Apolipoprotein E isoform dependently affects Tat-mediated HIV-1 LTR transactivation.

BACKGROUND: Apolipoprotein E (ApoE) is the major carrier protein that mediates the transport and delivery of cholesterol and other lipids in the brain. Three isoforms of ApoE (ApoE2, ApoE3, ApoE4) exist in humans, and their relative expression levels impact HIV-1 infection, HIV-1/AIDS disease progression, and cognitive decline associated with HIV-1-associated neurocognitive disorder. Because HIV-1 Tat, a viral protein essential for HIV-1 replication, can bind to low-density lipoprotein receptor-related protein 1 (LRP1) that controls ApoE uptake in the brain, we determined the extent to which different isoforms of ApoE affected Tat-mediated HIV-1 LTR transactivation. METHODS: Using U87MG glioblastoma cells expressing LTR-driven luciferase, we determined the extent to which LRP1 as well as ApoE2, ApoE3, and ApoE4 affected Tat-mediated HIV-1 LTR transactivation. RESULTS: A specific LRP1 antagonist and siRNA knockdown of LRP1 both restricted significantly Tat-mediated LTR transactivation. Of the three ApoEs, ApoE4 was the least potent and effective at preventing HIV-1 Tat internalization and at decreasing Tat-mediated HIV-1 LTR transactivation. Further, Tat-mediated LTR transactivation was attenuated by an ApoE mimetic peptide, and ApoE4-induced restriction of Tat-mediated LTR transactivation was potentiated by an ApoE4 structure modulator that changes ApoE4 into an ApoE3-like phenotype. CONCLUSIONS: These findings help explain observed differential effects of ApoEs on HIV-1 infectivity and the prevalence of HAND in people living with HIV-1 infection and suggest that ApoE mimetic peptides and ApoE4 structure modulator might be used as a therapeutic strategy against HIV-1 infection and associated neurocognitive disorders.

Role of LRP-1 in cancer cell migration in 3-dimensional collagen matrix.

The low-density lipoprotein receptor-related protein-1 (LRP-1) is a member of Low Density Lipoprotein Receptor (LDLR) family, which is ubiquitously expressed and which is described as a multifunctional endocytic receptor which mediates the clearance of various extracellular matrix molecules including serine proteinases, proteinase-inhibitor complexes, and matricellular proteins. Several studies showed that high LRP-1 expression promotes breast cancer cell invasiveness, and LRP-1 invalidation leads to cell motility abrogation in both tumor and non-tumor cells. Furthermore, our group has reported that LRP-1 silencing prevents the invasion of a follicular thyroid carcinoma despite increased pericellular proteolytic activities from MMP2 and uPA using a 2D-cell culture model. As the use of 3D culture systems is becoming more and more popular due to their promise as enhanced models of tissue physiology, the aim of the present work is to characterize for the first time how the 3D collagen type I matrix may impact the ability of LRP-1 to regulate the migratory properties of thyroid carcinoma using as a model FTC-133 cells. Our results show that inhibition of LRP-1 activity or expression leads to morphological changes affecting cell-matrix interactions, reorganizations of the actin-cytoskeleton especially by inhibiting FAK activation and increasing RhoA activity and MLC-2 phosphorylation, thus preventing cell migration. Taken together, our results suggest that LRP-1 silencing leads to a decrease of cell migratory capacity in a 3D configuration.

LRP1 modulates the microglial immune response via regulation of JNK and NF-κB signaling pathways.

