Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Studies performed at tissular (three-dimensional, 3-D) or cellular (two-dimensional, 2-D) levels showed that the loading pattern plays a crucial role in the osteoblastic physiology. In this study, we attempted to investigate the response of a 3-D osteoblastic culture submitted to either no external stress or static or dynamic stresses. Rat osteosarcoma cells (ROS 17/2.8) were embedded within collagen type I lattices and studied for 3 weeks. Entrapment and proliferation of cells within the hydrated collagen gel resulted in the generation of contractile forces, which led to contraction of the collagen gel. We used this ability to evaluate the influence of three modes of mechanical stresses on the cell proliferation and differentiation: (1) the freely retracted gels (FRG) were floating in the medium, (2) the tense gels (TG) were stretched statically and isometrically, with contraction prevented in the longitudinal axis, and (3) the dynamic gels (DG) were floating gels submitted to periodic stresses (50 or 25 rpm frequency). Gels showed maximum contraction at day 12 in 50 rpm DG, followed by 25 rpm DG, then FRG (88%, 81%, 70%, respectively) and at day 16 in TG (33%). The proliferation rate was greater in TG than in FRG (+52%) but remained low in both DGs. Gel dimensions were related to the collagen concentration and on a minor extent to cell number. Cells in DG appeared rounder and larger than in other conditions. In TG, cells were elongated and oriented primarily along the tension axis. Scanning electron microscopy (SEM) showed that tension exerted by cells in TG led to reorientation of collagen fibers which, in turn, determined the spatial orientation and morphology of the cells. Transmission electron microscopy (TEM) performed at maximum proliferation showed a vast majority of cells with a distended well-developed RER filled with granular material and numerous mitochondria. Alkaline phosphatase activity peaked close to the proliferation peak in FRG, whereas in TG, a biphasic curve was observed with a small peak at day 4 and the main peak at day 16. In DG, this activity was lower than in the two other conditions. A similar time course was observed for alkaline phosphatase gene expression as assessed by Northern blots. Regardless of the conditions, osteocalcin level showed a triphasic pattern: a first increase at day 2, followed by a decrease from day 4 to 14, and a second increase above initial values at day 18. Microanalysis-x indicated that mineralization occurred after 14 days and TEM showed crystals within the matrix. We showed that static and dynamic mechanical stresses, in concert with 3-D collagen matrices, played a significant role on the phenotypic modulation of osteoblast-like cells. This experimental model provided a tool to investigate the significance and the mechanisms of mechanical activity of the 3-D cultured osteoblast-like cells.
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PMID:Effects of static or dynamic mechanical stresses on osteoblast phenotype expression in three-dimensional contractile collagen gels. 1061 39

Cadherins are a family of cell surface adhesion molecules that play an important role in tissue differentiation. A limited repertoire of cadherins has been identified in osteoblasts, and the role of these molecules in osteoblast function remains to be elucidated. We recently cloned an osteoblast-derived N-cadherin gene from a rat osteoblast complementary DNA library. After in situ hybridization of rat bone and immunohistochemistry of human osteophytes, N-cadherin expression was localized prominently in well-differentiated (lining) osteoblasts. Northern blot hybridization in primary cultures of fetal rat calvaria and in human SaOS-2 and rat ROS osteoblast-like cells showed a relationship between N-cadherin messenger RNA expression and cell-to-cell adhesion, morphological differentiation, and alkaline phosphatase and osteocalcin gene expression. Treatment with a synthetic peptide containing the His-Ala-Val (HAV) adhesion motif of N-cadherin significantly decreased bone nodule formation in primary cultures of fetal rat calvaria and inhibited cell-to-cell contact in rat osteoblastic TRAB-11 cells. HAV peptide also regulated the expression of specific genes such as alkaline phosphatase and the immediate early gene zif268 in SaOS-2 cells. Transient transfection of SaOS-2 cells with a dominant-negative N-cadherin mutant (NCADdeltaC) significantly inhibited their morphological differentiation. In addition, aggregation of NCTC cells derived from mouse connective tissue stably transfected with osteoblast-derived N-cadherin was inhibited by either treatment with HAV or transfection with NCADdeltaC. Together, these results strongly support a role for N-cadherin, in concert with other previously identified osteoblast cadherins, in the late stages of osteoblast differentiation.
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PMID:A role for N-cadherin in the development of the differentiated osteoblastic phenotype. 1070 21

