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)

Space flight-induced bone loss has been attributed to a decrease in osteoblast function, without a significant change in bone resorption. To determine the effect of microgravity (MG) on bone, we used the Rotary Cell Culture System [developed by the National Aeronautics and Space Administration (NASA)] to model MG. Cultured mouse calvariae demonstrated a 3-fold decrease in alkaline phosphatase (ALP) activity and failed to mineralize after 7 d of MG. ALP and osteocalcin gene expression were also decreased. To determine the effects of MG on osteoblastogenesis, we cultured human mesenchymal stem cells (hMSC) on plastic microcarriers, and osteogenic differentiation was induced immediately before the initiation of modeled MG. A marked suppression of hMSC differentiation into osteoblasts was observed because the cells failed to express ALP, collagen 1, and osteonectin. The expression of runt-related transcription factor 2 was also inhibited. Interestingly, we found that peroxisome proliferator-activated receptor gamma (PPARgamma2), which is known to be important for adipocyte differentiation, adipsin, leptin, and glucose transporter-4 are highly expressed in response to MG. These changes were not corrected after 35 d of readaptation to normal gravity. In addition, MG decreased ERK- and increased p38-phosphorylation. These pathways are known to regulate the activity of runt-related transcription factor 2 and PPARgamma2, respectively. Taken together, our findings indicate that modeled MG inhibits the osteoblastic differentiation of hMSC and induces the development of an adipocytic lineage phenotype. This work will increase understanding and aid in the prevention of bone loss, not only in MG but also potentially in age-and disuse-related osteoporosis.
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PMID:Modeled microgravity inhibits osteogenic differentiation of human mesenchymal stem cells and increases adipogenesis. 1474 52

Runx2 (runt-related transcription factor 2) deficient mice lacked the mandibular condylar cartilage and the mandibular bone. The anlage of the condylar process consisted of mesenchymal condensation, which expressed Type I collagen mRNA and alkaline phosphatase activity, but not Type II collagen and aggrecan mRNAs. Therefore, the differentiation of the mandibular condylar cartilage stopped at the preosteoblast (skeletoblast) stage. The lateral pterygoid muscle was attached to this anlage, and relatively abundant mesenchymal condensations were also formed at the muscle-attaching sites, e.g. the anlage of the mandibular body, the angular and coronoid processes. Three-dimensional reconstruction models showed that each mesenchymal condensation was connected to one another, and roughly outlined the shape of the mandible. Meckel's cartilage in the Runx2-deficient mice had two ectopic cartilaginous processes to which the digastric and myohyoid muscles were attached. These findings indicate that Runx2 is essential for the formation of the mandibular condylar cartilage, as well as for normal development of Meckel's cartilage and that muscle tissues influence mandible morphology.
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PMID:Runx2-deficient mice lack mandibular condylar cartilage and have deformed Meckel's cartilage. 1515 1

Exposure to microgravity causes bone loss in humans, and the underlying mechanism is thought to be at least partially due to a decrease in bone formation by osteoblasts. In the present study, we examined the hypothesis that microgravity changes osteoblast gene expression profiles, resulting in bone loss. For this study, we developed an in vitro system that simulates microgravity using the Random Positioning Machine (RPM) to study the effects of microgravity on 2T3 preosteoblast cells grown in gas-permeable culture disks. Exposure of 2T3 cells to simulated microgravity using the RPM for up to 9 days significantly inhibited alkaline phosphatase activity, recapitulating a bone loss response that occurs in real microgravity conditions without altering cell proliferation and shape. Next, we performed DNA microarray analysis to determine the gene expression profile of 2T3 cells exposed to 3 days of simulated microgravity. Among 10,000 genes examined using the microarray, 88 were downregulated and 52 were upregulated significantly more than twofold using simulated microgravity compared with the static 1-g condition. We then verified the microarray data for some of the genes relevant in bone biology using real-time PCR assays and immunoblotting. We confirmed that microgravity downregulated levels of alkaline phosphatase, runt-related transcription factor 2, osteomodulin, and parathyroid hormone receptor 1 mRNA; upregulated cathepsin K mRNA; and did not significantly affect bone morphogenic protein 4 and cystatin C protein levels. The identification of gravisensitive genes provides useful insight that may lead to further hypotheses regarding their roles in not only microgravity-induced bone loss but also the general patient population with similar pathological conditions, such as osteoporosis.
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PMID:Simulated microgravity using the Random Positioning Machine inhibits differentiation and alters gene expression profiles of 2T3 preosteoblasts. 1568 15

