In vitro study of osteoblastic cells from patients with idiopathic osteoporosis and comparison with cells from non-osteoporotic controls [1]

We have examined bone cells derived from iliac crest trabecular explants of 30 patients with idiopathic osteoporosis and 45 control subjects in order to determine whether intrinsic abnormalities in osteoblast function may contribute to the decreased bone formation observed in this disease. Bone cells isolated from all subjects expressed several in vitro characteristics of the osteoblast phenotype including adenylate cyclase responsiveness to parathyroid hormone (PTH) and prostaglandin E₁ (PGE₁), basal and 1,25(OH)₂D₃-stimulated alkaline phosphatase activity and osteocalcin production. Results were compared amongst three subject groups: young controls less than 40 years old, older controls over 40 years old, and osteoporotics. Osteoporotic cells were found in general to be fully active in vitro. There were no differences between osteoporotic and control cells in their basal levels of adenylate cyclase, or alkaline phosphatase, in their growth rates, or cell morphology. The cyclic AMP (cAMP) response to PTH was significantly lower in osteoporotic cells (71%,p<0.01) and older control cells (64% ,p<0.005) relative to the response in cells from younger controls, suggesting that the decreased responsiveness in osteoporotic cells was due to subject age rather than the osteoporotic state. At the same time, the cAMP responses to PGE₁ and cholera toxin were similar in cells from all three subject groups. The response to forskolin was reduced to about 40% in osteoporotic cells compared with controls, but this was not mirrored by similar differences in the responses to PTH, PGE₁ or cholera toxin, suggesting that the availability of catalytic subunits is not rate-limiting in these cells. l,25 (OH)₂D₃-stimulated osteocalcin production was 220% higher in osteoporotics than in older controls, but the numbers tested were small and the difference did not reach significance. The one significant abnormality we observed in osteoporotic cells was in alkaline phosphatase activity: 1,25(OH)₂D₃-stimulated alkaline phosphatase activity was twofold higher in osteoporotics than in younger (p<0.05), older (p<0.05) and pooled controls (p<0.025). The significance of this finding is unknown, but we postulate that it may reflect an intrinsic abnormality in osteoblast function in patients with idiopathic osteoporosis.     

Modulation of PTH-stimulated cyclic AMP in cultured rodent bone cells: the effects of 1,25(OH)2 vitamin D3 and its interaction with glucocorticoids

Parathyroid hormone (PTH)-stimulated cyclic adenosine monophosphate (cAMP) in rat osteoblastlike (OB) cells has been shown to be modulated by steroid hormones; glucocorticoids are known to increase the level, while the effects of 1,25(OH)2D3 are inhibitory. In the present study, we found that the PTH-stimulated cAMP responses are similar in neonatal mouse and fetal rat OB cells. Dexamethasone (0.13-13 nM) augmented PTH-stimulated cAMP in both species. Mouse cells showed a higher maximal response to dexamethasone (100% increment) than rat cells (60-70% increment) with similar sensitivity to dexamethasone (ED50 approximately 1.0 nm). On the other hand, 1,25(OH)2D3 decreased PTH-stimulated cAMP, but the effect required pharmacological levels of hormone; mouse cells responded at a lower dose (1.3 nM) and were more sensitive than rat cells (responded at 13 nM) to 1,25(OH)2D3 treatment. Introduction of physiological concentrations of 1,25(OH)2D3 (0.013-1.3 nm) in addition to dexamethasone (13 nM) resulted in a synergistic enhancement of PTH-stimulated cAMP in rat cells. In contrast, a dose-dependent antagonistic effect was observed in mouse cells. In summary, our findings demonstrate species and concentration-dependent differences in hormonal responses to 1,25(OH)2D3 and a complex interplay among PTH, dexamethasone, and 1,25(OH)2D3.     

