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:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A variety of signal transduction pathways contribute to the regulation of transcription in mammalian cells. Several of these pathways ultimately rely upon the interaction of transcription factors with genetic sequences termed response elements in the promoter regions of some genes. The biochemical mechanisms that control the levels and state of activation of transcription factors are poorly understood. However, specific phosphorylation events mediated by protein kinase C, growth factor receptor-linked tyrosine kinases, and protein kinase A clearly participate in the regulation of these signal transduction pathways. To understand the relationship between activation and/or inhibition of these pathways and regulation of gene expression controlled by specific response elements, cell lines were prepared containing the TPA response element (TRE), serum response element (SRE), or cyclic AMP response element (CRE) fused to a gene encoding a secretable form of alkaline phosphatase (SEAP). These TRE-SEAP, SRE-SEAP, and CRE-SEAP cells exhibit dramatic increases in alkaline phosphatase (AP) activity following exposure to TPA, PDGF, or forskolin. Down regulation of protein kinase C or inhibition of tyrosine kinase activity blocked the stimulation of AP activity caused by TPA or PDGF. These cell lines can be used to characterize existing inhibitors, and to identify new agents that affect specific signal transduction pathways in mammalian cells.
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PMID:Mammalian cell lines engineered to identify inhibitors of specific signal transduction pathways. 171 Nov 89

We used the technique of in situ hybridization to determine if cells expressing PDGF B-chain mRNA can be detected in a model of mesangial proliferative nephritis in the rat induced with antibody directed against the Thy 1 antigen present on the mesangial cell membrane. The method involved hybridization with a digoxigenin-labeled cRNA probe for the murine PDGF B-chain followed by detection with an anti-digoxigenin-alkaline phosphatase conjugate and subsequent colorimetric reaction. In normal rats (N = 4), the majority of glomeruli (74%) were negative for PDGF B-chain mRNA, whereas 65% of glomeruli from rats with mesangial proliferative nephritis (N = 4) had segmental or diffuse staining for PDGF B-chain mRNA in a mesangial pattern. The difference, as measured using a semiquantitative scale, was significant (mean scores 0.4 +/- 0.2 vs. 1.9 +/- 0.2; scale 0 to 3+; P less than 0.001). The increase in PDGF B-chain mRNA positive cells localized to areas of hypercellularity and was associated with a significant increase in cells positive for PDGF B-chain by immunostaining with a specific monoclonal antibody (0.8 +/- 0.1 vs. 1.7 +/- 0.4, scale 0 to 3+, normal vs. diseased rats, P less than 0.005). Complement depletion, which prevents the mesangial cell proliferation, also prevented the increase in cells expressing PDGF B-chain mRNA and protein. Thus, this method of in situ hybridization can successfully detect cells expressing PDGF mRNA in active glomerulonephritis, and may be useful for detecting cells expressing genes for other growth factors and cytokines in both human and experimental models of glomerular injury.
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PMID:Demonstration of PDGF B-chain mRNA in glomeruli in mesangial proliferative nephritis by in situ hybridization. 172 21

In bone forming cartilage in vivo, cells undergo terminal differentiation, whereas most of the cells in normal articular cartilage do not. Chondrocyte hypertrophy can be induced also in vitro by diffusible signals. We have identified growth factors or hormones acting individually on 17-d chick embryo sternal chondrocytes cultured in agarose gels under strictly serum-free conditions. Insulin-like growth factor I or insulin triggered the first steps of chondrocyte maturation, i.e., cell proliferation and increased matrix deposition while the chondrocytic phenotype was maintained. However, cells did not progress to the hypertrophic stage. Proliferation and stimulated collagen production was preceded by a lag period, indicating that synthesis of other components was required before cells became responsive to insulin-like growth factor I or insulin. Very small amounts of FBS exerted effects similar to those of insulin-like growth factor I or insulin. However, FBS could act directly and elicited hypertrophy when constituting greater than 1% of the culture media. Basic FGF has been claimed to be the most potent chondrocyte mitogen, but had negligible effects under serum-free conditions. The same is true for PDGF, a major serum-mitogen. Under the direction of thyroxine, cells did not proliferate but became typical hypertrophic chondrocytes, extensively synthesizing collagen X and alkaline phosphatase.
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PMID:Induction of proliferation or hypertrophy of chondrocytes in serum-free culture: the role of insulin-like growth factor-I, insulin, or thyroxine. 173 18

