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PubMed1989

Rapid, simple identification of individual osteoblastic cells and their specific products by cell blotting assay.

Biochemical and molecular biological studies of osteoblastic cell function and hormonal regulation are frequently confounded by the inherent cellular heterogeneity and phenotypic instability of existing in vitro and in vivo model systems. A new technique (derived from Western blotting or antibody-based detection of protein molecules bound to nitrocellulose paper) is described for identification of individual cells which synthesize osteoblast-specific gene products (bone Gla-protein, type I collagen, and alkaline phosphatase) or produce cAMP in response to parathyroid hormone (PTH) or isoproterenol. Dispersed primary neonatal rat calvariae or osteogenic sarcoma cells were "plated" on Immobilon-P (a hydrophobic transfer membrane with very high protein-binding capacity) for 30 minutes to several hours, followed by agonist treatment, formalin fixation, hematoxylin staining, and immunostaining with a battery of antibodies specific for osteoblastic products. Individual cells and their secretory zones were visualized by light microscopy and counted. Treatment with PTH with or without isoproterenol resulted in increases in the percentages of osteoblastic cells elaborating cAMP, as well as the intensity of immunostaining, but had no effects on MCF-7 cells, a nonosteoblastic breast carcinoma control line. The percentage of cells within each primary osteoblastic cell population isolated or rat osteogenic sarcoma cell clone (G2 or C12) that elaborated bone-specific proteins or that generated cAMP in response to PTH varied with time and the individual cellular preparation, reconfirming the cellular heterogeneity of these systems. This method, in conjunction with techniques such as in vitro hybridization, should prove useful in characterizing discrete osteoblastic bone cell subpopulations and in clarifying mechanisms of hormonal regulation by local and systemic agents.

Alkaline Phosphatase↗

PubMed1991

Estrogen binding and estrogenic responses in normal human osteoblast-like cells.

A finite human cell line was established from trabecular bone explants obtained from a 48-year-old woman. These cells, designated BG688, were characterized as osteoblast-like in phenotype using the following independent criteria: (1) the presence of histochemically detectable alkaline phosphatase (AP) activity; (2) response to the calciotropic hormone 1,25-(OH)2D3 as assessed by increased AP activity; (3) synthesis and secretion of the osteoblast-specific marker bone gla protein; and (4) expression of alpha 1(I)-procollagen and alpha 1(III)-procollagen mRNAs in a pattern similar to that of other osteoblast-like cell lines. In addition to these classic osteoblast markers, BG688 cells also possess approximately 2400 high-affinity (Kd = 0.45 nM) 17 beta-estradiol (E2) binding sites per cell. The binding of E2 to these sites is specific, and of the steroid hormone agonists tested, E2 and diethylstilbestrol elicited the greatest amount of competition with radiolabeled E2. BG688 cells were also shown to respond to a physiologic concentration (10 nM) of E2. In vitro translation products of poly(A)+ RNA obtained from control and hormone-treated cells revealed a pleiotropic influence of E2 on the relative abundance of several mRNAs as assessed by two-dimensional gel electrophoretic analysis of their corresponding peptides. E2 also elicits a twofold increase in the steady-state concentration of alpha 1(I)-procollagen mRNA as demonstrated by northern blot hybridization. Thus, we here extend our previous data obtained in osteoblast-like osteosarcoma cells to indicate that a normal osteoblastic cell line is a target for the action of estrogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗

PubMed1991

Bradykinin-induced burst of prostaglandin formation in osteoblasts is mediated via B2 bradykinin receptors.