Comparative effects of a novel vitamin D analogue MC-903 and 1,25-dihydroxyvitamin D3 on alkaline phosphatase activity, osteocalcin and DNA synthesis by human osteoblastic cells in culture

MC-903 is a novel vitamin D analogue which has been shown to promote epidermal cell differentiation but is 100 times less active than 1,25 dihydroxyvitamin D3 (1,25(OH)2D) in causing hypercalcemia. In order to determine the activity of this compound on bone cells, we have compared the effects of MC-903 and 1,25 dihydroxyvitamin D3 (1,25(OH)2D) on parameters of cell proliferation and differentiation in cultured normal human osteoblastic cells derived by migration from trabecular bone fragments. Dose response curves showed that MC-903 was 10 to 100 times less effective than 1,25(OH)2D in stimulating the synthesis of the osteoblast specific protein osteocalcin by human bone cells depending on the basal osteocalcin production. In cells showing high basal osteocalcin synthesis, 1,25(OH)2D (10(-8) M) was 2- to 3-fold more potent than MC-903 (10(-8) M) in inducing osteocalcin from 48 to 96 h of treatment. The greater activity of 1,25(OH)2D over MC-903 was observed in human bone cell cultures with elevated basal osteocalcin levels, indicating that the response to 1,25(OH)2D but not to MC-903 was amplified in cells with the higher osteoblastic characteristics. The effects of MC-903 and 1,25(OH)2D on alkaline phosphatase activity were not markedly different. Transforming Growth Factor-beta (TGF beta) (0.5 ng/mL, 48 h) was found to completely suppress the osteocalcin synthesis induced by 1,25(OH)2D (10(-8) and 10(-9) M), whereas the MC-903-induced osteocalcin synthesis was not affected, suggesting a negative interaction between TGF beta and 1,25(OH)2D but not MC-903 on osteocalcin synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of PTH-stimulated cyclic AMP in cultured rodent bone cells: The effects of 1,25(OH)2 vitamin D3 and its interaction with glucocorticoids

Summary Parathyroid hormone (PTH)-stimulated cyclic adenosine monophosphate (cAMP) in rat osteoblastlike (OB) cells has been shown to be modulated by steroid hormones; glucocorticoids are known to increase the level, while the effects of 1,25(OH)₂D₃ are inhibitory. In the present study, we found that the PTH-stimulated cAMP responses are similar in neonatal mouse and fetal rat OB cells. Dexamethasone (0.13–13nM) augmented PTH-stimulated cAMP in both species. Mouse cells showed a higher maximal response to dexamethasone (100% increment) than rat cells (60–70% increment) with similar sensitivity to dexamethasone (ED₅₀ ∼ 1.0 nm). On the other hand, 1,25(OH)₂D₃ decreased PTH-stimulated cAMP, but the effect required pharmacological levels of hormone; mouse cells responded at a lower dose (1.3 nM) and were more sensitive than rat cells (responded at 13 nM) to 1,25(OH)₂D₃ treatment. Introduction of physiological concentrations of 1,25(OH)₂D₃ (0.013–1.3 nm) in addition to dexamethasone (13 nM) resulted in a synergistic enhancement of PTH-stimulated cAMP in rat cells. In contrast, a dose-dependent antagonistic effect was observed in mouse cells. In summary, our findings demonstrate species and concentration-dependent differences in hormonal responses to 1,25(OH)₂D₃ and a complex interplay among PTH, dexamethasone, and 1,25(OH)₂D₃.     

Isolation and hormonal responsiveness of primary cultures of human bone-derived cells: gender and age differences

We present a model for isolating human cell culture derived from biopsies obtained during orthopedic surgery. Four donor groups were defined by gender and age: pre- and postmenopausal women (<50 and >55 years, respectively), and younger (30–55 years) and older (>60 years) men. Bone-derived cells were identified as osteoblasts by major osteoblastic characteristics; that is, high alkaline phosphatase (ALP) activity, dose-dependent increase of ALP by 1,25-(OH)₂D₃, high levels of parathyroid hormone (PTH)-induced cyclic AMP, and 1,25-(OH)₂D₃-induced osteocalcin. In all cells, levels of osteocalcin were significantly elevated (p < 0.05 and 0.01). In cells derived from men, no significant age differences were found in ALP and osteocalcin values of basal activity and in fold stimulation 1,25(OH)₂D₃. Cells from postmenopausal women showed a nonsignificant lower basal ALP activity than premenopausal cells. In postmenopausal cells, ALP responded less to 1,25(OH)₂D₃ (33% increase, p < 0.05) than the premenopausal cells (100% increase, p < 0.05). In cells from either age group, ALP did not respond to the gonadal steroids 17β-estradiol (E₂) and dihydrotestosterone (DHT) or progesterone. Basal levels of osteocalcin were higher in cells of premenopausal origin as compared with postmenopausal cells (p = 0.05), but response to 1,25(OH)₂D₃ was the same. PTH significantly stimulated cAMP (p = 0.001) in all age and gender groups analyzed. In all groups, no differences were found in either basal activity or in PTH response. Unlike men, cells derived from the bone of women were more susceptible to age changes. We postulate that the postmenopausal cell population had a decreased number of osteoblasts, or cells in a lower differentiation stage. These results extend our knowledge of bone biology found in animal models and reveal that human osteoblasts from men do not show the same age-dependent differences observed in women.     

