Osteoblast-like Cell Line MG63 Research Articles

Gene expression of bone-associated cytokines in MG63 osteoblast-like cells incubated with acrylic bone cement extracts in minimum essential medium

This study examined the effects of in vitro challenge with four polymerized acrylic bone cements (Sulfix-60®, CMW 1®, CMW 2®, and CMW 3®) on the expression of interleukin-1β (IL-1β), interleukin-6 (IL-6), and transforming growth factor-β1 (TGF-β1) mRNAs in the osteoblastlike cell line MG63. The extracts of the cements in minimal essential medium (MEM) were tested following 1-h and 7-day curing. A semi-quantitative analysis of the cytokine-specific mRNAs was carried out by agarose gel densitometry and expression was compared with the GAPDH house-keeping gene. The ratio between cytokine gene expression and GAPDH expression was calculated. The mRNA specific for the bone-resorbing cytokines IL-1β and IL-6 was low in basal conditions. IL-1β mRNA increased only after incubation with the extract of CMW 1® following 1-h curing. The mRNA specific for the bone-resorbing cytokine IL-6 also increased after contact with CMW 1® at both curing times. Sulfix-60® and CMW 3® following 7-day curing, but not after 1 h, induced higher levels of IL-6 mRNA than the control. CMW 2® after 1-h curing constantly determined the expression of IL-6 mRNA, but at low levels. The mRNA specific for TGF-β1 was also expressed by the MG63 osteoblast-like cells in basal conditions. The levels increased after contact with Sulfix-60® after 7-day curing and with CMW 1® after 1-h curing. CMW 2® after 7-day curing decreased TGF-β1 mRNA. In conclusion, the highest expression of the cytokines IL-1β, IL-6, and TGF-β1 mRNA was determined by CMW 1®. If the results are confirmed in vivo, the increased expression of the osteolytic cytokines induced by the bone cement might result in loosening of the prosthesis, even with all the restrictions of an in vitro study on continuous cell lines.

Effect of four acrylic bone cements on transforming growth factor-beta1 expression by osteoblast-like cells MG63

Based on the hypothesis that bone cements cause changes in the production of transforming growth factor-beta 1 (TGF- β1) by bone cells, the effects of four acrylic bone cements (Sulfix-60, CMW 1, CMW 2 and CMW 3) were examined using the osteoblast-like cell line MG63. The extracts in MEM of the cements were tested, following 1 h- and 7 day-curing. MG63 cells seldom expressed mRNA specific for TGF- β1 in basal conditions. The cultures expressed mRNA constantly after incubation with the extract of CMW 1 cured for 1 h. TGF- β1 specific mRNA was seldom expressed after incubation with the other cement extracts. The release of TGF- β1 into the conditioned medium was increased significantly by CMW 1 extract at 1 h-curing, but was not changed significantly by CMW 1 extract at 7 day-curing and by the extracts of the other cements, at both curing times. The stimulating effect of CMW 1 on the secretion of TGF- β1, even with all the restrictions of an in vitro study of continuous cell lines, if confirmed in vivo, might favor the development of the synovial-like membrane around the implant, and therefore impair the chance of success of the prosthesis.

Interleukin-6 expression by osteoblast-like MG63 cells challenged with four acrylic bone cements

Periprosthetic osteolysis is a major clinical problem in total hip and total knee arthroplasty and polymethylmethacrylate (PMMA) is a possible etiologic factor. Recently, increasing importance was ascribed to interleukin-6 (IL-6) as an agent favouring bone resorption. The aim of the present study was to investigate the role of bone cements on IL-6 production by MG63. The effect of four acrylic bone cements (Sulfix-60®, CMW 1®, CMW 2®, and CMW 3®) on the protein release and mRNA expression of IL-6 in osteoblast-like cell line MG63 was examined using IL-1 β (0.2 μg ml-1) as the positive control. The extracts in minimum essential medium (MEM) of the cements were tested, following 1-h and 7-day curing. CMW 1® and CMW 2® significantly increased the IL-6 release into the culture media (p < 0.01). The cells incubated with Sulfix-60® and CMW 3® produced no significantly different levels of IL-6 than the basal production. A positive correlation was found between the concentration of IL-6 and the contents of benzoylperoxide (p = 0.0003) and barium sulphate (p < 0.0001). MG63 expressed IL-6 mRNA constitutively, as demonstrated by the positivity of the negative controls too. We conclude that CMW 1® and CMW 2® increase the production of IL-6 in MG63 cells. The response to Sulfix-60® and CMW 3® is not significantly greater than the negative control.