BACKGROUND: Neuroinflammation is characterized by microglial activation and the increased levels of cytokines and chemokines in the central nervous system (CNS). Recent evidence has implicated both beneficial and toxic roles of microglia when over-activated upon nerve injury or in neurodegenerative diseases, including Alzheimer's disease (AD). The low-density lipoprotein receptor-related protein 1 (LRP1) is a major receptor for apolipoprotein E (apoE) and amyloid-ß (Aß), which play critical roles in AD pathogenesis. LRP1 regulates inflammatory responses in peripheral tissues by modulating the release of inflammatory cytokines and phagocytosis. However, the roles of LRP1 in brain innate immunity and neuroinflammation remain unclear. METHODS: In this study, we determined whether LRP1 modulates microglial activation by knocking down Lrp1 in mouse primary microglia. LRP1-related functions in microglia were also assessed in the presence of LRP1 antagonist, the receptor-associated protein (RAP). The effects on the production of inflammatory cytokines were measured by quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). Potential involvement of specific signaling pathways in LRP1-regulated functions including mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) were assessed using specific inhibitors. RESULTS: We found that knocking down of Lrp1 in mouse primary microglia led to the activation of both c-Jun N-terminal kinase (JNK) and NF-κB pathways with corresponding enhanced sensitivity to lipopolysaccharide (LPS) in the production of pro-inflammatory cytokines. Similar effects were observed when microglia were treated with LRP1 antagonist RAP. In addition, treatment with pro-inflammatory stimuli suppressed Lrp1 expression in microglia. Interestingly, NF-κB inhibitor not only suppressed the production of cytokines induced by the knockdown of Lrp1 but also restored the down-regulated expression of Lrp1 by LPS. CONCLUSIONS: Our study uncovers that LRP1 suppresses microglial activation by modulating JNK and NF-κB signaling pathways. Given that dysregulation of LRP1 has been associated with AD pathogenesis, our work reveals a critical regulatory mechanism of microglial activation by LRP1 that could be associated with other AD-related pathways thus further nominating LRP1 as a potential disease-modifying target for the treatment of AD.

Evidence that LDL receptor-related protein 1 acts as an early injury detection receptor and activates c-Jun in Schwann cells.

Schwann cells (SCs) detect injury to peripheral nerves and transform phenotypically to respond to injury and facilitate repair. Cell-signaling pathways and changes in gene expression that drive SC phenotypic transformation in injury have been described; however, the SC receptors that detect peripheral nervous system (PNS) injury have not been identified. LDL receptor-related protein 1 (LRP1) is a receptor for numerous ligands, including intracellular proteins released by injured cells and protein components of degenerated myelin. In certain cell types, including SCs, LRP1 is a cell-signaling receptor. Here, we show that binding of the LRP1 ligand, tissue-type plasminogen activator (tPA), to cultured rat SCs induces c-Jun phosphorylation, a central event in activation of the SC repair program. The response to tPA was blocked by the LRP1 antagonist, receptor-associated protein. c-Jun phosphorylation was also observed when cultured rat SCs were treated with a recombinant derivative of matrix metalloproteinase-9 that contains the LRP1 recognition motif (PEX). The ability of LRP1 to induce c-Jun phosphorylation and ERK1/2 activation was confirmed using cultures of human SCs. When tPA or PEX was injected directly into crush-injured rat sciatic nerves, c-Jun phosphorylation and ERK1/2 activation were observed in SCs in vivo. The ability of LRP1 to bind proteins released in the earliest stages of PNS injury and to induce c-Jun phosphorylation support a model in which SC LRP1 functions as an injury-detection receptor in the PNS.

Generation of a Potent Low Density Lipoprotein Receptor-related Protein 1 (LRP1) Antagonist by Engineering a Stable Form of the Receptor-associated Protein (RAP) D3 Domain.

The low density lipoprotein receptor-related protein 1 (LRP1) is a member of the low density lipoprotein receptor family and plays important roles in a number of physiological and pathological processes. Expression of LRP1 requires the receptor-associated protein (RAP), a molecular chaperone that binds LRP1 and other low density lipoprotein receptor family members in the endoplasmic reticulum and traffics with them to the Golgi where the acidic environment causes its dissociation. Exogenously added RAP is a potent LRP1 antagonist and binds to LRP1 on the cell surface, preventing ligands from binding. Following endocytosis, RAP dissociates in the acidic endosome, allowing LRP1 to recycle back to the cell surface. The acid-induced dissociation of RAP is mediated by its D3 domain, a relatively unstable three-helical bundle that denatures at pH <6.2 due to protonation of key histidine residues on helices 2 and 3. To develop an LRP1 inhibitor that does not dissociate at low pH, we introduced a disulfide bond between the second and third helices in the RAP D3 domain. By combining this disulfide bond with elimination of key histidine residues, we generated a stable RAP molecule that is resistant to both pH- and heat-induced denaturation. This molecule bound to LRP1 with high affinity at both neutral and acidic pH and proved to be a potent inhibitor of LRP1 function both in vitro and in vivo, suggesting that our stable RAP molecule may be useful in multiple pathological settings where LRP1 blockade has been shown to be effective.

