Tag Archives: in vitro

Inhibition of osteoclast differentiation and bone resorption by sauchinone.

Abstract

Inhibition of osteoclast differentiation and bone resorption by sauchinone.

Osteoclasts are bone-specific multinucleated cells generated by differentiation of monocyte/macrophage lineage precursors. Regulation of osteoclast differentiation is considered an effective therapeutic approach to the treatment of bone-lytic diseases. In this study, we investigated effects of sauchinone, a lignan from Saururus chinensis, on osteoclastogenesis induced by the differentiation factor RANKL (receptor activator of nuclear factor kappa B ligand). Sauchinone strongly inhibited the osteoclastogenesis from primary bone marrow-derived macrophages (BMMs). This effect was accompanied by a significant decrease in the level of carbonic anhydrase II, calcitonin receptor, MMP9, and TRAP, which are normally upregulated during osteoclast differentiation. For the induction of osteoclastogenesis-associated genes, RANKL activates multiple transcription factors through mechanisms involving mitogen-activated protein kinases (MAPK) and reactive oxygen species (ROS). Sauchinone greatly attenuated the activation of ERK and, less prominently, that of p38 MAPKs by RANKL. The RANKL-stimulated induction of c-Fos and NFATc1 transcription factors was also abrogated by sauchinone. In addition, the activation of AP-1, NFAT, and NF-kappaB transcription factors was alleviated in sauchinone-treated cells. Sauchinone also diminished the RANKL-stimulated increase of ROS production in BMMs. Consistent with the in vitro anti-osteoclastogenic effect, sauchinone inhibited bone destruction and osteoclast formation caused by lipopolysaccharide in an animal model. Taken together, our data demonstrate that sauchinone inhibits RANKL-induced osteoclastogenesis by reducing ROS generation, which attenuates MAPK and NF-kappaB activation, ultimately leading to the suppression of c-Fos and NFATc1 induction. Also the in vivo effect of sauchinone on bone erosion strengthens the potential usefulness of this compound for diseases involving bone resorption.

Han KY, Yang D, Chang EJ, Lee Y…
Biochem. Pharmacol. Sep 2007
PMID: 17662251

Saurolactam Inhibits Osteoclasts In Vitro

Abstract

Saurolactam inhibits osteoclast differentiation and stimulates apoptosis of mature osteoclasts.

The receptor activator of nuclear factor-kappaB ligand (RANKL) plays a critical role in the differentiation and bone resorptive activity of osteoclasts. Recently, the development of anti-resorptive agents from natural substances has become a subject of interest. Therefore, we evaluated the effects of 222 natural compounds on the RANKL-induced tartrate-resistance acid phosphatase (TRAP; a marker for osteoclast differentiation) activity and multinucleated osteoclast formation in RAW264.7 murine macrophage cells. We found that saurolactam was one of the compounds inhibiting the RANKL-induced osteoclastogenesis; it significantly inhibited the RANKL-induced TRAP activity and formation of multinucleated osteoclasts without any cytotoxicity. Interestingly, saurolactam prevented RANKL-induced activation of MAP kinases and NF-kappaB, and mRNA expression of osteoclast-related genes and transcription factors (c-Fos, Fra-2, and NFATc1). We also observed the inhibitory effect of saurolactam on the differentiation of mouse bone marrow-derived macrophages into osteoclasts. Furthermore, saurolactam inhibited the bone resorptive activity of mature osteoclasts with the induction of apoptotic signaling cascade and the inhibition of survival signaling pathways such as c-Src/PI3K/Akt, Ras/ERK, and JNK/c-Jun. In conclusion, although further studies are needed to determine the precise mechanism and biological efficacy of saurolactam in osteoclast-mediated bone disorders, our results demonstrate that saurolactam potentially inhibits osteoclast differentiation by preventing the activation of MAP kinases and transcription factors that consequently affect the regulation of genes required for osteoclastogenesis, and the bone resorptive activity of mature osteoclasts by inhibiting osteoclast survival-related signaling pathways and triggering the apoptotic signaling cascade.

Kim MH, Ryu SY, Choi JS, Min YK…
J. Cell. Physiol. Dec 2009
PMID: 19653230

CoQ10 Inhibits Osteoclasts and Enhances Osteoblasts In Vitro

Abstract

Coenzyme q10 regulates osteoclast and osteoblast differentiation.

