Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cells derived from embryonic rat calvariae were immortalized by retroviral delivery of cDNA for the SV-40 large T antigen and the bacterial neomycin resistance gene. After selection with G418, cells were cloned by limiting dilution and screened for expression of osteoblast characteristics. One clone (RCT-3), derived from cells collected during the third period of enzymatic digestion, showed high constitutive expression of alkaline phosphatase (ALP), synthesized type I collagen in the virtual absence of type III and exhibited a parathyroid hormone (PTH)-responsive adenylate cyclase (EC50, 10 nM). Messenger RNAs for osteonectin and osteopontin were present in RCT-3 cells and osteopontin mRNA was enhanced by 1,25 (OH)2 vitamin D3 treatment. The other cell line (RCT-1), derived from cells released during the first 10 min of digestion, expressed osteoblast features only after 3 d treatment with 1 microM retinoic acid (RA). ALP activity increased from 0.003 to 0.25 mumole/min/mg protein, there was a substantial increase in the steady-state level of type I collagen mRNA and a dose-dependent and saturable response to PTH was induced (EC50, 10 nM). Osteopontin mRNA was induced by 1,25 (OH)2D3. This study has provided two new cell lines which may be useful models for studies of differentiation-related gene expression in bone cells.
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PMID:SV-40 large-T immortalization of embryonic bone cells: establishment of osteoblastic clonal cell lines. 261 49

The relationship of proliferation to the developmental sequence associated with bone cell differentiation was examined in primary osteoblast cultures derived from fetal rat and embryonic chick calvaria. A reciprocal and functional relationship exists between the decline in proliferative activity which occurs during the initial stages of the developmental sequence and the induction of genes encoding osteoblast phenotype proteins associated with matrix maturation and mineralization. This relationship is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phosphatase (AP) and osteopontin (OP) genes immediately following the proliferative period and expression of osteocalcin with the onset of mineralization, and 2) increases in AP and OP when DNA synthesis is inhibited. By determining cellular mRNA levels and rates of mRNA synthesis in isolated nuclei, we found that the down-regulation of cell growth-related genes is modified at both the levels of transcription and mRNA stability. For a histone gene where down-regulation is transcriptionally mediated, we have observed that the shutdown of osteoblast proliferation is associated with the selective loss of the interaction of a promoter binding factor (HiNF-D) with a proximal regulatory element (Site II). A relationship between Site II occupancy by HiNF-D and the onset of osteoblast differentiation is supported by the persistence of Site II-HiNF-D interactions when proliferating rat osteoblasts are growth arrested under conditions that do not induce differentiation; and additionally, by the loss of Site II-HiNF-D interactions during the shut-down of proliferation when HL60 promyelocytic leukemia cells are induced to differentiate into monocytes. Our results are consistent with a requirement of proliferation for expression of genes involved with production, deposition and possibly organization of the osteoblast extracellular matrix. It is also reasonable to postulate that properties of the mineralizing matrix are related to the shut-down of proliferation.
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PMID:The onset and progression of osteoblast differentiation is functionally related to cellular proliferation. 261 61

The non-collagen proteins of bone are a complex set of molecules that arise from local or exogenous sources. Because bone mineral is an excellent adsorbent, many circulatory and/or cell surface proteins bind to bone, where they may have immediate or subsequent effects. These include the alpha 2-HS-glycoprotein from blood and the potent growth factors TGF-beta, PDGF, IGF-1, FGF-a and -b, and IL-1, derived from both bone and non-bone cells. Furthermore, bone cell membrane proteins such as alkaline phosphatase may be cleaved from the cell surface and entrapped in the bone matrix. Bone is enriched in a variety of enzymes and their inhibitors by similar adsorption processes. Even osteocalcin, a bone cell product, is adsorbed to bone via mineral-binding (Gla) groups. The bone sialoproteins (BSP-I or osteopontin and BSP-II) also bind to the mineral via acidic groups. Because of this phenomenon it is difficult to distinguish whether a given protein's presence in bone is advantageous or merely fortuitous. The bone matrix proper consists of type I collagen and other osteoblast products such as osteonectin (a phosphorylated glycoprotein) and small proteoglycans (PG-I and/or PG-II) which are incorporated into bone collagen fibrils. These proteins may have additional roles in tissue morphogenesis and/or differentiation.
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PMID:Non-collagen proteins in bone. 306 9

