Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To determine if activation of the glucocorticoid receptor involves covalent charge modification of the steroid-binding protein, unactivated and activated IM-9 cell glucocorticoid receptors were examined by high resolution two-dimensional gel electrophoresis. As previously reported (Smith, A. C., and Harmon, J. M. (1985) Biochemistry 24, 4946-4951), two-dimensional electrophoresis of immunopurified, [3H]dexamethasone mesylate-labeled, steroid-binding protein from unactivated receptors resolves two 92-kDa isoforms (pI congruent to 5.7 and 6.0-6.5). After activation, the apparent pI of neither isoform was altered, indicating that there had been no covalent charge modification of the steroid-binding protein. Thus, the physicochemical changes observed after activation of the steroid receptor cannot be explained by dephosphorylation or other models which involve covalent charge modification of the steroid-binding protein. This conclusion was consistent with the observation that treatment of immunopurified, affinity-labeled receptors with calf intestine alkaline phosphatase did not alter the apparent pI values or distribution of the steroid-binding protein isoforms. However, chromatography of activated steroid-receptor complexes on DNA-cellulose revealed that only the more basic of the two steroid-binding protein isoforms bound to DNA. Therefore, the charge heterogeneity of the steroid-binding protein may be important in regulating the ability of the steroid-binding protein to interact with DNA.
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PMID:Analysis of glucocorticoid receptor activation by high resolution two-dimensional electrophoresis of affinity-labeled receptor. 375 64

Dexamethasone increased alkaline phosphatase levels up to 7-fold in the osteoblast-like rat osteosarcoma cell line ROS 17/2.8. This effect was associated with reduced cell growth and took place over several days in culture. The increase in enzyme activity was dose dependent, (half-maximum near 1 nM, with a hormone specificity suggesting glucocorticoid receptor mediation). Dexamethasone also increased enzyme activity in ROS 2/3 cells, but not in two nonosteoblastic osteosarcoma cell lines, indicating that among these cell lines, the effect is specific for osteoblast-like cells. Moreover, enzyme activity in both control and dexamethasone-treated cells correlated directly with levels of radioimmunoassayable bone-type isoenzyme. Increases in alkaline phosphatase activity in response to dexamethasone were detectable after about 5 h and were inhibited by both actinomycin D and cycloheximide. Thus glucocorticoids appear to increase de novo enzyme synthesis in ROS 17/2.8 cells. Finally, the cAMP-elevating agents PTH, isoproterenol, and 8-bromo-cAMP, which were previously shown to reduce alkaline phosphatase activity in osteoblast-like cells, antagonized the effects of dexamethasone. Moreover, in the presence of dexamethasone, lower concentrations of these agents were required for inhibitory effects on alkaline phosphatase.
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PMID:Glucocorticoid regulation of alkaline phosphatase in the osteoblastic osteosarcoma cell line ROS 17/2.8. 385 55

We have examined the effect of Na+,K+-ATPase on 3H-triamcinolone acetonide binding capacity of cytosol glucocorticoid receptors from rat brain and liver. Preincubation of the brain or liver cytosol with Na+,K+-ATPase (10 units/ml) at 30 degrees C resulted in a rapid loss of specific 3H-triamcinolone acetonide binding, with a half-life of approximately 7 min. The ATPase effect could be prevented by the addition of 10(-5) M ouabain, or substantially reduced by the omission of Na+,K+ or Mg+2. The cytosol receptor bound with 3H-triamcinolone acetonide was totally resistant to the inactivation by the ATPase. Since there is some evidence that ATP may bind to glucocorticoid receptor, our findings indicate that an ATP-receptor complex may be essential for steroid binding. The effects of the ATPase in the inactivation of the receptor are very similar to those of alkaline phosphatase reported by others. This raises doubts about the proposal based on the phosphatase inactivation that the cytosol glucocorticoid receptor may be phosphorylated.
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PMID:Inactivation by Na+,K+-ATPase of cytosol glucocorticoid receptors from rat brain and liver. 630 15

