Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The differential expression of Rho family of low molecular weight GTP-binding proteins and protein kinase C (PKC) isozymes were examined during differentiation of rat C6 glial cells to astrocytic phenotypes induced by dibutyryl cAMP (dbcAMP)/theophylline. The cells showed rapid and distinct morphological changes, resembling stellate astrocytes at 12 h after the treatment. The treated cells had a round cell body that extended several long processes each with a beaded appearance. In addition to morphological changes, Western blot analysis revealed that S-100 protein, known as a glial cell differentiation marker, increased and reached the maximal level (approximately 6-fold increase) at 24 h following the addition of dbcAMP. In the control experiments with cells cultured in the absence of serum but also without dbcAMP/theophylline, morphological changes were marginal and apparent increases of S-100 protein were not observed by Western blotting. In response to dbcAMP/theophylline treatment, RhoA showed increases in the mRNA level followed by the protein level, as inferred by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting, respectively. Rac1 and Cdc42 proteins were undetectable by Western blot analyses. In PKC isozymes, increases were observed in PKC beta 1, epsilon, and zeta by RT-PCR, and in beta 1 and epsilon by Western blotting. Among them, PKC epsilon showed the most distinct changes. Its mRNA level transiently increased from 3 to 6 h and then decreased even below the basal level at 18 h after the treatment. In contrast, Western blot analysis revealed that PKC epsilon gradually increased time-dependently to 24 h (approximately 6-fold increase), and remained elevated until 48 h. These results suggested that RhoA and PKC epsilon, and probably also PKC beta 1 and PKC zeta, were closely implicated in C6 cell differentiation.
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PMID:Differential expression of Rho family GTP-binding proteins and protein kinase C isozymes during C6 glial cell differentiation. 910 74

We have attempted to determine whether muscarinic stimulation induces RhoA/ROCK-mediated Ca2+ sensitization of contractions in chicken gizzard smooth muscles. rhoA is a small GTP-binding protein, and ROCK is a rhoA-associated coiled coil-forming serine/threonine kinase. The relationship between the cytosolic Ca2+ level ([Ca2+]i) and muscle force in the presence of a high K+ concentration was not different from that in the presence of carbachol. Verapamil inhibited muscle force in proportion to the decrease in [Ca2+]i in both the muscle stimulated with high K+ and that stimulated with carbachol. In addition, Y-27632 (10 microM), a ROCKs inhibitor, had no effect on the contractions. In the alpha-toxin-permeabilized muscles, Ca2+ induced a greater contraction in the presence of guanosine 5'-O-(3-thiotriphosphapte) (GTP[gamma-S]), whereas carbachol with GTP was not effective. The GTP[gamma-S]-induced Ca2+ sensitization was completely inhibited by Clostridium botulinum exoenzyme C3. Western blot analysis revealed both rhoA and ROCKII in the muscle extract. In addition, reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed the expression of both ROCKI and ROCKII mRNAs. These results suggest that Ca2+ sensitization in the chicken gizzard is elicited via a rhoA/ROCKs pathway, and that this pathway may be responsible for the augmentation of contraction by GTP[gamma-S] in the permeabilized muscles. If such a pathway does exist, however, carbachol-induced contraction may not be coupled to it, which explains the absence of Ca2+ sensitization in the intact chicken gizzard stimulated by carbachol.
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PMID:Muscarinic stimulation does not induce rhoA/ROCK-mediated Ca2+ sensitization of the contractile element in chicken gizzard smooth muscle. 1121 Nov 3

