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Y Khew-Goodall

Publications and source records attributed to Y Khew-Goodall.

13 recordsLinked to original sources
PubMed2000

Translocation of protein tyrosine phosphatase Pez/PTPD2/PTP36 to the nucleus is associated with induction of cell proliferation.

Pez is a non-transmembrane tyrosine phosphatase with homology to the FERM (4.1, ezrin, radixin, moesin) family of proteins. The subcellular localisation of Pez in endothelial cells was found to be regulated by cell density and serum concentration. In confluent monolayers Pez was cytoplasmic, but in cells cultured at low density Pez was nuclear, suggesting that it is a nuclear protein in proliferating cells. This notion is supported by the loss of nuclear Pez when cells are serum-starved to induce quiescence, and the rapid return of Pez to the nucleus upon refeeding with serum to induce proliferation. Vascular endothelial cells normally exist as a quiescent confluent monolayer but become proliferative during angiogenesis or upon vascular injury. Using a 'wound' assay to mimic these events in vitro, Pez was found to be nuclear in the cells that had migrated and were proliferative at the 'wound' edge. TGFbeta, which inhibits cell proliferation but not migration, inhibited the translocation of Pez to the nucleus in the cells at the 'wound' edge, further strengthening the argument that Pez plays a role in the nucleus during cell proliferation. Together, the data presented indicate that Pez is a nuclear tyrosine phosphatase that may play a role in cell proliferation.

Antibodies↗
PubMed1999

Stat6 activation is essential for interleukin-4 induction of P-selectin transcription in human umbilical vein endothelial cells.

Chronic upregulation of P-selectin expression on the surface of the endothelium has been observed in and likely contributes to a number of chronic inflammatory diseases, including atherosclerosis. Agonists of P-selectin expression fall into 2 categories: those that induce a very rapid, transient increase, lasting only hours, and those that induce prolonged upregulation lasting days. It is the latter group, which includes interleukin-4 (IL-4), that is likely to be a mediator of chronic P-selectin upregulation. The increase in P-selectin expression induced by IL-4 results from increased transcriptional activation of the P-selectin gene. The aim of this study was to deduce the postreceptor signaling pathway(s) giving rise to the prolonged increase in P-selectin expression induced by IL-4. We demonstrate the existence of 2 functional signal transducer and activator of transcription 6 (Stat6) binding sites on the P-selectin promoter and further demonstrate, by functional analysis of the P-selectin promoter, that binding of activated Stat6 to at least 1 site is essential for IL-4-induction of P-selectin transcription. Site 1 (nucleotide[nt] -142) bound Stat6 with a higher affinity than did site 2 (nt -229), and this difference was reflected functionally as constructs in which only site 1 was functional showed full IL-4 inducibility, whereas constructs in which only site 2 was functional showed only 40% of maximal IL-4 inducibility. IL-4 also induced prolonged activation of Stat6, which was contingent on the continuous presence of IL-4. The sustained activation of Stat6 induced by IL-4 is likely to be a key factor leading to the prolonged activation of the P-selectin promoter, thereby resulting in prolonged P-selectin upregulation.

Base Sequence↗
PubMed1998

Tumor necrosis factor-alpha induces adhesion molecule expression through the sphingosine kinase pathway.

The signaling pathways that couple tumor necrosis factor-alpha (TNFalpha) receptors to functional, especially inflammatory, responses have remained elusive. We report here that TNFalpha induces endothelial cell activation, as measured by the expression of adhesion protein E-selectin and vascular adhesion molecule-1, through the sphingosine kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with TNFalpha resulted in a rapid SKase activation and sphingosine 1-phosphate (S1P) generation. S1P, but not ceramide or sphingosine, was a potent dose-dependent stimulator of adhesion protein expression. S1P was able to mimic the effect of TNFalpha on endothelial cells leading to extracellular signal-regulated kinases and NF-kappaB activation, whereas ceramide or sphingosine was not. Furthermore, N, N-dimethylsphingosine, an inhibitor of SKase, profoundly inhibited TNFalpha-induced extracellular signal-regulated kinases and NF-kappaB activation and adhesion protein expression. Thus we demonstrate that the SKase pathway through the generation of S1P is critically involved in mediating TNFalpha-induced endothelial cell activation.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1998

A splice-isoform of vesicle-associated membrane protein-1 (VAMP-1) contains a mitochondrial targeting signal.

