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)

The AML/CBFA family of runt homology domain (rhd) transcription factors regulates expression of mammalian genes of the hematopoietic lineage. AML1, AML2 and AML3 are the three AML genes identified to date which influence myeloid cell growth and differentiation. Recently AML-related proteins were identified in an osteoblast-specific promoter binding complex that functionally modulates bone-restricted transcription of the osteocalcin gene. In the present study we demonstrate that in primary rat osteoblasts AML-3 is the AML family member present in the osteoblast-specific complex. Antibody specific for AML-3 completely supershifts this complex, in contrast to antibodies with specificity for AML-1 or AML-2, AML-3 is present as a single 5.4 kb transcript in bone tissues. To establish the functional involvement of AML factors in osteoblast differentiation, we pursued antisense strategies to alter expression of rhd genes. Treatment of osteoblast cultures with rhd antisense oligonucleotides significantly decreased three parameters which are linked to differentiation of normal diploid osteoblasts: the representation of alkaline phosphatase-positive cells, osteocalcin production, and the formation of mineralized nodules. Our findings indicate that AML-3 is a key transcription factor in bone cells and that the activity of rhd proteins is required for completion of osteoblast differentiation.
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PMID:Runt homology domain proteins in osteoblast differentiation: AML3/CBFA1 is a major component of a bone-specific complex. 921 22

Cell differentiation is determined by a certain set of transcription factors such as MyoD in myogenesis. However, transcription factors that play a positive role in phenotypic gene expression in skeletal cells are largely unknown, except the recently identified CBFA1. Scleraxis is a helix-loop-helix-type transcription factor whose transcripts are expressed in sclerotome and in a certain set of skeletal cells; however, nothing is known about its function with regard to the regulation of cell function. To examine possible roles of scleraxis, we overexpressed scleraxis in osteoblastic ROS17/2.8 cells, which express low levels of scleraxis. Scleraxis overexpression enhanced expression of the aggrecan gene, which is not normally expressed at high levels in these osteoblastic cells. Overexpression of scleraxis also increased mRNA levels of type II collagen and osteopontin while suppressing expression of osteoblast phenotype-related genes encoding type I collagen and alkaline phosphatase. Transient transfection experiments indicated that scleraxis enhanced the chloramphenicol acetyltransferase activity of the reporter construct AgCAT-8, which contained an 8-kilobase pair (kb) fragment of the aggrecan gene including both the promoter and its first intron. Deletion analysis identified a 1-kb region that is responsive to scleraxis within the aggrecan gene. This region contains two adjacent E-box sequences. A 29-base pair DNA fragment (AgE) containing these E-box sequences bound to proteins in the ROS17/2.8 cell nuclear extracts as well as to in vitro translated scleraxis. This binding was competed with unlabeled AgE, but not with a mutated E-box DNA sequence (mAgE), indicating the specificity of the binding activity. The AgE binding activity in the ROS17/2.8 cell nuclear extracts was enhanced in the cells overexpressing scleraxis and was supershifted by the antiserum raised against scleraxis. Furthermore, AgE, but not mAgE, conferred responsiveness to scleraxis overexpression to a heterologous promoter. Finally, replacement mutation of the AgE sequence within the 2.5-kb AgCAT-1 construct significantly reduced its responsiveness to scleraxis. These results indicate that overexpression of a single helix-loop-helix-type transcription factor, scleraxis, enhances aggrecan gene expression via binding to the E-box-containing AgE sequence in ROS17/2.8 cells.
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PMID:Overexpression of a single helix-loop-helix-type transcription factor, scleraxis, enhances aggrecan gene expression in osteoblastic osteosarcoma ROS17/2.8 cells. 936 62

Soluble and membrane-associated variants of the orphan T1-receptor, a homolog of interleukin-1 receptor type I, are expressed in proliferating preosteoblasts in differentiating bone. Recent evidence reveals that T1-receptor synthesis is retained in osteogenic osteosarcoma cells. Here we report that the suppression of T1-receptor expression by mouse osteosarcoma cells using a T1 -antisense expression vector results in the abrogation of the osteogenic potential of the tumor cells. T1-antisense-expressing tumor cells formed anaplastic tumors in vivo and failed to express the osteoblast-specific genes collagen type 1, alkaline phosphatase, and osteocalcin when cultured in a 3-dimensional collagen type I matrix in vitro. Suppression of T1-receptor synthesis did not affect the expression of the essential bone cell-specific transcription factor AML3/CBFA1 in the osteosarcoma cells. These data provide the first evidence that T1-receptor plays a key role in osteogenic differentiation.
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PMID:Suppression of T1-receptor expression by antisense RNA abrogates differentiation of osteogenic osteosarcoma cells. 1033 64

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.
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PMID:Further characterization of cells expressing STRO-1 in cultures of adult human bone marrow stromal cells. 1045 67

