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)

A growing interest in the understanding of the ontogeny and mineralization of fish skeleton has emerged from the recent implementation of fish as a vertebrate model, particularly for skeletal development. Whereas several in vivo studies dealing with the regulation of bone formation in fish have been published, in vitro studies have been hampered because of a complete lack of fish-bone-derived cell systems. We describe here the development and the characterization of two new cell lines, designated VSa13 and VSa16, derived from the vertebra of the gilthead sea bream. Both cell types exhibit a spindle-like phenotype and slow growth when cultured in Leibovitz's L-15 medium and a polygonal phenotype and rapid growth in Dulbecco's modified Eagle medium (D-MEM). Scanning electron microscopy and von Kossa staining have revealed that the VSa13 and VSa16 cells can only mineralize their extracellular matrix when cultured in D-MEM under mineralizing conditions, forming calcium-phosphate crystals similar to hydroxyapatite. We have also demonstrated the involvement of alkaline phosphatase, a marker of bone formation in vivo, and Gla proteins (osteocalcin and matrix Gla protein, MGP) in the process of mineralization. Finally, we have shown that VSa13 and VSa16 cell lines express osteocalcin and MGP in a mutually exclusive manner. Thus, both cell lines are capable of mineralizing in vitro and of expressing genes found in chondrocyte and osteoblast cell lineages, emphasizing the suitability of these new cell lines as valuable tools for analyzing the expression and regulation of cartilage- and bone-specific genes.
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PMID:Development of two bone-derived cell lines from the marine teleost Sparus aurata; evidence for extracellular matrix mineralization and cell-type-specific expression of matrix Gla protein and osteocalcin. 1476 9

Osteoblast differentiation is controlled by multiple transcription factors, Runx2, AJ18, Osterix, Dlx5 and Msx2. The mechanisms of regulation of AJ18 mRNA expression by the transforming growth factor beta (TGF-beta) superfamily remain poorly understood. However, it is known that BMP-2 induces differentiation of C26 cells into more mature osteoblastic cells. The present study, using Northern blot and real-time reverse transcription polymerase chain reaction analyses, investigated the effects of bone morphogenetic protein-2 (BMP-2) and TGF-beta1 on mRNA expression of AJ18 and Runx2 in a clonal osteoblast precursor cell line ROB-C26 (C26) cultured for 3, 6 or 9 days in the presence or absence of BMP-2. Although mRNA expression of Osterix and bone sialoprotein (BSP) was undetectable in the C26 culture, BMP-2 induced Osterix expression on days 3-9, but not BSP expression. BMP-2 also stimulated significantly Dlx5 expression on days 3-9, Msx2 and matrix Gla protein expressions on days 3 and 6, Runx2, alkaline phosphatase and osteocalcin expressions on days 6 and 9 in the culture. Furthermore, BMP-2 increased significantly Smad5 mRNA in the culture on day 3, indicating BMP-2 involvement in the regulation of Smad5 mRNA expression. In contrast, the inhibitory effects of BMP-2 on AJ18 mRNA expression were significant on days 3-9, indicating that a decrease in AJ18 mRNA expression is essential for the increased osteoblastic differentiation. Furthermore, TGF-beta1 (0, 0.1, 1.0 and 5.0 ng/ml) treatment of C26 cells cultured for 6 days in the presence or absence of BMP-2 for 24h stimulated mRNA levels of AJ18 and Runx2, maximal stimulation occurring principally at 1.0 ng/ml. These observations indicate that the expression of AJ18 and Runx2 mRNAs in C26 cells is under the control of BMP-2 and TGF-beta1, which exert different effects on AJ18 mRNA expression, but are potent stimulators of Runx2 mRNA expression during osteoblast differentiation.
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PMID:Effects of bone morphogenetic protein-2 and transforming growth factor beta1 on gene expression of transcription factors, AJ18 and Runx2 in cultured osteoblastic cells. 1532 53

