Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mechanism of regulation of the plasminogen activator inhibitor type-1 (PAI-1) gene by transforming growth factor-beta1 (TGF-beta1) was studied in vitro and in vivo in endothelial cells. We constructed adenovirus vectors containing PAI-1 5'-flanking sequences driving expression of a beta-galactosidase (beta-gal) reporter gene. Cultured bovine endothelial cells were transduced with the vectors and treated with TGF-beta1. beta-Gal expression was up-regulated 10-20-fold by TGF-beta1 when vectors contained 799-base pair (bp) of 5'-flanking sequence, but only minimally (2-3-fold) from a vector containing only 82-bp of 5' PAI-1 flanking sequence. TGF-beta1 up-regulated beta-gal expression at the mRNA level, congruently with TGF-beta1 up-regulation of expression of the endogenous PAI-1 gene. The constructs were transduced into intact rat carotid endothelium, and TGF-beta1 was injected systemically. In vivo, TGF-beta1 up-regulated endothelium-specific expression of beta-gal 3-fold (p < 0.03) from a vector containing the 799-bp sequence, but did not alter expression from a vector containing the 82-bp sequence. The sequence between -799 and -82 mediates up-regulation of reporter gene expression by TGF-beta1 in endothelial cells in vitro and in vivo. This general method permits the elucidation of mechanisms of gene regulation by physiologic stimuli delivered to the endothelium of intact animals.
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PMID:Identification of a cis-acting sequence in the human plasminogen activator inhibitor type-1 gene that mediates transforming growth factor-beta1 responsiveness in endothelium in vivo. 893 42

After a limited number of population doublings (PDs), cultures of normal mammalian diploid cells undergo an irreversible growth arrest known as replicative senescence [1]. As well as contributing to cellular ageing, senescence is viewed as an important mechanism of tumour suppression by preventing the emergence of immortal cell clones [2-4]. Senescent cells have a number of characteristics that distinguish them from cycling or quiescent cells including elevated levels of two cyclin-dependent kinase (Cdk) inhibitors, p16INK4a and p21CIP1 [5-11]. Here, we demonstrate that both of these Cdk inhibitors, as well as other members of their protein families (the INK4 and CIP/KIP families, respectively [12]), induce several facets of the senescent phenotype when ectopically expressed in young human diploid fibroblasts. These include a reduced proliferative capacity, an altered size and shape, the presence of underphosphorylated retinoblastoma protein (pRb), increased expression of plasminogen activator inhibitor (PAI-1) and the appearance of senescence-associated beta-galactosidase (SA-beta-gal) activity [2,3,13-15]. A 20 amino acid peptide from p16INK4a that inhibits Cdks active in the G1 phase of the cell cycle [16] produces similar effects in a dose-dependent manner suggesting that, in primary fibroblasts, inhibition of G1-specific Cdk activity is sufficient to induce phenotypic changes that normally occur at the end of their finite lifespan.
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PMID:Inhibitors of cyclin-dependent kinases induce features of replicative senescence in early passage human diploid fibroblasts. 951 19

Platelet plasminogen activator inhibitor I (PAI-1), a trace alpha-granule protein, is a key physiological regulator of fibrinolysis. Because information on the packaging of PAI-1 into alpha-granules during megakaryocytopoiesis may reveal novel approaches for controlling hemostasis, this study investigated basal, plasmid-mediated, and alphavirus-mediated PAI-1 packaging into alpha-granules-like structures in the megakaryocytic cell line MEG-01. Differentiation of MEG-01 cells with phorbol myristate acetate (PMA) was observed to result in a four-fold increase in both secreted and cell-associated PAI-1 antigen over a four day period. Subcellular fractionation of PMA-treated MEG-01 cells on 45% self-forming Percoll gradients was employed to separate low density membrane and Golgi-rich fractions from a high density granule-containing region. A subsequent 30-60% pre-formed Percoll gradient was employed to remove contaminating lysosomes from the PAI-1/glycoprotein IIbIIIa-containing granules. Electron microscopy showed that these MEG-01 granules share a similar size distribution (350-600 nm) and morphology to platelet alpha-granules. PAI-1 (40 ng/mg protein) in isolated MEG-01 storage granules was approximately 10% of the levels present in isolated platelet alpha-granules. To elevate PAI-1 production/storage, two expression systems were investigated. Experiments with plasmids encoding PAI-1 and beta-galactosidase resulted in low transfection efficiency (0.001%). In contrast, Semliki Forest virus (SFV)-mediated gene transfer increased cellular PAI-1 by 31-fold (1,200 ng/10(6) cells at 10 MOI) in comparison to mock-infected cells. Pulse-chase experiments demonstrated that SFV/PAI-1 mediated gene expression could enhance PAI-1 storage 6-9-fold, reaching levels present within platelets. To document the ability of PAI-1 to be stored in a rapidly releasable form in MEG-01 cells, we isolated platelet-like particles from the media conditioned by the cells and examined secretagogue-induced release of PAI-1. Particles from SFV/PAI-1 infected cells display a 5-fold enhanced secretion of PAI-1 following treatment with ADP in comparison to particles incubated in the absence of secretagogue. These results suggest that SFV mediated gene expression in MEG-01 cells provides a useful framework for analyzing the production and storage of alpha-granule proteins.
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PMID:Recombinant Semliki Forest virus enhanced plasminogen activator inhibitor 1 expression and storage in the megakaryocytic cell line MEG-01. 1152 53

