Reciprocal modulation of aconitase activity and RNA-binding activity of iron regulatory factor by nitric oxide.
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Aconitate Hydratase↗
Biomedical subjects
J Wietzerbin
Publications and source records attributed to J Wietzerbin.
Modulation of murine and human interferon-gamma receptor expression by their ligands or phorbol ester.
IFN-gamma receptor expression on murine leukaemic L1210-cells has been studied. With the help of a transfected cell-line expressing the heterologous human receptor it was possible to discern receptor-specific properties like internalization from those regulating their expression on the surface. Recombinant IFN-gamma binds specifically to its homologous receptor at 4 degrees C and is rapidly internalized at physiologic temperatures. For this effect to occur, ligand binding to its receptor at 37 degrees C is necessary and sufficient. This notion is confirmed since a reduction in the number of heterologous human IFN-gamma receptors on the murine cell surface occurred exclusively after treatment with human IFN-gamma. Even weak doses of ligand, insufficient to occupy all receptors, led to a pronounced disappearance of binding sites. However, both receptors are simultaneously up-regulated in the presence of TPA, indicating a separate pathway which is not species-specific. Our findings imply that similar elements of the intracellular signal transduction machinery are involved in the control of MuIFN-gamma and HuIFN-gamma receptor expression. The results indicate also that factors involved in binding, internalization, and regulation of receptor gene expression are not species-specific.
Animals↗
Characterization of specific functional receptors for HuIFN-alpha on a human megakaryocytic cell line (Dami): expression related to differentiation.
Interferon-alpha (IFN-alpha) treatment has been shown to be highly effective in inhibiting human megakaryocytopoiesis and controlling thrombocytosis in patients with myeloproliferative disorders. These observations suggest that IFN-alpha might play some role in the biological feature of the megakaryocytic lineage and led us to investigate the presence of specific receptors for IFN-alpha on human megakaryocytic cells, i.e. the Dami cell line, and to study the regulation of their expression. Our study demonstrates that [125I]-recombinant human IFN-alpha ([125I]rHu-IFN-alpha) binds to high-affinity specific receptor on these cells. Scatchard analysis of binding data indicates the presence of homogeneous binding sites estimated in the range of 3000-5000, with an apparent equilibrium dissociation constant, Kd, of 1-2 x 10(-9) M. Also, [125I]rHuIFN-alpha binding capacity decreased in Dami cells incubated with unlabelled rHuIFN-alpha. This down-regulation which was dose-dependent appeared to result from a reduction of IFN-alpha cell surface receptors and was observed at doses that elicited antiproliferative effects in Dami cells. Cross-linking of [125I]rHuIFN-alpha to Dami membrane proteins using a bifunctional reagent yielded to a radioactive complex of approximately 150,000 kD on SDS-PAGE. Furthermore, in response to PMA, which induces the differentiation/maturation of the Dami cells as evaluated by surface marker and ploidy analysis, a 3-fold increase of the number of specific membrane receptors for IFN-alpha was observed, without any modification of either the affinity or the M(r) value of the cross-linked complex. Such an increase appeared to be restricted to IFN-alpha receptors; actually it was not observed in [125I]IFN-gamma binding experiments. Transcript analysis indicated that down-regulation and increased expression of the IFN-alpha receptor after PMA treatment are post-transcriptional events.
Antigens, Surface↗
Involvement of the transcription factor PU.1/Spi-1 in myeloid cell-restricted expression of an interferon-inducible gene encoding the human high-affinity Fc gamma receptor.
Induction by gamma interferon (IFN-gamma) of the gene encoding the human high-affinity Fc gamma receptor (Fc gamma R1) in myeloid cells requires an IFN-gamma response region (GRR) and a myeloid cell-activating transcription element (MATE). GRR and MATE interact with factors to form, respectively, an IFN-gamma-activating complex (GIRE-BP), depending on the phosphorylation of the 91-kDa protein (subunit of ISGF3), and a cell-type-specific complex (MATE-BP). Although GIRE-BP is detected in cells of different origins after IFN-gamma treatment, the presence of MATE-BP was found to be restricted to B- and myeloid cell lines. Sequence analysis of a cDNA encoding a polypeptide recognizing specifically the MATE motif led to the identification of this product as the proto-oncogene PU.1/Spi-1, a transcriptional activator expressed in myeloid and B cells. Expression of this factor in nonhematopoietic cells allowed IFN-gamma-induced expression of a reporter gene under control of the GRR and MATE sequences. The presence of these motifs in other gene promoters indicates that the binding of PU.1/Spi-1 and IFN regulatory proteins to their respective motifs could be part of a general mechanism leading to cell-type-restricted and IFN-induced gene expression.