Over 24-h culture with hydrocortisone (400 nM), activity of brush-border alkaline phosphatase, alpha-glucosidase, and leucyl-2-naphthylamidase and cytoplasmic-mitochondrial malate dehydrogenase increased (P less than 0.05) by 80-133% compared with controls. Uptake of 3-O-methyl-D-[14C]glucose after 24-h culture was increased (P less than 0.05) by 30% compared with cultures without hydrocortisone. Labeling of protein with L-[14C]tyrosine and glycoprotein with D-[3H]glucosamine increased (P less than 0.05) by 40 and 88%, respectively, with hydrocortisone. The effects of hydrocortisone were dose dependent at normal serum concentrations (100-600 nM) and not further stimulated by larger concentrations. Cytoplasmic lactate dehydrogenase and lysosomal hexosaminidase activity, specific radioactivity of soluble precursor pools for protein and glycoprotein labeling, incorporation of [3H]thymidine into DNA, and morphology were unaffected by hydrocortisone. Inhibitors of glucocorticoid receptor binding (progesterone), mRNA transcription (alpha-amanitin), and protein synthesis (cycloheximide) prevented the effects of hydrocortisone. We suggest that hydrocortisone maintains the digestive, absorptive, and cellular function of cultured human jejunum. These protective effects were associated with increased protein synthesis and glycosylation and dependent on a classical steroid-hormone mechanism.
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PMID:Protection of epithelial function in human jejunum cultured with hydrocortisone. 634 19

Glucocorticoids induce alkaline phosphatase activity in HeLa S3 cells only during the Late G1 and S phases of the cell cycle. In order to determine why these cells respond to glucocorticoids only during these phases, we have determined the physicochemical characteristics of the glucocorticoid receptor (GR)1 in synchronized HeLa S3 cells. GR isolated from HeLa S3 cells in the Late G1 and S phases elute from DEAE cellulose as a single, major peak at a competing [KCl] of 0.15 M (Form I). In contrast, GR isolated from cells in the G2 and Early G1 phases show a biphasic elution pattern; a more highly acidic GR form elutes at a [KCl] or 0.25 M (Form II). Similar elution patterns are observed when GR is chromatographed on hydroxylapatite (HAP). In these studies a [K2HPO4] of 0.125 M elutes the major GR form in Late G1 and S phase cells, whereas a [K2HPO4] of 0.15 M elutes a second binding species from G2 and Early G1 phase cells. GR sediments through 5-20% sucrose density gradients as an approximately 4.6S species under 0.15 M KCl buffer conditions in all phases of the cell cycle; thus the altered receptor charge is not accompanied by a change in receptor size or shape. These data suggest that HeLa S3 cell GR are susceptible to modification during the cell cycle. Conversion of GR to acidic forms may relate to the receptors' ability to transmit hormonal signals.
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PMID:Identification of modified forms of human glucocorticoid receptors during the cell cycle. 707 56

