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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The expression of the major protein kinase C substrate MARCKS (myristoylated alanine-rich C kinase substrate) is controlled by the stability of its mRNA. While the MARCKS mRNA is long living in quiescent fibroblasts (t1/2 = 14 h), its half-life time is drastically reduced (t1/2 = 2 h) in cells treated with phorbol esters to activate protein kinase C (PKC) or treated with growth factors. In a first step to study the underlying mechanism we identified both a cis-element on the MARCKS mRNA and the corresponding trans-acting factors. Fusing the complete 3'-UTR or specific regions of the 3'-UTR of the MARCKS gene to a luciferase reporter gene caused a drastic decrease in luciferase expression to as low as 5-10% of controls. This down-regulation was a result of destabilization of the chimeric transcript as shown by RNA run-off and Northern blot-assays. By RNase/EMSA and UV-cross-linking experiments, we identified a stretch of 52 nucleotides [(CUUU)11(U)8] in the 3'-UTR of the MARCKS mRNA specifically recognized by two RNA-binding proteins, HuD and HuR. These trans-acting factors are members of the ELAV gene family and bind the MARCKS CU-rich sequence with high affinity. Overexpression of HuD and HuR in murine fibroblasts caused a striking stabilization of the endogenous MARCKS mRNA even under conditions when the MARCKS mRNA is normally actively degraded, i.e. after treating cells with phorbol ester. These data imply, that the identified CU-rich cis-element of the MARCKS 3'-UTR is involved in conferring instability to mRNAs and that members of the ELAV gene family oppose this effect. Based on its structural and functional properties, the (CUUU)11(U)8 sequence described here can be grouped into class III of AU-rich elements.
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PMID:The 3'-UTR of the mRNA coding for the major protein kinase C substrate MARCKS contains a novel CU-rich element interacting with the mRNA stabilizing factors HuD and HuR. 1260 86

Inactivation of protein kinase C (PKC)alpha plays an important role in modulating hepatic failure and/or apoptosis during sepsis. To determine whether and how PKCalpha inactivation mediates the apoptosis, PKCalpha was suppressed by antisense treatment or transiently transfection in Clone-9 rat hepatic epithelial cell line. Apoptosis was evaluated by cell survival rate, poly-adenyl ribonuclease polymerase (PARP) cleavage, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling stain. The expressions of PKCalpha and Bcl-xL were quantified by Western blot analysis after antisense treatment. In the transfection studies, cells were co-transfected with green fluorescent protein cDNA as a transfection marker. The expressions of PKCalpha and Bcl-xL were detected by immunohistochemical staining with second antibody conjugated with Texas red. Apoptosis was evaluated by tetramethyl-rhodamine labeling of DNA strand breaks and immunostaining of 85-kDa fragment of PARP. The results showed that cytosolic and membrane-associated PKCalpha were decreased by 54.5% and 41.4%, respectively, after PKCalpha antisense treatment. The apoptotic incidence and percentage of PARP cleavage were significantly increased, whereas protein expression of Bcl-xL was decreased after PKCalpha-antisense treatment. In the transfection studies, the results showed that most of the cells expressing green fluorescent protein revealed less PKCalpha and Bcl-xL protein contents and more in situ PARP cleavage and DNA strand breaks. These findings indicated that decrease of PKCalpha declines the Bcl-xL content and leads to the vulnerability of apoptosis in hepatic epithelial cells. Taken together, our data provide evidence that suppression of PKCalpha plays a critical role in triggering caspase-dependent apoptosis, which may act through modulating the Bcl-xL expression.
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PMID:Suppression of protein kinase Calpha triggers apoptosis through down-regulation of Bcl-xL in a rat hepatic epithelial cell line. 1278 16

Calcitonin gene-related peptide (CGRP) is synthesized in dorsal root ganglion (DRG) neurons and released from primary afferent neurons to mediate hemodynamic effects and neurogenic inflammation. The effect of the proinflammatory cytokine interleukin-1 (IL-1)-beta on CGRP release from these sensory neurons was investigated. The results showed that IL-1beta (1 ng/ml) could directly induce CGRP release following prolonged incubation (24 hr) with these neurons. Treatment with IL-1beta (0.1-1.0 ng/ml) significantly increased CGRP release in a concentration-dependent manner. In addition, pretreatment of DRG cells with actinomycin D at 1 microM or cyclohexamide at 10 microM for 30 min inhibited 1 ng/ml IL-1beta-induced CGRP release in DRG neurons of neonatal rats. The inhibitors of PKC, JNK MAPK and NF-kappaB, but not p38 or ERK1/2 MAPK, blocked IL-1beta-induced CGRP release. RNase protection assay showed that IL-1beta could cause alpha-CGRP mRNA increase in a time- and concentration-dependent manner, although the level of beta-CGRP mRNA was not affected. These results indicate that IL-1beta may activate PKC, which in turn initiates JNK MAPK and activates NF-kappaB and finally induces alpha-CGRP gene expression and release from these sensory neurons.
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PMID:Mechanism of interleukin-1 beta-induced calcitonin gene-related peptide production from dorsal root ganglion neurons of neonatal rats. 1283 61