B-Lymphocytes↗
Investigating hairy cell leukemia dysregulations. Looking for interferon alpha site of action in hairy cells.
One of the most striking characteristics of hairy cell leukemia (HCL) is its remarkable responsiveness to alpha-interferon (IFN-alpha) therapy. IFN-alpha is a multifunctional cytokine able to regulate the growth, differentiation and recirculation of normal and malignant B lymphocytes. These activities have been extensively studied in hairy cells, but none of these properties has been shown to fully account for IFN-alpha responsiveness. Numerous data now support the idea that IFN-alpha exerts its therapeutic effect both indirectly, through stimulation of immunocompetent cells, and directly on the hairy cells. Our approach to investigate the mode action of IFN-alpha in HCL has been to identify abnormalities which occur in these tumor cells and then to ascertain whether these abnormalities can be rectified by IFN-alpha treatment. A high level of free Ca2+ in the cytoplasm of hairy cells was identified. Increases in cytosolic Ca2+ are believed to be a pivotal signal in regulating cell proliferation, cell differentiation and cell death. These high Ca2+ levels in hairy cells could be reduced upon treatment with IFN-alpha either in vitro or in vivo, probably acting by reducing Ca2+ influx into the leukemic cells. Moreover, the effect of IFN-alpha on [Ca2+]i seems to be correlated with alteration in the pattern of kinase/phosphatase activities and with down-regulation of CD20 phosphorylation, a B cell specific phosphoprotein involved in Ca2+ influx across the plasma membrane.
B-Lymphocytes↗
Non-cooperative effects of glucose and 2-deoxyglucose on their metabolism in Saccharomyces cerevisiae studied by 1H-NMR and 13C-NMR spectroscopy.
The reciprocal effects of 2-deoxy-D-glucose (dGlc) and glucose (Glc) on the aerobic metabolism of Glc and dGlc in Glc-grown repressed Saccharomyces cerevisiae were studied at 30 degrees C in a standard pyrophosphate medium containing about 4.5 x 10(7) cells/ml. 1H-, 13C-NMR and biochemical techniques were used for quantitative evaluation of Glc and dGlc metabolites. The detection of intracellular dGlc and the determination of the intracellular dGlc6P/dGlc ratio were realised using [1-13C]dGlc and 13C-NMR spectroscopy. The rates of Glc consumption in the absence and in the presence of 5 mM dGlc and 20 mM dGlc were 29 +/- 0.03 mM/min (n = 3), 16 +/- 0.02 mM/min (n = 3), and 0.08 +/- 0.01 mM/min (n = 3), respectively. This means that the Glc consumption is reduced about 41% and 70% in the presence of 5 mM and 20 mM dGlc, respectively. When dGlc is the unique carbon source, only alpha and beta anomers of dGlc6P were formed. Their quantities are equivalent and reach the maximum values within 1 h of incubation and then decrease gradually. By contrast, Glc favours the consumption of dGlc and the synthesis of dGlc6P, dideoxy-trehalose (dGlc-dGlc), deoxy-trehalose (dGlc-Glc). In the presence of Glc, dGlc6P reaches a plateau after 1 h or 2 h of incubation while the quantities of trehalose (Glc-Glc), dGlc-dGlc, dGlc-Glc, which are small at 1 h, rapidly increase with time. The reasons why dGlc and Glc exert opposite effects on their metabolism are discussed. The production of Glc-Glc decreases with increasing the external dGlc concentration or the dGlc/Glc ratio. The effect of dGlc on the biosynthesis of Glc-Glc can be explained by the competition of dGlc and Glc with respect to hexokinase. Although Glc favours the synthesis of dGlc6P, the maximum concentration of dGlc6P shows little dependence on the external dGlc concentration as long as glucose is not exhausted. The internal dGlc6P/dGlc ratio at equilibrium, about 4.7 +/- 0.7, is also found to be independent of the dGlc concentration in the suspension. Only a small fraction of dGlc6P disappears to give rise to the formation of dGlc-dGlc and dGlc-Glc. At equilibrium the inverse reaction from dGlc6P to dGlc may be important to compensate for the fast reaction of dGlc phosphorylation by hexokinase. At least nine series of experiments were conducted and showed that, in pyrophosphate media and for incubation times less than 4 h, dGlc-dGlc was not observed in the absence of Glc.(ABSTRACT TRUNCATED AT 400 WORDS)
Carbohydrate Sequence↗
Inactivation of interleukin-6 in vitro by monoblastic U937 cell plasma membranes involves both protease and peptidyl-transferase activities.
