Showing papers on "Alkaline phosphatase published in 1999"

Roles of Bone Morphogenetic Protein Type I Receptors and Smad Proteins in Osteoblast and Chondroblast Differentiation

Abstract: The biological effects of type I serine/threonine kinase receptors and Smad proteins were examined using an adenovirus-based vector system. Constitutively active forms of bone morphogenetic protein (BMP) type I receptors (BMPR-IA and BMPR-IB; BMPR-I group) and those of activin receptor-like kinase (ALK)-1 and ALK-2 (ALK-1 group) induced alkaline phosphatase activity in C2C12 cells. Receptor-regulated Smads (R-Smads) that act in the BMP pathways, such as Smad1 and Smad5, also induced the alkaline phosphatase activity in C2C12 cells. BMP-6 dramatically enhanced alkaline phosphatase activity induced by Smad1 or Smad5, probably because of the nuclear translocation of R-Smads triggered by the ligand. Inhibitory Smads, i.e., Smad6 and Smad7, repressed the alkaline phosphatase activity induced by BMP-6 or the type I receptors. Chondrogenic differentiation of ATDC5 cells was induced by the receptors of the BMPR-I group but not by those of the ALK-1 group. However, kinase-inactive forms of the receptors of the ALK-1 and BMPR-I groups blocked chondrogenic differentiation. Although R-Smads failed to induce cartilage nodule formation, inhibitory Smads blocked it. Osteoblast differentiation induced by BMPs is thus mediated mainly via the Smad-signaling pathway, whereas chondrogenic differentiation may be transmitted by Smad-dependent and independent pathways.

Alkaline phosphatase knock-out mice recapitulate the metabolic and skeletal defects of infantile hypophosphatasia

Abstract: Hypophosphatasia is an inborn error of metabolism characterized by deficient activity of the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP) and skeletal disease due to impaired mineralization of cartilage and bone matrix. We investigated two independently generated TNSALP gene knock-out mouse strains as potential models for hypophosphatasia. Homozygous mice (−/−) had < 1% of wild-type plasma TNSALP activity; heterozygotes had the predicted mean of ~50%. Phosphoethanolamine, inorganic pyrophosphate, and pyridoxal 5′-phosphate are putative natural substrates for TNSALP and all were increased endogenously in the knock-out mice. Skeletal disease first appeared radiographically at ~10 days of age and featured worsening rachitic changes, osteopenia, and fracture. Histologic studies revealed developmental arrest of chondrocyte differentiation in epiphyses and in growth plates with diminished or absent hypertrophic zones. Progressive osteoidosis from defective skeletal matrix mineralization was noted but not associated with features of secondary hyperparathyroidism. Plasma and urine calcium and phosphate levels were unremarkable. Our findings demonstrate that TNSALP knock-out mice are a good model for the infantile form of hypophosphatasia and provide compelling evidence for an important role for TNSALP in postnatal development and mineralization of the murine skeleton.

Differential Cell Surface Expression of the STRO-1 and Alkaline Phosphatase Antigens on Discrete Developmental Stages in Primary Cultures of Human Bone Cells

Abstract: Human osteoblast-like cells can be readily cultured from explants of trabecular bone, reproducibly expressing the characteristics of cells belonging to the osteoblastic lineage. Dual-color fluorescence-activated cell sorting was employed to develop a model of bone cell development in primary cultures of normal human bone cells (NHBCs) based on the cell surface expression of the stromal precursor cell marker STRO-1 and the osteoblastic marker alkaline phosphatase (ALP). Cells expressing the STRO-1 antigen exclusively (STRO-1+/ALP-), were found to exhibit qualities preosteoblastic in nature both functionally by their reduced ability to form a mineralized bone matrix over time, as measured by calcium release assay, and in the lack of their expression of various bone-related markers including bone sialoprotein, osteopontin, and parathyroid hormone receptor based on reverse trancriptase polymerase chain reaction (PCR) analysis. The majority of the NHBCs which expressed the STRO-1-/ALP+ and STRO-1-/ALP- phenotypes appeared to represent fully differentiated osteoblasts, while the STRO-1+/ALP+ subset represented an intermediate preosteoblastic stage of development. All STRO-1/ALP NHBC subsets were also found to express the DNA-binding transcription factor CBFA-1, confirming that these cultures represent committed osteogenic cells. In addition, our primer sets yielded four distinct alternative splice variants of the expected PCR product for CBFA-1 in each of the STRO-1/ALP subsets, with the exception of the proposed preosteoblastic STRO-1+/ALP- subpopulation. Furthermore, upon re-culture of the four different STRO-1/ALP subsets only the STRO-1+/ALP- subpopulation was able to give rise to all of the four subsets yielding the same proportions of STRO-1/ALP expression as in the original primary cultures. The data presented in this study demonstrate a hierarchy of bone cell development in vitro and facilitate the study of bone cell differentiation and function.

