Growth on type I collagen promotes expression of the osteoblastic phenotype in human osteosarcoma MG-63 cells.

Using MG-63 cells as a model system capable of partial osteoblastic differentiation, we have examined the effect of growth on extracellular matrix. MG-63 cell matrix and purified type I collagen induced a morphological change characterized by long cytoplasmic processes reminiscent of those seen in osteocytes. Concurrent biochemical changes involving bone marker proteins included increased specific activity of cell-associated alkaline phosphatase and increased secretion of osteonectin (up to 2.5-fold for each protein); all changes occurred without alterations in the growth kinetics of the MG-63 cells. The increase in alkaline phosphatase activity was maximal on days 6-8 following seeding; increased osteonectin secretion was most prominent immediately following seeding; all changes decreased as cells reached confluence. Growing cells on type I collagen resulted in an increased induction of alkaline phosphatase activity by 1,25(OH)2D3 (with little change in the 1,25(OH)2D3 induction of osteonectin and osteocalcin secretion), and increased TGF-beta induction of alkaline phosphatase activity as well (both TGF-beta 1 and TGF-beta 2). Both the 1,25(OH)2D3 and TGF-beta effects appeared to be synergistic with growth on type I collagen. These studies support the hypothesis that bone extracellular matrix may play an important role in osteoblastic differentiation and phenotypic expression.     

Mechanical stimulation effects on functional end effectors in osteoblastic MG-63 cells

Receptor activator of Nf-kappaB ligand (RANKL) and osteoprotegerin (OPG) have been implicated in bone metabolism. Specifically, the balance of these factors in conjunction with receptor activator of Nf-kappaB (RANK) is believed to be key in determining the rate of osteoclastogenesis and the net outcome of bone formation/resorption. While it is well accepted that mechanical loading in vivo affects bone formation/resorption and that alterations in the responsiveness of bone cells to mechanical loading have been implicated in metabolic bone diseases, the effect of in vitro mechanical loading on osteoblastic production of OPG and RANKL has not been extensively studied. Thus, in the current study, we developed an in vitro model to load human osteoblasts and studied levels of OPG, RANKL, PGE(2) and macrophage colony stimulating factor (M-CSF). We hypothesized that stimulating osteoblastic cells would increase the release of soluble OPG relative to RANKL favoring a bone-forming (and resorption-inhibiting) event. To accomplish this, we developed a small-scale loading machine that imparts via bending, well-defined substrate deformation to bone cells cultured on artificial substrates. Following 2h of loading and a 1h incubation period, media was collected and levels of soluble OPG, RANKL, PGE(2) and M-CSF were quantified using ELISA and western blotting. We found that mechanical loading significantly increased soluble OPG levels relative to RANKL at this 3h time point. Levels of soluble and cellular RANKL detected were not significantly affected by mechanical stimulation. The relative shift in abundance of OPG over RANKL associated with applied mechanical stimulation suggests the soluble OPG:RANKL ratio may be important in load-induced coupling mechanisms of bone cells.     

Nobiletin promotes osteogenic differentiation of human osteoblastic cell line (MG-63) through activating the BMP-2/RUNX-2 signaling pathway

Nobiletin (NOB) is polymethoxy flavonoids, which plentifully there in Citrus depressa and they demonstrate numerous pharmacological effects. NOB has an anti-proliferative effect, attenuates ovalbumin-treated eosinophilic airway inflammation and Type II collagen treated arthritis. NOB noticeably inhibits bone resorption and renovates bone loss in mice model, but role of NOB in bone metabolism is unclear. Human bone is a important organ that sustains its homeostasis among bone resorpting osteoclasts and bone developing osteoblasts. The balances of among these two kind of cell outcomes are implicated in bone remodeling. The current study designed to explore possessions of NOB on differentiation and proliferation of MG-63 cells and contribution of morphogenetic protein signaling. Cell proliferation was analyzed by MTT, mineralization analysis by alizarin red staining and morphogenetic signaling protein by RT-PCR. No stimulus outcome of NOB on cell proliferation was found at days of 1, 3 and 7. Accumulation of calcium was augmented after that treatment of NOB. The mRNA expression of BMP-2, COL-I, ALP, OCN, RUNX2 and COL1A1 augmented markedly with NOB supplement. Hence, NOB can stimulate osteogenic differentiation of MG-63, almost certainly by promoting RUNX2 and BMP-2 signaling and this result might provide to its action on stimulation of osteoblast development, differentiation and augments of bone mass.     