The receptor subtype mediating the bradykinin (Bk)-induced burst of prostaglandin formation in osteoblasts has been studied. Bk, but not des-Arg9-Bk, induced the formation of prostaglandin E2 and prostacyclin in neonatal mouse calvarial bones incubated for 30 minutes. Bk-induced prostaglandin synthesis is neonatal mouse calvarial bones was significantly inhibited by the B2 Bk receptor antagonist D-Arg0[Hyp3,Thi5,8,D-Phe7]-Bk. The B2 Bk receptor agonists Bk and Lys-Bk, but not the B1 Bk receptor agonist des-Arg9-Bk, caused a rapid burst (5 minutes) of prostaglandin E2 and prostacyclin formation in isolated osteoblast-like cells from neonatal mouse calvarial bones and in the murine osteoblastic cell lineage MC3T3-E1. When comparing the relative potency of different kinin analogs on their stimulatory effect on prostaglandin formation in isolated osteoblast-like cells and in MC3T3-E1 cells, we found that Bk = Lys-Bk greater than Met-lys-Bk much much greater than Des-Arg9-Bk. Bk-induced prostaglandin synthesis in isolated osteoblast-like cells and in MC3T3-E1 cells was inhibited by D-Arg0[Hyp3,Thi5,8,D-Phe7]-Bk, whereas the B1 Bk receptor antagonist des-Arg9-Leu8-Bk had no effect. Total binding of 3H-Bk (3-20 nM; 4 degrees C) to whole MC3T3-E1 cells reached a maximum after 4-5 h. An excess of nonradioactive Bk (1 microM) reduced cell-associated radioactivity by 20-30%. The B2 Bk receptor agonist Bk and the B2 Bk receptor antagonist D-Arg0[Hyp3,Thi5,8,D-Phe7]-Bk were able to reduce specific binding, but the B1 Bk receptor agonist des-Arg9-Bk was unable to reduce the specific binding of 3H-Bk to whole MC3T3-E1 cells. These findings indicate the presence of B2 Bk receptors on osteoblasts coupled to enhanced prostaglandin synthesis.

Amino Acid Sequence↗

PubMed1991

Effects of ipriflavone and its metabolites on a clonal osteoblastic cell line.

Protective effects of ipriflavone, an isoflavone derivative, in osteoporosis are believed to be caused by the inhibitory action on bone resorption. A direct effect of ipriflavone on bone formation is as yet unknown. Ipriflavone and four of its metabolites (I, II, III, and V) were examined for their effects on parathyroid hormone response, collagen synthesis, alkaline phosphatase activity, and cell proliferation in a clonal cell population of rat osteoblastic cells. Pretreatment of osteoblasts with high concentrations of ipriflavone for 48 h significantly inhibited the cAMP response to parathyroid hormone, producing a shift in the dose-response curve; at lower concentrations metabolites II and III potentiated the cAMP accumulation induced by low doses of parathyroid hormone. The 48 h treatment with metabolite V at the 1 nM dose significantly stimulated collagen synthesis in osteoblastic cells. Ipriflavone and metabolite I showed a biphasic stimulatory action on the alkaline phosphatase activity of osteoblasts, with a maximal effect at the 0.1 and 1 nM doses, respectively. A similar biphasic response was observed with ipriflavone and metabolite I on osteoblastic cell growth, with a maximal effect at the 0.1 nM concentration. These results suggest a direct role of ipriflavone in modulating the synthetic and growth properties of osteoblast-like cells.

Alkaline Phosphatase↗

PubMed1992

Relationship between collagen synthesis and expression of the osteoblast phenotype in MC3T3-E1 cells.

The MC3T3-E1 mouse calvaria-derived cell line has been used to study the role of collagen synthesis in osteoblast differentiation. MC3T3-E1 cells, like several previously characterized osteoblast culture systems, expressed osteoblast markers and formed a mineralized extracellular matrix only after exposure to ascorbic acid. Mineralization was stimulated further by beta-glycerol phosphate. Ultrastructural observations indicated that the extracellular matrix produced by ascorbic acid-treated cells was highly organized and contained well-banded collagen fibrils. Expression of osteoblast markers followed a clear temporal sequence. The earliest effects of ascorbic acid were to stimulate type I procollagen mRNA and collagen synthesis (24 h after ascorbate addition), followed by induction of alkaline phosphatase (48-72 h) and osteocalcin (96-144 h) mRNAs. Procollagen mRNA, which was expressed constitutively in the absence of ascorbate, increased only twofold after vitamin C addition. In contrast, alkaline phosphatase and osteocalcin mRNAs were undetectable in untreated cultures. Actions of ascorbic acid on osteoblast marker gene expression are mediated by increases in collagen synthesis and/or accumulation because (1) parallel dose-response relationships were obtained for ascorbic acid stimulation of collagen accumulation and alkaline phosphatase activity, and (2) the specific collagen synthesis inhibitors, 3,4-dehydroproline and cis-4-hydroxyproline, reversibly blocked ascorbic acid-dependent collagen synthesis and osteoblast marker gene expression.