Number of osteoprogenitor cells in human bone marrow markedly decreases after skeletal maturation.

Abstract: Pluripotent mesenchymal stem cells in bone marrow differentiate to osteoblast progenitor cells. When the bone marrow cells are cultured in vitro, they form colony-forming units-fibroblastic (CFU-Fs) with exhibiting osteoblastic features such as expression of alkaline phosphatase (ALP) and formation of calcified nodules ex vivo. This article describes the effect of growth, maturation, and aging of the skeleton on human CFU-Fs harvested from human iliac bone marrow. Human bone marrow cells were harvested from the ilia of 49 women, and were cultured ex vivo for examination. The 49 subjects ranged in age from 4 to 88 years and were without metabolic bone disease. These aspirated bone marrow cells from human ilium exhibited osteoblastic phenotype such as alkaline phosphatase (ALP) activity, expression of osteocalcin (OSC) and parathyroid hormone-receptor (PTH-R) mRNA, and the formation of calcified nodules in vitro. The number of ALP-positive CFU-Fs and the ALP activity were quantified. The highest levels of ALP-positive CFU-Fs were observed in the young group, particularly in those under 10 years of age. The levels of ALP-positive CFU-Fs declined sharply after 10 years of age; those above 20 years of age exhibited a lower number of ALP-positive CFU-Fs, with a gradual decline with increasing age. These results indicate that change in the number of ALP-positive CFU-Fs may be associated with skeletal growth and maturation. The results also show that osteoblastic features such as ALP activity and capability of formation of calcification nodules were maintained even in the older subjects. These findings suggest that decreased activity of bone formation in the aged subjects could be, in part, caused by the decreased number of osteoprogenitor cells differentiating into osteoblasts because the number of ALP-positive CFU-Fs was one of the indices exhibiting bone-forming activity in the human marrow stromal cells.

Engineered pluripotent mesenchymal cells integrate and differentiate in regenerating bone: a novel cell-mediated gene therapy.

Abstract: Background Among the approximately 6.5 million fractures suffered in the United States every year, about 15% are difficult to heal. As yet, for most of these difficult cases there is no effective therapy. We have developed a mouse radial segmental defect as a model experimental system for testing the capacity of Genetically Engineered Pluripotent Mesenchymal Cells (GEPMC, C3H10T1/2 clone expressing rhBMP-2), for gene delivery, engraftment, and induction of bone growth in regenerating bone. Methods Transfected GEPMC expressing rhBMP-2 were further infected with a vector carrying the lacZ gene, that encodes for β-galactosidase (β-gal). In vitro levels of rhBMP-2 expression and function were confirmed by immunohistochemistry, and bioassay. Differentiation was assayed using alkaline phosphatase staining. GEPMC were transplanted in vivo into a radial segmental defect. The main control groups included lacZ clones of WT-C3H10T1/2-LacZ, and CHO-rhBMP-2 cells. New bone formation was measured quantitatively via fluorescent labeling, X-ray analysis and histomorphometry. Engrafted mesenchymal cells were localized in vivo by β-gal expression, and double immunofluorescence. Results In vitro, GEPMC expressed rhBMP-2, β-gal and spontaneously differentiated into osteogenic cells expressing alkaline phosphatase. Detection of transplanted cells revealed engrafted cells that had differentiated into osteoblasts and co-expressed β-gal and rhBMP-2. Analysis of new bone formation revealed that at fout to eight week post-transplantation, GEPMS significantly enhanced segmental defect repair. Conclusions Our study shows that cell-mediated gene transfer can be utilized for growth factor delivery to signaling receptors of transplanted cells (autocrine effect) and host mesenchymal cells (paracrine effect) suggesting the ability of GEPMC to engraft, differentiate, and stimulate bone growth. We suggest that our approach should lead to the designing of mesenchymal stem cell based gene therapy strategies for bone lesions as well as other tissues. Copyright © 1999 John Wiley & Sons, Ltd.

Melatonin stimulates proliferation and type I collagen synthesis in human bone cells in vitro.

