Fetal Rat Calvaria Cells Research Articles

Inhibition of osteoblastic cell differentiation by conditioned medium derived from the human prostatic cancer cell line PC-3 in vitro

Human prostatic carcinoma frequently metastasizes to bone tissue and activates bone metabolism, especially bone formation, at the site of metastasis. It has been reported that an extract of prostatic carcinoma and conditioned medium (CM) of a human prostatic carcinoma cell line, PC-3, established from a bone metastastic lesion, stimulate osteoblastic cell proliferation. However, there is little information about the effect of PC-3 CM on the differentiation of osteoblastic cells. In this study, we investigated the effect of PC-3 CM on the differentiation of two types of osteoblastic cells, primary fetal rat calvaria (RC) cells containing many undifferentiated osteoprogenitor cells, and ROS 17/2.8, a well-differentiated rat osteosarcoma cell line. PC-3 CM inhibited bone nodule formation and the activity of alkaline phosphatase (ALPase), an osteoblastic marker enzyme, on days 7, 14, and 21 (RC cells) or 3, 6, and 9 (ROS 17/2.8 cells) in a dose-dependent manner (5-30% CM). However, the CM did not affect cell proliferation or cell viability. PC-3 CM was found to markedly block the gene expression of ALPase and osteocalcin (OCN) mRNAs but had no effect on the mRNA expression of osteopontin (OPN), the latter two being noncollagenous proteins related to bone matrix mineralization. These findings suggest that PC-3 CM contains a factor that inhibits osteoblastic cell differentiation and that this factor may be involved in the process of bone metastasis from prostatic carcinoma.

Osteogenic Protein-1 and Insulin-Like Growth Factor I Synergistically Stimulate Rat Osteoblastic Cell Differentiation and Proliferation1

Previous studies have shown that osteogenic protein-1 (OP-1; also known as BMP-7) alters the steady state levels of messenger RNA (mRNA) encoding insulin-like growth factor I (IGF-I), IGF-II, and IGF-binding proteins (IGFBPs) in primary cultures of fetal rat calvaria (FRC) cells. In the present study, the effects of exogenous IGF-I on bone cell differentiation and mineralized bone nodule formation induced by OP-1 were examined. Exogenous IGF-I synergistically and dose dependently enhanced OP-1 action in stimulating [3H]thymidine incorporation, alkaline phosphatase activity, PTH-dependent cAMP level, and bone nodule formation. Maximal synergism between OP-1 and IGF-I was observed when both factors were added simultaneously. Synergism was not observed when FRC cells were pretreated with IGF-I for 24 h, followed by OP-1 treatment. These findings suggest that IGF-I acted on OP-1-sensitized FRC cells. To examine the mechanism(s) by which this sensitization may occur, levels of mRNA encoding OP-1 receptor, IGF-I receptor, and IGFBPs were measured. The mRNA levels of both type I and II OP-1 receptors were elevated by OP-1, but were not changed further by combined OP-1 and IGF-I treatment. IGF-I receptor gene expression was not changed by OP-1, IGF-I, or a combination of both factors. OP-1 alone or together with IGF-I increased the steady state IGFBP-3 mRNA level and reduced the steady state mRNA levels of IGFBP-4, -5, and -6. IGF-I alone did not change the steady state mRNA levels of IGFBP-3, -4, and -6, but elevated that of IGFBP-5. Des(1-3)-IGF-I, which has a lower affinity for IGFBPs, was more effective than the full-length IGF-I in enhancing the OP-1-induced alkaline phosphatase activity. Exogenous IGFBP-5 inhibited the OP-1-induced alkaline phosphatase activity and reduced the synergistic stimulatory effect of IGF-I and OP-1. These findings strongly suggest that the OP-1-induced down-regulation of IGFBPs, especially that of IGFBP-5, is an important mechanism by which OP-1 and IGF-I synergize to stimulate FRC cell differentiation.

Sequential expression of bone matrix proteins during rat calvaria osteoblast differentiation and bone nodule formation in vitro.