Alkaline Phosphatase↗

PubMed1992

Identification of proteins secreted by human osteoblastic cells in culture.

To better understand the biochemistry of matrix-forming cells, we developed a simple and reproducible procedure for the isolation and identification by N-terminal sequencing of proteins secreted by cells into culture medium and applied this procedure to the analysis of the major Coomassie blue-staining proteins under 100 kD that are secreted from three different human osteoblastic cell cultures. The major proteins secreted by normal human osteoblasts from adult trabecular bone were identified by N-terminal sequencing to be gelatinase, osteonectin, the C-terminal propeptides of the alpha 1 and alpha 2 chains of type I collagen, tissue inhibitor of metalloproteinase 1 (TIMP-1), and beta 2-microglobulin. The amounts of each of these proteins secreted into medium over a 24 h interval did not change over the 7 consecutive days of culture under serum-free conditions, which indicates that this pattern of protein secretion is not significantly affected by the serum-free conditions needed for protein identification by this method. In addition, radioimmunoassay for bone gla protein (BGP), a marker for osteoblast phenotype, revealed that BGP secretion remained high over 7 days of culture under serum-free conditions and was comparable to the rate of BGP secretion in control cultures with 10% serum. The major proteins secreted by MG-63 cells were identified by N-terminal sequencing to be gelatinase, a novel 40 kD human bone protein we termed YKL-40, TIMP-1, the recently discovered TIMP-2, and beta 2-microglobulin. Further studies revealed that YKL-40 is the only protein detectable by Coomassie staining of SDS gels of MG-63 media proteins that is induced by extended time at confluence or by treatment with 1,25-(OH)2D3. The apparent absence of detectable Coomassie-stained bands corresponding to the C-terminal propeptides of collagen in the medium of MG-63 cells suggests that these transformed cells may not be a good model for bone matrix formation. The major proteins secreted by normal fetal osteoblastic cells were identified by N-terminal sequencing to be osteonectin and the C-terminal propeptides of the alpha 1 and alpha 2 chains of type I collagen. Gelatinase and TIMP could not be detected among the conditioned medium proteins by these methods. These observations indicate that fetal osteoblasts primarily express proteins that are matrix constituents and adult human osteoblasts secrete, in addition to these, proteins that could function in matrix turnover.

Amino Acid Sequence↗

PubMed1992

Adhesion molecules in skeletogenesis: I. Transient expression of neural cell adhesion molecules (NCAM) in osteoblasts during endochondral and intramembranous ossification.

We report that neural cell adhesion molecules (NCAM) are expressed transiently in developing chicken osteoblasts during osteogenesis using immunostaining on cryostat sections. NCAM is strongly expressed in most osteoblasts along bone trabeculae that coincide with the presence of collagen I and alkaline phosphatase activity. In endochondral ossification, NCAM is highly expressed in osteogenic buds as seen in the epiphysis and diaphysis of tibia and vertebrae. In intramembranous ossification, NCAM is seen in osteogenic condensation of calvaria and in the periosteum of tibial diaphysis. The expression is transient because NCAM is not expressed in mesenchymal cells before osteogenic condensation and NCAM expression is lost in osteocytes in later stages. The staining pattern suggests that NCAM is present on the cell membrane of osteoblasts. Using a specific monoclonal antibody, the osteoblast NCAM is shown to contain polysialic acid, which is enriched in embryonic brain. Northern blot analysis using chicken brain NCAM cDNA as probes showed two major sizes of mRNA at 6.4 and 4.2 kb in calvarial mRNA as opposed to bands at 7.2, 6.4, and 4.2 kb in the brain. An immunoblot showed major proteins at Mr 165 and 110 kd, unlike brain NCAM, which are 180, 140, and 120 kD. That NCAM is involved in bone morphogenesis is consistent with the general hypothesis that NCAM plays pivotal roles in mesenchymal condensation, as shown in the formation of muscle, kidney, skin, and cartilage. The results establish NCAM as a cell surface molecule expressed transiently during osteoblast lineage. The implication that NCAM may mediate osteoblast interaction and regulate skeletal morphogenesis is discussed.

