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

Mesenchymal stem cells (MSCs) are well known to possess multipotential differentiation and are becoming a good tool for clinical research. However, specific markers for their purification and the mechanism of their osteogenic differentiation remain to be elucidated. In the present study, we compared the expression of CD106, and osteogenic differentiation-related proteins and genes in human bone marrow (BM)-derived MSCs, before and after differentiation by FACS, histochemical staining, immunohistochemical staining, RT-PCR, and real-time PCR. It was found that MSCs were positive for CD13, CD29, CD44, CD73, CD90, CD105, and CD166, but negative for CD14, CD31, CD34, CD62E, CD45, and GlyA. Notably, CD106 was detected before osteogenic induction, but its expression was downregulated 10 fold after 2 weeks of osteogenic differentiation as determined by flow cytometry. The results of RT-PCR and real-time PCR revealed that the expression of CD106 mRNA in MSCs significantly decreased by 7.1-, 4.2-, and 5.1-fold, respectively after osteogenic, chondrogenic, and adipogenic differentiation. In contrast, other MSC-positive markers described above did not change significantly even after differentiation. Compared to levels in control cells, after 2 weeks of osteogenic differentiation, mRNA levels of alkaline phosphatase, bone sialoprotein, osteocalcin, and transcript factors RUNX2 and Osterix showed more than 2-fold, 5-fold, 1.5-fold, 2-fold, and 5-fold increase, respectively. Thus, we speculate that CD106 might be a useful surface marker for BMMSCs. Moreover, alkaline phosphatase, type I collagen, osteonectin, osteopontin, and biglycin were expressed in the early stages of osteogenic differentiation before bone sialoprotein and osteocalcin. The present study should help to provide a novel marker for isolating purified MSCs and characterizing osteogenic differentiation.
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PMID:Changes in the expression of CD106, osteogenic genes, and transcription factors involved in the osteogenic differentiation of human bone marrow mesenchymal stem cells. 1860 Mar 96

BMP2 signaling and RUNX2 regulatory pathways converge for transcriptional control of bone formation in vivo. SMAD proteins are recruited to RUNX2 regulatory complexes via an overlapping nuclear matrix targeting signal/Smad interacting domain sequence (391-432) in Runx2. To establish the contribution of RUNX2-SMAD interaction to osteoblastogenesis, we characterized a number of point mutants. Only a triple mutation of amino acids 426-428 (HTY-AAA) results in loss of RUNX2 interactions with either BMP2- or TGF-beta- responsive SMADs and fails to integrate the BMP2/TGF-beta signal on target gene promoters. In a Runx2 null cell reconstitution assay, the HTY mutant did not activate the program of osteoblast differentiation (alkaline phosphatase, collagen type 1, osteopontin, bone sialoprotein and osteocalcin) in response to BMP2 signaling. Thus, subnuclear targeting function and formation of a RUNX2-SMAD osteogenic complex are functionally inseparable. Taken together, these studies provide direct evidence that RUNX2 is essential for execution and completion of BMP2 signaling for osteoblast differentiation.
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PMID:Specific residues of RUNX2 are obligatory for formation of BMP2-induced RUNX2-SMAD complex to promote osteoblast differentiation. 1872 44