We investigated the expression of osteocalcin (OC), bone sialoprotein (BSP), osteonectin (ON), and alkaline phosphatase (ALP) during cell differentiation and bone nodule formation by fetal rat calvaria cells, using immunofluorescent and immunogold techniques at light and electron microscopic levels. Six hours after plating all proteins were expressed in calvaria cells. However, expression was not detected during the proliferation phase after plating. Cell morphological modifications were observed in osteoblastic cells expressing ALP, OC, and BSP, but not ON. During the matrix formation phase, all proteins were expressed with various intensities and OC was limited to differentiated osteoblastic cells. EM observations demonstrated that BSP was selectively associated with clusters of needle-like crystals, but not with collagen fibers, in mineralization foci and in the mineralized matrix. OC was localized intracellularly and in all the extracellular compartments, and was concentrated at the mineralization front. ON was distributed uniformly throughout the osteoid and mineralized matrix, which was intensely labeled. The results show that the expression of bone matrix proteins during differentiation of calvaria cells and nodule formation in vitro duplicate what is observed during osteogenesis in vivo.

Expression and regulation of galectin 3 in rat osteoblastic cells.

Galectin 3 is an endogenous soluble beta-galactoside-specific lectin originally identified and termed epsilon BP or IgE-binding protein in rat basophilic leukemia cells, but its wide tissue distribution and the multiple contexts in which it has been isolated have suggested that its function may not be limited to IgE binding but may include a role in cell growth regulation and differentiation, neoplastic transformation, and cell adhesion (Liu, 1990, Crit. Rev. Immunol., 10:289-306; Barondes et al., 1994, J. Biol. Chem., 269:20807-20810). After immunoscreening of a lambda gt11 cDNA expression library made from bone-nodule forming cultures of fetal rat calvaria (RC) cells with an antibody raised against osteoblastic cells (Turksen et al., 1992, J. Histochem. Cytochem., 40:1339-1352), three cDNA clones were isolated and sequenced; the sequence matched that of rat galectin 3. Galectin 3 mRNA was detected in various fetal and adult rat tissues, including calvaria and cultured RC cells. In RC cells and the rat osteosarcoma cell line ROS 17/2.8, galectin 3 mRNA expression increased with time in culture, in contrast to its behavior in fetal rat skin fibroblasts (RSF) in which its expression decreased with time in culture. In a second rat osteosarcoma line, UMR 106.01, galectin 3 mRNA was almost nondetectable. The synthetic glucocorticoid dexamethasone (Dex) enhanced galectin 3 expression in RSF cell cultures, while 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) had no significant effect. In contrast, Dex downregulated and 1,25(OH)2D3 upregulated galectin 3 expression in RC and ROS 17/2.8 cells, especially at later time points in culture when expression of osteoblast-associated differentiation markers by these cell types is most marked. Immunolabeling with an antibody against rat galectin 3 to identify galectin 3 protein showed that cells labelled within both the ROS 17/2.8 and RC populations but with marked intercellular heterogeneity of intensity. Our data support the conclusion that galectin 3 is a previously unrecognized product of osteoblastic cells, that galectin 3 mRNA and protein expression increases with time in vitro concomitant with other markers of osteogenesis, including formation of bone nodules and expression of osteoblast-associated markers such as alkaline phosphatase, bone sialo-protein, and osteocalcin, and that its expression is regulated by hormones such as glucocorticoids and 1,25(OH)2D3 that modulate other aspects of the osteoblast phenotype.

Prostate-derived soluble factors block osteoblast differentiation in culture.

Bone metastasis is a common event and a major cause of morbidity in prostate cancer patients. After colonization of bone, prostate cells induce an osteoblastic reaction which is not associated with marrow fibrosis (i.e., osteoblast but not fibroblast proliferation). In the present study we test the hypothesis that the tumoral prostatic cell line (PC-3) secretes factors that block the osteoblast differentiation process, resulting in an increase of the relative size of the proliferative cell pool. Our results, using fetal rat calvaria cells in culture, show that conditioned medium from PC-3 cells (PC-3 CM) stimulates osteoblast proliferation and inhibits both alkaline phosphatase (AP) activity (an early differentiation marker) and the mineralization process, measured as calcium accumulation (late differentiation marker). The inhibition of the expression of AP and mineralization depends on the presence of PC-3 CM during the proliferative phase of culture and suggests that both processes occur in a nonsimultaneous fashion. The inhibitory effect of PC-3 CM was not reverted by dexamethasone, which would indicate that prostatic-derived factors and the glucocorticoid do not share a common site of action. Measurement of the proliferative capacity of subcultures from control and treated cells demonstrates that PC-3 CM treatment induces the maintenance of the proliferative potential that characterizes undifferentiated precursor cells.