Primary human embryo lung fibroblasts and adult diploid fibroblasts infected by the human cytomegalovirus (HCMV) display beta-galactosidase (beta-Gal) activity at neutral pH (senescence-associated beta-Gal [SA-beta-Gal] activity) and overexpression of the plasminogen activator inhibitor type 1 (PAI-1) gene, two widely recognized markers of the process designated premature cell senescence. This activity is higher when cells are serum starved for 48 h before infection, a process that speeds and facilitates HCMV infection but that is insufficient by itself to induce senescence. Fibroblasts infected by HCMV do not incorporate bromodeoxyuridine, a prerequisite for the formal definition of senescence. At the molecular level, cells infected by HCMV, beside the accumulation of large amounts of the cell cycle regulators p53 and pRb, the latter in its hyperphosphorylated form, display a strong induction of the cyclin-dependent kinase inhibitor (cdki) p16(INK4a), a direct effector of the senescence phenotype in fibroblasts, and a decrease of the cdki p21(CIP1/WAF). Finally, a replicative senescence state in the early phases of infection significantly increased the number of cells permissive to virus infection and enhanced HCMV replication. HCMV infection assays carried out in the presence of phosphonoformic acid, which inhibits the virus DNA polymerase and the expression of downstream genes, indicated that immediate-early and/or early (alpha) genes are sufficient for the induction of SA-beta-Gal activity. When baculovirus vectors expressing HCMV IE1-72 or IE2-86 proteins were inoculated into fibroblasts, the increase of p16(INK4a) (observed predominantly with IE2-86) was similar to that observed with the whole virus, as was the induction of SA-beta-Gal activity, suggesting that the viral IE2 gene leads infected cells into senescence. Altogether our results demonstrate for the first time that HCMV, after arresting the cell cycle and inhibiting apoptosis, triggers the cellular senescence program, probably through the p16(INK4a) and p53 pathways.
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PMID:Cell cycle arrest by human cytomegalovirus 86-kDa IE2 protein resembles premature senescence. 1241 54

