Human Osteoblast-like Cell Line MG-63 Research Articles

Design, synthesis and biological evaluation of inhibitors of cathepsin K on dedifferentiated chondrocytes.

Selective proteinase inhibitors have demonstrated utility in the investigation of cartilage degeneration mechanisms and may have clinical use in the management of osteoarthritis. The cysteine protease cathepsin K (CatK) is an attractive target for arthritis therapy. Here we report the synthesis of two cathepsin K inhibitors (CKIs): racemic azanitrile derivatives CKI-E and CKI-F, which have better inhibition properties on CatK than the commercial inhibitor odanacatib (ODN). Their IC50 values and inhibition constants (Ki) have been determined in vitro. Inhibitors demonstrate differential selectivity for CatK over cathepsin B, L and S in vitro, with Ki amounting to 1.14 and 7.21 nM respectively. We analyzed the effect of these racemic inhibitors on viability in different cell types. The human osteoblast-like cell line MG63, MOVAS cells (a murine vascular smooth muscle cell line) or murine primary chondrocytes, were treated either with CKI-E or with CKI-F, which were not toxic at doses of up to 5 µM. Primary chondrocytes subjected to several passages were used as a model of phenotypic loss of articular chondrocytes, occurring in osteoarthritic cartilage. The efficiency of CKIs regarding CatK inhibition and their specificity over other proteases were validated in primary chondrocytes subjected to several passages. Racemic CKI-E and CKI-F at 0.1 and 1 µM significantly inhibited CatK activity in dedifferentiated chondrocytes, even better than the commercial CatK inhibitor ODN. The enzymatic activity of other proteases such as matrix metalloproteinases or aggrecanases were not affected. Taken together, these findings support the possibility to design CatK inhibitors for preventing cartilage degradation in different pathologies.

Silicon bioceramic loaded with vancomycin stimulates bone tissue regeneration.

Porous ceramics doped with silicon and pure β-TCP were analyzed in terms of internal microstructure, cell behavior, and the percentage of newly formed bone. Additionally the materials were tested to determine which of the two had better properties to load and release vancomycin hydrochloride. Internal pore distribution and porosity were determined through high pressure mercury porosimetry and the specific surface area was measured by the Brunauer Emmet-Teller method. The proliferation and viability of the human osteoblast-like cell line MG-63 was studied to validate both materials. The materials were tested on eight New Zealand rabbits which created defects, 10 mm in diameter, in the calvaria bone. After 8 and 12 weeks a histological and histomorphometric analysis was performed. Si-β-TCP showed a higher porosity and specific surface area. The cytocompatibility test revealed acceptable results in terms of proliferation and viability whereas the percentage of new bone was higher in Si-β-TCP with a two-time study being statistically significant with 12 weeks of healing (p < 0.05).The vancomycin loaded within the ceramic scaffolds were burst released and the material had the ability to inhibit bacterial growth. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2307-2315, 2018.

A new iron calcium phosphate material to improve the osteoconductive properties of a biodegradable ceramic: a study in rabbit calvaria

β-tricalcium phosphate (β-TCP) is an osteoconductive and biodegradable material used in bone regeneration procedures, while iron has been suggested as a tool to improve the biological performance of calcium phosphate-based materials. However, the mechanisms of interaction between these materials and human cells are not fully understood. In order to clarify this relationship, we have studied the iron role in β-TCP ceramics. Iron-containing β-TCPs were prepared by replacing CaCO3 with C6H5FeO7 at different molar ratios. X-ray diffraction analysis indicated the occurrence of β-TCP as the sole phase in the pure β-TCP and iron-containing ceramics. The incorporation of iron ions in the β-TCP lattice decreased the specific surface area as the pore size was shifted toward meso- and/or macropores. Furthermore, the human osteoblastlike cell line MG-63 was cultured onto the ceramics to determine cell proliferation and viability, and it was observed that the iron-β-TCP ceramics have better cytocompatibility than pure β-TCP.Finally, in vivo assays were performed using rabbit calvaria as a bone model. The scaffolds were implanted for 8 and 12 weeks in the defects created in the skullcap with pure β-TCP as the control. The in vivo behavior, in terms of new bone formed, degradation, and residual graft material were investigated using sequential histological evaluations and histomorphometric analysis. The in vivo implantation of the ceramics showed enhanced bone tissue formation and scaffold degradation for iron-β-TCPs. Thus, iron appears to be a useful tool to enhance the osteoconductive properties of calcium phosphate ceramics.