The negative effect of acidosis on the skeleton has been known for almost a century. Bone mineral serves an important pathophysiologic role as a reserve of hydroxyl ions to buffer systemic protons if the kidneys and lungs are unable to maintain acid-base balance within narrow physiologic limits. Extracellular hydrogen ions are now thought to be the primary activation signal for osteoclastic bone resorption, and osteoclasts are very sensitive to small changes in pH within the pathophysiologic range. Herein, we investigated the effects of acidosis on osteoblast function by using mineralized bone nodule-forming primary osteoblast cultures. Osteoblasts harvested from neonatal rat calvariae were cultured up to 21 days in serum-containing medium, with ascorbate, beta-glycerophosphate and dexamethasone. pH was manipulated by addition of 5 to 30 mmol/L HCl and monitored by blood gas analyzer. Abundant, matrix-containing mineralized nodules formed in osteoblast cultures at pH 7.4, but acidification progressively reduced mineralization of bone nodules, with complete abolition at pH 6.9. Osteoblast proliferation and collagen synthesis, assessed by 3H-thymidine and 3H-proline incorporation, respectively, were unaffected by pH in the range 7.4 to 6.9; no effect of acidification on collagen ultrastructure and organization was evident. The apoptosis rate of osteoblasts, assessed by the enrichment of nucleosomes in cell lysates, was also unaffected by pH within this range. However, osteoblast alkaline phosphatase activity, which peaked strongly near pH 7.4, was reduced eight-fold at pH 6.9. Reducing pH to 6.9 also downregulated messenger ribonucleic acid (mRNA) for alkaline phosphatase, but upregulated mRNA for matrix Gla protein, an inhibitor of mineralization. The same pH reduction is associated with two-and four-fold increases in Ca2+ and PO4(3-) solubility for hydroxyapatite, respectively. Our results show that acidosis exerts a selective, inhibitory action on matrix mineralization that is reciprocal with the osteoclast activation response. Thus, in uncorrected acidosis, the deposition of alkaline mineral in bone by osteoblasts is reduced, and osteoclast resorptive activity is increased in order to maximize the availability of hydroxyl ions in solution to buffer protons.
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PMID:Acidosis inhibits bone formation by osteoblasts in vitro by preventing mineralization. 1607 62

Runx2 is essential for osteoblast differentiation and gene expression of bone matrix proteins, however, little is known about the mechanism regulating its activity. In this study, the role of Runx2 on gene expression of transcription factors, AJ18, Msx2, and Dlx5, was examined in vitro. It is known that AJ18 and Msx2 act as repressors to inhibit activity of Runx2, whereas Dlx5 promotes its activity. An expression vector inserted Runx2 cDNA was transiently overexpressed in a rat multipotential mesenchymal cell line, ROB-C26 (C26). Real time reverse transcription-PCR analysis showed that, in exogenous Runx2-overexpressing C26 cells (C26-Rx), AJ18 expression increased 1.8-fold, Msx2 expression increased 3.0-fold, and Dlx5 expression increased 2.7-fold compared to the cells transfected with vector alone (C26-Co). Luciferase assay also showed that, in C26-Rx, AJ18 promoter activity increased 2.1-fold compared to C26-Co. Furthermore, gene expression of alkaline phosphatase (ALP) and bone matrix proteins including type I collagen (Col1), osteocalcin (OC), osteopontin (OPN), and matrix Gla protein (MGP) was examined. In C26-Rx, MGP expression increased 1.8-fold, and OPN expression increased 1.4-fold compared to C26-Co. However, no significant difference in Col1, ALP, and OC expressions was detected between C26-Rx and C26-Co. These results suggest that the existence of autoregulatory feed back loops, which inhibit Runx2 activity through the interaction of AJ18, Dlx5, and Msx2 cooperating with that of MGP and OPN, interferes with the differentiation of C26 cells toward mature osteoblasts.
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PMID:Autoregulatory mechanism of Runx2 through the expression of transcription factors and bone matrix proteins in multipotential mesenchymal cell line, ROB-C26. 1641 64

Vascular calcification is present in many pathological conditions and is recognized as a strong predictor of future cardiovascular events. Current evidence suggests that it is a regulated process involving inducing and inhibitory molecules. Glucocorticoids have great clinical importance as antiinflammatory drugs and can act as potent inducers of osteogenic differentiation in vitro. The effect of glucocorticoids on vascular cells in vivo remains obscure. Pericytes are pluripotent cells that can differentiate into osteoblasts, and recent evidence suggests that they could participate in vascular calcification. We hypothesized that the synthetic glucocorticoid dexamethasone would enhance the rate of pericyte differentiation and mineralization in vitro with a concomitant suppression of calcification-inhibitory molecules. Three weeks of dexamethasone treatment induced a 2-fold increase in (1) alkaline phosphatase activity, (2) calcium deposition, and (3) the number of nodules formed in vitro; and a reduction in the expression of matrix Gla protein (MGP), osteopontin (OPN), and vascular calcification-associated factor (VCAF) mRNAs. The glucocorticoid receptor antagonist Org 34116 abolished dexamethasone-accelerated pericyte differentiation, nodule formation, and mineralization. Data obtained using Org 34116, the transcription inhibitor actinomycin D, and the protein synthesis inhibitor cyclohexamide suggest that MGP, OPN, and VCAF mRNA abundance are controlled at different and multiple levels by dexamethasone. This is the first report showing that dexamethasone enhances the osteogenic differentiation of pericytes and downregulates genes associated with inhibition of mineralization. Our study highlights the need for further investigation into the long-term consequences of prolonged glucocorticoid therapy on vascular calcification.
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PMID:Dexamethasone downregulates calcification-inhibitor molecules and accelerates osteogenic differentiation of vascular pericytes: implications for vascular calcification. 1662 86

