Rationale for active vitamin D analog therapy in senile osteoporosis.
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Aged↗
Biomedical subjects
K H Lau
Publications and source records attributed to K H Lau.
Fluoride at mitogenic doses induces a sustained activation of p44mapk, but not p42mapk, in human TE85 osteosarcoma cells.
Fluoride, at micromolar concentrations, stimulates bone cell proliferation in vitro. In this study, we sought to test whether fluoride at mitogenic doses increases the tyrosyl phosphorylation level and specific activity of a mitogen-activated protein kinase (MAPK) in human TE85 osteosarcoma cells. Analysis by immunoprecipitation with antiphosphotyrosine antibody followed by Western analysis using an anti-pan extracellular signal-regulated kinase antibody revealed that fluoride at the optimal mitogenic dose (i.e. 100 mumol/L) induced a time-dependent increase in the steady state tyrosyl phosphorylation level of p44mapk, but not p42mapk, with the maximal increase (4- to 13-fold) after 1-3 h fluoride treatment. The effect was sustained in that a 9-fold increase was seen after 12 h of the fluoride treatment. The sustained nature of the effect is consistent with an inhibition of dephosphorylation rather than a direct stimulation of phosphorylation. The fluoride effect on the tyrosyl phosphorylation level of p44mapk was dose dependent, with the optimal dose being 100 mumol/L fluoride. The mitogenic dose of fluoride also increased the specific activity and the in-gel kinase activity of p44mapk, but not that of p42mapk, in a time-dependent manner similar to the effect on the p44mapk tyrosyl phosphorylation level. Fluoride at the same micromolar doses did not increase cell proliferation, tyrosyl phosphorylation, or specific activity of any MAPK in human skin foreskin fibroblasts, which are fluoride-nonresponsive cells. Consistent with the interpretation that the effect of fluoride on the steady state tyrosyl phosphorylation level of p44mapk is a consequence of an inhibition of a phosphotyrosyl phosphatase (PTP), mitogenic doses of orthovanadate, a bone cell mitogen and a PTP inhibitor, also increased the steady state tyrosyl phosphorylation level of p44mapk, but not p42mapk, in a time-dependent sustained manner similar to that observed with fluoride. Together, these findings support the concept that inhibition of a PTP activity in bone cells could lead to an activation of MAPK activity.
Bone and Bones↗
Molecular cloning and expression of a unique rabbit osteoclastic phosphotyrosyl phosphatase.
Tyrosyl phosphorylation plays an important regulatory role in osteoclast formation and activity. Phosphotyrosyl phosphatases (PTPs), in addition to tyrosyl kinases, are key determinants of intracellular tyrosyl phosphorylation levels. To identify the PTP that might play an important regulatory role in osteoclasts, we sought to clone an osteoclast-specific PTP. A putative full-length clone encoding a unique PTP (referred to as PTP-oc) was isolated from a 10-day-old rabbit osteoclastic cDNA library and sequenced. A single open reading frame predicts a protein with 405 amino acid residues containing a putative extracellular domain, a single transmembrane region, and an intracellular portion. PTP-oc is structurally unique in that, unlike most known transmembrane PTPs, it has a short extracellular region (eight residues), lacks a signal peptide proximal to the N-terminus, and contains only a single 'PTP catalytic domain'. The PTP catalytic domain shows 45-50% sequence identity with the catalytic domain of human HPTP beta and with the first catalytic domain of LCA. The PTP-oc gene exists as a single copy in the rabbit genome. The corresponding mRNA (3.8 kb) is expressed in osteoclasts but not in other bone-derived cells (e.g. osteoblasts and stromal cells). The 3.8 kb PTP-oc mRNA transcript was also expressed in the rabbit brain, kidney and spleen. However, the brain and kidney, but not osteoclasts or spleen, also expressed a larger transcript (6.5 kb). The PTP catalytic domain of PTP-oc was expressed as a GST-cPTP-oc fusion protein. In vitro phosphatase assays indicated that the purified fusion protein exhibited phosphatase activities at neutral pH values toward p-nitrophenyl phosphate, phosphotyrosyl Raytide, and phosphotyrosyl histone, whereas it had no appreciable activity toward phosphoseryl casein. In summary, we have: (a) cloned and sequenced the putative full-length cDNA of a unique PTP (PTP-oc) from rabbit osteoclasts; (b) shown that the mature 3.8 kb PTP-oc mRNA was expressed primarily in osteoclasts and the spleen; and (c) shown that the PTP-oc fusion protein exhibited a phosphotyrosine-specific phosphatase activity. In conclusion, PTP-oc represents a structurally unique subfamily of transmembrane PTPs.