Animals↗

PubMed1993

Secretion of osteocalcin and its propeptide from human osteoblastic cells: dissociation of the secretory patterns of osteocalcin and its propeptide.

Specific immunoassay systems for intact human osteocalcin (I-OC) and its 26-residue propeptide have been newly developed to assess their usefulness as biochemical markers of bone metabolism. Using human cultured osteoblastic periosteal cells, we monitored 24 h secretion of these molecules from the osteoblastic cells and also examined the deposition of Ca, P, and I-OC on the extracellular matrix. At day 5, both I-OC and its propeptide were secreted by osteoblastic cells in a concentration-dependent manner by treatment with 1,25-(OH)2D3. This propeptide was not detected in the serum of adult subjects but was detected in the serum of normal children, which confirmed this in vitro result of propeptide secretion. The secretion of I-OC into medium transiently decreased at day 11, when the rapid accumulation of I-OC, Ca, and P, namely mineralization, was observed on the extracellular matrix of osteoblastic cells, although secretion of the propeptide constantly increased throughout the culture period. Therefore, the ratio of the amount of propeptide to I-OC in the supernatant markedly increased when mineralization started. These data demonstrate the superior specificity of propeptide as a marker of osteoblastic function in vitro compared with I-OC and that monitoring the changes in propeptide to I-OC ratios in the culture supernatant may be useful for predicting the timing of mineralization on the extracellular matrix of osteoblastic cells.

Adolescent↗

PubMed1993

Regulation of cytokine expression in osteoblasts by parathyroid hormone: rapid stimulation of interleukin-6 and leukemia inhibitory factor mRNA.

PTH and other hormones that stimulate resorption affect osteoclasts indirectly by modulating cytokine production by osteoblasts. However, the identity and role of the osteoblast-derived cytokines involved in this process are unclear. To examine which cytokines are regulated by PTH, we assessed cytokine mRNA levels in osteoblasts using the reverse transcription-polymerase chain reaction technique. Of the 16 cytokines we examined, unstimulated MC3T3-E1 osteoblastic cells expressed mRNA for interleukins 5, 6, and 7, macrophage and granulocyte-macrophage colony-stimulating factors, transforming growth factor beta 1, and leukemia inhibitory factor. PTH specifically increased expression of interleukin-6 (approximately 50-fold) and leukemia inhibitory factor (approximately 10-fold). Levels of both IL-6 and LIF mRNA peaked 30-60 minutes after addition of PTH and returned to baseline by 4-6 h. This rapid and transient mRNA response, which resembles that of immediate early genes, was also observed in primary rat osteoblasts. The transient mRNA response was accompanied by increased secretion of IL-6 protein. Lipopolysaccharide, another stimulator of resorption, increased mRNA levels of a group of cytokines that were not induced by PTH, namely interleukin-1 alpha, tumor necrosis factor alpha, and granulocyte-macrophage and granulocyte colony-stimulating factors. We conclude that osteoblasts produce complex networks of cytokines that (1) are regulated by bone-resorptive agents and (2) may be involved in controlling bone resorption.

3T3 Cells↗

PubMed1993

Expression of annexin I, II, V, and VI by rat osteoblasts in primary culture: stimulation of annexin I expression by dexamethasone.