Human promonocytic U937 cells have previously been shown to possess at their cell surface specific transmembrane serine proteases and N-terminal amino acid proteases as well as associated enzymes including elastase and cathepsin G. In this study, purified plasma membranes from U937 cells are reported to degrade the recombinant 21-kDa 125I-interleukin-6 (125I-IL-6) into 8-kDa products with loss of biological activity, as monitored by polyacrylamide gel electrophoresis and a cell-proliferation bioassay. Degradation of 125I-IL-6 by plasma membranes was completely prevented by the serine-protease inhibitor diisopropyl fluorophosphate, but was only partially impaired by alpha 1-protease inhibitor and antibody against cathepsin G. A similar incubation of 125I-IL-6 with cathepsin G purified from U937 cells caused hydrolysis of the cytokine into similar inactive 8-kDa fragments, whereas incubation with purified U937 cell elastase failed to degrade the peptide. These findings indicate that U937 cells hydrolyze IL-6 using cell-associated serine-protease activity and that cathepsin G partially participates in this degradation. Prolonged incubation of 8-kDa 125I-IL-6 fragments with purified U937 plasma membranes, led to a complete loss of IL-6 activity related to the transformation of the 8-kDa forms into a higher-molecular-mass complex (16 kDa). This complex was stable in SDS and 2-mercaptoethanol at 100 degrees C and was not dissociated by hydroxylamine treatment, indicating the formation of a covalent non-ester bond between the 8-kDa 125I-IL-6-derived peptide and an undetermined acceptor. An initial oxidative treatment of 125I-IL-6 partially prevented complex formation, suggesting the presence of one or more oxidizable methionine residues at the binding site of 8-kDa 125I-IL-6 peptide. The kinetics of complex formation (time dependence and plasma-membrane-concentration dependence), as well as its inhibition by a specific inhibitor of N-amino-peptidase activity, bestatin, suggest the participation of peptidyl-transferase activity in complex formation. Finally, a plasma-membrane fraction, corresponding to a molecular mass > or = 30 kDa, was able to convert the 8-kDa 125I-IL-6 forms into the 125I-labeled 16-kDa complex, suggesting that a > or = 30-kDa peptidyl-transferase enzyme catalyzes the reaction and provides the 125I-labeled 16-kDa peptide by dimerization of 8-kDa 125I-IL-6-derived intermediates. Further identification of the plasma-membrane-associated peptidyl transferase as a regulator of IL-6 proteolysis may be of physiological relevance for the control of IL-6 biological activity.
Aminopeptidases↗
Phosphorylation of CD20 in cells from a hairy cell leukemia cell line. Evidence for involvement of calcium/calmodulin-dependent protein kinase II.
