Overexpression of OSBP-related protein 2 (ORP2) in CHO cells induces alterations of phospholipid species composition

2005 ◽  
Vol 83 (5) ◽  
pp. 677-683 ◽  
Author(s):  
Reijo Käkelä ◽  
Kimmo Tanhuanpää ◽  
Saara Laitinen ◽  
Pentti Somerharju ◽  
Vesa M Olkkonen
Keyword(s):  
Fatty Acid ◽  
Neutral Lipid ◽  
Cho Cells ◽  
Cholesterol Metabolism ◽  
Chinese Hamster ◽  
Related Protein ◽  
Compensatory Response ◽  
Oxysterol Binding Protein ◽  
Phospholipid Species

We have previously shown that overexpression of human OSBP-related protein 2 (ORP2) in Chinese hamster ovary (CHO) cells results in increased efflux and reduced esterification of cholesterol. The ORP2-expressing cells also have a reduced level of triacylglycerols. We investigated the effects of ORP2 expression on the phospholipid (PL) molecular species and the neutral lipid (NL) fatty acid composition of CHO cells cultured in the presence or absence of serum lipoproteins. In the presence of lipoproteins, ORP2/CHO cells display an increase in polyunsaturated PL species, and polyunsaturated fatty acids (PUFA) in the diminished NL pool are reduced. The increase of polyunsaturated PL may represent a compensatory response to alterations in cholesterol metabolism. Upon lipoprotein deprivation, the ORP2/CHO cells display a drop in polyunsaturated and an increase in mono and diunsaturated PL species. Our results suggest that this is due to defective recycling of PUFA from the diminished NL pool to PL. Furthermore, the PL PUFA, which are elevated in ORP2/CHO cells, are most likely subject to more rapid turnover than the NL-associated pool. The results provide evidence for a delicate integration of cholesterol, PL, and NL metabolism and a role of ORP2 as a regulator of the cellular lipidome.Key words: cholesterol metabolism, mass spectrometry, neutral lipid, oxysterol binding protein, phospholipid, polyunsaturated fatty acid.

scholarly journals Overexpression of OSBP-related protein 2 (ORP2) induces changes in cellular cholesterol metabolism and enhances endocytosis

2005 ◽  
Vol 390 (1) ◽  
pp. 273-283 ◽  
Author(s):  
Riikka Hynynen ◽  
Saara Laitinen ◽  
Reijo Käkelä ◽  
Kimmo Tanhuanpää ◽  
Sari Lusa ◽  
...  
Keyword(s):  
Hela Cells ◽  
Cho Cells ◽  
Cholesterol Metabolism ◽  
Reductase Activity ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Receptor Expression ◽  
Cholesterol Depletion ◽  
Related Protein ◽  
Cellular Cholesterol

ORP2 [OSBP (oxysterol-binding protein)-related protein 2] belongs to the 12-member mammalian ORP gene/protein family. We characterize in the present study the effects of inducible ORP2 overexpression on cellular cholesterol metabolism in HeLa cells and compare the results with those obtained for CHO cells (Chinese-hamster ovary cells) that express ORP2 constitutively. In both cell systems, the prominent phenotype is enhancement of [14C]cholesterol efflux to all extracellular acceptors, which results in a reduction of cellular free cholesterol. No change was observed in the plasma membrane cholesterol content or distribution between raft and non-raft domains upon ORP2 expression. However, elevated HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase activity and LDL (low-density lipoprotein) receptor expression, as well as enhanced transport of newly synthesized cholesterol to a cyclodextrin-accessible pool, suggest that the ORP2 expression stimulates transport of cholesterol out of the endoplasmic reticulum. In contrast with ORP2/CHO cells, the inducible ORP2/HeLa cells do not show down-regulation of cholesterol esterification, suggesting that this effect represents an adaptive response to long-term cholesterol depletion in the CHO cell model. Finally, we provide evidence that ORP2 binds PtdIns(3,4,5)P3 and enhances endocytosis, phenomena that are probably interconnected. Our results suggest a function of ORP2 in both cholesterol trafficking and control of endocytic membrane transport.

