In vitro bone resorption is dependent on physiological concentrations of zinc

The addition of physiological concentrations of zinc (25-200 (Μg/dL) to Dulbecco’s Modified Eagle’s Medium containing tibiae from 19-d chick embryos resulted in a concentration-dependent increase in tibial content of tartrate-resistant acid phosphatase (TRAP) and an increase in bone resorption, as measured by tibial calcium release. This increase in bone resorption was additive to the resorptive effect resulting from the addition of 10^-9-10^-7 M parathyroid hormone (PTH), but was not additive to similar effects produced by the addition of 10^-9-10^-7 M prostaglandin E₂ (PGE₂). An inhibitor of prostaglandin synthesis, flurbiprofen (10^-6 M), did not influence the effect of zinc on bone resorption. However, the addition of 2,6-pyridinedicarboxylic acid (10^-3 M, 2,6-PDCA), a chelator of zinc, did attenuate the effects of zinc, as did the addition of an inhibitor of DNA replication (hydroxyurea, 10^-3 M). Hydroxyurea also attenuated the bone resorptive response to PGE², but had no influence on the effects of PTH. These results indicate that physiological concentrations of zinc alter bone resorptive rates in vitro by a mechanism that is dependent on DNA replication.     

Characterization of bone protein components with polyacrylamide gel electrophoresis: effects of zinc and hormones in tissue culture

An attempt was made to clarify the molecular characterization of zinc-induced bone protein synthesis in tissue culture. Calvaria were removed from weanling rat (3-week-old male) and cultured for periods up to 48 hr in Dulbecco's Modified Eagle Medium (high Glucose, 4500 mg/dl) supplemented with antibiotics and bovine serum albumin. When calvaria cultured in the presence of 10^–5 to 10^–4 M zinc were pulsed with [3H] leucine, zinc caused a significant increase in the incorporation of [³H] leucine into the acid-insoluble residues of bone tissue. The soluble fraction obtained from cultured bone was analyzed with SDS-polyacrylamide gel electrophoresis (SIDS-PAGE). The major components in the fraction obtained from control bone were 68 killo-dalton (kDa) and 45 kDa proteins. These components were clearly increased by the presence of zinc (10^–4 M). The effect of zinc was completely abolished by the coexistence of 10^–6 M cycloheximide. Meanwhile, 10^–9 M estrogen or 10^–8 M insulin, which can stimulate bone formation, did not enhance the effect of zinc to increase bone 68 and 45 kDa proteins. The present findings suggest that zinc increases many bone protein components, especially 68 and 45 kDa proteins.     

Stimulation of bone formation by prostaglandin E2

We examined the effect of prostaglandin E₂ (PGE₂), in the presence or absence of cortisol, on bone formation in 21-day fetal rat calvaria maintained in organ culture for 24 to 96 h. [³H]Thymidine and [³H] proline incorporation were used to assess DNA and collagen synthesis, respectively. Changes in dry weight and DNA content were assessed after 96 h.PGE₂ (10⁻⁷ M) stimulated both DNA and collagen synthesis in calvaria. The effect on DNA synthesis was early (24 h), transient and limited to the periosteum. Collagen synthesis was stimulated at a later time (96 h), predominantly in the central bone. Cortisol (10⁻⁷ M) inhibited DNA and collagen synthesis. The addition of PGE₂ reversed the inhibitory effects of cortisol on DNA synthesis and content and increased collage synthesis in central bone to levels above control untreated cultures.We conclude that PGE₂ has stimulatory effects on bone formation and can reverse the inhibitory effects of cortisol. Hence the effects of cortisol may be mediated in part by their ability to reduce the endogenous production of prostaglandins.     

