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:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The stimulation of osteocalcin synthesis by human osteoblast-like cells in response to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is antagonised by several bone regulatory agents. We have shown that agents which activate adenylate cyclase inhibit this action of 1,25(OH)2D3 on human osteoblast-like cells. Activation of adenylate cyclase, either via the stimulatory GTP-binding protein using cholera toxin, or directly at the catalytic via the stimulatory GTP-binding protein using cholera toxin, or directly at the catalytic subunit using forskolin, results in a suppression of osteocalcin synthesis. Whilst the activation of adenylate cyclase induces this inhibitory response, neither exogenous dibutyryl cyclic AMP nor the phosphodiesterase inhibitor, IBMX, exerted any apparent effect on the production of osteocalcin. The tumour promoting phorbol ester, 4 beta-phorbol 12,13-dibutyrate, also inhibited 1,25(OH)2D3-stimulated osteocalcin production. This was not apparent in response to the non-tumour promoting phorbol ester 4 beta-phorbol suggesting the involvement of protein kinase C.
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PMID:Agents affecting adenylate cyclase activity modulate the stimulatory action of 1,25-dihydroxyvitamin D3 on the production of osteocalcin by human bone cells. 248 Jan 11

Osteoblasts have been reported to produce tissue-type (t) plasminogen activator (PA), which may be involved in the initiation of bone resorption via plasmin-metalloproteinase degradation of adjacent extracellular matrix. To investigate this cAMP-activated gene, we characterized the PTH regulation of tPA messenger RNA (mRNA) in neonatal rat osteoblast cultures before and after differentiation in vitro. RNA was purified from cultures at confluence or after treatment with glucocorticoid for 1 week and BGJ/ascorbic acid/beta-glycerophosphate for a second week. Northern blots of total or poly(A)+ RNA were hybridized simultaneously with an oligonucleotide or complementary RNA probe for rat tPA and an oligomeric DNA probe for cyclophilin (CYP), an abundant control gene. Differentiation was monitored by expression of rat osteocalcin mRNA and protein. Both bovine PTH1-34 and forskolin caused an increase in tPA/CYP ratio in the presence of phosphodiesterase inhibitor (IBMX) and cycloheximide (CHX). The effect was maximal (16- to 21-fold increase in tPA mRNA and 6- to 8-fold increase in tPA/CYP ratio) with 25 nM hormone for 6 h and was half-maximally stimulated by 0.75-2.5 nM PTH. The tPA response to PTH was present in first passage osteoblast cultures at confluence and after 1 to 2 weeks of glucocorticoid treatment. Exposure of the differentiated cultures of 1,25-dihydroxyvitamin D (10 nM) for 2 days markedly stimulated osteocalcin mRNA while having no effect on tPA. In Northern blots of poly(A)+ RNA from cultures not treated with CHX, IBMX and PTH (2.5 h) independently stimulated tPA mRNA with no significant effect on CYP mRNA levels. The tPA/CYP ratio increased in five consecutive experiments and the effect of IBMX and PTH were additive. These data indicate that PTH acts via cAMP to stimulate tPA expression by a mechanism that is independent of protein synthesis. The enhancement of PTH action by CHX is compatible with feedback inhibition of tPA transcription by a hormone-activated repressor (which has been proposed to occur in granulosa cells) but effects of CHX on tPA mRNA stability may also occur. Expression of tPA mRNA before and after differentiation may indicate that the enzyme has more than one function.
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PMID:Increased expression of tissue plasminogen activator messenger ribonucleic acid is an immediate response to parathyroid hormone in neonatal rat osteoblasts. 811 83