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.

Glucocorticoids induce a near-total suppression of hyaluronan synthase mRNA in dermal fibroblasts and in osteoblasts: a molecular mechanism contributing to organ atrophy

Glucocorticoid (GC) administration induces atrophy of skin, bone, and other organs, partly by reducing tissue content of glycosaminoglycans, particularly hyaluronic acid (HA). We took advantage of the recent cloning of the three human hyaluronan synthase (HAS) enzymes (HAS1, HAS2 and HAS3), to explore the molecular mechanisms of this side effect. Northern and slot blots performed on RNA extracted from cultured dermal fibroblasts and the MG-63 osteoblast-like osteosarcoma cell line indicated that HAS2 is the predominant HAS mRNA in these cells. Incubation of both cell types for 24 h in the presence of 10-6 M dexamethasone (DEX) resulted in a striking 97-98% suppression of HAS2 mRNA levels. Time-course studies in fibroblasts demonstrated suppression of HAS2 mRNA to 28% of control by 1 h, and to 1.2% of control by 2 h, after addition of DEX. Dose-response studies in fibroblasts indicated that the majority of the suppressive effect required concentrations characteristic of cell-surface GC receptors, a point confirmed by persistent DEX-induced suppression in the presence of RU486, an antagonist of classic cytosolic steroid hormone receptors. Nuclear run-off experiments showed a 70% suppression of HAS2 gene transcription in nuclei from DEX-treated fibroblasts, which is unlikely to fully explain the rapid 50-80-fold reduction in message levels. Experiments with actinomycin D (AMD) demonstrated that the message half-life was 25 min in cells without DEX, whereas the combination of AMD with DEX dramatically increased the half-life of HAS2 mRNA, suggesting that DEX acts by inducing a short-lived destabilizer of the HAS2 message. Direct assessment of HAS2 mRNA stability by wash-out of incorporated uridine label established a half-life of 31 min in cells without DEX, which substantially shortened in the presence of DEX. In conclusion, GCs induce a rapid and sustained, near-total suppression of HAS2 message levels, mediated through substantial decreases in both gene transcription and message stability. These effects may contribute to the loss of HA in GC-treated organs.

Glucocorticoids induce a near-total suppression of hyaluronan synthase mRNA in dermal fibroblasts and in osteoblasts: a molecular mechanism contributing to organ atrophy.

