Differential effects of arachidonic acid and docosahexaenoic acid on cell biology and osteoprotegerin synthesis in osteoblast-like cells

The purpose of the study was to elucidate the mechanisms by which polyunsaturated fatty acids (PUFAs) prevent bone loss. MG-63 human osteoblasts and MC3T3-E1 murine osteoblasts were exposed to the n-6 PUFA arachidonic acid (AA) and the n-3 PUFA docosahexaenoic acid (DHA) as well as oestrogen (E2) and parathyroid hormone (PTH) and the effects thereof tested on a variety of biological parameters characteristic of osteoblasts. These parameters included prostaglandin E2 (PGE2) synthesis, proliferation, differentiation to mature mineralising osteoblasts as well as osteoprotegerin (OPG) and receptor activator of nuclear factor êB ligand (RANKL) secretion. Results showed that AA stimulates PGE2 production significantly in both cell lines. Stimulated PGE2 production by MC3T3-E1 cells however, was significantly higher, which might be attributed to auto-amplification by PGE2 itself in this cell line. Pre-incubation of the MG-63 cells with cyclo-oxygenase (COX)-blockers inhibited PGE2 production significantly, suggesting that both COX enzymes were involved in PGE2 synthesis. The number of functional osteoblasts is important for bone formation therefore in vitro osteoblastic cell proliferation was investigated. In contrast to the hormones E2 and PTH, both AA and DHA inhibited proliferation significantly. The AA-mediated anti-proliferative effect is possibly independent of PGE2 production, as PGE2 per se had little effect on proliferation. DHA inhibited proliferation of MG-63 cells more severely, which might be attributed to the osteosarcoma nature of the MG-63 cells. The anti-proliferative effect of these PUFAs might be attributed to modulation of cell cycle progression or anti-mitotic effects of PUFA peroxidation products. Morphological studies showed apoptotic cells after DHA exposure in MG-63 cells. There is a reciprocal relationship between reduced proliferation and the subsequent induction of cell differentiation in vitro. High basal levels of alkaline phosphatase (ALP) activity, a marker of the mature mineralising osteoblastic phenotype, were detected in MC3T3-E1 cells. Long-term exposure to AA inhibited ALP activity in these cells. This process might be PGE2-mediated. Exposure to PUFAs, however, did not compromise the ability of the MC3T3-E1 cells to differentiate to mature mineralising osteoblasts. In contrast with MC3T3-E1 cells, MG-63 cells demonstrated low basal ALP activity and were unable to differentiate to mature mineralising osteoblasts. In the absence of osteogenic-inducing supplements, PUFAs induced adipocyte-like features that might be due to the expression of high levels of PPARã in this cell line. Lipid-filled vacuoles were absent in the MC3T3-E1 cells suggesting that the MC3T3-E1 cell line may not express PPARã mRNA. The study furthermore demonstrated that PUFAs are able to modulate OPG and RANKL secretion in osteoblasts. AA inhibited OPG secretion dose-dependently in both cell lines, this could be PGE2-mediated. AA dose-dependently stimulated soluble RANKL (sRANKL) secretion in MC3T3-E1 cells thereby affecting the OPG/RANKL ratio in a negative way, supporting various reports that AA and PGE2 do cause bone resorption. No sRANKL could be detected after exposing the MC3T3-E1 cells to DHA suggesting that DHA could be protective to bone. In conclusion, contrary to in vivo evidence, this in vitro study could not indisputably demonstrate protective effects of PUFAs on the osteoblastic cell lines tested.Thesis (PhD (Physiology))--University of Pretoria, 2006.Physiologyunrestricte

'n Kritiese evaluering na die effektiwiteit van bestuurstrategieë vir ekstensiewe wolskaapboerdery in die groter Burgersdorp omgewing

The main objectives of this study can be divided into three categories. In the first place it refers to the determination of production- and reproduction norms for the different main production areas in the greater Burgersdorp area, to promote production. In the second place it refers to the determination of the different management styles of sheep farmers in the greater Burgersdorp area. In the third place it refers to the calculation of gross margins for sheep farming in the greater Burgersdorp area, to establish the sustainable economic position. The survey area (the greater Burgersdorp area) includes the magisterial districts of Burgersdorp, Molteno, Steynsburg and Venterstad. The four magisterial districts were divided into two main production areas namely Stormberg Highveld and Burgersdorp Lowveld. Stormberg Highveld includes Molteno and the high-lying area (highveld) of Burgersdorp. Burgersdorp Lowveld comprises Steynsburg, Venterstad and the low-lying area (lowveld) of Burgersdorp. The management strategies of sheep farmers in the survey area were determined by means of a questionnaire and statistically analysed. The economic analyses of the sheep industry (wool and meat) were performed with the Geyer computer model. In this study it showed that the Dorper farmers obtained the highest gross margin per sheep. The lowest gross margin per sheep resulted from the Vleismerino farmers. Although the composition of the gross production value and the directly allocatable variable costs compare favourably, each main production area has unique management strategies for the different sheep breeds. The different management strategies can serve as the norm for extensive sheep farming, applicable to the specific main production area