The effects of insulin-like growth factor-I (IGF-I) and IGF-II on the human osteoblast cell-line OHS-4 were investigated. Both IGF-I and IGF-II stimulated cell proliferation at nanomolar concentrations and alkaline phosphatase activity was decreased in a dose-dependent manner with either IGF-I or IGF-II. The production of the bone-specific protein osteocalcin was not influenced by either IGF-I or IGF-II. However, they acted synergistically with 1,25-dihydroxy-vitamin D3 at concentrations ranging from 10 to 100 nmol/l. Neither IGF-I nor IGF-II had an effect on either the basal or the parathyroid hormone-stimulated level of adenylate cyclase activity, and likewise they had no effect on phosphodiesterase activity. Binding and cross-linking experiments confirmed the presence of both type-I and type-II IGF receptors on the OHS-4 cells. The present study shows that IGF-I and IGF-II have similar effects on the parameters studied in these osteoblastic cells. They influenced both proliferation and differentiation markers.
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PMID:Comparison of the effects of insulin-like growth factors-I and -II on the human osteosarcoma cell line OHS-4. 842 72

Parathyroid hormone (PTH)-mediated gene activation was assessed in the osteoblast-like rat cell line ROS17/2.8 with two PTH fragments harboring distinct activating domains: PTH-(1-34) and PTH-(28-48). The PTH response of genes expressed immediate early in the cell cycle or in the osteoblast developmental sequence was investigated. In addition, subtractive cloning was used to identify genes in ROS17/2.8 cells that are activated by the two PTH domains. PTH-(1-34) immediately increased the transcript levels of c-fos and c-jun at a considerably higher rate than PTH-(28-48). A significant immediate PTH effect on osteoblastic marker genes could not be detected, with the exception of elevated ornithine decarboxylase transcript levels. However, continuous application of PTH-(1-34) increased transcript levels of the osteoblast-specific osteocalcin gene and reduced those of other osteoblastic marker genes including alkaline phosphatase and the PTH/PTH-related peptide receptor. By subtractive cloning, nine cDNAs were isolated corresponding to mRNAs directly up-regulated by PTH-(1-34) or PTH-(28-48). Among these were a cyclic phosphodiesterase, a (cytosine 5)-methyltransferase, an 80-kDa protein kinase C substrate, junB, and a novel GC-binding protein. Three cDNAs are unknown at present. Interestingly, in all cases, the efficiency of gene activation by PTH-(28-48) was substantially lower in comparison with PTH-(1-34). PTH-mediated protein kinase C signaling in ROS17/2.8 cells may therefore constitute a minor pathway in comparison with the dominant cAMP/protein kinase A cascade.
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PMID:Domain-specific gene activation by parathyroid hormone in osteoblastic ROS17/2.8 cells. 870 88

We have investigated the effect of 1-(5-oxohexyl)-3,7-dimethylxanthine or pentoxifylline (PeTx), a nonselective phosphodiesterase inhibitor, on osteoblastic differentiation in vitro by using two mesenchymal cell lines, C3H10T1/2 and C2C12, which are able to acquire the osteoblastic phenotype in the presence of bone morphogenetic protein-2 (BMP-2). PeTx induced the osteoblastic markers, osteocalcin and Osf2/Cbfa1, in C3H10T1/2 and C2C12 cells and enhanced BMP-2-induced expression of osteocalcin, Osf2/Cbfa1, and alkaline phosphatase. This activity was partially attributed to the fact that PeTx is able to enhance BMP-2-induced Smad1 transcriptional activity. Although PeTx clearly stimulates PKA in these cells, neither pretreatment of cells with the PKA inhibitor H89 nor transfection with the specific PKA inhibitor PKI prevented the induction or enhancement of osteoblast markers by PeTx, demonstrating that these effects were independent of PKA activation. On the other hand, PeTx induced the activation of ERK1/2 and p38 kinase pathways independently of the activation of PKA. Selective inhibitors of these MAPK cascades prevented the induction of osteoblastic markers in cells treated with PeTx, suggesting that the activation of these two pathways plays a role in the effect of PeTx on osteoblastic differentiation.
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PMID:1-(5-oxohexyl)-3,7-Dimethylxanthine, a phosphodiesterase inhibitor, activates MAPK cascades and promotes osteoblast differentiation by a mechanism independent of PKA activation (pentoxifylline promotes osteoblast differentiation). 1160 32

