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)

Interaction of type I collagen (COL(I)) with alpha2beta1 integrin causes differentiation and transforming growth factor (TGF)-beta receptor down-regulation in osteoblastic cells (Takeuchi, Y., Nakayama, K., and Matsumoto, T. (1996) J. Biol. Chem. 271, 3938-3644). The TGF-beta receptor down-regulation enables cells to escape from the inhibition of differentiation by TGF-beta. To clarify how the cell-matrix interaction regulates these phenotypic changes, signaling pathways were examined in murine MC3T3-E1 cells. Attachment of cells to COL(I) stimulated tyrosine phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), a mitogen-activated protein kinase (MAPK), and enhanced MAPK activity. Inhibition of tyrosine kinase by herbimycin A, destruction of focal adhesion by cytochalasin D, or overexpression of antisense FAK mRNA prevented the activation of ERK/MAPK and the increase in alkaline phosphatase (ALP) activity. Transient expression of a MAPK-specific phosphatase, CL100, also suppressed the elevation of ALP activity. In addition, introduction of a constitutively active MAPK kinase enhanced ALP activity in the absence of collagen production. TGF-beta receptor down-regulation was abrogated by treatments that inactivate FAK, whereas the expression of CL100 had no effect. These results demonstrate that COL(I)-alpha2beta1 integrin interaction facilitates differentiation and down-regulates TGF-beta receptors via the activation of FAK and its diverse downstream signals. These signaling pathways may play an important role in the sequential differentiation of osteoblasts during bone formation.
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PMID:Differentiation and transforming growth factor-beta receptor down-regulation by collagen-alpha2beta1 integrin interaction is mediated by focal adhesion kinase and its downstream signals in murine osteoblastic cells. 936 Oct 11

To clarify the function of osteopontin in osteoblast differentiation, we have examined the signal transduction pathway in an osteoblastic cell line (UMR106-6) bound to osteopontin, fibronectin, vitronectin and collagen type I surfaces. This was done by investigating the production and autophosphorylation of focal adhesion kinase (FAK) and the expression of alkaline phosphatase (ALP) at the transcription level. Results suggest that osteopontin was not only responsible for the autophosphorylation of FAK but regulated the expression of ALP, which was strongly correlated with FAK activity. These results suggest that osteopontin might act as a trigger in the early differentiation of osteoblasts.
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PMID:Osteopontin involvement in integrin-mediated cell signaling and regulation of expression of alkaline phosphatase during early differentiation of UMR cells. 945 May 60

Cytostatin, which is isolated from a microbial cultured broth as a low molecular weight inhibitor of cell adhesion to extracellular matrix (ECM), has anti-metastatic activity against B16 melanoma cells in vivo. In this study, we examined a target of cytostatin inhibiting cell adhesion to ECM. Cytostatin inhibited tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin upon B16 cell adhesion to fibronectin. While the amount of FAK was not affected by cytostatin, electrophoretically slow-migrating paxillin appeared. Alkaline phosphatase treatment diminished cytostatin-induced slow-migrating paxillin. Furthermore, cytostatin increased intracellular serine/threonine-phosphorylated proteins and was found to be a selective inhibitor of protein phosphatase 2A (PP2A). Cytostatin inhibited PP2A with an IC(50) of 0.09 microgram/ml in a non-competitive manner against a substrate, p-nitrophenyl phosphate, but it had no apparent effect on other protein phosphatases including PP1, PP2B and alkaline phosphatase even at 100 microgram/ml. On the contrary, dephosphocytostatin, a cytostatin analogue, without inhibitory effect on PP2A did not affect B16 cell adhesion including FAK and paxillin. These results indicate that cytostatin inhibits cell adhesion through modification of focal contact proteins such as paxillin by inhibiting a PP2A type protein serine/threonine phosphatase. This is the first report that describes a drug with anti-metastatic ability that inhibits PP2A selectively.
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PMID:Cytostatin, an inhibitor of cell adhesion to extracellular matrix, selectively inhibits protein phosphatase 2A. 1055 74