Abstract: The pineal secretory product melatonin reportedly regulates release of growth hormone in humans and prevents phototherapy-induced hypocalcemia in newborn rats, suggesting that melatonin affects bone metabolism. Little is known about the effects of melatonin on bone in vitro or in vivo. The present study was undertaken to examine whether melatonin acts directly on normal human bone cells (HOB-M cells) and human osteoblastic cell line (SV-HFO cells) to affect osteogenic action in vitro. The effect of melatonin on bone cell proliferation was determined using the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carbo xanilide (XTT) assay after a 24 hr incubation with melatonin. Melatonin significantly and dose-dependently increased the proliferation in HOB-M cells and SV-HFO cells by 215 +/- 22.1%, and 193 +/- 6.4%), respectively, with a maximal effect at a concentration of 50 microM. To evaluate the effect of melatonin on bone cell differentiation, alkaline phosphatase (ALP) activity, osteocalcin secretion and procollagen type I c-peptide (PICP) production (a measure of type I collagen synthesis) were measured after a 48 hr treatment. While melatonin at micromolar concentrations did not significantly affect either the ALP activity or the osteocalcin secretion, it significantly and dose-dependently increased the PICP production in HOB-M cells and SV-HFO cells by 983 +/- 42.2%, and 139 +/- 4.2%, respectively, with the maximal stimulatory doses between 50 and 100 microM. These results provide new evidence that melatonin stimulates the proliferation and type I collagen synthesis in human bone cells in vitro, suggesting that melatonin may act to stimulate bone formation.

Further characterization of cells expressing STRO-1 in cultures of adult human bone marrow stromal cells.

Abstract: Primitive cells of the osteoblast lineage are not well characterized but are known to be present within the STRO-1+ fraction of adult human bone and marrow. A survey of human osteosarcoma cell lines revealed that STRO-1 is expressed by MG-63 but not SaOS-2. Among murine cell lines tested, expression of STRO-1 was detected in the bipotential (adipocyte/osteoblast) line BMS-2 but not the committed osteoblast precursor MC3T3-E1. A proportion of cultured adult human bone marrow stromal cells (BMSCs) consistently expressed the STRO-1 antigen. The expression of a range of cell surface antigens was studied in relation to STRO-1 by flow cytometry and several, including the bone/liver/kidney isoform of alkaline phosphatase (ALP), were found to subtype the STRO-1+ population of BMSCs. Further, BMSCs dual-labeled with antibodies recognizing STRO-1 and ALP could be assigned to one of four fractions: STRO-1-/ALP-, STRO-1+/ALP-, STRO-1+/ALP+, and STRO-1-/ALP+. Cells from each fraction could be isolated in high purity and, when recultured, remained viable and exhibited a limited degree of phenotypic stability. Using reverse transcriptase-polymerase chain reaction, cells in the four fractions were found to express different levels of transcripts for the parathyroid hormone receptor (PTHr) and bone sialoprotein (BSP). The expression of transcripts for the nuclear transcription factor core-binding factor alpha 1/osteoblast-specific factor-2 (CBFA1/OSF2) was restricted to those fractions expressing STRO-1 and/or ALP. Treatment with 10 nM dexamethasone consistently increased the proportion of cells present in those fractions which expressed the highest levels of transcripts for PTHr and BSP (STRO-1+/ALP+ and STRO-1-/ALP+) while simultaneously decreasing the proportion present in the STRO-1+/ALP- fraction. In conclusion, the expression of STRO-1 in vitro remains a characteristic of less well differentiated cells of the osteoblast lineage; in cultures of BMSCs and in established human osteosarcoma cell lines, there is an inverse association between the expression of STRO-1 and ALP; dual labeling of BMSCs with monoclonal antibodies recognizing STRO-1 and ALP permits the identification and isolation of cells of the osteoblast lineage at different stages of differentiation.

Estrogen receptor-alpha detected on the plasma membrane of aldehyde-fixed GH3/B6/F10 rat pituitary tumor cells by enzyme-linked immunocytochemistry.

Abstract: A population of estrogen receptor-alpha (ER alpha) proteins, located at the plasma membrane, is postulated to mediate the rapid, nongenomic responses of GH3/B6/F10 pituitary cells to estrogen. To demonstrate the presence of ER alpha at the plasma membrane and to distinguish this receptor population from that in the nucleus, GH3/B6/F10 cells were first prepared in 2% paraformaldehyde/0.1% glutaraldehyde in PBS (P/G) without detergent, then exposed to one of several antibodies (Abs) raised against nuclear ER alpha. Ab binding was visualized as a fluorescent/chromagenic reaction product catalyzed by avidin-biotin-complexed alkaline phosphatase. With P/G fixation, Abs could only access antigens at the cell surface, as evidenced by the inability of 70K mol wt dextrans to permeate cells and the absence of intracellular staining by Abs to cytoplasmic or nuclear antigens. ER alpha Abs generated membrane, but not nuclear, staining in P/G-fixed cells; nuclear receptor labeling could only be detected in detergent-treated cells. Specificity of staining for ER alpha was confirmed by three approaches: first, treatment with an antisense oligodeoxynucleotide to nuclear ER alpha mRNA reduced immunolabeling of both membrane and nuclear ER alpha; second, labeling by two Abs raised against different ER alpha oligopeptides was neutralized by competing peptide; third, six Abs (ER21, H226, R4, H222, MC20, and C542) that recognize unique epitopes on rodent ER alpha produced immunolabeling, but neither primate-specific ER alpha Ab nor Ab to ER beta caused staining. In addition to demonstrating the plasma membrane ER alpha in GH3/B6/F10 cells, this method should be applicable to other cell types that exhibit nongenomic responses to estrogen or other steroid hormones.