Amino Acid Sequence↗
Osteogenic actions of phenytoin in human bone cells are mediated in part by TGF-beta 1.
We have recently demonstrated that phenytoin, a widely used therapeutic agent for seizure disorders, has osteogenic effects in rats and in humans in vivo, and in human bone cells in vitro. The goal of the present study was to determine the mechanism of the osteogenic action of phenytoin in normal human mandible-derived bone cells. Because many osteogenic agents increased bone cell proliferation through mediation by growth factors, we tested the hypothesis that the osteogenic effects of phenytoin involved the release of a growth factor by measuring the mRNA level of several bone cell growth factors and insulin-like growth factor (IGF) binding proteins with Northern blots using specific cDNA probes. Treatment with 5-50 microM phenytoin reproducibly and markedly increased (up to 6-fold, p < 0.001) the mRNA of transforming growth factor (TGF)-beta 1, but not that of other growth factors (i.e., IGF-II, platelet-derived growth factor-A [PDGF-A], PDGF-B, and TGF-beta 2) and IGF binding proteins (i.e., IGFBP-3, -4, and -5). The stimulation was dose dependent, with an optimal dose of 10-50 microM. Maximal increase was seen after 1 h of phenytoin treatment. The release of biologically active TGF-beta activity in conditioned media was measured with the mink lung cell proliferation inhibition assay. Twenty-four hours of phenytoin treatment significantly increased the production of biologically active TGF-beta (2-fold, p < 0.05) with the optimal dose between 5-50 microM. Comparisons between the in vitro osteogenic effects of phenytoin and those of TGF-beta 1 reveal that these two agents at their respective optimal doses had similar maximal stimulatory effects on [3H]thymidine incorporation, alkaline phosphatase (ALP)-specific activity, and type I alpha-2 collagen mRNA expression in human bone cells. The stimulatory effects of phenytoin on [3H]thymidine incorporation and ALP-specific activity were completely blocked by a neutralizing anti-TGF-beta antibody. In conclusion, these findings demonstrate for the first time that at least some of the osteogenic actions of phenytoin in human bone cells could be in part mediated by TGF-beta 1.
Anticonvulsants↗
Mitogenic action of hydrochlorothiazide on human osteoblasts in vitro: requirement for platelet-derived growth factor.
Long-term use of hydrochlorothiazide (HCTZ), a common diuretic agent for hypertension, has been associated with increased bone density and reduced hip fracture rates in patients. In this study, we sought to examine whether HCTZ has an anabolic effect on the proliferation of human osteoblasts (derived from either vertebrate or rib bone samples) in vitro. Cell proliferation was determined by [3H]thymidine incorporation and cell number counting. In medium supplemented with 1% bovine calf serum, HCTZ significantly and reproducibly increased [3H]thymidine incorporation and cell number. The stimulatory effect was dose dependent in a biphasic manner, with the maximal stimulation (approximately 60% above control, P < 0.001) seen at 1 microM HCTZ. In fresh serum-free medium, HCTZ was ineffective as a bone cell mitogen, indicating that the bone cell mitogenic activity of HCTZ required a serum growth factor (GF). HCTZ at doses greater than 10 microM was inhibitory in the presence or the absence of serum, presumably because of the cytotoxic effects. The serum requirement for the bone cell mitogenic activity of HCTZ could be replaced with a conditioned medium (conditioned with normal human osteoblasts for 24 hours), or with a mitogenic dose (1 ng/ml) of PDGF. The GF requirement was specific for PDGF, because other bone cell-derived growth factors (i.e., TGFbeta, IGF-I, IGF-II, and bFGF) were unable to replace serum for the bone cell mitogenic activity of HCTZ. In summary, this study shows that (1) HCTZ stimulated the proliferation of normal, untransformed, human osteoblasts in vitro; (2) the bone cell mitogenic effect of HCTZ required the presence of a serum GF; (3) the serum requirement could be replaced with a bone cell GF in conditioned medium; (4) the GF requirement was specific for PDGF. In conclusion, we have demonstrated for the first time that HCTZ has a direct anabolic effect on human osteoblasts in vitro, and that the mitogenic activity is dependent on the presence of PDGF. Because increased bone cell proliferation is a key determinant of bone formation, these observations raise the interesting possibility that HCTZ could act directly on bone cells to stimulate bone formation in patients.