Round embryonic mesenchymal cells have the potential to differentiate into smooth muscle (SM) cells upon spreading/elongation (Yang, Y., K.C. Palmer, N. Relan, C. Diglio, and L. Schuger. 1998. Development. 125:2621-2629; Yang, Y., N.K. Relan, D.A. Przywara, and L. Schuger. 1999. Development. 126:3027-3033; Yang, Y., S. Beqaj, P. Kemp, I. Ariel, and L. Schuger. 2000. J. Clin. Invest. 106:1321-1330). In the developing lung, this process is stimulated by peribronchial accumulation of laminin (LN)-2 (Relan, N.K., Y. Yang, S. Beqaj, J.H. Miner, and L. Schuger. 1999. J. Cell Biol. 147:1341-1350). Here we show that LN-2 stimulates bronchial myogenesis by down-regulating RhoA activity. Immunohistochemistry, immunoblotting, and reverse transcriptase-PCR indicated that RhoA, a small GTPase signaling protein, is abundant in undifferentiated embryonic mesenchymal cells and that its levels decrease along with SM myogenesis. Functional studies using agonists and antagonists of RhoA activation and dominant positive and negative plasmid constructs demonstrated that high RhoA activity was required to maintain the round undifferentiated mesenchymal cell phenotype. This was in part achieved by restricting the localization of the myogenic transcription factor serum response factor (SRF) mostly to the mesenchymal cell cytoplasm. Upon spreading on LN-2 but not on other main components of the extracellular matrix, the activity and level of RhoA decreased rapidly, resulting in translocation of SRF to the nucleus. Both cell elongation and SRF translocation were prevented by overexpression of dominant positive RhoA. Once the cells underwent SM differentiation, up-regulation of RhoA activity induced rather than inhibited SM gene expression. Therefore, our studies suggest a novel mechanism whereby LN-2 and RhoA modulate SM myogenesis.
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PMID:High RhoA activity maintains the undifferentiated mesenchymal cell phenotype, whereas RhoA down-regulation by laminin-2 induces smooth muscle myogenesis. 1187 60

Enhanced expression of (pro)matrix metalloproteinase-9 (MMP-9) is associated with human tumor invasion and/or metastasis. COH cells derived from a highly invasive and metastatic Ewing's sarcoma constitutively express proMMP-9. Transfection of a double stranded RNA that targets the MMP-9 mRNA into COH cells depleted the corresponding mRNA and protein as demonstrated by reverse transcriptase-PCR, enzyme-linked immunosorbent assay, and gelatin zymography. proMMP-9 extinction resulted in the following: (i) decreased spreading on extracellular matrix (fibronectin, laminin, collagen IV)-coated surfaces, (ii) inhibition of migration toward fibronectin, and (iii) induced aggregation, which was specifically disrupted by a function-blocking E-cadherin antibody. MMP-9 knockdown concomitantly resulted in increased levels of surface E-cadherin, redistribution at the plasma membrane of beta-catenin, and its physical association with E-cadherin. Moreover, induction of E-cadherin-mediated adhesion was associated with RhoA activation and changes in paxillin cytoskeleton. Finally, an inhibitor of gelatinolytic activity of pro-MMP9 did not reduce COH cell migration confirming that the enzymatic property of COH MMP-9 was not required for migration toward fibronectin. Overall, our observations define a novel critical role for proMMP-9 in providing a cellular switch between stationary and migratory cell phases.
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PMID:Matrix metalloproteinase-9 silencing by RNA interference triggers the migratory-adhesive switch in Ewing's sarcoma cells. 1284 1

Thrombin, a serine protease, plays an important role in the progression of atherosclerosis. How atorvastatin could limit the pro-inflammatory response to thrombin was studied in cultured rat aortic smooth muscle cells. The variations in expression of interleukin-6, heme oxygenase-1, p(22phox) and Mox-1 mRNAs were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). Interleukin-6 release was determined using the B9 cell assay. Nuclear factor-kappa B (NF-kappaB) translocation was analysed by electrophoretic mobility shift assay (EMSA) and RhoA protein translocation by Western blot. Thrombin activated interleukin-6 secretion and mRNA expression in smooth muscle cells in a dose-dependent manner. The greatest effect on mRNA expression was obtained after 1 h of stimulation. Preincubation (72 h) of the cells with various concentrations of atorvastatin prevented this effect. Simultaneous addition of mevalonate overcame this statin effect. Thrombin was without effect on p(22phox) and heme oxygenase-1 mRNA expression but, after 3 h of stimulation, induced a two-fold increase in that of Mox-1. Preincubation with atorvastatin dose-dependently downregulated this Mox-1 mRNA expression. In addition, thrombin induced NF-kappaB translocation and membrane translocation of RhoA in smooth muscle cells which were both prevented by pre-treatment of the cells by atorvastatin. These data demonstrate the ability of atorvastatin to prevent the induction by thrombin of a pro-inflammatory phenotype in smooth muscle cells.
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PMID:Atorvastatin limits the pro-inflammatory response of rat aortic smooth muscle cells to thrombin. 1292 59