Nrf2 (nuclear factor E2 p45-related factor 2) is believed to be a transcription factor essential for the regulation of many detoxifying and antioxidative genes in different tissues. In the present study, we investigated the role of Nrf2 in the regulation of osteoblastic differentiation. nrf2 mRNA expression was significantly up-regulated in femur isolated from ovariectomized mice, whereas in situ hybridization analysis revealed that up-regulation of nrf2 mRNA was mainly found in osteoblasts attached on cancellous bone in femur of ovariectomized mice. Expression of Nrf2 protein was also seen in osteoblasts in neonatal mouse tibia and calvaria. In osteoblastic MC3T3-E1 cells stably transfected with nrf2 expression vector, significant inhibition was seen in the maturation-dependent increase in alkaline phosphatase activity as well as the mineralized matrix formation. Stable overexpression of nrf2 significantly impaired Runx2 (runt-related transcription factor 2)-dependent stimulation of osteocalcin promoter activity and recruitment of Runx2 on osteocalcin promoter without affecting the expression of runx2 mRNA. Coimmunoprecipitation and mammalian two-hybrid assay revealed a physical interaction between Runx2 and Nrf2, whereas cellular distribution of endogenous Runx2 was not apparently changed by nrf2 overexpression in MC3T3-E1 cells. Alternatively, Nrf2 bound to antioxidant-responsive element-like-2 sequence of osteocalcin promoter. The inhibition by nrf2 on runx2-dependent osteocalcin promoter activity was partially prevented by the introduction of reporter of deletion mutant for ARE-like-2 sequence of osteocalcin promoter. These data suggest that Nrf2 may negatively regulate cellular differentiation through inhibition of the Runx2-dependent transcriptional activity in osteoblasts.
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PMID:Nrf2 negatively regulates osteoblast differentiation via interfering with Runx2-dependent transcriptional activation. 1661 47

To study cellular characteristics of human cementoblasts using a cellular model is important for understanding the mechanisms of homeostasis and regeneration of periodontal tissues. However, at present no immortalized human cementoblast cell line has been established due to limitation of the life span. In the present study, therefore, we attempted to establish human cementoblast-like cell lines by transfection with telomerase catalytic subunit hTERT gene. Two stable clones (HCEM-1 and -2) with high telomerase activity were obtained and they grew over 200 population doublings without significant growth retardation. The expression of mRNA for differentiation markers, type I collagen, alkaline phosphatase (ALP), runt-related transcription factor 2, osteocalcin, bone sialoprotein and cementum-derived protein was revealed in these clones by RT-PCR. Moreover, these cells showed high ALP activity and calcified nodule formation in vitro. Interestingly, HCEM-2 showed cementum like formation on the surface of hydroxyapatites granules by subcutaneous transplantation into immunodeficient mice with hydroxyapatite granules. Thus, we established human cementoblast-like cell lines. We suggest that HCEM cell lines can be useful cell models for investigating the characteristics of human cementoblasts.
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PMID:Characterization of established cementoblast-like cell lines from human cementum-lining cells in vitro and in vivo. 1685 33