Mechanical Behavior of Bioactive TiC Nanocomposite Thin Films

Carbon-based nanocomposite thin films have large application potential because they possess unique mechanical properties, especially high hardness, high elasticity, and a low widely adjustable friction coefficient. In this work, relatively easy preparation of the nanocomposite Ti and C system with good mechanical properties and bioactivity was showed. Formation of physical and mechanical processes, relationship between the evolving structure and other properties of TiC films were studied. The films were deposited on oxidized silicon substrates by dc magnetron sputtering of Ti and C targets in argon and nitrogen at different temperatures between 25°C and 800°C. The composite films consisted of metallic nanocrystalls embedded in a carbon matrix. Highest hardness ~ 18 GPa and reduced modulus of elasticity ~ 205 GPa were obtained when the crystalline nanoparticles were separated by 2-3 nm thin carbon matrix consisting of amorphous and graphite-like carbon phases. In these films the H/E ratio in the both cases is ~ 0,1. Bioactivity studies were carried out on human osteoblast-like cell line MG-63. The number of initially adhering cells on day 7 after seeding was significantly higher on the TiC surface than on the control culture dishes. Good biocompatibility and bioadhesion of these surfaces are attained by a favourable combination of surface roughness and chemistry.

Hijikia fusiforme protects against ovariectomy-induced bone loss in rats.

The prophylactic effects of Hijikia fusiforme on bone metabolism were examined using in vitro indices of bone formation and resorption. As the indices of bone formation, osteoblast proliferation and differentiation were measured through mitochondrial enzyme activity [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay] and bone marker alkaline phosphatase (ALP) activity. The aqueous extract of H. fusiforme stimulated the proliferation of the human osteoblast-like cell line MG63 and the ALP activity of the mouse osteoblast-like cell line MC3T3-E1. Moreover, extracellular matrix mineralization was accelerated by the addition of H. fusiforme. As the indices of bone resorption, differentiation of the murine macrophage/osteoclast precursor cell line RAW 264.7 by receptor activator of nuclear factor-κB ligand (RANKL) was measured as tartrate-resistant acid phosphatase-positive multinucleated cells, which were suppressed by H. fusiforme. Additionally, H. fusiforme lowered the secreted amount of RANKL that is required for the osteoclastic differentiation and activation, but the amount of osteoprotegerin as a decoy receptor for RANKL was not affected. The bone-protective effects of H. fusiforme in estrogen-deficient ovariectomized rats were also investigated. Osteoporosis was induced in female Sprague-Dawley rats by ovariectomy for 15 weeks, and then H. fusiforme was orally administered at a dose of 100 mg/kg of body weight every day for 6 weeks. Bone mineral density in the group orally administered H. fusiforme was increased, compared with ovariectomized rats, but not significantly (P>.05). Oral administration of H. fusiforme improved microarchitecture of bone in terms of bone volume (bone volume/total volume ratio) and trabecular separation (P<.05). The amounts of osteocalcin and C-telopeptide type I collagen in serum were measured as the biomarkers for bone formation and resorption. The level of osteocalcin in serum was increased, but not significantly. However, the level of C-telopeptide type I collagen in serum was significantly decreased (P<.05). When the results are taken together, the present study indicates that H. fusiforme might be useful in the treatment of osteoporosis.

