Differential expression of glycosylphosphatidylinositol-anchored proteins in a murine T cell hybridoma mutant producing limiting amounts of the glycolipid core. Implications for paroxysmal nocturnal hemoglobinuria.

A T cell hybridoma mutant, which expressed a markedly reduced level of glycosylphosphatidylinositol (GPI)-anchored proteins on the cell surface, was characterized. The surface expression level of Thy-1 was approximately 17% of the wild-type level, whereas the surface expression of Ly-6A was approximately 2.4% of the wild-type level. We show here that these cells synthesized limiting amounts of the GPI core and that the underlying defect in these cells was an inability to synthesize dolichyl phosphate mannose (Dol-P-Man) at the normal level. The defect in Ly-6A expression could be partially corrected by tunicamycin, which blocked the biosynthesis of N-linked oligosaccharide precursors and shunted Dol-P-Man to the GPI pathway. Full restoration of Thy-1 and Ly-6A expression, however, required the stable transfection of a yeast Dol-P-Man synthase gene into the mutants. These results revealed that when the GPI core is limiting, there is a differential transfer of the available GPI core to proteins that contain GPI-anchor attachment sequences. Our findings also have implications for the elucidation of the defects in paroxysmal nocturnal hemoglobinuria.     

Glycoinositol phospholipid-anchored interleukin 2 but not secreted interleukin 2 inhibits melanoma tumor growth in mice

Whereas cancer immunotherapy with interleukin (IL) 2 and/or other cytokines has proved effective in activating immune responses against tumor cells, the major obstacle with the use of these cytokines in cancer patients is their severe side effects when delivered systemically at high doses. In an effort to overcome this problem, in the present study, a fusion protein containing human IL-2 and a glycoinositol phospholipid (GPI) anchor sequence of decay accelerating factor was generated. When expressed by transfected cells, these fusion proteins were presented on the cell surface in the GPI-anchored form as demonstrated by fluorescence-activated cell sorter and ELISA analyses. This GPI-anchored IL-2 is highly functional as indicated by significantly increased T-cell infiltration in tumor masses. Immunohistochemical analysis of tumor cells isolated from experimental tumors indicated that a local high level of IL-2 was achieved by GPI-anchored IL-2. More importantly, when injected into mice i.v., the growth of these B16F0 melanoma cells that were engineered to express this fusion protein was significantly inhibited. In contrast, the inhibition of secreted IL-2 on tumor growth was not observable in this study. These studies may provide a novel approach to locally deliver high doses of cytokines for cancer immunotherapy.     

Internalization of monomeric lipopolysaccharide occurs after transfer out of cell surface CD14.

Lipopolysaccharide (LPS) fluorescently labeled with boron dipyrromethane (BODIPY) first binds to the plasma membrane of CD14-expressing cells and is subsequently internalized. Intracellular LPS appears in small vesicles near the cell surface and later in larger, punctate structures identified as the Golgi apparatus. To determine if membrane (m)CD14 directs the movement of LPS to the Golgi apparatus, an mCD14 chimera containing enhanced green fluorescent protein (mCD14-EGFP) was used to follow trafficking of mCD14 and BODIPY-LPS in stable transfectants. The chimera was expressed strongly on the cell surface and also in a Golgi complex-like structure. mCD14-EGFP was functional in mediating binding of and responses to LPS. BODIPY-LPS presented to the transfectants as complexes with soluble CD14 first colocalized with mCD14-EGFP on the cell surface. However, within 5-10 min, the BODIPY-LPS distributed to intracellular vesicles that did not contain mCD14-EGFP, indicating that mCD14 did not accompany LPS during endocytic movement. These results suggest that monomeric LPS is transferred out of mCD14 at the plasma membrane and traffics within the cell independently of mCD14. In contrast, aggregates of LPS were internalized in association with mCD14, suggesting that LPS clearance occurs via a pathway distinct from that which leads to signaling via monomeric LPS.     