To investigate potential effects of endogenous parathyroid hormone-related peptide (PTHrP) on osteoblast function, ROS 17/2.8 cells were transfected with full-length PTHrP cDNA in a sense or antisense orientation to alter PTHrP production. Compared with vector-transfected control cells, PTHrP-overproducing (sense-transfected) cells showed increased DNA synthesis ([(3)H]-thymidine incorporation) and increased growth (cell number). The extent of apoptosis was compared for the different clones using the terminal deoxynucleotide-mediated dUTP nick-end-labeling assay (TUNEL) and Hoechst staining. No differences in percentages of apoptotic cells were found under basal culture conditions or after 3 days of serum deprivation, which, itself, markedly increased numbers of apoptotic cells. The effect of PTHrP on osteoblast differentiation was assessed by examining two protein markers of differentiation, alkaline phosphatase, and bone morphogenetic protein (BMP)-2. Alkaline phosphatase activity was decreased in sense-transfected cells and increased in antisense-transfected cells, compared with cells transfected with empty vector. PTHrP-overproducing cells also showed decreased numbers of BMP-2-positive cells, whereas antisense-transfected cells showed no difference compared with vector control. The results indicate that: (a) endogenously produced PTHrP can increase growth of these osteoblastic cells by stimulating proliferation while not affecting apoptosis; and (b) the increased cell proliferation produced by PTHrP was accompanied by a reduction in activity or amount of two proteins normally expressed by differentiated osteoblasts.
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PMID:Endogenous parathyroid hormone-related peptide enhances proliferation and inhibits differentiation in the osteoblast-like cell line ROS 17/2.8. 1077 81

A gap junction-deficient cell line was utilized to test whether intercellular coupling plays a significant role in modulating the influence of biophysical stimuli such as extracellular electrical currents. ROS 17/2.8 cells, an osteosarcoma cell line, along with a control transfected cell line and a connexin 43-gap junction-deficient cell line, were exposed to a time-changing magnetic flux (30 Hz, 1.8 milliTesla) sufficient to induce an electric field in the cultures on the order of 2 mV/m. Field exposure inhibited cell growth independent of gap junctional coupling, while alkaline phosphatase activity was found to be dependent on gap junctional coupling. These findings can be interpreted to suggest that magnetic and electric field exposures have differential effects on cell cultures, with magnetic field exposure inhibiting cell growth through a mechanism independent of gap junctional coupling, while the alteration in enzyme activity appears to be stimulated by the induced electric field in a gap junction-dependent manner.
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PMID:Osteoblastic networks with deficient coupling: differential effects of magnetic and electric field exposure. 1091 15

Previous studies have shown that neonatal rat calvaria osteoblasts elaborate substantial amounts of extracellular material with bone-like characteristics when cultured on porous bioactive glass substrates in vitro. However, the osteoblastic response to this material has not been fully characterized. The objective of this study was to characterize osteoblast response to porous bioactive glass substrates following the expression of the classical markers for osteoblast differentiation. In this study we synthesized porous bioactive glass substrates, seeded them with osteoblast-like cells (ROS 17/2.8) and followed the temporal expression of alkaline phosphatase (AP) activity, as well as the expression of mRNA for collagen type I (Coll-1), osteonectin (OSN), osteopontin (OPN), osteocalcin (OCN), and bone sialoprotein (BSP). The data confirm that porous bioactive glass substrates are capable of supporting the in vitro growth and maturation of osteoblast-like cells. At a porosity of 42% and an average pore size of 80 microm, the substrates promote the expression and maintenance of the osteoblastic phenotype. The results additionally suggest that there is both a solution-mediated and a surface-controlled effect on cell activity.
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PMID:Evaluation of osteoblast response to porous bioactive glass (45S5) substrates by RT-PCR analysis. 1094 Nov 97

