Vishwa Deepak

Effects of ω3- and ω6-Polyunsaturated Fatty Acids on RANKL-Induced Osteoclast Differentiation of RAW264.7 Cells: A Comparative in Vitro Study

Polyunsaturated fatty acids (PUFAs) have been reported to have an anabolic effect on bone in vivo, but comparative studies to identify inhibitors of osteoclast formation amongst ω3- and ω6-PUFAs are still lacking. Here we assessed the effects of the ω3-PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and the ω6-PUFAs, arachidonic acid (AA) and γ-linolenic acid (GLA) on a RAW264.7 osteoclast differentiation model. The effects of PUFAs on RANKL-induced osteoclast formation were evaluated by counting tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells. PUFAs significantly inhibited RANKL-induced osteoclast formation in a dose-dependent manner with AA- and DHA-mediated inhibition being the strongest. Furthermore, RANKL-induced mRNA- and protein expression of the key osteoclastogenic genes cathepsin K and TRAP were inhibited by AA and more potently by DHA. Owing to the attenuated osteoclastogenesis by DHA and AA, actin ring formation and bone resorptive activity of these cells as evaluated on bone-mimetic plates were severely compromised. Hence, of the tested PUFAs, AA and DHA were found to be the most effective in inhibiting RANKL-induced osteoclast formation with the latter providing the strongest inhibitory effects. Collectively, the data indicates that these PUFAs may play an important role in regulating bone diseases characterized by excessive osteoclast activity.

Arachidonic Acid and Docosahexaenoic Acid Suppress Osteoclast Formation and Activity in Human CD14+Monocytes, In vitro

An unbalanced diet can have adverse effects on health. Long chain polyunsaturated fatty acids (LCPUFAs) have been the focus of research owing to their necessity of inclusion in a healthy diet. However, the effects of LCPUFAs on human osteoclast formation and function have not been explored before. A human CD14+ monocyte differentiation model was used to elucidate the effects of an ω-3 LCPUFA, docosahexaenoic acid (DHA), and an ω-6 LCPUFA, arachidonic acid (AA), on osteoclast formation and activity. CD14+ monocytes were isolated from peripheral blood of healthy donors and stimulated with macrophage colony stimulating factor and receptor activator of nuclear factor kappa-B ligand to generate osteoclasts. Data from this study revealed that both the LCPUFAs decreased osteoclast formation potential of CD14+ monocytes in a dose-dependent manner when treated at an early stage of differentiation. Moreover, when exposed at a late stage of osteoclast differentiation AA and DHA impaired the bone resorptive potential of mature osteoclasts without affecting osteoclast numbers. AA and DHA abrogated vitronectin receptor expression in differentiating as well as mature osteoclasts. In contrast, the degree of inhibition for calcitonin receptor expression varied between the LCPUFAs with only AA causing inhibition during osteoclast differentiation. Furthermore, AA and DHA down regulated the expression of key osteoclast-specific genes in differentiating as well as mature osteoclasts. This study demonstrates for the first time that LCPUFAs can modulate osteoclast formation and function in a human primary osteoclast cell line.

Inhibitory effects of eugenol on RANKL-induced osteoclast formation via attenuation of NF-κB and MAPK pathways

Abstract Bone loss diseases are often associated with increased receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation. Compounds that can attenuate RANKL-mediated osteoclast formation are of great biomedical interest. Eugenol, a phenolic constituent of clove oil possesses medicinal properties; however, its anti-osteoclastogenic potential is unexplored hitherto. Here, we found that eugenol dose-dependently inhibited the RANKL-induced multinucleated osteoclast formation and TRAP activity in RAW264.7 macrophages. The underlying molecular mechanisms included the attenuation of RANKL-mediated degradation of IκBα and subsequent activation of NF-κB pathway. Furthermore, increase in phosphorylation and activation of RANKL-induced mitogen-activated protein kinase pathways (MAPK) was perturbed by eugenol. RANKL-induced expression of osteoclast-specific marker genes such as TRAP, cathepsin K (CtsK) and matrix metalloproteinase-9 (MMP-9) was remarkably downregulated by eugenol. These findings provide the first line of evidence that eugenol mediated attenuation of RANKL-induced NF-κB and MAPK pathways could synergistically contribute to the inhibition of osteoclast formation. Eugenol could be developed as therapeutic agent against diseases with excessive osteoclast activity.