The human trabecular meshwork (TM) expresses many genes that have been associated with physiological (bone, cartilage, teeth) and pathological (vascular systems, kidney) calcification. In particular, the TM highly expresses the inhibitor of calcification Matrix Gla (MGP) gene, which encodes a vitamin K-dependent protein that requires post-translational activation to inhibit the formation of calcium precipitates. TM cells have high activity of the activating gamma-carboxylase enzyme and produce active MGP. Silencing MGP increases the activity of alkaline phosphatase (ALP), an enzyme of the matrix vesicles and marker of calcification. Overexpressing MGP reduces the ALP activity induced by bone morphogenetic 2 (BMP2), a potent inducer of calcification. In this review we gathered evidence for the existence of a mineralization process in the TM. We selected twenty regulatory calcification genes, reviewed their functions in their original tissues and looked at their relative abundance in the TM by heat maps derived from existing microarrays. Although results are not yet fully conclusive and more experiments are needed, examining TM expression in the light of the calcification literature brings up many similarities. One such parallel is the role of mechanical forces in bone induction and the high levels of mineralization inhibitors found in the constantly mechanically stressed TM. During the next few years, examination of other calcification-related regulatory genes and pathways, as well as morphological examination of knockout animals, would help to elucidate the relevance of a calcification process to TM's overall function.
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PMID:Evidence for a calcification process in the trabecular meshwork. 1908 18

In order to study the effects of photoperiod on fish bone, Atlantic salmon (Salmo salar L.) were exposed to two light regimes (natural and continuous light) from January until June. During the experimental period, several parameters related to the inorganic (minerals) and organic (osteoid) phases were measured. Changes in the organic phase were related to mechanical strength (yield-load) and the expression of the genes sonic hedgehog (shh) and collagen type I alpha 2 (col I). Co-variation between yield-load and the expression of both shh and col I were detected in both groups. It was also shown that fish on the continuous light regime had delayed activation of osteoid incorporation. Mineralization properties were measured with stiffness, mineral incorporation per day and expression of alkaline phosphatase (alp) and matrix Gla protein (mgp). Stiffness, mineral incorporation and gene expression followed the same trend in both light groups in late spring, whereas an increase in the expression of mgp and alp was detected in April, followed by significantly higher stiffness at last sampling in both light groups. These results indicate that constant light affects mineralization and delays osteoid incorporation in Atlantic salmon during the spring. However, in this experiment light treatment did not promote the development of vertebral deformities. Our results also suggest that shh can be used as a marker of osteoblast proliferation and col I a marker of osteoid incorporation, and that both alp and mgp expression could be associated with a rapid increase in mineralization in Atlantic salmon vertebrae.
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PMID:Continuous light affects mineralization and delays osteoid incorporation in vertebral bone of Atlantic salmon (Salmo salar L.). 1921 16