Geraniol attenuates osteoclast differentiation by suppressingNF-kB activity and expression of osteoclastogenic genes

Osteoporotic patients have lower bone mass due to increased bone resorption by osteoclasts. The aim of this study was to investigate the cytotoxic and antiosteoclastogenic effects of geraniol, a natural monoterpene on human CD14+ monocytes (ex vivo) and murine RAW264.7 macrophages (in vitro) using alamar blue and tartrate resistant acid phosphatase staining respectively. The anti-osteoclastogenic activity of geraniol was further explored by analyzing its effects on actin ring formation and bone resorptive function of osteoclasts. Geraniol significantly (p < 0.001) inhibited osteoclast formation in CD14+ monocytes and RAW264.7 macrophages without cytotoxicity. Moreover, reduced osteoclastogenesis in these cells led to an arrest in actin ring formation and diminished bone resorption. Analysis of underlying molecular mechanisms revealed that geraniol alleviated NF-kB activity, an indispensable upstream modulator of osteoclast formation. Furthermore, expression of key osteoclastogenic genes such as dendritic cell-specific transmembrane protein (DC-STAMP) involved in cell-cell fusion and nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1), a master transcription factor essential for osteoclast differentiation was downregulated by geraniol. These observations indicate that inhibition of osteoclast differentiation is presumably one of the pharmacological properties of geraniol.Grants from the University of Pretoria Vice Chancellor’s Postdoctoral Research Fellowship; RESCOM, University of Pretoria and the University of Pretoria’s Strategic Institutional Research Theme in Food, Nutrition and Well-being.http://link.springer.com/journal/442017-12-31hb2016Food ScienceHuman NutritionPhysiolog

In vitro effects of arachidonic acid and prostaglandin E2 on proliferation, osteoclast formation and bone resoption in RAW 264.7 murine macrophages

This study investigated the effects of arachidonic acid and prostaglandin E2 on osteoclast formation and bone resorption in RAW 264.7 murine pre-osteoclasts. Data obtained suggests an inhibitory effect of these compounds on osteoclastogenesis and bone resorption in the cell line tested.Die Mediese Navorsingsraad en die Navorsingsontwikkelingsprogram (Universiteit van Pretoria)http://www.satnt.ac.z

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.S1 Fig. Effects of AA and DHA on cell viability. CD14+ monocytes were treated with indicated concentrations of AA and DHA for 48 h and cell viability was measured by alamar blue assay. The results are representative of two independent experiments conducted in triplicate and expressed as percentage cell viability relative to the control.The South African Medical Research Council (MRC), the University of Pretoria School of Medicine Research Committee (RESCOM) and the University of Pretoria Postgraduate Study Abroad Programme.http://www.plosone.orgam201

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

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.The Vice Chancellor’s Postdoctoral Research Fellowship and Institute for Food, Nutrition and Well-being, University of Pretoria.http://informahealthcare.com/journal/cts2016-06-30hb201

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, we investigated the effects of piperine, an alkaloid present in Piper nigrum L. on osteoclast formation. Piperine inhibited tartrate-resistant acid phosphatase (TRAP)-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 (MAPK) pathway activation, while the extracellular-signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) 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 suggest that piperine inhibits osteoclast differentiation by suppressing the p38/NFATc1/c-Fos signaling axis.RESCOM, University of Pretoria, the University of Pretoria Vice Chancellor’s Postdoctoral Research Fellowship, the University of Pretoria’s Strategic Institutional Research Theme in Food, Nutrition and Well-being and the Struwig-Germeshuysen Research Trust, South Africa.http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1872-80812016-11-30hb201

Ellagic acid inhibits RANKL-induced osteoclast differentiation by suppressing the p38 MAP kinase pathway

