anti-LRP Antibody Research Articles

UPA impairs cerebrovasodilation after hypoxia/ischemia through LRP and ERK MAPK

We have previously observed that soluble urokinase plasminogn activator receptor (suPAR) prevents impairment of cerebrovasodilation induced by hypercapnia and hypotension after hypoxia/ischemia (H/I) in the newborn pig. In this study, we investigated the role of low-density lipoprotein-related protein (LRP) receptor and the ERK isoform of mitogen activated protein kinase (MAPK) in uPA-mediated impairment of vasodilation after H/I in piglets equipped with a closed cranial window. CSF uPA increased from 9 ± 2 to 52 ± 8 and 140 ± 21 ng/ml at 1 and 4 h after H/I, respectively. The LRP antagonist receptor associated protein (RAP) and anti-LRP antibody blunted the increase in CSF uPA at 1 h (17 ± 2 ng/ml) but not 4 h post insult. uPA detectable in sham-treated cortex by immunhistochemistry was markedly elevated 4 h after H/I. Phosphorylation (activation) of CSF ERK MAPK was detected at 1 and 4 h post H/I and blocked by RAP. Exogenous uPA administered at 4 h post H/I further stimulated ERK MAPK phosphorylation, which was blocked by RAP. Pre-treatment of piglets with RAP, anti-LRP, and suPAR completely prevented, and the ERK MAPK antagonist U 0126 partially prevented, impaired responses to hypotension and hypercapnia post H/I, but none of these antagonists affected the response to isoproterenol. These data indicate that uPA is upregulated after H/I through an LRP-dependent process and that the released uPA impairs hypercapnic and hypotensive dilation through an LRP- and ERK MAPK dependent pathway. These data suggest that modulation of uPA upregulation and/or uPA-mediated signal transduction may preserve cerebrohemodynamic control after hypoxia/ischemia.

UPA contributes to impairment of hypercapnic and hypotensive cerebrovasodilation in an LRP and ERK MAPK dependent process

Cerebral hypoxia/ischemia (H/I) impairs pial artery dilation (PAD) to hypercapnia and hypotension but not isoproterenol in the newborn pig. We investigated the role of endogenous urokinase plasminogen activator (uPA), the low-density lipoprotein related protein (LRP) receptor, and the ERK isoform of mitogen activated protein kinase (MAPK) in vasodilator impairment after H/I in piglets equipped with a closed cranial window. CSF uPA increased from 9 ± 2 to 52 ± 8 and 140 ± 21 ng/ml at 1 and 4h after H/I, respectively. The LRP antagonist receptor associated protein (RAP) and anti-LRP antibody blunted the increase in CSF uPA at 1h (17 ± 2 ng/ml) but not 4h post insult. uPA detectable in sham-treated piglet parietal cortex by immunocytochemistry was markedly elevated 4h after H/I. Phosphorylation (activation) of ERK MAPK was detected 1 and 4h post H/I and blocked by RAP. Exogenous uPA administered at 4h post H/I further stimulated ERK MAPK phosphorylation, blocked by RAP. Pre-treatment with RAP, anti-LRP, and soluble uPA receptor completely prevented and the ERK MAPK antagonist U 0126, partially prevented the impairment to hypotension and hypercapnia post-H/I, but none of these antagonists affected the response to isoproterenol. These data indicate that uPA is released after H/I through an LRP-initiated process and that the released uPA impairs hypercapnic and hypotensive PAD in an LRP- and ERK MAPK-dependent pathway. These data suggest that modulation of uPA expression and/or action may preserve cerebrohemodynamic control after H/I.

Selective uptake and efflux of cholesteryl linoleate in LDL by macrophages expressing 12/15-lipoxygenase