Exposure to chronic hypoxia (CH) induces a sustained pulmonary hypertension associated with structural and functional changes in the pulmonary arterial bed, including alterations of contractile properties. The small G-protein RhoA and its effector Rho kinase play a major role in the sustained rise in tension induced by vasoconstrictors. The aim of this study was to analyze the effect of CH on the RhoA/Rho kinase signaling pathway in the rat pulmonary artery. Maximal contraction of pulmonary artery rings to endothelin-1, noradrenaline, and the thromboxane A2 analog U46619 was markedly decreased in rats exposed to CH (10% O2, 2 weeks). This CH-induced decrease response to agonists was attributable to the abolition of RhoA-mediated Ca2+ sensitization of the contraction. Real-time reverse transcriptase-polymerase chain reaction and Western blot analysis revealed a decrease in RhoA mRNA (79.4+/-6.0%, n=4) and RhoA (81.1+/-8.0%, n=4) expression in the main pulmonary artery from CH rats, whereas RhoA expression was not modified in arterial smooth muscle cells and arteries exposed to hypoxia and high intraluminal pressure, respectively. Treatment of rats with sildenafil (25 mg/kg per day) throughout 2 weeks of exposure to CH prevented CH-induced downregulation of RhoA, reduction of contraction, and pulmonary artery remodeling. These findings indicate that CH-induced downregulation of RhoA expression, leading to the abolition of RhoA/Rho kinase-mediated Ca2+ sensitization of contraction, is responsible for the decreased responses to contracting agonists in the pulmonary artery of CH rats. These alterations are prevented by sildenafil, indicating a major role of the NO/cyclic GMP pathway in CH-induced altered RhoA signaling in the pulmonary artery.
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PMID:Sildenafil prevents change in RhoA expression induced by chronic hypoxia in rat pulmonary artery. 1294 46

Sphingosine 1-phosphate (S1P) is a lipid agonist that regulates smooth muscle cell (SMC) and endothelial cell functions by activating several members of the S1P subfamily of G-protein-coupled Edg receptors. We have shown previously that SMC differentiation is regulated by RhoA-dependent activation of serum response factor (SRF). Because S1P is a strong activator of RhoA, we hypothesized that S1P would stimulate SMC differentiation. Treatment of primary rat aortic SMC cells with S1P activated RhoA as measured by precipitation with a glutathione S-transferase-rhotekin fusion protein. In SMC and 10T1/2 cells, S1P treatment up-regulated the activities of several transiently transfected SMC-specific promoters, and these effects were inhibited by the Rho-kinase inhibitor, Y-27632. S1P also increased smooth muscle alpha-actin protein levels in SMC but had no effect on SRF binding to the smooth muscle alpha-actin CArG B element. Quantitative reverse transcriptase-PCR showed that S1P treatment of SMC or 10T1/2 cells did not increase the mRNA level of either of the recently identified SRF co-factors, myocardin or myocardin-related transcription factor-A (MRTF-A). MRTF-A protein was expressed highly in SMC and 10T1/2 cultures, and importantly the effects of S1P were inhibited by a dominant negative form of MRTF-A indicating that S1P may regulate the transcriptional activity of MRTF-A. Indeed, S1P treatment increased the nuclear localization of FLAG-MRTF-A, and the effect of MRTF-A overexpression on smooth muscle alpha-actin promoter activity was inhibited by dominant negative RhoA. S1P also stimulated SMC growth by activating the early growth response gene, c-fos. This effect was not attenuated by Y-27632 but could be inhibited by the MEK inhibitor, UO126. S1P enhanced SMC growth through ERK-mediated phosphorylation of the SRF co-factor, Elk-1, as measured by gel shift and Elk-1 activation assays. Taken together these results demonstrate that S1P activates multiple signaling pathways in SMC and regulates proliferation by ERK-dependent activation of Elk-1 and differentiation by RhoA-dependent activation of MRTF-A.
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PMID:Sphingosine 1-phosphate stimulates smooth muscle cell differentiation and proliferation by activating separate serum response factor co-factors. 1529 66

