Cell Surface Tf Research Articles

Disruption of endocytic transport by transthyretin aggregates

The cytotoxicity of amyloidogenic proteins such as transthyretin (TTR) has implications for neurodegeneration and other pathologies, but is not well understood. In the current study, potential effects of misfolded, aggregated TTRs (agTTR) upon a major cell membrane function—endocytosis—were assessed. Internalization of transferrin (Tf), a ligand whose receptor-mediated endocytosis is well characterized, was analyzed in different cell types after treatment with agTTR. The results indicate disruption of Tf endocytosis: 20–25% inhibition by agTTR relative to the same concentrations of normal soluble TTR, or relative to another control protein, albumin (p<0.05 for agTTR relative to controls). No statistically significant difference was observed for cell surface Tf binding between agTTR-treated and control cells. This is the first evidence for endocytic disruption by agTTR, and presents a novel cytotoxicity mechanism that may account for previously reported inhibitory effects of amyloidogenic TTR on neuronal growth.

Tissue factor is induced by interleukin-33 in human endothelial cells: a new link between coagulation and inflammation.

Tissue factor (TF) is the primary trigger of coagulation. Elevated levels of TF are found in atherosclerotic plaques, and TF leads to thrombus formation when released upon plaque rupture. Interleukin (IL)-33 was previously shown to induce angiogenesis and inflammatory activation of endothelial cells (ECs). Here, we investigated the impact of IL-33 on TF in human ECs, as a possible new link between inflammation and coagulation. IL-33 induced TF mRNA and protein in human umbilical vein ECs and coronary artery ECs. IL-33-induced TF expression was ST2- and NF-κB-dependent, but IL-1-independent. IL-33 also increased cell surface TF activity in ECs and TF activity in ECs-derived microparticles. IL-33-treated ECs reduced coagulation time of whole blood and plasma but not of factor VII-deficient plasma. In human carotid atherosclerotic plaques (n = 57), TF mRNA positively correlated with IL-33 mRNA expression (r = 0.691, p < 0.001). In this tissue, IL-33 and TF protein was detected in ECs and smooth muscle cells by immunofluorescence. Furthermore, IL-33 and TF protein co-localized at the site of clot formation within microvessels in plaques of patients with symptomatic carotid stenosis. Through induction of TF in ECs, IL-33 could enhance their thrombotic capacity and thereby might impact on thrombus formation in the setting of atherosclerosis.

Differential ability of tissue factor antibody clones on detection of tissue factor in blood cells and microparticles

IntroductionTissue factor (TF), the primary initiator of coagulation in vivo, plays a major role in both thrombosis and hemostasis. The expression of TF in monocytes is well documented, but its presence in other blood cells has been disputed, possibly due to methodological variations among different studies. Materials and methodsWe studied TF expression on platelets, monocytes, lymphocytes and microparticles (MPs) by flow cytometry (FCM) with five commercially available mouse anti-human TF antibodies (HTF-1, TF9-10H10, CLB/TF-5, VIC7 and VD8). The ability of different TF antibodies to inhibit cell surface TF activity was explored by incubating LPS-stimulated monocytes and MPs derived from LPS-stimulated monocytes (MMPs) with TF antibodies followed by measuring TF activity. ResultsHTF-1 detected TF only on LPS-stimulated monocytes, whereas, TF9-10H10 and VD8 detected TF associated with MPs and MMPs in addition to LPS stimulated monocytes. Surprisingly, CLB/TF-5 and VIC7 detected TF on platelets, monocytes even under unstimulated conditions, in addition to MPs and MMPs. CLB/TF-5 also detected TF on unstimulated lymphocytes. Inhibitory studies showed that at a final concentration of 10μg/mL, HTF-1, CLB/TF-5 and VD8 inhibited monocyte TF activity by 81-84% and MMP TF activity by 92-96%; whereas TF9-10H10 had no inhibitory effect on TF activity in monocytes and MMPs. ConclusionsOur results suggest non-specific binding by the CLB/TF-5 and VIC7 antibodies in a FCM test system and explain at least some of the reports on TF presence in blood cells, particularly TF associated with platelets and MPs. TF9-10H10 and VD8 are more suitable to detect TF on MPs by FCM.

