Biomedical subjects

M Jaye

Publications and source records attributed to M Jaye.

At least 19 recordsLinked to original sources

PubMed2004

REDD2 gene is upregulated by modified LDL or hypoxia and mediates human macrophage cell death.

OBJECTIVE: Cholesterol accumulation in macrophages is known to alter macrophage biology. In this article we studied the impact of macrophage cholesterol loading on gene expression and identified a novel gene implicated in cell death. METHODS AND RESULTS: The regulated in development and DNA damage response 2 (REDD2) gene was strongly upregulated as THP-1 macrophages are converted to foam cells. These results were confirmed by Northern blot of RNA from human monocyte-derived macrophages (HMDM) treated with oxidized LDL (oxLDL). Human REDD2 shares 86% amino acid sequence identity with murine RTP801-like protein, which is 33% identical to RTP801, a hypoxia-inducible factor 1-responsive gene involved in apoptosis. Treatment of HMDM with desferrioxamine, a molecule that mimics the effect of hypoxia, increased expression of REDD2 in a concentration-dependent fashion. Transfection of U-937 and HMEC cells with a REDD2 expression vector increased the sensitivity of the cells for oxLDL-induced cytotoxicity, by inducing a shift from apoptosis toward necrosis. In contrast, suppression of mRNA expression using siRNA approach resulted in increased resistance to oxLDL treatment. CONCLUSIONS: We showed that stimulation of REDD2 expression in macrophages increases oxLDL-induced cell death, suggesting that REDD2 gene might play an important role in arterial pathology.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Thioredoxin reductase 1 is upregulated in atherosclerotic plaques: specific induction of the promoter in human macrophages by oxidized low-density lipoproteins.

Uptake of modified low-density lipoproteins (LDLs) by macrophages in the arterial wall is an important event in atherogenesis. Indeed, oxidatively modified LDLs (oxLDLs) are known to affect various cellular processes by modulating oxidation-sensitive signaling pathways. Here we found that the ubiquitous 55 kDa selenoprotein thioredoxin reductase 1 (TrxR1), which is a key enzyme for cellular redox control and antioxidant defense, was upregulated in human atherosclerotic plaques and expressed in foam cells. Using reverse transcription polymerase chain reaction analysis, we also found that oxLDLs, but not native LDLs (nLDLs), dose-dependently increased TrxR1 mRNA in human monocyte-derived macrophages (HMDMs). This stimulating effect was specific for oxLDLs, as pro-inflammatory factors, such as lipopolysaccharides (LPSs), interleukin-1beta (IL-1beta), interleukin-6 (Il-6), and tumor necrosis factor alpha (TNFalpha), under the same conditions, failed to induce TrxR1 mRNA levels to the same extent. Moreover, phorbol ester-differentiated THP-1 cells or HMDMs transiently transfected with TrxR1 promoter fragments linked to a luciferase reporter gene allowed identification of a defined promoter region as specifically responding to the phospholipid component of oxLDLs (p <.05 vs. phospholipid component of nLDLs). Gel mobility shift analyses identified a short 40-nucleotide stretch of the promoter carrying AP-1 and HoxA5 consensus motifs that responded with an altered shift pattern in THP-1 cells treated with oxLDLs, however, without evident involvement of either the Fos, Jun, Nrf2 or HoxA5 transcription factors.

Base Sequence↗

PubMed2001

Therapeutic modulation of transcription factor activity.

Aberrant gene expression is a fundamental cause of many disease-associated pathophysiologies. The pharmacological modulation of transcription factor activity therefore represents an attractive therapeutic approach to such disorders. With the exception of nuclear receptors, which are the direct targets of pharmaceuticals, other known classes of transcription factors are largely regulated indirectly by drugs that impact upon those signal transduction cascades that alter transcription factor phosphorylation and dephosphorylation and/or nuclear import. However, recent advances in drug discovery technologies now enable high-throughput screens that can identify molecules that act directly at the level of transcription factor complexes.