Erratum to: hRpn13, a newly identified component of the 19S particle, regulates proliferation, differentiation, and function in the human osteoblast-like cell line MG63

Erratum to: hRpn13, a newly identified component of the 19S particle, regulates proliferation, differentiation, and function in the human osteoblast-like cell line MG63

HRpn13, a newly identified component of the 19S particle, regulates proliferation, differentiation, and function in the human osteoblast-like cell line MG63

The 26S proteasome is a key component of the ubiquitin-proteasome system, a process responsible for the majority of cellular protein degradation. The function of the proteasomal ubiquitin receptor hRpn13, a component of the 26S proteasome, is not completely understood. To investigate the role of hRpn13 in the ubiquitin-proteasome system in osteoblasts, the effects of suppressing and overexpressing the hRpn13 gene on proliferation, differentiation, and function of human osteoblast-like MG63 cells were examined. After knockdown of hRpn13 by small interfering RNA, changes in osteoblast proliferation were evaluated by methyl-thiazolyl-tetrazolium assay. There was an increase in markers for osteoblast proliferation, specifically alkaline phosphatase activity, and elevated protein levels of osteocalcin, proliferating cell nuclear antigen (PCNA), and ubiquitin. Furthermore, hRpn13 knockdown also resulted in a decrease in the ratio between the gene expressions of RANKL and OPG, key players in the pathogenesis of bone diseases that influence the normal balance between bone formation and resorption. In contrast, overexpression of hRpn13 inhibited the proliferation of MG63 cells, and decreased alkaline phosphatase activity as well as protein levels of osteocalcin, PCNA, and ubiquitin while the ratio of RANKL to OPG expression increased. To confirm the function of hRpn13 in the ubiquitin-proteasome pathway, osteoblast proliferation enhancement and ubiquitin accumulation after hRpn2 knockdown was assessed. The results suggest that overexpression of hRpn13 negatively influences proliferation and osteogenic differentiation in MG63 cells. The evidence implies that hRpn13 modulates the influence of osteoblasts on osteoclasts by controlling the stability of regulatory proteins in osteoblasts. In summary, overexpression of hRpn13 promoted the activity of the ubiquitin-proteasome system.

Effect of thyroid hormones on the expression of thyroid hormone receptor isoforms in human osteogenic sarcoma cell line MG-63

Objective To examine the expression of thyroid hormone receptor isoforms (TR α1, α2, β1, and β2) in human osteoblast-like cell line MG-63 at the mRNA level and the effect of thyroid hormone (T3 or T4 ) on the expression. Methods Realtime quantitative PCR was performed. Results The expression of TRα1 mRNA was the highest, that was 10. 70± 0.45, TRβ1 was 5.75 ± 0. 10, TRβ2 was 3.34 ± 0. 08, and TRα2 was very low, only (3.66 ±0. 59) × 10-2. Only the expression of TRαl and TRα2 mRNA was down regulated significantly by the treatment of 10-10 ～ 10-6 mol/L T3, and there was a negative correlation between the expression of TRα1 or TRα2 mRNA and the concentration of T3. Conclusion TRα1 plays a primary role in mediating the effects of thyroid hormones in skeletal development. Key words: Thyroid hormones; Thyroid hormone receptors; Human osteoblast-like cell line MG-63; Gene expression

Forkhead Proteins Are Critical for Bone Morphogenetic Protein-2 Regulation and Anti-tumor Activity of Resveratrol

Osteoporosis is a major public health problem and the most obvious preventive strategy, hormone replacement therapy, has lost favor due to recent findings of the Women's Health Initiative regarding increased risks of breast cancer and cardiovascular disease. Resveratrol, a naturally occurring compound possessing estrogenic activity, is thought to have considerable potential for therapy of osteoporosis. In the present study, resveratrol was found to exhibit bone-protective effects equivalent to those exerted by hormone replacement therapy and decrease the risk of breast cancer in the in vivo and in vitro models. Forkhead proteins were found to be essential for both effects of resveratrol. The bone-protective effect was attributable to induction of bone morphogenetic protein-2 through Src kinase-dependent estrogen receptor activation and FOXA1 is required for resveratrol-induced estrogen receptor-dependent bone morphogenetic protein-2 expression. The tumor-suppressive effects of resveratrol were the consequence of Akt inactivation-mediated FOXO3a nuclear accumulation and activation. Resveratrol is therefore anticipated to be highly effective in management of postmenopausal osteoporosis without an increased risk of breast cancer.