2'-5' Oligoadenylate (2-5A)-dependent RNase L is one of the key enzymes involved in the molecular mechanisms of interferon (IFN) function. Although the regulation of RNase L by 2-5A has been studied extensively, relatively little is known about how RNase L is controlled by posttranslational processes. Here, we report that phorbol-12-myristate-13-acetate (PMA) treatment of mouse L929 fibroblasts caused rapid degradation of RNase L in a dose-dependent and time-dependent manner. RNase L levels were decreased to 40% of control levels after only 5 min exposure of cells to PMA, suggesting the involvement of protein kinase C (PKC). After PMA treatment for 1 h, RNase L levels decreased to 18% of the pretreatment levels. Decay of RNase L was measured by 2-5A binding assay, ribonuclease activity, and protein levels in Western blots probed with antibody to murine RNase L. PMA treatment caused decreases in the levels of RNase L in both cytoplasm and nucleus. To explore the mechanism of RNase L degradation, we treated cells with the selective proteasome inhibitors, ALLN, MG132, and PSI, prior to PMA treatment. These inhibitors completely blocked the degradation of RNase L caused by PMA. Our results show a novel regulatory pathway for RNase L that could have an impact on its antitumor and antiviral functions.
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PMID:Proteasome-mediated degradation of RNase L in response to phorbol-12-myristate-13-acetate (PMA) treatment of mouse L929 cells. 1458 96

The presence of phospholipids as a component of chromatin is now well documented and many enzymes such as sphingomyelinase, sphingomyelin-synthase, reverse sphingomyelin-synthase and phosphatidylcholine-dependent phospholipase C have been described and characterised. Other lipids were demonstrated inside the nucleus especially plasmalogens and cholesterol. The chromatin phospholipids, comprising 10% of that present in the nucleus, show a different metabolism with respect to those present in either microsomes or in nuclear membranes; they increase also during the DNA duplication as shown during both liver regeneration and cell maturation. They appear localised near newly synthesized RNA in decondensed chromatin. Digestion of chromatin with RNase, but not with DNase, causes a loss of phospholipids. The composition of the chromatin phospholipid fraction shows an enrichment in sphingomyelin and phosphatidylserine. In this review the behaviour of single lipids in relation to cell proliferation, cell differentiation and apoptosis is described. Sphingomyelin, the lipid most represented in chromatin with respect to microsomes and nuclear membranes, is localised near to newly synthesized RNA, its presence appearing to protect RNA from RNase digestion. This effect is reversed by sphingomyelinase which digests sphingomyelin and, as a consequence, RNA may be hydrolysed. The amount of sphingomyelin is restored by sphingomyelin-synthase. Sphingomyelin increases during the differentiation process and apoptosis. An increase of sphingomyelinase with consequent decrease in sphingomyelin is observed at the beginning of S-phase of the cell cycle. A possible role in stabilising the DNA double helix is indicated. Phosphatidylserine behaves similarly during differentiation and appears to stimulate both RNA and DNA polymerases. Phosphatidylcholine is implicated in cell proliferation through the activation of intranuclear phosphatidylcholine-dependent phospholipase C and diacylglycerol production. The increase in diacylglycerol stimulates phosphatidylcholine synthesis through the major pathway from cytidyltriphosphate. An inhibition of phosphatidylcholine synthesis is responsible for the initiation of apoptosis. The presence of reverse sphingomyelin-synthase favours the formation of phosphatidylcholine, the donor of phosphorylcholine, from sphingomyelin. Little information has been reported for phospatidylethanolamine, but phosphtidylinositol appears to influence cell differentiation and proliferation. This last effect is due to the action of two enzymes: PI-PLCss1 having a role in the onset of DNA synthesis and PC-PLCgamma1 acting in G2 transit. Phosphoinositides also may have an important role: in membrane-stripped nuclei isolated from mitogen stimulated cells a decrease in PIP and PIP2 followed by an increase in diacylglycerol and a translocation of protein kinase C inside the nucleus is observed. On the other hand, overexpression of the enzyme inositol polysphosphate-1-phosphatase reduced DNA synthesis by 50%. Nevertheless, an enhanced rate of phosphorylation has been demonstrated in cells induced to differentiate. These molecules probably favour RNA transcription, counteracting the inhibition of H1 on RNA polymerase II. Plasmalogens were demonstrated in the nucleus and their increase favours the increased activity of phosphatidylcholine-dependent phospholipase C when DNA synthesis starts. Moreover, two forms of cholesterol has been described in chromatin: one, a less soluble sphingomyelin-linked form and a free fraction. Cholesterol increases during liver regeneration, first as a linked fraction and then, when DNA synthesis starts, as a free fraction. The changes of these components have been summarised in relation to cell function in order to give an overview of their possible roles in the different phases of cell duplication and their influence on cell differentiation and during apoptosis. Finally, the relevance of these molecules as intranuclear signals is discussed and future directions are indicated in clarifying pathological process such as tumour cell transformation and the possibility in finding new therapeutic tools.
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PMID:The role of intranuclear lipids. 1551 99

