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)

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

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

Bone morphogenetic protein-2 (BMP-2) is an important regulator of osteoblast differentiation. However, the regulation of osteoblast apoptosis by BMP signaling remains poorly understood. Here we examined the role of type I BMP receptor (BMP-RI) in osteoblast apoptosis promoted by BMP-2. Despite undetectable BMP-RIB expression in OHS4 cells, BMP-2 or BMP-2 overexpression increased osteoblast differentiation similarly as in SaOS2 cells which express BMP-RIB, as shown by alkaline phosphatase and CBFA1/RUNX2 expression. In contrast to SaOS2 cells, however, BMP-2 or BMP-2 overexpression did not increase caspase-9 and caspases-3, -6, and -7 activity and DNA fragmentation in OHS4 cells. Consistently, BMP-2 increased protein kinase C (PKC) activity, and PKC inhibition suppressed BMP-2-induced caspase activity in SaOS2 but not in OHS4 cells that lack BMP-RIB. A dominant negative BMP-RIB inhibited BMP-2-induced caspase activity, whereas wild-type BMP-RIB promoted caspase activity induced by BMP-2 in SaOS2 and MC3T3-E1 cells. Wild-type BMP-RIB rescued the apoptotic response to BMP-2, and a constitutively active BMP-RIB restored the apoptotic signal in OHS4 cells, supporting an essential role for BMP-RIB in osteoblast apoptosis. We also assessed whether BMP-2-induced apoptosis occurred independently of osteoblast differentiation. General inhibition of caspases did not abolish BMP-2-induced alkaline phosphatase and CBFA1/RUNX2 expression in SaOS2 cells. Furthermore, broad caspases inhibition increased matrix mineralization but did not reverse the BMP-2 effect on mineralization in MC3T3-E1 cells. These results indicate that BMP-2-induced apoptosis was mediated by BMP-RIB in osteoblasts and occurred independently of BMP-2-induced osteoblast differentiation, which provides additional insights into the dual mechanism of BMP-2 action on osteoblast fate.
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PMID:Bone morphogenetic protein receptor IB signaling mediates apoptosis independently of differentiation in osteoblastic cells. 1457 67

Apert syndrome is an autosomal dominant disease characterized by craniosynostosis and bony syndactyly associated with point mutations (S252W and P253R) in the fibroblast growth factor receptor (FGFR) 2 that cause FGFR2 activation. Here we investigated the role of the S252W mutation of FGFR2 on osteoblastic differentiation. Osteoblastic cells derived from digital bone in two Apert patients with the S252W mutation showed more prominent alkaline phosphatase activity, osteocalcin and osteopontin mRNA expression, and mineralized nodule formation compared with the control osteoblastic cells derived from two independent non-syndromic polydactyly patients. Stable clones of the human MG63 osteosarcoma cells (MG63-Ap and MG63-IIIc) overexpressing a splice variant form of FGFR2 with or without the S252W mutation (FGFR2IIIcS252W and FGFR2IIIc) showed a higher RUNX2 mRNA expression than parental MG63 cells. Furthermore MG63-Ap exhibited a higher osteopontin mRNA expression than did MG63-IIIc. The enhanced osteoblastic marker gene expression and mineralized nodule formation of the MG63-Ap was inhibited by the conditioned medium from the COS-1 cells overexpressing the soluble FGFR2IIIcS252W. Furthermore the FGF2-induced osteogenic response in the mouse calvarial organ culture system was blocked by the soluble FGFR2IIIcS252W. These results show that the S252W mutation in the FGFR2 gene enhances the osteoblast phenotype in human osteoblasts and that a soluble FGFR2 with the S252W mutation controls osteoblast differentiation induced by the S252W mutation through a dominant negative effect on FGFR2 signaling in Apert syndrome.
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PMID:A soluble form of fibroblast growth factor receptor 2 (FGFR2) with S252W mutation acts as an efficient inhibitor for the enhanced osteoblastic differentiation caused by FGFR2 activation in Apert syndrome. 1531 Jul 57

Seathre-Chotzen syndrome (SCS) is an autosomal dominant craniosynostosis syndrome, associated with loss-of-function mutations in the basic helix-loop-helix transcription factor, TWIST1. The biologic activity of TWIST1 has been implicated in the inhibition of differentiation of multiple cell lineages. Therefore, premature fusion of cranial sutures (craniosynostosis) in SCS may be mediated by altered differentiation of calvarial osteoblasts. In this study, we evaluated osteoblasts derived from calvarial bone of three patients with SCS and three unaffected individuals as controls to investigate the principle stages of osteoblast differentiation: (1) proliferation, (2) matrix maturation, and (3) mineralization. Using a BrdU-Hoechst flow cytometry assay, we found that the percent of proliferating cells was significantly reduced in cells derived from patients with SCS compared with those derived from controls (P < or = 0.05). In the matrix maturation stage, alkaline phosphatase (ALP) enzyme activity and the expression of extracellular matrix genes, collagen I alpha 2 (COL1A2), osteopontin (OPN), osteocalcin (OC), and the runt-related transcription factor RUNX2 were examined by enzymatic assay and real-time quantitative RT-PCR, respectively. We identified no significant differences in the expression of matrix related transcripts. However, we found significant reductions in ALP activity on days 3 and 7 and in RUNX2 expression on days 14 and 21 (P < or = 0.05). Quantitative alizarin red S mineralization assays showed a trend toward increased mineralization in osteoblasts derived from patients with SCS at days 21 and 28, although not statistically significant. Our results demonstrated that loss-of-function mutations of TWIST1 led to reduced proliferation regardless of the functional domain affected. We did not find any conclusive differences in matrix maturation or mineralization in these primary osteoblasts. It is plausible that mutations in different functional domains of TWIST1 have divergent effects on these later stages of differentiation.
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PMID:In vitro differentiation profile of osteoblasts derived from patients with Saethre-Chotzen syndrome. 1578 Oct 3