Platelet-derived growth factor (PDGF) exists as a homodimer or a heterodimer comprising either PDGF-A or PDGF-B subunits, and each isoform occurs in various tissues, including bone. Although the stimulatory effects of PDGF-BB have been studied in cultures of bone cells and intact bone fragments, the influence of other isoforms that may arise locally or systematically in vivo, has not been reported. Therefore recombinant human PDGF-BB, PDGF-AB, and PDGF-AA were evaluated in osteoblast-enriched cultures from fetal rat bone. Within 24 hours these factors produced a graded response in bone cell DNA and protein synthesis, with half-maximal effects at approximately 0.6, 2.1, and 4.8 nM PDGF-BB, PDGF-AB, and PDGF-AA, respectively. Increases in collagen and noncollagen protein synthesis were abrogated when DNA synthesis was blocked with hydroxyurea. Furthermore, each factor reduced alkaline phosphatase activity, PDGF-BB being the most inhibitory. Binding studies with 125I-PDGF-BB or 125I-PDGF-AA and each unlabeled PDGF isoform produced discrete ligand binding and displacement patterns: 125I-PDGF-BB binding was preferentially displaced by PDGF-BB (Ki approximately 0.7 nM), less by PDGF-AB (Ki approximately 2.3 nM) and poorly by PDGF-AA. In contrast, 125I-PDGF-AA binding was measurably reduced by PDGF-AA (Ki approximately 4.0 nM), but was more effectively displaced by PDGF-BB or PDGF-AB (each with Ki approximately 0.7 nM). These studies indicate that each PDGF isoform produces biochemical effects proportional to binding site occupancy and suggest that receptors that favor PDGF-B subunit binding preferentially mediate these results in osteoblast-enriched bone cell cultures.
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PMID:Relative binding and biochemical effects of heterodimeric and homodimeric isoforms of platelet-derived growth factor in osteoblast-enriched cultures from fetal rat bone. 206 62

A cell line, called MG-63.3A, was selected for its resistance to detachment from cell culture by a synthetic peptide containing the fibronectin cell-attachment sequence, Arg-Gly-Asp-Ser. The mechanism of this resistance is probably the 6-fold overproduction of the cell surface fibronectin receptor in MG-63.3A cells (Dedhar et al, J. Cell. Biol. 105, 1175-1182, (1987]. Compared to the parental, tumorigenic MG-63 cells, the non-tumorigenic MG-63.3A cells display strikingly different properties. These include an altered morphology, a slower proliferation rate, ability to form a calcified matrix in vitro, increased synthesis of type I collagen and expression of bone type alkaline phosphatase activity. Studies with purified growth factors indicate that the MG-63 and MG-63.3A cell lines respond to differentially to growth factors; the growth of MG-63 cells if stimulated by PDGF and GM-CSF and inhibited IL-1 beta, whereas the growth of MG-63.3A is unaffected by GM-CSF and IL-1 beta but is stimulated by PDGF and estradiol. We conclude from these data that the MG-63.3A cells may represent a more differentiated cell type with osteoblast-like properties. Studies are currently underway to further characterize, by electron microscopy, the calcified matrix formation by MG-63.3A cells.
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PMID:The osteoblast-like differentiated phenotype of a variant of MG-63 osteosarcoma cell line correlated with altered adhesive properties. 253 54

We have previously reported that the J774A.1 macrophage-like tumor cell line produces two potent monokines which stimulate the growth of osteoblasts and chondrocytes. These growth factors, which have an affinity for heparin-agarose, have been termed HEP I (a 30 Kd PDGF-like molecule) and HEP II (an approximately 20 Kd molecule), respectively, based on their elution profile. Unlike HEP I, HEP II does not stimulate the growth of fibroblasts. Extensive biological and chromatographic studies disclosed that HEP II appears to be a unique bone cell mitogen unlike any known growth factor, including the FGFs, IL-1s, and TNFs, EGF, IGF-I and -II, TGF-beta, beta 2 microglobulin, G-CSF, CSF-1 and GM-CSF. To characterize more fully the effects of the macrophage-derived monokines on osteoblast growth and function, clones were derived from calvaria explant cultures. Two clones, SDFRC-2.05 and SDFRC-3, were developed and found to exhibit osteoblastic characteristics, including high levels of alkaline phosphatase, synthesis of type I but not type III collagen, and an increased intracellular cAMP production in response to PTH. The SDFRC-3 cells exhibited a polygonal morphology like that of the explant-derived cells while SDFRC-2.05 cells exhibited a more fibroblastic morphology. When tested on the explant cultures and clones, HEP I and HEP II were found to stimulate DNA synthesis and increase protein per culture, but decreased alkaline phosphatase activity. Clone SDFRC-3 was found to be more responsive to HEP II than clone SDFRC-2.05. Both monokines were found to be more potent mitogens for bone cells than TGF-beta. HEP II, but not HEP I or TGF-beta, induced a transformation of bone cells from a polygonal to a fibroblastic morphology, suggesting the induction of migration prior to proliferation. Thus, macrophages may be responsible not only for bone repair but also for ensuring the linkage of bone formation to resorption during physiological remodeling.
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PMID:Monokines produced by macrophages stimulate the growth of osteoblasts. 263 Jan 69

