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

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Omeprazole is an inhibitor of gastric H+,K(+)-ATPase. Although the major proton transport of osteoclast is mediated by a vacuolar-type H(+)-ATPase which is different from the gastric H+,K(+)-ATPase, in vitro studies have demonstrated that omeprazole inhibits bone resorption. In this study, the effect of omeprazole on bone resorption was evaluated in patients who had a history of gastric ulcer and were treated with maintenance doses of H2 blocker without any gastric complaints at the study time. H2-blocker administration was changed to omeprazole treatment in the study group and to no treatment in the control group. Urinary excretion of hydroxyproline and calcium decreased after omeprazole treatment in the study group. Serum intact PTH, alkaline phosphatase, osteocalcin, and tartrate-resistant acid phosphatase (TRAP) increased in this group. In the control group, there were not any changes in these parameters. The discrepancy between serum TRAP and urinary excretion of hydroxyproline and calcium in the study group was thought to be due to the suppression of bone resorption by omeprazole, which probably interfered the acidification at resorption lacunae and resulted in the inactivation of TRAP and other lysosomal enzymes. The results of our study suggest the possibility that the specific inhibitors of the osteoclastic proton pump (such as bafilomycins) will more effectively suppress bone resorption and be useful for the treatment of metabolic bone diseases with increased bone resorption.
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PMID:Effect of omeprazole, an inhibitor of H+,K(+)-ATPase, on bone resorption in humans. 810 18

The present studies evaluated the feasibility of establishing a conditionally immortalized osteoprecursor cell line derived from human fetal bone tissue. Primary cultures were transfected with a plasmid in which the Mx-1 promoter drives the expression of SV40 T-antigen when activated by human A/D interferon. Several neomycin (G418)-resistant colonies were characterized for cell growth and alkaline phosphatase (ALP) enzyme activity. The clone, designated OPC1 (osteoblastic precursor cell line 1), which exhibited the highest ALP enzyme activity at passage 10 (P10), was selected for additional osteogenic phenotypic characterization. Reverse transcription-polymerase chain reaction (RT-PCR) phenotyping revealed abundant mRNA for osteocalcin (OC), osteonectin (ON), osteopontin (OP), parathyroid hormone receptor (PTHr), ALP, and procollagen type I (ProI). In addition, the levels of quantitative RT-PCR product of ON, OP, PTHr, and ProI mRNAs exhibited a marked up-regulation when maintained in medium containing an osteogenic supplement (OS). The ability to stimulate osteogenic differentiation was characterized in postconfluent OPC1 cells maintained in tissue culture medium supplemented with recombinant human bone morphogenetic protein-2 (rhBMP-2) either with or without an OS. All treatment groups exhibited a striking up-regulation of ALP enzyme activity that coincided with ALP histochemical observations. Postconfluent cells also exhibited the ability to form mineralized nodules under all treatments (confirmed by von Kossa histochemical staining and calcium deposition). An enzyme immunosorbent assay (EIA) was utilized to measure intact human OC from the OPC1 line under the various treatments. Abundant OC was evident in the tissue culture medium indicating de novo sythesis and release from the OPC1 line under appropriate conditions. The clonal human-derived OPC1 line represents a homogeneous osteogenic cell line that not only has maintained a consistent bone phenotype from P10 to at least P30, but has also exhibited the capacity to generate programmed differentiation in the presence of low dose rhBMP-2 (10 ng/ml). Thus, the OPC1 line is a human-derived osteoprecursor that provides a sensitive in vitro cell culture system to evaluate bone development, cell/biomaterial interactions, and may be a useful screen for putative bone differentiating factors.
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PMID:Establishing an immortalized human osteoprecursor cell line: OPC1. 1049 Dec 20

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

Previously recognized intracellular proteins with an affinity for vitamin D metabolites include the vitamin D receptor and the cytochrome P-450-based vitamin D metabolizing mixed-function oxidases. We recently characterized a third set of high-capacity, intracellular vitamin D binding proteins (IDBPs) in the inducible heat shock protein-70 (hsp-70) family. Here we report the cloning and expression of cDNAs coding for two IDBPs. The full-length cDNAs for IDBP-1 and IDBP-2 demonstrated 95% and 94% nucleotide homology, respectively, with the cDNAs for human constitutively expressed heat shock protein 70 (hsc-70) and hsp-70. Transient expression of the IDBP cDNAs in a vitamin D-responsive primate cell line increased extractable 25-hydroxylated vitamin D metabolite-IDBP-binding 25-fold. Transfection experiments also demonstrated that the majority of the constitutively expressed 25-hydroxylated vitamin D metabolite binding activity was attributable to expression of the hsc-70-related IDBP-1 and that metabolite binding activity sublocalized to the highly conserved ATP-binding/ATPase domain of hsp-70s. Stable overexpression of IDBP-1 in wild-type cells enhanced vitamin D-directed responsiveness of endogenous vitamin D-24-hydroxylase, osteopontin, and osteocalcin genes by several-fold over that observed in cells transfected with an empty vector. These results suggest that IDBP-1 facilitates the intracellular localization of active vitamin D metabolites and vitamin D receptor-mediated transactivation.
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PMID:Intracellular vitamin D binding proteins: novel facilitators of vitamin D-directed transactivation. 1097 17