Ecto-alkaline phosphatase considered as levamisole-sensitive phosphohydrolase at physiological pH range during mineralization in cultured fetal calvaria cells

Alkaline phosphatase (ALP) activity expressed on the external surface of cultured fetal rat calvaria cells and its relationship with mineral deposition were investigated under pH physiological conditions. After replacement of culture medium by assay buffer and addition of p-nitrophenyl phosphate (pNPP), the rate of substrate hydrolysis catalyzed by whole cells remained constant for up to seven successive incubations of 10 min and was optimal over the pH range 7.6-8.2. It was decreased by levamisole by a 90% inhibition at 1 mM which was reversible within 10 min, dexamisole having no effect. Values of apparent Km for pNPP were close to 0.1 mM, and inhibition of pNPP hydrolysis by levamisole was uncompetitive (Ki = 45 microM). Phosphatidylinositol-specific phospholipase C (PI-PLC) produced the release into the medium of a p-nitrophenyl phosphatase (pNPPase) sensitive to levamisole at pH 7.8. The released activity whose rate was constant up to 75 min represented after 15 min 60% of the value of ecto-pNPPase activity. After 75 min of PI-PLC treatment the ecto-pNPPase activity remained unchanged despite the 30% decrease in Nonidet P-40-extractable ALP activity. High levels of 45Ca incorporation into cell layers used as index of mineral deposition were decreased by levamisole in a stereospecific manner after 4 h, an effect which was reversed within 4 h after inhibitor removal, in accordance with ecto-pNPPase activity variations. These results evidenced the levamisole-sensitive activity of a glycosylphosphatidylinositol-anchored pNPPase consistent with ALP acting as an ecto-enzyme whose functioning under physiological conditions was correlated to 45Ca incorporation and permit the prediction of the physiological importance of the enzyme dynamic equilibrium at the cell surface in cultured fetal calvaria cells.

ラット胎児頭蓋冠由来細胞培養系におけるプロスタグランディンＥ２の石灰化促進作用

ラット胎児頭蓋冠由来細胞培養系におけるプロスタグランディンＥ２の石灰化促進作用

Aluminum inhibits both initiation and progression of mineralization of osteoid nodules formed in differentiating rat calvaria cell cultures

Osteoid nodules form in cultures of fetal rat calvaria (RC) cells grown in medium containing 10% fetal bovine serum (FBS) and 50 microns/ml of ascorbic acid. When 10 mM beta-glycerophosphate (beta-GP) is added, the nodules mineralize in two phases: an initiation phase that is dependent upon alkaline phosphatase activity for cleavage of beta-GP to inorganic phosphate (P(i)) and a progression phase that proceeds independently of the activity of alkaline phosphatase and does not require exogenous phosphate. We have used this system to investigate the effects of aluminum (Al3+)on mineralization. When AlCl3 was added to culture medium at concentrations of 0, 3, 10, 30, 100, and 300 muM, the total concentrations of aluminum were 0.98, 6.07, 16.82, 40.19, 88.45, and 284.52 muM, respectively. The corresponding free Al3+ concentrations, assessed after ultrafiltration, were found to be 1.11, 1.75, 3.40, 6.22, 5.38, and 12.11 muM. In cultures in which osteoid was formed and mineralization initiated in the presence of added Al+ (3-300 muM), a dose-dependent inhibition of mineralization occurred. Osteoid formed in the presence of added Al3+ mineralized normally when Al3+ was removed from cultures at the time of initiation of mineralization with beta-GP. In osteoid nodules grown in the absence of Al3+, addition of Al3+ (3-300 muM) at the start of the initiation phase of mineralization resulted in a dose-dependent inhibition of mineralization. Addition of Al3+ to cultures after mineralization had been initiated in the absence of Al3+ inhibited progression of mineralization at added Al3+ concentrations of 10 muM and above. Al3+ did not decrease the conversion of beta-GP to P(i) and caused a small but significant increase in alkaline phosphatase activity at added concentrations of 100 muM or greater. The data show that Al3+ inhibits both the initiation and progression phases of mineralization starting at added concentrations of 3-10 muM (approximately 1.7-3.4 muM free Al3+) and that mineralization of osteoid formed in the presence of Al3+ is unaffected if Al3+ is removed prior to the initiation of mineralization.