Osteogenic potential in vitro of human bone marrow cells cultured on macroporous biphasic calcium phosphate ceramic.

Abstract: Calcium phosphate ceramics are synthetic bone substitutes that promote bone formation by osteoconduction. However, they have shown an osteogenic potential in vivo in animal models when associated with bone marrow cells. In order to develop an osteogenic human “hybrid material,” we studied the in vitro osteogenic potential of human bone marrow cells cultured on macroporous biphasic calcium phosphate (BCP) pellets in the presence of dexamethasone with or without 1α,25 dihydroxyvitamin D3. We were examining, in particular, their mesenchymal stem cell proliferation and hematopoietic potential. Osteogenic differentiation was evaluated in terms of alkaline phosphatase activity and immunological characterization of the extracellular matrix formed by these cells. The specimens were observed by scanning and transmission electron microscopy. Human mesenchymal stem cells proliferated on macroporous BCP ceramic, with a doubling time delayed at the beginning of the culture as compared to the cultures realized on plastic. These cells maintained a hematopoietic activity identical to that of cultures performed in plastic wells. The proliferating mesenchymal stem cells expressed an osteoblastic phenotype, as shown by alkaline phosphatase activity and the synthesis in ceramic macropores of an extracellular matrix composed of fibronectin, osteocalcin, and collagen I (but not collagen III). In addition, numerous microcrystals of apatite precipitated on the extracellular matrix, producing a mineralized fibrillar network within the ceramic not observed with cell cultures realized on plastic. These results demonstrate that human mesenchymal stem cells cultured on macroporous BCP ceramic express and conserve their osteoblastic phenotype even after one month of culture and that these osteogenic cells are able to form new bone matrix in a BCP ceramic in vitro. © 1999 John Wiley & Sons, Inc. J Biomed Mater Res, 44, 98–108, 1999.

Development of the osteoblast phenotype in primary human osteoblasts in culture: comparison with rat calvarial cells in osteoblast differentiation.

Abstract: In rat osteoblast-like cells, a time-dependent sequence of growth and differentiation-dependent genes has been identified and a model of osteoblast differentiation in culture suggested. We investigated the expression of the bone matrix-associated proteins osteonectin and procollagen I and of the bone cell phenotype-related proteins alkaline phosphatase and osteocalcin during cell culture in primary human osteoblast like cells. Primary human explant cultures from nine young healthy donors were established under highly standardized conditions. Cells in the second passage were analyzed on different days from day 1 to 32, comparing cells growing under the influence of ascorbate with controls. Gene expression was determined by Northern blot analysis or polymerase chain reaction. Osteocalcin expression was also investigated after 1,25-(OH)(2)D(3) stimulation. On the protein level, newly synthesized collagen I, alkaline phosphatase activity, and secretion of osteocalcin were analyzed at all time points. On comparing our findings to the pattern of gene expression suggested for the rat calvarial osteoblast system, we found a similar developmental sequence for the so-called "proliferation" as well as a similar, but lengthened, sequence for the "matrix maturation stage." During "matrix maturation," we found an ongoing proliferation despite increased alkaline phosphatase and decreased procollagen I gene expression. Our study, therefore, shows that in pHOB the gene expression profile proceeded to the "matrix maturation stage," as defined by Owen and colleagues, independent of ongoing proliferation. We were unable to observe the mineralization period as demonstrated by the missing increase of osteocalcin expression and lack of nodule formation in our human osteoblast model. In contrast to the rat system, we found a proliferation stimulating influence of ascorbate, suggesting species-specific differences in response to differentiation factors. From these data, we conclude that general considerations on physiology and pathophysiology of bone cell differentiation have to be confirmed in the human osteoblastic cell system.