Macrophage expression of LRP1, a receptor for apoptotic cells and unopsonized erythrocytes, can be regulated by glucocorticoids.

Macrophage phagocytosis of apoptotic cells, or unopsonized viable CD47(-/-) red blood cells, can be mediated by the interaction between calreticulin (CRT) on the target cell and LDL receptor-related protein-1 (LRP1/CD91/α2-macroglobulin receptor) on the macrophage. Glucocorticoids (GC) are powerful in treatment of a range of inflammatory conditions, and were shown to enhance macrophage uptake of apoptotic cells. Here we investigated if the ability of GC to promote macrophage uptake of apoptotic cells could in part be mediated by an upregulation of macrophage LRP1 expression. Using both resident peritoneal and bone marrow-derived macrophages, we found that the GC dexamethasone could dose- and time-dependently increase macrophage LRP1 expression. The GC receptor-inhibitor RU486 could dose-dependently prevent LRP1 upregulation. Dexamethasone-treated macrophages did also show enhanced phagocytosis of apoptotic thymocytes as well as unopsonized viable CD47(-/-) red blood cells, which was sensitive to inhibition by the LRP1-agonist RAP. In conclusion, these data suggest that GC-stimulated macrophage uptake of apoptotic cells may involve an upregulation of macrophage LRP1 expression and enhanced LRP1-mediated phagocytosis.

Inhibition of Abeta aggregation and neurotoxicity by the 39-kDa receptor-associated protein.

Aggregation of beta-amyloid protein (Abeta) to form oligomers is considered to be a key step in generating neurotoxicity in the Alzheimer's disease brain. Agents that bind to Abeta and inhibit oligomerization have been proposed as Alzheimer's disease therapeutics. In this study, we investigated the binding of fluorescein-labeled Abeta(1-42) (FluoAbeta(1-42)) to SH-SY5Y neuroblastoma cells and examined the effect of the 39-kDa receptor-associated protein (RAP), on the Abeta cell interaction. FluoAbeta(1-42) bound to the cells in a punctate pattern. Surprisingly, when RAP was added to the incubations, FluoAbeta(1-42) and RAP were found to be co-localized on the cell surface, suggesting that RAP and Abeta may bind to each other. Experiments using the purified proteins confirmed that a RAP-Abeta complex was stable and resistant to sodium dodecyl sulfate. RAP also inhibited Abeta oligomerization. We next examined whether RAP could inhibit the neurotoxic effects of Abeta. Addition of Abeta(1-42) to SH-SY5Y cells caused an increase in intracellular Ca2+ that was inhibited by treatment of the Abeta peptide with RAP. RAP also blocked an Abeta-induced inhibition of long-term memory consolidation in 1-day-old chicks. This study demonstrates that RAP binds to Abeta and is an inhibitor of the neurotoxic effects of Abeta.

Low-density lipoprotein receptors regulate microglial inflammation through c-Jun N-terminal kinase.

Apolipoprotein E (apoE) has been implicated in modulating the central nervous system (CNS) inflammatory response. However, the molecular mechanisms involved in apoE-dependent immunomodulation are poorly understood. We hypothesize that apoE alters the CNS inflammatory response by signaling via low-density lipoprotein (LDL) receptors in glia. To address this hypothesis, we used a small bioactive peptide formed from the receptor-binding domain of apoE, apoE peptide (EP), to study LDL receptor signaling in microglia. To model glial activation, we treated primary mouse microglia and the microglial cell line BV2 with lipopolysaccharide (LPS) and studied two inflammatory responses: an increase in nitric oxide production (NO) and a decrease in apoE production. We found that treatment of primary microglia and BV2 cells with EP attenuated LPS-induced NO accumulation and apoE reduction in a dose-dependent manner. Using the receptor-associated protein to block ligand binding to members of the LDL receptor family, we found that EP attenuated both of these LPS-induced inflammatory responses via LDL receptors. We studied two intracellular signaling cascades associated with apoE: c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). LPS induced both ERK and JNK activation, whereas EP induced ERK activation, but drastically reduced JNK activation. Inhibition of JNK with SP600125 reduced LPS-induced NO production and apoE reduction in a dose-dependent manner. Treatment of microglia with suboptimal EP in combination with JNK inhibitor enhanced attenuation of LPS-induced NO production. These data suggest that microglial LDL receptors regulate JNK activation, which is necessary for apoE modulation of the inflammatory response.