Animals↗

PubMed2001

PPAR-alpha and PPAR-gamma activators induce cholesterol removal from human macrophage foam cells through stimulation of the ABCA1 pathway.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that regulate lipid and glucose metabolism and cellular differentiation. PPAR-alpha and PPAR-gamma are both expressed in human macrophages where they exert anti-inflammatory effects. The activation of PPAR-alpha may promote foam-cell formation by inducing expression of the macrophage scavenger receptor CD36. This prompted us to investigate the influence of different PPAR-activators on cholesterol metabolism and foam-cell formation of human primary and THP-1 macrophages. Here we show that PPAR-alpha and PPAR-gamma activators do not influence acetylated low density lipoprotein-induced foam-cell formation of human macrophages. In contrast, PPAR-alpha and PPAR-gamma activators induce the expression of the gene encoding ABCA1, a transporter that controls apoAI-mediated cholesterol efflux from macrophages. These effects are likely due to enhanced expression of liver-x-receptor alpha, an oxysterol-activated nuclear receptor which induces ABCA1-promoter transcription. Moreover, PPAR-alpha and PPAR-gamma activators increase apoAI-induced cholesterol efflux from normal macrophages. In contrast, PPAR-alpha or PPAR-gamma activation does not influence cholesterol efflux from macrophages isolated from patients with Tangier disease, which is due to a genetic defect in ABCA1. Here we identify a regulatory role for PPAR-alpha and PPAR-gamma in the first steps of the reverse-cholesterol-transport pathway through the activation of ABCA1-mediated cholesterol efflux in human macrophages.

ATP Binding Cassette Transporter 1↗

PubMed2001

Hypoxia induces the expression of the pro-apoptotic gene BNIP3.

It has been shown that oxygen deprivation results in apoptotic cell death, and that hypoxia inducible factor 1 (HIF1) and the tumor suppressor p53 play key roles in this process. However, the molecular mechanism through which hypoxia and HIF1 induce apoptosis is not clear. Here we show that the expression of pro-apoptotic gene BNIP3 is dramatically induced by hypoxia in various cell types, including primary rat neonatal cardiomyocytes. Overexpression of HIF1alpha, but not p53, induces the expression of BNIP3. Overexpression of BNIP3 leads to a rather unusual type of apoptosis, as no cytochrome c leakage from mitochondria was detected and inhibitors of caspases were unable to prevent cell death. Taken together, these data suggest that HIF1-dependent induction of BNIP3 may play a significant role during hypoxia-induced cell death.

Animals↗

PubMed2000

Endothelial lipase: a new member of the triglyceride lipase gene family.

The triglyceride lipase gene family plays a central role in intestinal lipid absorption, energy homeostasis, lipoprotein metabolism, and atherosclerosis. A new member of this gene family, termed endothelial lipase, was recently reported. The presence of key functional motifs, the endothelial synthesis, the enzymatic profile, and the in-vivo metabolic effects of endothelial lipase suggest that, like other members of this gene family, endothelial lipase may play a role in energy delivery to tissues and in modulating lipoprotein metabolism, and could impact on atherogenesis.

Arteriosclerosis↗

PubMed1999

Solution structure of human acidic fibroblast growth factor and interaction with heparin-derived hexasaccharide.

Fibroblast growth factors (FGFs) bind to extracellular matrices, especially heparin-like carbohydrates of heparan-sulfate proteoglycans which stabilize FGFs to protect against inactivation by heat, acid, proteolysis and oxidation. Moreover, binding of FGFs to cell surface proteoglycans promotes to form oligomers, which is essential for receptor oligomerization and activation. In the present study, we determined the solution structure of acidic FGF using a series of triple resonance multi-dimensional NMR experiments and simulated annealing calculations. Furthermore, we prepared the sample complexed with a heparin-derived hexasaccharide which is a minimum unit for aFGF binding. From the chemical shift differences between free aFGF and aFGF-heparin complex, we concluded that the major heparin binding site was located on the regions 110-131 and 17-21. The binding sites are quite similar to those observed for bFGF-heparin hexasaccharide complex, showing that both FGFs recognize heparin-oligosaccharides in a similar manner.

Amino Acid Sequence↗

PubMed1999

A novel endothelial-derived lipase that modulates HDL metabolism.

High-density lipoprotein (HDL) cholesterol levels are inversely associated with risk of atherosclerotic cardiovascular disease. At least 50% of the variation in HDL cholesterol levels is genetically determined, but the genes responsible for variation in HDL levels have not been fully elucidated. Lipoprotein lipase (LPL) and hepatic lipase (HL), two members of the triacylglyerol (TG) lipase family, both influence HDL metabolism and the HL (LIPC) locus has been associated with variation in HDL cholesterol levels in humans. We describe here the cloning and in vivo functional analysis of a new member of the TG lipase family. In contrast to other family members, this new lipase is synthesized by endothelial cells in vitro and thus has been termed endothelial lipase (encoded by the LIPG gene). EL is expressed in vivo in organs including liver, lung, kidney and placenta, but not in skeletal muscle. In contrast to LPL and HL, EL has a lid of only 19 residues. EL has substantial phospholipase activity, but less triglyceride lipase activity. Overexpression of EL in mice reduced plasma concentrations of HDL cholesterol and its major protein apolipoprotein A-I. The endothelial expression, enzymatic profile and in vivo effects of EL suggest that it may have a role in lipoprotein metabolism and vascular biology.