Physiological concentrations of retinoic acid suppress the osteoblastic differentiation of fetal rat calvaria cells in vitro

The effects of retinoic acid (RA) on osteoblastic differentiation and activity were studied in fetal rat calvaria cells cultured for up to 24 days. Fetal bovine serum used for the experiments was treated with an anion-exchange resin to remove endogenous RA. The depletion of RA in the treated serum was confirmed by high-performance liquid chromatography and tritiated RA tracing. Under the culture conditions employed, the continuous presence of RA for 14 days at 10(-9) mol/l or higher decreased both alkaline phosphatase (ALP) activity on day 12 and the number of bone nodules on day 14 in a dose-dependent manner. Short-term (24 h) exposure to RA at 10(-8) mol/l, which is a physiological concentration, decreased and increased the levels of ALP and osteopontin mRNA on day 6, respectively. Retinoic acid at 10(-8) mol/l also increased the level of osteocalcin mRNA on day 12. However, these effects were not obvious at later stages (days 18 and 24). At a high concentration (10(-6) mol/l), RA increased the level of osteopontin mRNA on day 6 and decreased the levels of ALP and osteocalcin mRNA irrespective of culture period. These results suggest that, at physiological concentrations, RA suppresses the differentiation of osteoprogenitor cells and regulates osteoblastic functions.

Osteoblast and chondroblast differentiation.

Recognition of discrete commitment and differentiation stages requires characterization of changes in proliferative capacity together with the temporal acquisition or loss of expression of molecular and morphological traits. Both cell lines and primary cultures have been useful for analysis of transitional steps in the chondroblast (CB) and osteoblast (OB) lineages. One striking feature is that OBs and CBs share expression of some molecules, including newer markers such as epsilon BP (galectin-3), while also having unique markers. The fact that hypertrophic chondrocytes appear able to downregulate cartilage markers and upregulate OB markers also points to an interesting lineage relationship that needs to be explored further. Recently, we have focused on the osteoprogenitors that divide and differentiate into mature OBs forming bone nodules in fetal rat calvaria cell cultures. We use cellular, immunocytochemical, and molecular approaches, including PCR on small numbers of cells, to discriminate stages. Nodule formation is characterized by loss of proliferative capacity and sequential increased marker expression, that is, alkaline phosphatase (AP), followed by bone sialoprotein (BSP), and osteocalcin. Upregulation of collagen type I and biphasic expression of osteopontin, with two peaks corresponding to proliferation and differentiation stages, also occurs. A variety of other molecules are also upregulated in the mature OB, including epsilon BP and CD44s. By replica plating and PCR, we have begun to study the expression of the messenger RNAs (mRNAs) for potential regulatory molecules (e.g., PTHrP) and their receptors (e.g., PTHR, FGFR-1, and PDGFR alpha) and have found all to be modulated during the progression from committed osteoprogenitor to mature OB.(ABSTRACT TRUNCATED AT 250 WORDS)

Tri-iodothyronine (T3) and dexamethasone interact to modulate osteoprogenitor cell differentiation in fetal rat calvaria cell cultures