Anabolic effect of genistein and genistin on bone metabolism in the femoral-metaphyseal tissues of elderly rats: The genistein effect is enhanced by zinc

The effect of genistein and genistin on bone components in the femoral-metaphyseal tissues obtained from elderly female rats was investigated in vitro. The metaphyseal tissues were cultured for 24 h in a medium containing either vehicle, genistein (10^-8-10^-5 M) or genistin (10^-7-10^-5 M). The presence of genistein or genistin caused a significant increase in alkaline phosphatase activity, deoxyribonucleic acid (DNA) and calcium contents in the metaphyseal tissues. The effect of genistein was greater than that of genistin. The bone components increased by genistein (10^-5 M) or genistin (10^-5 M) were completely blocked by the presence of cycloheximide (10^-6 M). The presence of zinc sulfate (10^-5 M) caused a significant increase in the genistein (10^-5 M)-elevated alkaline phosphatase activity, DNA and calcium contents. The enhancement with zinc was not seen by genistin (10^-5 M). The stimulatory effect of zinc on the genistein-induced increase in bone components of the metaphyseal tissues was completely blocked by the presence of cycloheximide (10^-6 M). The present results suggest that genistein and genistin have an anabolic effect on bone metabolism in the femoral-metaphyseal tissues of elderly rats, and that the genistein effect is enhanced by zinc, an essential trace element.     

Effect of beta-D-xylosides on collagen synthesis in cultured fibroblasts.

Cultured normal human skin fibroblasts were incubated with [¹⁴C]proline in the presence and absence of 1.0 mM p-nitrophenyl-β-D-xylose. Formation of non-dialyzable hydroxyproline was used as a measure of collagen synthesis. Although total [¹⁴C]proline incorporation was similar in the two cultures, [¹⁴C]hydroxyproline formation was significantly decreased in the β-xyloside-treated cultures. Increasing the period of incubation increased the radioactivity of the insoluble collagen fraction in untreated fibroblasts, however, in β-xyloside-treated cultures no such increase was observed. In contrast to the decreased production of collagen, growth of cells in the presence of the β-xyloside induced the synthesis of high levels of soluble glycosaminoglycans as measured by ³⁵SO₄ incorporation into isolated polysaccharide.     

Stimulatory effect of menaquinone-7 (vitamim K2) on osteoblastic bone formation in vitro

Menaquinone-7, which is vitamin K₂ (menatetrenone) with seven isoprene units, is highly contained in the fermented soybean. The effect of menaquinone-7 (MK-7) on osteoblastic bone formation was investigated. Femoral-diaphyseal and metaphyseal tissues of young male rats (4 weeks old) were cultured for 48 h in a medium containing either vehicle or MK-7 (10^–7–10^–5 M). Calcium content, alkaline phosphatase activity, and deoxyribonuclic acid (DNA) content in the diaphyseal and metaphyseal tissues was significantly increased in the presence of MK-7 (10^–6 and 10^–5 M). The effect of MK-7 in increasing the diaphyseal and metaphyseal calcium content and alkaline phosphatase activity was completely prevented in the presence of cycloheximide (10^–6 M), an inhibitor of protein synthesis. Moreover, osteoblastic MC3T3-E1 cells after subculture were cultured for 24 h in a serum-free medium containing MK-7 (10^–7–10^–5 M). Protein content, alkaline phophatase activity, osteocalcin and DNA content in the cells was significantly increased in the presence of MK-7 (10^–6 and 10^–5 M). The effect of MK-7 in increasing protein content, alkaline phosphatase activity, and osteocalcin production in the cells was completely blocked by cycloheximide. This study demonstrates that MK-7 has an anabolic effect on bone tissue and osteoblastic MC3T3-E1 cells in vitro, suggesting that the compound can stimulate osteoblastic bone formation.     