Clonal osteogenic cell lines express myogenic and adipocytic developmental potential

Summary Clonal osteoblastic cell lines were isolated from neonatal rat calvariae and characterized with regard to a number of features associated with authentic osteoblasts. These included elevated alkaline phosphatase activity (relative to fibroblasts), PTH and PGE₂-stimulated increases in cAMP, the predominant synthesis of type 1 collagen, and the production of a mineralized matrixin vitro. By these criteria, five clones with osteoblast-like phenotypes were identified (ROB-C8a, C11, C20, C23, and C26) which varied somewhat in shape, levels of alkaline phosphatase activity, and in responsiveness to PTH and PGE₂, C11, C20, and C23 responded to both effector substances, whereas C8a only responded to PTH and C26 only responded strongly to PGE₂. Upon further examination, two of the clones (C23 and C26) were also found to exhibit significant muscle myotube formation after reaching confluence, and three of the clones (C8a, C11, and C26) showed marked adipocyte differentiation after treatment with dexamethasone. Overall, these data add further supporting documentation to (1) the suspected ontogenetic relationships of osteoblasts to other connective tissue cells, and (2) the concept that osteoblastic cells associated with neonatal rat calvariae are in various stable stages of differentiation and developmental commitment.     

Age-dependent expression of osteoblastic phenotypic markers in normal human osteoblasts cultured long term in the presence of dexamethasone

We have previously shown that osteoblasts derived from trabecular bone explants and cultured long term in 10 nM dexamethasone ((HOB+DEX) cells) exhibited properties consistent with a more differentiated phenotype compared with those grown in the absence of dexamethasone ((HOB-DEX) cells). To characterize these two cell models further, we measured the steady-state mRNA levels of the phenotypic markers alkaline phosphatase (ALP), collagen type I (COLL) and osteocalcin (OC), OC production, and the activities of ALP and parathyroid hormone (PTH)-stimulated adenylate cyclase. These findings were then correlated with the age and sex of the bone donors. Long-term culture in dexamethasone significantly increased ALP and OC mRNA levels and the activities of ALP and PTH-stimulated adenylate cyclase but not OC production, in (HOB+DEX) compared with (HOB-DEX) cells (p<0.05). When the data were examined with respect to the age of the bone donor, age-dependent differences in the expression and responses to dexamethasone were apparent. ALP and PTH-stimulated adenylate cyclase activities decreased with increasing age of the bone donor in (HOB-DEX) and (HOB+DEX) cells (p<0.05). There were no significant correlations between phenotypic marker mRNA levels and bone donor age in (HOB-DEX) and (HOB+DEX) cells. All age-dependent decreases in ALP and PTH-stimulated cyclase activities were enhanced in the (HOB+DEX) cells. However, when the data were examined according to the sex of the bone donor, there were no differences in mRNA levels, OC production, or ALP and cyclase activities between cells from male and female donors. These results indicate an age dependence in the expression of osteoblastic markers in human bone cells at different stages of differentiation: thus osteoblastic cultures derived from older donors are likely to contain fewer osteoprogenitor cells, lower levels of glucocorticoid receptors or represent more differentiated osteblasts compared with those derived from younger donors.     

Effect of Polyethylene on Osteocalcin, Alkaline Phosphatase and Procollagen Secretion by Human Osteoblastic Cells

We have studied the direct effects of polyethylene particles on osteoblastic function in primary human bone cell cultures. The cells were obtained from trabecular bone fragments of patients undergoing knee reconstructive surgery. When the cells reached confluency, they were subcultured into two flasks, one untreated (control culture) and the other treated with polyethylene particles, and incubated until confluency. Osteoblastic function was evaluated by assaying osteocalcin, alkaline phosphatase, and C-terminal procollagen type I, with and without 1,25(OH)₂D stimulation, in the cell-conditioned medium. We found that addition of polyethylene to these osteoblastic cell cultures induced higher levels of secreted osteocalcin after 1,25(OH)₂D stimulation. Alkaline phosphatase levels increased whereas C-terminal procollagen type I levels decreased in the cell conditioned medium after polyethylene was added to the cultures. Treatment of the control cultures with 1,25(OH)₂D stimulated alkaline phosphatase levels and decreased C-terminal procollagen type I. However, these osteoblastic markers in 1,25(OH)₂D-treated cells did not change in cultures with polyethylene. This study demonstrates that polyethylene particles have a direct effect on osteoblastic markers in human bone cells in culture. Received: 2 May 1996 / Accepted: 17 June 1997     

Human osteoblastlike cells do not respond to interleukin-6.