Amino Acid Sequence↗

PubMed1998

Transforming growth factor-beta-mediated apoptosis in the Ramos B-lymphoma cell line is accompanied by caspase activation and Bcl-XL downregulation.

Upon transforming growth factor-beta (TGF-beta) treatment, Ramos cells, a B-cell lymphoma cell line, undergo apoptosis, as measured by annexin V labeling, DNA fragmentation, and propidium iodide staining. Apoptosis could be observed by 24 h after TGF-beta exposure and occurred before the development of a significant blockage of cell cycle progression. TGF-beta-mediated apoptosis was also accompanied by a strong induction of caspase-3 subfamily activity. Incubation of cells with the caspase inhibitor Z-VAD.FMK at 20 microM, but not at 10 microM, prevented TGF-beta-induced apoptosis from occurring. By comparison, caspase-3 subfamily activity was 87% inhibited at 10 microM Z-VAD.FMK and completely inhibited at 20 microM. Because of TGF-beta's well-established role of regulating gene transcription, the mRNA levels for proteins associated with apoptosis (Fas- and Fas-associated proteins, Bcl-2 family members, IAP proteins, and I kappa B) were also studied. After 24 h of TGF-beta treatment, the most significant mRNA changes occurred with Bcl-XL (two-fold decrease) and Bik (twofold increase). TGF-beta treatment also resulted after 48 h in a fivefold decrease in Bcl-XL protein levels, based on immunoblotting analysis. Therefore, TGF-beta-mediated apoptosis involves the activation of caspases. In addition, TGF-beta transcriptionally regulates Bcl-2 family members, Bcl-XL and Bik, to further influence the apoptosis process.

Amino Acid Chloromethyl Ketones↗

PubMed1998

Structure of a heparin-linked biologically active dimer of fibroblast growth factor.

The fibroblast growth factors (FGFs) form a large family of structurally related, multifunctional proteins that regulate various biological responses. They mediate cellular functions by binding to transmembrane FGF receptors, which are protein tyrosine kinases. FGF receptors are activated by oligomerization, and both this activation and FGF-stimulated biological responses require heparin-like molecules as well as FGF. Heparins are linear anionic polysaccharide chains; they are typically heterogeneously sulphated on alternating L-iduronic and D-glucosamino sugars, and are nearly ubiquitous in animal tissues as heparan sulphate proteoglycans on cell surfaces and in the extracellular matrix. Although several crystal structures have been described for FGF molecules in complexes with heparin-like sugars, the nature of a biologically active complex has been unknown until now. Here we describe the X-ray crystal structure, at 2.9 A resolution, of a biologically active dimer of human acidic FGF in a complex with a fully sulphated, homogeneous heparin decassacharide. The dimerization of heparin-linked acidic FGF observed here is an elegant mechanism for the modulation of signalling through combinatorial homodimerization and heterodimerization of the 12 known members of the FGF family.

Animals↗

PubMed1998

Cloning and characterization of human Jak-2 kinase: high mRNA expression in immune cells and muscle tissue.

We report on the cloning and sequence analysis of the mRNA coding for full-length human Janus kinase 2 (Jak2). The human form of Jak2 is 1132 amino acids in length with a M(r) of 131 KDa. It has 95% sequence similarity to pig and rat Jak2. The highest level of mRNA expression was found in the spleen, peripheral blood leukocytes, and testis. Also a significantly high level of Jak2 mRNA was found in heart and skeletal muscle. Northern blot analysis showed three mRNA species in all tissues tested, except heart and skeletal muscle, of 7.6, 5.9, and 4.8 Kb. In skeletal muscle and heart, three mRNA species of 7.6, 4.8, and 3.9 Kb were identified. The catalytic domain of the human Jak2 was expressed and its specificity for phosphorylating peptide substrates derived from the gp130, STAT, and Jak3 molecules was determined and compared to that for human Jak1 and Jak3.