Arginine vasopressin increases the rate of protein synthesis in isolated perfused adult rat heart via the V1 receptor

The effect of daidzein on cortical bone in vitro was investigated. Femoral-diaphyseal tissues obtained from elderly female rats were cultured for 24 h in Dulbecco's modified Eagle's medium (high glucose, 4.5%) supplementation with antibiotics and bovine serum albumin. The experimental cultures contained 10-7 to 10-5 M daidzein. The presence of daidzein (10-6 and 10-5 M) caused a significant increase of alkaline phosphatase activity, deoxyribonucleic acid (DNA) and calcium contents in bone tissues. This effect was equal to that of genistein (10-6 and 10-5 M). Daidzein (10-5 M) or genistein (10-5 M)-induced increase of calcium content and alkaline phosphatase activity in bone tissues was completely prevented by cycloheximide (10-6 M), an inhibitor of protein synthesis. Anabolic effect of daidzein and genistein on bone components was equal to that of 17-estradiol (10-8 M). The effect of isoflavohoids was not enhanced by the addition of 17-estradiol. The combination of daidzein and genistein did not have an additive effect. These findings indicate that daidzein has an anabolic effect on bone metabolism in tissue culture in vitro, and that this effect is equal to genistein effect. Isoflavonoids may stimulate bone formation and mineralization.     

Inhibition of cell metabolism by a smokeless tobacco extract: tissue and species specificity.

Smokeless tobacco contains a nonnicotine inhibitor of posttranslational modification of collagen (hydroxylation of [3H]proline) by cultured chick embryo tibias and osteoblasts. This study was undertaken to determine whether a methanol extract of smokeless tobacco (STE) containing the inhibitor has similar effects on collagen-producing cells and tissues other than bone. Its effects on DNA synthesis and cell proliferation (incorporation of [3H]thymidine) were also determined. Frontal bone, aorta, and cartilage were incubated for 2 days in medium containing STE. Glycolysis (lactate production) was stimulated by 80% in cartilage, but was not affected in the other tissues; medium alkaline phosphatase activity was unaffected. In frontal bone and cartilage, [3H] hydroxyproline content was decreased 88% and 57%, respectively, and [3H]proline content was decreased 68% and 37%, respectively; neither was affected in the aorta. Confluent cultures of collagen-producing mouse fibroblasts or primary osteoblasts obtained from chick embryo calvarias were incubated for 2 days in medium containing increasing concentrations of STE. Glycolysis and DNA synthesis were not affected. Cell proliferation was unaffected in fibroblasts, but was inhibited (34%) at the highest STE concentration in osteoblasts. AIPase activity was not detectable in fibroblast medium, but was decreased up to 72% in osteoblast medium. Inhibition of collagen synthesis by STE was concentration related in both cell types. At the highest concentration, [3H] hydroxyproline and [3H]proline contents in the cell layers were decreased to the following respective values: fibroblasts 56% and 45% and osteoblasts 50% and 29%, respectively. When incubation with STE was discontinued for 1 day, recovery did not occur. These findings suggest that inhibition of collagen synthesis by STE is not specific for bone, that collagen-producing cells are directly affected, and that recovery is not immediate. This inhibitor could contribute to the periodontal disease often seen in users of smokeless tobacco. Its identification and removal would produce a safer product.     

Inhibition of formation of protein-bound hydroxyproline by free hydroxyproline in Avena coleoptiles

Free hydroxyproline inhibits the formation of protein-bound hydroxyproline from proline to a considerably greater extent than it does the incorporation of proline into protein of auxin-treated Avena coleoptiles. This inhibition is greater in the wall than in the cytoplasmic fraction. In the absence of auxin, free hydroxyproline exerts little or no inhibition of hydroxyproline formation. Furthermore free hydroxyproline has no effect on respiration, RNA synthesis or the incorporation of leucine into protein. Hydroxyproline is not a general inhibitor of metabolism or protein synthesis in Avena coleoptiles.

These results suggest that free hydroxyproline may inhibit auxin-induced cell elongation by blocking the formation or utilization of a particular hydroxyproline-rich protein which must be incorporated into the cell wall during auxin-induced wall extension.