Osteoblasts differentiate from mesodermal progenitors and play a pivotal role in bone formation and mineralization. Several transcription factors including runt-related transcription factor 2 (RUNX2), Osterix (OSX), and activating transcription factor4 (ATF4) are known to be crucial for the process, whereas the upstream signal transduction controlling the osteoblast differentiation sequence is largely unknown. Here, we explored the role of c-jun N-terminal kinase (JNK) in osteoblast differentiation using in vitro differentiation models of primary osteoblasts and MC3T3-E1 cells with ascorbic acid/beta-glycerophosphate treatment. Terminal osteoblast differentiation, represented by matrix mineralization, was significantly inhibited by the inactivation of JNK with its specific inhibitor and exogenous overexpression of MKP-M (MAP kinase phosphatase isolated from macrophages), which preferentially inactivates JNK. Conversely, enhanced mineral deposition was observed by inducible overexpression of p54(JNK2), whereas it was not observed by the overexpression of p46(JNK1) or p46(JNK2), indicating a distinct enhancing role of p54(JNK2) in osteoblast differentiation. Inactivation of JNK significantly inhibited late-stage molecular events of osteoblast differentiation, including gene expression of osteocalcin (Ocn) and bone sialoprotein (Bsp). In contrast, earlier differentiation events including alkaline phosphatase (ALP) activation and osteopontin (Opn) expression were not inhibited by JNK inactivation. Although the expression levels of two transcription factor genes, Runx2 and Osx, were not significantly affected by JNK inactivation, induction of Atf4 mRNA during osteoblast differentiation was significantly inhibited. Taken together, these data indicate that JNK activity is specifically required for the late-stage differentiation events of osteoblasts.
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PMID:JNK activity is essential for Atf4 expression and late-stage osteoblast differentiation. 1901 86

The osteoblastic cell-line hFOB 1.19 with the potential to proliferate and differentiate revealed that cellular differentiation is not affected by material and roughness on newly developed zirconia implant materials. Materials under investigation were surfaces machined titanium (Ti-m), modified titanium (TiUnite, machined zirconia (TZP-A-m), modified zirconia (ZiUnitemachined alumina-toughened zirconia (ATZ-m) and modified alumina-toughened zirconia (ATZ-mod). After surface description by scanning electron microscopy (SEM) and atomic force microscopy (AFM), cellular proliferation (EZ4U, Casy1) and differentiation were examined after days 1, 3, 7, 14, 21, and 28. Osteogenic differentiation was visualized by alkaline phosphatase staining, mineralization assay (alizarin red) and by expression analysis (RT-PCR) of bone- and extracellular matrix-related genes. Proliferation on rough surfaces was reduced on both titanium and zirconia. Cell-attachment and cytoskeleton organization documented by confocal laser scanning microscopy (CLSM) elucidated attenuated cell attachment within the first 4h to be the reason for impaired proliferation. A specific up-regulation of m-RNAs in an early event (RUNX2, NELL-1, RUNX3, and BMP7) and a late event (Integrin B3) could be observed on TiUnite and ZiUnite. For titanium an up-regulation of IBSP and Integrin B1 could be described at day 21. In total, differentiation was neither affected by material nor by roughness.
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PMID:The gene-expression and phenotypic response of hFOB 1.19 osteoblasts to surface-modified titanium and zirconia. 1902 46

The compliance of the extracellular matrix (ECM) regulates osteogenic differentiation by modulating extracellular signal-regulated kinase (ERK) activity. However, the molecular mechanism linking ECM compliance to the ERK-mitogen-activated protein kinase (MAPK) pathway remains unclear. Furthermore, RhoA has been widely implicated in integrin-mediated signaling and mechanotransduction. We studied the relationship between RhoA and ERK-MAPK signaling to determine their roles in the regulation of osteogenesis by ECM compliance. Inhibition of RhoA and ROCK in MC3T3-E1 pre-osteoblasts cultured on substrates of varying compliance reduced ERK activity, whereas constitutively active RhoA enhanced it. The expression of RUNX2, a potent osteogenic transcription factor, was increased on stiffer matrices and correlated with elevated ERK activity. Inhibition of RhoA, ROCK, or the MAPK pathway diminished RUNX2 activity and delayed the onset of osteogenesis as shown by altered osteocalcin (OCN) and bone sialoprotein (BSP) gene expression, alkaline phosphatase (ALP) activity, and matrix mineralization. These data establish that one possible mechanism by which ECM rigidity regulates osteogenic differentiation involves MAPK activation downstream of the RhoA-ROCK signaling pathway.
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PMID:ECM compliance regulates osteogenesis by influencing MAPK signaling downstream of RhoA and ROCK. 1911 8