We have previously shown that several stressful situations associated with tissue injury determine a decrease in serum osteocalcin concentration. Since reduced osteocalcin production is a marker of decreased osteoblastic activity, this finding could be related to the pathogenesis of osteoporosis secondary to some diseases. Endogenous opioids are involved in stress response. Proenkephalin-derived peptides have been shown to inhibit alkaline phosphatase activity, another marker of bone formation, in the murine cell line ROS-17/2.8. On the other hand, serum osteocalcin has been reported as being low in heroin abusers. We have therefore thought it of interest to study the presence of opioid receptors in the human osteoblast-like cell line MG-63, and to evaluate the effects of different opioid agonists on the secretion of alkaline phosphatase and osteocalcin by these cells. The presence of opioid receptors was studied by means of RT-PCR and immunohistochemistry. RT-PCR studies suggested the presence of specific mRNA for the three types of receptors, and immunohistochemistry clearly showed their occurrence. Osteocalcin synthesis was significantly inhibited by high concentrations of the mu agonists morphine and (D-Ala(2), N-MePhe(4),Gly(5)-ol)-enkephalin although no changes were seen with the delta agonist (D-Ala(2),D-leu(5))-enkephalin. Morphine-induced osteocalcin inhibition was abolished when osteoblastic cells were incubated simultaneously with naloxone, whereas it was potentiated when cells were preincubated with naloxone. None of the opioid agonists modified the secretion of alkaline phosphatase. In conclusion, human osteoblast-like cells MG-63 express the three types of opioid receptors. Endogenous opioids may be involved in the reduction of osteocalcin observed in stressful situations associated with tissue injury.
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PMID:Expression of opioid receptors in osteoblast-like MG-63 cells, and effects of different opioid agonists on alkaline phosphatase and osteocalcin secretion by these cells. 1102 13

The objective of this study was to determine the effect of porous bioactive glass (45S5) substrate characteristics on the expression and maintenance of the osteoblastic phenotype. We cultured ROS 17/2. 8 cells on substrates with different pore size and porosity for periods up to 14 days and analyzed the characteristics of the cells and extracellular matrix. Results of the study show that the glass substrates supported the proliferation and growth of osteoblast-like cells. Although the morphologies of the cells differed on the various substrates, their shape and the extent of membrane ruffling suggested that they maintained high levels of metabolic activity. Cells on all substrates expressed high levels of alkaline phosphatase activity and produced extracellular matrices that mineralized to form nonstoichiometric, carbonated, calcium-deficient apatites. An important finding was that at a given porosity of 44%, the pore size neither directed nor modulated the in vitro expression of the osteoblastic phenotype. In contrast, porosity did affect cellular function. We noted that at an average pore size of 92 microm, as the porosity increased from 35 to 59%, osteoblast activity was reduced. As designed in this experiment, an increase in the porosity led to a corresponding increase in total surface area of the specimens. With increasing porosity and surface area, glass reactions in the media may persist for longer durations at higher intensities, thereby affecting local media composition. As such, we suggest that extensive conditioning treatments before cell seeding can reduce this effect. Our results also revealed that the expression of the osteoblastic phenotype is enhanced by the ongoing glass dissolution. The reaction pathway at the origin of this effect still needs to be elucidated. Taken together, the findings support the overall hypothesis that in vitro cell activity can be controlled by a careful selection of substrate properties.
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PMID:Effect of varying physical properties of porous, surface modified bioactive glass 45S5 on osteoblast proliferation and maturation. 1103 62