Parathyroid hormone-related protein (PTHrP) has been implicated in regulating tooth eruption and/or development. Formation of cementum, a mineralized tissue covering the tooth root surface, is a critical biological event for tooth root development. To test the hypothesis that PTHrP targets cementoblasts (CMs) and acts to regulate cementogenesis, CM cell lines were established and their responsiveness to PTHrP stimulation was determined, in vitro. First, subclones were derived from two immortalized murine cell populations that contained CMs; SV-CM/periodontal ligament (PDL) cells were obtained from the root surface of first mandibular molars of CD-1 mice and immortalized with SV40 T-antigen (TAg), and OC-CM cell population was established from OC-TAg transgenic mice in which their cells harbor an osteocalcin (OC and/or OCN) promoter-driving immortal gene SV40 TAg. Based on our previous in situ studies, CM subclones were identified as cells expressing bone sialoprotein (BSP) and OCN transcripts, while PDL cell lines were designated as cells lacking BSP and OCN messenger RNA (mRNA). CMs exhibited a cuboidal appearance and promoted biomineralization, both in vitro and in vivo. In contrast, PDL cells (PDL subclones) displayed a spindle-shaped morphology and lacked the ability to promote mineralized nodule formation, both in vitro and in vivo. Next, using these subclones, the effect of PTHrP on cementogenesis was studied. CMs, not PDL cells, expressed PTH/PTHrP receptor mRNA and exhibited PTHrP-mediated elevation in cyclic adenosine monophosphate (cAMP) levels and c-fos gene induction. PTHrP stimulation repressed mRNA expression of BSP and OCN in CMs and blocked CM-mediated mineralization, in vitro. Collectively, these data suggest that CMs possess PTH/PTHrP receptors and, thus, are direct targets for PTHrP action during cementogenesis and that PTHrP may serve as an important regulator of cementogenesis.
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PMID:Parathyroid hormone-related protein regulates extracellular matrix gene expression in cementoblasts and inhibits cementoblast-mediated mineralization in vitro. 1109 95

Altered divalent cation homeostasis with bone mineral loss, hypercalciuria, and hypomagnesemia have been associated consistently with human diabetes mellitus. This study investigated functional, molecular, and biochemical determinants that accompany this condition in chronically (2 wk) streptozotocin (STZ)-diabetic rats. Catheterized, conscious, diabetic rats on servo-controlled fluid replacement exhibited an increased GFR (+70%) and a substantially raised urinary calcium output (+568%) when compared with control rats. In addition, fractional calcium reabsorption was reduced, indicating that the hypercalciuria was not due solely to an osmotic effect but may involve an actual tubular defect. The expression of proteins involved in renal distal Ca2+ and water transport in STZ-diabetic rats were then studied by Western analysis and immunofluorescence microscopy to investigate the molecular basis of the hypercalciuria. Extracellular Ca2+-sensing receptor abundance was reduced to 52% of control in STZ-diabetes, whereas thiazide-sensitive NaCl cotransporter expression was increased by 192%. Subcutaneous insulin implant rectified both functional and molecular parameters. The levels of calbindin D(28k), plasma membrane Ca2+ ATPase, and aquaporin 1 in whole kidney and of aquaporin 2 in inner medulla were unchanged in diabetic and/or insulin replacement. Blood levels of 1,25(OH)(2)D(3) were reduced in diabetes as were levels of osteocalcin, a marker of bone formation. It is concluded that diabetic hypercalciuria in rats involves elevated GFR with raised urinary output, reduced Ca2+ reabsorption, and impaired bone deposition. Changes in Ca2+-sensing receptor and NaCl cotransporter protein expression could account for the altered divalent cation homeostasis seen during diabetes mellitus.
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PMID:Functional, molecular, and biochemical characterization of streptozotocin-induced diabetes. 1127 39