Estrogen increases 1,25-dihydroxyvitamin D receptors expression and bioresponse in the rat duodenal mucosa

Abstract: Menopause and estrogen deficiency are associated with apparent intestinal resistance to vitamin D, which can be reversed by estrogen replacement. The in vivo influence of estrogens on duodenal vitamin D receptor (VDR) was studied in three groups of rats: ovariectomized (OVX), sham-operated, and ovariectomized rats treated daily with estrogen (40 microg/kg BW) for 2 weeks (OVX + E). Estrogen administration to OVX rats resulted in a 2-fold increase in VDR messenger RNA transcripts. 1,25(OH)2D3 was shown to bind specifically to one class of receptors in duodenal mucosal extracts, with a dissociation constant of 0.03 nM. Binding was significantly increased in duodenal extracts from OVX + E rats, compared with OVX rats (735 +/- 81 vs. 295 +/- 26 fmol/mg protein; P < 0.001); a comparable, 1.5- to 2-fold increase in VDR protein expression was observed in Western blot analyzes of the duodenal mucosa. Markers of VDR activity were increased in estrogen-exposed rats: calbindin-9k messenger RNA transcript content was 1.4- to 1.6-fold higher, and alkaline phosphatase activity was 1.4- to 3-fold higher in sham-operated and OVX + E, respectively, compared with OVX. 25(OH)D, 1,25(OH)2D, or PTH levels were not altered by estrogen treatment. Cumulatively, these findings suggest that estrogen up-regulates VDR expression in the duodenal mucosa and concurrently increases the responsiveness to endogenous 1,25(OH)2D. Modulation of intestinal VDR activity by estrogen, and subsequent influence on intestinal calcium absorption, could be one of the major protective mechanisms of estrogen against osteoporosis.

Phosphate Stress in Cultures and Field Populations of the Dinoflagellate Prorocentrum minimum Detected by a Single-Cell Alkaline Phosphatase Assay

Abstract: Alkaline phosphatase activity is a common marker of phosphate stress in many phytoplankton, but it has been difficult to attribute alkaline phosphatase activity to specific organisms or groups of phytoplankton in the field with traditional biochemical procedures. A new alkaline phosphatase substrate, ELF-97 (enzyme-labeled fluorescence), shows promise in this regard. When a phosphate group is cleaved from the ELF-97 reagent, the remaining molecule precipitates near the site of enzyme activity, thus fluorescently tagging cells with alkaline phosphatase activity. We characterized ELF-97 labeling in axenic cultures of a common dinoflagellate, Prorocentrum minimum, in order to understand ELF-97 labeling dynamics when phosphate nutrition varies. Enzyme activity, as detected by ELF-97 labeling, appears to be induced in late-log- or early-stationary-phase cultures if cells are grown in low-phosphate media and is lost when phosphate-stressed cells are refed with phosphate. ELF-97 appears to label an inducible intracellular alkaline phosphatase in P. minimum based on confocal microscopy studies. This may limit the use of this reagent to organisms that lack high levels of constitutive intracellular phosphatases. After laboratory cultures were characterized, ELF-97 was used to assay field populations of P. minimum in Narragansett Bay during two 1-week periods, and 12 to 100% of the P. minimum cells were labeled. The level of cell labeling was reduced by 3 days of incubation with added inorganic phosphate. Our results indicate that ELF-97 is an excellent new tool for monitoring phytoplankton phosphate stress in the environment when the data are supported by appropriate laboratory studies.

Characterization of osteoblastic differentiation of stromal cell line ST2 that is induced by ascorbic acid

Abstract: The stromal cell line ST2, derived from mouse bone marrow, differentiated into osteoblast-like cells in response to ascorbic acid. Ascorbic acid induced alkaline phosphatase (ALPase) activity, the expression of mRNAs for proteins that are markers of osteoblastic differentiation, the deposition of calcium, and the formation of mineralized nodules by ST2 cells. We investigated the mechanism whereby ascorbic acid induced the differentiation of ST2 cells. Inhibitors of the formation of collagen triple helices completely blocked the effects of ascorbic acid on ST2 cells, an indication that matrix formation by type I collagen is essential for the induction of osteoblastic differentiation of ST2 cells by ascorbic acid. We furthermore examined the effects of bone morphogenetic proteins (BMPs) on the differentiation of ST2 cells induced by ascorbic acid. Ascorbic acid had no effect on the expression of mRNAs for BMP-4 and the BMP receptors. However, a soluble form of BMP receptor IA inhibited the induction of ALPase activity by ascorbic acid. These results suggest that ascorbic acid might promote the differentiation of ST2 cells into osteoblast-like cells by inducing the formation of a matrix of type I collagen, with subsequent activation of the signaling pathways that involve BMPs.