New aspects of the distribution and developmental appearance of the 44-kDa bone phosphoprotein (44K BPP, also called sialoprotein I or osteopontin) and bone gamma-carboxyglutamic acid (Gla)-containing protein (BGP, also called osteocalcin) during osteogenesis and dentinogenesis were investigated with immunocytochemical techniques using monospecific, affinity-purified polyclonal antibodies. Sections from newborn rat incisors and from various bone anlagen of newborn animals and fetuses were processed for detection of 44K BPP or BGP antigenicity. In addition, histochemical reactions for detection of alkaline phosphatase or calcium salts were performed on a number of the sections. The 44K BPP appears to be synthesized and secreted by chondrocytes only in the areas of cartilage-to-bone transition; these cells could participate indirectly in the process of bone formation by providing a suitable scaffold onto which primary marrow osteoblasts attach and spread. During osteogenesis, 44K BPP is found in bone-forming cells almost concomitantly with the appearance of alkaline phosphatase and before osteoid deposition, whereas BGP is still absent during early stages of mineralization. We hypothesize that this dramatic difference between the developmental appearance of 44K BPP and BGP reflects the delayed expression of the BGP gene relative to that of 44K BPP. In long-term cultures of bone marrow from adult mice, some fibroblastic cells expressed the 44K BPP phenotype; these cells could represent early osteogenic progenitor cells. Some experiments also suggested that, as with BGP, 44K BPP or an immunologically related protein is synthesized by some odontoblasts and secreted into predentin, prior to the onset of mineralization.
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PMID:Developmental expression of 44-kDa bone phosphoprotein (osteopontin) and bone gamma-carboxyglutamic acid (Gla)-containing protein (osteocalcin) in calcifying tissues of rat. 326 Aug 80

Intratypic osteosarcoma hybrids were constructed by fusing the human osteoblast-like osteosarcoma SaOS-2 with the rat osteoblast-like osteosarcoma UMR-106. Both of these osteosarcomas express liver/bone/kidney alkaline phosphatase (ALPL), but only the UMR-106 cell line expresses osteopontin (OPN), a gene expressed during later stages of osteoblast differentiation. Analysis of osteoblast gene expression in these hybrids demonstrated that ALPL continued to be expressed; however, OPN steady-state mRNA levels were dramatically reduced in four hybrids. Quantitative measurements indicated that OPN steady-state mRNA levels were extinguished by a factor of 20- to 1000-fold. Since SaOS-2 chromosomes are preferentially lost from these hybrids, subclones of extinguished hybrids were isolated that reexpressed OPN mRNA at levels similar to the UMR-106 parental line. These data indicate that trans-acting negative regulatory factors, expressed from the SaOS-2 genome, are responsible for OPN extinction. This report provides the first demonstration of the negative regulation of OPN gene expression and also provides additional evidence that extinction plays a role in the regulation of osteoblast gene expression.
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PMID:Regulation of osteoblast gene expression in intratypic osteosarcoma hybrid cells. 749 36