Hairy cell leukemia is an uncommon B cell lymphoproliferative disease of unknown etiology. We previously observed that CD20, a membrane protein involved in B cell activation, is hyperphosphorylated on hairy cells and that these cells have unusually high levels of intracellular free Ca2+. Therefore, we used a hairy cell line, HCLL-7876, to study the potential involvement of Ca(2+)-activated protein kinases in CD20 phosphorylation. Addition of the Ca2+ ionophore, ionomycin, increased CD20 phosphorylation both in activated B cells and in cells from the hairy cell line; addition of EGTA to either cell type decreased basal levels of CD20 phosphorylation. Ionomycin treatment of these cells resulted in increased kinase activity of cytosolic extracts toward syntide-2, a synthetic peptide substrate for calcium/calmodulin-dependent kinase II (CaM-KII), with kinetics similar to those of CD20 phosphorylation in the cell line. CD20 isolated from the cell line was a substrate for purified CaM-KII in vitro. Phosphopeptide maps of CD20 from untreated hairy cells or ionomycin-treated HCLL-7876 cells were similar to maps of CD20 that had been phosphorylated in vitro by CaM-KII. These results suggest that the unusually high levels of intracytoplasmic Ca2+ in hairy cells may enhance the phosphorylation of key surface proteins.
Amino Acid Sequence↗
Interferon-gamma modulates retinoblastoma gene mRNA in monocytoid cells.
To study the effect of interferon gamma (IFN-gamma) on the expression of the retinoblastoma (RB) susceptibility gene, we performed Northern-blot analysis on RNA extracted from Wish, HEL and monocytoid cell lines U-937 and THP-1 treated with 1,000 IU/ml of recombinant IFN-gamma. In U-937 and THP-1 cells, IFN-gamma increased the abundance of RB mRNA. In Wish and HEL cells, co-treatment with cycloheximide was required for IFN-gamma to increase the level of RB mRNA. Pre-treatment of THP-1 cells with cycloheximide prior to IFN-gamma treatment augmented the effects of IFN-gamma on RB gene expression. The effect of IFN-gamma in THP-1 cells was observed after 3 hr of treatment, being more pronounced after 6 hr and persisting until at least 18 hr, although at a lower level. These results suggest that IFN-gamma regulates the level of RB mRNA by different mechanisms in the different cell types. This cytokine increases the abundance of RB mRNA in monocytoid cell lines, reinforced by prior treatment with cycloheximide. Inhibition of protein synthesis is required in Wish and HEL cell lines before IFN-gamma has an effect on RB gene expression.
Actins↗
Biosynthesis of nitric oxide activates iron regulatory factor in macrophages.
Biosynthesis of nitric oxide (NO) from L-arginine modulates activity of iron-dependent enzymes, including mitochondrial acontiase, an [Fe-S] protein. We examined the effect of NO on the activity of iron regulatory factor (IRF), a cytoplasmic protein which modulates both ferritin mRNA translation and transferrin receptor mRNA stability by binding to specific mRNA sequences called iron responsive elements (IREs). Murine macrophages were activated with interferon-gamma and lipopolysaccharide to induce NO synthase activity and cultured in the presence or absence of NG-substituted analogues of L-arginine which served as selective inhibitors of NO synthesis. Measurement of the nitrite concentration in the culture medium was taken as an index of NO production. Mitochondria-free cytosols were then prepared and aconitase activity as well as IRE binding activity and induction of IRE binding activity were correlated and depended on NO synthesis after IFN-gamma and/or LPS stimulation. Authentic NO gas as well as the NO-generating compound 3-morpholinosydnonimine (SIN-1) also conversely modulated aconitase and IRE binding activities of purified recombinant IRF. These results provide evidence that endogenously produced NO may modulate the post-transcriptional regulation of genes involved in iron homeostasis and support the hypothesis that the [Fe-S] cluster of IRF mediates iron-dependent regulation.
Aconitate Hydratase↗
Influence of glucose on the deoxyglucose metabolism in S cerevisiae: detection and identification of deoxyglucose and trehalose derivatives by 1H- and 13C-NMR spectroscopy.
The metabolism of 2-deoxy-D-glucose (DG) in glucose grown repressed Saccharomyces cerevisiae cells was studied in the absence and presence of glucose (Glc) at 30 degrees C in a standard pyrophosphate medium containing 4.5 x 10(7) cells/ml. 1H- and 13C-NMR spectroscopy were successfully used to distinguish and identify several derivatives of DG and trehalose. Using [1-13C]DG, alpha- and beta-DG, alpha- and beta-DG6P, dideoxy-trehalose (DG-DG) and deoxy-trehalose (DG-Glc) can be simultaneously observed in the intracellular medium. The [DG6P]/[DG] ratio is about 5-6. The results seem to indicate the existence of an equilibrium between DG and DG6P, which limits the production of DG6P in cells. Glucose was found to exert a great influence on the metabolism of DG. It favours the formation of DG-DG and DG-Glc.