Because regulation of the differentiation to osteoblasts and adipocytes from a common progenitor in bone marrow stroma is poorly understood, we assessed effects of bone morphogenetic protein-2 (BMP-2) on a conditionally immortalized human marrow stromal cell line, hMS(2-6), which is capable of differentiation to either lineage. BMP-2 did not affect hMS(2-6) cell proliferation but enhanced osteoblast differentiation as assessed by a 1.8-fold increase in expression of OSF2/CBFA1 (a gene involved in commitment to the osteoblast pathway), by increased mRNA expression and protein secretion for alkaline phosphatase (ALP), type I procollagen and osteocalcin (OC) (except for OC protein), and by increased mineralized nodule formation. Transient transfection with Osf2/Cbfa1 antisense oligonucleotide substantially reduced BMP-2-stimulated expression of ALP mRNA and protein. The effects of BMP-2 on adipocyte differentiation varied: expression of peroxisome proliferator-activated receptor gamma2 (a gene involved in commitment to the adipocyte pathway) was unchanged, mRNA expression of the early differentiation marker, lipoprotein lipase, was increased, and mRNA and protein levels of the late differentiation marker, leptin, and the formation of cytoplasmic lipid droplets were decreased. Thus, by enhancing osteoblast commitment and by inhibiting late adipocyte maturation, BMP-2 acts to shunt uncommitted marrow stromal precursor cells from the adipocyte to the osteoblast differentiation pathway.
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PMID:Differentiation of human marrow stromal precursor cells: bone morphogenetic protein-2 increases OSF2/CBFA1, enhances osteoblast commitment, and inhibits late adipocyte maturation. 1046 80

Core-binding factor A1 (Cbfa1), also called Pebp2 alpha A/AML3, is a transcription factor that belongs to the runt-domain gene family. Cbfa1-deficient mice are completely incapable of both endochondral and intramembranous bone formation, indicating that Cbfa1 is indispensable for osteogenesis. Maturation of chondrocytes in these mice is also disorganized, suggesting that Cbfa1 may also play a role in chondrogenesis. The aim of this study was to examine the expression and regulation of Pebp2 alpha A/AML3/Cbfa1 expression in the chondrocyte-like cell line, TC6. Northern blot analysis indicated that Cbfa1 mRNA was constitutively expressed as a 6.3 kb message in TC6 cells and the level of Cbfa1 expression was enhanced by treatment with bone morphogenetic protein-2 (BMP2) in a time- and dose-dependent manner. This effect was blocked by an RNA polymerase inhibitor, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole, but not by a protein synthesis inhibitor, cycloheximide. Western blot analysis of the cell lysates using polyclonal antibody raised against Cbfa1 indicated that BMP2 treatment increased the Cbfa1 protein level in TC6 cells. In TC6 cells, BMP2 treatment enhanced expression of alkaline phosphatase and type I collagen mRNAs but suppressed that of type II collagen mRNA. In addition to TC6 cells, Cbfa1 mRNA was also expressed in primary cultures of chondrocytes and BMP2 treatment enhanced Cbfa1 mRNA expression in these cells similarly to its effect on TC6 cells. These data indicate that the Pebp2 alpha A/AML3/Cbfa1 gene is expressed in a chondrocyte-like cell line, TC6, and its expression is enhanced by treatment with BMP.
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PMID:An osteogenesis-related transcription factor, core-binding factor A1, is constitutively expressed in the chondrocytic cell line TC6, and its expression is upregulated by bone morphogenetic protein-2. 1082 41

The regulation of human bone marrow stromal precursor cell differentiation toward the chondrocyte, osteoblast or adipocyte lineages is not known. In this study, we assessed the lineage-specific differentiation and conversion of immortalized clonal F/STRO-1(+) A human fetal bone marrow stromal cells under the control of dexamethasone (Dex), indomethacin/insulin (Indo/Ins) and linoleic acid (LA). Under basal conditions, F/STRO-1(+) A cells expressed markers mRNAs or proteins of the osteoblast lineage [CBFA1, osteocalcin (OC), alkaline phosphatase (ALP), type 1 collagen], of the chondrocyte lineage (aggrecan, types 2, 9 and 10 collagen), and of the adipocyte lineage (PPARgamma2, C/EBPalpha, aP2, G3PDH, lipoprotein lipase, leptin). Treatment with Dex increased CBFA1, OC and ALP mRNA and protein levels. Exposure to LA enhanced expression of adipocytic genes and cytoplasmic triglycerides accumulation, and suppressed the Dex-induced stimulation of osteoblast marker genes. Indo/Ins stimulated the synthesis of aggrecan and type 2 collagen and increased types 9 and 10 collagen mRNA levels, and suppressed both basal and Dex-promoted expression of osteoblast markers. Conversely, stimulation of osteoblastogenesis by Dex suppressed both basal and Indo/Ins-stimulated chondrocyte genes. Thus, the clonal human fetal bone marrow stromal F/STRO-1(+) A cell line is a lineage-unrestricted common progenitor that expresses tripotential adipocyte, osteoblast or chondrocyte characteristics. Our data also show that differentiation towards one pathway in response to Dex, Indo/Ins and LA restricts expression of other lineage-specific genes, and provide evidence for a controlled reciprocal regulation of osteoblast/chondroblast and osteoblast/adipocyte differentiation of clonal F/STRO-1(+) human bone marrow stromal cells.
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PMID:Reciprocal control of osteoblast/chondroblast and osteoblast/adipocyte differentiation of multipotential clonal human marrow stromal F/STRO-1(+) cells. 1118 Mar 95