Some years ago we showed that the Pasteurella multocida toxin (PMT) is a potent mitogen for cells in culture. It is an intracellularly acting toxin that stimulates several signal transduction pathways. The heterotrimeric G-protein, Gq, is stimulated, which in turn causes activation of protein kinase C and an increase in inositol trisphosphates. The Rho GTPase is also activated, leading via the Rho kinase, to activation of the focal adhesion kinase and to cytoskeletal rearrangements. Analysis of the PMT sequence suggested the presence of three domains that encode receptor binding, translocation and catalytic domains. The location of all three domains has been confirmed directly. Competitive binding assays confirmed that the N-terminus of PMT encoded the receptor-binding domain, while cytoplasmic microinjection of expressed PMT fragments identified the location of the C-terminal catalytic domain. Recently, we have demonstrated the presence of key amino acids that affect membrane insertion within the putative transmembrane domain. Several lines of evidence suggest that PMT activates Galphaq, and that this is one potential molecular target for the toxin. Galphaq is known to be tyrosine phosphorylated when activated normally via a G-protein-coupled receptor (GPCR), and it has been suggested that this is an essential part of the activation process. We have shown that PMT induces Galphaq tyrosine phosphorylation, but that this is not essential for activation of the G-protein. Furthermore, a totally inactive mutant of PMT stimulates Galpha phosphorylation without leading to its activation. Phosphorylation of Galphaq triggered by the inactive mutant potentiates activation of Gq via a GPCR, demonstrating that phosphorylation of Gq cannot lead to receptor uncoupling. Natural or experimental infection of animals with toxigenic P. multocida, or injection with purified recombinant PMT causes loss of nasal turbinate bone. The effects on bone have been analysed in vitro using cultures of osteoblasts--cells that lay down bone. PMT blocks the formation of mature calcified bone nodules and the expression of differentiation markers such as CBFA-1, alkaline phosphatase and osteocalcin. These effects can be partially prevented by inhibitors of Rho or Rho kinase function, implicating this pathway in osteoblast differentiation. Indeed, inhibitors of Rho stimulate the formation of bone nodules in vitro. In summary, PMT is a novel toxin that acts via signalling pathways to promote proliferation in many cells, while specifically inhibiting differentiation in osteoblast cells.
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PMID:The pasteurella multocida toxin interacts with signalling pathways to perturb cell growth and differentiation. 1514 25

The interactions of osteoblasts with their surrounding extracellular matrix (ECM) are essential for skeletal development, homeostasis, and maintenance of the mature osteoblastic phenotype. Integrins are the principal transducers of ECM signals that regulate this process of osteoblast commitment and differentiation. Several studies indicate that the alpha(2)beta(1) integrin interaction with type I collagen is a crucial signal for the induction of osteoblastic differentiation and matrix mineralization. Integrin alpha(2)beta(1) recognizes the Gly-Phe-Hyp-Gly-Glu-Arg (GFOGER) motif in residues 502-507 of the alpha(1)[I] chain of type I collagen. This study demonstrates that an alpha(2)beta(1) integrin-specific GFOGER peptide triggers the activation of focal adhesion kinase and alkaline phosphatase in MC3T3-E1 murine immature osteoblast-like cells, two events that have been implicated in the osteoblastic differentiation pathway. These GFOGER-peptide surfaces also support the expression of multiple osteoblast-specific genes, including osteocalcin and bone sialoprotein, and induce matrix mineralization in a manner similar to type I collagen. This triple-helical peptide represents a promising surface modification strategy for the design of collagen-mimetic bioadhesive surfaces that support osteoblastic differentiation.
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PMID:Alpha2beta1 integrin-specific collagen-mimetic surfaces supporting osteoblastic differentiation. 1516