5'-Deoxypyridoxal, a vitamin B-6 analogue, increased the rate of dissociation of [3H]dexamethasone from HeLa S3 cytoplasmic glucocorticoid receptor complexes in vitro. This effect was achieved at millimolar concentrations of 5'-deoxypyridoxal, suggesting a low-affinity interaction of 5'-deoxypyridoxal with receptor. Loss of [3H]dexamethasone-receptor binding in the presence of 5'-deoxypyridoxal was pH dependent, and a plot of Kdiss vs. pH fit a simple sigmoidal titration curve with an inflection point at pH 7.8, suggesting that deprotonation of a single functional group on 5'-deoxypyridoxal increases Kdiss. Loss of [3H]dexamethasone binding in the presence or absence of unlabeled steroid also increased with pH, but no inflection point occurred over the range of pH tested. A titration of 5'-deoxypyridoxal indicated a pK of 7.94 for the pyridinium proton, suggesting deprotonation of the pyridinium nitrogen may account for the pH dependence of Kdiss of dexamethasone from receptor. 5'-Deoxypyridoxal also caused a decrease in nuclear [3H]-dexamethasone-receptor binding when incubated with whole HeLa S3 cells at 37 degrees C. Furthermore, 5'-deoxypyridoxal was effective in reducing nuclear binding of dexamethasone when added either simultaneously with [3H]dexamethasone or after achievement of equilibrium of steroid with receptor. The reduction in nuclear [3H]dexamethasone binding is highly specific for 5'-deoxypyridoxal. Several analogues of this compound, including 5'-deoxypyridoxamine, were ineffective. In addition, this effect was reversible following removal of extracellular 5'-deoxypyridoxal. Under these conditions, 5'-deoxypyridoxal was competitive with dexamethasone for binding to nuclear receptor, with KI = 8.1 X 10(-6) M. Scatchard plot analysis of dexamethasone-receptor binding in the presence or absence of 5'-deoxypyridoxal was consistent with an apparent reduced affinity of [3H]dexamethasone for receptor, which again suggests competitive interaction or allosteric interaction mediated dissociation. Glucocorticoids are known to stimulate alkaline phosphatase activity within HeLa S3 cells. In whole cell incubations, 5'-deoxypyridoxal was effective in reducing the dexamethasone-induced increase in alkaline phosphatase activity by 60% under conditions in which cell viability and cell growth were not affected.
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PMID:5'-deoxypyridoxal interaction with dexamethasone receptor: a new probe for structure and function of steroid receptors. 717 76

The co-transfection assay is a novel functional assay using cells transiently transfected with plasmids encoding intracellular receptors and corresponding reporter genes. Using this assay, natural product extracts were tested to identify compounds that modulate intracellular receptor activity, measured as changes in reporter gene activity. A crude extract of the marine alga Cymopolia barbata was found to inhibit progesterone-stimulated reporter gene expression in cells transfected with the human progesterone receptor (hPR) and an appropriate reporter construct. Purification of the active constituents of the extract, guided by the co-transfection assay, yielded two diastereomers of cyclocymopol monomethyl ether, possessing opposing pharmacological activities with the hPR. The antagonist (3R)-cyclocymopol monomethyl ether (LG100127) blocked 1 nM progesterone-stimulated reporter gene expression with an IC50 value of 549 +/- 55 nM in the co-transfection assay. The agonist (3S)-cyclocymopol monomethyl ether (LG100128) had efficacy similar to that of progesterone and an EC50 value of 35 +/- 2 nM. Stimulation by progesterone of the hPR in the human breast cancer cell line T-47D results in enhanced expression of alkaline phosphatase; LG100127 blocked alkaline phosphatase expression stimulated either by progesterone or by LG100128, and LG100128 mimicked progesterone in this assay. Both diastereomers displaced [3H]progesterone from baculovirus-expressed hPR. LG100127 and LG100128 each interacted with the human androgen receptor but did not interact with the human glucocorticoid receptor, estrogen receptor, vitamin D receptor, or retinoid receptors. In summary, these in vitro studies describe the first nonsteroidal pharmacophores for the hPR and demonstrate the use of the co-transfection assay in their discovery.
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PMID:Nonsteroidal human progesterone receptor modulators from the marine alga Cymopolia barbata. 770 Feb 60