We have evaluated effects of a phosphodiesterase (PDE) 4 inhibitor on retinoic acid-increased alkaline phosphatase activity in the mouse fibroblastic C3H10T1/2 clone 8 (10T1/2) cell line. 10T1/2 cells were cultured in minimum essential medium (MEM) and 10% fetal bovine serum with or without 1 microM retinoic acid and/or the PDE 4 inhibitor, rolipram, and harvested at specific intervals before measurement of alkaline phosphatase activity, cAMP production in response to parathyroid hormone, osteocalcin synthesis and expression, and phosphodiesterase activity. Retinoic acid-increased alkaline phosphatase activity, and slightly enhanced cAMP production in response to parathyroid hormone. However, it did not affect osteocalcin synthesis and expression. In the presence of retinoic acid, PDE 4 activity was not changed. A PDE 4 inhibitor, rolipram, and cAMP analog, 8-bromo-cAMP dramatically increased retinoic acid's ability to induce alkaline phosphatase activity. This is the first report that PDE 4 may be involved in regulation of retinoic acid-increased alkaline phosphatase activity.
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PMID:Effect of the phosphodiesterase 4 inhibitor, rolipram, on retinoic acid-increased alkaline phosphatase activity in the mouse fibroblastic C3H10T1/2 cell line. 1261 43

The second messenger molecule cyclic adenosine monophosphate (cAMP) plays an important role in the hormonal regulation of bone metabolism. cAMP is inactivated by the cyclic nucleotide phosphodiesterases (PDEs), a superfamily of enzymes divided into 11 known families designated PDE 1-11. The aim of this study was to investigate the effect of PDE7 and PDE8 inhibition on the gene expression and differentiation of human osteoblasts. Osteoblasts differentiated from human mesenchymal stem cells (hMSC) were cultured and treated with short interfering RNAs (siRNAs) generated from PDE7 and PDE8 PCR products. Total RNA was isolated from the cells, and gene expression was assayed with cDNA microarray and quantitative real-time PCR. bALP measurements were assayed during differentiation, and mineralization was determined by quantitative Alizarin red S staining. PDE7 and PDE8 inhibition by RNA interference decreased the gene expression of PDE7A by 60-70%, PDE7B by 40-50%, and PDE8A by 30%. PDE7 silencing increased the expression of beta-catenin, osteocalcin, caspase-8, and cAMP-responsive element-binding protein 5 (CREB-5) genes and decreased the expression of the 1, 25-dihydroxyvitamin D3 receptor gene. PDE8A silencing increased the expression of anti-apoptotic genes, but decreased the expression of osteoglycin (osteoinductive factor) and bone morphogenetic protein 1 (BMP-1). PDE7 silencing increased bALP and mineralization up to three-fold compared to controls. Treatment with the PDE7-selective PDE inhibitor BRL-50481 had similar effects on mineralization as the gene silencing. The PDE7 silencing also increased forskolin stimulated cAMP response, but had no effect on the proliferation rate. Furthermore, osteocalcin expression was increased by PDE7 silencing by a mechanism dependent on protein kinase A. Our results show that specific gene silencing with the RNAi method is a useful tool for inhibiting the gene expression of specific PDEs and that PDE7 silencing upregulates several osteogenic genes and increases mineralization. PDE7 may play an important role in the regulation of osteoblastic differentiation.
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PMID:Effects of phosphodiesterase 7 inhibition by RNA interference on the gene expression and differentiation of human mesenchymal stem cell-derived osteoblasts. 1842 Apr 79

PTH is a potent bone anabolic agent in vivo but anabolic effects on osteoblast differentiation in vitro are difficult to demonstrate. This study examined the role of cyclooxygenase (COX)-2 and prostaglandin (PG) production in the effects of PTH on osteoblast differentiation in vitro using marrow stromal cell (MSC) and calvarial osteoblast (COB) cultures from COX-2 knockout (KO) and wild type (WT) mice. Cells were treated with PTH (10 nM) or vehicle throughout culture. Alkaline phosphatase (ALP) and osteocalcin (OCN) mRNA levels were measured at days 14 and 21, respectively, and mineralization at day 21. cAMP concentrations were measured in the presence of a phosphodiesterase inhibitor. PTH did not stimulate differentiation in cultures from WT mice but significantly increased ALP and OCN mRNA expression 6- to 7-fold in KO MSC cultures and 2- to 4-fold in KO COB cultures. PTH also increased mineralization in both KO MSC and COB cultures. Effects in KO cells were mimicked in WT MSC cultures treated with NS-398, an inhibitor of COX-2 activity. PTH increased cAMP concentrations similarly in WT and KO COBs. Differential gene responses to PTH in COX-2 KO COBs relative to WT COBs included greater fold-increases in the cAMP-mediated early response genes, c-fos and Nr4a2; increased IGF-1 mRNA expression; and decreased mRNA expression of MAP kinase phosphatase-1. PTH inhibited SOST mRNA expression 91% in COX-2 KO MSC cultures compared to 67% in WT cultures. We conclude that endogenous PGs inhibit the anabolic responses to PTH in vitro, possibly by desensitizing cAMP pathways.
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PMID:Anabolic effects of PTH in cyclooxygenase-2 knockout osteoblasts in vitro. 1850 Nov 88