A simple culture procedure and assay conditions are described which have permitted us to quantify the synthesis of proteins which are associated with an osteoblastic phenotype, by rat calvarial periosteal cells grown on particulate materials. The main feature of the method is the use of an adhesive which does not permit cells to attach to itself but allows attachment and growth of cells on material particles embedded in it on glass coverslips. Cells were cultured for 27 d on hydroxyapatite particle-coated coverslips. Alkaline phosphatase, osteopontin and collagen type I were monitored in cell lysates from d 10 to d 20. After Western blotting, osteopontin and collagen type I were quantified using specific antisera and enhanced chemiluminescence. Maximum levels coincided with peak alkaline phosphatase activity, after 10 and 17 d. The procedures described will be generally applicable to the comparison of cell behaviour on particulate substrata.
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PMID:Novel culture procedure permitting the synthesis of proteins by rat calvarial cells cultured on hydroxyapatite particles to be quantified. 752 93

The effects of retinoic acid (RA) on the expression of osteoblastic-related cell markers was examined. A marrow stromal osteogenic cell line, MBA-15, was analyzed by Northern blotting for the expression of bone matrix proteins. These cells constitutively express mRNA encoding for procollagen alpha 2 (I), osteonectin, osteopontin, biglycan, and alkaline phosphatase (ALK-P). Gene expression was unchanged in response to RA triggering for 24 hr. Furthermore, cell growth and enzymatic activities of ALK-P and neutral endopeptidase (CD10/NEP) were studied. These parameters were examined in MBA-15 and clonal populations representing different stages of differentiation. The cell's growth rate was unchanged, while ALK-P activity was greatly increased during the culture period under RA treatment in MBA-15 and in the clonal cell lines examined while CD10/NEP activity displayed a different pattern. MBA-15.4, a preosteoblast cell line, exhibited an inhibition in CD10/NEP activity at the beginning of the culture period, reaching basal level with time. This activity was greatly increased over control level in MBA-15.6, a mature stage of osteoblasts. Furthermore, the response of cell lines to various growth factors was tested subsequent to priming the cultures with RA. A synergistic effect was monitored for ALK-P activity in MBA-15.4 and MBA-15.6 cells under rh-bone morphogenic protein (BMP-2) and purified osteogenin (BMP-3), and an antagonist effect was measured when cells were exposed to transforming growth factor beta (TGF beta). Contrarily, BMP-2 and BMP-3 inhibited the CD10/NEP activity that had remained unchanged following priming of the cell with RA. Insulin-like growth factor I (IGF-I) and basic fibroblast growth factors (bFGF) did not affect either ALK-P nor CD10/NEP activities in both cloned cells. Cellular response to bone-seeking hormone, parathyroid hormone (PTH), and prostaglandin E2 (PGE2) was monitored by activation of intracellular cAMP. Treatment with RA caused a dramatic decrease in MBA-15.6 cell responses to PTH and PGE2, but no significant effects could be observed in other clonal lines.
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PMID:Differential effects of retinoic acid and growth factors on osteoblastic markers and CD10/NEP activity in stromal-derived osteoblasts. 752 53

Osteopontin (OPN) gene expression and alkaline phosphatase activity were evaluated in the epiphyseal growth plates of normal chickens and in diet-induced tibial dyschdroplasia (TD)-afflicted chickens. In the normal growth plate, OPN gene was expressed by a) cells of the subperichondrial zone surrounding the articular cartilage, b) a narrow layer of hypertrophic chondrocytes at the hypertrophic zone, and c) lower hypertrophic chondrocytes at the zone of matrix calcification and endochondral bone formation. The latter two layers were separated by OPN-negative chondrocytes. Osteopontin gene was not expressed throughout the zone of articular cartilage in the nonhypertrophic or upper hypertrophic portions of the growth plate cartilage. Only at sites of calcification of the lower hypertrophic zone was the expression of the OPN gene associated with alkaline phosphatase activity. In all TD lesions, regardless of the induction procedure, the layer of chondrocytes of the lower hypertrophic zone expressing the OPN gene and the layer of OPN-negative cells separating the two areas of OPN-expressing cells were grossly enlarged. This resulted in a wide discontinuity between the chondrocytes of the lower hypertrophic zone expressing the OPN gene and the cells expressing the OPN gene that are associated with mineralization. In TD, no alkaline phosphatase activity was detected within the growth plate cartilage, but normal OPN gene expression was observed at the subperichondrium zone and at the zone of endochondral bone formation. The results of this study suggest that in the epiphyseal growth plate, OPN expression is not restricted to sites of bone calcification.
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PMID:Osteopontin gene expression and alkaline phosphatase activity in avian tibial dyschondroplasia. 754