We investigated the role of 3,5,3'-tri-iodothyronine (T3) in regulating differentiation of osteoprogenitor cells and also studied the effects of the glucocorticoid hormone dexamethasone (Dex) on the T3-induced effects on osteoprogenitor populations. This was done by determining the effects of either hormone alone, or of combinations of the two hormones, on the number of bone nodules formed in long-term cultures of rat calvaria cells. In this system, Dex has been shown to increase bone nodule formation, the maximal effective dose being 10 nM (Bellows et al. Endocrinology 121: 1985-1992; 1987). In standard culture medium containing 15% fetal bovine serum FBS), low concentrations of T3 (0.001-0.1 nM) had no effect on the number of bone nodules, while higher concentrations of 1-100 nM inhibited. However, in culture medium containing 10 nM Dex, the lower concentrations of T3 markedly increased the number of nodules. Short term pulse experiments with these low concentrations of T3 in the presence of Dex indicated that stimulation of nodule formation occurred only when T3 was present prior to confluency. Higher concentrations of T3 (1-100 nM) decreased nodule number whether or not Dex was added. We then cultured cells in medium containing FBS from which T3 and T4 were removed by treatment with AG-1 chi-10 resin. In both + or - Dex conditions, bone nodule formulation was increased 1.5 to 2-fold in T3, T4-depleted medium when compared with cultures maintained in standard culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Leukemia inhibitory factor inhibits osteogenic differentiation in rat calvaria cell cultures.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine with both anabolic and catabolic effects on bone tissue. To investigate the effect of LIF on bone formation in the absence of a resorption cycle, we used fetal rat calvaria cell cultures and quantified bone nodule production, which provides a colony assay to analyze the effects of factors on osteoprogenitor differentiation and bone formation. In these cultures, dexamethasone (Dex) stimulates bone nodule formation. In dose-response experiments, LIF inhibited bone nodule formation by cells cultured with (+Dex; ID50 = 250 U/ml) or without (-Dex; ID50 = 30 U/ml) 10(-8) M Dex. Residual nodules were small and poorly mineralized. Continuous exposure to LIF (500 U/ml) up to day 25 did not affect either the growth rate or saturation density of the cultures, but decreased alkaline phosphatase activity and bone nodule production, with greater inhibition in -Dex cultures. Exposure to LIF (500 U/ml) for 3 days early during nodule formation (about day 10) reduced bone nodule numbers to the same extent as continuous treatment in -Dex cultures and significantly, but less markedly, in +Dex cultures; earlier and later pulses had no effect. Northern blot analysis of expression of messenger RNAs of bone related proteins in cultures pulsed (-Dex) at various stages of development showed marked inhibition of alkaline phosphatase, bone sialoprotein, and osteocalcin; slight inhibition of type I collagen; early stimulation of osteopontin; and no effect on Secreted Protein, Acidic and Rich in Cysteine/osteonectin. These results suggest that LIF is an inhibitor of bone nodule formation in these cultures, acting at a stage when late osteoprogenitors and/or early osteoblasts are present, and that Dex may modulate the effects of LIF by shifting effective doses to higher concentrations.

1α,25-Dihydroxyvitamin D3 down-regulates pleiotrophin messenger RNA expression in osteoblast-like cells

Pleiotrophin (PTN)[heparin-binding-growth-associated molecule (HB-GAM), heparin-binding neurite-promoting factor (HBNF)] is a recently identified polypeptide that stimulates growth of fibroblasts and enhances neurite extension. PTN is expressed in many tissues but relatively high level of expression has been observed in brain and bone. We examined hormonal regulation of PTN mRNA expression in several osteoblast-like cell lines including MC3T3-E1 and ROS17/2.8. The levels of PTN mRNA in these cells was significantly reduced by treatment with 10(-8) M: 1α,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) for 24 h. However, PTN mRNA levels were increased when the non-osteoblastic cell line, ROS 25/1, was treated with 1,25(OH)(2)D(3). These effects were observed in a dose-dependent manner in a dose range between 10(-11) M: to 10(-8) M: . This effect was specific to 1,25(OH)(2)D(3), since PTN mRNA levels were not affected by other steroids such as retinoic acid and dexamethasone in MC3T3-E1 or ROS17/2.8 cells. Similar 1,25(OH)(2)D(3) down-regulation of PTN mRNA was also observed in primary cultures of osteoblast-enriched fetal rat calvaria cells as well as cultures of MC3T3-E1 and ROS17/2.8 cells. These observations suggest that PTN expression in osteoblasts is regulated by the calcitropic hormone, 1,25(OH)(2)D(3), and that PTN may play a role in vitamin D-dependent regulation of bone metabolism.

Thyroid hormone suppresses the differentiation of osteoprogenitor cells to osteoblasts, but enhances functional activities of mature osteoblasts in cultured rat calvaria cells

The effects of thyroid hormone on osteoblastic differentiation and activity were studied in fetal rat calvaria (RC) cells cultured for up to 30 days in medium supplemented with thyroid hormone-depleted serum. In this condition, the cells proliferated and differentiated to form mineralized bone nodules (BN) and expressed osteoblastic markers such as alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). The continuous presence of triiodothyronine (T3) at 10(-9)-10(-8) M in the medium inhibited the osteoblastic differentiation: 34% decrease in ALP activity on day 12 and 60% decrease in BN formation on day 15 at 10(-8) M. T3 at these doses had no effect on the DNA content of RC cells at confluence (day 6). Short-term (48-h) exposure of T3 at 10(-9) M or higher decreased ALP activity when RC cells were differentiating (days 7-11). However, when BN formation by the cells had already reached a plateau (day 28), the activity was increased by treatment with T3 at 10(-7)-10(-6) M. OCN production was increased dose dependently by this treatment with T3 (2.1-fold and 1.3-fold of control at 10(-8) M on days 11 and 28, respectively). Similar increases were observed in the levels of OCN mRNA. In addition, increases in phosphorylated OPN in the medium (day 11) and mineralized matrix (day 28) were observed (1.5-fold at 10(-8)-10(-6) M), while OPN synthesis and the level of its mRNA were depressed by T3 (60-70% of control at 10(-8) M). These results suggest that T3 regulates osteoblastic differentiation and activity depending on the state of cell differentiation: T3 suppresses the differentiation of osteoprogenitor cells to osteoblasts, but enhances the functional activity of mature osteoblasts.