Oxidation of low density lipoprotein (LDL) is a critical step for atherogenesis, and the role of the 12/15-lipoxygenase (12/15-LOX) as well as LDL receptor-related protein (LRP) expressed in macrophages in this process has been suggested. The oxygenation of cholesteryl linoleate in LDL by mouse macrophage-like J774A.1 cells overexpressing 12/15-LOX was inhibited by an anti-LRP antibody but not by an anti-LDL receptor antibody. When the cells were incubated with LDL double-labeled by [ 3H]cholesteryl linoleate and [ 125I]apoB, association with the cells of [ 3H]cholesteryl linoleate expressed as LDL protein equivalent exceeded that of [ 125I]apoB, indicating selective uptake of [ 3H]cholesteryl linoleate from LDL to these cells. An anti-LRP antibody inhibited the selective uptake of [ 3H]cholesteryl ester by 62% and 81% with the 12/15-LOX-expressing cells and macrophages, respectively. Furthermore, addition of LDL to the culture medium of the [ 3H]cholesteryl linoleate-labeled 12/15-LOX-expressing cells increased the release of [ 3H]cholesteryl linoleate to the medium in LDL concentration- and time-dependent manners. The transport of [ 3H]cholesteryl linoleate from the cells to LDL was also inhibited by an anti-LRP antibody by 75%. These results strongly suggest that LRP contributes to the LDL oxidation by 12/15-LOX in macrophages by selective uptake and efflux of cholesteryl ester in the LDL particle.

Sorting nexin 17 facilitates LRP recycling in the early endosome

The low-density lipoprotein (LDL) receptor-related protein (LRP) is a multiligand endocytic receptor and a member of the LDL receptor family. Here we show that sorting nexin 17 (Snx 17) is part of the cellular sorting machinery that regulates cell surface levels of LRP by promoting its recycling. While the phox (PX) domain of Snx 17 interacts with phosphatidylinositol-3-phosphate for membrane association, the FERM domain and the carboxyl-terminal region participate in LRP binding. Immunoelectron microscopy shows that the membrane-bound fraction of Snx 17 is localized to the limiting membrane and recycling tubules of early endosomes. The NPxY motif, proximal to the plasma membrane in the LRP cytoplasmic tail, is identified as the Snx 17-binding motif. Functional mutation of this motif did not interfere with LRP endocytosis, but decreased LRP recycling from endosomes, resulting in increased lysosomal degradation. Similar effects are found after knockdown of endogenous Snx 17 expression by short interfering RNA. We conclude that Snx 17 binds to a motif in the LRP tail distinct from the endocytosis signals and promotes LRP sorting to the recycling pathway in the early endosomes.

The Low Density Lipoprotein Receptor Regulates the Level of CentralNervous System Human and Murine Apolipoprotein E but Does Not Modify AmyloidPlaque Pathology in PDAPPMice

Apolipoprotein E (apoE), a chaperone for the amyloid beta (Abeta) peptide, regulates the deposition and structure of Abeta that deposits in the brain in Alzheimer disease (AD). The primary apoE receptor that regulates levels of apoE in the brain is unknown. We report that the low density lipoprotein receptor (LDLR) regulates the cellular uptake and central nervous system levels of astrocyte-derived apoE. Cells lacking LDLR were unable to appreciably endocytose astrocyte-secreted apoE-containing lipoprotein particles. Moreover, cells overexpressing LDLR showed a dramatic increase in apoE endocytosis and degradation. We also found that LDLR knock-out (Ldlr-/-) mice had a significant, approximately 50% increase in the level of apoE in the cerebrospinal fluid and extracellular pools of the brain. However, when the PDAPP mouse model of AD was bred onto an Ldlr-/- background, we did not observe a significant change in brain Abeta levels either before or after the onset of Abeta deposition. Interestingly, human APOE3 or APOE4 (but not APOE2) knock-in mice bred on an Ldlr-/- background had a 210% and 380% increase, respectively, in the level of apoE in cerebrospinal fluid. These results demonstrate that central nervous system levels of both human and murine apoE are directly regulated by LDLR. Although the increase in murine apoE caused by LDLR deficiency was not sufficient to affect Abeta levels or deposition by 10 months of age in PDAPP mice, it remains a possibility that the increase in human apoE3 and apoE4 levels caused by LDLR deficiency will affect this process and could hold promise for therapeutic targets in AD.