Vascular calcification is associated with increased cardiovascular risk and occurs by osteochondrogenic differentiation of vascular cells. Many of the same regulatory factors that control skeletal mineralization, including the complex metabolic pathway controlling levels of the activator, inorganic phosphate, and the potent inhibitor, pyrophosphate, also govern vascular calcification. We previously found that the cAMP/PKA signaling pathway mediates in vitro vascular cell calcification induced by inflammatory factors including tumor necrosis factor-alpha 1 and oxidized phospholipids. In this report, we tested whether this signaling pathway modulates phosphate and pyrophosphate metabolism. Treatment of primary murine aortic cells with the PKA activator, forskolin, significantly induced osteoblastic differentiation markers, including alkaline phosphatase (ALP), osteopontin, and osteocalcin as well as the pyrophosphate generator, ectonucleotide-pyrophosphatase/phosphodiesterase-1 (Enpp1) and the pyrophosphate transporter, ankylosis protein, but not the sodium/phosphate cotransporter, Pit-1. In the presence of a substrate for ALP, beta-glycerophosphate, which generates inorganic phosphate, forskolin also enhanced matrix mineralization. Inhibitors of ALP or Pit-1 abrogated forskolin-induced osteopontin expression and mineralization but not forskolin-induced osteocalcin or ALP. These results suggest that phosphate is necessary for PKA-induced calcification of vascular cells and that the extent of PKA-induced calcification is controlled by feedback induction of the inhibitor, pyrophosphate.
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PMID:Phosphate and pyrophosphate mediate PKA-induced vascular cell calcification. 1865 72

Human skeletal progenitors were engineered to stably express R201C mutated, constitutively active Gs alpha using lentiviral vectors. Long-term transduced skeletal progenitors were characterized by an enhanced production of cAMP, indicating the transfer of the fundamental cellular phenotype caused by activating mutations of Gs alpha. Like skeletal progenitors isolated from natural fibrous dysplasia (FD) lesions, transduced cells could generate bone but not adipocytes or the hematopoietic microenvironment on in vivo transplantation. In vitro osteogenic differentiation was noted for the lack of mineral deposition, a blunted upregulation of osteocalcin, and enhanced upregulation of other osteogenic markers such as alkaline phosphatase (ALP) and bone sialoprotein (BSP) compared with controls. A very potent upregulation of RANKL expression was observed, which correlates with the pronounced osteoclastogenesis observed in FD lesions in vivo. Stable transduction resulted in a marked upregulation of selected phosphodiesterase (PDE) isoform mRNAs and a prominent increase in total PDE activity. This predicts an adaptive response in skeletal progenitors transduced with constitutively active, mutated Gs alpha. Indeed, like measurable cAMP levels, the differentiative responses of transduced skeletal progenitors were profoundly affected by inhibition of PDEs or lack thereof. Finally, using lentiviral vectors encoding short hairpin (sh) RNA interfering sequences, we demonstrated that selective silencing of the mutated allele is both feasible and effective in reverting the aberrant cAMP production brought about by the constitutively active Gs alpha and some of its effects on in vitro differentiation of skeletal progenitors.
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PMID:Transfer, analysis, and reversion of the fibrous dysplasia cellular phenotype in human skeletal progenitors. 1987 99


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