Cryptic Vs. Active Tissue Factor: Cystine186-Cystine 209 Disulfide Bond Switch Is Not the Answer.

Abstract 333▪▪This icon denotes an abstract that is clinically relevant.Tissue factor (TF) plays the key role in initiation of blood coagulation by allosterically activating coagulation factor VIIa (FVIIa). TF is essential for hemostasis but aberrant expression would lead to thrombotic disorders. TF on cell surfaces resides mostly in a cryptic state. At present it is not entirely clear how cryptic TF differs from procoagulant active TF and how de-encryption occurs. Recent studies have suggested that cryptic and active TF may have different conformations, i.e., cryptic TF contains unpaired cysteine thiols at Cys 186 and Cys 209 in the membrane proximal domain whereas procoagulant active TF contains the oxidized Cys186-Cys209 disulfide bond. It has been suggested that the catalytic switching of this disulfide bond between reduced and oxidized states by protein disulfide isomerase (PDI) might modulate the activity status of TF. Although the validity of this hypothesis has been questioned, more recent reports have supported the contention that PDI-catalyzed disulfide bond formation of Cys186-Cys209 in TF plays a role in regulation of TF activity both in vitro and in vivo. To reconcile the conflicting data in the recent literature, we re-examined in the present study the central dogma on which the hypothesis was originated, i.e., a TF mutant lacking Cys186-Cys 209 disulfide bond is functionally inactive (cryptic) and that TF activation requires formation of the Cys186-Cys209 disulfide bond. CHO cells were transfected with wild type TF or TF mutants that selectively preclude Cys186-Cys209 disulfide bond formation, i.e., TFC186S, TFC209S, or TFC186S/C209S, and TF procoagulant and antigen expression levels at the cell surface were quantified accurately by measuring TF procoagulant activity in a factor × activation assay and TF antigen levels in radioligand binding studies utilizing two different TF mAb (10H10 and 5G9) and FVIIa. CHO cells transfected with TFC186S, TFC209S or TFC186S/C209S expressed very little procoagulant activity (less than 1% of CHO cells transfected with wild-type TF under identical experimental conditions). However, TF mAb and FVIIa binding studies revealed that CHO cells transfected with TFC186S, TFC209S or TFC186S/C209S expressed very little TF protein on the cell surface (∼ 1 to 2% of CHO cells transfected with wild-type TF). Similar results were obtained whether we mutated Cys 186 or Cys 209 to Ser or Ala or whether we used transient or stable transfections. To confirm that the lower expression of TFC186S, TFC209S or TFC186S/C209S is neither an artifact nor limited to CHO cell model system, we extended these studies to endothelial cells. Human umbilical vein endothelial cells (HUVECs) were transduced with adenovirus particles (25 moi/cell) encoding wild-type TF, TFC186S, TFC209S or TFC186S/C209S and TF antigen levels at the cell surface and in cell lysates were measured using multiple methods - confocal microscopy, FACS, TF antigen assay, immuno blot analysis, TF mAb binding and FVIIa binding. HUVEC transduced with adenoviral particles encoding TFC186S, TFC209S or TFC186S/C209S expressed very low levels of TF antigen both at the cell surface ( 1 to 5%) as well as in total (10 to 15%) (TFC186S > TFC209S >TFC186S/C209S) compared to HUVEC transduced with the same number of adenovirus particles expressing wild-type TF. Cell surface TF activity measured in the presence of saturating concentrations of FVIIa revealed that the TF procoagulant activity of the mutants correlate well with the amount of TF antigen present on the cell surface, i.e., there were no significant differences in the specific activity of TF mutants and wild-type TF (TF procoagulant activity/amount of TF antigen, pM FXa formed/femto mole TF: wild-type TF, 68 ± 6; TFC186S, 55 ± 10; TFC209S, 57 ± 17; and TFC186S/C209S, 64 ± 0.2). More importantly, treatment of HUVEC with the thiol-oxidizing agent HgCl2 or with ionomycin increased the cell surface TF activity to the same extent (200 to 400% increase over HUVEC treated with control vehicle) in HUVEC expressing TFC186S/C209S or wild-type TF. In summary the present data provide clear evidence for that TF lacking Cys186-Cys209 bond is coagulantly active and a lower TF procoagulant activity in cells expressing the mutant TF that precludes Cys186-Cys209 disulfide bond formation is due to severe impairment in TF protein synthesis/processing and not because the mutant TF assumes the cryptic conformation. Our data also show that TF de-encryption does not require the formation of Cys186-Cys209 disulfide bond. Disclosures:No relevant conflicts of interest to declare.