To determine whether rat osteoblasts synthesize proteins of the annexin family and to evaluate the extent to which glucocorticoids modulate the expression of annexins by these cells, osteoblasts were grown in primary cultures in the absence or presence of dexamethasone, and the expression of annexins was evaluated by immunoblotting using polyclonal antibodies against human annexins. Four different annexins (I, II, V, and VI) were found to be expressed by rat osteoblasts. The expression of annexin I, but not the other annexins studied, was increased in osteoblasts cultured in the presence of dexamethasone (173 +/- 33% increase comparing untreated cells and cells treated for 10 days with 5 x 10(-7) M dexamethasone). Increased expression of annexin I was observed after the third day of exposure to dexamethasone and rose thereafter until day 10; annexin I expression increased with dexamethasone concentrations above 10(-10) M throughout the range of concentrations studied. The increase in annexin I protein was associated with an increase in annexin I mRNA and was completely blocked by the concomitant addition of the glucocorticoid receptor antagonist RU 38486. The increase in annexin I content following dexamethasone treatment was associated with an increase in alkaline phosphatase activity and PTH-induced cAMP stimulation, whereas phospholipase A2 activity in the culture medium was reduced to undetectable levels. The finding that four annexins are expressed in rat osteoblasts in primary culture raises the possibility that these proteins could play an important role in bone formation by virtue of their ability to bind calcium and phospholipids, serve as Ca2+ channels, interact with cytoskeletal elements, and/or regulate phospholipase A2 activity. In addition, the dexamethasone-induced increase in annexin I may represent a mechanism by which glucocorticoids modify osteoblast function.

Alkaline Phosphatase↗

PubMed1994

Cancellous bone turnover in growing rats: time-dependent changes in association between calcein label and osteoblasts.

Calcein was given to growing male rats to label mineralizing bone, and groups of animals were sacrificed from 6 h to 12 days later. Fluorochrome-labeled perimeter and osteoblast perimeter were determined in a growth-adjusted sampling site in the secondary spongiosa of the proximal tibial metaphysis. The best correspondence between calcein-labeled perimeter and osteoblast-lined perimeter was during the initial 24 h following administration of the label. This association decreased progressively thereafter because of (1) cessation of bone formation on fluorochrome-labeled surfaces and (2) initiation of new foci of bone formation. The time-dependent decline in osteoblast-lined trabeculae with adjacent calcein label exceeded the increase in osteoblast perimeter on nonfluorochrome-labeled trabeculae, indicating that there was a net decrease in osteoblast number. Fluorochrome-labeled perimeter and bone area showed parallel decreases with time after labeling, as a result of bone resorption. Interestingly, the decrease in cancellous bone was caused exclusively by a reduction in trabecular number. There was no change in trabecular thickness. The findings suggest that the decreased osteoblast number and progressive bone resorption resulted in complete erosion of trabeculae most distal to the growth plate. As a result of the nearly equal growth and destruction of trabeculae, there was no change in the length of the cancellous metaphysis with time. It is concluded that cancellous bone turnover in growing rats represents a maturation process that differs fundamentally from bone turnover in adults.

Animals↗

PubMed1995

Cell proliferation of human fibroblasts and osteoblasts in osteogenesis imperfecta: influence of age.

Clinical studies indicate that as a group, osteogenesis imperfecta (OI) subjects are shorter than age- and sex-matched controls. Not only somatic growth, but also cellular growth appears to be impaired, and these may be related to defects in extracellular matrix common to this disorder. We have investigated the growth characteristics of dermal fibroblasts and trabecular osteoblasts isolated from patients with OI and control subjects of various ages. Cell growth curves and cell doubling times were determined by measuring cell number using crystal violet dye binding. Growth curves were modeled by a modified logistic function, the three parameters of which are markers for biologically relevant growth parameters: the plateau value or upper asymptote, which reflects the maximum cell density upon confluence; the maximal growth rate (microM); and the lag time. Both normal human fibroblasts and osteoblasts showed an age-dependent decrease in microM. Normal fibroblasts exhibited no age-dependence to their upper asymptote or lag time. Fibroblasts derived from patients with OI did not have significantly different upper asymptote values microM, or lag times when compared with normal fibroblasts. Normal osteoblasts had a decrease in upper asymptote, decrease in microM, but a relatively constant lag time with increasing age. In contrast, OI osteoblast microM was decreased relative to that of normal subjects. For osteoblasts from OI patients, decreased microM appeared unrelated to the age of the subject, whereas OI fibroblasts did exhibit an age-dependent decrease in microM. The percentage of collagenase-digestible protein (a measure of collagen synthesis) produced by normal human fibroblasts correlated well with microM. Treating normal human osteoblasts with the proline analogue 3,4-dehydroproline, which destabilizes collagen triple helix formation and alters collagen synthesis, secretion, and turnover, also decreased microM. A dose response to varying concentrations of 3,4-dehydroproline was observed for normal human bone cell microM. These data suggest a link between type I collagen synthesis and cellular proliferation.