Tissue-like self-assembly in cocultures of endothelial cells and osteoblasts and the formation of microcapillary-like structures on three-dimensional porous biomaterials

The survival and functioning of a bone biomaterial requires a rapid and stable vascularization after implantation. However, the mechanisms involved in the context of the complex healing microenvironment are poorly understood. To evaluate the vascularization potential of bone biomaterials, angiogenic stimuli were added to human dermal microvascular endothelial cells (HDMEC) growing on three-dimensional (3-D) bone biomaterials consisting of porous hydroxyapatite, porous calcium phosphate, porous nickel–titanium, successfully being used in humans, and also silk fibroin nets. HDMEC did not migrate to form microcapillary-like structures as they did on cell culture plastic. In cocultures of HDMEC and primary human osteoblast cells (HOS) or the human osteoblast-like cell line MG-63 on these biomaterials, a tissue-like self-assembly of cells occurred with time, with endothelial cells forming microcapillary-like structures containing a lumen and giving a strong PECAM-1 expression at cell interfaces. These microcapillary-like structures were intertwined between cell layers of osteoblasts and did not form when exogenous angiogenic stimuli were added to these cocultures. The life span of HDMEC was also significantly enhanced by coculture; with HDMEC being present for up to at least 42 days, compared to the monoculture where cells began to die rapidly after 1 week without passage. This coculture system may be applicable to a prevascularization strategy for biomaterials prior to implantation. Irrespective of this, the coculture model holds promise for studies to deepen our understanding of bone regeneration on 3-D substrates. Most importantly, these data raise important questions concerning the exact nature of pro-angiogenic drug- or gene-delivery systems to be incorporated into scaffolds. Our results underline the necessity to take into account the in situ production of growth factors by invading mesenchymal cells in the regenerative niche.

Taurine transporter is expressed in osteoblasts

Taurine influences bone metabolism and is taken up by cells via a specific transport system, the taurine transporter (TAUT). We report a link between taurine and bone homeostasis by demonstrating transcription and translation of TAUT in bone-forming cells. TAUT was expressed in human primary osteoblasts, the human osteosarcoma osteoblast-like cell line MG63, and the mouse osteoblastic cell line MC3T3-E1. Immunostaining with polyclonal antibodies also demonstrated the presence of TAUT in both human and murine osteoblasts. TAUT mRNA expression and [(3)H]taurine uptake increased during differentiation of MG63 cells in culture. Supplementation of culture medium with taurine enhanced alkaline phosphatase activity and osteocalcin secretion. The regulation and detailed function of taurine and TAUT in bone remain unclear, but our findings suggest a functional role for them in bone homeostasis.

Fraxetin inhibits the induction of anti-Fas IgM, tumor necrosis factor-α and interleukin-1β-mediated apoptosis by Fas pathway inhibition in human osteoblastic cell line MG-63

The survival of osteoblast cells is one of the determinants of the development of osteoporosis in patients with inflamed synovium, such as in rheumatoid arthritis (RA). By means of alkaline phosphatase (ALP) activity and osteocalcin ELISA assay, we have shown that fraxetin exhibits a significant induction of differentiation in the human osteoblast-like cell line MG-63. In addition, we also assessed whether fraxetin affects inflammatory cytokine-mediated apoptosis in osteoblast cells. TNF-α or IL-1β enhance apoptotic DNA fragmentation in anti-Fas IgM-treated MG-63 cells by increasing Fas receptor expression. However, TNF-α or IL-1β treatment alone does not induce apoptosis. Treatment of MG-63 cells with fraxetin not only inhibited anti-Fas IgM-induced apoptosis, but also blocked the synergetic effect of anti-Fas IgM with TNF-α or IL-1β on cell death. The apoptotic inhibition of fraxetin is associated with inhibition of TNF-α and IL-1β-mediated Fas expression and enhancement of FLIP expression, resulting in a decrease of caspase-8 and caspase-3 activation. These results indicate a potential use of fraxetin in preventing osteoporosis by inhibiting inflammatory cytokine-mediated apoptosis in osteoblast cells.