Interleukin 6 (IL-6) exerts well-established effects on cells of the immune system as well as on various other cell types. It has been implicated in the control of connective tissue cells in such conditions as rheumatoid arthritis and osteoporosis. We have investigated the effects of recombinant human interleukin-6 (rhIL-6) on human osteoblastlike cells derived from explants of trabecular bone. ROS 17/2.8 cells were used as an additional osteoblastlike cell model system. We were unable to identify any effects of rhIL-6 (5-5000 pg/ml) on the proliferation, alkaline phosphatase activity. osteocalcin production, or release of cytokines or prostaglandins by either osteoblastlike cell model system. Since we have shown previously that these cells release IL-6 in culture, we used a sheep anti-human IL-6 antibody to investigate the possibility that (1) action of added exogenous IL-6 could be masking endogenous production, and (2) endogenous IL-6 may regulate the effects of osteotropic agents on the osteoblastlike cells. Presence of the antibody exerted no detectable effects on 1,25-(OH)2D3-stimulated alkaline phosphatase or on proliferation or TNF production enhanced by IL-1. Thus IL-6 does not appear to be involved in the regulation of osteoblast activity.     

Differential regulation of prostaglandin E2 synthesis and phospholipase A2 activity by 1,25-(OH)2D3 in three osteoblast-like cell lines (MC-3T3-E1, ROS 17/2.8, and MG-63).

Both 1,25-(OH)2D3 and prostaglandin E2 (PGE2) stimulate alkaline phosphatase activity in MC-3T3-E1 cells. Previous studies, demonstrating a correlation between 1,25-(OH)2D3-dependent alkaline phosphatase and phospholipase A2 activities in matrix vesicles isolated from growth cartilage chondrocyte cultures, suggest that one mechanism of vitamin D action may be via autocrine or paracrine action of PGE2. Since most PGE2 is derived from arachidonic acid released by the action of phospholipase A2, we examined whether 1,25-(OH)2D3 stimulates phospholipase A2 activity in three osteoblastic cell lines: ROS 17/2.8 cells, MC-3T3-E1 cells, and MG-63 cells. 1,25-(OH)2D3-dependent alkaline phosphatase and phospholipase A2 activity were correlated with production of PGE2 and PGE1 in the MC-3T3-E1 cells. Alkaline phosphatase specific activity was enriched in the matrix vesicles produced by all three cell types and was stimulated by 1,25-(OH)2D3 at 10(-8) to 10(-7) M. Although phospholipase A2 specific activity was enriched in the matrix vesicles produced only by the ROS 17/2.8 cell cultures, stimulation of this enzyme activity was observed only in the MC-3T3-E1 cell cultures. The effects of 1,25-(OH)2D3 on phospholipase A2 were dose-dependent and were significant at 10(-8) to 10(-7) M. There was a significant increase in PGE2 production in the MC-3T3-E1 cell cultures only. Indomethacin reduced PGE2 production to base line values. Even at baseline, MC-3T3-E1 cells produced ten times more PGE2 than did the ROS 17/2.8 or MG-63 cell cultures. The effects of 1,25-(OH)2D3 on PGE1 were comparable to those on PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of osteoblast-like cells from normal adult rat femoral trabecular bone

Summary Osteoblast-like cell cultures have been established from the trabecular surfaces of normal adult rat femoral trabecular bone. The cultured cells responded to stimulation by parathyroid hormone (rPTH), with a rise in intracellular cAMP in excess of 25-fold while failing to respond to incubation with sCT. Furthermore, the osteoblast-like cells exhibited a high level of alkaline phosphatase expression, both histochemically and biochemically. Incubations with 1,25(OH)₂ vitamin D₃ increased the alkaline phosphatase activity by 50% and stimulated bone Gla-protein (BGP) synthesis. When the cell layers were supplemented with both 50 g/ml ascorbic acid and 10 mM -glycerophosphate and allowed to grow past confluency for 3 weeks, they formed calcified ridges and multilayered nodules. Confirmation of the mineralization of an extracellular matrix was made by von Kossa staining. This simple isolation technique now facilitates the availability of normal adult rat osteoblastic cells for investigation of bone and mineral metabolism.     