Glucocorticoid (GC) administration induces atrophy of skin, bone, and other organs, partly by reducing tissue content of glycosaminoglycans, particularly hyaluronic acid (HA). We took advantage of the recent cloning of the three human hyaluronan synthase (HAS) enzymes (HAS1, HAS2 and HAS3), to explore the molecular mechanisms of this side effect. Northern and slot blots performed on RNA extracted from cultured dermal fibroblasts and the MG-63 osteoblast-like osteosarcoma cell line indicated that HAS2 is the predominant HAS mRNA in these cells. Incubation of both cell types for 24 h in the presence of 10(-6) M dexamethasone (DEX) resulted in a striking 97--98% suppression of HAS2 mRNA levels. Time-course studies in fibroblasts demonstrated suppression of HAS2 mRNA to 28% of control by 1 h, and to 1.2% of control by 2 h, after addition of DEX. Dose-response studies in fibroblasts indicated that the majority of the suppressive effect required concentrations characteristic of cell-surface GC receptors, a point confirmed by persistent DEX-induced suppression in the presence of RU486, an antagonist of classic cytosolic steroid hormone receptors. Nuclear run-off experiments showed a 70% suppression of HAS2 gene transcription in nuclei from DEX-treated fibroblasts, which is unlikely to fully explain the rapid 50--80-fold reduction in message levels. Experiments with actinomycin D (AMD) demonstrated that the message half-life was 25 min in cells without DEX, whereas the combination of AMD with DEX dramatically increased the half-life of HAS2 mRNA, suggesting that DEX acts by inducing a short-lived destabilizer of the HAS2 message. Direct assessment of HAS2 mRNA stability by wash-out of incorporated uridine label established a half-life of 31 min in cells without DEX, which substantially shortened in the presence of DEX. In conclusion, GCs induce a rapid and sustained, near-total suppression of HAS2 message levels, mediated through substantial decreases in both gene transcription and message stability. These effects may contribute to the loss of HA in GC-treated organs.

Flow cytometry for assessing biocompatibility.

Flow cytometry (FCM) was examined as a possible procedure for measuring in vitro the biocompatibility of implant materials for orthopedic and dental surgery. The human osteoblast-like cell line MG63 was grown on hydroxyapatite (HA) and P2O5 glass-reinforced HA composite discs and compared with the same cells grown on polystyrene culture dishes. While morphological observation at the light and electron microscopic levels showed no major deleterious effects, FCM indicated that cell size was somewhat reduced, particularly by growth on the HA composite. Morever, this material also appeared to delay the progression of the cells from the G0/G1 into the S phase of the cell cycle. In addition to this low level of inhibition of cell growth relative to control cultures, FCM analysis also demonstrated that the glass-reinforced HA caused some down-regulation of the expression of osteocalcin and fibronectin, two antigens which play a vital part in the integrity and function of bone and soft connective tissue, respectively. These results thus show, first, that although HA and the HA composite used in these experiments were generally biocompatible, they nevertheless had certain suboptimal effects on the cells; and second, that FCM could be a highly useful procedure for effectively screening and evaluating important biological responses to implant materials.

Flow cytometry for assessing biocompatibility

Flow cytometry (FCM) was examined as a possible procedure for measuring in vitro the biocompatibility of implant materials for orthopedic and dental surgery. The human osteoblast-like cell line MG63 was grown on hydroxyapatite (HA) and P2O5 glass-reinforced HA composite discs and compared with the same cells grown on polystyrene culture dishes. While morphological observation at the light and electron microscopic levels showed no major deleterious effects, FCM indicated that cell size was somewhat reduced, particularly by growth on the HA composite. Moreover, this material also appeared to delay the progression of the cells from the G0/G1 into the S phase of the cell cycle. In addition to this low level of inhibition of cell growth relative to control cultures, FCM analysis also demonstrated that the glass-reinforced HA caused some down-regulation of the expression of osteocalcin and fibronectin, two antigens which play a vital part in the integrity and function of bone and soft connective tissue, respectively. These results thus show, first, that although HA and the HA composite used in these experiments were generally biocompatible, they nevertheless had certain suboptimal effects on the cells; and second, that FCM could be a highly useful procedure for effectively screening and evaluating important biological responses to implant materials. © 1998 John Wiley & Sons, Inc. J Biomed Mater Res, 41, 649–656, 1998.