IL-6, a proinflammatory cytokine, has been implicated in the development of vascular diseases. We previously demonstrated that mechanical stress can initiate signaling pathways leading to smooth muscle cell (SMC) proliferation and apoptosis, but little is known concerning cyclic stress-induced inflammatory response. To explore the role of stretch in the upregulation of cytokine expression in SMCs we performed RNase protection assay for a panel of cytokines and found that mechanical stress resulted in a time-dependent induction of IL-6 mRNA but not other cytokines, e.g., IL-1alpha, IL-1beta, IL-6, IL-10, IL-12p35, IL-12p40, IL-18, IFN-gamma, and macrophage migration inhibitory factor (MIF). This induction also correlated with elevated IL-6 protein levels in the supernatant. Pretreatment of the cells with NF-kappaB inhibitors inhibited NF-kappaB activity and resulted in marked inhibition (50%) of IL-6 protein. Moreover, SMC lines stably expressing dominant-negative Ras (RasN17) or Rac (RacN17) exhibited a remarkable decrease in p38 MAPK activity and IL-6 mRNA induction by mechanical stress. Furthermore, a significant inhibition of 30 and 40% in IL-6 protein was observed in SMCs pretreated with inhibitors of p38 MAPK and ERK1/2, respectively, but not JNK. Interestingly, SMCs isolated from PKC-delta-deficient mice exhibited higher levels of IL-6 compared with wild-type cells. Finally, high levels of IL-6 expression were observed in atherosclerotic lesions of vein bypass grafts, which are related to altered biomechanical stress. Our findings demonstrate that biomechanical stress-induced IL-6 expression occurs via a mechanism that involves Ras/Rac/p38 MAPK/NF-kappaB/NF-IL6 signaling pathways, which is downregulated by PKC-delta, and suggest that modulation of this event contributes to the pathogenesis of atherosclerosis.
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PMID:Biomechanical stress induces IL-6 expression in smooth muscle cells via Ras/Rac1-p38 MAPK-NF-kappaB signaling pathways. 1568 96

Nitric oxide (NO) derived from endothelial NO synthase (eNOS) is a powerful vasodilator and possesses vasoprotective effects. Therefore, augmentation of eNOS expression and -activity by pharmacological means could provide protection against cardiovascular disease. However, this concept has been questioned recently, because in several disease models, eNOS upregulation was associated with a dysfunctional enzyme (referred to as eNOS uncoupling). In contrast, the present study demonstrates that an eNOS gene expression-enhancing compound with additional protein kinase C (PKC) inhibitory properties can upregulate eNOS while preserving its enzymatic function. Apolipoprotein E-knockout mice were treated for 7 days with midostaurin (4'-N-benzoyl staurosporine, compound CGP 41251, 50-125 mg/kg/day), a PKC inhibitor previously shown to increase eNOS expression and NO production in cultured human endothelial cells. Midostaurin treatment enhanced eNOS mRNA expression (RNase protection assay) in mouse aorta, kidney, and heart in a dose-dependent fashion. In the dorsal skinfold microcirculation, midostaurin produced an arteriolar vasorelaxation (intravital microscopy), which could be prevented by the NOS inhibitor L-NAME, indicating that the upregulated eNOS remained functional. In organ chamber experiments, the aorta from midostaurin-treated mice showed an enhanced NO-mediated relaxation in response to acetylcholine. Accordingly, serum levels of nitrite/nitrate (NO-Analyzer) were increased, and the production of reactive oxygen species in the aorta (L-012 chemiluminescence) was reduced by midostaurin. Thus, in mice in vivo, midostaurin treatment results in enhanced expression of eNOS with preserved enzyme function and enhanced production of bioactive NO. Given the beneficial effects of endothelial-derived NO, vasoprotective and anti-atherosclerotic effects are likely to ensue.
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PMID:Midostaurin upregulates eNOS gene expression and preserves eNOS function in the microcirculation of the mouse. 1589 May 50

