Similar additive role of Rspo 1 and Wnt3a was apparent in alkaline phosphatase (ALP) activity analysis of human primary cells. Moreover, a reduction in ALP activity was observed with knock-down of Rspo 1 by transfected shRNA in hFOB 1.19 cells. These results suggested the possibility of autocrine regulation by Rspo 1 on the osteogenic activities in human in vitro osteoblast models. Furthermore, these results were corroborated in MC3T3-E1, murine osteoblast cell model. Osteoblastic differentiation was induced by transfection of Rspo 1 which was confirmed by increased ALP staining and qRT-PCR analysis of osteogenic markers, such as Runx2 and osteocalcin. In conclusion, present study highlights the role of Rspo 1 in bone remodeling where it activates Wnt signaling to induce differentiation, as shown in human as well murine in vitro osteoblast cell models.

Lysophosphatidylcholine increases neutrophil bactericidal activity by enhancement of azurophil granule-phagosome fusion via glycine.GlyR alpha 2/TRPM2/p38 MAPK signaling.

Neutrophils are the first-line defense against microbes. Enhancing the microbicidal activity of neutrophils could complement direct antimicrobial therapy for controlling intractable microbial infections. Previously, we reported that lysophosphatidylcholine (LPC), an endogenous lipid, enhances neutrophil bactericidal activity (Yan et al. 2004. Nat. Med. 10: 161-167). In this study we show that LPC enhancement of neutrophil bactericidal activity is dependent on glycine, and is mediated by translocation of intracellularly located glycine receptor (GlyR) alpha2 to the plasma membrane, and subsequent increase in azurophil granule-phagosome fusion/elastase release. LPC induced GlyRalpha2-mediated [Cl(-)](i) increase, leading to transient receptor potential melastatin (TRPM)2-mediated Ca(2+) influx. Studies using human embryonic kidney 293 cells heterologously expressing TRPM2 and neutrophils showed that TRPM2 channel activity is sensitive to [Cl(-)](i). Finally, LPC induced p38 MAPK phosphorylation in an extracellular calcium/glycine dependent manner. SB203580, a p38 MAPK inhibitor, blocked LPC-induced enhancement in Lucifer yellow uptake, azurophil granule-phagosome fusion, and bactericidal activity. These results propose that enhancement of azurophil granule-phagosome fusion via GlyRalpha2/TRPM2/p38 MAPK signaling is a novel target for enhancement of neutrophil bactericidal activity.

Effect of protein concentrations of bovine serum albumin and γ-globulin on the frictional response of a cobalt-chromium femoral head.

The study aims to identify the concentration-dependent role of bovine serum albumin (BSA) and γ-globulin in the lubricating ability of a cobalt-chromium femoral head. The frictional coefficients of the cobalt-chromium femoral head decreased with increasing BSA concentrations from 10 to 40 mg/ml and showed statistical differences between any of the BSA concentration groups, except between the 30 and 40 mg/ml concentration groups. In γ-globulin, the frictional coefficients significantly decreased at concentrations of 2.5 and 5.0 mg/ml as compared with the PBS control group, but significant increases were observed at 7.5 and 12.5 mg/ml. These results suggest that the friction of the cobalt-chromium femoral head is dependent on the concentration of both BSA and γ-globulin. However, there is a maximum concentration for BSA to act as an effective boundary lubricant, while the lubricating ability of γ-globulin is most effective in the physiological concentration range within human synovial fluid.

Therapeutic effects of lysophosphatidylcholine in experimental sepsis.

Sepsis represents a major cause of death in intensive care units. Here we show that administration of lysophosphatidylcholine (LPC), an endogenous lysophospholipid, protected mice against lethality after cecal ligation and puncture (CLP) or intraperitoneal injection of Escherichia coli. In vivo treatment with LPC markedly enhanced clearance of intraperitoneal bacteria and blocked CLP-induced deactivation of neutrophils. In vitro, LPC increased bactericidal activity of neutrophils, but not macrophages, by enhancing H(2)O(2) production in neutrophils that ingested E. coli. Incubation with an antibody to the LPC receptor, G2A, inhibited LPC-induced protection from CLP lethality and inhibited the effects of LPC in neutrophils. G2A-specific antibody also blocked the inhibitory effects of LPC on certain actions of lipopolysaccharides (LPS), including lethality and the release of tumor necrosis factor-alpha (TNF-alpha) from neutrophils. These results suggest that LPC can effectively prevent and treat sepsis and microbial infections.

