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  • 1
    Electronic Resource
    Electronic Resource
    Skeletal Alkaline Phosphatase Activity Is Primarily Released from Human Osteoblasts in an Insoluble Form, and the Net Release Is Inhibited by Calcium and Skeletal Growth Factors (1998)
    Springer
    Calcified tissue international 62 (1998), S. 332-340 
    Details
    ISSN: 1432-0827
    Keywords: Key words: Skeletal alkaline phosphatase — Osteoblasts — Calcium — Growth factors
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract. Skeletal alkaline phosphatase (ALP) is anchored to membrane inositol-phosphate on the outer surface of osteoblasts. Although skeletal ALP activity in serum is, essentially, all in an anchorless (soluble) form, in vitro studies indicate that ALP can be released in either an anchorless, soluble form (e.g., by a phospholipase) or an anchor-intact, insoluble form (e.g., by vesicle exocytosis). The current studies were intended to define the contributions of each of these putative processes of ALP release and to assess the significance of regulation by calcium (Ca) and skeletal effectors. ALP activity was measured in serum-free medium from replicate cultures of human osteosarcoma (SaOS-2) cells and normal human bone cells. Temperature-sensitive phase distribution (in Triton X-114) allowed separation of soluble from insoluble ALP activity. Our studies revealed that most of the ALP activity released from SaOS-2 cells was in an insoluble form (78% ± 8%), a percentage that was constant between 2 and 96 hours. A similar result was seen for normal human bone cells. Calcium had a negative, biphasic dose-dependent effect on net release of ALP activity: r=−0.85, P 〈 0.001 at 24 hours, with KIapparent values for biphasic inhibition of 20 and 300 μmol/l Ca. Of the skeletal effectors tested, insulin-like growth factor-II (IGF-II) had the greatest effect, decreasing the net release of ALP activity in a dose-dependent manner (r=−0.82, P 〈 0.005). Neither Ca nor IGF-II affected the distribution of soluble/insoluble ALP activity by more than 9%. IGF-II had no effect on extracellular ALP stability, but the addition of Ca to Ca-free cultures resulted in parallel losses of extracellular ALP activity and ALP immunoreactive protein (P 〈 0.001 for each). A similar effect was seen when Ca was added to Ca-free, cell-free, conditioned medium, but not when Ca was added to purified ALP, which is consistent with the general hypothesis that a Ca-dependent protease might be present in the cell-conditioned medium. Together, these data suggest that most of the ALP activity released from osteoblasts is insoluble (and, presumably, anchorless), net release of ALP activity is negatively regulated by Ca and skeletal growth factors, the effect of Ca may reflect Ca-dependent protease activity, and an exogenous (e.g., serum) phospholipase may be responsible for releasing ALP from its insoluble anchor.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002239900441
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  • 2
    Electronic Resource
    Electronic Resource
    Skeletal Response to Dietary Zinc in Adult Female Mice (1998)
    Springer
    Calcified tissue international 62 (1998), S. 309-315 
    Details
    ISSN: 1432-0827
    Keywords: Key words: Zinc — Alkaline phosphatase — Tartrate-resistant acid phosphatase — Female mice.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract. The current studies were intended to assess dose- and time-dependent effects of dietary zinc (Zn) on alkaline phosphatase (ALP) activity and tartrate-resistant acid phosphatase (TRAP) activity in adult female mice. In the first study, mice were given 0, 1×, 2×, 3×, or 4× normal dietary Zn for 2 weeks, 4 weeks, or 6 weeks. In the second study, mice were given 0, 1×, 2×, 3×, 4×, and 5× normal dietary Zn for 4 weeks. Sera were collected for measurements of ALP and (in the second study) osteocalcin. Tibiae and calvaria were extracted for measurements of ALP, protein, and TRAP. The first study showed positive correlations between dietary Zn and serum ALP (4 and 6 weeks, P 〈 0.001), Zn and tibial ALP (2, 4, and 6 weeks, P 〈 0.03), and Zn and tibial protein (2, 4, and 6 weeks, P 〈 0.001), as well as a negative correlation between dietary Zn and tibial TRAP (2, 4, and 6 weeks, P 〈 0.001). Covariant analyses showed that serum ALP, tibial ALP, tibial protein, and tibial TRAP were affected by the dose of Zn (P 〈 0.005) and by the treatment time (P 〈 0.03). Supplemental studies showed that (1) the dose-dependent effect of dietary Zn on serum ALP (at 6 weeks) was proportional to the effects on tibial ALP and calvarial ALP, but not to the effects of Zn on renal, hepatic, or intestinal ALP; (2) 6 weeks of dietary Zn caused dose-dependent increases in ALP specific activity in the tibia, calvaria, and liver, but not kidneys or intestines; and (3) Zn increased ALP activity and cell layer protein and decreased TRAP activity in monolayer cultures of the murine osteoblastic cell line, MC3T3-E1. The second dietary study confirmed the results of the first: 4 weeks of treatment with Zn caused significant increases in serum ALP, calvarial ALP, and tibial ALP activities, and a significant decrease in tibial TRAP (P 〈 0.05–0.005 for each). This study also revealed an effect of Zn to increase serum osteocalcin (P 〈 0.03 at 2× normal Zn). Together, these data indicate that incremental increases in dietary Zn are associated with increases in ALP activity in serum and in bone. The effect of Zn to decrease TRAP activity in osteoblast-line cells precludes the interpretation of a Zn-dependent decrease in tibial TRAP activity as evidence of decreased bone resorption.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002239900437