Aging↗

PubMed1995

Fibronectin gene expression, synthesis and accumulation during in vitro differentiation of chicken osteoblasts.

A well-defined chicken osteoblast culture system(18) has been used to examine fibronectin (FN) mRNA levels, synthesis, and accumulation during in vitro differentiation and matrix mineralization. Immunofluorescent staining of cells after 6 or 18 days in culture revealed that FN was initially associated with the cell surface and in partial coalignment with cytoskeletal elements while at the latter time most FN was associated with the extracellular matrix as a ubiquitous fibrillar network. Western blot analysis of total cell-associated proteins also detected FN at all culture times. However, when results were normalized to cellular DNA, FN levels increased until 12-16 and remained relatively constant thereafter. Similarly, FN synthesis as measured by [35S]-methionine labeling, and immunoprecipitation was greatest in early cultures (culture day 3) and then declined such that synthesis decreased 60% at day 18 and 94% after 24-31 days. FN mRNA levels as measured by Northern blot analysis were well correlated with FN synthesis. These results clearly show that FN is made by primary osteoblasts during their in vitro maturation. In contrast to other osteoblast markers such as alkaline phosphatase, osteocalcin, and osteopontin, whose expression increases as cells differentiate, FN accumulates in the matrix during periods of early cell growth and attachment and then remains proportional to cell number. Results with FN differ from those obtained with collagen which continues to accumulate in the extracellular matrix during osteoblast maturation. These results are consistent with FN being important for the initial attachment of early osteoblasts or osteoblast precursors to the pericellular matrix.

Actins↗

PubMed1996

Prostaglandin E2 stimulates osteoclast-like cell formation and bone-resorbing activity via osteoblasts: role of cAMP-dependent protein kinase.

Prostaglandin E2 (PGE2) is an important local regulator in bone. The present study was performed to investigate the effect of PGE2 on osteoclast-like cell formation and bone-resorbing activity of mature osteoclasts in the presence or absence of osteoblasts, PGE2 (10(-8) to 10(-6) M) significantly stimulated osteoclast-like cell formation in osteoblast-containing mouse bone cell cultures, although it did not affect osteoclast-like cell formation from hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor in osteoblast-free mouse spleen cell cultures. The conditioned medium from osteoblastic UMR-106 cells pretreated with PGE2 (10(-8) and 10(-6) M) significantly stimulated osteoclast-like cell formation from hemopoietic blast cells. PGE2 also significantly stimulated the bone-resorbing activity of mature osteoclasts in osteoblast-containing mouse bone cell cultures. In contrast, PGE2 significantly inhibited the bone-resorbing activity and osteopontin mRNA expression in isolated rabbit osteoclasts. Rp-cAMPS, a direct protein kinase (PKA) antagonist, significantly inhibited PGE2-stimulated osteoclast-like cell formation and the bone-resorbing activity of mature osteoclasts, although protein kinase C inhibitors, dantrolene (an inhibitor of calcium release from the intracellular calcium pool) and voltage-dependent calcium channel blockers did not affect PGE2-stimulated osteoclast-like cell formation. In conclusion, PGE2 stimulated osteoclast-like cell formation and bone-resorbing activity in mouse bone cell cultures presumably through osteoblasts. The activation of PKA is linked to PGE2-stimulated osteoclast-like cell formation and bone-resorbing activity.

Animals↗

PubMed1996

Calcitonin prevents bone loss but decreases osteoblastic activity in ovariohysterectomized beagle dogs.