Carbon Isotopes↗
Divergent regulation of cell surface protease expression in HL-60 cells differentiated into macrophages with granulocyte macrophage colony stimulating factor or neutrophils with retinoic acid.
Taking advantage of the recently demonstrated presence of N-aminopeptidases and the serine protease dipeptidyl aminopeptidase IV (DPP IV) at the surface of human myeloblastic HL-60 cells, the regulation of these protease activities in HL-60 cell differentiation has been assessed using combined spectrophotometric and flow cytometric assays. Addition of human recombinant granulocyte macrophage colony stimulating factor (rHu-GM-CSF) to HL-60 cells to induce differentiation into macrophages led to a time- and dose-dependent increase in both cell surface N-aminopeptidase and DPP IV activities. Protease up-regulation was due to an enhancement in cell surface protease number, associated with a slight rise in apparent affinities of the enzymes for their substrates. In contrast, in HL-60 cells induced to differentiate into neutrophils in the presence of retinoic acid, expression of cell surface N-aminopeptidases was almost completely abolished in a time- and dose-dependent fashion, and this down-regulation was accompanied by a weak but significant decrease in affinity. However, no noticeable difference was seen in serine DPP IV expression between retinoic acid-treated and untreated HL-60 cells. Retinoic acid treatment also reduced soluble protease activity in vitro indicating that down-regulation of membrane aminopeptidases was not due to their proteolytic clip. No modulation in the activity of any of the enzymes tested was seen with human recombinant tumor necrosis factor-alpha or retinol which do not induce HL-60 cell differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)
Blood Cells↗
Two cis-DNA elements involved in myeloid-cell-specific expression and gamma interferon (IFN-gamma) activation of the human high-affinity Fc gamma receptor gene: a novel IFN regulatory mechanism.
The human high-affinity receptor for the constant region of immunoglobulin G (human Fc gamma R1) is encoded by two mRNAs induced selectively by gamma interferon (IFN-gamma) and expressed in cells of myeloid lineage. The cis-DNA element (GRR) previously found to confer IFN-gamma responsiveness to this gene acts as an inducible enhancer and is the target of an IFN-gamma-activated factor(s) (GIRE-BP) in cells of different origins. Although the GRR motif is not related to the DNA elements involved in the regulation of other IFN-stimulated genes, GIRE-BP binding depends on the IFN-gamma-dependent activation of the 91-kDa protein known to be one of the factors of a transcriptional complex activated by IFN-alpha. Deletions of the Fc gamma R1 promoter allowed us to identify a 25-bp element, downstream from the GRR motif, conferring cell-type-specific expression. This element, called MATE (myeloid activating transcription element), is the DNA target for constitutive factors forming two complexes, MATE-BP1 and MATE-BP2. In accordance with the functional analysis, MATE-BP binding activities were detected in extracts prepared from myeloid cell lines such as THP-1, HL-60, and U-937 but not in HeLa cell extracts. The MATE motif is present not only in the promoter of other Fc receptor genes but also in several promoters of genes whose expression is restricted to monocytic cells. Our results suggest that human Fc gamma R1 gene expression in myeloid cells is initiated by the interaction of IFN-gamma-activated factors with cell-type-specific factors through their binding to the GRR and MATE motifs.
Animals↗
Interferon-alpha downregulates the abnormal intracytoplasmic free calcium concentration of tumor cells in hairy cell leukemia.