Osteoblasts are derived from mesenchymal/stromal cells in bone marrow, and gain the ability to support osteoclastogenesis during differentiation though the expression of receptor activator of NF-kappaB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF). However, the properties (differentiation stage and expression of osteoblast marker genes) of stromal or osteoblastic cells that have the capacity to support osteoclast differentiation are unclear. Therefore, we sought to establish and characterize bone marrow-derived stromal cell lines (TSB) from temperature-sensitive SV40 T-antigen transgenic mice to define them at the clonal level. Of the 24 randomly selected cell lines, only 2 cell lines, TSB13 and TSB20, could support osteoclast differentiation in the presence of 1alpha,25(OH)(2)D(3). In both cell lines, RANKL mRNA was induced and osteoprotegerin (OPG) mRNA was decreased in response to treatment with 1alpha,25(OH)(2)D(3) for 2 days. Other RNA expression analyses of osteoblast-specific marker genes demonstrated the following characteristics of TSB13 and TSB20: (1) alkaline phosphatase (ALP) and type I collagen genes are expressed; (2) osteocalcin and osteopontin genes are expressed at low levels, and their expression levels are upregulated after induction of differentiation by a temperature shift from 33 degrees C to 37 degrees C, or 1alpha,25(OH)(2)D(3) treatment. Consequently, the long-term culture of TSB13 and TSB20 cell lines strongly stimulated osteocalcin expression and effectively induced calcified nodule formation in the presence of phosphate. The results suggest that the supportive cells for osteoclastogenesis are restricted to a specialized population of bone marrow stromal cells, and the high ratio of RANKL vs. OPG expression found in this population after 1alpha,25(OH)(2)D(3) treatment might be a general property of osteoclast-supporting cells.
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PMID:Identification and characterization of mouse bone marrow stromal cell lines immortalized by temperature-sensitive SV40 T antigen: supportive activity for osteoclast differentiation. 1155 67

Human cementifying fibroma (HCF) is a benign fibro-osseous neoplasm of periodontal ligament (PDL) origin containing varying amounts of mineralized material resembling cementum. In the present study, we established cell lines from HCF, which were detected in the mandible of a 54-year-old Japanese man. To obtain immortalized cell clones, we undertook transfection with temperature-sensitive simian virus-40 (SV40) T-antigen and hTERT into HCF cells. Cells transfected with SV40 T-antigen entered "crisis" state between passages 22 and 35, but activation of telomerase by transfection with hTERT in the SV40-transformed HCF cells resulted in bypass of the crisis and maintenance over passage 200. HCF cell lines decreased the expression of SV40 T-antigen and the activity of cell proliferation at a nonpermissive temperature (39 degrees C) in comparison with that at a permissive temperature (33 degrees C). High activities of alkaline phosphatase and mineralization and the expression of type I collagen, osteocalcin, osteopontin, and bone sialoprotein by reverse transcription-polymerase chain reaction (RT-PCR) were observed in HCF cells at 39 degrees C. Overall, these findings suggest that: (i) HCF cell lines may represent a novel in vitro human cell model for the study of the regulatory mechanism of differentiation and proliferation of the human PDL; and (ii) transfection of plasmids encoding the temperature-sensitive SV40 T-antigen gene and hTERT gene may be useful for obtaining immortalized cell lines from benign human tumor and, probably, nonneoplastic human tissues.
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PMID:Establishment of human cementifying fibroma cell lines by transfection with temperature-sensitive simian virus-40 T-antigen gene and hTERT gene. 1199 9

Defining the regulatory mechanisms promoting differentiation and proliferation of cementoblasts has not been well understood, because of the lack of cell models in vitro. To establish an in vitro cell model for the cementoblasts, extracted rat molars obtained from 8-week-old rats were used. Cells lining the root surface (cemetoblasts) were obtained by an enzymatic digestion method, and immediately immortalized by transfection of thermolabile SV40 T-antigen gene. The transfected cementum lining cell clones, RCM-C3 and -C4, were maintained for more than 200 population doublings (PD), while the original cells stopped their growth at 60 PD. Thus, immortalized cell lines decreased expression of SV40 T-antigen and subsequently cell proliferation at non-permissive temperature (39 degrees C). Reverse-transcribed-polymerase chain reaction indicated expression of gene for type I collagen, alkaline phosphatase (ALP), osteopontin, and osteocalcin mRNA at both permissive (33 degrees C) and non-permissive (39 degrees C) temperatures. RCM-C4 expressed higher bone siaploprotein (BSP) mRNA than RCM-C3, and further RCM-C4 showed higher BSP mRNA at 39 degrees C than 33 degrees C. High ALP activity and mineralized nodule formation were observed at 39 degrees C in both cell lines. These findings suggested that the cell lines, RCM-C3 and -C4, are useful model for studying the regulatory mechanisms of differentiation and proliferation of cementoblasts.
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PMID:Establishment of cementoblast cell lines from rat cementum lining cells by transfection with temperature-sensitive simian virus-40 T-antigen gene. 1598 73


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