Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human osteoblast-like cells were examined for the presence of the Ca2+-Mg2+ ATPase pump. The osteoblast-like cells had characteristic features of the osteoblast phenotype, including the presence of osteonectin, bone GLA protein, and type I collagen. The cells were able to mineralize matrix, their production of cAMP increased in response to PTH, and their alkaline phosphatase activity increased in response to 1,25-dihydroxyvitamin D3. Immunocytochemical staining of the osteoblast-like cells with a monoclonal antibody against human red cell Ca2+-Mg2+ ATPase demonstrated the presence of an epitope of the Ca2+-Mg2+ ATPase in these cells; staining of paraffin-embedded osteoblast-like cell sections demonstrated anti-Ca2+-Mg2+ ATPase staining only in cell plasma membranes. Western blot analysis of osteoblast-like cell homogenates showed that the monoclonal antibody to human erythrocyte Ca2+-Mg2+ ATPase bound to a major band at 140,000 mol wt, similar to the mol wt of known plasma membrane Ca2+-Mg2+ ATPases. The presence in the osteoblast-like cells of a Ca2+-Mg2+ ATPase similar to the human red cell calcium pump suggests that this enzyme may play a role in osteoblast intracellular calcium homeostasis.
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PMID:Epitopes of the human erythrocyte Ca2+-Mg2+ ATPase pump in human osteoblast-like cell plasma membranes. 246 88

In the embryo, epithelia give rise to mesenchyme at specific times and places. Recently, it has been reported (Greenburg, G., and E. D. Hay. 1986. Dev. Biol. 115:363-379; Greenberg, G., and E. D. Hay. 1988. Development (Camb.). 102:605-622) that definitive epithelia can give rise to fibroblast-like cells when suspended within type I collagen gels. We wanted to know whether Madin-Darby canine kidney (MDCK) cells, an epithelial line, can form mesenchyme under similar conditions. Small explants of MDCK cells on basement membrane were suspended within or placed on top of extracellular matrix gels. MDCK cells on basement membrane gel are tall, columnar in shape, and ultrastructurally resemble epithelia transporting fluid and ions. MDCK explants cultured on type I collagen gel give rise to isolated fusiform-shaped cells that migrate over the gel surface. The fusiform cells extend pseudopodia and filopodia, lose cell membrane specializations, and develop an actin cortex around the entire cell. Unlike true mesenchymal cells, which express vimentin and type I collagen, fusiform cells produce both keratin and vimentin, continue to express laminin, and do not turn on type I collagen. Fusiform cells are not apically-basally polarized, but show mesenchymal cell polarity. Influenza hemagglutinin and virus budding localize to the front end or entire cell surface. Na,K-ATPase occurs intracellularly and also symmetrically distributes on the cell surface. Fodrin becomes diffusely distributed along the plasma membrane, ZO-1 cannot be detected, and desmoplakins distribute randomly in the cytoplasm. The loss of epithelial polarity and acquisition of mesenchymal cell polarity and shape by fusiform MDCK cells on type I collagen gel was previously unsuspected. The phenomenon may offer new opportunities for studying cytoplasmic and nuclear mechanisms regulating cell shape and polarity.
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PMID:Type I collagen gel induces Madin-Darby canine kidney cells to become fusiform in shape and lose apical-basal polarity. 253 38

Arterial basement-membrane-like material was isolated from rabbit aortic myomedial cell cultures by sonication and differential centrifugation. Isolated basement-membrane-like material was shown to be free of both cellular and matrix contaminants, on the basis of determinations of DNA, RNA, cholesterol, phosphorus and (Na+ + K+)-activated ATPase, combined with electron microscopy. Amino acid analyses showed that arterial basement-membrane-like material was composed of predominantly non-collagenous amino acids. Evaluated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, reduced basement-membrane-like material comprised six major and about 30 minor components in the Mr range 10 000-600 000. One of the major peptides (Mr 225 000) was disulphide-linked. Periodic acid-Schiff staining of gels indicated that most high-molecular-weight components were glycoproteins. Two-dimensional gel electrophoresis resolved reduced basement-membrane-like material into more than 100 components, with pI from 5 to 7. The disulphide-linked Mr-225 000 peptide appeared heterogeneous, with pI of 5.6-6.0, and was considered to represent fibronectin. All major peptides were of non-collagenous nature, on the basis of their susceptibility to pepsin and resistance to collagenase. Purified myomedial basement-membrane-like material contained collagenous peptides, as indicated by the presence of hydroxyproline and hydroxylysine. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of pepsin-treated and reduced basement-membrane-like material revealed five high-molecular-weight collagenous components appearing in the Mr range 105 000-375 000 relative to type I collagen standards.
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PMID:Arterial basement-membrane-like material isolated and characterized from rabbit aortic myomedial cells in culture. 687 Aug 38