CRH-binding protein (CRH-BP) binds CRH with high affinity and inhibits CRH-mediated ACTH release from anterior pituitary corticotrope-like cells in vitro. In female mouse pituitary, CRH-BP is localized not only in corticotropes, but is also expressed in gonadotropes and lactotropes. To investigate the functional significance of gonadotrope CRH-BP, we examined the molecular mechanisms underlying GnRH-regulated CRH-BP expression in alphaT3-1 gonadotrope-like cells. CRH-BP is endogenously expressed in alphaT3-1 cells, and quantitative real-time RT-PCR and ribonuclease protection assays demonstrate that GnRH induces a 3.7-fold increase in CRH-BP mRNA levels. GnRH also induces intracellular CRH-BP (2.0-fold) and secreted CRH-BP (5.3-fold) levels, as measured by [125I]CRH:CRH-BP chemical cross-linking. Transient transfection assays using CRH-BP promoter-luciferase constructs indicate that GnRH regulation involves protein kinase C-, ERK- and calcium-dependent signaling pathways and is mediated via a multipartite GnRH response element that includes activator protein 1 and cAMP response element (CRE) sites. The CRE site significantly contributes to GnRH responsiveness, independent of protein kinase A, representing a unique form of multipartite GnRH regulation in alphaT3-1 cells. Furthermore, EMSAs indicate that alphaT3-1 nuclear proteins specifically bind at activator protein 1 and CRE sites. These data demonstrate novel regulation of pituitary CRH-BP, highlighting the importance of the pituitary gonadotrope as a potential interface between the stress and reproductive axes.
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PMID:Gonadotropin-releasing hormone (GnRH) positively regulates corticotropin-releasing hormone-binding protein expression via multiple intracellular signaling pathways and a multipartite GnRH response element in alphaT3-1 cells. 1597 7

Human monocytic THP-1 cells can be induced to differentiate to macrophages when treated with phorbol 12-myristate 13-acetate (PMA). It is understood that before initiating cell differentiation, PMA treatment must first induce an inhibition of cell growth. Since the initial biochemical and molecular events that are associated with this growth inhibition have not been characterized, the present study was carried out to elucidate the molecular mechanisms associated with the PMA-induced growth arrest of THP-1 cells. Our results indicate that PMA inhibits THP-1 cells at G1-phase of the cell cycle, via a complex mechanism associated with the modulation of the expression of several cell cycle regulators, initiated by the cellular generation of reactive oxygen species (ROS). Both p21WAF1/CIP1 mRNA and protein were upregulated 24 h post PMA treatment as demonstrated by ribonuclease protection assay and Western blotting, respectively. Because these cells lack functional p53, this effect was independent of p53 activity. Electrophoretic mobility shift assay showed that the PMA-induced activation of the p21WAF1/CIP1 promoter was driven by the specific protein 1 (Sp1) transcription factor through Sp1-binding sites. Additionally, our study demonstrates that PMA-induces the upregulation of p21 through a protein kinase C (PKC)-mediated ROS-dependent signaling mechanism involving MAP kinase activation.
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PMID:Signal transduction of phorbol 12-myristate 13-acetate (PMA)-induced growth inhibition of human monocytic leukemia THP-1 cells is reactive oxygen dependent. 1597 37

Mechanical load and chemical factors as stimuli for the different pattern of the extracellular matrix (ECM) could be responsible for cardiac dysfunction. Since fibroblasts can both synthesize and degrade ECM, ventricular fibroblasts from adult rat hearts underwent cyclical mechanical stretch (CMS; 0.33 Hz) by three different elongations (3%, 6%, 9%) and four different serum concentrations (0%, 0.5%, 5%, 10%) within 24 h. Expression of collagen I and III, as well as matrix metalloproteinase-2 (MMP-2), tissue inhibitor of MMP-2 (TIMP-2), and colligin were analyzed by RNase protection assay. In the absence of serum, 9% CMS increased the mRNA of collagen I by 1.70-fold and collagen III by 1.64-fold. This increase was prevented by the inhibition either of PKC or of tyrosine kinase but not of PKA. Inhibition of PKC or tyrosine kinase itself reduced the expression of collagen I and collagen III mRNA. The mRNA of MMP-2, TIMP-2, and colligin showed the same tendency by stretch. Combined with 10% serum, 6% CMS reduced the mRNA of collagen I (0.62-fold) and collagen III (0.79-fold). Inhibition of PKC or tyrosine kinase, but not of PKA, prevented the reduction of collagen I and collagen III mRNA in 10% serum. The results show that the response of fibroblasts to CMS depends on the serum concentration. At least two signaling pathways are involved in the stretch-induced ECM regulation. Myocardial fibrosis due to ECM remodeling contributes to the dysfunction of the failing heart, which might be attributed to changes in hemodynamic loading.
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PMID:Cyclical mechanical stretch modulates expression of collagen I and collagen III by PKC and tyrosine kinase in cardiac fibroblasts. 1768 80


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