Apoptosis through Fas/Fas ligand (FasL) is an important regulator of immune system homeostasis but its role in bone homeostasis is elusive. We systematically analyzed: 1) the expression of Fas/FasL during osteoblastogenesis and osteoclastogenesis in vitro, 2) the effect of FasL on apoptosis and osteoblastic/osteoclastic differentiation, and 3) osteoblastogenesis and osteoclastogenesis in mice deficient in Fas or FasL. The expression of Fas increased with osteoblastic differentiation. Addition of FasL weakly increased the proportion of apoptotic cells in both osteoclastogenic and osteoblastogenic cultures. In a CFU assay, FasL decreased the proportion of osteoblast colonies but did not affect the total number of colonies, indicating specific inhibitory effect of Fas/FasL on osteoblastic differentiation. The effect depended on the activation of caspase 8 and was specific, as addition of FasL to osteoblastogenic cultures significantly decreased gene expression for runt-related transcription factor 2 (Runx2) required for osteoblastic differentiation. Bone marrow from mice without functional Fas or FasL had similar osteoclastogenic potential as bone marrow from wild-type mice, but generated more osteoblast colonies ex vivo. These colonies had increased expression of the osteoblast genes Runx2, osteopontin, alkaline phosphatase, bone sialoprotein, osteocalcin, and osteoprotegerin. Our results indicate that Fas/FasL system primarily controls osteoblastic differentiation by inhibiting progenitor differentiation and not by inducing apoptosis. During osteoclastogenesis, the Fas/FasL system may have a limited effect on osteoclast progenitor apoptosis. The study suggests that Fas/FasL system plays a key role in osteoblastic differentiation and provides novel insight into the interactions between the immune system and bone.
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PMID:The Fas/Fas ligand system inhibits differentiation of murine osteoblasts but has a limited role in osteoblast and osteoclast apoptosis. 1733 32

Plastic compression of hyperhydrated collagen gels produces tissue-like scaffolds of enhanced biomechanical properties. By increasing collagen density, these scaffolds could be developed into highly Biomimetic cell-seeded templates. When utilizing three-dimensional (3-D) scaffold systems for tissue repair, and indeed when investigating the cytocompatibility of two-dimensional (2-D) surfaces, the cell seeding density is often overlooked. In this study, we investigated this potentially critical parameter using MG-63 cells seeded in the dense collagen scaffolds. This is conducted within the overall scope of developing these scaffolds for bone repair. Cell proliferation, osteoblastic differentiation, and matrix remodelling capacity in relation to various seeding densities, ranging from 10(5) to 10(8) cells/ml compressed collagen, were evaluated in vitro. This was performed using the AlamarBlue assay, quantitative polymerase chain reaction (qPCR), and tensile mechanical analysis respectively. Variations in cell seeding density significantly influenced cell proliferation where lower initial seeding density resulted in higher proliferation rates as a function of time in culture. Gene transcription levels for alkaline phosphatase (ALPL), runt-related transcription factor 2 (RUNX2), and osteonectin (SPARC) were also found to be dependent on the cell density. While ALPL transcription was down-regulated with culturing time for all seeding densities, there was an increase in RUNX2 and SPARC transcription, particularly for scaffolds with cell densities in the range 10(6)-10(7) cells/ml collagen. Furthermore, this range of seeding density affected cell capacity in conducting collagenous matrix degradation as established by analyzing matrix metalloproteinase 1 (MMP1) transcription and scaffold mechanical properties. This study has shown that the seeded cell population in the three-dimensional dense collagen scaffolds clearly affected the degree of osteoblastic cell proliferation, differentiation, and some aspects of matrix remodelling activity. The seeding density played a major role in influencing the corresponding cell differentiation and cell-matrix interaction.
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PMID:Effect of cell density on osteoblastic differentiation and matrix degradation of biomimetic dense collagen scaffolds. 1809 52

Bone cells and their precursors are sensitive to changes in their biomechanical environment. The importance of mechanical stimuli has been observed in bone homeostasis and osteogenesis, but the mechanisms responsible for osteogenic induction in response to mechanical signals are poorly understood. We hypothesized that compressive forces could exert an osteogenic effect on osteoblasts and act in a dose-dependent manner. To test our hypothesis, electrospun poly(epsilon-caprolactone) (PCL) scaffolds were used as a 3-D microenvironment for osteoblast culture. The scaffolds provided a substrate allowing cell exposure to levels of externally applied compressive force. Pre-osteoblasts adhered, proliferated and differentiated in the scaffolds and showed extensive matrix synthesis by scanning electron microscopy (SEM) and increased Young's modulus (136.45+/-9.15 kPa) compared with acellular scaffolds (24.55+/-8.5 kPa). Exposure of cells to 10% compressive strain (11.81+/-0.42 kPa) resulted in a rapid induction of bone morphogenic protein-2 (BMP-2), runt-related transcription factor 2 (Runx2), and MAD homolog 5 (Smad5). These effects further enhanced the expression of genes and proteins required for extracellular matrix (ECM) production, such as alkaline phosphatase (Akp2), collagen type I (Col1a1), osteocalcin/bone gamma carboxyglutamate protein (OC/Bglap), osteonectin/secreted acidic cysteine-rich glycoprotein (ON/Sparc) and osteopontin/secreted phosphoprotein 1 (OPN/Spp1). Exposure of cell-scaffold constructs to 20% compressive strain (30.96+/-2.82 kPa) demonstrated that these signals are not osteogenic. These findings provide the molecular basis for the experimental and clinical observations that appropriate physical activities or microscale compressive loading can enhance fracture healing due in part to the anabolic osteogenic effects.
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PMID:Compressive forces induce osteogenic gene expression in calvarial osteoblasts. 1819 Nov 37