Bone cell responsiveness to transforming growth factor beta, parathyroid hormone, and prostaglandin E2 in normal and postmenopausal osteoporotic women

We have shown previously that the decreased trabecular bone formation in osteoporotic postmenopausal women results from a reduced ability of osteoblastic cells to proliferate. In this study we have tested the possibility that bone cells from osteoporotic women with low bone formation have an abnormal responsiveness to hormonal or local mitogenic factors. Primary cultures of bone cells with osteoblastic characteristics were obtained by migration from the trabecular bone surface in osteoporotic postmenopausal women with high (n = 7) or low (n = 7) bone formation as evaluated histomorphometrically by the extent of double tetracycline-labeled surface (DLS). Control bone cells were obtained under identical conditions from eight normal age-matched postmenopausal women. Parameters of osteoblastic differentiation (alkaline phosphatase activity and osteocalcin production) were found to be normal and similar in bone cells from osteoporotic women with low or high DLS. In contrast, cell replication as evaluated by [3H]thymidine into DNA was 3.4-fold lower in the low DLS group compared to the high DLS group, confirming our previous findings. Treatment of quiescent bone cells with TGF-beta (0.5-1 ng/ml) for 24 h significantly stimulated DNA synthesis in osteoblastic cells from normal women and in bone cells from osteoporotic patients with low or high DLS, indicating a normal responsiveness to TGF-beta in these patients. We have compared the effect of parathyroidhormone (PTH) on bone cells from normal and osteoporotic women. Basal cAMP levels and the cAMP accumulation in response to (1-34)-hPTH were similar in bone cells from patients with low or high DLS and were not different from normal values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of endosteal osteoblastic cells isolated from mouse caudal vertebrae

We have developed a reliable procedure for isolating endosteal osteoblasts from mouse trabecular bone. Endosteal osteoblasts were obtained by migration and proliferation of the cells from the metaphyseal bone surface of caudal vertebrae onto nylon meshes. The isolated cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. The cell population consisted of 95% alkaline-phosphatase-positive cells. The cell level of alkaline phosphatase was elevated (1.19 +/- 0.26 (SD) mumol PNP/mn/mg protein) and the enzyme activity was heat-inhibitable, indicating its skeletal origin. Light and electron microscopic observation revealed that cells have morphologic and ultrastructural appearance of typical osteoblasts with high protein synthesis activity. Osteoblasts grown in multilayers in the presence of 50 micrograms/ml ascorbic acid produced within 4 days an abundant fibrous intercellular collagenous matrix forming nodules in which osteocyte-like cells were embedded. Immunolabeling revealed synthesis of type I collagen but no detectable type III collagen. In presence of 7 mM beta-glycerophosphate the matrix became mineralized after 14-21 days of culture. Mineralization could not be induced by mouse skin fibroblasts cultured under similar conditions. The mineral deposits were closely associated with the collagen matrix, consisted of EDTA-removable, Von Kossa and alizarin red S stainable material and were composed of hydroxyapatite crystals identified by X-ray electron probe microanalysis. The isolated endosteal osteoblasts also displayed an intense (+457%) increase in intracellular cAMP production in response to human (1-34) PTH (2 x 10(-8) M) stimulation. The confluent cells responded to 20 nM 1,25(OH)2D3 by a significant 45% reduction in heat labile alkaline phosphatase activity. This procedure allowed us to isolate from trabecular bone a cell population that differentiates into osteoblasts in vitro, respond to calcitropic hormones and that retains its capacity to form a calcified bone tissue in culture. This method provided us a culture system for investigating the differentiation and metabolism of endosteal osteoblastic bone forming cells.     

