Showing papers on "Alkaline phosphatase published in 1992"
TL;DR: Two other crystal structures of enzymes involved in phosphate ester hydrolysis have been completed that show a triad of closely spaced zinc ions present at their active centers, and suggest that there may be a significant dissociative character to each of the two displacements at phosphorous catalyzed by alkaline phosphatase.
Abstract: Alkaline phosphatase was the first zinc enzyme to be discovered in which three closely spaced metal ions (two Zn ions and one Mg ion) are present at the active center. Zn ions at all three sites also produce a maximally active enzyme. These metal ions have center-to-center distances of 3.9 A (Zn1-Zn2), 4.9 A (Zn2-Mg3), and 7.1 A (Zn1-Mg3). Despite the close packing of these metal centers, only one bridging ligand, the carboxyl of Asp51, bridges Zn2 and Mg3. A crystal structure at 2.0-A resolution of the noncovalent phosphate complex, E.P, formed with the active center shows that two phosphate oxygens form a phosphate bridge between Zn1 and Zn2, while the two other phosphate oxygens form hydrogen bonds with the guanidium group of Arg166. This places Ser102, the residue known to be phosphorylated during phosphate hydrolysis, in the required apical position to initiate a nucleophilic attack on the phosphorous. Extrapolation of the E.P structure to the enzyme-substrate complex, E.ROPO4(2-), leads to the conclusion that Zn1 must coordinate the ester oxygen, thus activating the leaving group in the phosphorylation of Ser102. Likewise, Zn2 appears to coordinate the ester oxygen of the seryl phosphate and activate the leaving group during the hydrolysis of the phosphoseryl intermediate. Both of these findings suggest that there may be a significant dissociative character to each of the two displacements at phosphorous catalyzed by alkaline phosphatase. A water molecule (or hydroxide) coordinated to Zn1 following formation of the phosphoseryl intermediate appears to be the nucleophile in the second step of the mechanism. Dissociation of the product phosphate from the E.P intermediate is the slowest, 35 s-1, and therefore the rate-limiting, step of the mechanism at alkaline pH. Since the determination of the initial crystal structure of alkaline phosphatase, two other crystal structures of enzymes involved in phosphate ester hydrolysis have been completed that show a triad of closely spaced zinc ions present at their active centers. These enzymes are phospholipase C from Bacillus cereus (structure at 1.5-A resolution) (43) and P1 nuclease from Penicillium citrinum (structure at 2.8-A resolution) (74). Both enzymes hydrolyze phosphodiesters. Substrates for phospholipase C are phosphatidylinositol and phosphatidylcholine, while P1 nuclease is an endonuclease hydrolyzing single stranded ribo- and deoxyribonucleotides. P1 nuclease also has activity as a phosphomonoesterase against 3'-terminal phosphates of nucleotides. The Zn ions in both enzymes form almost identical trinuclear sites.(ABSTRACT TRUNCATED AT 400 WORDS)
836 citations
TL;DR: Evaluation of hOP-1 effects on cell growth and collagen synthesis in rat osteoblast-enriched bone cell cultures showed that both cell proliferation andcollagen synthesis were stimulated in a dose-dependent manner and increased 3-fold in response to 40 ng of hop-1/ml.
698 citations
TL;DR: Results indicate that rhBMP-2 induces the expression of several markers associated with the osteoblast phenotype in W-20-17 cells and raises the possibility that B MP-2 may be involved in the differentiation of osteoblasts from progenitor cells resident in bone marrow.
Abstract: To better understand the in vivo bone-inductive properties of recombinant human (rh) BMP-2, we examined the ability of the protein to alter the phenotype of a bone marrow stromal cell line. W-20-17. rhBMP-2 increased alkaline phosphatase activity in W-20-17 cells in a dose-responsive manner in the absence of an effect on proliferation. The induction of alkaline phosphatase activity was not apparent until 12 h after rhBMP-2 treatment had begun and was effectively eliminated by cotreatment with cycloheximide, suggesting a requirement for protein synthesis. Continued treatment of W-20-17 cells with rhBMP-2 for 8 days resulted in a significant increase, compared to control cultures, in the production of cellular cAMP in response to a PTH challenge. In addition, 4-day treatment with rhBMP-2 induced osteocalcin levels in W-20-17 cells. These results indicate that rhBMP-2 induces the expression of several markers associated with the osteoblast phenotype in W-20-17 cells and raises the possibility that BMP-2 may ...