ICAM-1-CD18 interaction mediates neutrophil cytotoxicity through protease release

1998 ◽  
Vol 274 (6) ◽  
pp. C1634-C1644 ◽  
Author(s):  
Carlton C. Barnett ◽  
Ernest E. Moore ◽  
Gary W. Mierau ◽  
David A. Partrick ◽  
Walter L. Biffl ◽  
...  
Keyword(s):  
Cho Cells ◽  
Chinese Hamster ◽  
Cell Lysis ◽  
Polymorphonuclear Neutrophil ◽  
Intercellular Adhesion Molecule ◽  
Wild Type ◽  
Myristate Acetate ◽  
Intercellular Adhesion Molecule 1 ◽  
Eglin C

Interaction of the β2-integrin complex on the polymorphonuclear neutrophil (PMN) with intercellular adhesion molecule-1 (ICAM-1) has been implicated in PMN-mediated cytotoxicity. This study examined interaction of the CD11a, CD11b, and CD18 subunits of the β2-integrin with ICAM-1, transfected into Chinese hamster ovarian (CHO) cells to avoid effects of other adhesion molecules. Incubation of quiescent PMNs with wild-type and ICAM-1-transfected CHO cells produced nominal cell lysis. Similarly, when phorbol myristate acetate (PMA)-activated PMNs were incubated with wild-type CHO cells, minimal cytotoxicity was produced. However, when ICAM-1-transfected CHO cells were incubated with PMA-activated PMNs, 40% cell lysis occurred. Blockade with a monoclonal antibody (MAb) to ICAM-1 or MAbs to CD11a, CD11b, or CD18 reduced PMN-mediated cytotoxicity to baseline. To examine the role of adhesion in cytotoxicity, we studied β2-integrin-mediated PMN adhesion to ICAM-1-transfected CHO cells and found that MAbs for CD11a, CD11b, and CD18 all abrogated PMN cytotoxicity despite disparate effects on adhesion. To assess the role of CD18, β2-integrin subunits were cross-linked, and CD18 alone mediated protease release. Moreover, ICAM-1 was immunoprecipitated from transfected CHO cells and incubated with PMNs. This soluble ICAM-1 provoked elastase release, similar to PMA, which could be inhibited by MAbs to CD18 but not MAbs to other β2-integrin subunits. In addition, coincubation with protease inhibitors eglin C and AAPVCK reduced PMN-mediated cytotoxicity to control levels. Finally, ICAM-1-transfected CHO cells were exposed to activated PMNs from a patient with chronic granulomatous disease that caused significant cell lysis, equivalent to that of PMNs from normal donors. Collectively, these data suggest that ICAM-1 provokes PMN-mediated cytotoxicity via CD18-mediated protease release.

scholarly journals Opsonization Modulates Rac-1 Activation during Cell Entry by Leishmania amazonensis

2002 ◽  
Vol 70 (8) ◽  
pp. 4571-4580 ◽  
Author(s):  
J. Morehead ◽  
I. Coppens ◽  
N. W. Andrews
Keyword(s):  
Cho Cells ◽  
Chinese Hamster ◽  
Cytochalasin D ◽  
Leishmania Amazonensis ◽  
Cell Entry ◽  
Butanedione Monoxime ◽  
Respiratory Burst Oxidase ◽  
Insight Into ◽  
Host Tissues

ABSTRACT Lesions caused by Leishmania amazonensis normally heal, but relapses occur due to parasite persistence in host tissues. It has been proposed that infection of fibroblasts plays an important role in this process by providing the parasites with a safe haven in which to replicate. However, most previous studies have focused on the entry of Leishmania into macrophages, a process mediated by serum opsonins. To gain insight into a possible role of nonopsonic entry in the intracellular persistence of amastigotes, we examined the invasion of Chinese hamster ovary (CHO) cells. Amastigotes entered CHO cells by a cytochalasin D, genistein, wortmannin, and 2,3-butanedione monoxime-sensitive pathway and replicated within phagolysosomes. However, unlike most phagocytic processes described to date, amastigote internalization in CHO cells involved activation of the GTPases Rho and Cdc42 but not Rac-1. When uptake was mediated by fibronectin or when amastigotes were opsonized with immunoglobulin G and internalized by Fc receptor-expressing CHO cells, Rac-1 activation was restored and found to be required for parasite internalization. Given the essential role of Rac in assembly of the respiratory burst oxidase, invasion through this nonopsonic, Rac-1-independent pathway may play a central role in the intracellular survival of Leishmania in immune hosts.