Interstitial flow in and around tumor tissue affects the mechanical microenvironment to modulate tumor cell growth and metastasis. We investigated the roles of flow-induced shear stress in modulating cell cycle distribution in four tumor cell lines and the underlying mechanisms. In all four cell lines, incubation under static conditions for 24 or 48 h led to G(0)/G(1) arrest; in contrast, shear stress (12 dynes/cm(2)) induced G(2)/M arrest. The molecular basis of the shear effect was analyzed, and the presentation on molecular mechanism is focused on human MG63 osteosarcoma cells. Shear stress induced increased expressions of cyclin B1 and p21(CIP1) and decreased expressions of cyclins A, D1, and E, cyclin-dependent protein kinases (Cdk)-1, -2, -4, and -6, and p27(KIP1) as well as a decrease in Cdk1 activity. Using specific antibodies and small interfering RNA, we found that the shear-induced G(2)/M arrest and corresponding changes in G(2)/M regulatory protein expression and activity were mediated by alpha(v)beta(3) and beta(1) integrins through bone morphogenetic protein receptor type IA-specific Smad1 and Smad5. Shear stress also down-regulated runt-related transcription factor 2 (Runx2) binding activity and osteocalcin and alkaline phosphatase expressions in MG63 cells; these responses were mediated by alpha(v)beta(3) and beta(1) integrins through Smad5. Our findings provide insights into the mechanism by which shear stress induces G(2)/M arrest in tumor cells and inhibits cell differentiation and demonstrate the importance of mechanical microenvironment in modulating molecular signaling, gene expression, cell cycle, and functions in tumor cells.
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PMID:Tumor cell cycle arrest induced by shear stress: Roles of integrins and Smad. 1831 Mar 19

To investigate the cascade of matrix mineralization, cells expressing high and low alkaline phosphatase (ALP) were separated from human osteoblast-like (HOS) cells by fluorescence-activated cell sorting with an ALP antibody. After these cells had been recloned from single cells and then cultured under osteogenic conditions, high-ALP-expressing HOS (H-HOS) cells showed matrix mineralization, but low-ALP-expressing HOS (L-HOS) cells did not. The interaction among osteogenic-related genes, such as runt-related transcription factor 2 (RUNX2), collagen type I alpha1 chain (COL1A1), tissue non-specific ALP, and osteocalcin (OCN), is well known as being related to matrix mineralization. Quantitative real-time polymerase chain reaction revealed that the gene expression of ALP was higher in H-HOS cells than in L-HOS, whereas the gene expression of RUNX2, COL1A1, and OCN was lower in H-HOS cells than in L-HOS cells. When small interfering RNAs (siRNAs) of these osteogenic-related genes were introduced into H-HOS cells by transfection, only ALP siRNA inhibited matrix mineralization. Furthermore, the expression of not only the ALP gene, but also the COL1A1 and RUNX2 genes was influenced by the inhibition of ALP, although the expression of OCN was not affected by the inhibition of ALP. We have been able to confirm that the ALP gene is a strong candidate as the trigger of matrix mineralization. These results indicate the usefulness of cloned osteogenic cells in investigating the molecular mechanisms of matrix mineralization, the function of which can be modulated by using a variety of siRNAs.
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PMID:Small interfering RNA of alkaline phosphatase inhibits matrix mineralization. 1831 13


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