Tibial dyschondroplasia (TD) is a metabolic cartilage disease of young poultry in which endochondral bone formation is disrupted leading to the retention of a non-calcified, avascular plug of cartilage in the tibial growth plate. Chicks aged 7 days were fed either a control diet or one containing thiram 100 ppm for 48 h to induce TD. Cell multiplication in the growth plate was determined thereafter with bromodeoxyuridine (BrdU) labelling, and metabolic changes by measuring alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), and glutathione (GSH) activities. The effect on chondrocyte maturation was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of gene expression. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) and DNA fragmentation were used to determine the effects of thiram on cell survival. The results showed that thiram-induced TD was not due to the multiplication of cells in the post-proliferative zones. Thiram did not affect ALP activity, which would have indicated a loss of calcification potential, but it reduced both TRAP and the glutathione concentrations, suggesting that the growth plate metabolism and remodelling functions were adversely affected. Thiram appeared to have no effect on the expression of type X collagen, transglutaminase, RUNX2, or matrix metalloproteinase-2 (MMP) genes suggesting that it did not alter the maturation potential of chondrocytes. On the contrary, the expressions of MMP-13 and vascular endothelial growth factor (VEGF) genes were "up-regulated," suggesting that thiram has pro-angiogenic activity. However, TUNEL assay showed that thiram induced endothelial cell apoptosis in the capillary vessels of the growth plates, as early as 10 days of age, when TD was not visually evident. The vascular death increased on subsequent days accompanied by massive death of chondrocytes in the transition zone of the growth plate. The induction of apoptosis in the growth plate was also demonstrated by DNA fragmentation. It was concluded that thiram induced TD not through an increase in the multiplication of chondrocytes in the transition zone and not by altering the expression of genes causing the arrest of chondrocytes in a prehypertrophic state, but by creating a metabolic dysfunction which led to the destruction of blood capillaries in the transition zone chondrocytes.
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PMID:Changes in the tibial growth plates of chickens with thiram-induced dyschondroplasia. 1589 90

Decreased bone formation contributes to the development of bone lesions in multiple myeloma (MM) patients. In this study, we have investigated the effects of myeloma cells on osteoblast formation and differentiation and the potential role of the critical osteoblast transcription factor RUNX2/CBFA1 (Runt-related transcription factor 2/core-binding factor Runt domain alpha subunit 1) in the inhibition of osteoblastogenesis in MM. We found that human myeloma cells suppress the formation of human osteoblast progenitors in bone marrow (BM) cultures. Moreover, an inhibitory effect on osteocalcin, alkaline phosphatase, collagen I mRNA, protein expression, and RUNX2/CBFA1 activity by human preosteoblastic cells was observed in cocultures with myeloma cells. The inhibitory effect was more pronounced in the cell-to-cell contact conditions compared with those without the contact and involved the very late antigen 4 (VLA-4) integrin system. Among the soluble osteoblast inhibitors screened, we show the potential contribution of interleukin-7 (IL-7) in the inhibitory effect on osteoblast formation and RUNX2/CBFA1 activity by human myeloma cells in coculture. Finally, our in vitro results were supported in vivo by the finding of a significant reduction in the number of Runx2/Cbfa1-positive cells in the BM biopsies of patients with MM who had osteolytic lesions compared with those who did not have bone lesions, suggesting the critical involvement of RUNX2/CBFA1 in the decreased bone formation in MM.
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PMID:Myeloma cells block RUNX2/CBFA1 activity in human bone marrow osteoblast progenitors and inhibit osteoblast formation and differentiation. 1593 61