Hairy cell leukemia (HCL) is a B-cell tumor affecting the preplasma stage of B-cell differentiation. One important feature of the disease is its exquisite sensitivity to interferon-alpha (IFN-alpha) therapy. Because we showed earlier that the CD20 molecule is consistently hyperphosphorylated in hairy cells and because previous studies showed that CD20 is involved in regulating intracytoplasmic free calcium concentrations ([Ca2+]i) in normal B lymphocytes, we measured [Ca2+]i in tumor cell samples from patients with HCL and studied the effect of IFN-alpha on this parameter. Using the Ca(2+)-sensitive fluorophore fura-2, we observed that hairy cells display a slightly but consistently higher [Ca2+]i than normal 48-hour-activated B cells or other leukemic cells. Furthermore, both in vitro preincubation of cell samples with IFN-alpha and in vivo administration of this cytokine reduced the [Ca2+]i in hairy cells. This effect was observed together with a decrease in transmembrane Ca2+ influx. However, preincubation with IFN-gamma had no effect. The in vivo correlation between the diminution of CD20 phosphorylation and [Ca2+]i in tumor cell samples from patients at the beginning of IFN-alpha therapy suggests that these two parameters are connected.
Antigens, CD↗
Tumor necrosis factor-alpha and IL-6 up-regulate IFN-gamma receptor gene expression in human monocytic THP-1 cells by transcriptional and post-transcriptional mechanisms.
IFN-gamma is a potent activator of monocytic cell functions, including the stimulation of TNF-alpha and the control of IL-6 synthesis. These cytokines might act as autocrine or paracrine factors together with IFN-gamma in inducing biologic responses. In this study, we addressed the question of whether or not TNF-alpha and IL-6 play a role in modulating of IFN-gamma binding. Expression of IFN-gamma surface receptor was measured in the human monocytic THP-1 cells. The capacity of these cells to bind IFN-gamma increased with time after treatment with each cytokine. The number of IFN-gamma R per cell rose by three- and fourfold after 16 h of treatment with TNF-alpha and IL-6, respectively. Scatchard analysis of binding data showed that neither TNF-alpha nor IL-6 affected the affinity of IFN-gamma for its receptor. The increased surface expression of IFN-gamma R induced by TNF-alpha and IL-6 correlated with the rise in IFN-gamma mRNA receptor levels. TNF-alpha-mediated up-regulation of IFN-gamma R was due to an increase in transcriptional activity of the IFN-gamma R gene, as shown by run-on experiments. No significant modulation of IFN-gamma R gene transcription was observed in nuclei from cells treated with IL-6, whose effect appeared to be related to IFN-gamma mRNA receptor stabilization. Taken together, the present results provide, to our knowledge, the first evidence for cytokine-mediated up-regulation of IFN-gamma R in human monocytic cells. This up-regulation by TNF-alpha and IL-6, which are both produced by monocytes, occurs through different mechanisms, and may be of physiologic relevance in host defense.
Cell Line↗
Comparative study of the effects of amphotericin B on the glucose metabolism in Saccharomyces cerevisiae in K(+)- and Na(+)-rich media.
In order to elucidate the effects of amphotericin B (AMB) on the glycolytic pathway, the metabolism of [1-13C]glucose in glucose-grown repressed Saccharomyces cerevisiae was studied. The cells were aerobically suspended in pyrophosphate solutions of high potassium concentration with or without 10(-6) M amphotericin B and measurements were made using 1H-, 13C-NMR spectroscopy and biochemical methods. The results were compared with those obtained under the same experimental conditions but in a medium rich in sodium salts containing the same antibiotic concentration. In general the presence of 10(-6) M AMB reduces the glucose consumption and the ethanol production while favouring the glycerol and trehalose formation. These effects are greatly reduced when a high K+ concentration was used. The AMB effects on the glucose consumption and the production of ethanol, glycerol and trehalose, observed in a suspension rich in Na+, can be fairly well explained by the leakage of K+ through AMB membrane channels. This outflux induces a substantial decrease in the activity of some K(+)-dependent enzymes, such as aldolase, phosphofructokinase and pyruvate kinase. The intensities of the glutamate C2 and C4 signals are higher with a suspension rich in Na+ than with a suspension rich in K+, suggesting that the Krebs cycle operates more effectively in a solution rich in Na+. In the absence of AMB, the passive diffusion of glycerol through the cell membrane is relatively slow and apparently depends on the ionic external medium: it is more efficient in solutions with a high K+ than with a high Na+ concentration. In the presence of 10(-6) M AMB, the glycerol C1,3 resonance drastically decreases at 20 min and then disappears in the noise. This rapid disappearance suggests that glycerol can easily pass through the pores arising from the interaction of AMB with the membrane sterols. However, the rate of pore formation is slow, independent of the external medium (Na+ or K+) and this process is not completed within 20 min.