Transcriptional Activation of the p21WAF1,CIP1,SDI1 Gene by Interleukin-6 Type Cytokines: A PREREQUISITE FOR THEIR PRO-DIFFERENTIATING AND ANTI-APOPTOTIC EFFECTS ON HUMAN OSTEOBLASTIC CELLS

The cyclin-dependent kinase inhibitor p21(WAF1,CIP1,SDI1) plays a critical role in cell differentiation, and it has been shown to confer resistance to apoptosis. Based on this, and on evidence that activation of the gp130/signal transducer and activator of transcription (STAT) signal transduction pathway by interleukin (IL)-6 type cytokines promotes differentiation and prevents apoptosis in osteoblastic cells, we have investigated the possibility that p21 is a downstream effector of this signaling pathway in osteoblasts. We report that either oncostatin M (OSM) or IL-6 plus soluble IL-6 receptor increased the levels of p21 mRNA and protein in the osteoblast-like human osteosarcoma cell line MG63 and stimulated the activity of a 2.4-kilobase pair segment of the human p21 gene promoter. Further, nuclear extracts from cytokine-stimulated MG63 cells formed protein-DNA complexes with a 19-base pair nucleotide fragment of the p21 promoter containing a single STAT response element. The identity of the binding proteins as Stat3 and Stat1 was demonstrated with specific antibodies. In addition, and in support of a mediating role of STATs in the activation of the p21 promoter, overexpression of Stat3 potentiated the cytokine effect on the p21 promoter; whereas a dominant negative Stat3, or a mutation of the STAT response element on the promoter, significantly reduced the cytokine effect. Finally, antisense oligonucleotides complementary to p21 mRNA inhibited OSM-induced stimulation of alkaline phosphatase expression and antagonized the protective effect of OSM on anti-Fas-induced apoptosis. These results demonstrate that p21 is a downstream effector of gp130/Stat3 activation and a critical mediator of the pro-differentiating and anti-apoptotic effects of IL-6 type cytokines on human osteoblastic cells.

Effect of desferrioxamine and deferiprone (L1) on the proliferation of MG-63 bone cells and on phosphatase alkaline activity.

In the present work, we studied: (i) the effect of different doses of desferrioxamine and deferiprone on the proliferation of osteoblasts and the possible role of iron in this; and (ii) the effect of both chelators on the metabolic activity of these cells, using the quantification of alkaline phosphatase as an enzymatic marker of cellular activity or differentiation. Cellular proliferation was investigated at different concentrations of deferiprone and desferrioxamine, and the effect of the addition of iron citrate on this proliferation was measured. The production of alkaline phosphatase after incubation with deferiprone (150 and 300 microM) and desferrioxamine (20 and 100 microM) was also studied. Cellular proliferation was completely inhibited with 100 microM of desferrioxamine and 300 microM of deferiprone. In both cases, this effect was corrected by means of co-incubation with iron citrate. In the second phase, using the same dose that inhibited the proliferation, it was observed that after 24 h, both chelators slightly decreased alkaline phosphatase activity, while at 48 and 96 h they increased alkaline phosphatase activity. These results demonstrate that both desferrioxamine and deferiprone inhibit the proliferation of the osteoblast-like cell line MG-63; the effect seems to be related to the chelation of some fraction of available iron. In spite of the effect on bone cell proliferation, the chelators do not impair cellular activity.

Proteolytic fragments of insulin-like growth factor binding protein-3: N-terminal sequences and relationships between structure and biological activity

Insulin-like growth factors (IGF-I and IGF-II) in biological fluids bind to high-affinity binding proteins (IGFBP-1 to -6), which transport them and regulate their activities. Limited proteolysis of certain IGFBPs plays a major role in this regulation. IGFBP-3 is proteolysed in vivo and in several cell lines by serine proteases, including plasmin. In earlier studies we reproduced this proteolysis in vitro using recombinant human non-glycosylated IGFBP-3. Two major fragments were obtained, the larger retaining weak affinity for IGF-I and weakly inhibiting IGF I mitogenic effects. The smaller fragment, though lacking affinity for IGFs, is a potent growth inhibitor. These proteolytic fragments were isolated by HPL C and their N-terminal amino acids sequenced. Both major fragments contain the N-terminal region of the intact protein, the larger form corresponding to residues 1–160, and the smaller form, to residues 1–95. Kinetics experiments using the MG-63 osteoblast-like cell line showed that the larger peptide is generated before the smaller peptide, the latter probably being a product of secondary proteolysis of the former. Our data suggest that proteolysis of IGFBP-3 is intimately linked to its biological function. We propose a model for its action at cellular level.