Clinical practice reveals that osteoporotic women treated with BPs (bisphosphonates) show an increased bone mass density and a reduced risk of fractures. However, the mechanisms leading to these beneficial effects of BPs are still poorly understood. We hypothesized that ZOL (zoledronic acid), a potent third-generation BP, may induce the expression of proteins associated with the bone-forming potential of osteoblastic cells such as BSP (bone sialo-protein). Expression of BSP gene is up-regulated by hormones that promote bone formation and has been associated with de novo bone mineralization. Using real-time reverse transcriptase-PCR and Western-blot analysis, we demonstrated that ZOL increased BSP expression in Saos-2 osteoblast-like cells. Nuclear run-on and mRNA decay assays showed no effect at the transcriptional level but a stabilization of BSP transcripts in ZOL-treated cells. ZOL effect on BSP expression occurred through an interference with the mevalonate pathway since it was reversed by either mevalonate pathway intermediates or a Rho GTPase activator. We showed that ZOL impaired membrane localization of RhoA in Saos-2 cells indicating reduced prenylation of this protein. By the use of small interfering RNAs directed to RhoA and Rac1, we identified both Rho GTPases as negative regulators of BSP expression in Saos-2 cells. Our study demonstrates that ZOL induces BSP expression in osteoblast-like cells through inactivation of Rho GTPases and provides a potential mechanism to explain the favourable effects of ZOL treatment on bone mass and integrity.
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PMID:Zoledronic acid up-regulates bone sialoprotein expression in osteoblastic cells through Rho GTPase inhibition. 1532 9

The zebrafish genomic sequence database was analyzed for the presence of genes encoding members of the Rho small GTPases. The analysis shows the presence of 32 zebrafish Rho genes representing one or more homologs of the human RHOA, RND3, RHOF, RHOG, RHOH, RHOJ, RHOU, RHOV, CDC42, RAC1, RAC2, RAC3, RND1, RHOBTB1, RHOBTB2, RHOBTB3, and RHOT1 genes. By expression analysis using reverse transcriptase-PCR we show that at least 20 of the predicted zebrafish small GTPase genes are expressed in the adult stage. Interestingly, only 5 of these were found to be expressed at early embryonic stages, including rhoab, rhoad, cdc42a, cdc42c, and rac1a. We observed a strong upregulation of zebrafish rhogb expression after Mycobacterium marinum infection of adult fish. This complete annotation study provides a firm basis for the use of zebrafish as a model for analysis of Rho GTPase function in vertebrate development and the innate immune system.
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PMID:Genomic annotation and expression analysis of the zebrafish Rho small GTPase family during development and bacterial infection. 1589 57

Rho GTPases play an essential role in the control of various cellular functions. Accumulating evidence suggests that RhoA overexpression contributes to human cancer development. However, the activation states of RhoA are poorly defined in cancer cells. In this study, we examined both the expression levels and the activation states of RhoA in various lung cancer cells by quantitative real-time reverse transcriptase-polymerase chain reaction and in vivo Rho guanine nucleotide exchange assay, respectively. Moreover, we dissected the signaling pathway from the cell surface receptors to RhoA using a broad-spectrum G protein coupled receptor (GPCR) antagonist, [D-Arg1,D-Trp5,7,9,Leu11]Substance P (SP), and a recently reported Galphaq/11-selective inhibitor, YM-254890. We found that RhoA was expressed highly in large cell carcinoma cells but only weakly in adenocarcinoma cells. The activation states of RhoA are considerably different from its expression profiles. We found that four of six small cell lung carcinoma (SCLC) cell lines exhibited a moderate to high activation rate of RhoA. The addition of [D-Arg1,D-Trp5,7,9,Leu11]SP reduced RhoA activity by almost 60% in H69 SCLC cells. The addition of YM-254890 had no effect on RhoA activity in H69 cells. Our results suggest that RhoA is activated in various lung cancer cells independent of its expression levels, and the high activation state of RhoA in SCLC cells mainly depends on a neuroendocrine peptide autocrine system which signals through Galpha12 coupled GPCR to RhoA. This study provides new insights into RhoA signaling in lung cancer cells and may help in developing novel therapeutic strategies against lung cancer.
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PMID:Diverse activation states of RhoA in human lung cancer cells: contribution of G protein coupled receptors. 1727 73


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