Coenzyme Q10 (CoQ10), a powerful antioxidant, is a key component in mitochondrial bioenergy transfer, generating energy in the form of ATP. Many studies suggest that antioxidants act as inhibitors of osteoclastogenesis and we also have previously demonstrated the inhibitory effect of CoQ10 on osteoclast differentiation. Despite the significance of this effect, the molecular mechanism when CoQ10 is present at high concentrations in bone remodeling still remains to be elucidated. In this study, we investigated the inhibitory effect of CoQ10 on osteoclastogenesis and its impact on osteoblastogenesis at concentrations ranging from 10 to 100 μM. We found that nontoxic CoQ10 markedly attenuated the formation of receptor activator of nuclear factor κB ligand (RANKL)-induced tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in both bone-marrow-derived monocytes (BMMs) and RAW 264.7 cells. Osteoclastogenesis with CoQ10 was significantly suppressed the gene expression of NFATc1, TRAP, and osteoclast-associated immunoglobulin-like receptor, which are genetic markers of osteoclast differentiation and scavenged intracellular reactive oxygen species, an osteoclast precursor, in a dose-dependent manner. Furthermore, CoQ10 strongly suppressed H2 O2 -induced IκBα, p38 signaling pathways for osteoclastogenesis. In bone formation study, CoQ10 acted to enhance the induction of osteoblastogenic biomarkers including alkaline phosphatase, type 1 collagen, bone sialoprotein, osteoblast-specific transcription factor Osterix, and Runt-related transcription factor 2 and, also promoted matrix mineralization by enhancing bone nodule formation in a dose-dependent manner. Together, CoQ10 acts as an inhibitor of RANKL-induced osteoclast differentiation and an enhancer of bone-forming osteoblast differentiation. These findings highlight the potential therapeutic applications of CoQ10 for the treatment of bone disease.

Moon HJ, Ko WK, Jung MS, Kim JH…
J. Food Sci. May 2013
PMID: 23582186

CoQ10, Selenite, and Curcumin, Inhibit Bone Resorption via Antioxidation

Abstract

Antioxidants, like coenzyme Q10, selenite, and curcumin, inhibited osteoclast differentiation by suppressing reactive oxygen species generation.

Coenzyme Q10 (CoQ10), selenium, and curcumin are known to be powerful antioxidants. Osteoclasts are capable of resorbing mineralized bone and excessive bone resorption by osteoclasts causes bone loss-related diseases. During osteoclast differentiation, the reactive oxygen species (ROS) acts as a secondary messenger on signal pathways. In this study, we investigated whether antioxidants can inhibit RANKL-induced osteoclastogenesis through suppression of ROS generation and compared the relative inhibitory activities of CoQ10, sodium selenite, and curcumin on osteoclast differentiation. We found that antioxidants markedly inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in both bone marrow-derived monocytes (BMMs) and RAW 264.7 cells. Antioxidants scavenged intracellular ROS generation within osteoclast precursors during RANKL-stimulated osteoclastogenesis. These also acted to significantly suppress the gene expression of NFATc1, TRAP, and osteoclast-associated immunoglobulin-like receptor (OSCAR), which are genetic markers of osteoclast differentiation in a dose-dependent manner. These antioxidants also suppressed ROS-induced IκBα signaling pathways for osteoclastogenesis. Specially, curcumin displayed the highest inhibitory effect on osteoclast differentiation when concentrations were held constant. Together, CoQ10, selenite, and curcumin act as inhibitors of RANKL-induced NFATc1 which is a downstream event of NF-κB signal pathway through suppression of ROS generation, thereby suggesting their potential usefulness for the treatment of bone disease associated with excessive bone resorption.

Moon HJ, Ko WK, Han SW, Kim DS…
Biochem. Biophys. Res. Commun. Feb 2012
PMID: 22252298

Glucosamine Reduces Anabolic and Catabolic Processes in Chondrocytes In Vitro

Abstract

Glucosamine reduces anabolic as well as catabolic processes in bovine chondrocytes cultured in alginate.

To investigate the working mechanism of glucosamine (GlcN) by studying the effect of different GlcN derivatives on bovine chondrocytes in alginate beads under anabolic and catabolic culture conditions.
Bovine chondrocytes seeded in alginate beads were treated with different concentrations of glucosamine-sulfate (GlcN-S), glucosamine-hydrochloride (GlcN-HCl) or N-acetyl-glucosamine (GlcN-Ac). Culture conditions were anabolic, 3 day pre-culture followed by 14 days’ treatment; catabolic, extracellular matrix (ECM) breakdown induced by 10ng/ml interleukin-1beta (IL-1beta); or a situation with balance between ECM breakdown and synthesis, 24 days’ pre-culture followed by 14 days’ treatment. The outcome measurements were total glycosaminoglycan (GAG) and DNA content per bead.
In the situation with balance between ECM breakdown and synthesis, GlcN-Ac had a small stimulatory effect on total GAG content. GlcN-S and GlcN-HCl had no effect. Under anabolic condition 5mM GlcN-S and GlcN-HCl significantly reduced total GAG content. GlcN-Ac did not show this effect. IL-1beta induced catabolic effects were prevented by adding 5mM GlcN-HCl. Interference of GlcN with glucose (Gluc) was demonstrated by adding extra Gluc to the medium in the anabolic culture conditions. Increasing extracellular Gluc concentrations diminished the effect of GlcN.
GlcN-S and GlcN-HCl, but not GlcN-Ac, reduce anabolic and catabolic processes. For anabolic processes this was demonstrated by decreased ECM synthesis, for catabolic processes by protection against IL-1beta mediated ECM breakdown. This might be due to interference of GlcN with Gluc utilization. We suggest that the claimed structure modifying effects of GlcN are more likely based on protection against ECM degradation than new ECM production.