Expression of extracellular calcium-sensing receptor in human osteoblastic MG-63 cell line

We have previously shown the expression of the extracellular calcium (Ca2+o)-sensing receptor (CaR) in osteoblast-like cell lines, and others have documented its expression in sections of murine, bovine, and rat bone. The existence of the CaR in osteoblasts remains controversial, however, since some studies have failed to document its expression in the same osteoblast-like cell lines. The goals of the present study were twofold. 1) We sought to determine whether the CaR is expressed in the human osteoblast-like cell line, MG-63, which has recently been reported by others not to express this receptor. 2) We investigated whether the CaR, if present in MG-63 cells, is functionally active, since most previous studies have not proven the role of the CaR in mediating known actions of Ca2+o on osteoblast-like cells. We used immunocytochemistry and Western blotting with the specific, affinity-purified anti-CaR antiserum 4637 as well as Northern blot analysis and RT-PCR using a riboprobe and PCR primers specific for the human CaR, respectively, to show readily detectable CaR protein and mRNA expression in MG-63 cells. Finally, we employed the patch-clamp technique to show that an elevation in Ca2+o as well as the specific, allosteric CaR activator NPS R-467 (0.5 microM), but not its less active stereoisomer NPS S-467 (0.5 microM), activate an outward K+ channel in MG-63 cells, strongly suggesting that the CaR in MG-63 cells is not only expressed but is functionally active.     

Induction of osteogenic differentiation of osteoblast-like cells MG-63 during cultivation on fibroin microcarriers

We have developed microcarriers made from silk fibroin. Microcarriers can be used as a substrate for cell cultivation and cell delivery during cell-based therapy and for the construction of bioengineered tissue. Fibroin microcarriers were mineralized, which led to the appearance of calcium phosphate crystals on their surface. The ability of mineralized and nonmineralized microcarriers to support osteogenic differentiation of the osteoblast-like cell line MG-63 was estimated by alkaline phosphatase activity, an early marker of bone formation. The experiment showed cells actively proliferating on the surface of both mineralized and nonmodified microcarriers. Culturing MG-63 on the surface of fibroin microcarriers resulted in an increase of alkaline phosphatase activity indicative of osteogenic differentiation of MG-63 cells in the absence of inductors. The level of alkaline phosphatase was higher when mineralized microcarriers were used. Alkaline phosphatase activity of MG-63 cells cultivated using traditional two-dimensional approaches were close to zero. As opposed to conventional monolayer culturing, microcarrier culture cells are in a three-dimensional environment that is closer to physiological conditions. This can have a significant impact on their morphology and functional properties. During this study, we also characterized mechanical properties of porous scaffolds used for microcarriers.     

Comparative effect of seeds of Rhynchosia volubilis and soybean on MG-63 human osteoblastic cell proliferation and estrogenicity

The seeds of Rhynchosia volubilis (SRV) (Leguminosae) and soybean have been used in oriental folk medicine to prevent postmenopausal osteoporosis. Their beneficial effects are caused by a high content of isoflavone, which function as partial agonists or antagonists of estrogen. To compare the estrogenic effects of SRV and soybean on the MG-63 osteoblastic cell proliferation, 70% methanol extracts of SRV or soybean were treated on MG-63 cells. Although biphasic over a concentration range of 0.001 mg/ml-0.1 mg/ml, both SRV and soybean extracts increased MG-63 cell proliferation. However SRV was more effective at increasing the cell proliferation that paralleled with the greater estrogenic effects as determined by estrogen receptor alpha (ERalpha) expression, an estrogenic response element (ERE)-luciferase activity and the selective expression of insulin-like growth factor-I (IGF-I). SRV-induced IGF-I expression resulted from increases in the mRNA levels. Despite the increased expression of ERbeta, ERE activity and IGF-I expression by soybean were lower than those by SRV. Furthermore, the comparable estrogenic effects between SRV and the combined treatment of genistein and daidzein standards at 0.5 x 10(-8) M, which is a concentration of these two isoflavones similar to that of SRV at 0.001 mg/ml, demonstrate that the greater estrogenicity of SRV for MG-63 cell proliferation is mediated by the synergism of low levels of isoflavones for the selective expression of IGF-I.     

Changes of nuclear matrix proteins following the differentiation of human osteosarcoma MG-63 cells

Human osteosarcoma MG-63 cells were induced into differentiation by 5 mmol/L hexamethylene bisacetamide （HMBA）. Their nuclear matrix proteins （NMPs） were selectively extracted and subjected to two-dimensional gel electrophoresis analysis. The results of protein patterns were analyzed by Melanie software. The spots of differentially expressed NMPs were excised and subjected to in situ digestion with trypsin. The maps of peptide mass fingerprinting were obtained by MALDI-TOF-MS analysis, and were submitted for NCBI database searches by Mascot tool. There were twelve spots changed remarkably during the differentiation induced by HMBA, nine of which were identified. The roles of the regulated proteins during the MG-63 differentiation were analyzed. This study suggests that the induced differentiation of cancer cells is accompanied by the changes of NMPs, and confirms the presence of some specific NMPs related to the cancer cell proliferation and differentiation. The changed NMPs are potential markers for cancer diagnosis or targets for cancer therapy.     