The effects of titanium and polymethylmethacrylate particles on osteoblast phenotypic stability

Wear particles generated following total joint arthroplasty interact with cells at the periprosthetic margin and induce an inflammatory response that contributes to osteolysis, aseptic loosening, and implant failure. This study examined the long-term effects of particles from two commonly implanted materials, titanium (Ti) and polymethylmethacrylate (PMMA), on cell viability and metabolism over a 21-day time course, using the human osteoblast-like cell line MG-63. Addition of particles was not associated with increased cell death or nitric oxide production at the particle concentration chosen. Collagen production was increased with exposure to titanium particles, whereas alkaline phosphatase and osteocalcin expression remained unchanged following exposure to both types of particles. The data show that titanium but not PMMA particles shifts bone cell metabolism to preferentially produce fibrous tissue rather than bone.

Polyetherimide membrane formation by the cononsolvent system and its biocompatibility of MG63 cell line

Polyetherimide (PEI) membrane could be prepared by immersion–precipitation process by using the N-methyl-2-pyrrolidone (NMP)–methylene chloride (MC) cononsolvent as the coagulation medium. The membrane, via scanning electron microscopy (SEM), showed a typically asymmetrical structure consisting of a thin and dense skin layer, sponge morphology in the cross-section and porous bottom surface. Trend expected on the basis of the phase diagram of the use of two solvents serving as a cononsolvent system for the membrane formation, replacing the traditional solvent–nonsolvent pair, was in reasonable agreement with the observed membrane morphology. Therefore, the principles of membrane formation established for the ternary systems with polymer–solvent–nonsolvent can be applied to a polymer–cononsolvent (solvent–solvent) system. Furthermore, the biocompatibility of the PEI membrane top (dense) and bottom (porous) surfaces was assessed in an in vitro model using the human osteoblast-like cell line MG63. Compared to cells grown on tissue culture polystyrene (TCPS), cells cultured on the PEI membranes had lower cell growth, but every single cell on the PEI membrane showed similar viability to control cells. SEM analysis showed comparable adhesion and confluence characteristics on all substrates. In addition, the levels of alkaline phosphotase (ALP), prostaglandin E2 (PGE 2) and transforming growth factor β1 (TGF-β1) cytokines produced by MG63 cells on the prepared PEI membranes, regardless of the top or bottom surface, were similar to those on TCPS. The results of this study suggest that the PEI membrane prepared from a cononsolvent system is a non-toxic, biocompatible substrate for the proliferation of human bone cells in vitro.

Myricetin inhibits the induction of anti-Fas IgM-, tumor necrosis factor-α- and interleukin-1β-mediated apoptosis by Fas pathway inhibition in human osteoblastic cell line MG-63

The survival of osteoblast cells is one of the determinants of the development of osteoporosis in patients with inflamed synovium, such as in rheumatoid arthritis (RA). By means of alkaline phosphatase (ALP) activity and osteocalcin ELISA assay, I have shown that myricetin exhibits a significant induction of differentiation in the human osteoblast-like cell line MG-63. In addition, I also assessed whether myricetin affects inflammatory cytokines-mediated apoptosis in osteoblast cells. TNF-α or IL-1β enhances apoptotic DNA fragmentation in anti-Fas IgM-treated MG-63 cells by increasing Fas receptor expression. However, TNF-α or IL-1β treatment alone does not induce apoptosis. Treatment of MG-63 cells with myricetin not only inhibited anti-Fas IgM-induced apoptosis, but also blocked the synergetic effect of anti-Fas IgM with TNF-α or IL-1β on cell death. The apoptotic inhibition of myricetin is associated with inhibition of TNF-α and IL-1β-mediated Fas expression and enhancement of FLIP expression, resulting in a decrease of caspase-8 and caspase-3 activation. These results indicate a potential use of myricetin in preventing osteoporosis by inhibiting inflammatory cytokines-mediated apoptosis in osteoblast cells.