The development of age-related proliferative disorders of the prostate gland is supported by transdifferentiation and cellular senescence processes in the stroma. Both processes are involved in remodeling of stromal tissue, as observed in benign prostatic hyperplasia (BPH), and in "reactive stroma" adjacent to prostate cancer (PCa). It has been assumed that TGF-beta1 plays a key role in the aging prostate by inducing premature senescence and favoring myofibroblast differentiation. Therefore, we evaluated the stromal cell phenotypes of human primary adult prostatic fibroblasts (n=3) and the molecular and cellular mechanisms of growth arrest after treatment with TGF-beta1 and of in vitro cellular senescence. Microarray analysis, quantitative PCR, immunofluorescence and western blot revealed that cellular senescence and transdifferentiation of fibroblasts have distinct underlying mechanisms, pathways and gene and protein expression profiles in human PrSCs. In clear contrast to senescent cells, TGF-beta1-treated cells morphologically transdifferentiated into myofibroblasts with dense cytoskeletal fibers and increased expression of smooth muscle cell alpha-actin, calponin and tenascin. TGF-beta1 induced neither expression of senescence-associated markers nor genes involved in terminal growth arrest, such as senescence-associated beta-galactosidase and cyclin-dependent kinase (cdk) inhibitors p16(Ink4A) and p21(Cip1) but increased p15(Ink4B) protein expression. Differentiation inhibitor (Id-1) protein level down-regulation was observed under both conditions. Genes specifically up-regulated by transdifferentiation but not by cellular senescence of PrSCs were metalloproteinase 1 tissue inhibitor (Timp1), transgelin (Tagln), gamma 2 actin (Actg2), plasminogen activator inhibitor 1 (Serpinel), insulin-like growth factor binding protein 3 (Igfbp3), parathyroid hormone-like hormone (Pthlp), Tgfb-1, four and a half LIM domains 2 (Fhl-2), hydrogen peroxide-inducible clone 5 (Hic5) and cartilage oligomeric matrix protein (Comp). Other genes, such as Cdc28 protein kinase 1 (Cks1b), v-myb myeloblastosis viral oncogene homolog (MybL2), pyruvate kinase, muscle 2 (Pkm2) and Forkhead box M1 (FoxM1), were down-regulated only upon TGF-beta1 treatment but not by cellular senescence. Pyruvate dehydrogenase kinase 3 (Pdk3) and connective tissue growth factor (Ctgf) were up-regulated and hyaluronan synthase 3 (Has3) down-regulated under both conditions. Moreover, GageC1, a prostate/testis-specific protein overexpressed in symptomatic BPH and PCa was induced in transdifferentiated stromal cells. Genes such as GageC1 could be promising targets for therapeutic inhibitors of stromal tissue remodeling and progression of BPH and PCa.
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PMID:Profiling molecular targets of TGF-beta1 in prostate fibroblast-to-myofibroblast transdifferentiation. 1561 Jul 63

Upregulation of plasminogen activator inhibitor type 1 (PAI-1) expression is a critical mechanism through which transforming growth factor-beta1 (TGF-beta1) accelerates intimal growth. The aim of this study was to identify signaling pathways through which TGF-beta1 upregulates PAI-1 expression in endothelial cells (EC) and test interventions for blocking these pathways. We transduced cultured bovine EC with an adenoviral vector containing the PAI-1 promoter fused to a beta-galactosidase reporter gene. We used these cells, along with vectors expressing potential modifiers of TGF-beta1 signaling and pharmacologic antagonists of mitogen-activated protein kinase (MAPK) pathways to identify key mediators of basal and TGF-beta1-regulated PAI-1 expression. Basal activity of the PAI-1 promoter was directly correlated with Ras activation and was blocked by a dominant negative (DN) type I TGF-beta receptor. TGF-beta1-stimulated activity of the PAI-1 promoter did not require Ras activation, and was lessened or eliminated by expression of either DN type I or type II TGF-beta receptors and by inhibition of either of two MAPKs: MEK and p38. Our results suggest unanticipated pathways of TGF-beta1 signaling in EC and point to new strategies to limit TGF-beta1-induced vascular disease.
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PMID:Identification of intracellular pathways through which TGF-beta1 upregulates PAI-1 expression in endothelial cells. 1613 37

The induction of senescence-like growth arrest has emerged as a putative contributor to the anticancer effects of chemotherapeutic agents. Clinical trials are underway to evaluate the efficacy of inhibitors for class I and II histone deacetylases to treat malignancies. However, a potential antiproliferative effect of inhibitor for Sirt1, which is an NAD(+)-dependent deacetylase and belongs to class III histone deacetylases, has not yet been explored. Here, we show that Sirt1 inhibitor, Sirtinol, induced senescence-like growth arrest characterized by induction of senescence-associated beta-galactosidase activity and increased expression of plasminogen activator inhibitor 1 in human breast cancer MCF-7 cells and lung cancer H1299 cells. Sirtinol-induced senescence-like growth arrest was accompanied by impaired activation of mitogen-activated protein kinase (MAPK) pathways, namely, extracellular-regulated protein kinase, c-jun N-terminal kinase and p38 MAPK, in response to epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I). Active Ras was reduced in Sirtinol-treated senescent cells compared with untreated cells. However, tyrosine phosphorylation of the receptors for EGF and IGF-I and Akt/PKB activation were unaltered by Sirtinol treatment. These results suggest that inhibitors for Sirt1 may have anticancer potential, and that impaired activation of Ras-MAPK pathway might take part in a senescence-like growth arrest program induced by Sirtinol.
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PMID:Sirt1 inhibitor, Sirtinol, induces senescence-like growth arrest with attenuated Ras-MAPK signaling in human cancer cells. 1617 Mar 53