Amino Acid Sequence↗

PubMed1998

hUBC9 associates with MEKK1 and type I TNF-alpha receptor and stimulates NFkappaB activity.

hUBC9, an E2 ubiquitin conjugating enzyme, was identified by yeast two-hybrid screening and coprecipitation studies to interact with MEKK1 and the type I TNF-alpha receptor, respectively. Because both of these proteins regulate NFkappaB activity, the role of hUBC9 in modulating NFkappaB activity was investigated. Overexpression of hUBC9 in HeLa cells stimulated the activity of NFkappaB as determined by NFkappaB reporter and IL-6 secretion assays. hUBC9 also synergized with MEKK1 to activate NFkappaB reporter activity. Thus, hUBC9 modulates NFkappaB activity which, at least in part, can be attributed to its interaction with MEKK1 and the type I TNF-alpha receptor.

Antigens, CD↗

PubMed1997

Ligand-binding specificity of human fibroblast growth factor receptor-3 IIIc.

Earlier studies indicated that human fibroblast growth factor receptor (FGFR)-3 IIIc was activated equally well by both FGF-1 and FGF-2. In contrast, murine FGFR-3 IIIc was preferentially activated by FGF-1. To address this issue, we determined the ligand-binding specificity of human FGFR-3 IIIc in comparison with human FGFR-1 IIIc. By equilibrium binding human FGFR-3 IIIc preferentially bound FGF-1 with high affinity, whereas FGFR-1 IIIc bound both FGF-1 and -2 with high affinity. By competition binding using FGF-1, -2, -4, or -6, FGF-1 competed more efficiently than the other FGFs. These results suggest that like the murine FGFR-3 III, FGF-1 is a preferred ligand for human FGFR-3 IIIc.

Animals↗

PubMed1997

Monocyte chemoattractant protein-2 is a potent agonist of CCR2B.

The binding and functional activity of the CC chemokines monocyte chemoattractant protein-1 (MCP-1), MCP-2, and MCP-3 have been characterized using Chinese hamster ovary DXB-11 cells transfected with the chemokine receptor CCR2B. Receptor binding studies demonstrated that 125I-labeled MCP-1 bound to a single class of high-affinity receptors with a Kd of 0.14 (0.07-0.32) nM. In competition studies MCP-1, MCP-2, and MCP-3 completely inhibited 125I-labeled MCP-1 binding with Ki values of 0.3 (0.16-0.46), 8.8 (3.4-26), and 12.2 (0.6-22) nM, respectively. In calcium mobilization studies, MCP-1 and MCP-3 induced robust elevations in intracellular calcium concentrations, whereas MCP-2 was only weakly active. In contrast, using changes in extracellular acidification rate as a functional readout, all three chemokines were identified as potent agonists of CCR2B. These data demonstrate that MCP-2, in addition to MCP-1 and MCP-3, is a potent agonist of CCR2B and furthermore that MCP-2 activates either different or a subset of the signaling pathways activated by MCP-1 and MCP-3.

Animals↗

PubMed1996

Identification of six novel autophosphorylation sites on fibroblast growth factor receptor 1 and elucidation of their importance in receptor activation and signal transduction.

Fibroblast growth factor receptor (FGFR) activation leads to receptor autophosphorylation and increased tyrosine phosphorylation of several intra cellular proteins. We have previously shown that autophosphorylated tyrosine 766 in FGFR1 serves as a binding site for one of the SH2 domains of phospholipase Cy and couples FGFR1 to phosphatidylinositol hydrolysis in several cell types. In this report, we describe the identification of six additional autophosphorylation sites (Y-463, Y-583, Y-585, Y-653, Y-654 and Y-730) on FGFR1. We demonstrate that autophosphorylation on tyrosines 653 and 654 is important for activation of tyrosine kinase activity of FGFR1 and is therefore essential for FGFR1-mediated biological responses. In contrast, autophosphorylation of the remaining four tyrosines is dispensable for FGFR1-mediated mitogen-activated protein kinase activation and mitogenic signaling in L-6 cells as well as neuronal differentiation of PC12 cells. Interestingly, both the wild-type and a mutant FGFR1 (FGFR1-4F) are able to phosphorylate Shc and an unidentified Grb2-associated phosphoprotein of 90 kDa (pp90). Binding of the Grb2/Sos complex to phosphorylated Shc and pp90 may therefore be the key link between FGFR1 and the Ras signaling pathway, mito-genesis, and neuronal differentiation.