Complete Removal of N-Linked Glycans from Platelet GPIbα Impairs Platelet Agglutination and von Willebrand Factor Binding In Vitro.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1535-1535
Author(s):  
Suzana M. Zorca ◽  
Emma C. Josefsson ◽  
Viktoria Rumjantseva ◽  
John H. Hartwig ◽  
Karin M. Hoffmeister
Keyword(s):  
Flow Cytometry ◽  
Von Willebrand Factor ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Lectin Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We previously reported that the lectin domain of the αMβ2 receptor on macrophages mediates the rapid clearance of transfused washed murine platelets which have been refrigerated for 2 hrs in the absence of plasma. The clearance is mediated by the recognition of exposed βN-acetylglucosamine (β-GlcNAc) residues on N-linked glycans of clustered platelet GPIbα molecules. Covering the exposed β-GlcNAc residues on GPIbα N-linked glycans via galactosylation prevents the clearance of chilled murine platelets from the circulation. The role of N-linked glycans in platelet function and survival is unclear. To dissect the role of N-linked glycosylation of GPIbα on the binding of von Willebrand factor (vWf), we use human platelets and Chinese Hamster Ovary (CHO) cells, stably expressing human GPIbα/βand GPIX. Deglycosylation of platelet GPIbα N-liked glycans was achieved using the enzyme peptide-N-glycosidase F (PGNaseF), specific for complex N-linked glycans. In agglutination assays using platelets incubated with and without PNGaseF for 16hrs at 37°C, we observed 30-40 % less agglutination in response to ristocetin for platelets depleted of N-linked glycans with PNGaseF. Additionally, a 30 % reduction in vWf binding to PNGaseF-treated platelets compared with control platelets was measured by flow cytometry, using a FITC-conjugated mAb that detects surface-bound vWf. In CHO cells, GPIbα N-linked oligosaccharides were manipulated by adding swainsonine or tunicamycin, two inhibitors of N-linked oligosaccharide synthesis in the Golgi. vWf binding to platelets or to CHO cells was studied by aggregometry or by light microscopy to establish the fraction of CHO-cell aggregates. As was the case with platelets, vWf-dependent aggregation of CHO cells expressing GPIb-IX decreased three fold in response to botrocetin, but only following complete N-linked glycans depletion with tunicamycin. In contrast, partial N-linked carbohydrate modification with swainsonine did not significantly alter aggregate formation in CHO- cells expressing GPIb-IX. Complete inhibition of N-linked glycosylation decreased botrocetin-induced vWf binding to CHO- cells expressing GPIb-IX by ~50%, as determined by flow cytometry. No change was observed following swainsonine treatment. Surface expression of GP1bα remained unchanged after both tunicamycin and swainsonine treatment, and with PGNaseF treatment of platelets. These results confirm that 1) N-linked glycans are not required for GPIbα surface expression, and 2) indicate that N-linked glycans likely play a role in vWf binding to platelet GPIbα.

scholarly journals Fatty Acid Remodeling of GPI-anchored Proteins Is Required for Their Raft Association

2007 ◽  
Vol 18 (4) ◽  
pp. 1497-1506 ◽  
Author(s):  
Yusuke Maeda ◽  
Yuko Tashima ◽  
Toshiaki Houjou ◽  
Morihisa Fujita ◽  
Takehiko Yoko-o ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Lipid Rafts ◽  
Cho Cells ◽  
Membrane Fraction ◽  
Double Mutant ◽  
Chinese Hamster ◽  
Wild Type ◽  
Saturated Chain ◽  
Mutant Cells ◽  
Detergent Resistant Membrane

Whereas most of the cellular phosphatidylinositol (PI) contain unsaturated fatty chains and are excluded from rafts, GPI-anchored proteins (APs) unusually contain two saturated fatty chains in their PI moiety, and they are typically found within lipid rafts. However, the origin of the saturated chains and whether they are essential for raft association are unclear. Here, we report that GPI-APs, with two saturated fatty chains, are generated from those bearing an unsaturated chain by fatty acid remodeling that occurs most likely in the Golgi and requires post-GPI-attachment to proteins (PGAP)2 and PGAP3. The surface GPI-APs isolated from the PGAP2 and -3 double-mutant Chinese hamster ovary (CHO) cells had unsaturated chains, such as oleic, arachidonic, and docosatetraenoic acids in the sn-2 position, whereas those from wild-type CHO cells had exclusively stearic acid, a saturated chain, indicating that the sn-2 chain is exchanged to a saturated chain. We then assessed the association of GPI-APs with lipid rafts. Recovery of unremodeled GPI-APs from the double-mutant cells in the detergent-resistant membrane fraction was very low, indicating that GPI-APs become competent to be incorporated into lipid rafts by PGAP3- and PGAP2-mediated fatty acid remodeling. We also show that the remodeling requires the preceding PGAP1-mediated deacylation from inositol of GPI-APs in the endoplasmic reticulum.