Cell Division↗
Dexamethasone enhances the osteogenic effects of fluoride in human TE85 osteosarcoma cells in vitro.
The in vitro osteogenic effects of fluoride have not always been consistently observed in human bone cells. The present study sought to test if dexamethasone (Dex) could potentiate the action of fluoride to increase the detectability of the stimulatory effects of fluoride on [3H]thymidine incorporation, alkaline phosphatase (ALP) specific activity, collagen synthesis, and osteocalcin secretion in human TE85 osteosarcoma cells. Neither Dex at 10(-10)-10(-6) M or fluoride at a mitogenic dose (100 microM) had any consistent stimulatory effects on thymidine incorporation. When the cells were treated with both agents simultaneously, significant and highly reproducible stimulations were observed. The mitogenic effects of the two agents were confirmed with cell number counting. Analysis of variance (ANOVA) revealed a significant interaction (P < 0.001) between fluoride and Dex on cell proliferation. The enhancing effect of Dex on [3H]thymidine incorporation was not due to a shift of the optimal dose response of fluoride. Though fluoride alone or Dex alone also had no consistent effect on ALP specific activity, the co-treatment with fluoride and Dex for 24 hours produced significant (P < 0.001, ANOVA) stimulation in ALP specific activity. Fluoride alone had no consistent effect on collagen synthesis and on 1, 25(OH)2D3-dependent osteocalcin secretion, whereas Dex treatment consistently inhibited these two osteoblastic parameters in a dose-dependent manner. However, both the collagen synthesis and osteocalcin secretion rates were significantly higher (P < 0.001 ANOVA for each) when the cells were co-treated with Dex and fluoride (100 microM) than when they were treated with Dex alone. Thus, these data indicate that the response in collagen synthesis and osteocalcin secretion to fluoride stimulation was more readily observed in the presence of Dex than in its absence. ANOVA analysis revealed that the interaction between fluoride and Dex on collagen synthesis, but not the 1,25(OH)2D3-dependent osteocalcin secretion, was significant (P < 0.02). In summary, we have demonstrated for the first time that in TE85 cells (1) Dex potentiated the effects of fluoride on cell proliferation, ALP specific activity, and collagen synthesis; (2) while Dex at 10(-7)-10(-6) M alone inhibited the collagen synthesis and at 10(-9)-10(-6) M reduced osteocalcin secretion, Dex at 10(-8)-10(-6) M significantly stimulated the proliferation of TE85 cells; and (3) Dex interacted with fluoride to increase the percentage of experiments showing an osteogenic action of fluoride. In conclusion, the in vitro osteogenic actions of fluoride in human TE85 cells are more consistently observed in the presence than in the absence of Dex.
Alkaline Phosphatase↗
Fluoride at mitogenic concentrations increases the steady state phosphotyrosyl phosphorylation level of cellular proteins in human bone cells.