Targeting of cell receptors and gene transfer efficiency: a balancing act

Vectors conjugated with ligands recognized by cell surface receptors are of interest for cystic fibrosis gene therapy since these vectors would allow cell-specific targeting. However, an efficient and specific uptake may be abrogated by a subsequent intracellular trafficking leading to an inefficient gene transfer. This has been shown for polylysine substituted with mannose residues. While mannose-specific membrane lectins are predominantly expressed at the surface of airway cells and mannosylated complexes are the most efficiently incorporated glycosylated complexes in these cells, mannosylated complexes lead to a low gene transfer efficiency because of an inefficient exit from endosomal compartments, a high accumulation in lysosomes and an inefficient nuclear import. In contrast, the entry of low amounts of lactosylated complexes is balanced by more efficient intracellular trafficking, leading to an efficient gene transfer. This emphasizes that for a successful gene transfer, it is necessary to find the balance between efficient and specific uptake, and intracellular trafficking that overcomes the various cellular barriers and enables the plasmid to reach the nucleus.     

Pathways followed by membrane recovered from the surface of plasma cells and myeloma cells

Evidence for recovery of surface membrane and its fusion with Golgi cisternae has been obtained previously in several glandular cells. This study was conducted to determine whether or not membrane is similarly retrieved from the surfaces of plasma cells from lymph nodes (of rats immunized with horseradish peroxidase [HRP]) and mouse myeloma cells (RPC 5.4 and X63 Ag 8 cell lines). Electron-dense tracers (cationic and anionic ferritin, HRP) were used to trace the pathways followed by surface membrane recovered by endocytosis, and immunocytochemistry was used to identify the secretory compartments. When plasma cells or myeloma cells were incubated with cationized ferritin (CF), it bound to the cell surfaces and was taken up in endocytic vesicles, for the most part bound to the vesicle membrane. After 30-60 min, it was found increasingly within lysosomes and in several secretory compartments- notably in multiple stacked Golgi cisternae and secretory vacuoles. By immunocytochemistry the secretory product (immunoglobulins) and CF could be demonstrated in the same Golgi components. When myeloma cells were incubated with native (anionic) ferritin or in HRP, these tracers were taken up in much smaller amounts, primarily within the contents of endocytic vesicles. With continued incubation, they appeared only in lysosomes. When cells were doubly incubated, first in CF and then in HRP, both tracers were taken up (often within the same endocytic vesicle), but they maintained their same destinations as when incubated in a single tracer alone: the content marker, HRP, was localized exclusively within the lysosomal system, whereas the membrane marker, CF, was found within elements along the secretory pathway as well as within lysosomes. The findings demonstrate the existence of considerable membrane traffic between the cell membrane and the Golgi cisternae and lysosomes in both normal plasma cells and myeloma cells. Because myeloma cells behave like the glandular cells studied previously with regard to pathways of retrieved surface membrane, they represent a suitable and promising system for further studies of mechanisms and pathways of membrane retrieval and recycling in secretory cells.     

Paroxysmal nocturnal hemoglobinuria from bench to bedside

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematologic disease that presents with protean manifestations. Clinical and laboratory investigation over the past 25 years has uncovered most of the basic science underpinnings of PNH and has led to the development of a highly effective targeted therapy. PNH originates from a multipotent hematopoietic stem cell (HSC) that acquires a somatic mutation in a gene called phosphatidylinositol glycan anchor biosynthesis, class A (PIG-A). The PIG-A gene is required for the first step in glycosylphosphatidylinositol (GPI) anchor biosynthesis. Failure to synthesize GPI anchors leads to an absence of all proteins that utilize GPI to attach to the plasma membrane. Two GPI-anchor proteins, CD55 and CD59, are complement regulatory proteins; their absence on the surface of PNH cells leads to complement-mediated hemolysis. The release of free hemoglobin leads to scavenging of nitric oxide and contributes to many clinical manifestations, including esophageal spasm, fatigue, and possibly thrombosis. Aerolysin is a pore-forming toxin that binds GPI-anchored proteins and kills normal cells, but not PNH cells. A fluorescinated aerolysin variant (FLAER) binds GPI-anchor and serves as a novel reagent diagnosing PNH. Eculizumab, a humanized monoclonal antibody against C5, is the first effective drug therapy for PNH.     