433 citations
Journal Article•
TL;DR: S-allyl cysteine sulphoxide (SACS), a sulphur containing amino acid of garlic which is the precursor of allicin and garlic oil, has been found to show significant antidiabetic effects in alloxan diabetic rats.
Abstract: S-allyl cysteine sulphoxide (SACS), a sulphur containing amino acid of garlic which is the precursor of allicin and garlic oil, has been found to show significant antidiabetic effects in alloxan diabetic rats. Administration of it at a dose of 200 mg/kg body weight decreased significantly the concentration of serum lipids, blood glucose and activities of serum enzymes like alkaline phosphatase, acid phosphatase and lactate dehydrogenase and liver glucose-6-phosphatase. It increased significantly liver and intestinal HMG CoA reductase activity and liver hexokinase activity.
319 citations
TL;DR: Induction of increased activities of lysozyme and complement may, at least in part, explain the mechanisms by which glucan induces protection against bacterial pathogens in salmon.
313 citations
TL;DR: The findings demonstrate that the capillary or microvessel pericyte exhibits phenotypic expressions in vitro that are similar to that of in vitro bone cells, and these expressions may be somewhat oxygen dependent.
Abstract: Bovine capillary and microvessel pericytes were grown in monolayer in standard tissue culture medium supplemented with 10% newborn calf serum at various oxygen tensions for up to ten weeks. The pericytes synthesized alkaline phosphatase and formed colonies that mineralized. Energy dispersive X-ray spectrometry revealed the presence of calcium and phosphate, showed positive staining for collagen and glycosaminoglycan, and, most importantly, demonstrated the synthesis of osteocalcin. Cell proliferation, hydroxyproline production, and alkaline phosphatase synthesis were greatest in 3% oxygen, whereas osteocalcin production was least in 3% oxygen. These findings demonstrate that the capillary or microvessel pericyte exhibits phenotypic expressions in vitro that are similar to that of in vitro bone cells, and these expressions may be somewhat oxygen dependent. It is suggested from these findings that the capillary or microvessel pericyte may be an osteoblast precursor cell.
276 citations
TL;DR: The cell-mediated component of JCa during acidosis in vitro appears to result from a combination of inhibited osteoblastic and stimulated osteoclastic activity.
Abstract: Metabolic acidosis induces net calcium flux (JCa) from cultured neonatal mouse calvariae through physicochemical and cell-mediated mechanisms. To determine the role of osteoblasts in acid-induced JCa, collagen synthesis and alkaline phosphatase activity were assessed in calvariae incubated in reduced pH and bicarbonate medium, a model of metabolic acidosis (Met), and compared with controls (Ctl). Collagen synthesis fell from 30.5 +/- 1.1 in Ctl to 25.1 +/- 0.4% with Met, and alkaline phosphatase decreased from 403 +/- 25 in Ctl to 298 +/- 21 nmol Pi.min-1.mg protein-1 with Met. During acidosis JCa was correlated inversely with percent collagen synthesis (r = -0.743, n = 11, P = 0.009) and with alkaline phosphatase activity (r = -0.453, n = 22, P = 0.034). To determine the role of osteoclasts in acid-induced JCa, osteoclastic beta-glucuronidase activity was determined in Ctl and Met in the absence or presence of the osteoclastic inhibitor calcitonin (CT, 3 x 10(-9) M). Met increased beta-glucuronidase (5.9 +/- 0.2) compared with Ctl (4.6 +/- 0.3 micrograms phenolphthalein released.bone-1.h-1), whereas CT inhibited beta-glucuronidase in both Ctl and Met (3.1 +/- 0.2 and 3.5 +/- 0.3, respectively). During acidosis JCa was correlated directly with beta-glucuronidase activity (r = 0.683, n = 42, P less than 0.001). Thus the cell-mediated component of JCa during acidosis in vitro appears to result from a combination of inhibited osteoblastic and stimulated osteoclastic activity.