This study was designed to test the hypothesis that treatment of human bone cells with mitogenic concentrations of fluoride would lead to an increase in the steady state level of tyrosyl phosphorylation of specific cellular proteins. With an immunoblot assay method, it was found that mitogenic concentrations of fluoride (i.e. 50-200 mumol/L) induced a dose- and time-dependent increase in the level of tyrosyl phosphorylation of at least 13 cellular proteins in both normal human bone cells and human TE85 osteosarcoma cells. Time-course studies revealed that a statistically significant increase in tyrosyl phosphorylation of these 13 cellular proteins in human bone cells was observed after 3-6 h of fluoride treatment and was sustained for up to 24 h. This time course was not compatible with a direct activation of tyrosyl kinases, as epidermal growth factor, which activates tyrosyl kinase activity, induced an immediate and acute response that was rapidly reversible within 1 h. Although fluoride increased the steady state tyrosyl phosphorylation of the cellular proteins in human bone cells, the same micromolar doses of fluoride had no effect on human skin fibroblasts, which are fluoride-nonresponsive cells. The effects of fluoride were rapidly reversible in the absence of fluoride and could be acutely potentiated by pretreatment with epidermal growth factor. In summary, we have shown for the first time that mitogenic concentrations (i.e. 50-200 mumol/L) of fluoride increased the steady state level of tyrosyl phosphorylation of at least 13 cellular proteins in human bone cells, and that the increases were relatively show in onset and sustained. In conclusion, these findings are consistent with the hypothesis that the osteogenic actions of fluoride are mediated at least in part by an inhibition of the activity of one or more fluoride-sensitive phosphotyrosyl protein phosphatases in human bone cells.
Bone and Bones↗
Pretreatment with low doses of norethindrone potentiates the osteogenic effects of fluoride on human osteosarcoma cells.
We recently reported that picomolar doses of norethindrone (NET), a synthetic analog of 19-nortestosterone, significantly stimulated human TE85 osteosarcoma cell proliferation, differentiation, and activity in vitro. In the present study, we investigated the possibility that NET interacts with another osteogenic agent, i.e., fluoride, to stimulate human TE85 osteosarcoma cell proliferation, differentiation, and activities. Bone cell proliferation was measured by the stimulation in [3H]thymidine incorporation. Differentiation was monitored by the increase in alkaline phosphatase-specific activity. Osteoblastic activity was assessed by the stimulations in collagen synthesis and in osteocalcin secretion (in the presence of 1 nM 1,25-dihydroxyvitamin D3). When the human TE85 cells were incubated with mitogenic doses of NET and fluoride concurrently, the stimulatory effects of the two agents on these parameters exhibited no significant interaction. The enhancing effect of NET on the osteogenic effect of fluoride was not due to a shift of the fluoride dose response curve. Pretreatment with NET for 24 h followed by a treatment with a mitogenic dose (i.e., 100 microM) of fluoride for an additional 24 h significantly and synergistically potentiated the effects of fluoride on the [3H]thymidine incorporation, alkaline phosphatase-specific activity, collagen synthesis, and osteocalcin secretion, compared with those with the subsequent vehicle (0.05% ethanol) treatments. In contrast, pretreatment with fluoride for 24 h before the addition of NET for 24 h did not produce significant synergistic stimulations in the test parameters. Pretreatment of TE85 cells with the same doses of dihydrotestosterone or progesterone prior to treatment with fluoride under the same conditions did not induce synergistic potentiation of fluoride in [3H]thymidine incorporation, suggesting that the synergistic interaction with fluoride is probably not a common property of anabolic sex steroids. In summary, we found that: (1) the osteogenic effects of fluoride and NET were additive when cells were treated with both agents concurrently; (2) a 24-h pretreatment with picomolar doses of NET potentiated the osteogenic actions of fluoride in human TE85 osteosarcoma cells; and (3) pretreatment with NET produced a subsequent fluoride response that was synergistic. In conclusion, these findings led us to speculate that the osteogenic actions of NET and fluoride act through different mechanisms, and that NET at low doses has a permissive effect on the osteogenic effects of fluoride, and as such NET may be used in concert with fluoride to increase osteoblast proliferation, differentiation, and activity.
Alkaline Phosphatase↗
Expression of the rat alpha 1 thyroid hormone receptor ligand binding domain in Escherichia coli and the use of a ligand-induced conformation change as a method for its purification to homogeneity.