Mechanosensitivity of human osteosarcoma cells and phospholipase C β2 expression

Bone adapts to mechanical load by osteosynthesis, suggesting that osteoblasts might respond to mechanical stimuli. We therefore investigated cell proliferation and phospholipase C (PLC) expression in osteoblasts. One Hertz uniaxial stretching at 4000 μstrains significantly increased the proliferation rates of human osteoblast-like osteosarcoma cell line MG-63 and primary human osteoblasts. However, U-2/OS, SaOS-2, OST, and MNNG/HOS cells showed no significant changes in proliferation rate. We investigated the expression pattern of different isoforms of PLC in these cell lines. We were able to detect PLC β1, β3, γ1, γ2, and δ1 in all cells, but PLC β2 was only detectable in the mechanosensitive cells. We therefore investigated the possible role of PLC β2 in mechanotransduction. Inducible antisense expression for 24 h inhibited the translation of PLC β1 in U-2/OS cells by 35% and PLC β2 in MG-63 by 29%. Fluid shear flow experiments with MG-63 lacking PLC β2 revealed a significantly higher level of cells losing attachment to coverslips and a significantly lower number of cells increasing intracellular free calcium.

Na+/Ca2+ Exchanger Activity Modulates Connective Tissue Growth Factor mRNA Expression in Transforming Growth Factor β1- and Des-Arg10-kallidin-stimulated Myofibroblasts

Transforming growth factor (TGF)-beta and des-Arg(10)-kallidin stimulate the expression of connective tissue growth factor (CTGF), a matrix signaling molecule that is frequently overexpressed in fibrotic disorders. Because the early signal transduction events regulating CTGF expression are unclear, we investigated the role of Ca(2+) homeostasis in CTGF mRNA expression in TGF-beta1- and des-Arg(10)-kallidin-stimulated human lung myofibroblasts. Activation of the kinin B1 receptor with des-Arg(10)-kallidin stimulated a rise in cytosolic Ca(2+) that was extracellular Na(+)-dependent and extracellular Ca(2+)-dependent. The des-Arg(10)-kallidin-stimulated increase of cytosolic Ca(2+) was blocked by KB-R7943, a specific inhibitor of Ca(2+) entry mode operation of the plasma membrane Na(+)/Ca(2+) exchanger. TGF-beta1 similarly stimulated a KB-R7943-sensitive increase of cytosolic Ca(2+) with kinetics distinct from the des-Arg(10)-kallidin-stimulated Ca(2+) response. We also found that KB-R7943 or 2',4'-dichlorobenzamil, an amiloride analog that inhibits the Na(+)/Ca(2+) exchanger activity, blocked the TGF-beta1- and des-Arg(10)-kallidin-stimulated increases of CTGF mRNA. Pretreatment with KB-R7943 also reduced the basal and TGF-beta1-stimulated levels of alpha1(I) collagen and alpha smooth muscle actin mRNAs. These data suggest that, in addition to regulating ion homeostasis, Na(+)/Ca(2+) exchanger acts as a signal transducer regulating CTGF, alpha1(I) collagen, and alpha smooth muscle actin expression. Consistent with a more widespread role for Na(+)/Ca(2+) exchanger in fibrogenesis, we also observed that KB-R7943 likewise blocked TGF-beta1-stimulated levels of CTGF mRNA in human microvascular endothelial and human osteoblast-like cells. We conclude that Ca(2+) entry mode operation of the Na(+)/Ca(2+) exchanger is required for des-Arg(10)-kallidin- and TGF-beta1-stimulated fibrogenesis and participates in the maintenance of the myofibroblast phenotype.

Fatty acids and cytokine mRNA expression in human osteoblastic cells: a specific effect of arachidonic acid

Epidemiological, clinical and experimental evidence suggests that fatty acids have a modulatory effect on bone metabolism in animals and humans. To investigate this hypothesis, we evaluated the effects of three different fatty acids, arachidonic acid (AA), eicosapentaenoic acid (EPA) and oleic acid (OA), on the expression of cytokines involved in bone remodelling. Cytokine mRNAs in the human osteoblast-like cell line MG-63 were quantified by reverse transcription-PCR. AA induced increased expression of interleukin-1alpha, interleukin-1beta, tumour necrosis factor-alpha and macrophage colony-stimulating factor mRNAs in a time- and dose-dependent manner. EPA and OA had no stimulatory effects, but instead caused a significant inhibition of AA-induced cytokine mRNA expression. Cell treatment with calphostin C, an inhibitor of protein kinase C (PKC), and cellular PKC down-regulation experiments independently resulted in significant inhibition of AA-induced cytokine expression, suggesting that a PKC-dependent mechanism accounts for the effects of AA on cytokine production. In conclusion, our study demonstrates specific effects of fatty acids on cytokine gene expression in human osteoblast-like cells. The clinical relevance of our findings requires further investigation.