Selective effect of thiazides on the human osteoblast-like cell line MG-63

Thiazide diuretics have been shown to decrease bone-loss rate and to improve bone mineral density in patients using this medication. However, the exact role of thiazides on bone cells is still debated. In the present work, we studied whether thiazides could affect the normal features of osteoblasts using the human model cell line MG-63. Hydrochlorothiazide (HCTZ) did not affect cell growth nor DNA synthesis in these cells, yet slightly increased alkaline phosphatase activity in these cells at pharmacologically relevant concentrations. Under similar conditions, HCTZ dose-dependently inhibited 1,25(OH)2D3-induced osteocalcin secretion by these cells (maximal effect, -40 to 50%, P < 0.005). However, HCTZ did not inhibit the basal production of osteocalcin in MG-63 cells (without 1,25(OH)2D3 induction), which was very low to undectable. Two different thiazide derivatives, chlorothiazide and cyclothiazide, and two structurally related sulfonamides with selective inhibition of carbonic anhydrase (Acetazolamide) or hyperglycemic effects (Diazoxide) were also tested. Chlorothiazide (1000 microM) inhibited osteocalcin secretion (-42 +/- 12.7%) at doses 10-fold higher than HCTZ (100 microM) while cyclothiazide was effective at doses of 1 microM (-27 +/- 3.6%), and hence 100-fold lower than HCTZ, compatible with the relative natriuretic effect in vivo of these compounds. Acetazolamide (10 microM) poorly affected osteocalcin secretion at doses 100-fold higher than those needed in vivo to inhibit carbonic anhydrase. Likewise, Diazoxide (100 microM) poorly affected osteocalcin secretion at doses known to promote its biological effect. Higher doses of acetazolamide and diazoxide induced cell death. Neither Acetazolamide nor Diazoxide affected alkaline phosphatase, whereas chlorothiazide had a weak positive effect on this enzymatic activity. The production of macrophage colony-stimulating factor (M-CSF) was stimulated in the presence of 1,25(OH)2D3 (50 nM), TNF-alpha (2 ng/ml) or both in MG-63 cells. HCTZ (25 microM, 24 hr of preincubation) did not modify basal M-CSF production and did not reduce the response to 1,25(OH)2D3 alone. In contrast, HCTZ inhibited the response to TNF-alpha alone (P < 0.05), and also reduced the response to a combination of 1,25(OH)2D3 and TNF-alpha (P < 0.01). In conclusion, these results indicate that thiazide diuretics show a selective inhibition of osteocalcin secretion and M-CSF production by MG-63 cells unlike structurally related drugs. Therefore, these features may explain, in part, the positive effect of thiazides on bone mineral density.

Characterization of an antiproliferative surface-associated protein from Actinobacillus actinomycetemcomitans which can be neutralized by sera from a proportion of patients with localized juvenile periodontitis.

The gentle agitation of suspensions of Actinobacillus actinomycetemcomitans serotype a, b, or c in saline resulted in the release of a proteinaceous surface-associated material (SAM) which produced a dose-dependent inhibition of tritiated thymidine incorporation by the osteoblast-like cell line MG63 in culture. This cell line was sensitive to low concentrations of SAM (50% inhibitory concentration, 200 ng/ml for serotype c). Immunoglobulin G antibodies to constituents of the SAM were found in the blood of patients with localized juvenile periodontitis (LJP). Sera from 9 of 16 patients with LJP significantly neutralized the antiproliferative activity of the SAM, while sera from 15 controls, with no evidence of periodontal disease, were unable to neutralize this activity. Neutralization was not directly related to the patient's antibody titer to the whole SAM. Characterization of the antiproliferative activity in the SAM demonstrated that it was not cytotoxic and was heat and trypsin sensitive. The active component separated in a well-defined peak in anion-exchange high-performance liquid chromatography (HPLC) which, when further analyzed by size exclusion HPLC, revealed a single active peak, which had an apparent molecular mass of approximately 8 kDa. The lipopolysaccharide from A. actinomycetemcomitans was only weakly active. SAM from Porphyromonas gingivalis W50 and Eikenella corrodens NCTC 10596 did not exhibit any antiproliferative activity with this cell line, even at concentrations as high as 10 micrograms/ml. This study has shown that SAM from A. actinomycetemcomitans contains a potent antiproliferative protein whose activity can be neutralized by antibodies in the sera from some patients with LJP.