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

Treatment of the U-2 OS human osteosarcoma cell line with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) dramatically decreased the rate of DNA synthesis. This decrease in proliferation as well as the change in morphology of the TPA-treated cells can be blocked by the protein kinase C inhibitor GF 109203X. The U-2 OS cells are known to express the c-sis oncogene [platelet-derived growth factor (PDGF) B-chain], PDGF-A, and receptors for PDGF, thus providing a potential autocrine loop of growth stimulation. TPA was found to induce the expression of both the PDGF-A and the PDGF-B chains. However, the levels of the PDGF receptor beta subunits and of the PDGF-BB inducable tyrosine phosphorylation of the PDGF receptor were markedly reduced. The TPA treatment of the U-2 OS cells also induced changes typical for maturing bone cells, such as increased expression levels of alkaline phosphatase and osteopontin. The expression levels of type I collagen and bone sialoprotein were reduced. The results show a TPA-dependent down-regulation of the PDGF receptor beta subunits that correlates with an increased expression of osteoblast phenotypic markers.
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PMID:Phenotypic modification of human osteosarcoma cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. 779 13

Mesenchymal progenitors cells can be isolated from rat bone marrow and mitotically expanded in vitro. When these cells, which we operationally call mesenchymal stem cells (MSCs), are placed in an appropriate environment, they have the capacity to differentiate into bone and/or cartilage. This capacity is called osteochondrogenic potential. In this study, preconfluent MSCs were exposed in vitro to 5 ng/ml transforming growth factor-beta 1 (TGF-beta 1) or platelet-derived growth factor, isoform BB (PDGF-BB) for a pulse of 48 h and assayed for cell proliferation, alkaline phosphatase activity, and osteochondrogenic potential; untreated MSC's served as controls. In these cell culture conditions, TGF-beta 1 or PDGF-BB had similar effects on proliferation and alkaline phosphatase activity. Both growth factors increased cell proliferation and decreased alkaline phosphatase activity of MSCs. Sister cultures of TGF-beta 1- or PDGF-BB-treated MSCs and untreated MSCs were trypsinized. For each type of culture, the trypsinised MSCs were split in two parts: one part was replated in an osteogenic medium to assess its in vitro osteogenic potential, whereas the other part was seeded into porous calcium phosphate ceramics and implanted subcutaneously in syngeneic rats to assess its in vivo osteochondrogenic potential. PDGF-pretreated MSCs showed no difference in in vivo and in vitro osteochondrogenesis from that of control MSCs, while TGF-beta 1 pretreatment blocked the osteochondrogenic potential of MSCs when assayed in vitro for bone nodule formation. However, when tested in vivo, TGF-beta 1-pretreated MSCs were able to form bone and cartilage. These data show that measurements of proliferation and alkaline phosphatase activity of preconfluent MSCs immediately after exposure to growth factor were not predictive of their subsequent osteochondrogenic potential. Moreover, the variation of the osteochondrogenic potential of MSCs after exposure to growth factor was further modulated by the environment in which the MSCs were assayed.
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PMID:Osteochondrogenic potential of marrow mesenchymal progenitor cells exposed to TGF-beta 1 or PDGF-BB as assayed in vivo and in vitro. 886 1

We have recently demonstrated that phenytoin, a widely used therapeutic agent for seizure disorders, has osteogenic effects in rats and in humans in vivo, and in human bone cells in vitro. The goal of the present study was to determine the mechanism of the osteogenic action of phenytoin in normal human mandible-derived bone cells. Because many osteogenic agents increased bone cell proliferation through mediation by growth factors, we tested the hypothesis that the osteogenic effects of phenytoin involved the release of a growth factor by measuring the mRNA level of several bone cell growth factors and insulin-like growth factor (IGF) binding proteins with Northern blots using specific cDNA probes. Treatment with 5-50 microM phenytoin reproducibly and markedly increased (up to 6-fold, p < 0.001) the mRNA of transforming growth factor (TGF)-beta 1, but not that of other growth factors (i.e., IGF-II, platelet-derived growth factor-A [PDGF-A], PDGF-B, and TGF-beta 2) and IGF binding proteins (i.e., IGFBP-3, -4, and -5). The stimulation was dose dependent, with an optimal dose of 10-50 microM. Maximal increase was seen after 1 h of phenytoin treatment. The release of biologically active TGF-beta activity in conditioned media was measured with the mink lung cell proliferation inhibition assay. Twenty-four hours of phenytoin treatment significantly increased the production of biologically active TGF-beta (2-fold, p < 0.05) with the optimal dose between 5-50 microM. Comparisons between the in vitro osteogenic effects of phenytoin and those of TGF-beta 1 reveal that these two agents at their respective optimal doses had similar maximal stimulatory effects on [3H]thymidine incorporation, alkaline phosphatase (ALP)-specific activity, and type I alpha-2 collagen mRNA expression in human bone cells. The stimulatory effects of phenytoin on [3H]thymidine incorporation and ALP-specific activity were completely blocked by a neutralizing anti-TGF-beta antibody. In conclusion, these findings demonstrate for the first time that at least some of the osteogenic actions of phenytoin in human bone cells could be in part mediated by TGF-beta 1.
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PMID:Osteogenic actions of phenytoin in human bone cells are mediated in part by TGF-beta 1. 897 Aug 89


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