Phospholipid-anchored and transmembrane versions of either decay- accelerating factor or membrane cofactor protein show equal efficiency in protection from complement-mediated cell damage

Decay-accelerating factor (DAF) is a glycosyl-phosphatidylinositol (GPI)-anchored membrane protein that protects cells from complement-mediated damage by regulation of the C3 convertase. To investigate the role of the GPI anchor in the function of DAF, the cDNA encoding human DAF was expressed by transfection in Chinese hamster ovary (CHO) cells. Testing of these DAF transfectants in an antibody plus human complement-mediated cytotoxicity assay demonstrated that DAF protects these cells from cytotoxicity, and that the level of protection increases with expression of surface DAF. A cDNA construct encoding a transmembrane version of DAF (DAF-TM) protects CHO transfectants from cytotoxicity with equal efficiency to DAF. This DAF-TM construct used the TM and cytoplasmic domains of membrane cofactor protein (MCP); an alternate TM version of DAF constructed with the TM and cytoplasmic domains of HLA-B44 showed equivalent protection. The protection from cytotoxicity involved a decrease in the deposition of C3 on the cell, consistent with the effect of DAF on the C3 convertase. A second pair of anchor variants, MCP and a GPI-anchored construct, MCP-PI, were also equivalent in their complement protection. The equivalent function of GPI-anchored and TM versions of a protein was not expected based on the hypothesized increased lateral mobility of GPI-anchored proteins, which should confer a functional advantage in contacting ligand, in this case, C3b or C4b, on the cell surface. These data suggest either that GPI-anchored and TM versions of a protein have equal lateral mobility in the membrane, or else that increased lateral mobility is not advantageous to DAF or MCP in carrying out their complement inhibitory roles. Furthermore, DAF and MCP demonstrated approximately equal protection of cells from complement-mediated cytotoxicity, suggesting that DAF and MCP provide overlapping levels of protection to cells against damage mediated by the complement system.     

Tissue factor and tissue factor pathway inhibitor: a potential role in pregnancy and obstetric vascular complications?

Tissue factor (TF) is the principal cellular initiator of normal blood coagulation. As a result it is considered to be a major regulator of haemostasis and thrombogenesis. In vivo TF activity is regulated by specific circulating inhibitor known as "tissue factor pathway inhibitor (TFPI)". TF is also essential for other cellular processes including embryogenesis and angiogenesis as well as in implantation where it is particularly important in the first trimester. TF is highly expressed in syncytiotrophoblasts (STB) while TFPI is expressed in human umbilical vein endothelial cells (HUVEC). TFPI may be internalized via an endocytic pathway and recycled to the cell surface. The procoagulant tendency of STB may reflect a physiological need for immediate inhibition of hemorrhage in the placental intervillous spaces. Furthermore, the haemostatic balance involving STB and HUVEC may be critical for normal placental function and pregnancy outcome. Homozygous knockouts of both TF and TFPI are generally lethal in fetal mice; heterozygotes survive but with altered coagulation parameters. Despite their apparent association with placental microcirculation-thrombi-formation only few studies have addressed the role of TF and TFPI in the pathogenesis of gestational vascular complications. In this context, detailed studies could provide clinically relevant information.     

Acid sphingomyelinase: relation of 93lysine residue on the ratio of intracellular to secreted enzyme activity

Acid sphingomyelinase (ASM) is the lysosomal enzyme responsible for the hydrolysis of sphingomyelin to ceramide and phosphocholine. An inherited deficiency of this enzymatic activity results in the Type A and B forms of Niemann-Pick disease (NPD). ASM is also readily secreted from cultured cells and can rapidly move from lysosomes to the cell surface upon stimulation by cytokines and other factors. Recent interest has focused on the role of this secreted/cell surface enzyme in ceramide-mediated signal transduction. We therefore sought to understand the mechanism(s) that might regulate intracellular targeting and secretion of this important hydrolase. Most lysosomal proteins are targeted to lysosomes in mammalian cells via the mannose 6-phosphate recognition system. Using cultured skin fibroblasts from I-cell disease patients, in which one of the enzymes responsible for mannose phosphorylation, GlcNAc-phosphotransferase, is deficient, we determined ASM activities in cell homogenates and media. The ratio of secreted to intracellular activity was approximately 8-fold greater in I-cell than in normal cells, indicating that mannose phosphorylation is important in the trafficking of this hydrolase. Most of the secreted activity required Zn+2 for full activity, supporting the concept that intracellular exposure of ASM to zinc within lysosomes is required for enzymatic activation. The recognition of lysosomal proteins by GlcNAc-phosphotransferase is mediated by protein structure, and a specific three-dimensional arrangement of lysine residues exposed on the surface of several enzymes has been shown to be critical for mannose phosphorylation. Alanine scanning mutagenesis of thirteen lysine residues in ASM demonstrated that 93lysine residue plays a critical role in ASM targeting since the K93A mutant had reduced intracellular activity, but enhanced secreted activity that was zinc responsive.     