Screening of a library derived from primary human endothelial cells revealed a novel human isoform of vesicle-associated membrane protein-1 (VAMP-1), a protein involved in the targeting and/or fusion of transport vesicles to their target membrane. We have termed this novel isoform VAMP-1B and designated the previously described isoform VAMP-1A. VAMP-1B appears to be an alternatively spliced form of VAMP-1. A similar rat splice variant of VAMP-1 (also termed VAMP-1B) has recently been reported. Five different cultured cell lines, from different lineages, all contained VAMP-1B but little or no detectable VAMP-1A mRNA, as assessed by PCR. In contrast, brain mRNA contained VAMP-1A but no VAMP-1B. The VAMP-1B sequence encodes a protein identical to VAMP-1A except for the carboxy-terminal five amino acids. VAMP-1 is anchored in the vesicle membrane by a carboxy-terminal hydrophobic sequence. In VAMP-1A the hydrophobic anchor is followed by a single threonine, which is the carboxy-terminal amino acid. In VAMP-1B the predicted hydrophobic membrane anchor is shortened by four amino acids, and the hydrophobic sequence is immediately followed by three charged amino acids, arginine-arginine-aspartic acid. Transfection of human endothelial cells with epitope-tagged VAMP-1B demonstrated that VAMP-1B was targeted to mitochondria whereas VAMP-1A was localized to the plasma membrane and endosome-like structures. Analysis of C-terminal mutations of VAMP-1B demonstrated that mitochondrial targeting depends both on the addition of positive charge at the C terminus and a shortened hydrophobic membrane anchor. These data suggest that mitochondria may be integrated, at least at a mechanistic level, to the vesicular trafficking pathways that govern protein movement between other organelles of the cell.

Alternative Splicing↗
PubMed1996

Transforming growth factor-beta 1 inhibits the production of IL-8 and the transmigration of neutrophils through activated endothelium.

A central mechanism of inflammation is the activation of vascular endothelium by the inflammatory cytokines TNF-alpha and IL-1. These cytokines induce the expression of adhesion molecules, the elaboration of chemokines, and the transendothelial migration of white cells. TGF-beta 1 has anti-inflammatory properties, is expressed in the vessel wall, and has previously been shown to inhibit leukocyte adhesiveness to the endothelium at least in part by inhibiting the expression of E-selectin. We now show that TGF-beta 1 also inhibits the migration of neutrophils through endothelial monolayers activated by TNF-alpha. At a dose of 10 U/ml TNF-alpha, the transmigration of neutrophils was inhibited 42.7 +/- 7.9% (n = 8) by 0.2 ng/ml TGF-beta 1. Furthermore, TGF-beta 1 inhibited, in a time- and dose-dependent fashion, the elaboration of IL-8 by TNF-activated endothelial cells by between 33 and 78% (TNF doses from 100 down to 0.1 U/ml) and the elaboration of mRNA for IL-8 by 69%. TGF-beta 1 treatment did not significantly alter the TNF-induced IL-8 mRNA stability, suggesting that the mechanism of action of TGF-beta 1 is on gene transcription. Neutrophil transmigration through cytokine-activated endothelium involves both IL-8-dependent and IL-8-independent mechanisms. Using an anti-IL-8 Ab, we show that TGF-beta 1 inhibits only the IL-8-dependent pathway, but does not affect the IL-8-independent transendothelial migration mechanism. These and our previous results show that TGF-beta1, achieves its anti-inflammatory properties by inhibiting the expression of at least two genes, E-selectin and IL-8, which are essential in the inflammatory pathway.

Cell Movement↗
PubMed1996

Chronic expression of P-selectin on endothelial cells stimulated by the T-cell cytokine, interleukin-3.