We have previously shown that the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects bone growth, modelling and mechanical strength in vivo. In this study, we utilized differentiation of bone marrow stem cells to osteoblasts and osteoclasts as a model system to study the effects of TCDD on bones. Stem cells were isolated from bone marrow of femurs and tibias of rats and mice. Progress of osteoblastic differentiation was monitored by measuring mRNA expression levels of differentiation markers from control and TCDD-treated cells using quantitative RT-PCR. TCDD significantly and dose-dependently decreased the mRNA levels of RUNX2, alkaline phosphatase and osteocalcin. Also the activity of alkaline phosphatase was significantly inhibited in both rat and mice cells. In the case of osteoclasts, TCDD decreased the number of TRACP+ multinucleated cells, with corresponding decreases in the number of F-actin rings and the area of resorption. Studies in AHR-knockout mice indicated that TCDD has no effect on the expression of osteoblastic differentiation markers suggesting that TCDD mediates its effects by AHR. Both osteoblastic and osteoclastic effects took place at very low doses of TCDD, as in most cases 100 fM TCDD was enough to significantly affect the differentiation markers. Therefore, differentiation of osteoblasts and osteoclasts from bone marrow stem cells seems to be a very sensitive target for TCDD. Disrupting effects in osteoblastic cells, in addition to disturbed osteoclastogenesis, may thus play a role in adverse effects on bone quality in TCDD exposed animals.
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PMID:Dioxins interfere with differentiation of osteoblasts and osteoclasts. 1926 58

Mesenchymal stem cells are multipotent cells able to differentiate into different mesenchymal lineages. Studies in the past had suggested that two of these mesenchymal differentiation directions, the chondrogenic and the myogenic differentiation, are negatively regulated by the transcription factor NF-kappaB. Although osteogenic differentiation has been extensively studied, the influence of NF-kappaB on this differentiation lineage was not subject of detailed analyses in the past. We have analyzed the consequences of TNF-alpha treatment and genetic manipulation of the NF-kappaB pathway for osteogenic differentiation of hMSCs. Treatment of hMSCs during differentiation with TNF-alpha activates NF-kappaB and this results in enhanced expression of osteogenetic proteins like bone morphogenetic protein2 (BMP-2) and alkaline phosphatase (ALP). In addition, enhanced matrix mineralization was observed. The direct contribution of the NF-kappaB pathway was confirmed in cells that express a constitutively active version of the NF-kappaB-inducing kinase IKK2 (CA-IKK2). The IKK2/NF-kappaB-induced BMP-2 up-regulation results in the enhancement of RUNX2 and Osterix expression, two critical regulators of the osteogenic differentiation program. Interestingly, a genetic block of the NF-kappaB pathway did not interfere with osteogenic differentiation. We conclude that TNFalpha mediated NF-kappaB activation, although not absolutely required for BMP-2 expression and matrix mineralization nevertheless supports osteogenic differentiation and matrix mineralization by increasing BMP-2 expression. Our results therefore suggest that NF-kappaB activation may function in lineage selection during differentiation of hMSCs by fostering osteogenic differentiation at the expense of other differentiation lineages.
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PMID:TNFalpha promotes osteogenic differentiation of human mesenchymal stem cells by triggering the NF-kappaB signaling pathway. 1941 75