Molecular cloning of gp42, a cell-surface molecule that is selectively induced on rat natural killer cells by interleukin 2: glycolipid membrane anchoring and capacity for transmembrane signaling

We have previously shown that in vitro culture of rat natural killer (NK) cells in high concentrations of recombinant interleukin 2 (rIL-2) leads to the expression of a surface glycoprotein with a molecular mass of approximately 42 kD. This glycoprotein, gp42, is not induced on other lymphocytes and thus provides a lineage-specific marker for rIL-2-activated NK cells. We here present the nucleotide sequence for gp42 cDNA. The open reading frame encodes 233 amino acids with three potential sites for N-linked glycosylation. The deduced amino acid sequence lacks an apparent transmembrane domain and instead contains a hydrophobic COOH terminus that is characteristic of glycosylphosphatidylinositol (GPI)-anchored surface proteins. Consistent with this, gp42 is cleaved from the NK-like cell line, RNK-16, by phosphatidylinositol-specific phospholipase C (PI-PLC), as is gp42 expressed on CHO cells that have been transformed with gp42 cDNA. On rIL-2-activated NK cells, gp42 is resistant to PI-PLC, though our studies suggest that gp42 on these cells is still expressed as a GPI-anchored molecule. Antibody to gp42 stimulates in RNK-16 cells an increase in inositol phosphates and in intracellular calciu, signals that are associated with the activation of lymphocytes, including NK cells. rIL-2-activated NK cells, however, lack this response to gp42 as well as to other stimuli. Thus, gp42, the only NK-specific activation antigen, is a GPI-anchored surface molecule with the capacity to stimulate transmembrane signaling.     

Temporal expression of alternatively spliced forms of tissue factor pathway inhibitor in mice

Summary.  Background: Mouse tissue factor pathway inhibitor (TFPI) is produced in three alternatively spliced isoforms that differ in domain structure and mechanism for cell surface binding. Tissue expression of TFPI isoforms in mice was characterized as an initial step for identification of their physiological functions. Methods and Results: Sequence homology demonstrates that TFPIα existed over 430 Ma while TFPIβ and TFPIγ evolved more recently. In situ hybridization studies of heart and lung did not reveal any cells exclusively expressing a single isoform. Although our previous studies have demonstrated that TFPIα mRNA is more prevalent than TFPIβ or TFPIγ mRNA in mouse tissues, western blot studies demonstrated that TFPIβ is the primary protein isoform produced in adult tissues, while TFPIα is expressed during embryonic development and in placenta. Consistent with TFPIβ as the primary isoform produced within adult vascular beds, the TFPI isoform in mouse plasma migrates like TFPIβ in SDS-PAGE and mice have a much smaller heparin-releasable pool of plasma TFPIα than humans. Conclusions: The data demonstrate that alternatively spliced isoforms of TFPI are temporally expressed in mouse tissues at the level of protein production. TFPIα and TFPIβ are produced in embryonic tissues and in placenta while adult tissues produce almost exclusively TFPIβ.     