Human immunodeficiency virus protein Tat induces synapse loss via a reversible process that is distinct from cell death.

Human immunodeficiency virus (HIV)-1 infection of the CNS produces changes in dendritic morphology that correlate with cognitive decline in patients with HIV-1 associated dementia (HAD). Here, we investigated the effects of HIV-1 transactivator of transcription (Tat), a protein released by virus-infected cells, on synapses between hippocampal neurons using an imaging-based assay that quantified clusters of the scaffolding protein postsynaptic density 95 fused to green fluorescent protein (PSD95-GFP). Tat (24 h) decreased the number of PSD95-GFP puncta by 50 +/- 7%. The decrease was concentration-dependent (EC(50) = 6 +/- 2 ng/ml) and preceded cell death. Tat acted via the low-density lipoprotein receptor-related protein (LRP) because the specific LRP blocker, receptor associated protein (RAP), prevented the Tat-induced decrease in the number of PSD95-GFP puncta. Ca(2+) influx through the NMDA receptor was necessary for Tat-induced synapse loss. Expression of an ubiquitin ligase inhibitor protected synapses, implicating the ubiquitin-proteasome pathway. In contrast to synapse loss, Tat induced cell death (48 h) required activation of nitric oxide synthase. The ubiquitin ligase-inhibitor nutlin-3 prevented synapse loss but not cell death induced by Tat. Thus, the pathways diverged, consistent with the hypothesis that synapse loss is a mechanism to reduce excess excitatory input rather than a symptom of the neuron's demise. Furthermore, application of RAP to cultures treated with Tat for 16 h reversed synapse loss. These results suggest that the impaired network function and decreased neuronal survival produced by Tat involve distinct mechanisms and that pharmacologic targets, such as LRP, might prove useful in restoring function in HAD patients.

A unique function for LRP-1: a component of a two-receptor system mediating specific endocytosis of plasma-derived factor V by megakaryocytes.

BACKGROUND: Factor V is endocytosed by megakaryocytes from plasma via a specific, receptor-mediated, clathrin-dependent mechanism to form the unique platelet-derived FV pool. OBJECTIVE: The role of low-density lipoprotein (LDL) receptor-related protein-1 (LRP-1), or a related family member, in FV endocytosis by megakaryocytes was examined because of its known interactions with other proteins involved in hemostasis. METHODS: LRP-1 expression by megakaryocytes and its functional role in FV endocytosis was confirmed using reverse transcription polymerase chain reaction (RT-PCR) and specific antibodies. FV binding to megakaryocytes was performed under Ca(2+)-free conditions to quantify binding in the absence of endocytosis. RESULTS AND CONCLUSION: Cell surface expression of LRP-1 by CD34+ ex vivo-derived megakaryocytes and the megakaryocyte-like cell line CMK was confirmed using anti-LRP-1 antibodies and was consistent with the detection of LRP-1 message in these cells. All cells capable of endocytosing FV expressed LRP-1. Anti-LRP-1 antibodies and receptor-associated protein (RAP), a known antagonist of LDL receptor family members, displaced only 50% of the [(125)I]FV bound to megakaryocytes. FV binding to megakaryocytes showed positive cooperativity (Hill coefficient = 1.92 +/- 0.18) that was substantially reduced in the presence of RAP (1.47 +/- 0.26). As FV endocytosis is specific to this cofactor, a model is hypothesized where FV binding to a specific receptor facilitates binding and endocytosis of a second FV molecule by LRP-1, or a related family member. These combined observations describe a unique role for LRP-1 in endocytosis of a coagulation protein trafficked to alpha-granules and not destined for lysosomal degradation.

Triglyceride-rich lipoproteins prime aortic endothelium for an enhanced inflammatory response to tumor necrosis factor-alpha.