268 citations
TL;DR: Results indicate that the folding of PhoA in vivo is not spontaneous but catalyzed at least at the disulfide bond formation step, and is susceptible to degradation by proteases in vivo and in vitro.
Abstract: Tn5 insertion mutations of Escherichia coli were isolated that impaired the formation of correctly folded alkaline phosphatase (PhoA) in the periplasm. The PhoA polypeptide synthesized in the mutants was translocated across the cytoplasmic membrane but not released into the periplasmic space. It was susceptible to degradation by proteases in vivo and in vitro. The wild-type counterpart of this gene (named ppfA) has been sequenced and shown to encode a periplasmic protein with a pair of potentially redox-active cysteine residues. PhoA synthesized in the mutants indeed lacked disulfide bridges. These results indicate that the folding of PhoA in vivo is not spontaneous but catalyzed at least at the disulfide bond formation step.
265 citations
TL;DR: The results suggest that the phosphorylation sites not affected by phosphatase may be located in the 25-26 kDa C-terminal region ofPHF-tau and may play a role in structural stability of PHF.
240 citations
TL;DR: It is concluded that medium supplementation with β-GP or Pi should not exceed 2 mM; if this value is exceeded, then there will be nonphysiological mineral deposition in the bone cell culture.
Abstract: Experiments were performed to determine whether β-glycerophosphate (β-GP) promoted mineralization in vitro by modulating bone cell metabolic activity and/or serving as a local source of inorganic phosphate ions (Pi). Using MC3T3-E1, ROS 17/2.8, and chick osteoblast-like cells in the presence of β-GP or Pi, we examined mineral formation, lactate generation, alkaline phosphatase (AP) activity, and protein and phospholipid synthesis. Neither β-GP nor Pi modulated any of the major biosynthetic activities of the bone cells. Thus, we found no change in the levels of phospholipids, and the total protein concentration remained constant. Measurement of lactate synthesis showed that β-GP did not effect the rate of anaerobic glycolysis. Evaluation of medium Pi levels clearly indicated that β-GP was hydrolyzed by bone cells; within 24 hours, almost 80% of 10 mM β-GP was hydrolyzed. It is likely that this local increase in medium Pi concentration promoted rapid mineral deposition. Chemical, energy dispersive X-ray, and Fourier transform infrared analysis of the mineral formed in the presence of β-GP showed that it was nonapatitic; moreover, mineral particles were also seen in the culture medium itself. Experiments performed with a cell-free system indicated that mineral particles formed spontaneously in the presence of AP and β-GP and were deposited into a collagen matrix. We conclude that medium supplementation with β-GP or Pi should not exceed 2 mM. If this value is exceeded, then there will be nonphysiological mineral deposition in the bone cell culture.
218 citations
Book•
01 Jan 1992
TL;DR: Nucleic acid hybridization test formats, strategies and applications, C. Kessler nonradioactive labeling methods for nucleic acids, detection methods, E.E. Morrison energy transfer and fluorescence quenching methods.
Abstract: Nucleic acid hybridization test formats, strategies and applications, C. Kessler nonradioactive labeling methods for nucleic acids, detection methods, E. Templeton, et al alkaline phosphatase/time-resolved fluorescence, R.E. Geiger alkaline phosphatase/bioluminescence, A. Tumulo, et al alkaline phosphatase/chemiluminescence, A. Rashtchian alkaline phosphatase/colorimetry, I. Durant horseradish peroxidase/enhanced chemiluminescence, P.C.Verlander horseradish peroxidase/colorimetry, P. Balaguer, et al glucose 6-phosphate dehydrogenase/bioluminescence, T. Lovgren, et al lanthanide chelates/time-resolved fluorescence, E.P. Diamandis and T.K. Christopoulos lanthanide chelates and multiple labelling strategies/time-resolved fluorescence, N.C. Nelson, et al acridinium esters/ chemiluminescence, L.E. Morrison energy transfer and fluorescence quenching methods.