E1210, a new broad-spectrum antifungal, suppresses Candida albicans hyphal growth through inhibition of glycosylphosphatidylinositol biosynthesis

Continued research toward the development of new antifungals that act via inhibition of glycosylphosphatidylinositol (GPI) biosynthesis led to the design of E1210. In this study, we assessed the selectivity of the inhibitory activity of E1210 against Candida albicans GWT1 (Orf19.6884) protein, Aspergillus fumigatus GWT1 (AFUA_1G14870) protein, and human PIG-W protein, which can catalyze the inositol acylation of GPI early in the GPI biosynthesis pathway, and then we assessed the effects of E1210 on key C. albicans virulence factors. E1210 inhibited the inositol acylation activity of C. albicans Gwt1p and A. fumigatus Gwt1p with 50% inhibitory concentrations (IC(50)s) of 0.3 to 0.6 μM but had no inhibitory activity against human Pig-Wp even at concentrations as high as 100 μM. To confirm the inhibition of fungal GPI biosynthesis, expression of ALS1 protein, a GPI-anchored protein, on the surfaces of C. albicans cells treated with E1210 was studied and shown to be significantly lower than that on untreated cells. However, the ALS1 protein levels in the crude extract and the RHO1 protein levels on the cell surface were found to be almost the same. Furthermore, E1210 inhibited germ tube formation, adherence to polystyrene surfaces, and biofilm formation of C. albicans at concentrations above its MIC. These results suggested that E1210 selectively inhibited inositol acylation of fungus-specific GPI which would be catalyzed by Gwt1p, leading to the inhibition of GPI-anchored protein maturation, and also that E1210 suppressed the expression of some important virulence factors of C. albicans, through its GPI biosynthesis inhibition.     

Tissue factor pathway inhibitor-γ is an active alternatively spliced form of tissue factor pathway inhibitor present in mice but not in humans

Summary.  Background:  Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of tissue factor procoagulant activity produced as two alternatively spliced isoforms, TFPIα and TFPIβ, which differ in domain structure and mechanism for cell surface association. 3' Rapid amplification of cDNA ends was used to search for new TFPI isoforms. TFPIγ, a new alternatively spliced form of TFPI, was identified and characterized. Methods:  The tissue expression, cell surface association and anticoagulant activity of TFPIγ were characterized and compared to those of TFPIα and TFPIβ through studies of mouse and human tissues and expression of recombinant proteins in Chinese hamster ovary (CHO) cells. Results:  TFPIγ is produced by alternative splicing using the same 5'-splice donor site as TFPIβ and a 3'-splice acceptor site 276 nucleotides beyond the stop codon of TFPIβ in exon 8. The resulting protein has the first two Kunitz domains connected to an 18 amino acid C-terminal region specific to TFPIγ. TFPIγ mRNA is differentially produced in mouse tissues but is not encoded within the human TFPI gene. When expressed in CHO cells, TFPIγ is secreted into conditioned media and effectively inhibits tissue factor procoagulant activity. Conclusions:  TFPIγ is a third alternatively spliced form of TFPI that is widely expressed in mouse tissues but not made by human tissues. It contains the first two Kunitz domains and is a secreted, rather than a cell surface-associated, protein. It is a functional anticoagulant and may partially explain the resistance of mice to coagulopathy in tissue factor-mediated models of disease.     

Binding of pro-prion to filamin A disrupts cytoskeleton and correlates with poor prognosis in pancreatic cancer

The cellular prion protein (PrP) is a highly conserved, widely expressed, glycosylphosphatidylinositol-anchored (GPI-anchored) cell surface glycoprotein. Since its discovery, most studies on PrP have focused on its role in neurodegenerative prion diseases, whereas its function outside the nervous system remains unclear. Here, we report that human pancreatic ductal adenocarcinoma (PDAC) cell lines expressed PrP. However, the PrP was neither glycosylated nor GPI-anchored, existing as pro-PrP and retaining its GPI anchor peptide signal sequence (GPI-PSS). We also showed that the PrP GPI-PSS has a filamin A–binding (FLNa-binding) motif and interacted with FLNa, an actin-associated protein that integrates cell mechanics and signaling. Binding of pro-PrP to FLNa disrupted cytoskeletal organization. Inhibition of PrP expression by shRNA in the PDAC cell lines altered the cytoskeleton and expression of multiple signaling proteins; it also reduced cellular proliferation and invasiveness in vitro as well as tumor growth in vivo. A subgroup of human patients with pancreatic cancer was found to have tumors that expressed pro-PrP. Most importantly, PrP expression in tumors correlated with a marked decrease in patient survival. We propose that binding of pro-PrP to FLNa perturbs FLNa function, thus contributing to the aggressiveness of PDAC. Prevention of this interaction could provide an attractive target for therapeutic intervention in human PDAC.     