Uitterlinden EJ, Jahr H, Koevoet JL, Bierma-Zeinstra SM…
Osteoarthr. Cartil. Nov 2007
PMID: 17543549

Glucosamine Detrimental to Intervertebral Disc In Vitro

Abstract

The effects of glucosamine sulfate on intervertebral disc annulus fibrosus cells in vitro.

Glucosamine has gained widespread use among patients, despite inconclusive efficacy data. Inconsistency in the clinical literature may be related to lack of understanding of the effects of glucosamine on the intervertebral disc, and therefore, improper patient selection.
The goal of our study was to investigate the effects of glucosamine on intervertebral disc cells in vitro under the physiological conditions of inflammation and mechanical loading.
Controlled in vitro laboratory setting.
Intervertebral disc cells isolated from the rabbit annulus fibrosus were exposed to glucosamine sulfate in the presence and absence of interleukin-1β and tensile strain. Outcome measures included gene expression, measurement of total glycosaminoglycans, new proteoglycan synthesis, prostaglandin E2 production, and matrix metalloproteinase activity. The study was funded by NIH/NCCAM, and the authors have no conflicts of interest.
Under conditions of inflammatory stimulation alone, glucosamine demonstrated a dose-dependent effect in decreasing inflammatory and catabolic mediators and increasing anabolic genes. However, under conditions of mechanical stimulation, although inflammatory gene expression was decreased, PGE2 was not. In addition, matrix metalloproteinase-3 gene expression was increased and aggrecan expression decreased, both of which would have a detrimental effect on matrix homeostasis. Consistent with this, measurement of total glycosaminoglycans and new proteoglycan synthesis demonstrated detrimental effects of glucosamine under all conditions tested.
These results may in part help to explain the conflicting reports of efficacy, as there is biological plausibility for a therapeutic effect under conditions of predominate inflammation but not under conditions where mechanical loading is present or in which matrix synthesis is needed.

Sowa GA, Coelho JP, Jacobs LJ, Komperda K…
Spine J Dec 2013
PMID: 24361347

Glycation Suppresses Bone Formation

Abstract

Advanced glycation end products suppress osteoblastic differentiation of stromal cells by activating endoplasmic reticulum stress.

Advanced glycation end products (AGEs) are involved in bone quality deterioration in diabetes mellitus. We previously showed that AGE2 or AGE3 inhibited osteoblastic differentiation and mineralization of mouse stromal ST2 cells, and also induced apoptosis and decreased cell growth. Although quality management for synthesized proteins in endoplasmic reticulum (ER) is crucial for the maturation of osteoblasts, the effects of AGEs on ER stress in osteoblast lineage are unknown. We thus examined roles of ER stress in AGE2- or AGE3-induced suppression of osteoblastogenesis of ST2 cells. An ER stress inducer, thapsigargin (TG), induced osteoblastic differentiation of ST2 cells by increasing the levels of Osterix, type 1 collagen (Col1), alkaline phosphatase (ALP) and osteocalcin (OCN) mRNA. AGE2 or AGE3 suppressed the levels of ER stress sensors such as IRE1α, ATF6 and OASIS, while they increased the levels of PERK and its downstream molecules, ATF4. A reduction in PERK level by siRNA did not affect the AGEs-induced suppression of the levels of Osterix, Col1 and OCN mRNA. In conclusion, AGEs inhibited the osteoblastic differentiation of stromal cells by suppressing ER stress sensors and accumulating abnormal proteins in the cells. This process might accelerate AGEs-induced suppression of bone formation found in diabetes mellitus.

Tanaka K, Yamaguchi T, Kaji H, Kanazawa I…
Biochem. Biophys. Res. Commun. Aug 2013
PMID: 23933252

Arginine and Lysine Have Positive Effect on Osteoblasts In Vitro

Abstract

Human osteopenic bone-derived osteoblasts: essential amino acids treatment effects.