The toothless (tl) osteopetrotic mutation in the rat is characterized by generalized skeletal sclerosis, a severe reduction in the numbers of osteoclasts, monocytes, and macrophages, and absence of tooth eruption. Studies examining gene expression in bone-derived cells of tl rats and their normal littermates have shown that genes related to osteoblast function are aberrantly expressed in tl rats compared to normal littemates. We have previously shown that exogenous administration of colony stimulating factor-1 (CSF-1) to tl rats results in a dramatic reduction of the skeletal sclerosis and significant increases in the number of osteoclasts. Thus, we examined the effects of CSF-1 on osteoblast and osteoclast gene expression in tl rats as demonstrated by Northern blot analysis. While osteoblast-related gene expression as reflected by mRNA levels of alkaline phosphatase, osteocalcin, osteopontin, and type I collagen was normalized, osteoclast-related gene expression, as reflected by mRNA levels of carbonic anhydrase II and tartrate-resistant adenosine triphosphatase, remained significantly lower in CSF-1-treated tl rats compared to untreated normal littermates. Since previous studies have not demonstrated the CSF-1 receptor on osteoblasts, these results suggest that osteoblast abnormalities in tl rats are an effect of the osteopetrotic condition rather than the cause of the disease.
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PMID:Administration of colony stimulating factor-1 to toothless osteopetrotic rats normalizes osteoblast, but not osteoclast, gene expression. 766 37

Several extracellular matrix (ECM) configurations involving type I collagen and Matrigel were examined for their ability to support differentiated function and polarity of cultured adult rat hepatocytes. Collagen sandwich- and Matrigel-based cultures yielded superior and comparable albumin secretion for at least 2 weeks. In collagen sandwich, hepatocytes were polygonal, and formed multicellular arrays. Collagen sandwich was also found to promote in vivo-like polarization of F-actin, cell adhesion molecules (E-cadherin), and lateral (Na+, K(+)-ATPase, glucose transporter) and apical (dipeptidyl peptidase, aminopeptidase) membrane polarity markers, but not the expression of the gap junction protein connexin 32 and the epidermal growth factor (EGF) receptor. In contrast, hepatocytes cultured in or on Matrigel were more rounded and formed aggregates. Matrigel-based cultures also elicited detectable levels of connexin and EGF receptor and an altered distribution of F-actin, E-cadherin, and apical and lateral membrane proteins. Composite sandwich configurations containing collagen I and Matrigel restored markers lacking in the collagen sandwich, and showed a variable morphology and membrane polarity. Hepatocyte polarity could thus be manipulated by the overall ECM composition. Furthermore, in composite sandwich cultures, these manipulations can be effected largely independent of changes in hepatocyte morphology and albumin secretion.
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PMID:Culture matrix configuration and composition in the maintenance of hepatocyte polarity and function. 874 35

To examine the effect of extracellular matrix on osteoclast polarization, we focused on the actin organization in osteoclasts, using murine osteoclast-like multinucleated cells (OCLs) formed in cocultures of osteoblastic cells and bone marrow cells. When OCLs were cultured on either a plastic plate, calcified dentine, or calcium phosphate thin films in the presence of fetal bovine serum (FBS), they similarly formed ringed structures of F-actin dots (actin rings). However, OCLs placed on demineralized dentine or type I collagen gel matrix (collagen gel) failed to form actin rings. In the absence of FBS, actin ring formation in OCLs was induced on plastic plates coated with vitronectin, fibronectin, or type I collagen, but not on those coated with laminin, poly-L-lysine, or bovine serum albumin. Actin ring formation appeared to depend on integrins, since the GRGDS, but not the GRGES, peptide inhibited it in a dose-dependent manner. Moreover, immunoelectron microscopic examination revealed that vacuolar proton ATPase (V-ATPase) was localized along the apical membrane in much higher densities than the basolateral membrane in OCLs placed on plastic coverslips. In OCLs placed on collagen gel, however, V-ATPase was found to be distributed throughout the cytoplasm without polarity. These results suggest that actin ring formation in osteoclasts was dependent on matrix substrates, matrix proteins and integrins, and was closely related to osteoclast function.
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PMID:Chemical and physical properties of the extracellular matrix are required for the actin ring formation in osteoclasts. 897 Aug 88