Acidic and basic fibroblast growth factors suppress transcriptional activation of tissue factor and other inflammatory genes in endothelial cells.

Tissue factor (TF) is a transmembrane receptor that serves as a cofactor for factor VIIa and initiates the extrinsic pathway of blood coagulation. Under normal physiological conditions, TF is expressed in extravascular and perivascular cells but not in vascular endothelial cells and monocytes. TF can be induced in these cells by inflammatory regulators and other stimulators, such as LPS, thrombin, oxidized lipoproteins, and certain growth factors. An earlier study showed that growing primary cultures of human umbilical vein endothelial cells (HUVECs) with endothelial cell growth supplement (ECGS) and heparin had impaired the ability of monolayers to express surface membrane TF activity after perturbation. The mechanism by which ECGS suppressed TF activity was not known. In the present study, we investigated the effect of recombinant acidic and basic fibroblast growth factors (aFGF and bFGF) on the induction of TF in a HUVEC cell line and a fibroblast cell line. Both aFGF and bFGF suppressed the phorbol myristate acetate-induced expression of TF in endothelial cells but not the serum-induced expression of TF in fibroblast cells. Diminished expression of the cell surface TF activity observed in endothelial cells grown with aFGF or bFGF was due to the accumulation of a lower number of TF mRNA transcripts. TF mRNA stability was not altered in HUVECs grown with aFGF or bFGF. Nuclear run-on experiments revealed that the transcription of TF and several other genes that play an important role in inflammation and angiogenesis was reduced in the endothelial cells that were cultured with aFGF or bFGF. The diminished expression of TF may be part of a generalized response of endothelial cells to FGF that facilitates migration of endothelial cells during angiogenesis.

Resistance to the Antitumor Agent Gallium Nitrate in Human Leukemic Cells Is Associated with Decreased Gallium/Iron Uptake, Increased Activity of Iron Regulatory Protein-1, and Decreased Ferritin Production

The mechanism of drug resistance to gallium nitrate is not known. Since gallium can be incorporated into ferritin, an iron storage protein that protects cells from iron toxicity, we investigated whether ferritin expression was altered in gallium-resistant (R) CCRF-CEM cells. We found that the ferritin content of R cells was decreased, while heavy chain ferritin mRNA levels and iron regulatory protein-1 (IRP-1) RNA binding activity were increased. IRP-1 protein levels were similar in gallium-sensitive (S) and R cells, indicating that R cells contain a greater proportion of IRP-1 in a high affinity mRNA binding state. 59Fe uptake and transferrin receptor expression were decreased in R cells. In both S and R cells, gallium inhibited cellular 59Fe uptake, increased ferritin mRNA and protein, and decreased IRP-1 binding activity. Gallium uptake by R cells was markedly diminished; however, the sensitivity of R cells to gallium could be restored by increasing their uptake of gallium with excess transferrin. Our results suggest that R cells have developed resistance to gallium by down-regulating their uptake of gallium. In parallel, iron uptake by R cells is also decreased, leading to changes in iron homeostasis. Furthermore, since gallium has divergent effects on iron uptake and ferritin synthesis, its action may also include a direct effect on ferritin mRNA induction and IRP-1 activity.

Aluminum alters iron and manganese uptake and regulation of surface transferrin receptors in primary rat oligodendrocyte cultures