TL;DR: It is proposed that the glucocorticoid promote changes in gene expression involved in cell‐cell and cell‐extracellular matrix signaling mechanism that support the growth and differentiation of cells capable of osteoblast phenotype development and bone tissue‐like organization, while inhibiting the growth of cells that cannot progress to the mature osteoblow phenotype in fetal rat calvarial cultures.
Abstract: To understand the mechanisms by which glucocorticoids promote differentiation of fetal rat calvaria derived osteoblasts to produce bone-like mineralized nodules in vitro, a panel of osteoblast growth and differentiation related genes that characterize development of the osteoblast phenotype has been quantitated in glucocorticoid-treated cultures. We compared the mRNA levels of osteoblast expressed genes in control cultures of subcultivated cells where nodule formation is diminished, to cells continuously (35 days) exposed to 10(-7) M dexamethasone, a synthetic glucocorticoid, which promotes nodule formation to levels usually the extent observed in primary cultures. Tritiated thymidine labelling revealed a selective inhibition of internodule cell proliferation and promotion of proliferation and differentiation of cells forming bone nodules. Fibronectin, osteopontin, and c-fos expression were increased in the nodule forming period. Alkaline phosphatase and type I collagen expression were initially inhibited in proliferating cells, then increased after nodule formation to support further growth and mineralization of the nodule. Expression of osteocalcin was 1,000-fold elevated in glucocorticoid-differentiated cultures in relation to nodule formation. Collagenase gene expression was also greater than controls (fivefold) with the highest levels observed in mature cultures (day 35). At this time, a rise in collagen and TGF beta was also observed suggesting turnover of the matrix. Short term (48 h) effects of glucocorticoid on histone H4 (reflecting cell proliferation), alkaline phosphatase, osteopontin, and osteocalcin mRNA levels reveal both up or down regulation as a function of the developmental stage of the osteoblast phenotype. A comparison of transcriptional levels of these genes by nuclear run-on assays to mRNA levels indicates that glucocorticoids exert both transcriptional and post-transcriptional effects. Further, the presence of glucocorticoids enhances the vitamin D3 effect on gene expression. Those genes which are upregulated by 1,25(OH)2D3 are transcribed at an increased rate by dexamethasone, while those genes which are inhibited by vitamin D3 remain inhibited in the presence of dexamethasone and D3. We propose that the glucocorticoids promote changes in gene expression involved in cell-cell and cell-extracellular matrix signaling mechanisms that support the growth and differentiation of cells capable of osteoblast phenotype development and bone tissue-like organization, while inhibiting the growth of cells that cannot progress to the mature osteoblast phenotype in fetal rat calvarial cultures.
TL;DR: The SEAP gene has several advantages when compared to CAT or to other prevalent reporter genes, including very high stability, efficient secretion by all cells tested, and the availability of a simple, inexpensive, and highly quantitative assay that does not require any unusual equipment or reagents.
Abstract: Publisher Summary This chapter describes the use of a novel indicator gene, producing a secreted form of the human enzyme placental alkaline phosphate, that believes offers a number of advantages. These include very high stability, efficient secretion by all cells tested, and the availability of a simple, inexpensive, and highly quantitative assay that does not require any unusual equipment or reagents. A major focus of the current molecular biological research is the identification and functional dissection of the cis-acting sequences and trans-acting factors that regulate eukaryotic gene expression in vivo. A common method of addressing this question is to link the regulatory sequence of interest, e.g., an inducible promoter/enhancer element, to a reporter gene. Several alternate or complementary reporter gene systems have been proposed. Among the more prevalent are the genes for the intracellular enzymes β-galactosidase, luciferase and the secreted protein human growth hormone. This chapter describes SEAP, a novel indicator gene producing secreted alkaline phosphatase. The SEAP gene has several advantages when compared to CAT or to other prevalent reporter genes.