Transfection of a glycosylated phosphatidylinositol-anchored folate-binding protein complementary DNA provides cells with the ability to survive in low folate medium.

KB cells express a folate-binding protein that is anchored to the plasma membrane by a glycosylated phosphatidylinositol (GPI) tail and these cells can grow in medium containing a very low folate concentration (1 nM). In contrast, mouse 3T3 cells do not express a membrane-associated folate-binding protein and cannot grow under similar low folate conditions. In these studies, 3T3 cells were transfected with a vector containing the cDNA that codes for the KB cell folate-binding protein. In contrast to the wild-type 3T3 cells, the transfected 3T3 cells express a level of folate-binding protein similar to KB cells, 1 and 1.4 ng/micrograms protein, respectively. The capacity for binding [3H] folate to the surface of transfected 3T3 cells cultured in folate-deficient medium is 7.7 pmol/10(6) cells, and this is approximately 50% of the surface binding capacity of KG cells under similar culture conditions. Moreover, after treatment of the transfected 3T3 cells with phospholipase C specific for phosphatidylinositol, the binding of [3H] folate to the surface of these cells is reduced by 90%, indicating that, like the KB cells, the folate-binding protein is anchored to the plasma membrane by a GPI tail. Although the doubling time of wild-type 3T3 cells markedly increases after 13 d of culture in folate-deficient medium, the doubling time of both the transfected 3T3 cells and KB cells do not change. The results of these experiments indicate that the GPI-anchored folate-binding protein provides a mechanism to maintain a level of folate that permits the folate-dependent metabolic functions necessary for cell survival under low folate conditions.     

Comparison of cell-surface TFPIα and β

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is mainly produced by endothelial cells and alternative mRNA splicing generates two forms, TFPIalpha and TFPIbeta. A portion of expressed TFPI remains associated with the cell surface through both direct (TFPIbeta) and indirect (TFPIalpha) glycosylphosphatidyl-inositol (GPT)-mediated anchorage. OBJECTIVE: Compare the structure and properties of TFPIalpha and TFPIbeta. METHODS: TFPIalpha and TFPIbeta, with protein molecular masses of 36 and 28 kDa, respectively, migrate similarly (46 kDa) on SDS-PAGE. Experiments using specific glycosidases were carried out to determine the different glycosylation pattern of the two forms. ECV304 cells, a cell line with some endothelial properties, were stimulated with IL-lbeta, LPS, and TNFalpha for up to 24 hrs and mRNA levels and protein synthesis were determined. Stable clones of ECV304 cells that express reduced levels of TFPIalpha, TFPIbeta or both were produced using a plasmid-based small-interfering RNA technique. Surface TFPI activity was determined by a two-stage chromogenic assay based on the ability of each form to inhibit FXa activation by FVIIa on cells with comparable amount of tissue factor (TF). RESULTS AND CONCLUSIONS: The deglycosylation studies show that the difference in molecular masses is due to a greater degree of sialylation in O-linked carbohydrate in TFPIbeta. The mRNA and protein levels of neither form of TFPI were affected by stimulation of cells with inflammatory stimuli. Although TFPIalpha comprises 80% of the surface-TFPI, TFPIbeta was responsible for the bulk of the cellular FVIIa/TF inhibitory activity, suggesting a potential alternative role for cell surface TFPIalpha.     