Piperine alleviates osteoclast formation through the p38/c-Fos/NFATc1 signaling axis

Increased bone fracture is one of the health risk factors in patients with bone loss related disorders such as osteoporosis and breast cancer metastasis to bone. Over activity of osteoclasts leads to uncoupling of bone remodeling favoring bone loss over bone formation. Receptor activator of nuclear factor-κβ ligand (RANKL) triggers the differentiation pathway leading to multinucleated osteoclast formation. Modulation of RANKL or its downstream signaling pathways involved in osteoclast formation is of significant interest in the development of anti-resorptive agents. In this study, the effects of piperine, an alkaloid present in Piper nigrum L. on osteoclast formation was investigated. Piperine inhibited tartrate-resistant acid phosphatase-positive multinucleated osteoclast formation in murine RAW264.7 macrophages and human CD14+ monocytes induced by RANKL and breast cancer cells. Piperine attenuated the p38-mitogen activated protein kinase pathway activation, while the extracellular-signal-regulated kinase, c-Jun N-terminal kinase, or NF-κβ pathways downstream of RANKL remained unaffected. Concomitantly, expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), the key transcription factors involved in osteoclastogenesis were remarkably inhibited by piperine. Furthermore, piperine disrupted the actin ring structure and bone resorption, a characteristic hallmark of osteoclasts. Collectively, these results suggested that piperine inhibited osteoclast differentiation by suppressing the p38/NFATc1/c-Fos signaling axis..

Carvacrol Inhibits Osteoclastogenesis and Negatively Regulates the Survival of Mature Osteoclasts

Bone is a dynamic tissue that undergoes continuous remodeling coupled with the action of osteoblasts and osteoclasts. Osteoclast activity is elevated during osteoporosis and periodontitis resulting in excessive loss of trabecular and alveolar bone. Osteoclasts are formed in an inflammatory response to cytokine production receptor activator of nuclear factor-kappaB (NF-κB) ligand (RANKL) and bacterial challenge lipopolysaccharide (LPS). Carvacrol, a monoterpenic phenol present in Origanum vulgare and Thymus vulgaris, is a natural compound with known medicinal properties. We investigated the effects of carvacrol on osteoclast formation induced by RANKL and LPS. Carvacrol suppressed RANKL-induced formation of tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells in RAW264.7 macrophages and human CD14(+) monocytes. Furthermore, carvacrol inhibited LPS-induced osteoclast formation in RAW264.7 macrophages. Investigation of the underlying molecular mechanisms revealed that carvacrol downregulated RANKL-induced NF-κB activation in a dose-dependent manner. Furthermore, the suppression of NF-κB activation correlated with inhibition of inhibitor of kappaB (IκB) kinase (IKK) activation and attenuation of inhibitor of NF-κB (IκBa) degradation. Carvacrol potentiated apoptosis in mature osteoclasts by caspase-3 activation and DNA fragmentation. Moreover, carvacrol did not affect the viability of proliferating MC3T3-E1 osteoblast-like cells. Collectively, these results demonstrate that carvacrol mitigates osteoclastogenesis by impairing the NF-κB pathway and induction of apoptosis in mature osteoclasts.

Commercial Honeybush (Cyclopia spp.) Tea Extract Inhibits Osteoclast Formation and Bone Resorption in RAW264.7 Murine Macrophages—An in vitro Study

Honeybush tea, a sweet tasting caffeine-free tea that is indigenous to South Africa, is rich in bioactive compounds that may have beneficial health effects. Bone remodeling is a physiological process that involves the synthesis of bone matrix by osteoblasts and resorption of bone by osteoclasts. When resorption exceeds formation, bone remodeling can be disrupted resulting in bone diseases such as osteoporosis. Osteoclasts are multinucleated cells derived from hematopoietic precursors of monocytic lineage. These precursors fuse and differentiate into mature osteoclasts in the presence of receptor activator of NF-kB ligand (RANKL), produced by osteoblasts. In this study, the in vitro effects of an aqueous extract of fermented honeybush tea were examined on osteoclast formation and bone resorption in RAW264.7 murine macrophages. We found that commercial honeybush tea extract inhibited osteoclast formation and TRAP activity which was accompanied by reduced bone resorption and disruption of characteristic cytoskeletal elements of mature osteoclasts without cytotoxicity. Furthermore, honeybush tea extract decreased expression of key osteoclast specific genes, matrix metalloproteinase-9 (MMP-9), tartrate resistant acid phosphatase (TRAP) and cathepsin K. This study demonstrates for the first time that honeybush tea may have potential anti-osteoclastogenic effects and therefore should be further explored for its beneficial effects on bone.