Amphotericin B↗
IFN-gamma and transforming growth factor-beta 1 differently regulate fibronectin and laminin receptors of human differentiating monocytic cells.
The regulation of extracellular matrix (ECM) protein receptor expression was followed in the human promonocytic cell line U937 before and after stimulation either with PMA or various cytokines implicated in monocytopoiesis. On undifferentiated U937 cells, alpha-chains of very late Ag (VLA)-4, VLA-5, and VLA-6 were constitutively expressed whereas alpha-chains of VLA-2 (alpha 2) and vitronectin receptor (alpha V) were not. Maturation of U937 cells with PMA resulted in a marked decrease in alpha 4 expression (25% of control by day 5), and a small but significant increase in the expression of alpha 2 and alpha v over 4 days of stimulation. Unstimulated U937 cells attached to fibronectin (FN) but not to laminin (LM), collagens I/IV-coated surfaces. After PMA stimulation, U937 cells exhibited enhanced adherence on FN and expressed the ability to adhere to LM. PMA stimulation also promoted U937 spreading both on FN and LM. Adhesion on FN all along the maturation pathway was specifically and totally inhibited by anti-alpha 5 mAb but not by anti-alpha 4 mAb. Anti-beta 1, anti-alpha 6, anti-alpha 2, and anti-alpha v mAb, as well as Tyr-Ile-Gly-Ser-Arg and Arg-Gly-Asp synthetic peptides from LM, had no effect on adhesion of PMA-stimulated cells on LM, implying that U937 cell adherence to LM is mediated through hitherto distinct receptors. In the presence of rIFN-gamma, differentiating U937 cells did not adhere to LM and lost the capacity to bind to FN. Loss of adhesion to FN was correlated with the concomitant decrease in the expression of alpha 4 and alpha 5 integrin subunits. In contrast, TGF-beta 1 mimicked most of the effects of PMA by enhancing the attachment of maturating U937 cells on FN through alpha 5 receptors and by promoting adherence to LM. TGF-beta 1 stimulation also promoted U937 cell spreading on both FN- and LM-coated surfaces. The data suggest that inflammatory cytokines such as IFN-gamma and TGF-beta 1 may be critically important in the homing of monocytic cells at sites of inflammation by modulating cell-surface expression of ECM receptors.
Amino Acid Sequence↗
Human endothelial cells transfected by SV40 T antigens: characterization and potential use as a source of normal endothelial factors.
A putative role for the vascular endothelium as target for autoantibodies has been suggested in several autoimmune disorders and connective-tissue diseases. However, there are some difficulties linked to the use of cultured endothelial cells (EC) that limit considerably the extensive studies on the nature of endothelial target antigens involved. To overcome this problem, human EC, derived from umbilical veins, were transfected with recombinant plasmid pSV1 which contained the early genes of simian virus SV40. These transfected cells, called EC-pSV1, are able to grow without EC growth supplement and demonstrate a population doubling time of about 50 h. Among the EC properties, EC-pSV1 retain intracellular content of angiotensin-converting enzyme activity, exhibit constitutive production of interleukin 6 and of a growth-promoting activity on early passage EV, express intercellular adhesion molecule 1 (ICAM-1) and its up-regulation by tumor necrosis factor alpha, but have lost the expression of factor VIII-related antigen. Moreover, EC-pSV1 express a 55-kDa antigen found on EC and human platelets, and presumably acting as an antibody target in some cases of non-allergic asthma. However, at the 50-55th generation, morphological changes and altered growth behavior were visible. This work demonstrates that transfection of EC with SV40 T antigens may be of interest, particularly in areas of research including the study of EC targets involved in different human diseases.
Antigens, Polyomavirus Transforming↗