Impaired 1,25-dihydroxyvitamin D3 action underlies enthesopathy development in the Hyp mouse model of X-linked hypophosphatemia.

X-linked hypophosphatemia (XLH) is characterized by high serum fibroblast growth factor 23 (FGF23) levels, resulting in impaired 1,25-dihydroxyvitamin D3 (1,25D) production. Adults with XLH develop a painful mineralization of the tendon-bone attachment site (enthesis), called enthesopathy. Treatment of mice with XLH (Hyp) with 1,25D or an anti-FGF23 Ab, both of which increase 1,25D signaling, prevents enthesopathy. Therefore, we undertook studies to determine a role for impaired 1,25D action in enthesopathy development. Entheses from mice lacking vitamin D 1α-hydroxylase (Cyp27b1) (C-/-) had a similar enthesopathy to Hyp mice, whereas deletion of Fgf23 in Hyp mice prevented enthesopathy, and deletion of both Cyp27b1 and Fgf23 in mice resulted in enthesopathy, demonstrating that the impaired 1,25D action due to high FGF23 levels underlies XLH enthesopathy development. Like Hyp mice, enthesopathy in C-/- mice was observed by P14 and was prevented, but not reversed, with 1,25D therapy. Deletion of the vitamin D receptor in scleraxis-expressing cells resulted in enthesopathy, indicating that 1,25D acted directly on enthesis cells to regulate enthesopathy development. These results show that 1,25D signaling was necessary for normal postnatal enthesis maturation and played a role in XLH enthesopathy development. Optimizing 1,25D replacement in pediatric patients with XLH is necessary to prevent enthesopathy.

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.

Impaired 1,25 dihydroxyvitamin D3 action and hypophosphatemia underlie the altered lacuno-canalicular remodeling observed in the Hyp mouse model of XLH.

Osteocytes remodel the perilacunar matrix and canaliculi. X-linked hypophosphatemia (XLH) is characterized by elevated serum levels of fibroblast growth factor 23 (FGF23), leading to decreased 1,25 dihydroxyvitamin D3 (1,25D) production and hypophosphatemia. Bones from mice with XLH (Hyp) have enlarged osteocyte lacunae, enhanced osteocyte expression of genes of bone remodeling, and impaired canalicular structure. The altered lacuno-canalicular (LCN) phenotype is improved with 1,25D or anti-FGF23 antibody treatment, pointing to roles for 1,25D and/or phosphate in regulating this process. To address whether impaired 1,25D action results in LCN alterations, the LCN phenotype was characterized in mice lacking the vitamin D receptor (VDR) in osteocytes (VDRf/f;DMP1Cre+). Mice lacking the sodium phosphate transporter NPT2a (NPT2aKO) have hypophosphatemia and high serum 1,25D levels, therefore the LCN phenotype was characterized in these mice to determine if increased 1,25D compensates for hypophosphatemia in regulating LCN remodeling. Unlike Hyp mice, neither VDRf/f;DMP1Cre+ nor NPT2aKO mice have dramatic alterations in cortical microarchitecture, allowing for dissecting 1,25D and phosphate specific effects on LCN remodeling in tibial cortices. Histomorphometric analyses demonstrate that, like Hyp mice, tibiae and calvariae in VDRf/f;DMP1Cre+ and NPT2aKO mice have enlarged osteocyte lacunae (tibiae: 0.15±0.02µm2(VDRf/f;DMP1Cre-) vs 0.19±0.02µm2(VDRf/f;DMP1Cre+), 0.12±0.02µm2(WT) vs 0.18±0.0µm2(NPT2aKO), calvariae: 0.09±0.02µm2(VDRf/f;DMP1Cre-) vs 0.11±0.02µm2(VDRf/f;DMP1Cre+), 0.08±0.02µm2(WT) vs 0.13±0.02µm2(NPT2aKO), p<0.05 all comparisons) and increased immunoreactivity of bone resorption marker Cathepsin K (Ctsk). The osteocyte enriched RNA isolated from tibiae in VDRf/f;DMP1Cre+ and NPT2aKO mice have enhanced expression of matrix resorption genes that are classically expressed by osteoclasts (Ctsk, Acp5, Atp6v0d2, Nhedc2). Treatment of Ocy454 osteocytes with 1,25D or phosphate inhibits the expression of these genes. Like Hyp mice, VDRf/f;DMP1Cre+ and NPT2aKO mice have impaired canalicular organization in tibia and calvaria. These studies demonstrate that hypophosphatemia and osteocyte-specific 1,25D actions regulate LCN remodeling. Impaired 1,25D action and low phosphate levels contribute to the abnormal LCN phenotype observed in XLH.