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  • 3
    Electronic Resource
    Electronic Resource
    Effects of Zinc on Human Skeletal Alkaline Phosphatase Activity In Vitro (1999)
    Springer
    Calcified tissue international 64 (1999), S. 163-172 
    Details
    ISSN: 1432-0827
    Keywords: Key words: Inorganic phosphate — Alkaline phosphate — Zinc deficiency-SaOS-2 cells.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract. Inorganic phosphate (Pi) can regulate the level of skeletal alkaline phosphatase (ALP) activity in human osteoblast-like cells by stabilizing the enzyme (without affecting transcription, ALP release from the cell surface, or the amount of ALP protein). These observations suggest that Pi determines the level of ALP activity by modulating a process of irreversible inactivation. The current studies were intended to examine the hypothesis that this inactivation of ALP activity is caused by the dissociation of an active center Zn and that Pi inhibits that dissociation. Initial studies showed that Zn, like Pi, could increase ALP specific activity in human osteosarcoma SaOS-2 cells in a time- and dose-dependent manner (e.g., a 50% increase at 0.2 μmol/liter Zn, P 〈 0.005). This effect was specific for Zn (i.e., no similar effect was seen with Ca, Fe, Co, Mg, Mn, or Cu), but not for SaOS-2 cells. Zn also increased ALP specific activity in (human osteosarcoma) MG-63 cells and in cells derived from normal human vertebrae (P 〈 0.001 for each). The effect of Zn to increase ALP activity was not associated with parallel increases in total protein synthesis, collagen production, or tartrate-resistant acid phosphatase activity (no change in any of these indices), net IGF-2 synthesis (a Zn-dependent decrease, P 〈 0.005), or PTH-dependent synthesis of cAMP (a biphasic increase, P 〈 0.02). Kinetic studies of Pi and Zn as co-effectors of ALP activity showed that Zn was a mixed-type effector with respect to Pi, whereas Pi was competitive with respect to Zn. Mechanistic studies showed that (1) Zn reversed the effect of Pi withdrawal to decrease ALP activity, but not by reactivating inactive ALP protein (the process required protein synthesis, without increases in ALP mRNA or the level of ALP immunoreactive protein); (2) Zn increased the half-life of ALP activity in intact cells and after a partial purification; and (3) Pi inhibited the process of ALP inactivation by EDTA (which chelates active center Zn). All these findings are consistent with the general hypothesis that Pi increases the half-life of skeletal ALP by preventing the dissociation of active center Zn and with a mechanistic model of skeletal ALP activity in which active center Zn participates in Pi-ester binding and/or hydrolysis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002239900597
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  • 4
    Electronic Resource
    Electronic Resource
    On fluoride and bone strength (1995)
    Springer
    Calcified tissue international 56 (1995), S. 415-418 
    Details
    ISSN: 1432-0827
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00301613
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  • 5
    Electronic Resource
    Electronic Resource
    Phosphate regulates the stability of skeletal alkaline phosphatase activity in human osteosarcoma (SaOS-2) cells without equivalent effects on the level of skeletal alkaline phosphatase immunoreactive protein (1995)
    Springer
    Calcified tissue international 57 (1995), S. 371-378 
    Details
    ISSN: 1432-0827
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract Inorganic phosphate (Pi) can regulate the level of skeletal alkaline phosphatase (ALP) activity in human osteoblast-like cells, but not by means of changes in transcription or release from the cell surface. The current studies were intended to determine whether (1) Pi affected the inactivation of ALP activity in human osteosarcoma (SaOS-2) cells; and (2) Pi-dependent changes in ALP-specific activity were associated with equal, concomitant changes in the level of ALP immunoreactive protein. The results of these studies revealed that Pi increased the stability of skeletal ALP activity without equivalent effects on the level of ALP immunoreactive protein. An increase in Pi (from 0 to 1.8 mmol/liter) caused a time-dependent increase in the amount of skeletal ALP activity in the SaOS-2 cells, without a parallel increase in the amount of skeletal ALP immunoreactive protein, and a decrease in Pi (from 1.8 to 0 mmol/liter) caused a time-dependent decrease in the amount of ALP activity, without a significant decrease in the total cellular content of ALP immunoreactive protein. Together, these observations suggest that Pi may alter the level of skeletal ALP activity in SaOS-2 cells by inhibiting a process of irreversible inactivation that does not effect equal, concomitant changes in the level of skeletal ALP immunoreactive protein.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00302073
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  • 6
    Electronic Resource
    Electronic Resource
    Calcitonin acutely increases tyrosyl-phosphorylation of proteins in human osteosarcoma (SaOS-2) cells (1995)
    Springer
    Calcified tissue international 56 (1995), S. 268-273 
    Details
    ISSN: 1432-0827