The development of in vitro cell culture methods has made it possible to study bone cell metabolism and growth and obtain a deeper insight into the pathophysiology of common orthopedic diseases such as osteoporosis. After analyzing the effect of two essential amino acids, L-arginine (Arg) and L-lysine (Lys), in previous in vitro and in vivo studies, the present authors investigated the administration of Arg and Lys in osteoblasts derived from human osteopenic bone. After isolation, osteoblasts were cultured in DMEM supplemented with either Arg (0.625 mg/ml/day, Arg Group) or Lys (0.587 mg/ml/day, Lys Group), or both of them (Arg-Lys Group), whereas the Control Group was sham-treated. After 7 days the following parameters were tested in all groups: MTT proliferation test, Alkaline Phosphatase (ALP), Nitric Oxide (NO), Calcium (Ca), Phosphorus (P), Osteocalcin (OC), C-Terminal Procollagen type I (PICP), Interleukin-6 (IL-6), Transforming Growth Factor-beta 1 (TGF-beta 1), Platelet Derived Growth Factor (PDGF) and Insulin-Like Growth Factor-I (IGF-I). Results were compared with those obtained from human healthy bone to verify the effect of the amino acids on osteoblasts derived from pathological tissue. In addition, a comparison was also made with the results obtained from rat osteopenic bone to assess reliability of the in vitro model. The current results support previous findings and indicate that Arg and Lys stimulation has a positive effect on osteoblast proliferation, activation and differentiation. Therefore, administration of these amino acids may be useful in clinical treatment and prevention of osteoporosis.

Torricelli P, Fini M, Giavaresi G, Giardino R
Artif Cells Blood Substit Immobil Biotechnol Feb 2003
PMID: 12602815

Melatonin Induces Bone Sialoprotein In Vitro

Abstract

Melatonin regulates human bone sialoprotein gene transcription.

Melatonin is produced by the pineal gland and regulates various physiological processes including osteoblast differentiation and bone formation. Bone sialoprotein (BSP) is a mineralized connective tissue-specific protein expressed in the early stage of cementum and bone mineralization. To elucidate the effects of melatonin on human BSP gene expression, we utilized human Saos2 osteoblast-like cells. Melatonin (100 nM) increased the level of BSP mRNA at 3 h, and the level became maximal at 12 and 24 h. We then investigated the melatonin-induced transcriptional activity of luciferase constructs (between -84LUC and -868LUC) including different lengths of the human BSP gene promoter transfected into Saos2 cells. The effects of melatonin abrogated in constructs included 2-bp mutations in the two cAMP response elements (CRE1 and CRE2). The effects of melatonin were suppressed by protein kinase A, tyrosine kinase, ERK1/2 and phosphatidylinositol 3-kinase inhibitors. Gel mobility shift assays showed that melatonin increased the binding of nuclear proteins to CRE1 and CRE2, and antibodies against CRE binding protein 1 (CREB1), phospho-CREB1, c-Fos, c-Jun, JunD and Fra2 disrupted CRE1 and CRE2 protein complex formation. These data indicate that melatonin induces BSP transcription via the CRE1 and CRE2 elements in the human BSP gene promoter.

Matsumura H, Ogata Y
J Oral Sci 2014
PMID: 24739710 | Free Full Text

Chondroitin from Deer Antler may be Osteogenetic

Abstract

Characterization of chondroitin sulfate from deer tip antler and osteogenic properties.

Deer antler is a highly regenerative tissue that involves cellular differentiation, osteogenesis and ossification processes. Chondroitin sulfate is the major glycosaminoglycan contained in antler connective tissue and has been isolated from cartilaginous antler by 4 M GuHCl extraction, gradient ultracentrifugation and chromatography techniques. We examined the disaccharide composition by 2-AB labeling and anion exchange HPLC analysis of the three resultant fractions (high, medium and low density fractions). The high density fraction consists of A-unit and D-unit disaccharide in the ratio of 1:1, whereas, the CS disaccharide composition ratio of A- unit:C-unit:D-Unit:E-unit contained in medium and low density fractions are 3:4:3:1 and 2:2:2:1, respectively. The only intact CS oligosaccharides of the medium density fraction upregulated gene expression of bone-specific proteins of a human osteoblastic cell line (hFOB1.19). Thus, CS oligosaccharides from cartilaginous deer antler, with their oversulfated chondroitin sulfate composition, demonstrated the physiological properties and may be good candidates for osteogenetic agents in humans.

Pothacharoen P, Kodchakorn K, Kongtawelert P
Glycoconj. J. Oct 2011
PMID: 21894464