Effects of interleukin-4 on human osteoblast-like cells

Interleukin-4 (IL-4) modulates the activity of a variety of lymphoid, hemopoietic and mesenchymal cells, but little is known about its influence on bone cells. We have studied the effects of IL-4 on the human osteoblast-like cell line MG63. IL-4 (0.1-50 ng/ml) inhibited cell proliferation. The effect did not depend on cell density, but it was more marked in serum-free cultures than in the presence of serum. IL-4 also induced a dose-dependent increase in the expression of alkaline phosphatase stimulated by 1,25-dihydroxyvitamin D3, a marker of differentiated osteoblast activity. However, IL-4 did not modify the secretion of interleukin-6 or tumor necrosis factor. These results suggest that interleukin-4 may play a role as a modulator of osteoblast activity.

Regulation of osteocalcin secretion by human primary bone cells and by the human osteosarcoma cell line MG-63

We present evidence that the regulation of osteocalcin secretion by PTH and PGE 2 in normal human bone cells can be produced in the human osteoblast-like cell line MG-63. Both cell cultures showed time-and dose-dependent stimulation of osteocalcin secretion in response to 1,25(OH) 2D 3. Bovine parathyroid hormone (PTH) amino acid fragment 1–34 (40 nM) and prostaglandin E 2 (PGE 2, 5 nM) significantly inhibited 1,25(OH) 2D 3-induced osteocalcin secretion by these cells. The inhibition reached 20 and 36%, respectively. In contrast, PTH 3–34 had no effect on osteocalcin secretion. Both cell cultures produced cAMP in response to PTH. Dexamethasone (Dex) (100 nM) potentiated PTH-induced (40 nM) cAMP synthesis in subconfluent MG-63 cells (1.5-fold increase, P < 0.05). This treatment with Dex resulted in a greater inhibition of 1,25(OH) 2D 3-induced osteocalcin secretion (−30%, P < 0.005) by PTH in MG-63 cells as compared to cells exposed to PTH and 1,25(OH) 2D 3 alone. Pretreatment of subconfluent MG-63 cells with Dex (100 nM) for 48 h also increased 1,25(OH) 2D 3-induced osteocalcin secretion by 40% ( P < 0.025). In contrast, treatments of confluent MG-63 cells with Dex inhibited osteocalcin secretion regardless of the 1,25(OH) 2D 3 doses used. Forskolin (10 −7–10 −5 M) and dibutyryl cAMP (10 −6–10 −3 M) both reproduced the effects observed with PTH and PGE 2 in the two cell cultures. Forskolin's action was time-dependent: addition of forskolin (10 −6 M) 12 h after 1,25(OH) 2D 3 (50 nM) resulted in a progressively weaker inhibition of osteocalcin secretion. Increasing the extracellular calcium concentration of the incubation media resulted in a dose-dependent increase in osteocalcin secretion ( P < 0.01). These results indicate that PTH and PGE 2 inhibit osteocalcin secretion by a mechanism involving cAMP production. In contrast, an increase in extracellular calcium stimulated osteocalcin release. Thus the human osteosarcoma cell line MG-63 is a useful osteoblast-like cell model to study the regulation of osteocalcin secretion. Furthermore, a factor (or factors) between hormone-receptor coupling and gene induction can regulate the expression of the osteocalcin gene or affect pre- or posttranslational mechanisms implicated in osteocalcin synthesis and secretion.