The mouse submandibular gland (SMG) is an excellent model for the study of many important biological phenomena such as hormonal regulation of differentiation, neurotransmitter control of secretion, epithelial transport, exocytosis and endocytosis as well as the regulation of mouse SMG specific gene expression, in particular, NGF, EGF and renin. The postnatal development and sexual dimorphism of the mouse gland permits the isolation of male SMGs of different ages, corresponding to different stages of differentiation, particularly with respect to the cytodifferentiation of ductal cell types. We have immortalized SMG epithelial cell lines using mice transgenic for the large T antigen of SV40 or polyoma viruses. Epithelial clusters from the dissected glands were placed in culture and cell lines were established from the immortalized population. Two cell lines, SIMS and SIMP, which retain structural and functional characteristics, are described here. The cell lines are immortalised but not transformed, as judged by the absence of anchorage independent growth potential and the lack of tumour formation in athymic nude mice. Confocal and electron microscopy examination demonstrate that SIMP and SIMS cells express E-cadherin and ZO-1 and have features of polarised epithelial cells. In addition, they form spherical cysts with a wide lumen when grown in type I collagen gels. When grown on a filter support SIMS cells form a tight monolayer, exhibit vectorial transport function and show exclusive Na+, K(+)-ATPase localisation to the basolateral domain. We determined the cell type restricted expression of cytokeratin markers in the mouse SMG in vivo and we demonstrate that SIMS and SIMP cell lines express duct-specific cytokeratins. Finally, the expression of a set of differentiation markers, including EGF, NGF and renin, was detected by RT-PCR and by indirect immunofluorescence staining in these lines. Thus, these polarised ductal cell lines, as well as having important intrinsic properties, represent well characterised mouse epithelial models which, until now, have not been readily available for cellular studies.
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PMID:Immortalised mouse submandibular epithelial cell lines retain polarised structural and functional properties. 901 27

Studies in several cell types indicate that the actions of integrin receptors for extracellular matrix and receptors for growth factors are synergistic in regulating cellular differentiation and function. We studied the roles of the alpha1beta1 and alpha2beta1 integrin collagen receptors in regulating the differentiation of 2T3 osteoblastic cells in response to bone morphogenetic protein (BMP)-2. The immortalized 2T3 cell line was established from the calvaria of mice transgenic for a BMP-2 promoter driving SV40 T-antigen. These cells require exogenous BMP-2, as well as ascorbic acid and beta-glycerolphosphate, for expression of a mature osteoblast phenotype and formation of a mineralized matrix. To determine how integrin receptors for collagen-I affect BMP-2 signaling, function-perturbing anti-rat alpha1 and/or alpha2 integrin subunit, or anti-type I collagen (Col-I), antibodies were added to human recombinant (hr)BMP-2-treated 2T3 cultures at confluence (C0) or at 4 or 8 days postconfluence (C4, C8). After 4 days of exposure to the antibodies, cultures were assayed for alkaline phosphatase (ALP) mRNA levels and enzyme activity and for cAMP production in response to parathyroid hormone. Addition of anti-collagen-I or both anti-integrin-alpha1 and -alpha2 antibodies to C0 cultures blocked expression of these early osteoblast markers by more than 90%, and also blocked mineralization (0.5-1.8% control) of these cells. In all cases, adding anti-alpha1 or anti-alpha2 antibodies separately produced partial effects, while their combined effect approached that of anti-collagen-I. When antibodies were added to more differentiated 2T3 cells, the inhibitory effects decreased. 2T3 cells carrying constitutively active BMP receptor (caBMPR-IB) showed elevated ALP activity without hrBMP-2; this constitutive activity was also suppressed by alpha1 and alpha2 integrin antibodies and by anti-Col-I antibody. Together, our data suggest that a signal(s) from collagen integrin receptors regulates the response to BMP downstream of BMPR-IB and upstream of the regulation of ALP mRNA and other early markers of osteoblast differentiation.
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PMID:Collagen integrin receptors regulate early osteoblast differentiation induced by BMP-2. 1040 7