Stimulation by 1,25-dihydroxyvitamin D3 of in vitro mineralization induced by osteoblast-like MC3T3-E1 cells

Although vitamin D is essential for mineralization of bone, it is as yet unclear whether vitamin D has a direct stimulatory effect on the bone mineralization process. In the present study, the effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on in vitro mineralization mediated by osteoblast-like MC3T3-E1 cells was examined. MC3T3-E1 cells continued to grow after they reached confluency, and DNA content and alkaline phosphatase activity increased linearly until about 16 days of culture, whereas 45Ca accumulation into cell and matrix layer remained low. After this period, DNA content plateaued, and 45Ca accumulation increased sharply. Histological examination by von Kossa staining revealed that calcium was accumulated into extracellular matrix. In addition, needle-shaped mineral crystals similar to hydroxyapatite crystals could be demonstrated in between collagen fibrils by electron microscopy. Thus, MC3T3-E1 cells differentiate in vitro into cells with osteoblastic phenotype and exhibit mineralization. When MC3T3-E1 cells were treated with 1,25(OH)2D3 at this stage of culture, there was a dose-dependent stimulation of 45Ca accumulation by 1,25(OH)2D3, and a significant stimulation of 45Ca accumulation was observed with 3 x 10(-10) M 1,25(OH)2D3. Although 1,25(OH)2D3 enhanced alkaline phosphatase activity and collagen synthesis at the early phase of culture, it did not affect any of these parameters at the late phase when 1,25(OH)2D3 stimulated mineralization. Neither 24,25-dihydroxyvitamin D3 nor human PTH(1-34) affected mineralization in the presence or absence of 1,25(OH)2D3. These results demonstrate that 1,25(OH)2D3 stimulates matrix mineralization induced by osteoblastic MC3T3-E1 cells, and are consistent with the possibility that 1,25(OH)2D3 has a direct stimulatory effect on bone mineralization process.(ABSTRACT TRUNCATED AT 250 WORDS)     

1,25-dihydroxyvitamin D reduces parathyroid hormone receptor number in ROS 17/2.8 cells and prevents the glucocorticoid-induced increase in these receptors: relationship to adenylate cyclase activation.

We have previously shown that 1,25-dihydroxyvitamin D [1,25-(OH)2D3] and glucocorticoid modulate adenylate cyclase activation by PTH in osteoblast-like cells. Here we examine whether steroid effects on PTH receptor density explain the modulation of PTH action. Receptor assays were performed on late logarithmicphase monolayers of ROS 17/2.8 cells using human PTH-like peptide (hPLP) as radioligand. Kd and receptor density were computed from competition of tracer amounts of [125I-Tyr36] hPLP-(1-36) with unlabeled hPLP-(1-36) (0.1-30 nM). Steroid treatment had little or no effect on affinity for ligand. Pretreating cells with 10 nM 1,25-(OH)2D3 for 48 h decreased PTH receptor number to 17% of control values. Treating cells with 10 nM of the glucocorticoid triamcinolone acetonide (TRM) increased receptor number 10-fold, but simultaneous treatment with 1,25-(OH)2D3 (10 nM) completely prevented this receptor increase. Steroid effects required 13-18 h of treatment. Dose-response relationships for steroid modulation, determined from binding at 0.17 nM radioligand, indicated an EC50 of 0.3 nM for glucocorticoid augmentation of PTH receptor number and 0.02 nM for 1,25-(OH)2D3 reduction of receptor number in the presence of absence of the maximum TRM effect. The initial rate of cAMP production by receptor-saturating concentrations of PTH was 11,500 molecules per receptor per minute in untreated cells, comparable to reported turnover numbers for mammalian adenylate cyclase. Control experiments were validated measuring cAMP in intact cells as an indicator of adenylate cyclase activity. Cyclic AMP production was reduced 63% by 1,25-(OH)2D3 (10 nM) treatment. Glucocorticoid (10 nM) enhanced cAMP production twofold but reduced cAMP generation per receptor by 80%.(ABSTRACT TRUNCATED AT 250 WORDS)     

A newly established human osteosarcoma cell line with osteoblastic properties

A human osteosarcoma cell line, HuO9, was established from a tumor that was heterotransplanted into athymic nude mice. Antiserum against nude mouse spleen cells was added to the early passage cultures to eliminate the host fibroblastic cells. The cell line retained a high activity of liver/bone/kidney-type alkaline phosphatase (ALP) and secreted osteocalcin, i.e., bone gamma-carboxyglutamic acid-containing protein (BGP), into the medium. The addition of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) increased the ALP activity as well as the level of BGP secreted into the medium. The ALP of 1,25(OH)2D3-treated cells has the same inhibition characteristics to heat and amino acids as that of untreated cells. Synthetic human parathyroid hormone stimulated the production of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) approximately 100-fold within five minutes. However, the stimulation was not observed with a synthetic human thyrocalcitonin. When HuO9 cells were transplanted into the back of a nude mouse, a tumor with an abundant osteoid formation and mineralization was produced. The results indicate that the HuO9 cell line expresses well-differentiated osteoblastic phenotypes. HuO9 is the first established human cell line to produce BGP, and it provides a useful model for the studies of osteoblasts and the regulatory mechanisms of BGP production.     