The rat alpha 1 thyroid hormone receptor (rTR alpha 1) mediates hormone-dependent gene regulation by utilizing several distinct structural domains, including those containing DNA and ligand binding sites. Binding of the hormone to the ligand binding domain (TR-LBD) induces conformational changes in the receptor that are involved in affecting the receptor's transcriptional regulatory and other functions. A 33-kDa protein fragment (Met122-Val410) of rTR alpha 1, which includes the entire TR-LBD, was expressed in Escherichia coli, yielding typically 1.5 mg of soluble TR-LBD/liter of bacteria. The protein was purified to > 99% homogeneity with a final yield of 24% by hydrophobic interaction, DEAE anionic exchange, and heparin cationic exchange chromatographic steps. The Kd of the purified TR-LBD for 3,3',5-triiodo-L-thyronine (T3) was 0.06 nM, identical to that for full-length rTR alpha 1. T3 analogs had affinities consistent with values obtained for full-length rTR alpha 1. In all three chromatography steps, TR-LBD prebound to [125I]T3 eluted earlier than the unliganded TR-LBD, like the full-length receptor. These studies indicate that the binding affinity and specificity of the TR-LBD are similar to those of the intact rTR alpha 1 and that the ligand-induced conformational changes occur in the LBD itself. These studies also provide methodology for obtaining milligram quantities of protein useful for biochemical and biophysical studies of the thyroid hormone receptor and its ligand-induced changes.
Amino Acid Sequence↗
Low dose phenytoin is an osteogenic agent in the rat.
Long-term use of phenytoin for the treatment of epilepsy has been associated with increased thickness of craniofacial bones. The aim of the present study was to evaluate the possibility that low doses of phenytoin are osteogenic in vivo by measuring the effects of phenytoin administration on serum and bone histomorphometric parameters of bone formation in two rat experiments. In the first experiment, four groups of adult male Sprague-Dawley rats received daily I.P. injections of 0, 5, 50, or 150 mg/kg/day of phenytoin, respectively, for 47 days. Serum alkaline phosphatase (ALP) and osteocalcin were increased by 5 and 50 mg/kg/day phenytoin. The increases in osteocalcin and ALP occurred by day 7 and day 21, respectively. The tibial diaphyseal mineral apposition rate (MAR) at sacrifice (day 48) was significantly increased in rats receiving 5 mg/kg/day phenytoin. At a dose of 150 mg/kg/day, the increase in serum ALP, osteocalcin and MAR was reversed. No significant differences in serum calcium, phosphorus, or 1,25(OH)2D3 levels were seen. In a second experiment, three groups of rats received daily I.P. injection of lower doses of phenytoin (i.e., 0, 1, or 5 mg/kg/day, respectively) for 42 days. Phenytoin also did not affect the growth rate or serum calcium, phosphorus, and 25(OH)D3 levels. Daily injection of 5 mg/kg/day phenytoin significantly increased several measures of bone formation, i.e., serum ALP and osteocalcin bone ALP, periosteal MAR, and trabecular bone volume.(ABSTRACT TRUNCATED AT 250 WORDS)
Alkaline Phosphatase↗
Phenytoin and fluoride act in concert to stimulate bone formation and to increase bone volume in adult male rats.
We have recently demonstrated that phenytoin is an osteogenic agent at low doses. The present paper describes observations that a mitogenic dose (i.e., 20 microM in BGJb medium) of fluoride significantly augments the phenytoin-dependent stimulation of normal human bone cell proliferation and alkaline phosphatase (ALP) activity in cell culture. Additionally, the present study was designed to investigate whether fluoride and phenytoin would interact to increase bone formation in rats in vivo. Four groups of weight-matched adult male rats received daily I.P. injection of (1) vehicle (10% DMSO), (2) 5 mg/kg/day phenytoin, (3) 5 mg/kg/day phenytoin and 50 ppm NaF, and (4) 50 ppm NaF and vehicle, respectively, for 36 days. Sodium fluoride (NaF) was delivered in drinking water. Blood samples were drawn weekly and analyzed for serum osteocalcin, ALP, calcium, phosphorus, and 25(OH)D3. Rats were labeled with tetracycline at day 21 and 30 and histomorphometric analysis was carried out on the tibia at the end of the experiment. Neither agent by itself or together affected the serum calcium, phosphorus, or 25(OH)D3 levels. All measures of bone formation, i.e., serum osteocalcin level and ALP activity, bone ALP specific activity, mineral apposition rate, bone formation rate, and % bone formation surface, were increased by each agent. Fluoride and phenytoin together produced bigger increases in each parameter than did each agent alone. Trabecular bone volume was increased in the tibial metaphysis by fluoride or phenytoin alone; and when administered together, the two agents produced a greater increase.(ABSTRACT TRUNCATED AT 250 WORDS)
Alkaline Phosphatase↗
Calcitonin acutely increases tyrosyl-phosphorylation of proteins in human osteosarcoma (SaOS-2) cells.