IL1RN promotes osteoblastic differentiation via interacting with ITGB3 in osteoporosis

The occurrence and progress of osteoporosis(OP)are partially caused by impaired osteoblast differentiation.Interleukin-I receptor antagonist(IL1RN)is an immune ...     

Tetrameric peptide purified from hydrolysates of biodiesel byproducts of Nannochloropsis oculata induces osteoblastic differentiation through MAPK and Smad pathway on MG-63 and D1 cells

Ongoing efforts to search for bioactive substances for bone diseases have led to the discovery of natural products with substantial bioactive properties. In this present study, an osteoblast activating-peptide was isolated from biodiesel by-products of microalgae, Nannochloropsis oculata. To utilize biodiesel by-products of N. oculata and evaluate their beneficial effects, enzymatic hydrolysis was carried out using commercial enzymes such as alcalase, flavourzyme, neutrase, trypsin (PTN?), protamex and alcalase hydrolysate exhibited the highest osteoblastic differentiation activity. Using consecutive purification by liquid chromatographic techniques, an osteoblast-differentiatory peptide was purified and identified to be a peptide (MPDW, 529.2 Da) by the tandem MS analysis. The results showed that purified peptide promotes osteoblast differentiation by increasing expression of several osteoblast phenotype markers such as alkaline phosphatase (ALP), osteocalcin, collagen type I, BMP-2, BMP2/4 and bone mineralization in both human osteoblastic cell (MG-63) and murine mesenchymal stem cell (D1). In addition, the purified peptide induced phosphorylation of MAPK and Smad pathway in both cells. These results suggest that peptide possesses positive effects on osteoblast differentiation and may provide possibility for treating bone diseases.     

Downregulation of PKD1 by shRNA results in defective osteogenic differentiation via cAMP/PKA pathway in human MG-63 cells

Mutations and/or deletions of Pkd1 in mouse models resulted in attenuation of osteoblast function and defective bone formation; however, the function of PKD1 in human osteoblast and bone remains uncertain. In the current study, we used lentivirus-mediated shRNA technology to stably knock down PKD1 in the human osteoblastic MG-63 cell line and to investigate the role of PKD1 on human osteoblast function and molecular mechanisms. We found that a 53% reduction of PKD1 by PKD1 shRNA in stable, transfected MG-63 cells resulted in increased cell proliferation and impaired osteoblastic differentiation as reflected by increased BrdU incorporation, decreased alkaline phosphatase activity, and calcium deposition and by decreased expression of RUNX2 and OSTERIX compared to control shRNA MG-63 cells. In addition, knockdown of PKD1 mRNA caused enhanced adipogenesis in stable PKD1 shRNA MG-63 cells as evidenced by elevated lipid accumulation and increased expression of adipocyte-related markers such as PPARγ and aP2. The stable PKD1 shRNA MG-63 cells exhibited lower basal intracellular calcium, which led to attenuated cytosolic calcium signaling in response to fluid flow shear stress, as well as increased intracellular cAMP messages in response to forskolin (10 μM) stimulation. Moreover, increased cell proliferation, inhibited osteoblastic differentiation, and osteogenic and adipogenic gene markers were significantly reversed in stable PKD1 shRNA MG-63 cells when treated with H89 (1 μM), an inhibitor of PKA. These findings suggest that downregulation of PKD1 in human MG-63 cells resulted in defective osteoblast function via intracellular calcium-cAMP/PKA signaling pathway.     