A retinoic acid-inducible transgenic marker of sino-atrial development in the mouse heart.

Abstract: To study the specification of inflow structures in the heart we generated transgenic animals harboring the human alkaline phosphatase (HAP) gene driven by the proximal 840 bp of a quail SMyHC3 promoter. In transgenic mice, the SMyHC3-HAP reporter was expressed in posterior heart precursors at 8.25 dpc, in sinus venosa and in the atrium at 8.5 and 9.0 dpc, and in the atria from 10.5 dpc onwards. SMyHC3-HAP transgene expression overlapped synthesis and endogenous response to retinoic acid (RA) in the heart, as determined by antibodies directed against a key RA synthetic enzyme and by staining of RAREhsplacZ transgenic animals. A single pulse of all-trans RA administered to pregnant mice at 7.5, but not after 8.5, dpc induced cardiac dismorphology, ranging from complete absence of outflow tract and ventricles to hearts with reduced ventricles expressing both SMyHC3-HAP and ventricular markers. Blockade of RA synthesis with disulfiram inhibited RA-induced transcription and produced hearts lacking the atrial chamber. This study defines a novel marker for atrial-restricted transcription in the developing mouse heart. It also suggests that atrial-specific gene expression is controlled by localized synthesis of RA, and that exclusion of RA from ventricular precursors is essential for correct specification of the ventricles.

Effects of nicotine on periodontal ligament fibroblasts in vitro

Abstract: Cigarette smoking is associated with increased incidence of periodontal disease and poor response to therapy. In the present study, we examined the effects of nicotine on several functions of periodontal ligament fibroblasts (PDLF): proliferation, attachment, alkaline phosphatase production and chemotaxis. Nicotine concentrations varying from 5 ng/ml to 250 microg/ml were tested. Proliferation of cells was studied by the incorporation of 3H-thymidine, and a dose-dependent inhibition was observed with concentrations > or =100 ng/ml. Similar results were observed when studying the attachment of the cells on plastic surfaces, using a colorimetric method. The inhibition of attachment was even more evident after 6 h incubation of the cells with nicotine. The activity of alkaline phosphatase, as determined with the substrate p-nitrophenyl phosphate, in both conditioned medium (CM) and cellular extract (CE), was also significantly decreased in a concentration-related fashion. Finally, the chemotaxis of PDLE as examined by a modification of the Boyden's blind-well chamber technique, was inhibited in a dose-dependent manner. The degree of inhibition varied from 15% with the lowest concentration of nicotine (50 ng/ml), to almost 90% with the highest (5 microg/ml). The results show that nicotine can have direct adverse effects on various functions of the periodontal cells.

A general method for rapid purification of soluble versions of glycosylphosphatidylinositol-anchored proteins expressed in insect cells: an application for human tissue-nonspecific alkaline phosphatase.

Abstract: A soluble form of tissue-nonspecific alkaline phosphatase was purified to apparent homogeneity from the culture media of Sf9 cells which had been infected with recombinant baculoviruses encoding human tissue-nonspecific alkaline phosphatase (TNSALP). To facilitate purification, an oligonucleotide consisting of 6 tandem codons for histidine and a stop codon was engineered into the TNSALP cDNA. The molecular mass of the enzyme purified through a nickel-chelate column was estimated to be 54 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. That of the native enzyme was 90 kDa as estimated by gel filtration, indicating that the purified soluble TNSALP is dimeric. The enzyme was used for production of antibodies specific for human TNSALP. Immunoblotting analysis showed a single 80-kDa band in the cell homogenate prepared from Saos-2 (human osteosarcoma) cells. However, upon digestion with peptide: N-glycosidase F, the 80-kDa TNSALP of human origin and the soluble enzyme of insect origin migrated to the same position on SDS-polyacrylamide gel, indicating that the size difference between the two enzymes is ascribed to N-linked oligosaccharides. The antibodies prepared against the purified TNSALP were found to be useful also for immunoprecipitation and immunofluorescence studies.

Amyloid precursor protein, although partially detergent-insoluble in mouse cerebral cortex, behaves as an atypical lipid raft protein.