TL;DR: The present experiments show that beta-GP is rapidly and virtually completely degraded to Pi during the initiation phase of mineralization and that the addition of increased concentrations of Pi can replacebeta-GP in the initiationphase of Mineralization in the absence of non-specific 45Ca uptake or apparent cellular toxicity.
TL;DR: The results suggest that propionate, like butyrate, may play an important role in the physiology of the colon and could partially account for the protective effect of dietary fibers with respect to colon carcinogenesis.
Abstract: Short-chain fatty acids (SCFAs), namely butyrate, acetate and propionate, originate from the bacterial fermentation of dietary fibers and are the predominant anions present in the large bowel. Our study was carried out to investigate the effects of SCFAs on growth of the human adenocarcinoma cell line, HT29. The results show that, under our culture conditions, both propionate and butyrate inhibit growth of HT29 cells, whereas acetate has no significant effect. The antiproliferative effect of propionate or butyrate is associated with an inhibition of FCS-induced activation of ornithine decarboxylase (ODC), a key enzyme of polyamine metabolism. Inhibition of growth induced by either propionate or butyrate is not reversed by the addition of putrescine, which reveals that these SCFAs are not acting solely on the ODC/polyamine system. Our data show that propionate and butyrate, unlike acetate, induce an increase in alkaline phosphatase activity, which reflects a more differentiated phenotype than that of untreated control cells. Taken together, our results suggest that propionate, like butyrate, may play an important role in the physiology of the colon and could partially account for the protective effect of dietary fibers with respect to colon carcinogenesis.
TL;DR: TGF beta appears to modulate the differentiation effects of 1,25D3 on this human osteoblast-like cell and thereby retain the cell in a non-fully differentiated state.
TL;DR: Recognition that alkaline phosphatase belongs to the category of molecules that are localized to cell membranes through a COOH-terminal glycan-phosphatidylinositol anchor provides a basis for understanding the generation of isoforms observed in plasma in disease.
Abstract: Gene cloning and site-directed mutagenesis have had a profound effect on alkaline phosphatase research. Four distinct structural genes encoding placental, intestinal, and tissue-nonspecific isoenzymes have been cloned, sequenced, and mapped to human chromosomes. Differences in properties between the respective gene products are due to variations in primary structure involving only one, or a few, key amino acid residues. Recognition that alkaline phosphatase belongs to the category of molecules that are localized to cell membranes through a COOH-terminal glycan-phosphatidylinositol anchor provides a basis for understanding the generation of isoforms observed in plasma in disease. Isoforms produced by differential cleavage or preservation of the glycan-phosphatidylinositol anchor may offer new correlations with disease that are of diagnostic value. However, a more important contribution of alkaline phosphatase research to clinical chemistry may prove to be an increased understanding of disease processes at the molecular level.
TL;DR: The bound scFv(FRP5)–PhoA protein can be detected directly on tumor cells using a substrate for alkaline phosphatase, showing that the chimeric protein retains both binding and enzymatic activity.
Abstract: We have constructed genes expressing single-chain antigen binding proteins (scFv) which recognize the human erbB-2 receptor. These genes encode the heavy and light chain variable domains of an erbB-2 receptor specific monoclonal antibody, MAb FRP5, connected by a peptide linker. In order to express a bifunctional molecule, a bacterial alkaline phosphatase gene was fused 3' to the scFv gene. The scFv(FRP5) and scFv(FRP5)-alkaline phosphatase fusion protein (scFv(FRP5)-PhoA) expressed in E. coli specifically recognize the human erbB-2 protein and compete with MAb FRP5 for binding to the receptor. The bound scFv(FRP5)-PhoA protein can be detected directly on tumor cells using a substrate for alkaline phosphatase, showing that the chimeric protein retains both binding and enzymatic activity.
TL;DR: Phosphorylation of GBF1 resulted in stimulation of the G-box binding activity and formation of a slower migrating protein-DNA complex, which fully corresponded to the properties of casein kinase II, in particular its dependence on the known phosphate donors.