High levels of triglyceride-rich lipoproteins (TGRLs) in blood are linked to development of atherosclerosis, yet the mechanisms by which these particles initiate inflammation of endothelium are unknown. TGRL isolated from human plasma during the postprandial state was examined for its capacity to bind to cultured human aortic endothelial cells (HAECs) and alter the acute inflammatory response to tumor necrosis factor-alpha. HAECs were repetitively incubated with dietary levels of freshly isolated TGRL for 2 hours per day for 1 to 3 days to mimic postprandial lipidemia. TGRL induced membrane upregulation of the low-density lipoprotein family receptors LRP and LR11, which was inhibited by the low-density lipoprotein receptor-associated protein-1. TGRLs alone did not elicit inflammation in HAECs but enhanced the inflammatory response via a 10-fold increase in sensitivity to cytokine stimulation. This was reflected by increased mitogen-activated protein kinase activation, nuclear translocation of NF-kappaB, amplified expression of endothelial selectin and VCAM-1, and a subsequent increase in monocyte-specific recruitment under shear flow as quantified in a microfabricated vascular mimetic device.

Mesd binds to mature LDL-receptor-related protein-6 and antagonizes ligand binding.

Wnt co-receptors LRP5 and LRP6 are two members of the low-density lipoprotein receptor family. Receptor-associated protein is not only a specialized chaperone but also a universal antagonist for members of the low-density lipoprotein receptor family. Here we test whether Mesd, a newly identified chaperone for members of the low-density lipoprotein receptor family, also binds to mature receptors at the cell surface and antagonizes ligand binding. We found that Mesd binds to cell surface LRP5 and LRP6, but not to other members of the low-density lipoprotein receptor family. Scatchard analysis revealed that Mesd binds cell surface LRP6 with high affinity (K(d) approximately 3.3 nM). Interestingly, the C-terminal region of Mesd, which is absent in sequences from invertebrates, is necessary and sufficient for binding to mature LRP6, and is required for LRP6 folding. We also found that LRP6 is not a constitutively active endocytosis receptor and binding of the receptor-associated protein to LRP6 partially competes for Mesd binding. Finally, we demonstrated that Mesd antagonizes ligand binding to LRP6 at the cell surface. Together our results show that in addition to serving as a folding chaperone, Mesd can function as a receptor antagonist by inhibiting ligand binding to mature LRP6.

Lipoprotein lipase-dependent binding and uptake of low density lipoproteins by THP-1 monocytes and macrophages: possible involvement of lipid rafts.

Lipoprotein lipase (LPL) is produced by cells in the artery wall and can mediate binding of lipoproteins to cell surface heparan sulfate proteoglycans (HSPG), resulting in endocytosis (the bridging function). Active, dimeric LPL may dissociate to inactive monomers, the main form found in plasma. We have studied binding/internalization of human low density lipoprotein (LDL), mediated by bovine LPL, using THP-1 monocytes and macrophages. Uptake of (125)I-LDL was similar in monocytes and macrophages and was not affected by the LDL-receptor family antagonist receptor-associated protein (RAP) or by the phagocytosis inhibitor cytochalasin D. In contrast, uptake depended on HSPG and on membrane cholesterol. Incubation in the presence of dexamethasone increased the endogenous production of LPL by the cells and also increased LPL-mediated binding of LDL to the cell surfaces. Monomeric LPL was bound to the cells mostly in a heparin-resistant fashion. We conclude that the uptake of LDL mediated by LPL dimers is receptor-independent and involves cholesterol-enriched membrane areas (lipid rafts). Dimeric and monomeric LPL differ in their ability to mediate binding/uptake of LDL, probably due to different mechanisms for binding/internalization.

Role of LRP in TGFbeta2-mediated neuronal uptake of Abeta and effects on memory.

There is increasing evidence that soluble amyloid-beta peptide (Abeta) uptake into neurons is an early event in the pathogenesis of Alzheimer's disease (AD). Identification of the early events leading to neuronal dysfunction is key to developing therapeutic strategies, but relative roles of receptors and factors modulating uptake are poorly understood. Studies have shown that transforming growth factor beta (TGFbeta), particularly TGFbeta2, can influence the targeting of Abeta to cells in vitro. TGFbeta2 can target Abeta to neurons in organotypic hippocampal slice cultures (OHSC). We examine a specific mechanism for TGFbeta2-mediated targeting of Abeta to neurons. The receptor-associated protein (RAP), a low-density lipoprotein receptor-related protein (LRP) antagonist, can attenuate the cellular targeting of Abeta both in vitro and in vivo and prevent Abeta/TGFbeta2-induced memory retention deficits. Using both in vitro and in vivo methods, we identify LRP as playing a role in TGFbeta2-mediated Abeta uptake, neurodegeneration, and spatial memory impairment.