Vitamins A (VA) and D (VD) are metabolised by vertebrates to bioactive retinoic acid (RA) and calcitriol (CTR). RA and CTR involvement in bone metabolism requires fine-tuned regulation of their synthesis and breakdown. In mammals antagonism of VA and VD is observed, but the mechanism of interaction is unknown. We investigated VA-VD interactions in Atlantic salmon (Salmo salar L.) following i.p. injection of RA and/or CTR. VA metabolites, CTR, calcium (Ca), magnesium (Mg) and phosphorus (P) were determined in plasma. Expression of bone matrix Gla protein (mgp), collagen 1 alpha2 chain (col1a2) and alkaline phosphatase (alp) mRNA was quantified to reflect osteogenesis. Branchial epithelial Ca channel (ecac listed as trpv6 in ZFIN Database) mRNA levels and intestinal Ca and P influx were determined to study Ca/P handling targets of RA and CTR. RA-injection (with or without CTR) decreased plasma CTR-levels three- to sixfold. CTR injection did not affect RA metabolites, but lowered CTR in plasma 3 and 5 days after injection. Lowered plasma CTR correlated with decreased mgp and col1a2 expression in all groups and with decreased alp in CTR-injected fish. RA-treated salmon had enhanced alp expression, irrespective of reduced plasma CTR. Expression of ecac and unidirectional intestinal influx of Ca were stimulated following RA-CTR treatment. Plasma Ca, Mg and P were not affected by any treatment. The results suggest cross-talk of RA with the VD endocrine system in Atlantic salmon. Enhanced Ca flux and osteogenesis (alp transcription) in RA-treated fish and inhibition of mgp expression revealed unprecedented disturbance of Ca physiology in hypervitaminosis A.
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PMID:Retinoic acid cross-talk with calcitriol activity in Atlantic salmon (Salmo salar). 1953 80

Medial artery calcification, which does not accompany lipid or cholesterol deposit, preferentially occurs in elderly population, but its underlying mechanisms remain unclear. In the present study, we investigated the potential role of senescent vascular smooth muscle cells (VSMCs) in the formation of senescence-associated medial calcification. Replicative senescence was induced by the extended passages (until passages 11-13) in human primary VSMCs, and cells in early passage (passage 6) were used as control young cells. VSMC calcification was markedly enhanced in the senescent cells compared with that in the control young cells. We identified that genes highly expressed in osteoblasts, such as alkaline phosphatase (ALP) and type I collagen, were significantly upregulated in the senescent VSMCs, suggesting their osteoblastic transition during the senescence. Knockdown of either ALP or type I collagen significantly reduced the calcification in the senescent VSMCs. Of note, runt-related transcription factor-2 (RUNX-2), a core transcriptional factor that initiates the osteoblastic differentiation, was also upregulated in the senescent VSMCs. Knockdown of RUNX-2 significantly reduced the ALP expression and calcification in the senescent VSMCs, suggesting that RUNX-2 is involved in the senescence-mediated osteoblastic transition. Furthermore, immunohistochemistry of aorta from the klotho(-/-) aging mouse model demonstrated in vivo emergence of osteoblast-like cells expressing RUNX-2 exclusively in the calcified media. We also found that statin and Rho-kinase inhibitor effectively reduced the VSMC calcification by inhibiting P(i)-induced apoptosis and potentially enhancing matrix Gla protein expression in the senescent VSMCs. These findings strongly suggest an important role of senescent VSMCs in the pathophysiology of senescence-associated medial calcification, and the inhibition of osteoblastic transition could be a new therapeutic approach for the prevention of senescence-associated medial calcification.
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PMID:Replicative senescence of vascular smooth muscle cells enhances the calcification through initiating the osteoblastic transition. 1974 65

Alendronate, an aminobisphosphonate, is an effective reagent to reduce fracture risk in osteoporotic patients. Although several studies suggest that bisphosphonates affect osteoblast differentiation, how they affect the genes relating to the mineralization step remains unknown. The present study was performed to clarify the effects of alendronate on mineralization and its related genes in mouse osteoblastic MC3T3-E1 cells. Alendronate at 10 (-8) and 10 (-7) M induced mineralization in MC3T3-E1 cells. As for the genes that suppress mineralization, alendronate enhanced the level of PC-1 mRNA in a dose-dependent manner in 7-day cultures in semiquantitative RT-PCR, although it reduced the levels of PC-1 mRNA in 21-day cultures. On the other hand, alendronate did not affect the levels of ANK, osteopontin and matrix Gla protein mRNA in both 7- and 21-day cultures. Moreover, alendronate reduced the level of osteocalcin mRNA at 10 (-7) and 10 (-6) M in 14-day cultures of these cells. As for the expression of alkaline phosphatase (ALP), an important positive regulator of mineralization in osteoblasts, alendronate enhanced the levels of ALP mRNA and protein at 10 (-7)-10 (-5) M. In conclusion, low-dose alendronate induced mineralization in mouse osteoblastic cells. The regulation of PC-1, osteocalcin and ALP by alendronate might play some role in these effects.
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PMID:Alendronate induces mineralization in mouse osteoblastic MC3T3-E1 cells: regulation of mineralization-related genes. 2021


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