In order to test the hypothesis that salmon calcitonin has direct effects to modulate tyrosyl-protein phosphorylation in human osteosarcoma cells, SaOS-2 cells (with very high steady-state levels of skeletal alkaline phosphatase) were exposed to calcitonin, in duplicate serum-free cultures, at concentrations ranging from 10(-13) to 10(-9) mol/liter, for 0-60 minutes at 37 degrees C. Phospho-tyrosyl proteins were identified by autoradiography of Western blots after incubation with 125I-labeled antiphosphotyrosine antibodies (or with unlabeled antibodies and 125I-labeled protein A) and quantitated by laser densitometry. The results of these studies revealed (1) time-dependent effects of salmon calcitonin (sCt) (at 3 x 10(-12) mol/liter) to increase the level of tyrosylphosphorylation of at least six proteins, with apparent molecular weights of 20, 25, 27, 41, 48, and 135 kD (P < 0.05 for each); and (2) dose-dependent effects of sCt (during 15 minutes of exposure) to increase the level of tyrosyl-phosphorylation of at least 10 proteins with apparent molecular weights of 19, 20, 27, 35, 41, 102, 135, 195, 220, and 244 kD (P < 0.05 for each). A supplementary study of calcitonin effects on tyrosyl-protein phosphorylation in a subpopulation of SaOS-2 cells with very low steady-state levels of skeletal alkaline activity revealed similar responses--time and dose-dependent increases in the tyrosyl-phosphorylation of at least seven proteins with apparent molecular weights of 44, 48, 57, 62, 101, 244, and 280 kD (P < 0.05 for each).(ABSTRACT TRUNCATED AT 250 WORDS)
Alkaline Phosphatase↗
Phenytoin at micromolar concentrations is an osteogenic agent for human-mandible-derived bone cells in vitro.
The present study sought to test the hypothesis that phenytoin acts on normal human-mandible-derived bone cells to induce osteogenic effects. To test the effects of phenytoin on bone cell proliferation, we measured [3H]-thymidine incorporation into cell DNA during the final four hr of a 24-hour incubation with phenytoin. Phenytoin at micromolar concentrations significantly stimulated the [3H]-thymidine incorporation in a dose-dependent, biphasic, manner with a maximal effect at from 10 to 50 microM. We confirmed the proliferative effect of phenytoin by counting cell number. To evaluate the effects of phenytoin on osteoblastic differentiation, we determined alkaline-phosphatase specific activity and found that phenytoin at micromolar concentrations significantly increased that activity in a dose-dependent manner, with maximal stimulation at approximately 1 microM. To investigate the effects of phenytoin on mature osteoblastic activities, we measured de novo collagen synthesis and osteocalcin secretion. Mitogenic doses of phenytoin significantly increased collagen synthesis and osteocalcin secretion in a dose-dependent, biphasic, manner, with the maximal stimulatory dose at from 5 to 10 microM. In summary, phenytoin at micromolar ranges increased (a) [3H]-thymidine incorporation and cell number, (b) alkaline-phosphatase specific activity, (c) collagen synthesis, and (d) osteocalcin secretion in monolayer cultures of normal human-mandible-derived bone cells. These observations are consistent with the premise that low doses of phenytoin act on human craniofacial bone cells to stimulate cell proliferation, differentiation, and mature osteoblastic activities to stimulate bone formation.
Alkaline Phosphatase↗
Phenytoin increases markers of osteogenesis for the human species in vitro and in vivo.