Induction of osteoblast differentiation indexes by PTHrP in MG-63 cells involves multiple signaling pathways

Parathyroid hormone (PTH)-related peptide (PTHrP) can modulate the proliferation and differentiation of a number of cell types including osteoblasts. PTHrP can activate a G protein-coupled PTH/PTHrP receptor, which can interface with several second-messenger systems. In the current study, we have examined the signaling pathways involved in stimulated type I collagen and alkaline phosphatase expression in the human osteoblast-derived osteosarcoma cells, MG-63. By use of Northern blotting and histochemical analysis, maximum induction of these two markers of osteoblast differentiation occurred after 8 h of treatment with 100 nM PTHrP-(1-34). Chemical inhibitors of adenylate cyclase (H-89) or of protein kinase C (chelerythrine chloride) each diminished PTHrP-mediated type I collagen and alkaline phosphatase stimulation in a dose-dependent manner. These effects of PTHrP could also be blocked by inhibiting the Ras-mitogen-activated protein kinase (MAPK) pathway with a Ras farnesylation inhibitor, B1086, or with a MAPK inhibitor, PD-98059. Transient transfection of MG-63 cells with a mutant form of Galpha, which can sequester betagamma-subunits, showed significant downregulation of PTHrP-stimulated type I collagen expression, as did inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) by wortmannin. Consequently, the betagamma-PI 3-kinase pathway may be involved in PTHrP stimulation of Ras. Collectively, these results demonstrate that, acting via its G protein-coupled receptor, PTHrP can induce indexes of osteoblast differentiation by utilizing multiple, perhaps parallel, signaling pathways.     

Inhibition of Rac1 promotes BMP-2-induced osteoblastic differentiation

Small G proteins of the Rho family are pivotal regulators of several signaling networks. The Ras homolog family (Rho) and one of its targets, Rho-associated protein kinase (ROCK), participate in a wide variety of biological processes, including bone formation. A previous study has demonstrated that the ROCK inhibitor Y-27632 enhanced bone formation induced by recombinant human bone morphogenetic protein-2 (BMP-2) in vivo and in vitro. However, the effect of other Rho family members, such as Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division cycle 42 (Cdc42), on bone formation remains unknown. In this study, we investigated whether Rac1 also participates in BMP-2-induced osteogenesis. Expression of a dominant-negative mutant of Rac1 enhanced BMP-2-induced osteoblastic differentiation in C2C12 cells, whereas a constitutively active mutant of Rac1 attenuated that effect. Knockdown of T-lymphoma invasion and metastasis 1 (Tiam1), a Rac-specific guanine nucleotide exchange factor, enhanced BMP-2-induced alkaline phosphatase activity. Further, we demonstrated that BMP-2 stimulated Rac1 activity. These results indicate that the activation of Rac1 attenuates osteoblastic differentiation in C2C12 cells.     

Sortilin is upregulated during osteoblastic differentiation of mesenchymal stem cells and promotes extracellular matrix mineralization

Osteoblasts and adipocytes are derived from a common precursor in bone marrow, the mesenchymal stem cell (MSC). Factors driving human MSCs (hMSCs) to differentiate down the two lineages play important roles in determining bone density because it has been shown that bone volume loss associated with osteoporosis and aging is accompanied by reduced osteoblastic bone formation and increased marrow adipose tissue. The genes upregulated in hMSCs during osteogenic differentiation were screened using cDNA microarrays and were semi-quantitated by real-time RT-PCR. One of the genes identified was sortilin, which was upregulated one day after osteogenic induction and remained upregulated for a week. The overexpression of sortilin in hMSCs using an adenovirus vector resulted in the acceleration of mineralization during osteogenic differentiation without affecting alkaline phosphatase activity. Lipoprotein lipase (LPL), produced by adipocytes, is bound by sortilin, which may mediate its endocytosis. By adding LPL to osteogenic induction medium, osteoblastic mineralization was inhibited in a dose-dependent manner. Interestingly, sortilin overexpression abolished the LPL-mediated suppression of osteogenic differentiation. hMSCs exist in marrow where LPL-producing adipose cells are abundant and where osteogenesis is negatively regulated by LPL. Sortilin has a counter effect of promoting osteogenesis by acting as a scavenger of LPL.     

Taurine inhibits osteoblastic differentiation of vascular smooth muscle cells via the ERK pathway

Vascular calcification develops within atherosclerotic lesions and results from a process similar to osteogenesis. Taurine is a free β-amino acid and plays an important physiological role in mammals. We have recently demonstrated that vascular smooth muscle cells (VSMCs) express a functional taurine transporter. To evaluate the possible role of taurine in vascular calcification, we assessed its effects on osteoblastic differentiation of VSMCs in vitro. The results showed that taurine inhibited the β-glycerophosphate-induced osteoblastic differentiation of VSMCs as evidenced by both the decreasing alkaline phosphate (ALP) activity and expression of the core binding factor α1 (Cbfα1). Taurine also activated the extracellular signal-regulated protein kinase (ERK) pathway. Inhibition of ERK pathway reversed the effect of taurine on ALP activity and Cbfα1 expression. These results suggested that taurine inhibited osteoblastic differentiation of vascular cells via the ERK pathway.     

A human osteoblastic cell line, MG-63, produces two molecular types of macrophage-colony-stimulating factor.