     

Studies on the biochemistry and fine structure of silica shell formation in diatoms

In diatoms, the siliceous cell walls are enveloped by an organic component which includes 4-hydroxyproline and 3,4-dihydroxy-L-proline. The formation of these two amino acids were studied in Nitzschia angularis in Si-starvation synchrony. Both appear to arise from peptidyl proline. Its conversion to peptidyl hydroxyproline was shown in cell-free extracts and in kinetic studies using [¹⁴C]proline. Two lines of evidence indicate that dihydroxyproline does not arise from the further hydroxylation of peptidyl hydroxyproline: First, there was a lag of several minutes between the incorporation of [¹⁴C]proline into protein and the appearance therein of [¹⁴C]hydroxyproline but no such lag for the appearance of dihydroxyproline. Second, ,-dipyridyl blocked the formation of hydroxyproline, but not of dihydroxypyroline, from peptidyl proline. Cell walls made in the presence of dipyridyl differed little in overall chemical composition from walls made in its absence and were morphologically identical. [¹⁴C]dehydroproline was rapidly metabolized in the cells, with [¹⁴C]dihydroxyproline a prominent product. Studies of the conversion of [¹⁴C]proline to [¹⁴C]hydroxyproline at various stages of wall formation showed an increased synthesis of [¹⁴C]dihydroxyproline at the end of cell separation.     

Stimulation of bone formation by prostaglandin E2

We examined the effect of prostaglandin E2 (PGE2), in the presence or absence of cortisol, on bone formation in 21-day fetal rat calvaria maintained in organ culture for 24 to 96 h. [3H]Thymidine and [3H] proline incorporation were used to assess DNA and collagen synthesis, respectively. Changes in dry weight and DNA content were assessed after 96 h. PGE2 (10(-7) M) stimulated both DNA and collagen synthesis in calvaria. The effect on DNA synthesis was early (24 h), transient and limited to the periosteum. Collagen synthesis was stimulated at a later time (96 h), predominantly in the central bone. Cortisol (10(-7) M) inhibited DNA and collagen synthesis. The addition of PGE2 reversed the inhibitory effects of cortisol on DNA synthesis and content and increased collagen synthesis in central bone to levels above control untreated cultures. We conclude that PGE2 has stimulatory effects on bone formation and can reverse the inhibitory effects of cortisol. Hence the effects of cortisol may be mediated in part by their ability to reduce the endogenous production of prostaglandins.     

16, 16 Dimethyl prostaglandin E2 decreases the formation of collagen in fibrotic rat liver slices

The effect of 16, 16 dimethyl prostaglandin E₂ (DMPG) on fibrogenesis was studied in slices from normal and fibrotic rat liver. Rats received a cirrhogenic diet for seven months; supplemented controls received a diet with the deficient nutrients restored. Slices from fibrotic livers incorporated more ¹⁴C-proline and produced more ¹⁴C-hydroxyproline in TCA precipitable proteins than slices from control livers. DMPG (10^−10M) decreased the incorporation of labeled proline and the synthesis of labeled hydroxyproline in slices from fibrotic livers to the same extent, suggesting that DMPG did not affect the hydroxylation of proline per se. The magnitude of the DMPG induced decrease in labeled proline incorporation correlated with the hydroxyproline content in the liver (i.e. with increasing fibrosis there was a greater effect of DMPG; while in control rat liver slices, DMPG had no effect). DMPG did not change the size of the proline pool, its specific activity, or the activity of proline oxidase. We conclude that under these conditions of enhanced fibrogenesis, DMPG decreases the formation of collagen in vitro, possibly by lowering the incorporation of proline into collagen precursors. This may explain, at least in part, the inhibition of fibrogenesis by DMPG in vivo.     