Bone marrow stromal cells (BMSCs) have been shown to contribute to regeneration of numerous mesodermal tissue types including adipose, bone, and cartilage. Recent studies have shown that BMSCs migrate into damaged bone and help facilitate effects such as fracture healing. Although bone morphogenic proteins have been shown to stimulate bone repair, their levels remain low postfracture. Peripheral blood levels of transforming growth factor beta1 (TGF-beta1), on the other hand, rise dramatically within 2 weeks postfracture. Therefore, we investigated the role of TGF-beta1 on BMSC osteogenic differentiation in vitro. Murine BMSCs were freshly isolated from femurs, fluorescence-activated cell sorted for Sca-1, cultured in Iscove's modified Dulbecco's medium, and exposed to TGF-beta1. After 14 days, real-time reverse transcriptase-polymerase chain reaction and immunohistochemical staining were performed to examine the expression of self-renewal and terminal differentiation markers. Results showed that the treatment with TGF-beta1 reduced mRNA levels of self-renewal markers (Oct4, Stella, Nanos3, and Abcg2) by twofold and increased osteoblast differentiation markers (Runx2, Opn, and Col1) up to sevenfold compared with controls. We also observed decreased mRNA levels of adipogenic markers (Pparg2 and Adn) and an increase in alkaline phosphatase activity. Transcriptional coactivator with PDZ-binding motif (TAZ) mRNA and protein levels were elevated up to threefold following TGF-beta1 stimulation. In conclusion, our findings revealed an unexpected osteogenic differentiation pathway in murine BMSCs under the control of TGF-beta that is mediated by TAZ, which is known to increase RUNX2-dependent gene transcription while repressing PPARgamma2-dependent transcription. This is the first report demonstrating the upregulation of TAZ activity in BMSCs by a physiological growth factor present during acute bone injury.
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PMID:Transforming growth factor beta1 induces osteogenic differentiation of murine bone marrow stromal cells. 1976 30

Vascular calcification is implicated in many diseases including atherosclerosis and diabetes. Tumor necrosis factor-alpha (TNF-alpha) has been shown to promote vascular calcification both in vitro and in vivo. However, the molecular mechanism of TNF-alpha-mediated vascular calcification has not yet been fully defined. Therefore, in this study, we aimed to investigate whether MSX2 acts as a crucial regulator in TNF-alpha-induced vascular calcification and to define the regulatory mechanism of MSX2 induction in human vascular smooth muscle cells (VSMCs). TNF-alpha increased the expression of osteogenic marker genes including RUNX2, osterix, alkaline phosphatase (ALP), and bone sialoprotein, and it also promoted matrix mineralization in VSMCs. In addition, TNF-alpha enhanced MSX2 expression in a dose- and time-dependent manner. MSX2 over-expression alone induced ALP expression, whereas knockdown of MSX2 with small interfering RNA completely blocked TNF-alpha-induced ALP expression. New protein synthesis was dispensable for MSX2 induction by TNF-alpha, and the inhibition of NF-kappaB by BAY-11-7082 or by dominant negative IkappaBalpha abolished the TNF-alpha-directed induction of MSX2 expression. However, inhibition of NADPH oxidase did not affect MSX2 expression. In conclusion, our study suggests that TNF-alpha directly induces MSX2 expression through the NF-kappaB pathway, which in turn induces expression of ALP, a key molecule in mineralization, in VSMCs.
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PMID:Tumor necrosis factor-alpha increases alkaline phosphatase expression in vascular smooth muscle cells via MSX2 induction. 2000 46

Cleidocranial dysplasia (CCD) is a rare autosomal dominant skeletal dysplasia due to mutations causing haploinsufficiency of RUNX2, an osteoblast transcription factor specific for bone and cartilage. The classic form of CCD is characterized by delayed closure of the fontanels, hypoplastic or aplastic clavicles and dental anomalies. Clinical reports suggest that a subset of patients with CCD have skeletal changes which mimic hypophosphatasia (HPP). Mutations in RUNX2 are detected in approximately 65% of cases of CCD, and microdeletions occur in 13%. We present clinical and radiological features in a 6-year-old child with severe CCD manifested by absence of the clavicles marked calvarial hypomineralization, osteoporosis and progressive kyphoscoliosis. HPP features included Bowdler spurs, severe osteopenia, and low alkaline phosphatase. Following negative mutation analysis of RUNX2, comparative genomic hybridization (CGH) microarray was performed. The result revealed a microdeletion in RUNX2, disrupting the C-terminal part of the gene.
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PMID:Severe cleidocranial dysplasia and hypophosphatasia in a child with microdeletion of the C-terminal region of RUNX2. 2001 32


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