Osteocalcin secretion by the human osteosarcoma cell line MG-63

The human osteosarcoma cell line MG-63 has been used to study the production of the bone-specific protein, osteocalcin. In the absence of any stimuli, MG-63 cells secreted very low levels of osteocalcin. The secretion of osteocalcin started after a lag time of 10-12 h upon 1,25-(OH)2D3 treatment. Osteocalcin secretion was measured at doses as low as 0.03 nM (fourfold increase, p less than 0.05), and this activity increased further with higher doses of 1,25-(OH)2D3 to reach a plateau at 50 nM. The secretion increased transiently from very low levels in sparse cell cultures to peak values in subconfluent cultures (+/- 40%), two- to threefold above values obtained for confluent cells. Values for confluent cells average 55.9 +/- 2.0 ng/ml protein per 48 h. A similar behavior is observed for 1,25-(OH)2D3 receptor concentration under similar experimental conditions. Bmax increased transiently from sparse to subconfluent cell cultures (40-60% confluent) and reached values 50% lower in confluent cells. However, the receptor affinity was not affected by cell density. MG-63 cells also possessed an alkaline phosphatase isoenzyme of the bone-liver-kidney type that was stimulated by 1,25-(OH)2D3 treatment (two- to threefold) and inhibited by parathyroid hormone (40 nM, -25%, p less than 0.025). PTH and PGE2 increased cAMP production in a dose-dependent manner, but the cells were irresponsive to salmon calcitonin. Basal and PTH-responsive cyclic AMP production were also modulated by cell density. Dexamethasone pretreatment (100 nM, 48 h) stimulated the PTH-dependent cAMP production but failed to influence the response to PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hormonal Regulation of the Osteoblastic Phenotype Expression in Neonatal Murine Calvarial Cells

Osteoblastic cell cultures from fetal rat calvariae have provided a popular model for studying the effects of dexamethasone (DEX) and 1,25 dihydroxyvitamin D₃ [1,25(OH)₂D₃] on gene expression but data from murine calvarial cells are scarce. Species-specific responses of rat and mouse osteoblastic cells to these hormones have been reported previously. In the present study, we investigated the effects of DEX and 1,25(OH)₂D₃ on expression of the osteoblastic phenotype by mouse calvarial cells. These murine osteoblast-like (MOB) cells expressed alkaline phosphatase (ALP) activity and osteocalcin and formed calcified nodules. Unlike the rat calvarial cells, ALP activities and nodule formation in MOB were inhibited by DEX. 1,25(OH)₂D₃ enhanced and DEX lowered the amount of osteocalcin synthesized by MOB. 1,25(OH)₂D₃ did not affect the number of nodules, but increased their sizes. Treating the cells for 2 days with only DEX at the beginning of the culture enhanced the effect of 1,25(OH)₂D₃ on ALP. We found that in murine calvarial cells, DEX inhibits and 1,25(OH)₂D₃ enhances ALP activity, osteocalcin synthesis, and calcified nodule formation. This is in contrast to previous reports of rat calvarial cells where DEX is a positive and 1,25(OH)₂D₃ can be a negative regulator of the osteoblastic phenotype. These results suggest that profound species-specific differences exist between mice and rats in the regulation of the osteoblastic phenotype. Received: 15 October 1997 / Accepted: 16 June 1998     

Prostaglandins mediate the effects of 1,25-(OH)2D3 and 24,25-(OH)2D3 on growth plate chondrocytes in a metabolite-specific and cell maturation-dependent manner.