Rapid intracellular degradation of newly synthesized collagen by bone cells Effect of dichloromethylenebisphosphonate

Experiments were carried out to determine whether bone cells isolated from rat calvaria degrade newly synthesized collagen intracellularly prior to secretion and to assess the effect of dichloromethylenebisphosphonate, a compound shown to stimulate collagen synthesis during this event. The findings indicate that isolated bone cells grown in culture degraded a proportion (average 16%) of newly synthesizes collagen prior to secretion. This process was markedly reduced by exposure to dichloromethylenebisphosphonate in a dose-related manner. Concomitantly with the observed decrease of degradation, an increase of collagen synthesis was detected as determined by the incorporation of [³H]proline into collagenase-digestible proteins or by the conversion of [³H]proline into [³H]hydroxyproline. No similar enhancement on total non-collagenous protein synthesis was evident. Dichloromethylenebisphosphonate did not influence the extracellular degradation of collagen. Although the reduction in intracellular degradation accounted only for part of the bisphosphonate mediated increase in net collagen synthesis, it is conceivable that the rate of collagen synthesis is regulated, at least in part, by mechanisms that modulate the level of intracellular degradation.     

Extracts of bone contain a potent regulator of bone formation

We prepared aqueous extracts of whole femorae and tibiae of embryonic chicks. An amount of extract containing 25 μg of protein resulted in a 500% increase in DNA synthesis in calvarial cell cultures, and significant effects were detected with 5 μg (55%). The time course for stimulation of DNA synthesis showed a peak occurring 16–20 h after addition of the extract. This matrix factor is nondialyzable, and fractionation on a column of Sephadex G-100 indicated a molecular weight of 60–80 000. At the maximum dose used, [³H]proline incorporation into total protein of calvarial cells was increased by 55%, and thus far, all fractions active in promoting DNA synthesis have been found to increase collagen synthesis in culture chick tibiae. These data are consistent with an effect on osteoblasts as well as bone precursor cells. Extracts prepared from tibiae of 2-day-old chicks, from which the marrow had been removed, also stimulated DNA synthesis (280% increase), thus ruling out the possibility that the factor is a relatively nonspecific mitogen from the hematopoietic cell line. We conclude that bone matrix contains a substance which could regulate bone formation in vitro by control of mitosis in osteogenic precursors and/or stimulation of osteoblast activity.     

Effect of β-alanyl-L-histidinato zinc on differentiation of osteoblastic MC3T3-El cells: Increases in alkaline phosphatase activity and protein concentration

The effect of -alany-L-histidinato zinc (AHZ) on bone cell function was investigated in osteoblastic MC3T3-E1 cells. Cells were cultured for 3 days at 37°C in a CO₂ incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus AHZ (10^–7–10^–5 M) or other reagents, and the cells were cultured further for appropriate periods of time. The presence of AHZ (10^–7–10^–5 M) produced a remarkable increase of alkaline phosphatase activity and protein concentration in osteoblastic cells. Thus increases were seen with the prolonged cultivation (12–21 days). With the culture of 1, 3 and 12 days, the effect of AHZ (10^–6 M) to increase alkaline phosphatase activity and protein concentration was more intensive than the effect of zinc sulfate, (10^–6 M). The AHZ effects were completely abolished by the presence of cycloheximide (10^–6 M), indicating that AHZ stimulates protein synthesis in the cells. The present study suggests that AHZ has a stimulatory effect on cell differentiation, and that this effect is partly involved on protein synthesis in osteoblastic cells.     

Human skeletal growth factor stimulates collagen synthesis and inhibits proliferation in a clonal osteoblast cell line (MC3T3-E1)

Human skeletal growth factor (hSGF), an 11-kD polypeptide purified from human bone, has been proposed to be a local regulator of bone formation. To investigate the underlying cellular mechanisms in an in vitro model system, we examined the effects of hSGF on proliferation and collagen synthesis in cells of the clonal osteoblast cell line MC3T3-E1. This line was isolated from newborn mouse calvarial cells and retains many characteristics of mature osteoblasts (Sudo, H., et al., (1984) J. Cell Biol. 96:191). A 14-hr treatment with hSGF increased noncollagenous protein synthesis to 215% of unstimulated controls and increased collagen synthesis to 630% of controls as determined by [3H]proline incorporation and high-pressure liquid chromatographic separation of [3H]proline and [3H]hydroxyproline in acid hydrolysates of trichloroacetic acid-insoluble protein. HSGF did not increase cell number over a 48-hr period and caused a reversible inhibition of DNA synthesis. Half-maximal hSGF concentration for stimulation of [3H]proline incorporation and inhibition of [3H]thymidine incorporation was 100 ng/ml. HSGF also inhibited DNA synthesis in cells stimulated by serum. In contrast, hSGF stimulated both collagen synthesis and DNA synthesis in primary cultures of chick embryo bone cells, which may be developmentally less mature than MC3T3-E1 cells. The results suggest that hSGF directly stimulated mature osteoblast matrix synthetic activity and that hSGF has differential effects on proliferation of osteoblast progenitor cells and mature osteoblasts.     