Amino Acid Sequence↗

PubMed1995

Analysis of putative heparin-binding domains of fibroblast growth factor-1. Using site-directed mutagenesis and peptide analogues.

The contribution of individual basic amino acids within three putative "consensus sequences" for heparin binding of fibroblast growth factor-1 have been examined by site-directed mutagenesis. The results indicate that a significant reduction in the apparent affinity of fibroblast growth factor-1 for heparin is only observed when basic residues in one of the three regions are mutated. Mutation in the other regions are without affect on heparin binding. The heparin binding properties of synthetic peptides based on the three "consensus sequences" paralleled the mutagenesis results. That is, synthetic peptides corresponding to regions of the protein that were affected by mutagenesis with respect to heparin binding exhibited a relatively high affinity for immobilized heparin, whereas those corresponding to regions of similar charge density that were unaffected by mutagenesis did not. In addition, amino acid substitution of a nonbasic residue in the heparin-binding peptide could abolish its heparin binding capacity. The heparin-binding peptide could antagonize the mitogenic activity of FGF-1, probably because of the heparin dependence of this activity. Together these data demonstrate that the heparin binding properties of fibroblast growth factor-1 are dictated by structural features more complex than clusters of basic amino acids. The results of these and other studies indicate that consensus motifs for heparin-binding require further definition. More importantly, the results provide a basis for the design of peptide-based inhibitors of FGF-1.

3T3 Cells↗

PubMed1995

Activation of cytosolic phospholipase A2 by basic fibroblast growth factor via a p42 mitogen-activated protein kinase-dependent phosphorylation pathway in endothelial cells.

Basic fibroblast growth factor (FGF) stimulates the proliferation, differentiation, and motility of multiple cell types. Signal transduction by FGF is mediated by high affinity FGF receptors that have autophosphorylating tyrosine kinase activity and also elicit the release of low molecular weight signaling molecules, including inositol 1,4,5-trisphosphate, diacylglycerol, and arachidonate. We have shown previously that basic FGF-stimulated, phospholipase A2 (PLA2)-mediated arachidonate release regulates endothelial cell (EC) motility (Sa, G., and Fox, P.L. (1994) J. Biol. Chem. 269, 3219-3225). Here we identify the phospholipase responsible for basic FGF-mediated arachidonate release as cytosolic PLA2 (cPLA2) by demonstrating in EC lysates a requirement for micromolar Ca2+, dithiothreitol insensitivity, and inactivation by anti-cPLA2 antiserum. The role of cPLA2 is also indicated by the observed mechanisms of activation which show a requirement for p42 mitogen-activated protein kinase activity, cPLA2 phosphorylation, and cPLA2 translocation from cytosol to membranes. Phosphorylation of cPLA2, arachidonate release from prelabeled intact cells, and cell motility all have similar concentration dependencies on basic FGF. Since arachidonate release is required for basic FGF-stimulated motility of EC, our results show that p42 mitogen-activated protein kinase activation of cPLA2 may be a regulatory event in stimulation of cellular release of this important eicosanoid precursor during cellular responses to basic FGF.

Amino Acid Sequence↗

PubMed1995

Identification of novel peptide antagonists for von Willebrand factor binding to the platelet glycoprotein Ib receptor from a phage epitope library.

We have constructed a fusion phage epitope library in the filamentous bacteriophage fuse5. The library was made by inserting a degenerate oligonucleotide which encodes 15 variable amino acids into the NH2-terminal region of the phage gene III protein. This library, containing over 10(7) different epitope bearing phage, has been used in an attempt to identify inhibitors of the von Willebrand factor (vWF)-platelet Glycoprotein Ib interaction. The library was screened with a monoclonal antibody (RG46) that recognizes the GPIb binding domain of vWF (amino acids 445-733). A total of 30 clones falling into 8 classes have been identified that react with the RG46 antibody. Isolates from all 8 classes are positive by immunoblot analysis. The amino acid sequence of the gene III fusion protein from positive clones showed a strong homology to the known RG46 epitope. Peptides identified from the screen were synthesized and used to demonstrate that some of the synthetic peptides exhibited inhibitory activity towards ristocetin induced binding of vWF to the GPIb receptor. Thus, we have demonstrated that screening a fusion phage epitope library with a monoclonal antibody that inhibits vWF binding to the GPIb receptor can be a useful tool not only for mapping antibody recognizing determinants, but also can serve as a source for identifying novel peptides that are antagonists for vWF binding to the platelet GPIb receptor.

Amino Acid Sequence↗