P-selectin expressed on the surface of endothelium mediates leukocyte adhesion in vitro and rolling in vivo. Several inducers of cell-surface P-selectin expression on endothelial cells (EC) have previously been identified, all of which yield transient cell-surface expression of P-selectin lasting minutes to a few hours. We now show that a T-lymphocyte product, interleukin-3 (IL-3), stimulates the long-term endothelial cells (HUVEC). IL-3 induced cell-surface P-selectin expression in two phases. An initial peak at 10 minutes was followed by a prolonged upregulation beginning 16 hours after IL-3 addition and lasting at least 4 days. The level of P-selectin expression induced by IL-3 added for 48 hours was similar to that induced by treatment of HUVEC for 10 minutes with thrombin, and the effect of adding IL-3 for 48 hours followed by thrombin for 10 minutes was additive. Induction of cell-surface P-selectin expression by IL-3 was blocked by pretreatment of EC with a blocking monoclonal antibody against the IL-3 receptor alpha-chain. Lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF alpha) and a mutant form of IL-3 with decreased potency did not induce cell-surface P-selectin expression after 48 hours' incubation with HUVEC, suggesting that the effect was specific to IL-3. The increase in cell-surface P-selectin expression occurring after 16 hours of incubation with IL-3 was accompanied by a similar prolonged increase in the steady-state mRNA level that was not observed at 10 minutes after IL-3 addition. As T-lymphocyte infiltration is a hallmark of chronic inflammation, our observations suggest that the secretion of IL-3 by T lymphocytes may serve to maintain the inflammatory state during chronic inflammation.

Cells, Cultured↗
PubMed1993

Transforming growth factor-beta inhibits E-selectin expression on human endothelial cells.

Transforming growth factor-beta (TGF-beta), a pleiotropic cytokine that is elaborated in the active form upon co-culture of endothelial cells and pericytes or smooth muscle cells, has been shown to decrease the adhesiveness of endothelial cells for neutrophils, lymphocytes, and tumor cells. The mechanism whereby TGF-beta inhibits the adhesiveness of human endothelial cells was investigated. TGF-beta inhibited the basal E-selectin (formerly ELAM-1) expression by 55 +/- 7% and TNF-stimulated expression by 57 +/- 4%. Similar decreases of IL-1-stimulated expression were also seen. Peak inhibition was seen at TGF-beta doses between 0.2 and 2 ng/ml. Both TGF-beta 1 and -beta 2 were functional. The effectiveness of TGF-beta in inhibiting E-selectin expression was dependent on cell density and incubation time. TGF-beta also inhibited E-selectin mRNA levels in endothelial cells. TGF-beta had no effect on the expression of VCAM-1 and ICAM-1, but was additive with IL-4 in inhibiting the expression of E-selectin. The expression of E-selectin has been shown to mediate several aspects of the inflammatory response involving neutrophils and memory T lymphocytes. Perivascular TGF-beta appears to act as an inhibitor of the expression of the endothelium-specific selectin, E-selectin, and therefore of inflammatory responses involving neutrophils and (a subset of) lymphocytes.

Base Sequence↗
PubMed1991

Structure and transcriptional regulation of protein phosphatase 2A catalytic subunit genes.

The alpha and beta isoforms of the human protein phosphatase 2A catalytic subunit are encoded by distinct genes whose expression appears to be differentially regulated. To obtain a better understanding of the mechanism(s) that regulate(s) the expression of these two transcripts, we have cloned the genes encoding both isoforms. Both genes (each approximately 30 kbp) are composed of seven exons and six introns which intervene at identical locations, suggesting that they were derived from a common ancestral gene. However, the 5' upstream regions as well as the regions encoding the 5' and 3' untranslated sequences of each mRNA are different. The promoters of both genes are very G+C rich and lack the TATA and CCAAT sequences typical of many housekeeping genes. The C alpha gene contains several potential Sp1 binding sites and a potential cAMP-responsive element. Northern analysis using RNAs isolated from several different human cell lines showed that the steady-state C alpha mRNA was, in general, more abundant than the C beta mRNA. To determine whether the promoters regulate the differential C alpha and C beta RNA expression, they were fused to the reporter gene chloramphenicol acetyltransferase and transiently expressed in HeLa cells. Expression from the C alpha promoter was 7-10 times stronger than that from the C beta promoter, which paralleled the endogenous C alpha and C beta mRNA levels in HeLa cells. These data suggest that the steady-state levels of the C alpha and C beta mRNAs, are due, at least in part, to different promoter activities.