Lipoprotein-bound LPS induces cytokine tolerance in hepatocytes.

Bacterial endotoxin (LPS) elicits dramatic responses in the host including elevated plasma lipid levels due to the increased synthesis and secretion of triglyceride (TG)-rich lipoproteins by the liver. We postulate that this cytokine-induced hyperlipoproteinemia, clinically termed the 'lipemia of sepsis', represents an innate, non-adaptive host immune response to infection. Data in support of this hypothesis include the capacity of TG-rich lipoproteins (VLDL and chylomicrons, CM) to bind and neutralize LPS. Herein, we present evidence that CM-bound LPS attenuates the hepatocellular response to pro-inflammatory cytokines. Primary rodent hepatocytes pretreated with CM-LPS complexes for 2 h demonstrated a near 70% reduction in cytokine-induced NO production as compared to non-pretreated control cells (P > or = 0.04). Whereas hepatocytes were maximally tolerant to cytokine stimulation 6 h after CM-LPS pretreatment, the cells spontaneously regained cytokine responsiveness within 40 h. The induction of cytokine tolerance in hepatocytes follows the internalization of CM-LPS complexes and is a process regulated by the LDL receptor. CM-LPS complexes failed to induce cytokine tolerance in hepatocytes wherein lipoprotein receptor activity was inhibited with high dose receptor associated protein (30 microg/ml). Similarly, CM-bound LPS did not induce tolerance in hepatocytes from ldlr(-/-) mice. Thus, the biochemical or genetic inhibition of LDL receptor activity effectively prevented the CM-mediated induction of the cytokine tolerant phenotype. In conclusion, the lipemia of sepsis likely represents a mechanism by which the host combats sporadic, non-life-threatening episodes of endotoxemia. Also, it may indicate a negative regulatory mechanism for the hepatic response to sepsis, serving to effectively down-regulate the acute phase response. A better understanding of how TG-rich lipoproteins modulate the host response to LPS could yield novel biological insights with important clinical implications, including the development of lipid-based therapies for bacterial infections.

Internalization of beta-amyloid causes downregulation of apolipoprotein E mRNA expression in neuroblastoma cells.

Apolipoprotein (apo) E, like beta-amyloid (Abeta), is a key component of the senile plaques that characterize Alzheimer's disease (AD). Understanding how apoE participates in the formation of senile plaques is necessary to clarify the pathogenesis of AD; however, the mechanism remains unknown. In this study, we investigated the changes of cellular apoE and its mRNA level induced by addition of extracellular Abeta to neuroblastoma cells. The presence of > or = 1.0 micromol/L of Abeta induced a decrease of apoE mRNA expression and an increase in the immunofluorescence reactivity for intracellular apoE. Both Abeta and apoE were observed by electron-microscopy to be localized within lysosomes. The levels of intracellular apoE and its mRNA returned to the steady state time-dependently. These changes were attenuated by treatments with heparinase I or receptor-associated protein. These findings suggest that the internalized Abeta, along with cellular apoE, induces downregulation of apoE mRNA via a pathway possibly mediated by apoE receptors and heparin sulfate proteoglycans. A disorder of this physiological response could be linked to the development of AD.

Tissue factor pathway inhibitor induces expression of JUNB and GADD45B mRNAs.

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that regulates tissue factor-triggered blood coagulation. It has previously been reported that TFPI inhibits the proliferation of human umbilical vein endothelial cells (HUVECs), suggesting that TFPI may act as more than just a mediator of coagulation through changes in gene expression. By using DNA-array techniques and Northern blot analysis, we here revealed that TFPI transiently induced the mRNA expression of JUNB and GADD45B. The inducible effects were not observed in TFPIdeltaC (lacking the C-terminal basic region) or antithrombin (heparin-binding anticoagulant protease inhibitor). Moreover, the TFPI-induced expression of GADD45B was blocked by receptor-associated protein, which masks the ligand-binding domain of very low density lipoprotein receptor (VLDL-R). In conclusion, this is the first report to show an effect of TFPI on mRNA expression, and suggests that TFPI modulates cellular functions by inducing JUNB and GADD45B expression through binding to VLDL-R.