The roles of various soluble factors in promoting the osteogenic differentiation of adult mesenchymal stem cells (MSCs) have been widely studied, but little is known about how the extracellular matrix (ECM) instructs the phenotypic transition between growth and differentiation. To investigate this question, we cultured MSCs on purified vitronectin or type-I collagen, motivated by our earlier tissue engineering work demonstrating that MSC adhesion to polymer scaffolds is primarily mediated by the passive adsorption of these two ECM ligands from serum. Using alkaline phosphatase activity and matrix mineralization as indicators of the early and late stages of osteogenesis, respectively, we report here that both substrates supported differentiation, but the mechanism was substrate dependent. Specifically, osteogenesis on vitronectin correlated with enhanced focal adhesion formation, the activation of focal adhesion kinase (FAK) and paxillin, and the diminished activation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3 kinase (PI3K) pathways. By contrast, MSCs on type-I collagen exhibited reduced focal adhesion formation, reduced activation of FAK and paxillin, and increased activation of ERK and PI3K. Inhibition of ERK and FAK blocked mineral deposition on both substrates, suggesting that the observed differences in signaling pathways ultimately converge to the same cell fate. Understanding these mechanistic differences is essential to predictably control the osteogenic differentiation of MSCs and widen their use in regenerative medicine.
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PMID:Vitronectin and collagen I differentially regulate osteogenesis in mesenchymal stem cells. 1681 99

Human mesenchymal stem cell (hMSC) differentiation into osteoblasts and the signaling events involved are poorly understood. We recently established that contact with specific extracellular matrix (ECM) proteins, in particular laminin-5, is sufficient to induce an osteogenic phenotype in hMSC through an extracellular signal-related kinase (ERK)-dependent pathway. Activation of ERK 1/2 by laminin-5 induces phosphorylation of the runx2/cbfa-1 transcription factor that controls osteogenic gene expression. We hypothesized that focal adhesion kinase (FAK) mediated signaling pathways supply a link between cell surface integrin-ECM binding and activation of ERK 1/2, and that laminin-5 promotes its osteogenic effects through this pathway. To test this hypothesis, we plated hMSC on a laminin-5 matrix in the presence or absence of FAK-specific small inhibitory RNAs (siRNA), and assayed for phosphorylation of runx2/cbfa-1 as well as expression of established osteogenic differentiation markers (bone sialoprotein, osteocalcin, alkaline phosphatase, calcium deposition, and mineral:matrix ratio). We found that siRNA treatment reduced total endogenous FAK protein by approximately 40%, and reduced FAK phosphorylation on Y397 by approximately 33% in cells plated on laminin-5 for 30 min. SiRNA treated cells exhibited a decrease in ERK 1/2 phosphorylation after 1 h, and reduced serine/threonine phosphorylation of Runx2/Cbfa-1 after 8 days. Finally, FAK inhibition blocked osteogenic differentiation of hMSC, as assessed by lowered expression of osteogenic genes (RT-PCR), decreased alkaline phosphatase activity, greatly reduced calcium deposition, and a lower mineral:matrix ratio after 28 days in culture. These results establish FAK as an important mediator of laminin-5-induced osteogenic differentiation of hMSC.
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PMID:Activation of FAK is necessary for the osteogenic differentiation of human mesenchymal stem cells on laminin-5. 1692 79