Cleidocranial dysplasia (CCD), an autosomal dominant human bone disease, is thought to be caused by heterozygous mutations in RUNX2/PEBP2alphaA/CBFA1. To understand the mechanism underlying the pathogenesis of CCD, we studied a novel mutant of RUNX2, namely CCDalphaA376, originally identified in a CCD patient. The nonsense mutation, which resulted in a truncated RUNX2 protein, severely impaired RUNX2 transactivation activity. We showed that signal transducers of transforming growth factor (TGF)-beta and bone morphogenetic protein (BMP) receptors, Smads, interact with RUNX2 in vivo and in vitro and enhance transactivation ability. The truncated RUNX2 protein failed to interact with Smads, and was unable to induce the osteoblast-like phenotype in C2C12 myoblasts following stimulation with BMP. Exogenous expression of Smads 1 and 4 in C2C12 cells stably expressing RUNX2 showed alkaline phosphatase (ALP) activity, suggesting a possible link between Smads and RUNX2, while in C2C12 stably expressing CCDalphaA376, a detectable level of ALP activity failed to be induced. The results suggest that CCDalphaA376 inhibited RUNX2 function in a dominant negative fashion.
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PMID:A RUNX2/PEBP2alphaA/CBFA1 mutation in cleidocranial dysplasia revealing the link between the gene and Smad. 1136 5

Physical shock wave (SW) has shown effectiveness on promotion of bone growth. We have recently demonstrated that SW could promote bone marrow stromal cell differentiation toward osteoprogenitor associated with induction of TGF-beta1. We have further demonstrated that SW-induced membrane hyperpolarization and Ras activation acted an early signal for the osteogenesis in human bone marrow stromal cells. An optimal dose of SW treatment at 0.16 mJ/mm(2) for 500 impulses induced a rapid membrane hyperpolarization in 5 min, activation of Ras in 30 min, and cell proliferation in 2 days. The SW-promoted cell growth was related to osteogenesis as demonstrated by increase of bone alkaline phosphatase activity in 6 days and osteocalcin mRNA expression in 12 days. In support that SW-induced Ras activation mediated osteogenesis of human bone marrow stromal cells, we further demonstrated that transfection of bone marrow stromal cells with a dominant negative Ras mutant (Asn-17 ras(H)) abrogated the SW enhancement of osteogenic transcription factor (CBFA1) activation, osteocalcin mRNA expression, and bone nodule formations. These results suggest that physical SW promotes bone marrow stromal cell differentiation toward osteogenic lineage via membrane hyperpolarization, followed by Ras activation and specific osteogenic transcription factor CBFA1 expression. A link between physical SW and biomembrane perturbation-mediated Ras activation may highlight how noninvasive physical agents could be used to promote fracture healing and to rescue patients with osteoporosis and osteopenic disorders in the future.
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PMID:Physical shock wave mediates membrane hyperpolarization and Ras activation for osteogenesis in human bone marrow stromal cells. 1156 44

The remodeling of chromatin is required for tissue-specific gene activation to permit interactions of transcription factors and coregulators with their cognate elements. Here, we investigate the chromatin-mediated mechanisms by which the bone-specific osteocalcin (OC) gene is transcriptionally activated during cessation of cell growth in ROS 17/2.8 osteosarcoma cells and during normal osteoblast differentiation. Acetylation of histones H3 and H4 at the OC gene promoter was assayed during the proliferative and postproliferative stages of cell growth by using chromatin immunoprecipitation assays with antibodies that recognize different acetylated forms of histones H3 or H4. The results show that the promoter and coding regions of the OC gene contain very low levels of acetylated histones H3 and H4 during the proliferative period of osteoblast differentiation when the OC gene is inactive. Active expression of the OC gene in mature osteoblasts and confluent ROS 17/2.8 cells is functionally linked to preferential acetylation of histone H4 and, to a lesser extent, to acetylation of histone H3. Histone acetylation at the loci for RUNX2 (CBFA1), alkaline phosphatase, bone sialoprotein, osteopontin, and the cell growth regulator p21, which are expressed throughout osteoblast differentiation, is not altered postproliferatively. We conclude that acetylation of histones H3 and H4 is functionally coupled to the chromatin remodeling events that mediate the developmental induction of OC gene transcription in bone cells.
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PMID:Transcriptional induction of the osteocalcin gene during osteoblast differentiation involves acetylation of histones h3 and h4. 1255 83


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