Bone undergoes continuous remodeling by a coupled action between osteoblasts and osteoclasts. During osteoporosis, osteoclast activity is often elevated leading to increased bone destruction. Hence, osteoclasts are deemed as potential therapeutic targets to alleviate bone loss. Ellagic acid (EA) is a polyphenol reported to possess anticancer, antioxidant and anti-inflammatory properties. However, its effects on osteoclast formation and function have not yet been examined. Here, we explored the effects of EA on RANKL-induced osteoclast differentiation in RAW264.7 murine macrophages (in vitro) and human CD14+monocytes (ex vivo). EA dose-dependently attenuated RANKL-induced TRAP+ osteoclast formation in osteoclast progenitors with maximal inhibition seen at 1 µM concentration without cytotoxicity. Moreover, owing to perturbed osteoclastogenesis, EA disrupted actin ring formation and bone resorptive function of osteoclasts. Analysis of the underlying molecular mechanisms revealed that EA suppressed the phosphorylation and activation of the p38 MAP kinase pathway which subsequently impaired the RANKL-induced differentiation of osteoclast progenitors. Taken together, these novel results indicate that EA alleviates osteoclastogenesis by suppressing the p38 signaling pathway downstream of RANKL and exerts inhibitory effects on bone resorption and actin ring formation.RESCOM, University of Pretoria; the University of Pretoria’s Strategic Institutional Research Theme in Food, Nutrition and Well-being; and in part by the University of Pretoria Vice Chancellor’s Postdoctoral Research Fellowship.http://link.springer.com/journal/122722018-01-31hb2017Human NutritionPhysiolog

Palmitoleic acid inhibits RANKL-induced osteoclastogenesis and bone resorption by suppressing NF- B and MAPK signalling pathways

Osteoclasts are large,multinucleated cells that are responsible for the breakdown or resorption of bone during bone remodelling. Studies have shown that certain fatty acids (FAs) can increase bone formation, reduce bone loss, and influence total bone mass. Palmitoleic acid (PLA) is a 16-carbon, monounsaturated FA that has shown anti-inflammatory properties similar to other FAs. The effects of PLA in bone remain unexplored. Here we investigated the effects of PLA on receptor activator of nuclear factor kappa B (NF- B) ligand (RANKL)-induced osteoclast formation and bone resorption in RAW264.7 murine macrophages. PLA decreased the number of large, multinucleated tartrate resistant acid phosphatase (TRAP) positive osteoclasts and furthermore, suppressed the osteolytic capability of these osteoclasts. This was accompanied by a decrease in expression of resorption markers (Trap, matrix metalloproteinase 9 (Mmp9), cathepsin K (Ctsk)). PLA further decreased the expression of genes involved in the formation and function of osteoclasts. Additionally, PLA inhibited NF- B activity and the activation of mitogen activated protein kinases (MAPK), c-Jun N-terminal kinase (JNK) and extracellular signal–regulated kinase (ERK). Moreover, PLA induced apoptosis in mature osteoclasts. This study reveals that PLA inhibits RANKL-induced osteoclast formation in RAW264.7 murine macrophages through suppression of NF- B and MAPK signalling pathways. This may indicate that PLA has potential as a therapeutic for bone diseases characterized by excessive osteoclast formation.The South African Medical Research Council (SAMRC)—Grantholder M. Coetzee.http://www.mdpi.com/journal/nutrientsam2017Food SciencePhysiolog

Ferulic acid impairs osteoclast fusion and exacerbates survival of mature osteoclasts

Elevated bone loss induced by osteoclasts is a critical and most commonly observed pathological complication during osteolytic diseases such as osteoporosis. Hence, attenuation of osteoclast formation or function is a classical therapeutic approach to regulate bone loss. In this study, we found that ferulic acid (FA), a natural compound potently inhibited osteoclast formation in human CD14+ peripheral blood monocytes (PBMCs) ex vivo with an IC50 of 39 μM.Moreover, due to impaired differentiation of osteoclast progenitors, actin ring formation and bone resorption activity were also perturbed. Investigation of underlying molecular mechanisms revealed that FA inhibited the RANKL-induced expression of dendritic cell-specific transmembrane protein (DC-STAMP), a critical regulator of osteoclast fusion. In addition, expression of matrix metalloproteinase-9 (MMP-9) and cathepsin K (CTSK), the key osteoclast specific lysosomal proteases involved in bone matrix resorption were severely aggravated by FA. A significant reduction in mature osteoclast numbers was detected in the presence of FA accompanied by increased caspase-3 activity and DNA-fragmentation, a characteristic hallmark of apoptosis. Collectively, these results suggested that FA inhibited osteoclast fusion by suppressing the expression of DC-STAMP and induced apoptosis in mature osteoclasts by the caspase-3 pathway.Grants from RESCOM, University of Pretoria; the University of Pretoria’s Strategic Institutional Research Theme in Food, Nutrition and Well-being; and in part by the University of Pretoria Vice Chancellor’s Postdoctoral Research Fellowship.http://link.springer.com/journal/106162017-10-31hb2016Human NutritionPhysiolog