Evidence of functional modulation of the MEKK/JNK/cJun signaling cascade by the low density lipoprotein receptor-related protein (LRP).

Lipoprotein receptors, such as LRP, have been shown to assemble multiprotein complexes containing intracellular signaling molecules; however, in vivo, their signaling function is poorly understood. Using a novel LRP receptor fusion construct, a type I transmembrane protein chimera, termed sIgG-LRP (bearing the intracellular COOH-terminal tail of human LRP as recombinant fusion to a transmembrane region plus the extracellular IgG-F(c) domain), we here investigated LRP signal transduction specificity in intact cells. First and similar to activated alpha2-macroglobulin as agonist of endogenous LRP, expression of sIgG-LRP demonstrated significant apoptosis protection. Second and similar to alpha2-macroglobulin-induced endogenous LRP, sIgG-LRP is sufficient to negatively modulate mitogen-induced Elk-1 and cJun (but not NF-kappaB) transcriptional activity. Third, expression of sIgG-LRP also impaired cJun transactivation mediated by constitutive active mutants of Rac-1 and MEKK-1. Fourth and unexpectedly, sIgG-LRP expression was found to be associated with a marked enhancement of mitogen-induced cJun amino-terminal kinase (JNK) activation. Fifth, confocal microscopic examination and subcellular fractionation demonstrated that sIgG-LRP and JNK co-localize in transfected cells. Therefore, sIgG-LRP expression was found to significantly impair activation-induced translocation of JNK into the nucleus. Taken together, we here demonstrate that sIgG-LRP protein sequesters activated JNK into the plasma membrane compartment in intact cells, inhibiting nuclear activation of the JNK-dependent transcription factors Elk-1 and cJun.

Cutting edge: CD91-independent cross-presentation of GRP94(gp96)-associated peptides.

GRP94(gp96) elicits CD8(+) T cell responses against its bound peptides, a process requiring access of its associated peptides into the MHC class I cross-presentation pathway of APCs. Entry into this pathway requires receptor-mediated endocytosis, and CD91 (low-density lipoprotein receptor-related protein) has been reported to be the receptor mediating GRP94 uptake into APC. However, a direct role for CD91 in chaperone-based peptide Ag re-presentation has not been demonstrated. We investigated the contribution of CD91 to GRP94 cell surface binding, internalization, and trafficking in APCs. Whereas a clear role for CD91 in alpha(2)-macroglobulin binding and uptake was readily obtained, the addition of excess CD91 ligand, activated alpha(2)-macroglobulin, or receptor-associated protein, an antagonist of all known CD91 ligands, did not affect GRP94 cell surface binding, receptor-mediated endocytosis, or peptide re-presentation. These data identify a CD91-independent, GRP94 internalization pathway that functions in peptide Ag re-presentation.

IL-1beta down-regulates tissue-type plasminogen activator by up-regulating low-density lipoprotein receptor-related protein in AML 12 cells.

Interleukin-1 (IL-1) regulation of tPA in hepatocytes was studied in mouse hepatocyte line AML12. IL-1 induced transient accumulation of tPA mRNA as high as threefold by 2 h after the start of treatment. The cytokine also induced the mRNA for serum amyloid A, a typical acute-phase protein in mice, with more sustained kinetics in a time-dependent manner. In contrast to the induction of mRNA, tPA activity and protein levels in the harvested medium were dramatically diminished by IL-1. IL-1 stimulated the uptake of (125)I-tPA by AML 12. This uptake was inhibited by 39-kDa receptor-associated protein (RAP), but not by the sugar mannan. These results revealed that low-density lipoprotein receptor-related protein (LRP), which is known to be a receptor for tPA and to be blocked by RAP, was up-regulated by IL-1. We also demonstrated, for the first time, that IL-1 transiently increased the mRNA level of LRP threefold by 30 min after the start of IL-1 treatment of AML 12. The receptor-mediated endocytosis of tPA by hepatocytes may thus play a crucial role in the down-regulation of fibrinolysis during the acute-phase response.