We have established two clonal cell lines, designated SM1/9 and SM25/3 from the mandibular condyles of newborn BALB/c mice by immortalization with the SV40 large T antigen. These cells have a high proliferative activity and have been maintained in culture for over 50 passages. They are polygonal in shape. Electron microscopic studies indicate an immature phenotype for both clones and a lack of prominent intracellular filaments typical of fibroblasts. SM25/3 demonstrates different biological properties as compared to SM1/9, it is tumourigenic in nude mice, has a faster growth rate and exhibits less differentiated features. Both cell lines have low constitutive levels of alkaline phosphatase, and the activity of this enzyme is increased significantly in a dose and confluency dependent manner by retinoic acid and 1,25 (OH)2 vitamin D3. The cells express transcripts for retinoic acid receptors mRAR-alpha and mRAR-gamma but not for mRAR-beta. They also express mRNA for the 1,25 (OH)2 vitamin D3 receptor. They co-express transcripts for collagen types I, II, III. Expression of mRNA for extracellular matrix proteins such as biglycan, osteopontin, PAI-1 is detected. Cultured cells do not express mRNA for osteocalcin and this transcript is not inducible with 1,25 (OH)2 vitamin D3 or retinoic acid. Chondrocyte markers such as link protein and aggrecan are not detected. In vitro assays indicate that the cell lines have a limited capacity for osteogenic or chondrogenic differentiation. Similarly agarose culture experiments and extended treatment with retinoic acid indicate that they do not resemble dedifferentiated chondrocytes. Both the cell lines appear to express a phenotype intermediate to osteoblasts and chondroblasts and possibly represent transitional differentiation stages of the progenitor cells of the mandibular condyle. These cells could serve as useful models in elucidating the pathways of early mesenchymal cell differentiation.
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PMID:Establishment and characterization of two clonal cell lines derived from murine mandibular condyles. 757 May 75

Renal expression of alkaline phosphatase (AP) and osteopontin (OP) in rats of different age was examined. Northern blot hybridization showed that AP mRNA was reduced moderately, whereas OP mRNA was stimulated drastically in old rats. Dot-blot quantitation analysis showed that AP mRNA decreased 30% in 24-compared with 6-mo-old rats. In contrast, OP mRNA increased 3.1- and 9.1-fold, respectively, in 12- and 24-mo-old rats. beta-Actin mRNA did not change with age. Blood urea nitrogen (BUN) increased 47 and 187% in 12- and 24-mo-old rats, respectively. Correlation analysis showed that BUN correlated negatively with AP mRNA and positively with OP mRNA. No correlation was observed with beta-actin. The expression of these markers was also examined in femurs. AP and OP mRNAs were marginally reduced in old bones. To test whether the correlation also exists in other types of renal insufficiency, we examined these parameters in young rats infused with parathyroid hormone (PTH). BUN was elevated 3.5-fold, whereas AP mRNA decreased 48%, and OP mRNA increased 15.3-fold in kidneys of PTH-treated rats. To elucidate the possible mechanisms that lead to the overexpression of OP in kidney, we examined the expression of transforming growth factor-beta 1 (TGF-beta 1) mRNA. No significant differences in TGF-beta 1 expression were observed between young and old rats and control and PTH-treated young rats. Changes in the expression of OP were also visualized by immunostaining of renal sections. Alterations in the levels of OP and AP were validated by Western blot analysis and enzyme assay of homogenate, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Renal expression of osteopontin and alkaline phosphatase correlates with BUN levels in aged rats. 757 89


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