The Cytoplasmic Domain of Glycoprotein Ibβ Is Critical to Surface Trafficking of Glycoprotein Ib-IX Complex.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1531-1531
Author(s):  
Xi Mo ◽  
Jose A. Lopez ◽  
Renhao Li
Keyword(s):  
Cho Cells ◽  
Transmembrane Domain ◽  
Complex Structure ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Cytoplasmic Domain ◽  
Expression Level ◽  
Platelet Glycoprotein ◽  
Cellular Expression

Abstract Platelet glycoprotein (GP) Ib-IX-V complex mediates platelet adhesion to the subendothelium of damaged vessel walls under high shear conditions. Delineating the assembly process of the GP Ib-IX complex will aid our understanding of the complex structure and shed light on the signaling mechanism underlying platelet activation. The GP Ib-IX complex comprises three polypeptides, GP Ibα, GP Ibβ and GP IX, and efficient surface expression of the complex in transfected Chinese hamster ovary (CHO) cells requires all of its three subunits, indicating that the assembly of the complex in the endoplasmic reticulum is required for its subsequent trafficking to the plasma membrane. We recently showed that the interaction between the transmembrane domain of GP Ibβ and the other subunits is critical to efficient surface expression of the GP Ib-IX complex. Here, we have explored the role of the Ibβ cytoplasmic domain in the complex assembly and surface expression. In CHO cells transiently expressing the mutant Ib-IX complex in which the Ibβ cytoplasmic domain was deleted or replaced entirely with the IX counterpart, neither cellular expression of GP Ibα nor surface expression of GP Ibα and IX was detected. In contrast, deletion and/or replacement of the Ibα or IX cytoplasmic domains did not affect significantly the assembly and surface expression of the receptor complex. Furthermore, deletion of the last six residues in the Ibβ cytoplasmic domain did not affect the cellular expression level of GP Ibα but significantly decreased its surface expression level in CHO cells. Intriguingly, further deletion of the Ibβ cytoplasmic domain that removed the binding site for 14-3-3ζ restored surface expression of the GP Ib-IX complex to a level comparable to the wild type construct. Overall, our results demonstrated an important role of the Ibβ cytoplasmic domain in both assembly and trafficking of the GP Ib-IX complex. Critical residues in the Ibβ cytoplasmic domain have been identified and further characterization is currently underway.

scholarly journals Mechanisms and consequences of agonist-induced talin recruitment to platelet integrin αIIbβ3

2008 ◽  
Vol 181 (7) ◽  
pp. 1211-1222 ◽  
Author(s):  
Naohide Watanabe ◽  
Laurent Bodin ◽  
Manjula Pandey ◽  
Matthias Krause ◽  
Shaun Coughlin ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Living Cells ◽  
Bimolecular Fluorescence Complementation ◽  
Related Protein ◽  
Guanosine Triphosphate ◽  
Integrin Αiibβ3 ◽  
Adaptor Molecule ◽  
Thrombin Receptors

Platelet aggregation requires agonist-induced αIIbβ3 activation, a process mediated by Rap1 and talin. To study mechanisms, we engineered αIIbβ3 Chinese hamster ovary (CHO) cells to conditionally express talin and protease-activated receptor (PAR) thrombin receptors. Human PAR1 or murine PAR4 stimulation activates αIIbβ3, which was measured with antibody PAC-1, indicating complete pathway reconstitution. Knockdown of Rap1–guanosine triphosphate–interacting adaptor molecule (RIAM), a Rap1 effector, blocks this response. In living cells, RIAM overexpression stimulates and RIAM knockdown blocks talin recruitment to αIIbβ3, which is monitored by bimolecular fluorescence complementation. Mutations in talin or β3 that disrupt their mutual interaction block both talin recruitment and αIIbβ3 activation. However, one talin mutant (L325R) is recruited to αIIbβ3 but cannot activate it. In platelets, RIAM localizes to filopodia and lamellipodia, and, in megakaryocytes, RIAM knockdown blocks PAR4-mediated αIIbβ3 activation. The RIAM-related protein lamellipodin promotes talin recruitment and αIIbβ3 activity in CHO cells but is not expressed in megakaryocytes or platelets. Thus, talin recruitment to αIIbβ3 by RIAM mediates agonist-induced αIIbβ3 activation, with implications for hemostasis and thrombosis.