Abstract: To study the phosphorylation of one of the G-box binding factors from Arabidopsis (GBF1), we have obtained large amounts of this protein by expression in Escherichia coli. Bacterial GBF1 was shown to be phosphorylated very efficiently by nuclear extracts from broccoli. The phosphorylation activity was partially purified by chromatography on heparin-Sepharose and DEAE-cellulose and was characterized. It showed the essential features of casein kinase II activity: utilization of GTP in addition to ATP as a phosphate donor, strong inhibition by heparin, preference for acidic protein substrates, salt-induced binding to phosphocellulose, and salt-dependent deaggregation. The very low Km value for GBF1 (220 nM compared to approximately 10 microM for casein) was in the range observed for identified physiological substrates of casein kinase II. Phosphorylation of GBF1 resulted in stimulation of the G-box binding activity and formation of a slower migrating protein-DNA complex. The conditions of this stimulatory reaction fully corresponded to the properties of casein kinase II, in particular its dependence on the known phosphate donors. The DNA binding activity of the endogenous plant GBF was shown to be reduced by treatment with calf alkaline phosphatase; this reduction was diminished by addition of fluoride and phosphate or incubation in the presence of casein kinase II and ATP.
TL;DR: It is suggested that activin-A modulates the function of osteoblastic cells by being regulated by follistatin during differentiation.
TL;DR: The sequential expression of various genes implicated in the formation and removal of bone following marrow ablation in rats is reported, showing a lower expression of bone-related genes in older rats.
TL;DR: Female sex steroids may be involved in regulating bone mass in human adults via a direct anabolic action on the bone forming cells through their role in stimulating osteoblast growth.
TL;DR: A truncated placental alkaline phosphatase (SEAP) gene was inserted into the Autographa californica nuclear polyhedrosis virus genome under the transcriptional control of the polyhedrin gene promoter to evaluate insect tissue culture cells for their capacity to express, glycosylate and secrete foreign proteins.
Abstract: We have devised a simple and efficient baculovirus expression vector system to evaluate insect tissue culture cells for their capacity to express, glycosylate and secrete foreign proteins A truncated placental alkaline phosphatase (SEAP) gene was inserted into the Autographa californica nuclear polyhedrosis virus (AcMNPV) genome under the transcriptional control of the polyhedrin gene promoter Production levels, glycosylation, and secretion of the recombinant protein were examined in Thichoplusia ni (BTI–TN–5B1–4) and Spodoptera frugiperda (Sf9) cell lines The assay for SEAP activity, which is fast, inexpensive, and quantitative to concentrations of 20 picograms per milliliter, was used to assess cell–associated and secreted SEAP activity The proportion of SEAP which is modified with N–linked oligosaccharide can also be determined due to the difference in mobilities during SDS–PAGE between the glycosylated and nonglycosylated forms of the protein
TL;DR: It was shown that ALP-coupled DDS rapidly accumulated hydroxyapatite crystals, and direct evidence that ALP may play a crucial role in the induction of hydroxyAPatite deposition in collagenous matrices in vivo is presented.
Abstract: To determine whether alkaline phosphatase (ALP) can cause the mineralization of collagenous matrices in vivo, bovine intestinal ALP was covalently bound to slices of guanidine-extracted demineralized bovine dentin (DDS). The preparations were implanted subcutaneously over the right half of the rat skull. Control slices not treated with the enzyme were implanted over the left half of the skull of the same animals. Specimens were harvested after periods varying from 1 to 4 wk. It was shown that ALP-coupled DDS rapidly accumulated hydroxyapatite crystals. 4 wk after implantation, the content of calcium and phosphate per microgram of hydroxyproline amounted up to 80 and 60%, respectively, of that found in normal bovine dentin. Our observations present direct evidence that ALP may play a crucial role in the induction of hydroxyapatite deposition in collagenous matrices in vivo.
TL;DR: The hypothesis that bone extracellular matrix may play an important role in osteoblastic differentiation and phenotypic expression is supported.