Amino Acid Sequence↗
PubMed1991

Vomeromodulin, a putative pheromone transporter: cloning, characterization, and cellular localization of a novel glycoprotein of lateral nasal gland.

Vomeromodulin, a novel glycoprotein of lateral nasal gland, has been cloned by differential hybridization from nasal/olfactory tissue. The 2.2-kb mRNA directs the in vitro synthesis of a 60-kDa primary translation product in reticulocyte lysates. Differential sensitivity to endoglycosidases indicates that vomeromodulin is posttranslationally modified in vivo by N-glycosylation to form a 70-kDa glycoprotein of the complex type. Immunocytochemical localization with two different antipeptide antisera demonstrates that vomeromodulin is abundant in the lateral nasal glands and is also present in the posterior septal and vomeronasal glands. Most striking is the observation that it is highly concentrated in the mucus of the vomeronasal organ of Jacobson but is not detectable in the mucus of the main olfactory neuroepithelium. Evaluation of mRNA and protein distribution by Northern and Western analyses, respectively, indicates that vomeromodulin is absent from 15 other tissues. The glandular and mucosal distribution of this glycoprotein implies a transport function that may be related to the mechanisms by which pheromones of low volatility gain access to their receptors in the vomeronasal organ. These observations support our hypothesis that vomeromodulin participates in perireceptor events that facilitate the process of pheromone access and detection.

Amino Acid Sequence↗
PubMed1990

Expression and organization of protein phosphatase 2A catalytic subunit genes.

The two isoforms of protein phosphatase 2A catalytic subunit are expressed at different levels in all tissues and human cell lines analyzed. This differential expression suggests a specific function for the two isoforms. The structures of the C alpha and C beta genes are highly conserved. The most sequence divergence occurs in exon I and in the 3'-nontranslated region of exon VII. These divergent regions are highly conserved between species implying that they serve a specific function in terms of RNA regulation. Both promoter regions show characteristic features of "housekeeping" genes. This correlates well with a basal expression of both mRNAs observed in all cell lines and tissues. However, a differential expression of the two isoforms was observed. Analysis of the promoter activity in transiently transfected HeLa cells indicates that this differential expression is partially due to different promoter activities. It remains an interesting question whether the CRE in the alpha gene provides a mechanism for the transcriptional regulation by the cAMP signal transduction pathway. This would appear to be the first description where a protein kinase can modulate the mRNA levels of a protein phosphatase.

Base Sequence↗
PubMed1988

Tissue-specific expression of mRNAs encoding alpha- and beta-catalytic subunits of protein phosphatase 2A.

The mRNAs encoding the catalytic subunits of the type 1 phosphatase and the alpha and beta forms of phosphatase 2A (termed 2A alpha and 2A beta) were detected in all the porcine tissues examined (brain, heart, muscle, liver, kidney and ovaries). Total phosphatase 2A transcripts were about 10 times more abundant in brain and heart than in the other tissues while the distribution of phosphatase 1 mRNA showed significantly less variation. Phosphatase 2A alpha mRNA was about 10 times more abundant than phosphatase 2A beta mRNA in most tissues. Similarly, both the alpha and beta forms of the mRNA of the catalytic subunit of the phosphatase 2A were present in all the rat tissues examined. In addition to the 2 kb mRNA generally expressed, rat tissues also express a 2.8 kb mRNA related to 2A alpha and a 1.3 kb mRNA related to 2A beta.

Animals↗