Rab proteins mediate Golgi transport of caveola-internalized glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells

We recently showed that human skin fibroblasts internalize fluorescent analogues of the glycosphingolipids lactosylceramide and globoside almost exclusively by a clathrin-independent mechanism involving caveolae. In contrast, a sphingomyelin analogue is internalized approximately equally via clathrin-dependent and caveolar routes. Here, we further characterized the caveolar pathway for glycosphingolipids, showing that Golgi targeting of sphingolipids internalized via caveolae required microtubules and phosphoinositol 3-kinases and was inhibited in cells expressing dominant-negative Rab7 and Rab9 constructs. In addition, overexpression of wild-type Rab7 or Rab9 (but not Rab11) in Niemann-Pick type C (NP-C) lipid storage disease fibroblasts resulted in correction of lipid trafficking defects, including restoration of Golgi targeting of fluorescent lactosylceramide and endogenous GM(1) ganglioside, and a dramatic reduction in intracellular cholesterol stores. Our results demonstrate a role for Rab7 and Rab9 in the Golgi targeting of glycosphingolipids and suggest a new therapeutic approach for restoring normal lipid trafficking in NP-C cells.     

Disruption of transforming growth factor beta signaling by a novel ligand-dependent mechanism

Transforming growth factor (TGF)-beta is the prototype in a family of secreted proteins that act in autocrine and paracrine pathways to regulate cell development and function. Normal cells typically coexpress TGF-beta receptors and one or more isoforms of TGF-beta, thus the synthesis and secretion of TGF-beta as an inactive latent complex is considered an essential step in regula-ting the activity of this pathway. To determine whether intracellular activation of TGF-beta results in TGF-beta ligand-receptor interactions within the cell, we studied pristane-induced plasma cell tumors (PCTs). We now demonstrate that active TGF-beta1 in the PCT binds to intracellular TGF-beta type II receptor (TbetaRII). Disruption of the expression of TGF-beta1 by antisense TGF-beta1 mRNA restores localization of TbetaRII at the PCT cell surface, indicating a ligand-induced impediment in receptor trafficking. We also show that retroviral expression of a truncated, dominant-negative TbetaRII (dnTbetaRII) effectively competes for intracellular binding of active ligand in the PCT and restores cell surface expression of the endogenous TbetaRII. Analysis of TGF-beta receptor-activated Smad2 suggests the intracellular ligand-receptor complex is not capable of signaling. These data are the first to demonstrate the formation of an intracellular TGF-beta-receptor complex, and define a novel mechanism for modulating the TGF-beta signaling pathway.     

Activity and regulation of factor VIIa analogs with increased potency at the endothelial cell surface

BACKGROUND: Variants of recombinant factor VIIa (rFVIIa) with increased intrinsic activity have been developed to improve efficacy in the treatment of bleeding disorders in the future. The increased potency of FVIIa variants was demonstrated in limited in vitro and in vivo studies. However, further characterization of FVIIa variants is needed to evaluate their potential clinical use. METHODS: In the present study, we investigated the interactions of two FVIIa variants, FVIIa(Q) and FVIIa(DVQ), with plasma inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin (AT), and vascular endothelium. TF-FVIIa activity or its inhibition was measured directly in an amidolytic activity assay or for its ability to activate factor X. RESULTS: Both TFPI and AT/heparin inhibited the FVIIa variants more rapidly than the wild-type (WT) FVIIa in the absence of tissue factor (TF). In the presence of TF, TFPI, TFPI-Xa, and AT/heparin inhibited FVIIa and FVIIa variants at similar rates. Although the WT FVIIa failed to generate significant amounts of FXa on unperturbed endothelial cells, FVIIa variants, particularly FVIIa(DVQ), generated a substantial amount of FXa on unperturbed endothelium. Annexin V fully attenuated the FVIIa-mediated activation of FX on unperturbed endothelial cells. On stimulated human umbilical vein endothelial cells, FVIIa and FVIIa variants activated FX at similar rates, and annexin V blocked the activation only partly. AT/heparin and TFPI-Xa inhibited the activity of FVIIa and FVIIa variants bound to endothelial cell TF in a similar fashion. Interestingly, despite significant differences observed in FXa generation on unperturbed endothelium exposed to FVIIa and FVIIa analogs, no differences were found in thrombin generation when cells were exposed to FVIIa or FVIIa analogs under plasma mimicking conditions. CONCLUSION: Overall, the present data suggest that although FVIIa variants generate substantial amounts of FXa, they do not generate excessive thrombin on the surface of endothelium.