Extracellular pH modulates the activity of cultured human osteoblasts

The effect of medium pH on the activity of cultured human osteoblasts was investigated in this study. Osteoblasts derived from explants of human trabecular bone were grown to confluence and subcultured. The first-pass cells were incubated in Hepes-buffered media at initial pHs adjusted from 7.0 to 7.8. Osteoblast function was evaluated by measuring lactate production, alkaline phosphatase activity, proline hydroxylation, DNA content, and thymidine incorporation. Changes in medium pH were determined from media pHs recorded at the beginning and end of the final 48 h incubation period. As medium pH increased through pH 7.6, collagen synthesis, alkaline phosphatase activity, and thymidine incorporation increased. DNA content increased from pH 7.0 to 7.2, plateaued from pH 7.2 to 7.6, and increased again from pH 7.6 to 7.8. The changes in the medium pH were greatest at pHs 7.0 and 7.8, modest at pHs 7.4 and 7.6, and did not change at 7.2, suggesting that the pHs are migrating towards pH 7.2. Lactate production increased at pH 7.0 but remained constant from 7.2 to 7.8. These results suggest that in the pH range from 7.0–7.6 the activity of human osteoblasts increases with increasing pH, that this increase in activity does not require an increase in glycolytic activity, and that pH 7.2 may be the optimal pH for these cells. J. Cell. Biochem. 68:83–89, 1998. © 1998 Wiley-Liss, Inc.     

Collagen biosynthesis by human skin fibroblasts III. The effects of ascorbic acid on procollagen production and prolyl hydroxylase activity

Human skin fibroblasts were cultured under conditions optimized for collagen synthesis, and the effects of ascorbic acid on procollagen production, proline hydroxylation and the activity of prolyl hydroxylase were examined in cultures. The results indicated that addition of ascorbic acid to confluent monolayer cultures of adult human skin fibroblasts markedly increased tha amount of [³H]hydroxyproline syntehsized. Ascorbic acid, however, did not increase the synthesis of ³H-labeled collagenous polypeptides assayed independently of hydroxylation of proline residues, nor did it affect the amount of prolyl hydroxylase detectable by an in vitro enzyme assay. Also long-term cultures of the cells or initiation of fibroblast cultures in the presence of ascorbic acid did not lead to an apparent selection of a cell population which might be abnormally responsive to ascorbic acid. Thus, ascorbic acid appears to have one primary action on the synthesis of procollagen by cultured human skin fibroblasts: it is necessary for synthesis of hydroxyproline, and consequently for proper triple helix formation and selection of procollagen.     

Extracellular matrix metabolism by chondrocytes: VI. Concomitant depression by exogenous levels of proteoglycan of collagen and proteoglycan synthesis by chondrocytes

The synthesis of collagen and proteoglycans by cultured chondrocytes, as measured by the incorporation of L-[³H]proline into hydroxyproline and [³H]acetate into glycosaminoglycans, was shown to be depressed by 59% and 39%, respectively, by the addition of exogenous proteoglycan at a concetration of 10 mg/ml growth media. The incorporation of L-[³H]proline into acid-in-soluble protein remained unaltered in the presence of the proteoglycan. It was concluded that the effect was depressing the activity of the enzymatic steps, associated with the endoplasmic reticulum, which are responsible for the post-traslational modification of collagen and proteoglycan.