Abstract: Lipid rafts are regions of the plasma membrane that are enriched in cholesterol, glycosphingolipids and acylated proteins, and which have been proposed as sites for the proteolytic processing of the Alzheimer's amyloid precursor protein (APP). Lipid rafts can be isolated on the basis of their insolubility in Triton X-100 at 4 degrees C, with the resulting low-density, detergent-insoluble glycolipid-enriched fraction (DIG) being isolated by flotation through a sucrose density gradient. The detergent-insolubility of APP in mouse cerebral cortex relative to a variety of DIG marker proteins (alkaline phosphatase, flotillin, F3 protein and prion protein) and non-DIG proteins (alkaline phosphodiesterase I, aminopeptidase A and clathrin) has been examined. Alkaline phosphatase, flotillin, F3 protein and the prion protein were present exclusively in the DIG region of the sucrose gradient over a range of protein/detergent ratios used to solubilize the membranes and displayed a characteristic enrichment in the low-density fraction as the protein/detergent ratio was decreased. In contrast, most of the APP, alkaline phosphodiesterase I, aminopeptidase A and clathrin was effectively solubilized at all of the protein/detergent ratios examined. However, a minor proportion of these latter proteins was detected in DIGs at levels which remained constant irrespective of the protein/detergent ratio. When DIGs were isolated from the sucrose gradients and treated with excess Triton X-100, both the DIG marker proteins and APP, alkaline phosphodiesterase I and clathrin were predominantly resistant to detergent extraction at 37 degrees C. These results show that, although a minor proportion of APP is present in DIGs, where it is detergent-insoluble even at 37 degrees C, it behaves as an atypical lipid raft protein and raises questions as to whether lipid rafts are a site for its proteolytic processing.

Isoforms of bone alkaline phosphatase: characterization and origin in human trabecular and cortical bone.

Abstract: Alkaline phosphatase (ALP) is a glycoprotein and functions as an ectoenzyme attached to the cell membrane by a hydrophobic glycosyl-phosphatidylinositol (GPI) anchor. Three bone ALP (BALP) isoforms in human serum were separated and quantitated by high-performance liquid chromatography. B/I, a minor fraction, is composed on average of bone (70%) and intestinal (30%) ALP, and two major isoforms, B1 and B2. Treatment with GPI-specific phospholipase C (GPI-PLC) did not influence the activities or retention times for B1 and B2, indicating that the biochemical differences between B1 and B2 are likely to be due to different glycosylation patterns. The B/I fraction in serum, on average 4% of total ALP, was found to be composed of B1 and B2 isoforms, each with an intact hydrophobic GPI cell membrane anchor. We investigated the origin of these three BALP isoforms and osteocalcin in human femora from five healthy individuals (four males), mean age 51 years, obtained from a tissue bank. Bone was sampled from three sites: cortical bone, trabecular bone from the diaphysis, and trabecular bone from the greater trochanter. Trabecular bone, from both sites, had higher BALP activities compared with cortical bone. Conversely, the osteocalcin content of cortical bone was more than 3-fold greater than that of trabecular bone. Cortical bone had approximately 2-fold higher activity of B1 compared with B2, whereas trabecular bone had ∼2-fold higher activity of B2 compared with B1. We observed a previously undescribed BALP isoform (B1x) in all bone samples. B1x was also observed in sera from some patients (60%) with severe renal insufficiency and on chronic dialysis therapy (n = 20). The isoforms of BALP may provide information relating to bone metabolism within specific bone compartments.

Dexamethasone Enhances In Vitro Vascular Calcification by Promoting Osteoblastic Differentiation of Vascular Smooth Muscle Cells

Abstract: Vascular calcification is often associated with atherosclerotic lesions. Moreover, the process of atherosclerotic calcification has several features similar to the mineralization of skeletal tissue. Therefore, we hypothesized that vascular smooth muscle cells might acquire osteoblastic characteristics during the development of atherosclerotic lesions. In the present study, we investigated the effect of dexamethasone (Dex), which is well known to be a potent stimulator of osteoblastic differentiation in vitro, on vascular calcification by using an in vitro calcification model. We demonstrated that Dex increased bovine vascular smooth muscle cell (BVSMC) calcification in a dose- and time-dependent manner. Dex also enhanced several phenotypic markers of osteoblasts, such as alkaline phosphatase activity, procollagen type I carboxy-terminal peptide production, and cAMP responses to parathyroid hormone in BVSMCs. We also examined the effects of Dex on human osteoblast-like (Saos-2) cells and compared its effects on BVSMCs and Saos-2 cells. The effects of Dex on alkaline phosphatase activity and the cAMP response to parathyroid hormone in BVSMCs were less prominent than those in Saos-2 cells. Interestingly, we detected that Osf2/Cbfa1, a key transcription factor in osteoblastic differentiation, was expressed in both BVSMCs and Saos-2 cells and that Dex increased the gene expression of both transcription factors. These findings suggest that Dex may enhance osteoblastic differentiation of BVSMCs in vitro.