Transferrin (TO is a major transport protein for both iron (Fe) and aluminum (AI), as well as manganese (Mn) and it can mediate cellular uptake of these elements via cell surface Tf receptors. To study the effect of Al-Tf on Tf receptor regulation, primary oligodendrocyte cultures were prepared from cortices of newborn rats. The effects of Al-Tf on 54Mn and 59Fe uptake were compared to those of apo-, Fe-, or Mn-Tf (1.25 μM). To examine changes in cell surface Tf binding capacity, preincubation (4 h, 37°C) was performed with apo-, AI- or Fe-Tf and homologous receptor binding studies were subsequently conducted with 125I-Fe-Tf at 4°C. Incubation with Al-Tf, but not with equimolar amounts of Al chloride or Al citrate, led to dose-related increases in cellular Al. Incubation with either Al- or Fe-Tf decreased 59Fe uptake, while incubation with either Al- or Mn-Tf decreased 54Mn uptake. Surface Tf receptor sites/cell were 1.05, 0.60 and 0.46 x 10 5 after incubations with equivalent amounts of apo-, Fe-, and Al-Tf respectively. The data suggest that Al-Tf down-regulates surface Tf receptors on oligodendrocytes and can limit Fe and Mn uptake through this mechanism.

Evidence for nonvectorial, retrograde transferrin trafficking in the early endosomes of HEp2 cells.

We have previously characterized the trafficking of transferrin (Tf) through HEp2 human carcinoma cells (Ghosh, R. N., D. L. Gelman, and F. R. Maxfield, 1994. J. Cell Sci. 107:2177-2189). Early endosomes in these cells are comprised of both sorting endosomes and recycling compartments, which are distinct separate compartments. Endocytosed Tf initially appears in punctate sorting endosomes that also contain recently endocytosed LDL. After short loading pulses, Tf rapidly sorts from LDL with first-order kinetics (t1/2 approximately 2.5 min), and it enters the recycling compartment before leaving the cell (t1/2 approximately 7 min). Here, we report a second, slower rate for Tf to leave sorting endosomes after HEp2 cells were labeled to steady state with fluorescein Tf instead of the brief pulse used previously. We determined this rate using digital image analysis to measure the Tf content of sorting endosomes that also contained LDL. With an 11-min chase, the Tf in sorting endosomes was 24% of steady-state value. This was in excess of the amount expected (5% of steady state) from the rate of Tf exit after short filling pulses. The excess could not be accounted for by reinternalization of recycled cell surface Tf, implying that either some Tf was retained in sorting endosomes, or that Tf was delivered back to the sorting endosomes from the recycling compartment. The former is unlikely since nearly all sorting endosomes contain detectable Tf after an 11-min chase, even though more than one third of the sorting endosomes were formed during the chase time. Furthermore, while observing living cells by confocal microscopy, we saw vesicle movements that appeared to be fluorescent Tf returning from recycling compartments to sorting endosomes. The slow rate of exit after steady-state labeling was similar to the Tf exit rate from the cell, suggesting an equilibration of Tf throughout the early endosomal system by this retrograde pathway. This retrograde traffic may be important for delivering molecules from the recycling compartment, which is a long-lived organelle, to sorting endosomes, which are transient.

Characterization of Sorting Signals in the β-Amyloid Precursor Protein Cytoplasmic Domain

The beta-amyloid precursor protein (APP) is proteolytically processed to generate beta-amyloid protein, the principal protein component of neuropathological lesions characteristic of Alzheimer's disease. To investigate potential sorting signals in the cytoplasmic tail of APP, we transplanted APP cytoplasmic tail sequences into the cytoplasmic tail of the human transferrin receptor (TR) and showed that two sequence motifs from the APP cytoplasmic tail promote TR internalization. One sequence, GYENPTY, is related to the low density lipoprotein receptor internalization signal, FDNPVY, but also involves a critical glycine residue; the other, YTSI, conforms to the 4-residue tyrosine-based internalization signal consensus sequence. Furthermore, a chimeric molecule (APP-TR) consisting of the cytoplasmic domain of APP and the transmembrane and external domains of TR was rapidly internalized enabling the transport of iron into the cell at approximately 50% the rate of wild-type TR. Alanine scanning mutations indicated that the two sequences identified in transplantation experiments were required for internalization of the chimera. Metabolic pulse-chase experiments showed that the APP-TR chimeras were degraded in a post-Golgi membrane compartment within 2-4 h following normal glycosylation. Degradation was partially dependent upon the two internalization signals and was inhibited by ammonium chloride. A fraction of APP-TR chimeras traffic to a degradative endocytic compartment after appearing on the cell surface. Comparison of soluble APP released from cells expressing either full-length human APP or mutant APP with the sequence YENPTY deleted indicated that this sequence is required for sorting of full-length APP along similar trafficking pathways as the APP-TR chimera.