The antiresorptive effects of calcitonin are well documented. Recent in vitro and in vivo evidence points to an anabolic effect of calcitonin on osteoblasts. To assess the value of calcitonin in preventing the rapid and early bone loss after cessation of ovarian function and to investigate its effects on osteoblasts in vivo, 32 dogs were ovariohysterectomized (OHX) and 32 dogs were sham-operated (Sham). After the surgeries, half of the OHX and Sham dogs received every-other-day subcutaneous injections of human calcitonin (0.25 mg/dog/d), and the remaining dogs were given vehicle. Half of the animals had a bone biopsy at week 2 and were euthanized thereafter; the other half of the animals underwent a bone biopsy at month 1 and were euthanized at month 4. Blood drawings were done at baseline and at the time of each bone biopsy. Calcitonin prevented the increase in erosion depth seen in OHX animals and prevented the cancellous bone loss observed at 2 weeks and at 1 and 4 months. Calcitonin did not affect bone volume in Sham dogs. However, treatment with calcitonin induced a decrease in mineralizing surfaces and bone formation rates at the bone surface and cell level and an increase in mineralization lag time in both Sham and OHX animals without significantly affecting osteoblast number. This finding indicates that the negative effect of calcitonin on bone mineralization is not solely the result of a decrease in bone turnover. The data show that calcitonin, because of its antiresorptive effects, can prevent bone loss after cessation of ovarian function. However, short-term treatment with calcitonin does not stimulate osteoblast activity; on the contrary, it exerts a negative effect on osteoblastic bone formation and mineralization. Long-term studies are needed to investigate whether this unwanted effect of calcitonin on osteoblasts in vivo represents a transitory or persistent phenomenon.

Animals↗

PubMed2002

Phytoestrogen genistein stimulates the production of osteoprotegerin by human trabecular osteoblasts.

The anti-resorptive effects of estrogen on bone metabolism are thought to be mediated through modulation of paracrine factors produced by osteoblastic lineage cells that act on osteoclastic lineage cells. Receptor activator of nuclear factor-kappaB ligand (RANKL) is the essential factor for osteoclast formation and activation and enhances bone resorption. By contrast, osteoprotegerin (OPG), which is produced by osteoblastic lineage cells acts as a decoy receptor that neutralizes RANKL and prevents bone loss. Recently, 17 beta-estradiol was found to stimulate OPG mRNA levels and protein secretion in a human osteoblastic cell line through activation of the estrogen receptor (ER)-alpha. In this study, we assessed the effects of the phytoestrogen genistein on OPG mRNA steady state levels (by semiquantitative RT-PCR and Northern analysis) and protein production (by ELISA) in primary human trabecular osteoblasts (hOB) obtained from healthy donors. Genistein increased OPG mRNA levels and protein secretion by hOB cells by up to two- to six-fold in a dose- (P<0.0001) and time-dependent (P<0.0001) fashion with a maximum effect at 10(-7) M. Co-treatment with the pure ER antagonist ICI 182,780 completely abrogated the stimulatory effects of genistein on OPG protein secretion, indicating that these effects were specific and directly mediated through the ER. Pre-treatment with genistein partially prevented the inhibitory effects of the glucocorticoid dexamethasone on OPG mRNA and protein production. The stimulation of OPG mRNA levels by genistein was not affected by the protein synthesis inhibitor, cycloheximide and was shown to be due to enhancement of OPG gene transcription. In conclusion, our data suggest that the phytoestrogen genistein is capable of upregulating the production of OPG by human osteoblasts. Thus, dietary sources of phytoestrogens may help to prevent bone resorption and bone loss by enhanced osteoblastic production of OPG.

Adult↗

PubMed2002

Leptin stimulates human osteoblastic cell proliferation, de novo collagen synthesis, and mineralization: Impact on differentiation markers, apoptosis, and osteoclastic signaling.