Towards utilizing gene-targeted, repopulating mesenchymal stem cells (MSC) to increase osteogenesis, we evaluated the expression of bone-specific promoters during MSC differentiation. Multi-lineage potential of cultured MSC was confirmed by osteogenic, adipogenic and chondrogenic differentiation under controlled conditions. Recombinant adeno-associated virus (rAAV) encoding luciferase under the human cytomegalovirus (CMV), mouse alkaline phosphatase (ALP), Runx-2/cbfa1 (RUNX), osteopontin (OPN), collagen type 1a (COL), and osteocalcin (OCN) promoters was used to transduce mouse MSC. Replicate cultures were maintained undifferentiated or differentiated to osteoblast lineage. Luciferase expression was determined on days 1, 2, 3, 7, 14, or 21 as a measure of promoter activity. Expression of osteogenic markers and mineralization was determined as correlates of osteopoiesis. Results indicated expression from CMV promoter in undifferentiated and differentiated cultures at early stage. However, expression from COL and RUNX promoters was abundant only in differentiating cultures as early as 24 h but declined gradually. Expression from OPN and ALP promoters was evident 24 h following osteogenic differentiation and peaked gradually until 2 weeks before declining. Expression from OC promoter was evident only after 7 days of differentiation but remained until final analysis on day 21. That rAAV transduction of MSC does not induce differentiation was also confirmed by quantitative reverse-transcription polymerase chain reaction (QRT-PCR). The observed stage-specific expression of analyzed promoters was not significant when the MSC were differentiated to adipocytes. Thus, the use of RUNX2 or COL promoter to stably express osteoinductive factors in MSC may allow both self-renewal of modified MSC and enrichment of osteoblast commitment.
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PMID:Determination of osteoprogenitor-specific promoter activity in mouse mesenchymal stem cells by recombinant adeno-associated virus transduction. 1622 39

Therapeutic modalities aimed at bone regeneration are increasingly employing extracellular matrix (ECM) constituents to control bone marrow progenitor cell (BMPC) commitment, growth, and differentiation. However, the precise role these ECM elements play during stem cell differentiation remains unclear. (See also Salaszynk et al., Stem Cells Dev 14(6):608-620, 2005; and Schwartz et al., Stem Cells Dev. 14(6), 643-655, 2005, both in this issue.) Because bone formation ultimately begins with the recruitment and commitment of BMPCs into the osteogenic lineage, factors that enhance this process are clearly therapeutic targets. We hypothesized that BMPC attachment, proliferation, and osteogenic differentiation would be potentiated when cultured on ECM proteins normally found in the bone niche. To examine this, we cultured murine BMPCs on laminin-1, fibronectin, and collagen type-1 substrates for up to 14 days and assessed their homogeneity, attachment, proliferation, and expression of the specific bone lineage markers RUNX2, collagen-1, alkaline phosphatase, and osteocalcin. We found that freshly harvested mBMPCs contain a mixed population of progenitor cells and that the mesenchymal pool can be enriched by adherent culture in the presence of leucine methyl ester. Furthermore, mBMPCs attached to laminin, fibronectin, and collagen-1 with varying affinity up to 3 h (fibronectin>or=collagen>laminin), after which time no difference could be detected. Despite this, growth was unaffected; cells thereafter proliferated equally well on all substrates up to confluence (7 days). Notably, commitment to the osteoblast lineage (RUNX2) increased up to 14 days for cells cultured on the various substrates, yet no difference was observed at day 14 in the expression of collagen-1, alkaline phosphatase, or osteocalcin. We conclude that mBMPC differentiation down the osteoblastic lineage is time-dependent in osteogenic culture and that attachment to ECM matrices potentiates lineage commitment rather than growth.
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PMID:Substrate induction of osteogenesis from marrow-derived mesenchymal precursors. 1643 18

We have previously found that uremic human serum upregulates RUNX2 in vascular smooth muscle cells (VSMCs), and that RUNX2 is upregulated in areas of vascular calcification in vivo. To confirm the role of RUNX2, we transiently transfected a dominant-negative RUNX2 (DeltaRUNX2) construct in bovine vascular smooth muscle cells (BVSMCs). Blocking RUNX2 transcriptional activity significantly decreased uremic serum induced alkaline phosphatase (ALP) activity (268+/-34 vs 188+/-9.5 U/g protein, P<0.05) and osteocalcin expression (172+/-17 vs 125+/-9 ODU, P<0.05). To determine the mechanism by which uremic serum upregulates RUNX2, we examined cell signaling pathways. BVSMCs were incubated in the presence or absence of inhibitors and RUNX2 expression and ALP activity were determined. The results demonstrate that the cyclic AMP (cAMP)/protein kinase A (PKA), but not protein kinase C, signaling pathway is involved in uremic serum-induced RUNX2 expression and ALP activity in BVSMCs. To examine potential uremic 'toxins', we measured bone morphogenetic protein (BMP)-2 concentration and found that uremic serum contained increased BMP-2 (uremic serum=169+/-33 pg/ml, normal serum=117+/-15 pg/ml, P<0.05). The incubation of BVSMCs with noggin, an inhibitor of BMP, decreased RUNX2 expression. In addition, BMP-2 secretion progressively increased during calcification and uremic serum enhanced its secretion compared to normal serum. In conclusion, this study demonstrates that RUNX2 transcriptional activity is critical in uremic serum-induced bone matrix protein expression in BVSMCs and that the cAMP/PKA pathway is involved. BMP-2 is also increased in uremic serum and can upregulate RUNX2 and calcification in vitro in VSMCs.
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PMID:The mechanisms of uremic serum-induced expression of bone matrix proteins in bovine vascular smooth muscle cells. 1683 22


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