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract 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°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 3×10-12 mol/liter) to increase the level of tyrosyl-phosphorylation 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). Together, these studies demonstrate that sCt can have direct effects to modulate the level of tyrosyl-protein phosphorylation in human osteosarcoma cells, presumably by activation of tyrosyl-kinase activity and/or inhibition of phospho-tyrosyl-protein phosphatase activity.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00318045
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  • 7
    Electronic Resource
    Electronic Resource
    An age-related decrease in the concentration of insulin-like growth factor binding protein-5 in human cortical bone (1995)
    Springer
    Calcified tissue international 57 (1995), S. 206-212 
    Details
    ISSN: 1432-0827
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract The skeletal contents of insulin-like growth factor-2 (IGF-II), insulin-like growth factor binding protein-5 (IGFBP-5), and insulin-like growth factor binding protein-3 (IGFBP-3) were determined in duplicate samples of human femoral cortical bone obtained from 64 subjects (44 males and 20 females) between the ages of 20 and 64 years. The results of these quantitative measurements revealed an age-related decrease in the femoral cortical content of IGFBP-5 (r=-0.272, P=0.031) in the total population. Although the femoral cortical content of IGF-II did not show a similar decrease with age, it could be correlated to the femoral cortical content of IGFBP-5 (r=0.442, P〈0.001). In constrast, the femoral cortical content of IGFBP-3 did not decrease with age and could not be correlated to the femoral cortical contents of either IGFBP-5 or IGF-II. Comparisons of these results with previous measurements of insulin-like growth factor-1 (IGF-I) and transforming growth factor-β (TGF-β), in extracts of the same bones, showed significant cross-correlations between the femoral cortical contents of each of these growth factors and the femoral cortical contents of IGFBP-5 (r=0.625 for IGF-I versus IGFBP-5, r=0.554 for TGF-β versus IGFBP-5, P〈0.001 for each) but not IGFBP-3. Together, these data indicate average net losses of 60% and 29% of the femoral cortical contents of IGF-I and IGFBP-5, respectively, and apparent net losses (i.e., nonsignificant decreases) of 21% and 25% of the femoral cortical contents of IGF-II and TGF-β, respectively, between the third and the sixth decades (i.e., decreases from young adult values of 75.1 pmol/g of bone for IGF-I, 124.7 pmol/g of bone for IGF-II, 0.71 pmol/g of bone for TGF-β, 115.6 pmol/g of bone for IGFBP-5, and 26.2 pmol/g of bone for IGFBP-3).
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00310260
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  • 8
    Electronic Resource
    Electronic Resource
    Exercise and Mechanical Loading Increase Periosteal Bone Formation and Whole Bone Strength in C57BL/6J Mice but Not in C3H/Hej Mice (2000)
    Springer
    Calcified tissue international 66 (2000), S. 298-306 
    Details
    ISSN: 1432-0827
    Keywords: Key words: Exercise and mechanical stress — Genetics — Bone formation.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract. To identify the genes, and the mechanisms that account for the 53% higher peak bone density in C3H/HeJ (C3H) mice compared with C57BL/6J (B6) mice, we are performing quantitative trait locus and phenotypic analyses. The phenotypic studies revealed differences in bone formation and resorption, and showed that hindlimb immobilization (by sciatic neurectomy) caused a greater increase in endosteal resorption in the tibiae of B6 compared with C3H mice. The current studies were intended to examine the hypothesis that the bones of C3H mice are less sensitive to mechanical loading than the bones of B6 mice. To increase mechanical loading, 9-week-old female B6 and C3H mice (n = 10–13 mice/group) were subjected to a jumping exercise (20 jumps/day, 5 days/week, to heights of 20–30 cm) for a total of 4 weeks. Control mice did not jump. Osteocalcin, alkaline phosphatase (ALP) activity, and IGF-I were measured in serum. The left tibiae were used for histomorphometry (ground cross-sections prepared at the tibio-fibular junction) and the right tibiae and femora were used for determinations of bone breaking strength (3-point bending). The results of these studies revealed (1) significant effects of both mouse strain (B6 and C3H) and the jumping exercise on tibial strength; (2) an exercise-dependent increase in serum IGF-I in C3H, but not B6 mice; and (3) no effects on serum ALP or osteocalcin. The histomorphometric analyses showed no effect of exercise on C3H tibiae, but significant exercise-dependent increases in total bone area, periosteal perimeter, periosteal mineral apposition rate (MAR), and periosteal bone formation (P 〈 0.02 for each) in B6 tibiae. There were no effects of exercise on periosteal resorption or any endosteal measurement in either C3H or B6 mice. Since the jumping exercise was designed to cause a two–three fold increase in muscular-skeletal loading at the tibio-fibular junction, and the calculated stress (g/mm2) at this sampling site was only 16% greater for B6 compared with C3H mice, we had anticipated that both strains of mice would show exercise-dependent increases in periosteal bone formation, with a greater response in the B6 mice. The lack of a response in the C3H tibiae demonstrates that the bones of C3H mice are less sensitive to mechanical loading (and unloading) than the bones of B6 mice.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002230010060
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