Sclerostin-Mediated Impaired Osteogenesis by Fibroblast-Like Synoviocytes in the Particle-Induced Osteolysis Model.

Engineered biomaterials are envisioned to replace, augment, or interact with living tissues for improving the functional deformities associated with end-stage joint pathologies. Unfortunately, wear debris from implant interfaces is the major factor leading to periprosthetic osteolysis. Fibroblast-like synoviocytes (FLSs) populate the intimal lining of the synovium and are in direct contact with wear debris. This study aimed to elucidate the effect of Ti particles as wear debris on human FLSs and the mechanism by which they might participate in the bone remodeling process during periprosthetic osteolysis. FLSs were isolated from synovial tissue from patients, and the condition medium (CM) was collected after treating FLSs with sterilized Ti particles. The effect of CM was analyzed for the induction of osteoclastogenesis or any effect on osteogenesis and signaling pathways. The results demonstrated that Ti particles could induce activation of the NFκB signaling pathway and induction of COX-2 and inflammatory cytokines in FLSs. The amount of Rankl in the conditioned medium collected from Ti particle-stimulated FLSs (Ti CM) showed the ability to stimulate osteoclast formation. The Ti CM also suppressed the osteogenic initial and terminal differentiation markers for osteoprogenitors, such as alkaline phosphate activity, matrix mineralization, collagen synthesis, and expression levels of Osterix, Runx2, collagen 1α, and bone sialoprotein. Inhibition of the WNT and BMP signaling pathways was observed in osteoprogenitors after the treatment with the Ti CM. In the presence of the Ti CM, exogenous stimulation by WNT and BMP signaling pathways failed to stimulate osteogenic activity in osteoprogenitors. Induced expression of sclerostin (SOST: an antagonist of WNT and BMP signaling) in Ti particle-treated FLSs and secretion of SOST in the Ti CM were detected. Neutralization of SOST in the Ti CM partially restored the suppressed WNT and BMP signaling activity as well as the osteogenic activity in osteoprogenitors. Our results reveal that wear debris-stimulated FLSs might affect bone loss by not only stimulating osteoclastogenesis but also suppressing the bone-forming ability of osteoprogenitors. In the clinical setting, targeting FLSs for the secretion of antagonists like SOST might be a novel therapeutic approach for preventing bone loss during inflammatory osteolysis.

Differential Expression Patterns of Rspondin Family and Leucine-Rich Repeat-Containing G-Protein Coupled Receptors in Chondrocytes and Osteoblasts.

OBJECTIVE: Rspondins (RSPOs) are regarded as the significant modulators of WNT signaling pathway and they are expressed dynamically during developmental stages. Since in osteoarthritis (OA) both cartilage and subchondral bone suffer damages and WNT signaling pathway has a crucial role in their maintenance, the objective of the study was to analyze expression profile of RSPO family and its receptors [leucine-rich repeat-containing G-protein coupled receptors (LGRs)] in OA tissue samples as well as in differentiating chondrocytes and osteoblasts. MATERIALS AND METHODS: In this experimental study, human early and advanced stage of OA tissue samples were analyzed for the morphological changes of articular cartilage by hematoxylin and eosin (H and E) staining, safranin-O staining and lubricin immunostaining. RSPOs and LGRs expression were confirmed by immunohistochemistry. Human primary chondrocytes and human osteoblast cell line, SaOS-2, were cultured in differentiation medium till day 14 and they were analyzed in terms of expression of RSPOs, LGRs and specific marker for chondrogenesis and osteogenesis by western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR). RESULTS: Advanced stage OA tissue samples showed increased expression of RSPO1 and LGR6 in a region close to subchondral bone. While RSPO2 and LGR5 expression were seen overlapping in the deep region of articular cartilage. Differentiating chondrocytes demonstrated elevated expression of RSPO2 and LGR5 from day 7 to day 14, whereas, osteoblasts undergoing differentiation showed enhanced expression of RSPO1 and LGR6 from day 2 to day 14. Under tumor necrosis factor alpha (TNFα) stimulatory conditions, RSPO2 and RSPO1 recovered the suppressed expression of inflammatory, chondrogenic and osteogenic markers, respectively. A recovery in the stability of ß-catenin was also noticed in both cases. CONCLUSION: Spatial expression of RSPOs during progression of OA might be dynamically controlled by cartilage and subchondral bone. Interplay amid chondrocytes and osteoblasts, via RSPOs, might provide probable mechanisms for treating inflammatory pathogenic conditions like OA.