HOS-8603 is a newly established human osteosarcoma cell line with phenotypic characteristics of osteoblasts. When these cells were grown in monolayer culture in the presence of dexamethasone (Dex) or retinoic acid (RA), there was a significant inhibition of proliferation in a concentration-dependent manner. The combined effects of Dex and RA depended upon the concentrations: at low concentrations (< 10 nM) the effects of Dex and RA were additive, whereas at high concentrations the effects were antagonistic. Anchorage-independent growth studies performed in methylcellulose culture indicated that Dex or RA inhibited colony formation by HOS-8603 cells. Treatment of HOS-8603 cells with 100 nM Dex induced alkaline phosphatase activity in a time-dependent manner, reaching a maximum of about 6.5-fold over basal levels. All these effects of Dex on HOS-8603 cells could be reversed by RU 486, a potent antiglucocorticoid. Based upon saturation of specific binding and Scatchard plot analysis, we demonstrated that a saturable, high-affinity glucocorticoid receptor (GR) existed in HOS-8603 cells, suggesting that the effects of glucocorticoids on HOS-8603 cells are mediated by the specific GR. Finally, we further investigated the homologous and heterologous regulation of GR in HOS-8603 cells. Treatment of these cells with Dex led to a time-dependent decrease in GR concentrations. This homologous GR downregulation occurred not only at the level of hormone binding but also at the level of GR mRNA. In contrast, RA was capable of increasing GR concentrations in a concentration- and time-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of retinoic acid and dexamethasone on proliferation, differentiation, and glucocorticoid receptor expression in cultured human osteosarcoma cells. 799 82

To determine whether rat osteoblasts synthesize proteins of the annexin family and to evaluate the extent to which glucocorticoids modulate the expression of annexins by these cells, osteoblasts were grown in primary cultures in the absence or presence of dexamethasone, and the expression of annexins was evaluated by immunoblotting using polyclonal antibodies against human annexins. Four different annexins (I, II, V, and VI) were found to be expressed by rat osteoblasts. The expression of annexin I, but not the other annexins studied, was increased in osteoblasts cultured in the presence of dexamethasone (173 +/- 33% increase comparing untreated cells and cells treated for 10 days with 5 x 10(-7) M dexamethasone). Increased expression of annexin I was observed after the third day of exposure to dexamethasone and rose thereafter until day 10; annexin I expression increased with dexamethasone concentrations above 10(-10) M throughout the range of concentrations studied. The increase in annexin I protein was associated with an increase in annexin I mRNA and was completely blocked by the concomitant addition of the glucocorticoid receptor antagonist RU 38486. The increase in annexin I content following dexamethasone treatment was associated with an increase in alkaline phosphatase activity and PTH-induced cAMP stimulation, whereas phospholipase A2 activity in the culture medium was reduced to undetectable levels. The finding that four annexins are expressed in rat osteoblasts in primary culture raises the possibility that these proteins could play an important role in bone formation by virtue of their ability to bind calcium and phospholipids, serve as Ca2+ channels, interact with cytoskeletal elements, and/or regulate phospholipase A2 activity. In addition, the dexamethasone-induced increase in annexin I may represent a mechanism by which glucocorticoids modify osteoblast function.
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PMID:Expression of annexin I, II, V, and VI by rat osteoblasts in primary culture: stimulation of annexin I expression by dexamethasone. 825 57

The effect of retinoic acid (RA) on the expression of osteoblast-related genes as well as on steroid/vitamin D3 receptor contents was examined using cultured osteosarcoma cell line (BFO cells). Northern blot analysis revealed that mRNAs encoding osteocalcin, pro-alpha 1 (I) collagen and bone morphogenetic protein 4 (BMP-4) are expressed in BFO cells. Stimulation with RA, however, failed to alter their mRNA content, although the transcripts for retinoic acid receptor (RAR)-alpha and -gamma were present in BFO cells. In addition, alkaline phosphatase activity (AP) was significantly but modestly increased by RA treatment. These results suggest BFO cells have well differentiated osteoblastic properties. In contrast to the effects of RA on osteoblast-related gene regulation, RA was found to increase the quantity of estrogen receptor as well as of 1,25-dihydroxy vitamin D3 receptor (VDR) in BFO cells. The quantities, assessed by ligand binding assays, were approximately 200% more than those of the controls after 24 h stimulation with 10(-9)-10(-8) M RA. These RA effects on ER and VDR seem to be specific, since glucocorticoid receptor quantities were not affected by RA treatment. These results suggest that RA regulates ER and VDR quantities in BFO cells.
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PMID:Effects of retinoic acid on steroid and vitamin D3 receptors in cultured mouse osteosarcoma cells. 838 33


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