Soluble GTP-bound transglutaminase 2 (TG2) induces hypertrophic differentiation in chondrocyte cultures in a beta1 integrin-dependent fashion. beta1 integrin subfamily consists of 12 heterodimers with 12 different alpha subunits and a beta1 subunit. To identify the specific integrin heterodimer(s) responsible for this process, we specifically blocked individual beta1 integrins on the CH-8 immortalized human chondrocytes during hypertrophic differentiation. Blockade of alpha5beta1 inhibited matrix metalloproteinase 13 (MMP-13), type X collagen expression, alkaline phosphatase activity and matrix calcification by 30-50% associated with weak effects of anti-alpha3beta1 and -alpha4beta1. Anti-alpha1beta1, -alpha2beta1 and -alpha6beta1 had no effect. To examine whether the dominant effect of integrin alpha5beta1 was due to a direct interaction with TG2, we incubated the chondrocytic cells on plates coated with GTP-bound TG2. The immobilized GTP-bound TG2 induced hypertrophic differentiation to the same extent as the soluble GTP-bound TG2, which was also inhibited by anti-alpha5beta1. CH-8 cells grown on plates coated with GTP-bound TG2 demonstrated adherence associated with focal adhesion kinase phosphorylation. These properties were inhibited by anti-alpha5beta1. Furthermore, engagement of alpha5beta1 on CH-8 cells via anti-alpha5beta1 antibody did, in fact, induce differentiation. Although CH-8 cells adhered to GTP-free TG2 via integrin alpha5beta1, the cells failed to undergo hypertrophic differentiation. Thus, integrin alpha5beta1 is critical for the chondrocyte hypertrophic differentiation induced by GTP-bound TG2, and this induction is ligand dependent.
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PMID:The integrin alpha5beta1 regulates chondrocyte hypertrophic differentiation induced by GTP-bound transglutaminase 2. 1749 Aug 70

The overall mechanisms governing the role of laminins during osteogenic differentiation of human mesenchymal stem cells (hMSC) are poorly understood. We previously reported that laminin-332 induces an osteogenic phenotype in hMSC and does so through a focal adhesion kinase (FAK) and extracellular signal-related kinase (ERK) dependent pathway. We hypothesized that this is a result of integrin-ECM binding, and that it occurs via the known alpha3 LG3 integrin binding domain of laminin-332. To test this hypothesis we cultured hMSC on several different globular domains of laminin-332. hMSC adhered best to the LG3 domain, and this adhesion maximally activated FAK and ERK within 120 min. Prolonged culturing (8 or 16 days) of hMSC on LG3 led to activation of the osteogenic transcription factor Runx2 and expression of key osteogenic markers (osterix, bone sialoprotein 2, osteocalcin, alkaline phosphatase, extracellular calcium) in hMSC. LG3 domain binding did not increase matrix mineralization, demonstrating that the LG3 domain alone is not sufficient to induce complete osteogenic differentiation in vitro. We conclude that the LG3 domain mediates attachment of hMSC to laminin-332 and that this adhesion recapitulates most, but not all, of the osteogenic differentiation associated with laminin-5 binding to hMSC.
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PMID:Dissection of the osteogenic effects of laminin-332 utilizing specific LG domains: LG3 induces osteogenic differentiation, but not mineralization. 1820 71

Bone sialoprotein (BSP) is an abundant protein in the extracellular matrix of bone that has been suggested to have several different physiological functions, including the nucleation of hydroxyapatite (HA), promotion of cell attachment and binding of collagen. Studies in our lab have demonstrated that increased expression of BSP in osteoblast cells can increase expression of the osteoblast-related genes Runx2 and Osx as well as alkaline phosphatase and osteocalcin and increase matrix mineralization. To determine the molecular mechanisms responsible for the BSP-mediated increase in osteoblastic differentiation, several functional domain mutants of BSP were expressed in primary rat bone osteoblastic cells, including the contiguous glutamic acid sequences (polyGlu) and the arginine-glycine-aspartic acid (RGD) motif. Markers of osteoblast differentiation, including matrix mineralization and alkaline phosphatase staining, were increased in cells expressing BSP mutants of the polyGlu sequences but not in cells expressing RGD-mutated BSP. We also determined the dependence on integrin-associated pathways in promoting BSP-mediated differentiation responses in osteoblasts by demonstrating the activation of focal adhesion kinase, MAP kinase-associated proteins ERK1/2, ribosomal s6 kinase 2 and the AP-1 protein cFos. Thus, the mechanism regulating osteoblast differentiation by BSP was determined to be dependent on integrin-mediated intracellular signaling pathways.
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PMID:Activation of the mitogen-activated protein kinase pathway by bone sialoprotein regulates osteoblast differentiation. 1872 50


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