Abstract: Using MG-63 cells as a model system capable of partial osteoblastic differentiation, we have examined the effect of growth on extracellular matrix. MG-63 cell matrix and purified type I collagen induced a morphological change characterized by long cytoplasmic processes reminiscent of those seen in osteocytes. Concurrent biochemical changes involving bone marker proteins included increased specific activity of cell-associated alkaline phosphatase and increased secretion of osteonectin (up to 2.5-fold for each protein); all changes occurred without alterations in the growth kinetics of the MG-63 cells. The increase in alkaline phosphatase activity was maximal on days 6-8 following seeding; increased osteonectin secretion was most prominent immediately following seeding; all changes decreased as cells reached confluence. Growing cells on type I collagen resulted in an increased induction of alkaline phosphatase activity by 1,25(OH)2D3 (with little change in the 1,25(OH)2D3 induction of osteonectin and osteocalcin secretion), and increased TGF-beta induction of alkaline phosphatase activity as well (both TGF-beta 1 and TGF-beta 2). Both the 1,25(OH)2D3 and TGF-beta effects appeared to be synergistic with growth on type I collagen. These studies support the hypothesis that bone extracellular matrix may play an important role in osteoblastic differentiation and phenotypic expression.
TL;DR: The results of the present study show that this mineralizing rat osteoblastic cell culture system synthesizes collagen type I, V, and traces of type III, osteocalcin, and bone ALP isoenzyme, indicating that ALP expressed in this culture is bone specific.
Abstract: Rat calvaria bone cells isolated by collagenase digestion form a bone-like matrix which mineralizes in vitro in the presence of β-glycerophosphate, in less than 2 weeks. The purpose of this work was to investigate, in this mineralizing rat osteoblastic cell culture, the synthesis of collagen, osteocalcin, and bone alkaline phosphatase (ALP). The results obtained indicate (1) After 15 days in culture, the extracellular-matrix contains collagen type I, V, and to some extent type III. Metabolic labeling at day 14, during the phase of nodules mineralization as well as new nodules formation, shows that collagen types I and type V are synthesized; (2) During the phase of cell growth, no osteocalcin could be detected in the medium, however, at the point of nodule formation, the osteocalcin level reached values of 3.55±1.39 ng/ml, followed by a 30-fold increase after nodules became mineralized. At day 14, after metabolic labeling, de novo synthesized osteocalcin was chromatographed on an immunoadsorbing column. With urea-SDS PAGE the apparent molecular weight was determined to be 9,000 daltons. (3) Specific activity of ALP was found to be 10 nmol/min/mg of proteins at cell confluence. At day 15, when nodules are mineralized, this activity was increased by 40-fold. The Michaelis constant was 1.58 10-3 M/L. ALP was inhibited by L-homoarginine and levamisole but not by L-phenylalanine. ALP was shown to be heat sensitive at 56°C with two slopes of inhibition. On SDS-PAGE, apparent molecular weight of ALP showed one band at 116,000 daltons (d) when extracted at cell confluence and two bands at 116,000 and 140,000 d when extracted at the 15th day of culture. 32P-labeled subunit of the enzyme migrated as one band at 75,000 d. Sialic acid content was demonstrated by neuraminidase treatment either on the dimeric form or on the 32P-labeled subunit. These data indicate that ALP expressed in this culture is bone specific. The results of the present study show that this mineralizing rat osteoblastic cell culture system synthesizes collagen type I, V, and traces of type III, osteocalcin, and bone ALP isoenzyme. Medium osteocalcin was detected during nodule formation and increased during mineralization. Increase in ALP activity as well as the presence of an additional form of ALP occurred in the mineralization phase. Therefore, this culture may be a useful model for studying the functions of bone-specific proteins during the process of mineralization.
TL;DR: Results further support the view that cytokine-mediated osteoblastic regulation can be relatively selective.