11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) acts as a pre-receptor signaling mechanism for corticosteroids by regulating the access of active glucocorticoids to both glucocorticoid (GR) and mineralocorticoid receptors (MR). To examine the relationship between endogenous glucocorticoid metabolism and osteoblast function, we have characterized the expression of 11 beta-HSD isozymes in rat osteosarcoma cells. Analysis of mRNA from ROS 25/1, UMR 106 and ROS 17/2.8 cells revealed transcripts for both 11 beta-HSD type 1 (11 beta-HSD1) and type 2 (11 beta-HSD2) in all three cell lines. However, enzyme activity studies showed only high affinity dehydrogenase activity (inactivation of corticosterone (B) to 11-dehydrocorticosterone (A)), characteristic of 11 beta-HSD2; conversion of B to A was higher in ROS 25/1> UMR 106 cells>ROS 17/2.8. Although all three cell lines had similar numbers of GR (50,000/cell), glucocorticoid modulation of alkaline phosphatase activity and cell proliferation was only detectable in ROS 17/2.8 cells. Further studies showed that 11 beta-HSD2 activity in each of the cells was potently stimulated by both A and B, but not by synthetic dexamethasone. This effect was blocked by the 11 beta-HSD inhibitor, 18 beta-glycyrrhetinic acid (but not by GR or MR antagonists) suggesting direct, allosteric regulation of 11 beta-HSD2 activity. These data indicate that in osteosarcoma cells 11 beta-HSD2 plays a key role in controlling GR-mediated responses; cells with relatively high levels of 11 beta-HSD2 activity were insensitive to glucocorticoids, whilst cells with low levels showed functional responses to both dexamethasone and B. In addition to the established effects of 11 beta-HSD2 in protecting MR in the kidney and colon, our data suggest that 11 beta-HSD2 in bone represents an important pre-receptor mechanism in determining ligand availability to GR.
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PMID:Expression of 11 beta-hydroxysteroid dehydrogenase in rat osteoblastic cells: pre-receptor regulation of glucocorticoid responses in bone. 1125 28

The replacement of fetal bovine serum with rat serum in a culture medium brought about a marked increase in the formation of mineralized bone nodules (BN) in primary cultures of rat calvarial cells. These effects of rat serum were most prominent when added during the early phase of the culture, indicating that the serum factor mainly acts on the cells during the growing phase. A significant increase in BN formation was observable at final rat serum concentration as low as 1%, and the effect was dependent on serum concentration, at least up to 10%. The addition of rat serum also increased alkaline phosphatase (ALP) activity, collagen synthesis, and DNA synthesis in calvarial cells. BN formation stimulating activity was extractable with ethyl acetate. The ethyl acetate extract was purified by TSK-GEL OH-120 column chromatography by monitoring the stimulation of ALP activity in ROS 17/2.8 cells. The chromatographic behavior of the ALP activity was found to be identical to that of corticosterone, the major glucocorticoid in rodents and the preincubation of the purified fraction with anticorticosterone antibody abolished the ALP stimulating activity. These results suggest that BN formation stimulating activity in rat serum is mainly attributable to corticosterone. The concentration of serum corticosterone decreased with age in parallel with BN formation stimulating activity, which suggests that the physiological level of corticosterone may have a regulatory role in the maintenance of osteoblast function.
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PMID:Age-dependent decline in bone nodule formation stimulating activity in rat serum is mainly due to the change in the corticosterone level. 1125 37

Six days of microgravity (Bion10 mission) induced dramatic shape changes in ROS 17/2.8 osteoblasts (7). During the Foton 11 and 12 space flights, we studied the kinetics (0-4 days) of ROS 17/2.8 morphology and adhesion, the relationships between adhesion and cell cycle progression after 4 days in space, and osteoblastic growth and activity after 6 days in space. Quantitative analysis of high-resolution adhesion [focal adhesion area imaged by total interference reflection fluorescent microscopy (TIRFM)] and integrin-dependent adhesion (imaged on confocal microscope by vinculin and phosphotyrosine staining) as well as cell cycle phase classification [Ki-67 staining, S-G2, mitotic cells and G1 (postmitotic cells)] were performed using programs validated in parabolic flight and clinostat. We observed disorganization of the cytoskeleton associated with disassembling of vinculin spots and phosphorylated proteins within focal contacts with no major change in TIRFM adhesion after 2 and 4 days of microgravity. Postmitotic cells, alone, accounted for the differences observed in the whole population. They are characterized by immature peripheral contacts with complete loss of central spots and decreased spreading. Osteocalcin, P1CP and alkaline phosphatase, and proliferation were similar in flight cells and 1 g centrifuge and ground controls after 6 days. In conclusion, microgravity substantially affected osteoblastic integrin-mediated cell adhesion. ROS17/2.8 cells responded differently, whether or not they were cycling by reorganizing adhesion plaque topography or morphology. In ROS 17/2.8, this reorganization did not impair osteoblastic phenotype.
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PMID:Cell cycling determines integrin-mediated adhesion in osteoblastic ROS 17/2.8 cells exposed to space-related conditions. 1151 18


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