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.

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.

Fibroblast-Like-Synoviocytes Mediate Secretion of Pro-Inflammatory Cytokines via ERK and JNK MAPKs in Ti-Particle-Induced Osteolysis.

Biomaterials are designed to replace and augment living tissues in order to provide functional support to skeletal deformities. However, wear debris produced from the interfaces of metal implants initiates inflammatory bone loss, causing periprosthetic osteolysis. Lately, fibroblast-like synoviocytes (FLS) have been shown to play a role in wear-debris-induced osteolysis. Thus, here we have tried to understand the underlying mechanism of FLS involvement in wear-debris-induced osteolysis. Our results demonstrate that the effects of Ti particle (1:100 cell-to-Ti particle ratio) on FLS can induce Cox-2 expression and activate NFkB signaling. Moreover, the mRNA expression of pro-inflammatory cytokines such as IL-6, IL-8, IL-11, IL-1ß, and TNFα was found to be elevated. However, among these pro-inflammatory cytokines, the mRNA and protein levels of only IL-6, IL-1ß, and TNFα were found to be significantly higher. Ti particles activated extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) as an early response in FLS. Co-inhibition of ERK and JNK signaling pathways by their specific inhibitors (PD9805 and SP600125, respectively) resulted in the suppression of mRNA and protein levels of IL-6, IL-1ß, and TNFα in FLS. Taken together, targeting ERK and JNK MAPKs in FLS might provide a therapeutic option for reducing the secretion of bone-resorbing pro-inflammatory cytokines, thus preventing periprosthetic osteolysis.

Influence of single nucleotide polymorphisms (SNPs) in genetic susceptibility towards periprosthetic osteolysis.

Wear debris-induced inflammatory osteolysis remains a significant limiting factor for implant replacement surgeries. Hence, a comprehensive understanding of the complex network of cellular and molecular signals leading to these inflammatory responses is required. Both macrophages and monocytes have a critical role in the instigation of the inflammatory reaction to wear debris but differ in the extent to which they induce cytokine expression in patients. Lately, single nucleotide polymorphisms (SNPs) have been associated with genetic susceptibility among individual patients with implant failure. Studies have shown that SNPs in key pro-inflammatory cytokines and their receptors are associated with osteolytic susceptibility. Likewise, SNPs within several genes involved in the regulation of bone turnover have also been found to be associated with wear debris induced osteolysis. It is presumed that SNP variance might play a decisive role in the activation and signaling of macrophages, osteoblasts, chondrocytes, fibroblasts and other cells involved in inflammatory bone loss. Understanding the extent to which SNPs exist among genes that are responsible for inflammatory bone loss may provide potential targets for developing future therapeutic interventions. Herein, we attempt to summarize the various susceptible genes with possible SNP variance that could contribute to the severity of periprosthetic osteolysis in patients with implants.

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.

Anti-inflammatory effects of traditional mixed extract of medicinal herbs (MEMH) on monosodium urate crystal-induced gouty arthritis.

Korean oriental medicine prescription is widely used for the treatment of gouty diseases. In the present study, we investigated anti-inflammatory effects of modified Korean herbal formulation, mixed extract of medicinal herbs (MEMH), and its modulatory effects on inflammatory mediators associated with gouty arthritis. Both in vitro and in vivo studies were carried out to assess the anti-inflammatory efficacy of MEMH on monosodium urate (MSU) crystals-induced gouty inflammation. MSU crystals stimulated human chondrosarcoma cell line, SW1353, and human primary chondrocytes were treated with MEMH in vitro. The expression levels of pro-inflammatory mediators and metalloproteases were analyzed. The effect of MEMH on NFκB signaling pathway in SW1353 cells was examined. Effect of MEMH on the mRNA expression level of pro-inflammatory mediators and chemotactic factor from human monocytic cell line, THP-1, was also analyzed. The probable role of MEMH in the differentiation process of osteoblast like cells, SaOS-2, after MSU treatment was also observed. To investigate the effects of MEMH in vivo, MSU crystals-induced ankle arthritic model was established. Histopathological changes in affected joints and plasma levels of pro-inflammatory mediators (IL-1ß and TNFα) were recorded. MEMH inhibited NFκB signaling pathway and COX-2 protein expression in chondrocytes. MSU-induced mRNA expressions of pro-inflammatory mediators and chemotactic cytokines were suppressed by MEMH. In MSU crystals-induced ankle arthritic mouse model, administration of MEMH relieved inflammatory symptoms and decreased the plasma levels of IL-1ß and TNFα. The results indicated that MEMH can effectively inhibit the expression of inflammatory mediators in gouty arthritis, demonstrating its potential for treating gouty arthritis.