Abstract: Osteoblasts play a pivotal role during the bioresponse of bone to agents that stimulate bone n~sorption and/or inhibit bone formation including hormones, polypeptide growth factors, and cytokines. We examined the cytokines interleukin- 1-beta (IL-lfl) and tumor necrosis factor-alpha (TNF-a) for their effects on osteob- lastic proliferation and development and expression of alkaline phosphatase and the osteoblast-specific protein osteocalcin in a mineralizing environment. Primary rat osteoblast-like cells (Y, OB) and osteoblastic cell lines derived from rat (ROS 17/2.8) and human (MG-63) osteosarcomas were studied. IL-I~ and TNF-c~ were chosen because of their critical importance during the host response to local inflammatory stimuli. Qualitatively similar two- to threefold inhibition of osteocalcin synthesis by IL-lfl and TNF-a wel~e observed in all three postconfiuent bone-forming model sys- tems. Because of the readily measurable concentrations of osteocalcin produced in our culture protocol, it was not necessary to enhance osteoblastic synthesis of osteo- calcin by supplementation with 1,25(OH)2-vitamin D3, a treatment which exerts pleiotropic effects on ~steoblasts. Under the constraints of our protocol, where alka- line phosphatase and mineralization were already elevated at the 14-day onset of treatment, neither of these phenotypic properties was sensitive to a three-day cytokine exposure. Differences were noted in proliferation, where only TNF-a stimulated DNA synthesis in ROB cells, while both cytokines stimulated MG-63 cells. IL-lfl and TNF-c~ failed to alter ROS 17/2.8 DNA synthesis except at the highest doses (25 pM IL-lfl and 1 nlVl TNF-a) where inhibition was observed. These results further support the view tha~ cytokine-mediated osteoblastic regulation can be relatively selective.
TL;DR: The theory that hOB cells are target cells for glucocorticoids, and that as a primary event glucoc Corticoidoids rapidly regulate the expression of the nuclear oncoproteins Fos/Jun in these cells, is supported.
Abstract: In humans, glucocorticoids are known to have marked effects on bone metabolism and function, including the significant regulation of osteoblast cells. To aid in the understanding of the mechanism of glucocorticoid action on normal human osteoblasts (hOB), confluent cells were analyzed for the presence of glucocorticoid receptors (GR) as well as for the effects of the glucocorticoid dexamethasone (Dex) on the expression of both the rapid responding nuclear proto-oncogenes and the late responding structural genes for bone matrix proteins. The interactions between Dex and 1,25 dihydroxy vitamin D3 (1,25 D3) on the gene expression in these cells were also examined. Using a functional receptor assay, a mean of 11,600 functional nuclear bound glucocorticoid receptors (range 6,000-22,000) was measured in fifteen separate cell strains. Northern blot analysis with a cDNA probe to the human GR was used to demonstrate the presence of a 7Kb transcript which is a candidate mRNA for GR in these cells. In agreement with previous studies, treatment of the hOB cells with Dex increased the steady state mRNA levels for alkaline phosphatase (AP) but displayed little or no effect on the mRNA levels for osteocalcin (OC) and glyceraldehyde phosphate dehydrogenase (GAPDH). Interestingly, the 1,25 D3 inductions of mRNA levels for OC were blocked by Dex but enhanced for AP. The above effects of Dex on AP and OC gene expression, including the interaction with 1,25 D3, were also shown to occur at the level of protein. The effect of Dex on the mRNA levels of the nuclear proto-oncogenes c-myc, c-fos, and c-jun was also investigated, since the oncoproteins (Fos/Jun) appear to play a role in the delayed glucocorticoid regulation of structural genes. Interestingly, Dex increased the steady state levels of c-myc, c-fos, and c-jun mRNAs in nonproliferating (confluent) hOB cells by 3.5-, 10-, and 2.0-fold, respectively, over control (untreated cells) values within one h of steroid treatment. The Dex-induced mRNA levels were transient and returned to basal values within 24 h of the steroid treatment. A reduced but qualitatively similar pattern of response was found in proliferating hOB cells. The pattern of response of these genes to glucocorticoids in hOB cells mimics the response in avian liver cells but not in reproductive cells. These results support the theory that hOB cells are target cells for glucocorticoids, and that as a primary event glucocorticoids rapidly regulate the expression of the nuclear oncoproteins Fos/Jun in these cells.
TL;DR: The findings suggest that the proliferation of pulp cells may be stimulated mainly by PDGF and IGF-I, and the production of extracellular matrix proteoglycan may be enhanced by aFGF, IGF- I and IGF -II.