Osmotic activation of a Na(+)-dependent Cl-/HCO3- exchanger

1995 ◽  
Vol 268 (1) ◽  
pp. C147-C153 ◽  
Author(s):  
H. P. Reusch ◽  
J. Lowe ◽  
H. E. Ives
Keyword(s):  
Cho Cells ◽  
Chinese Hamster ◽  
Free Medium ◽  
Cell Shrinkage ◽  
Acetoxymethyl Ester ◽  
Hypertonic Medium ◽  
Kinase C ◽  
Protein Kinase C Activity ◽  
Induced Changes

In many systems, osmotically induced cell shrinkage activates the Na+/H+ exchanger. To assess the role of H(+)-extruding transporters in the response to osmotic shrinkage in vascular smooth muscle (VSM) and Chinese hamster ovary (CHO) cells, intracellular pH (pHi) was measured with 2',7'-bis(carboxy-ethyl)-5(6)- carboxyfluorescein-acetoxymethyl ester (BCECF-AM) after exposing cells to hypertonic medium. In nominally HCO(3-)-free medium, addition of 200 mM sucrose caused pHi to increase 0.33 pH unit on average in VSM cells but only 0.13 pH unit in CHO cells. Permeant solutes failed to increase pHi significantly. Cytochalasin B (1-20 microM), colchicine (1-10 microM), Ca2+ removal, and downregulation of protein kinase C activity did not affect osmotic activation of H+ extrusion in either cell type. Additional work was carried out to determine why osmotic activation of H+ extrusion was less in CHO than in VSM cells. In CHO cells, the osmotically induced delta pHi was only weakly sensitive to amiloride, suggesting that osmotic forces may activate an H+ transport system other than Na+/H+ exchange. In the presence of 10 mM HCO3-, osmotically induced delta pHi decreased by 60% in VSM cells but increased by 50% in CHO cells compared with the delta pHi in HCO(3-)-free medium. Lastly, removal of extracellular Cl- did not affect osmotically induced delta pHi in VSM cells but completely abolished the response in CHO cells. We conclude that in VSM cells osmotically induced changes in pHi are mediated by Na+/H+ exchange, whereas in CHO cells they are most likely mediated by a Na(+)-dependent Cl-/HCO3- exchanger.

scholarly journals A mammalian fatty acid hydroxylase responsible for the formation of α-hydroxylated galactosylceramide in myelin

2005 ◽  
Vol 388 (1) ◽  
pp. 245-254 ◽  
Author(s):  
Matthias ECKHARDT ◽  
Afshin YAGHOOTFAM ◽  
Simon N. FEWOU ◽  
Inge ZÖLLER ◽  
Volkmar GIESELMANN
Keyword(s):  
Fatty Acid ◽  
Functional Role ◽  
Time Course ◽  
Fluorescent Protein ◽  
Sequence Similarity ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Mammalian Brain ◽  
Fatty Acid Hydroxylase

Hydroxylation is an abundant modification of the ceramides in brain, skin, intestinal tract and kidney. Hydroxylation occurs at the sphingosine base at C-4 or within the amide-linked fatty acid. In myelin, hydroxylation of ceramide is exclusively found at the α-C atom of the fatty acid moiety. α-Hydroxylated cerebrosides are the most abundant lipids in the myelin sheath. The functional role of this modification, however, is not known. On the basis of sequence similarity to a yeast C26 fatty acid hydroxylase, we have identified a murine cDNA encoding FA2H (fatty acid 2-hydroxylase). Transfection of FA2H cDNA in CHO cells (Chinese-hamster ovary cells) led to the formation of α-hydroxylated fatty acid containing hexosylceramide. An EGFP (enhanced green fluorescent protein)–FA2H fusion protein co-localized with calnexin, indicating that the enzyme resides in the endoplasmic reticulum. FA2H is expressed in brain, stomach, skin, kidney and testis, i.e. in tissues known to synthesize fatty acid α-hydroxylated sphingolipids. The time course of its expression in brain closely follows the expression of myelin-specific genes, reaching a maximum at 2–3 weeks of age. This is in agreement with the reported time course of fatty acid α-hydroxylase activity in the developing brain. In situ hybridization of brain sections showed expression of FA2H in the white matter. Our results thus strongly suggest that FA2H is the enzyme responsible for the formation of α-hydroxylated ceramide in oligodendrocytes of the mammalian brain. Its further characterization will provide insight into the functional role of α-hydroxylation modification in myelin, skin and other organs.

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