Prior studies have shown that 1,25-(OH)2D3 stimulates alkaline phosphatase, phospholipase A2 (PLA2), and protein kinase C (PKC)-specific activities, and production of prostaglandin E2 (PGE2) in growth zone chondrocytes. In contrast, 24,25-(OH)2D3 stimulates alkaline phosphatase and PKC-specific activities but inhibits PLA2-specific activity and PGE2 production in resting zone cells. This indicates that different mechanisms are involved in the action of 1,25-(OH)2D3 and 24,25-(OH)2D3 on their respective target cells. In this study, we examined the hypothesis that differential regulation of prostaglandin production modulates the activity of PKC and alkaline phosphatase. To do this, we examined the effect of the cyclooxygenase inhibitor indomethacin (Indo) on alkaline phosphatase, PLA2, and PKC-specific activities in growth plate chondrocytes treated with these two vitamin D metabolites. In addition, we examined whether inhibition of PKC altered PGE2 production. In growth zone cells, Indo inhibited basal alkaline phosphatase and blocked the 1,25-(OH)2D3-dependent increase in alkaline phosphatase. This effect was due to inhibition of both plasma membrane and matrix vesicle alkaline phosphatase. In resting zone cells, Indo increased basal alkaline phosphatase activity in a dose-dependent manner, but it did not further enhance the 24,25-(OH)2D3-dependent stimulation of this enzyme. The effect of Indo was found in both plasma membranes and matrix vesicles. These data indicate that 1,25-(OH)2D3-dependent increases in alkaline phosphatase-specific activity in growth zone cells are mediated through increased prostaglandin production, whereas 24,25-(OH)2D3-mediated changes in enzyme activity in resting zone cells are mediated through decreased prostaglandin production. Regulation of PLA2 by either 1,25-(OH)2D3 or 24,25-(OH)2D3 in their target cells was unaffected by Indo, indicating that the effect of the vitamin D metabolites on this enzyme is not dependent on changes in PGE2 production. The rapid increase in 1,25-(OH)2D3-dependent PKC-specific activity in growth zone cells was inhibited by Indo, whereas there was a potentiation of the effect of 24,25-(OH)2D3 on PKC activity in resting zone cells. In addition, inhibition of PKC blocked the 1,25-(OH)2D3-dependent increase in PGE2 production in growth zone cells and the 24,25-(OH)2D3-dependent decrease in PGE2 production by resting zone cells. These data indicate that prostaglandins are involved in mediating the rapid effects of 1,25-(OH)2D3 on growth zone cells, and contribute to the effects of 24,25-(OH)2D3 on resting zone cells; in both instances, the vitamin D metabolites exert their effects on PKC through changes in arachidonic acid via the action of PLA2. In addition, PKC by itself may mediate the production of PGE2.     

Effect of phosphate supplementation on the expression of the mutant phenotype in murine X-linked hypophosphatemic rickets

The X-linked mouse, a murine homologue of X-linked hypophosphatemia in humans, is characterized by rachitic bone disease, hypophosphatemia, impaired renal brushborder membrane Na⁺-phosphate cotransport and abnormal regulation of renal vitamin D metabolism. We demonstrated that short-term phosphate supplementation decreases renal 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D) catabolism and increases serum 1,25-(OH)₂D levels in mice (Tenenhouse & Jones 1990). In the present study, we compared several other parameters in normal and mice fed control (1%) and high (1.6%) phosphate diets for 4 days. Phosphate supplementation significantly raised serum phosphate levels and decreased renal brush-border membrane Na⁺-phosphate, but not Na⁺-glucose, cotransport in both genotypes (67% of control diet, p < 0.05). However, under both dietary conditions, the phosphate/glucose transport ratio was significantly reduced in mice (58% of normal littermates, p < 0.05). Renal PTH-stimulated cAMP accumulation, which was significantly blunted in mice compared to normal mice under control dietary conditions (p < 0.05), was not altered by phosphate supplementation in either genotype. Serum alkaline phosphatase activity was significantly higher than normal in mice on the control diet and was further increased in mutants but not in normals fed the high phosphate diet (p < 0.05). Measurements of serum bilirubin and electrophoresis of serum alkaline phosphatase suggested that the elevation in serum alkaline phosphatase activity in phosphate-supplemented mice represents the bone-derived isozyme. The present study demonstrates that, in contrast to the abnormality in vitamin D metabolism, the abnormalities in renal brush-border membrane Na⁺-phosphate cotransport, PTH-stimulated cAMP accumulation and serum alkaline phosphatase activity are not corrected by phosphate supplementation of mice. We suggest that the increase in serum alkaline phosphatase activity in phosphatesupplemented mice represents a stimulation of osteoblastic activity in response to the increase in available phosphate and elevated serum 1,25-(OH)₂D levels.