Phenytoin therapy is a well recognized cause of gingival hyperplasia, a condition characterized by increased gingival collagen synthesis, and may also cause acromegalic-like facial features. Based on these clinical findings suggestive of anabolic actions, we sought to test the hypothesis that phenytoin acts on normal bone cells to induce osteogenic effects. To test the direct actions of phenytoin on human bone cells, we measured the dose responses to phenytoin for [3H]thymidine incorporation, cell number, alkaline phosphatase specific activity, and collagen synthesis in human hip bone-derived cells. Phenytoin significantly and reproducibly increased [3H]thymidine incorporation, cell number, alkaline phosphatase specific activity, and collagen synthesis in a biphasic manner with optimal stimulatory doses between 5-10 mumol/L. Thus, micromolar concentrations of phenytoin can act directly on human bone cells to stimulate osteoblast proliferation and differentiation. We next sought to test the hypothesis that phenytoin stimulates bone formation in humans in vivo. Accordingly, three serum biochemical markers of bone formation, i.e. osteocalcin, skeletal alkaline phosphatase, and procollagen C-terminal extension peptide, were measured in 39 male epileptic patients, 20-60 yr of age, with an average duration of phenytoin therapy of 10.5 +/- 1.62 yr (mean +/- SEM). In this group of patients, the mean serum phenytoin level was 9.56 +/- 0.90 mg/L (mean +/- SEM; equivalent to 34.9 +/- 3.3 mumol/L). Thirty apparently healthy male subjects of similar age and taking no medication were included as controls. Serum calcium, 25-hydroxyvitamin D3, and PTH levels in the phenytoin-treated patients were not significantly different from those in the age-matched controls and were within the clinical laboratory normal range of our hospitals, indicating that the patients did not develop hypocalcemia, vitamin D deficiency, or secondary hyperparathyroidism. Serum levels of osteocalcin, skeletal alkaline phosphatase, and procollagen peptide in the phenytoin-treated patients were significantly increased compared to those in the age-matched subjects; in each case these biochemical markers were significantly correlated with the serum phenytoin level, but not with the dose or duration of phenytoin treatment. These findings are consistent with the interpretation that phenytoin increases the bone formation rate in humans in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)
Adult↗
Picomolar norethindrone in vitro stimulates the cell proliferation and activity of a human osteosarcoma cell line and increases bone collagen synthesis without an effect on bone resorption.
To determine how progestins increase bone formation in vivo, the effects of the synthetic progestin norethindrone (NET), on aspects of bone formation in vitro were determined. NET at picomolar concentrations in vitro stimulated the proliferation of human TE85 osteosarcoma cells as assessed by the increase in [3H]thymidine incorporation into DNA and in cell number and also stimulated the release of osteocalcin in both the presence and absence of 10 nM 1,25-(OH)2D3. NET increased cellular alkaline phosphatase specific activity (an index of osteoblastic differentiation), but at much higher concentrations, that is, nanomolar. These findings suggest that low concentrations of NET act directly on human TE85 osteosarcoma cells to stimulate their proliferation, differentiation, and cell activity. Furthermore, mitogenic doses of NET stimulated bone collagen synthesis both in a chicken calvarial organ culture assay (assessed by the incorporation and hydroxylation of [3H]proline) and in a human TE85 osteosarcoma cell culture assay (determined by the incorporation of [3H]proline into collagenase-digestible proteins). In contrast, NET at 10(-6)-10(-12) M had no apparent effect on the rate of basal or PTH-stimulated release of 45Ca from prelabeled mouse calvariae in vitro. In summary, this study has demonstrated for the first time that picomolar NET acted directly on human TE85 osteosarcoma cells to increase (1) cell proliferation and differentiation, (2) osteoblastic activity (i.e., osteocalcin synthesis), and (3) bone collagen synthesis in vitro. The same doses of NET in vitro did not reduce the bone resorption rate under our assay conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Development of a heterologous radioimmunoassay for canine osteocalcin.