Simultaneous Detection of Multiple Bone-Related mRNAs and Protein Expression during Osteoblast Differentiation: Polymerase Chain Reaction and Immunocytochemical Studies at the Single Cell Level

Messenger RNA expression analyzed by in situ hybridization and Northern analysis and protein expression analyzed biochemically or immunocytochemically have been used to study the developmental expression of various osteoblast (OB)-associated molecules. These approaches have shown that over a time course of OB differentiation in vivo and in vitro, the expression of macromolecules associated with OB cells changes. However, ambiguities in data from different approaches and in populations representative of cells at different developmental stages are extant. To begin to discriminate differentiation stages with more precision and to address intercellular heterogeneity, fetal rat calvaria cells were grown at low densities under conditions in which bone nodules form and mineralize and colonies were classified morphologically as fibroblastic or osteoblastic (early, intermediate, or mature). Whole discrete colonies and single cells from individual colonies were analyzed molecularly by a random amplification poly(A)-polymerase chain reaction (PCR) and for protein expression by immunocytochemistry; we analyzed the expression of known bone-related macromolecules (collagen type I, alkaline phosphatase, osteopontin, bone sialoprotein, and osteocalcin). Both PCR and immunocytochemistry revealed that different colony types were reproducibly distinguishable in their expression of either general (collagen type I) or bone-associated (alkaline phosphatase, osteopontin, bone sialoprotein, and osteocalcin) macromolecules, such that fibroblastic colonies were distinguishable from osteoblastic colonies and the latter could be subdivided into less mature or more mature osteoblastic colonies. While some aspects of the temporal differentiation sequence defined earlier were confirmed, several additional features were evident from these single cell-single colony studies. First, different repertoires of OB-associated markers were expressed in different cells, suggesting variation in the switch-on of the OB differentiation program and heterogeneity in the OB phenotype. Second, among colonies classified as fibroblastic on the basis of morphology heterogeneity was also evident and there were some cells expressing features consistent with their being osteoprogenitor cells. Our data support the hypothesis that individual fibroblastic and osteoblastic cells are heterogeneous in expression of marker molecules. We also conclude that individual cells and colonies analyzed by poly(A)-PCR will be useful in lieu of mass populations to extend investigation of stages in the progression of OB differentiation.

Effects of transforming growth factor beta on bone nodule formation and expression of bone morphogenetic protein 2, osteocalcin, osteopontin, alkaline phosphatase, and type I collagen mRNA in long-term cultures of fetal rat calvarial osteoblasts.

Transforming growth factor beta (TGF-beta) is one of the most abundant of the known growth regulatory factors stored within the bone matrix. When bone is resorbed, TGF-beta is released in an active form and is a powerful bone growth stimulant. When injected into the subcutaneous tissue over the calvarial surface of rodents, it rapidly causes proliferation of the periosteal layer and accumulation of new woven bone. In this report, we describe the effects of TGF-beta 1 on first subcultures of fetal rat osteoblasts obtained from calvarial bones and cultured from confluence with ascorbic acid and beta-glycerophosphate. Under these conditions, nodules with characteristics of normal bone appear by day 8. Similar to experiments described by Antosz et al., TGF-beta added to confluent cultures inhibited the formation of bone nodules. Both the number and total area of the nodules were quantitated and shown to be completely inhibited by 2 ng/ml of TGF-beta 1. TGF-beta also impaired the expression of genes associated with bone formation, including type I collagen, alkaline phosphatase, osteopontin, and osteocalcin. TGF-beta also inhibited the expression of mRNA for the bone morphogenetic protein 2 (BMP-2). These results, showing suppression of markers representative of osteoblast differentiation, suggest that the effects of TGF-beta to stimulate bone formation in vivo are not likely a result of effects on differentiated mineralizing osteoblasts but, as suggested by previous studies, more likely are caused by effects on osteoblast precursors. These results also suggest that endogenous BMP-2 expression in fetal rat calvaria cells is important for bone cell differentiation.