Expression of Opioid Receptors in Osteoblast-Like MG-63 Cells, and Effects of Different Opioid Agonists on Alkaline Phosphatase and Osteocalcin Secretion by These Cells

We have previously shown that several stressful situations associated with tissue injury determine a decrease in serum osteocalcin concentration. Since reduced osteocalcin production is a marker of decreased osteoblastic activity, this finding could be related to the pathogenesis of osteoporosis secondary to some diseases. Endogenous opioids are involved in stress response. Proenkephalin-derived peptides have been shown to inhibit alkaline phosphatase activity, another marker of bone formation, in the murine cell line ROS-17/2.8. On the other hand, serum osteocalcin has been reported as being low in heroin abusers. We have therefore thought it of interest to study the presence of opioid receptors in the human osteoblast-like cell line MG-63, and to evaluate the effects of different opioid agonists on the secretion of alkaline phosphatase and osteocalcin by these cells. The presence of opioid receptors was studied by means of RT-PCR and immunohistochemistry. RT-PCR studies suggested the presence of specific mRNA for the three types of receptors, and immunohistochemistry clearly showed their occurrence. Osteocalcin synthesis was significantly inhibited by high concentrations of the mu agonists morphine and (D-Ala²,N-MePhe⁴,Gly⁵-ol)-enkephalin although no changes were seen with the delta agonist (D-Ala²,D-leu⁵)-enkephalin. Morphine-induced osteocalcin inhibition was abolished when osteoblastic cells were incubated simultaneously with naloxone, whereas it was potentiated when cells were preincubated with naloxone. None of the opioid agonists modified the secretion of alkaline phosphatase. In conclusion, human osteoblast-like cells MG-63 express the three types of opioid receptors. Endogenous opioids may be involved in the reduction of osteocalcin observed in stressful situations associated with tissue injury.

The Regulation of PGE2 Biosynthesis in MG-63 Osteosarcoma Cells by IL-1 and FGF Is Cell Density-Dependent

We investigated the molecular mechanisms by which treatment of the human osteoblast-like cell line MG-63 with interleukin 1β (IL-1) and/or fibroblast growth factor 1 (FGF-1) elicited prostaglandin biosynthesis. IL-1 induced a 5-fold increase in PGE2 production compared to controls. While treatment with FGF-1 alone did not affect PGE2 biosynthesis, it enhanced the formation of PGE2 by IL-1 by an additional 3- to 5-fold. IL-1-induced PGE2 biosynthesis accompanied increases in steady-state levels of mRNAs encoding cPLA2 (10- to 15-fold) and PGHS-2 (>3-fold) and concomitant increases in cPLA2 protein (>3-fold) and PGHS-2 protein (>1.5-fold). FGF-1 treatment did not affect PGHS-2 gene expression, but enhanced the effect of IL-1 on PGHS-2 expression by an additional 2- to 3-fold. FGF-1 alone enhanced cPLA2 expression (5-fold), and the combined effects of FGF-1 and IL-1 on cPLA2 expression were additive. There was no measurable effect of either agonist on PGHS-1 expression. We also discovered that induction of PGE2 biosynthesis in response to IL-1 or IL-1/FGF-1 was affected by the density of MG-63 cells in culture. Subconfluent cultures displayed a 3- to 10-fold greater response to IL-1 or IL-1/FGF-1 than confluent cultures. The decreased PGE2 induction by IL-1 in confluent cultures was associated with reduced IL-1 receptor expression. We conclude that the signaling pathways resulting in PGE2 biosynthesis in response to proinflammatory agents like IL-1 are subject to complex regulation by additional soluble mediators as well as cell–cell or cell–extracellular matrix interactions.