The aim of this study was to develop a routine and reliable radioimmunoassay (RIA) for dog osteocalcin. Two peaks of dog osteocalcin were purified to apparent homogeneity according to N-terminal sequence analysis. Amino acid composition analysis suggested that the second peak was intact dog osteocalcin whereas the first peak could be a truncated molecule. High titer (> 1:5,000) anti-dog osteocalcin antisera were produced in rabbits. The antiserum recognized dog and rat osteocalcins but not that in serum of human, bovine, rabbit, mouse, guinea pig, or goat. A homologous RIA using anti-dog osteocalcin as the antibody and dog osteocalcin as the tracer and standard was developed. Taking advantages of the facts that (1) anti-dog osteocalcin cross-reacted in parallel with rat osteocalcin and (2) purified rat osteocalcin is commercially available, we devised an approach that used rat osteocalcin as the tracer and standard, and anti-dog osteocalcin as the antibody to develop a heterologous RIA. This assay recognized dog serum osteocalcin and diluted in parallel with rat and dog osteocalcins. Quantitation was done using rat osteocalcin to construct standard curves, and results were expressed in ng/ml of rat osteocalcin-equivalent. The detection limit of the assay was 5 ng/ml rat osteocalcin-equivalent, and half-maximal displacement was seen at 30-40 ng/ml rat osteocalcin-equivalent. The inter- and intraassay variations were 16.1% and 8.5%, respectively. The assay accurately determined the amount of exogenously added dog osteocalcin in serum. The results quantitated with this RIA correlated well (r = 0.975, n = 86) with those obtained with the homologous RIA.(ABSTRACT TRUNCATED AT 250 WORDS)
Aging↗
Mechanism of mitogenic action of aluminum ion on human bone cells: potential involvement of the insulin-like growth factor regulatory system.
Aluminum ion at micromolar concentrations significantly stimulated the [3H]thymidine incorporation into human TE85 osteosarcoma cell DNA. Cells treated with mitogenic concentrations of aluminum ion for 48 h showed biphasic stimulation in secretion of IGFs (insulin-like growth factors) into the conditioned medium. Treatment of the human osteosarcoma TE85 cells with mitogenic doses of aluminum ion for 24 h also markedly and reproducibly increased the steady-state level of IGF-II mRNA in a dose-dependent, biphasic manner. The effect of aluminum ion on the steady-state level of IGF-I mRNA could not be determined since the IGF-I mRNA in these cells was not detectable with our oligodeoxynucleotide probes. To test whether the mitogenic effects of aluminum ion could be mediated through IGFs, the stimulation of [3H]thymidine incorporation of TE85 cells was evaluated in the presence and the absence of an inhibitory IGF binding protein (i.e., IGFBP-4). The presence of IGFBP-4 significantly reduced the stimulation in thymidine incorporation by a mitogenic concentration of aluminum ion. Western ligand blot analysis revealed that mitogenic concentrations of aluminum ion also inhibited the secretion of IGF-binding proteins, particularly the inhibitory IGFBP-4, which could lead to the potentiation of the overall activity of IGFs. In conclusion, these findings are consistent with the premise that the mitogenic action of aluminum ion on human bone cells is, in part, mediated by an increased local bone cell production and activity of IGFs.
Alkaline Phosphatase↗
Phosphotyrosyl protein phosphatases: potential regulators of cell proliferation and differentiation.
Tyrosyl phosphorylated proteins have been associated with the regulation of cellular activities, including proliferation, differentiation, and transformation. The overall cellular levels of tyrosyl phosphorylated proteins are regulated by the balance of the activities of both tyrosyl protein kinases and phosphotyrosyl protein phosphatases. Until recently, it was generally assumed that the kinase reaction was the key regulatory process for determining the level of tyrosyl phosphorylated proteins and that the dephosphorylation reaction was an unregulated event. However, recent evidence strongly suggest a regulatory role for phosphotyrosyl protein phosphatases for determination of the overall level of tyrosyl phosphorylated proteins. This review focuses on the discussion of the properties, the regulation, and the primary structure of phosphotyrosyl protein phosphatases. Evidence that supports an important role for phosphotyrosyl protein phosphatases on cell proliferation and differentiation also is presented.
Amino Acid Sequence↗