1,25-dihydroxyvitamin D3 stimulates adipocyte differentiation in cultures of fetal rat calvaria cells: comparison with the effects of dexamethasone

Progenitor cells for several mesenchymally derived cell types exist within freshly isolated fetal rat calvaria (RC) cell populations. We have characterized the effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on the differentiation of adipocytes from primary RC cells and compared these effects with those of dexamethasone (Dex). RC cells were plated at 3 x 10(4)/35-mm dish, and cultures were maintained for 14-19 days in alpha-Minimum Essential Medium containing 10% fetal bovine serum, 50 micrograms/ml ascorbic acid, 10 mM Na beta-glycerophosphate, and 0.1-100 nM 1,25-(OH)2D3 or 1-1000 nM Dex. Morphological (quantitation of adipocyte foci number and area after staining cultures with Sudan IV) and biochemical (glycerol-3-phosphate dehydrogenase activity) methods of assessing adipogenesis were used. In the presence of 1,25-(OH)2D3, adipocyte foci developed about 3 days after confluency as clusters of rounded or stellate cells. Stimulation of adipocyte foci development was dose dependent from 0.1-100 nM and was maximal with 10 nM 1,25-(OH)2D3; half-maximal stimulation occurred at about 1 nM. The presence of ascorbic acid and beta-glycerophosphate was not required for 1,25-(OH)2D3-induced stimulation of adipocytes, but both significantly increased the number of adipocyte foci in the presence of 1,25-(OH)2D3. The critical period for initiation of adipocyte differentiation with 1,25-(OH)2D3 was between 1-9 days, and once committed along the adipogenic pathway, adipocytes maintained their differentiated state in the absence of 1,25-(OH)2D3. Short term (48-h) pulses of 1,25-(OH)2D3 resulted in slight, but significant, increases in adipocyte formation. Other vitamin D3 metabolites were less effective than 1,25-(OH)2D3 in stimulating adipocyte differentiation. Dex (1-100 nM) also caused a dose-dependent increase in the differentiation of adipocyte foci in RC cell cultures. The adipocyte foci that developed in the presence of Dex frequently appeared earlier in culture, i.e. when cells reached confluency on days 6-7, and were more diffuse than those forming with 1,25-(OH)2D3. The stimulation of adipocyte differentiation by 1,25-(OH)2D3, however, was greater than that by Dex in mixed RC II-V cells. The combined effects of 1,25-(OH)2D3 and Dex were additive at low concentrations and synergistic at higher concentrations of either 1,25-(OH)2D3 or Dex. The data show that bone cell populations isolated from fetal RC contain adipocyte progenitors and that 1,25-(OH)2D3 as well as Dex are potent regulators of adipocyte differentiation within these bone cell populations.

1,25-dihydroxyvitamin D3 stimulates adipocyte differentiation in cultures of fetal rat calvaria cells: comparison with the effects of dexamethasone.

Progenitor cells for several mesenchymally derived cell types exist within freshly isolated fetal rat calvaria (RC) cell populations. We have characterized the effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on the differentiation of adipocytes from primary RC cells and compared these effects with those of dexamethasone (Dex). RC cells were plated at 3 x 10(4)/35-mm dish, and cultures were maintained for 14-19 days in alpha-Minimum Essential Medium containing 10% fetal bovine serum, 50 micrograms/ml ascorbic acid, 10 mM Na beta-glycerophosphate, and 0.1-100 nM 1,25-(OH)2D3 or 1-1000 nM Dex. Morphological (quantitation of adipocyte foci number and area after staining cultures with Sudan IV) and biochemical (glycerol-3-phosphate dehydrogenase activity) methods of assessing adipogenesis were used. In the presence of 1,25-(OH)2D3, adipocyte foci developed about 3 days after confluency as clusters of rounded or stellate cells. Stimulation of adipocyte foci development was dose dependent from 0.1-100 nM and was maximal with 10 nM 1,25-(OH)2D3; half-maximal stimulation occurred at about 1 nM. The presence of ascorbic acid and beta-glycerophosphate was not required for 1,25-(OH)2D3-induced stimulation of adipocytes, but both significantly increased the number of adipocyte foci in the presence of 1,25-(OH)2D3. The critical period for initiation of adipocyte differentiation with 1,25-(OH)2D3 was between 1-9 days, and once committed along the adipogenic pathway, adipocytes maintained their differentiated state in the absence of 1,25-(OH)2D3. Short term (48-h) pulses of 1,25-(OH)2D3 resulted in slight, but significant, increases in adipocyte formation. Other vitamin D3 metabolites were less effective than 1,25-(OH)2D3 in stimulating adipocyte differentiation. Dex (1-100 nM) also caused a dose-dependent increase in the differentiation of adipocyte foci in RC cell cultures. The adipocyte foci that developed in the presence of Dex frequently appeared earlier in culture, i.e. when cells reached confluency on days 6-7, and were more diffuse than those forming with 1,25-(OH)2D3. The stimulation of adipocyte differentiation by 1,25-(OH)2D3, however, was greater than that by Dex in mixed RC II-V cells. The combined effects of 1,25-(OH)2D3 and Dex were additive at low concentrations and synergistic at higher concentrations of either 1,25-(OH)2D3 or Dex. The data show that bone cell populations isolated from fetal RC contain adipocyte progenitors and that 1,25-(OH)2D3 as well as Dex are potent regulators of adipocyte differentiation within these bone cell populations.