Differences in transferrin response and numbers of transferrin receptors in rat and human mammary carcinoma lines of different metastatic potentials.

We previously found that transferrin (Tf) differentially stimulated the growth of highly metastatic variant lines of murine melanoma and that these highly metastatic cells also had greater numbers of Tf receptors on their cell surfaces. In the present study we found that highly metastatic rat mammary adenocarcinoma cell lines also responded differentially to Tf in proliferation assays, and cell monolayers bound Tf in relation to their metastatic potential (MTPaB10 > MTPaB5 > MTLn3 > MTLn2 > MTC > MTF7 > MTPa). The brain-colonizing lines PaB10 and PaB5 were the most responsive to Tf and had the highest numbers of Tf receptors. Different human breast cancer cell lines also responded differentially to Tf in proliferation assays and bound different amounts of Tf to their cell surface Tf receptors. Transferrin binding, but not growth response, correlated with metastatic and invasive properties of lines selected from the human MCF-7 series (MCF7/LCC2 > MCF7/LCC1 > MCF7). In examining the transferrin binding and growth response of lines from the human MDA series, the Tf binding and growth response was MDA231 > MDA435 > MDA468. The lines MDA435 and MDA231 were metastatic in nude mouse assays, whereas the line MDA468 was not. Scatchard analysis indicated the presence of a single class of receptor for Tf on the rat and human mammary cell lines. The results suggest that neoplastic cells displaying various metastatic properties may express differing numbers of Tf receptors and respond differently to growth factors such as Tf.

An Anti-Tissue Factor Monoclonal Antibody which Inhibits TF Vlla Complex Is a Potent Anticoagulant in Plasma

Tissue factor (TF) functions as the receptor and cofactor for factor VIIa (VIIa) to form a proteolytically active TF.VIIa complex on cell surfaces. We here demonstrate that most MAbs against human TF were poor inhibitors of TF function in plasma and that they inhibited preformed TF.VIIa complex at a slow rate which was dependent on dissociation of VIIa from the cell surface TF. An exception was defined by one MAb (TF8-5G9) which was an effective immediate anticoagulant in plasma. Binding of TF8-5G9 to TF.VIIa inhibited catalytic function prior to dissociation of the TF.VIIa complex. This analysis thus establishes two distinct mechanisms by which MAbs interfere with TF function. The MAb TF8-5G9 introduces a therapeutic principle for rapid arrest of inappropriate triggering of coagulation by TF as well as the TF.VIIa complex in vivo.

Functional expression of the human transferrin receptor cDNA in Chinese hamster ovary cells deficient in endogenous transferrin receptor.

Transferrin (Tf) receptor-variant Chinese hamster ovary cells have been isolated by selection for resistance to two Tf-toxin conjugates. The hybrid toxins contain Tf covalently linked to ricin A chain or a genetically engineered diphtheria toxin fragment. The Tf-receptor-variant (TRV) cells do not have detectable cell-surface Tf receptor; they do not bind fluorescein-Tf or 125I-Tf. TRV cells are at least 100-fold more resistant to the Tf-diphtheria toxin conjugate than are the parent cells. The TRV cells have retained sensitivity to native diphtheria toxin, indicating that the increased resistance to the conjugate is correlated with the loss of Tf binding. The endocytosis of fluorescein-labeled alpha 2-macroglobulin is normal in TRV cells, demonstrating that the defect does not pleiotropically affect endocytosis. Since these cells lack endogenous Tf receptor activity, they are ideally suited for studies of the functional expression of normal or altered Tf receptors introduced into the cells by cDNA transfection. One advantage of this system is that Tf binding and uptake can be used to monitor the behavior of the transfected receptor. A cDNA clone of the human Tf receptor has been transfected into TRV cells. In the stably expressing transfectants, the behavior of the human receptor is very similar to that of the endogenous Chinese hamster ovary cell Tf receptor. Tf binds to cell surface receptors, and is internalized into the para-Golgi region of the cell. Iron is released from Tf, and the apo-Tf and its receptor are recycled back to the cell surface. Thus, the TRV cells can be used to study the behavior of genetically altered Tf receptors in the absence of interfering effects from endogenous receptors.