Effects of zinc on human skeletal alkaline phosphatase activity in vitro.

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 micromol/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.

Inhibitory effects of activin-a on osteoblast differentiation during cultures of fetal rat calvarial cells

Abstract: Activin-A is a member of the transforming growth factor-beta (TGF-beta) superfamily and is expressed by osteoblasts. However, the role of activin-A on osteoblasts is not clearly understood. We examined the effects of activin-A on osteoblast proliferation or differentiation, and mineralization by the osteoblasts in the first subcultures of fetal rat osteoblasts obtained from calvarial bones. Exogenous activin-A led to impaired formation of bone nodules in a dose-dependent manner, although it did not influence cell proliferation using an MTT assay. This inhibitory effect depended upon the time at which activin-A was added to the culture media, and the effect was most significant when addition took place at the early phase of the culture. In addition, exogenous activin-A inhibited gene expression of type I procollagen, alkaline phosphatase, osteonectin, and osteopontin in the cultured cells using Northern blot analysis. The peak of osteocalcin mRNA was delayed. Gene expression for TGF-beta was not influenced by exogenous activin-A. The betaA subunit (activin-A) mRNA was detected during the early phase of this culture. These results indicate that activin-A inhibited early differentiation of the fetal rat calvarial cells, or osteoblasts.

Medium perfusion enhances osteogenesis by murine osteosarcoma cells in three-dimensional collagen sponges.

Abstract: In this study, we examined in vitro histogenesis by murine K8 osteosarcoma cells maintained in three-dimensional (3D) collagen sponges. We tested the hypothesis that perfusion of medium enhances cell viability and their biosynthetic activity as assessed by expression of the osteoblastic phenotype and mineral deposition. At intervals, samples were harvested and analyzed histologically, biochemically, and by Northern hybridization for type I collagen, osteopontin (OPN), osteocalcin (OC), and core binding factor alpha 1 (Cbfa1). Histologic evaluation showed greater viability, more alkaline phosphatase (ALP)-positive cells, and more mineralized tissue in the perfused sponges after 21 days. Immunohistological assessment of proliferating cell nuclear antigen revealed 5-fold more proliferating cells in the perfused sponges compared with the controls (p = 0.0201). There was 3-fold more ALP activity in the perfused sponges than the controls at 6 days and 14 days (p = 0.0053). The perfused sponges contained twice the DNA and eight times more calcium than the nonperfused controls after 21 days (p < 0.0001 for both). Northern hybridization analysis revealed more mRNA for collagen type I (2-fold) and 50% more for OC at 14 days and 21 days, whereas OPN and Cbfa1 mRNA expression remained unaffected by the medium perfusion. These results show that medium perfusion had beneficial effects on the proliferation and biosynthetic activity of this osteosarcoma cell line. This system mimics the 3D geometry of bone tissue and has the potential for revealing mechanisms of regulation of osteogenesis.

Inhibition of osteoblastic cell differentiation by lipopolysaccharide extract from Porphyromonas gingivalis.

Abstract: Lipopolysaccharide from Porphyromonas gingivalis (P-LPS), an important pathogenic bacterium, is closely associated with inflammatory destruction of periodontal tissues. P-LPS induces the release of cytokines and local factors from inflammatory cells, stimulates osteoclastic-cell differentiation, and causes alveolar bone resorption. However, the effect of P-LPS on osteoblastic-cell differentiation remains unclear. In this study, we investigated the effect of P-LPS extract prepared by the hot-phenol-water method, on the differentiation of primary fetal rat calvaria (RC) cells, which contain a subpopulation of osteoprogenitor cells, into osteoblastic cells. P-LPS extract significantly inhibited bone nodule (BN) formation and the activity of alkaline phosphatase (ALPase), an osteoblastic marker, in a dose-dependent manner (0 to 100 ng of P-LPS extract per ml). P-LPS extract (100 ng/ml) significantly decreased BN formation to 27% of the control value and inhibited ALPase activity to approximately 60% of the control level on days 10 to 21 but did not affect RC cell proliferation and viability. P-LPS extract time-dependently suppressed the expression of ALPase mRNA, with an inhibitory pattern similar to that of enzyme activity. The expression of mRNAs for osteocalcin and osteopontin, matrix proteins related to bone metabolism, was markedly suppressed by P-LPS extract. Furthermore, P-LPS extract increased the expression of mRNAs for CD14, LPS receptor, and interleukin-1beta in RC cells. These results indicate that P-LPS inhibits osteoblastic-cell differentiation and suggest that LPS-induced bone resorption in periodontal disease may be mediated by effects on osteoblastic as well as osteoclastic cells.