A human osteoblastic cell line, MG-63, mouse primary osteoblasts, and a mouse osteoblastic cell line, MC3T3-E1, were shown to produce macrophage-colony-stimulating factor (M-CSF) by bone-marrow-cell colony assay, using a specific neutralizing antibody for M-CSF. Immunoblot analysis of M-CSF, produced by MG-63 cells, revealed the presence of a higher-molecular-weight species of M-CSF, in addition to the 85-kDa M-CSF. The higher-molecular-weight species had a high affinity to the DEAE-Sephacel column and was sensitive to chondroitinase ABC and AC. These physico-chemical profiles were wholly compatible with those of the proteoglycan form of M-CSF (PG-M-CSF), which was recently identified by our group in the conditioned medium of Chinese hamster ovary cells transfected with the 4.0-kb cDNA of the M-CSF gene. Conditioned medium of MG-63 cells was fractionated by DEAE-Sephacel column chromatography, and the M-CSF of each fraction was measured by both enzyme-linked immunosorbent assay and bone-marrow-cell colony assay. The fractions eluted by 0.3-0.6 M NaCl, which were shown to contain only PG-M-CSF on immunoblot analysis, also have macrophage-colony-stimulating activity.     

Ferutinin promotes proliferation and osteoblastic differentiation in human amniotic fluid and dental pulp stem cells

AimsThe phytoestrogen Ferutinin plays an important role in prevention of osteoporosis caused by ovariectomy-induced estrogen deficiency in rats, but there is no evidence of its effect on osteoblastic differentiation in vitro. In this study we investigated the effect of Ferutinin on proliferation and osteoblastic differentiation of two different human stem cells populations, one derived from the amniotic fluid (AFSCs) and the other from the dental pulp (DPSCs).Main methodsAFSCs and DPSCs were cultured in a differentiation medium for 14 or 21 days with or without the addition of Ferutinin at a concentration ranging from 10^? 11 to 10^? 4 M. 17β-Estradiol was used as a positive drug at 10^? 8 M. Cell proliferation and expression of specific osteoblast phenotype markers were analyzed.Key findingsMTT assay revealed that Ferutinin, at concentrations of 10^? 8 and 10^? 9 M, enhanced proliferation of both AFSCs and DPSCs after 72 h of exposure. Moreover, in both stem cell populations, Ferutinin treatment induced greater expression of the osteoblast phenotype markers osteocalcin (OCN), osteopontin (OPN), collagen I, RUNX-2 and osterix (OSX), increased calcium deposition and osteocalcin secretion in the culture medium compared to controls. These effects were more pronounced after 14 days of culture in both populations.SignificanceThe enhancing capabilities on proliferation and osteoblastic differentiation displayed by the phytoestrogen Ferutinin make this compound an interesting candidate to promote bone formation in vivo.     

Steroid sulfatase mediated growth Sof human MG-63 pre-osteoblastic cells

Estrogen plays an important role in maintaining bone density. Postmenopausal women have low plasma estrogen, but have high levels of conjugated steroids, particularly estrone sulfate (E₁S) and dehydroepiandrosterone sulfate (DHEAS). Conversion of these precursors to active estrogens may help maintain bone density in postmenopausal women. The enzyme steroid sulfatase (STS) converts sulfated steroids into active forms in peripheral tissues. STS occurs in bone, but little is known about its role in bone function. In this study, we investigated STS activity and expression in the human MG-63 pre-osteoblastic cell line. We also tested whether sulfated steroids can stimulate growth of these cells. MG-63 cells and microsomes both possessed STS activity, which was blocked by the STS inhibitors EMATE and 667 Coumate. Further evidence for STS in these cells was provided by RT-PCR, using STS specific primers, which resulted in cDNA products of the predicted size. We then tested for growth of MG-63 cells in the presence of estradiol-17β, E₁S and DHEAS. All three steroids stimulated MG-63 cell growth in a steroid-free basal medium. We also tested whether the cell growth induced by sulfated steroids could be blocked using a STS inhibitor (667 Coumate) or using an estrogen receptor blocker (ICI 182,780). Both compounds inhibited E₁S-induced cell growth, indicating that E₁S stimulates MG-63 cell growth through a mechanism involving both STS and the estrogen receptor. Finally, we demonstrated using RT-PCR that MG-63 cells contain mRNA for both estrogen receptor alpha and estrogen receptor beta. Our data reveal that STS is present in human pre-osteoblastic bone cells and that it can influence bone cell growth by converting inactive sulfated steroids to estrogenic forms that act via estrogen receptor alpha or beta.