P8. Bone marrow stromal cells separated by buoyant density: an initial characterisation

Phenytoin (diphenylhydantoin, DPH), an anticonvulsant drug for epileptic patients, has several adverse effects, including calvarial thickening and coarsening of the facial features, which occur with chronic DPH therapy. While previous studies have demonstrated that DPH has an anabolic action on bone cells in vivo and in vitro, the basis of these effects is not fully understood. In this study, the effect of DPH on osteoblastic differentiation of fetal rat calvaria (RC) cells in culture was investigated by measuring bone nodule (BN) formation, cell growth, alkaline phosphatase (ALPase) activity, collagen synthesis, and expression of osteocalcin (OC) and osteopontin (OP) mRNAs. Continuous treatment of RC cells with DPH for 18 days dose-dependently increased the mineralized BN number by 1.2–1.7-fold at concentrations of 12.5–200 μmol/L DPH. Cell growth was not affected at the same concentrations of DPH. ALPase activity was stimulated by DPH (1.1–1.9-fold) dose-dependently and was maintained at higher levels in DPH-treated cells throughout the experimental period. DPH increased mineralized and unmineralized BN formations both in the presence and the absence of 10−8 mol/L dexamethasone (Dex). Expression of OC and OP mRNAs was markedly augmented by DPH on days 12–24 and on days 12–18, respectively. While control mRNA levels of OC and OP increased with time, the increases in DPH-treated cells were greater than those of the controls and the stimulatory effects were dose-dependent. Type I collagen was also influenced by DPH; mRNA level was enhanced and the percentage of collagen synthesized was increased significantly, by 200 μmol/L DPH. When DPH was added in three different culture stages, days 1–6 (growth), days 7–12 (matrix development), and days 13–18 (mineralization), BN formation was influenced primarily on days 1–6 and secondarily on days 7–12, but not on days 13–18, suggesting that DPH increased BN formation by enhancing not only the proportion of osteoprogenitor cells in the early stage but also the proportion of functional osteoblasts in the middle stage within mixed-cell populations. Moreover, such increases were detected in conditions of both Dex(+) and Dex(−). These findings demonstrate that DPH stimulates osteoblast-associated markers such as BNs, ALPase, OC, OP, and type I collagen by continuously affecting the stages of growth and matrix development in RC cells, and suggests that the stimulatory effects by DPH may possibly be induced independent of those by Dex.

Expression and characterization of parathyroid hormone receptors on fetal rat calvaria cells in culture

The expression of parathyroid hormone (PTH) receptors during osteoblastic differentiation was studied using long-term cultures of fetal rat calvaria (RC) cells. Scatchard and cross-linking studies with [125I]-labeled [Nle8,18, Tyr34] bovine PTH (1-34) amide indicated that RC cells expressed a single class of saturable, high affinity PTH receptors with a Kd of about 1.0 nM and a molecular mass of about 76 kDa. The number of receptors increased after plating, reached a maximum on day 15 (5.01 × 104 sites/cell) and decreased thereafter (3.82 × 104 sites/cell on day 20), whereas the osteoblastic markers, such as the alkaline phosphatase activity and number of bone nodules increased progressively up to day 20. These results suggest that PTH receptor expression changes during the osteoblastic lineage and that the highest number of receptors occurs at the differentiated stage prior to osteoblast maturation.