A human in vitro resorption assay has been developed using osteoclastoma-derived osteoclasts and used to evaluate novel antiresorptive agents including antagonists of the alphavbeta3 integrin, and inhibitors of cathepsin K and the osteoclast ATPase. The potency of novel compounds in the in vitro resorption assay correlates with functional assays for each class of inhibitor: the human alphavbeta3-mediated cell adhesion assay for the vitronectin receptor antagonists (r2 = 0.82), the chick osteoclast vacuolar ATPase enzyme assay for the H+-ATPase inhibitors (r2 = 0.77) and the recombinant human cathepsin K enzyme assay for the cathepsin K inhibitors (r2 = 0.80). Cell suspensions, rich in osteoclasts, are prepared by collagenase digestion of the tumor tissue. These cells can be stored long-term in liquid nitrogen and upon thawing maintain their bone-resorbing phenotype. The cryopreserved cells can be cultured on bovine cortical bone for 24-48 h and resorption can be measured by either confocal microscopy or biochemical assays. The resorptive activity of osteoclasts derived from a number of tumors can be inhibited reproducibly using a number of mechanistically unique antiresorptive compounds. In addition, the measurement of resorption pits by laser confocal microscopy correlates with the release of type I collagen C-telopeptides or N-telopeptides, as measured by enzyme-linked immunosorbent assay. Resorption can be measured reproducibly using a 48-h incubation of osteoclasts on bone slices, or a 24-h incubation with bone particles. This in vitro human osteoclast resorption assay provides a robust system for the evaluation of inhibitors of osteoclastic function that may be developed for the treatment of metabolic bone diseases such as osteoporosis.
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PMID:Development and characterization of a human in vitro resorption assay: demonstration of utility using novel antiresorptive agents. 1046 85

To clarify the mechanisms by which core-binding factor-alpha1 (Cbfa1), an essential transcription factor in osteogenesis, functions in osteoblast matrix formation, as well as in chondrocyte differentiation and osteoclastic bone resorption, Cbfa1-deficient embryonic mice were investigated ultrastructurally and histocytochemically at 18.5 days postcoitum. In homozygotic mice, both endochondral and intramembranous ossification were arrested, although bone tissue had already formed at this stage in the wild type. The tibiae of homozygotic mice were characterized by calcified cartilage and alkaline phosphatase (ALP)-positive perichondrium, whereas membranous structures indicating the presence of ALP activity in the lateral portion were observed in the calvariae, rather than the bone tissue. Most of the ALP-positive perichondrial cells in homozygotic tibiae possessed a spindle-shaped cell contour and small cytoplasm, the extracellular matrix of which contained neither type I collagen nor calcifying matrix vesicles. In contrast, some perichondrial cells at the very middle part of tibiae became flattened. In the vicinity of these cells, a thin layer of type I collagen-based calcified matrix, containing osteopontin, bone sialoprotein, or osteocalcin, was observed. In the cartilage of mutant mice, we observed a hypoplasic zone of proliferative chondrocytes, the flattening of hypertrophic chondrocyte-like cells, and calcified chondrocytes which, while not degraded, did display a high level of cell function. Mononuclear osteoclastic cells were found in the perichondrium, near calcified chondrocytes, in mutant mice. Multinuclear osteoclasts possessing H+-ATPase and ruffled borders were also present, although only in limited numbers. Neither the development of ruffled borders nor intracellular polarization was complete. Because the majority of osteogenic cells in Cbfa1-deficient mice can neither form nor calcify the bone matrix, Cbfa1 principally plays essential roles in osteoblastic differentiation and bone matrix formation. Cbfa1 also affects both the proliferation and the differentiation of chondrocytes, whereas its absence prevents normal osteoclast formation and related functions.
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PMID:Morphological characterization of skeletal cells in Cbfa1-deficient mice. 1059 8


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