Constitutive L-Sox5 overexpression delays differentiation of ATDC5 cells into chondrocytes and correlates with reduced expression of differentiation markers

L-Sox5 is a member of sex-determining region Y-type high mobility group box (SOX) family of transcription factors. We assessed the effects of retroviral overexpression of L-Sox5 on chondrocyte differentiation using the clonal murine cell line ATDC5. We observed a temporal-restricted expression pattern of L-Sox5 in insulin-induced ATDC5 cells differentiating toward chondrocyte lineage. The protein expression levels of L-Sox5 showed a drastic decrease in contrast to unaltered mRNA levels during differentiation. L-Sox5 delayed the differentiation of ATDC5 cells as evidenced by Alcian blue staining for proteoglycan synthesis. The mRNA levels of chondrocyte and hypertrophic/osteoarthritic markers were markedly decreased or delayed in L-Sox5 overexpressing cells. L-Sox5 abrogated the promoter activity of Runx2. These results suggest that L-Sox5 protein expression may diminish along with the progress of chondrogenic differentiation. L-Sox5 may act as a negative regulator if expressed aberrantly at least in part by regulating the critical fate of chondrogenesis.

In silico design and bioevaluation of selective benzotriazepine BRD4 inhibitors with potent antiosteoclastogenic activity

The bromodomain (BRD) and extra‐terminal domain (BET) protein family bind to acetylated histones on lysine residues and act as epigenetic readers. Recently, the role of this protein family in bone loss has been gaining attention. Earlier studies have reported that benzotriazepine (Bzt) derivatives could be effective inhibitors of BET proteins. In this study, using in silico tools we designed three Bzt analogs (W49, W51, and W52). By docking, molecular simulations, and chemiluminescent Alpha Screen binding assay, we show that the studied analogs were selective at inhibiting BRD4 when compared to BRD2. Furthermore, we tested the effectiveness of these analogs on osteoclast formation and function. Among the examined analogs, Bzt‐W49 and Bzt‐W52 were found to be the most potent inhibitors of osteoclastogenesis without cytotoxicity in murine RAW264.7 osteoclast progenitors. Both the compounds also inhibited osteoclast formation without affecting cell viability in human CD14+ monocytes. Moreover, owing to attenuated osteoclastogenesis, actin ring formation and bone resorptive function of osteoclasts were severely perturbed. In conclusion, these results suggest that the novel BRD4‐selective Bzt analogs designed in this study could be explored further for developing therapeutics against bone loss diseases characterized by excessive osteoclast activity.

Effects of Soy Phytoestrogens and New Zealand Functional Foods on Bone Health

New Zealand is a rich source of food components that may have bioactivity on bone. Docosahexaenoic acid (DHA) from fish oil has been shown to maintain bone in ovariectomised (OVX) rats. Kiwifruit, a source of fibre and carotenoids, may also affect bone via a prebiotic as well as direct cell-based mechanisms. We aimed to 1) ascertain the effects of DHA on two cell models, including interactions with soy isoflavones; 2) and investigate the specific effects of carotenoids from kiwifruit as well as whole kiwifruit in cell-based and rodent models as well as in a human study. RAW 264.7 mouse monocytes or mouse bone marrow was used to generate osteoclasts (OC). Cells were exposed to the agents between 5 and 21 d and formation and activity of OC measured, including molecular markers. DHA inhibited OC formation in both cell models, including expression of cathepsin K, NFATc1 as well as actin ring formation. Combination with isoflavones enhanced these effects. In OVX rats and mice fed with kiwifruit for 8 wk, green kiwifruit reduced the rate of bone loss after OVX, and in mice it reduced C-telopeptide of Type 1 collagen (CTX) levels and RANKL expression while in menopausal women, green kiwifruit affected blood lipids and bone markers positively.

Rooibos tea extracts inhibit osteoclast formation and activity through the attenuation of NF-κB activity in RAW264.7 murine macrophages

Rooibos tea is a naturally sweet and aromatic tea that is native to the Western Cape province of South Africa. Rooibos is usually fermented to produce the traditional reddish brown colour and has been found to have numerous health benefits. These include beneficial effects on osteoblasts; however, its effects on osteoclast formation and activity are unknown. Osteoclasts are large, multinucleated cells responsible for bone resorption. Binding of RANKL to its receptor on osteoclast precursors triggers the NF-κB signalling pathway leading to the formation of osteoclasts. Certain bone destructive diseases, such as osteoporosis, are characterised by overactive osteoclasts. The inhibition of osteoclasts may offer a potential mode to prevent these diseases. The polyphenol contents of both fermented and unfermented tea extracts were similar although the radical scavenging activity of fermented rooibos tea was lower. Both tea extracts were not cytotoxic and inhibited osteoclast formation. Fermented rooibos tea extract caused a greater reduction in osteoclast resorption and the associated gene expression when compared with unfermented rooibos tea. Both tea extracts were shown to attenuate NF-κB activity. Fermented rooibos was found to have a more potent inhibitory effect on osteoclasts than unfermented rooibos extract and therefore may have a beneficial effect on bone health.