Anabolic hormones, mechanical loading, and the obese protein leptin play separate roles in maintaining bone mass. We have previously shown that leptin, as well as its receptor, are expressed by normal human osteoblasts. Consequently, we have investigated how leptin affects proliferation, differentiation, and apoptosis of human osteoblasts. Iliac crest osteoblasts, incubated with either leptin (100 ng/ml), calcitriol (1,25(OH)(2)D(3); 10(-9) M) or 1-84 human parathyroid hormone (PTH; 10(-8) M), were cultured for 35 consecutive days and assayed for expression of various differentiation-related marker genes (as estimated by RT-PCR), de novo collagen synthesis, proliferation, in vitro mineralization, and osteoclast signaling. The effects of leptin on protection against retinoic acid (RA; 10(-7) M) induced apoptosis, as well as transition into preosteocytes, were also tested. Leptin exposure enhanced cell proliferation and collagen synthesis over both control condition and PTH exposure. Leptin inhibited in vitro calcified nodule production after 1-2 weeks in culture, however, subsequent to 4-5 weeks, leptin significantly stimulated mineralization. The mineralization profile throughout the entire incubation period was almost undistinguishable from the one induced by PTH. In comparison, 1,25(OH)(2)D(3) generally reduced proliferation and collagen production rates, whereas mineralization was markedly enhanced. Leptin exposure (at 2 and 5 weeks) significantly enhanced the expression of TGFbeta, IGF-I, collagen-Ialpha, ALP, and osteocalcin mRNA. Leptin also protected against RA-induced apoptosis, as estimated by soluble DNA fractions and DNA laddering patterns subsequent to 10 days of culture. The expression profiles of Bax-alpha and Bcl-2 mRNAs indicated that leptin per se significantly protected against apoptosis throughout the entire incubation period. Furthermore, the osteoblast marker OSF-2 was diminished, whereas the CD44 osteocyte marker gene expression was stimulated, indicating a transition into preosteocytes. In terms of osteoclastic signaling, leptin significantly augmented the mRNA levels of both interleukin-6 (IL-6) and osteoprotegerin (OPG). In summary, continuous leptin exposure of iliac crest osteoblasts, promotes collagen synthesis, cell differentiation and in vitro mineralization, as well as cell survival and transition into preosteocytes. Leptin may also facilitate osteoblastic signaling to the osteoclast.

Apoptosis↗

PubMed2002

Differential regulation of Cbfa1/Runx2 and osteocalcin gene expression by vitamin-D3, dexamethasone, and local growth factors in primary human osteoblasts.

Core binding factor alpha 1 (Cbfa1) is an osteoblast-specific transcription factor essential to develop a mature osteoblast phenotype. However, its exact role in the signaling of various osteotropic-differentiating agents is still unclear. In this study, we assessed the effects of 1,25-(OH)(2)-D3 (D3), ascorbic acid, bone morphogenetic protein-2 (BMP-2), dexamethasone (Dex), and transforming growth factor-beta (TGF-beta) on Cbfa1 and osteocalcin (OCN) mRNA steady state levels (by semiquantitative RT-PCR) in an in vitro model of osteoblast differentiation. TGF-beta increased Cbfa1 mRNA levels in normal primary human osteoblasts (pHOB) by 2.6-fold in a time-dependent fashion with maximum effect on day 28 (P < 0.001). Similarly, the glucocorticoid Dex enhanced Cbfa1 gene expression by pHOB in a time-dependent fashion by up to 4.6-fold (P < 0.001). In contrast, Dex inhibited OCN gene expression levels by 68% (P < 0.01). Treatment with BMP-2 resulted in an earlier enhancement of Cbfa1 and led to a 4.2-fold increase with a maximum on day 21 (P < 0.001). Ascorbic acid did not modulate Cbfa1 and OCN gene expression. The effect of vitamin D (D3) on Cbfa1 mRNA expression was influenced by the duration of treatment, being inhibitory after 1 h and having a stimulatory effect after 48 h. Time course experiments indicated a stimulatory effect of D3 on Cbfa1 mRNA levels (by 2.5-fold after 48 h; P < 0.01). Analysis of the late cellular differentiation marker osteocalcin revealed that D3 increased OCN gene expression by 14-fold (P < 0.001). In conclusion, in normal primary human osteoblasts, the rapid and pronounced increase of OCN after treatment with D3 seems not to be mediated by Cbfa1. These data imply that Cbfa1 gene expression is differentially regulated by various osteoblastic differentiating agents and is dependent on the stage of maturation.

Adult↗