Micro-Environmental Signature of The Interactions between Druggable Target Protein, Dipeptidyl Peptidase-IV, and Anti-Diabetic Drugs.

OBJECTIVE: Druggability of a target protein depends on the interacting micro-environment between the target protein and drugs. Therefore, a precise knowledge of the interacting micro-environment between the target protein and drugs is requisite for drug discovery process. To understand such micro-environment, we performed in silico interaction analysis between a human target protein, Dipeptidyl Peptidase-IV (DPP-4), and three anti-diabetic drugs (saxagliptin, linagliptin and vildagliptin). MATERIALS AND METHODS: During the theoretical and bioinformatics analysis of micro-environmental properties, we performed drug-likeness study, protein active site predictions, docking analysis and residual interactions with the protein-drug interface. Micro-environmental landscape properties were evaluated through various parameters such as binding energy, intermolecular energy, electrostatic energy, van der Waals'+H-bond+desolvo energy (EVHD) and ligand efficiency (LE) using different in silico methods. For this study, we have used several servers and software, such as Molsoft prediction server, CASTp server, AutoDock software and LIGPLOT server. RESULTS: Through micro-environmental study, highest log P value was observed for linagliptin (1.07). Lowest binding energy was also observed for linagliptin with DPP-4 in the binding plot. We also identified the number of H-bonds and residues involved in the hydrophobic interactions between the DPP-4 and the anti-diabetic drugs. During interaction, two H-bonds and nine residues, two H-bonds and eleven residues as well as four H-bonds and nine residues were found between the saxagliptin, linagliptin as well as vildagliptin cases and DPP-4, respectively. CONCLUSION: Our in silico data obtained for drug-target interactions and micro-environmental signature demonstrates linagliptin as the most stable interacting drug among the tested anti-diabetic medicines.

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.

Suppression of osteogenic activity by regulation of WNT and BMP signaling during titanium particle induced osteolysis.

Periprosthetic osteolysis remains the leading obstacle for total joint replacements. Primarily, it was thought that aseptic loosening is mainly caused by macrophage mediated inflammatory process arising from production of wear debris. The role of osteoclasts and its sequential bone resorption ability has been extensively studied, but little is known about impaired osteogenesis during osteolysis. In the current study, we have tried to delineate the regulatory mechanism of osteogenic signals by Ti particles in osteoprogenitor cells as well its participatory role in wear debris induced osteolysis. Implantation of Ti particles on mice calvaria induced pro-inflammatory response, elevated expression of COX2 and reduced the expression of Osterix. Treatment of Ti particles to MC3T3 E-1 cells displayed decreased osteogenic activity including ALP activity, mineralization and mRNA levels several osteogenic genes. Moreover, the basal activity of WNT and BMP signaling pathways was suppressed in MC3T3 E-1 cells treated with Ti particles. As an early response to Ti particles, MC3T3 E-1 cells showed activation of ERK and JNK. Co-inhibition of ERK and JNK with their specific inhibitors resulted in partial recovery of WNT and BMP signaling activity as well as ALP activity and collagen synthesis. Finally, LiCl mediated activation of WNT signaling pathway demonstrated rescue of Ti particle facilitated suppression of Osterix expression in mice calvaria. Our results provide evidences that WNT signaling pathway is regulated by ERK, JNK, and BMP signaling pathway during wear debris induced inflammatory osteolysis and may be considered as suitable therapeutic targets for the treatment. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 912-926, 2017.

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.