Effect of lung resistance-related protein on the resistance to cisplatin in human ovarian cancer cell lines

The mechanisms of drug-resistance in human ovarian cancer cells have not been entirely clarified. The purpose of this study was to investigate whether LRP is involved in the resistance of ovarian cancer cell lines to cisplatin and its molecular mechanism. Human ovarian cisplatin-resistant cancer cell lines (A2780/DDP and COC1/DDP) and their parental cisplatin-sensitive cell lines (A2780 and COC1), alone or transfected with antisense LRP-specific oligonucleotides (ODN) or sense ODN, were treated with cisplatin to induce differentiation. Expression of LRP was examined by RT-PCR and Western blot analysis. The sensitivities of cells to cisplatin were assessed using sulforhodamine B (SRB) assay and flow cytometry, and the accumulation and efflux of cisplatin in the cells and isolated nuclei were examined by high performance liquid chromatographic (HPLC) assay. The expressions of LRP in A2780/DDP and COC1/DDP cells were higher than those in A2780 and COC1 cells and conferred resistance to cisplatin. Transfection of LRP AsODN into A2780/DDP and COC1/DDP cells down-regulated LRP expression and reversed the resistance phenotype. Levels of cisplatin accumulating in cells were increased by LRP-specific AsODN and anti-LRP monoclonal antibody. Isolated nuclei from A2780 and COC1 cells or A2780/DDP and COC1/DDP cells incubated with anti-LRP antibody contained more cisplatin than the nuclei of A2780/DDP and COC1/DDP cells not treated with anti-LRP antibody. Efflux of cisplatin was greater from the nuclei of A2780/DDP and COC1/DDP cells than those of A2780 and COC1 cells, and was inhibited by anti-LRP monoclonal antibody. Thus, LRP was involved in the resistance of ovarian cancer cells to cisplatin and has an important role in the transport of cisplatin both in exocytotic vesicles and between the nucleus and cytoplasm.

Low-density Lipoprotein Receptor-related Protein Mediates the Endocytosis of Anionic Liposomes in Neurons

We have recently demonstrated that anionic liposomes efficiently introduce foreign DNA into postmitotic neurons and other cell types (Lakkaraju, A., Dubinsky, J. M., Low, W. C., and Rahman, Y.-E. (2001) J. Biol. Chem. 276, 32000-32007). To investigate the mechanism of liposome uptake, we followed the internalization of anionic liposome-encapsulated Cy3-labeled oligonucleotides (AL-Cy3ONs) by hippocampal neurons using confocal microscopy. Uptake of AL-Cy3ONs was widespread and time- and temperature-dependent, indicative of receptor-mediated endocytosis. The low-density lipoprotein receptor-related protein (LRP) was crucial for anionic liposome endocytosis because the receptor-associated protein or an anti-LRP antibody inhibited internalization, and fibroblasts lacking LRP did not internalize AL-Cy3ONs. Using selective endocytosis inhibitors, we found that liposome endocytosis and intracellular transport required clathrin, dynamin, an intact cytoskeletal network, and phosphatidylinositol 3-kinase activity. Cy3ONs did not significantly colocalize with recycling endosomal/lysosomal markers and entered neuronal nuclei within 1-3 h of incubation. Approximately 50% of the internalized liposomal phospholipids were recycled back to the cell surface, in keeping with the fluidity of their acyl chains. Liposome endocytosis did not require heparan sulfate proteoglycans or cause calcium influx into neurons. Thus, constitutive endocytosis of anionic liposomes by LRP utilizes only one component, in contrast to the more involved heparan sulfate proteoglycan-LRP pathway implicated in the pathogenesis of Alzheimer's disease.

Low Density Lipoprotein Receptor-related Protein Is Required for Macrophage-mediated Oxidation of Low Density Lipoprotein by 12/15-Lipoxygenase

The oxidative modification of low density lipoprotein (LDL) has been implicated in the early stage of atherosclerosis through multiple potential pathways, and 12/15-lipoxygenase is suggested to be involved in this oxidation process. We demonstrated previously that the 12/15-lipoxygenase overexpressed in mouse macrophage-like J774A.1 cells was required for the cell-mediated LDL oxidation. However, the mechanism of the oxidation of extracellular LDL by the intracellular 12/15-lipoxygenase has not yet been elucidated. In the present study, we found that not only the LDL receptor but also LDL receptor-related protein (LRP), both of which are cell surface native LDL-binding receptors, were down-regulated by the preincubation of the cells with cholesterol or LDL and up-regulated by lipoprotein-deficient serum. Moreover, 12/15-lipoxygenase-expressing cell-mediated LDL oxidation was decreased by the preincubation of the cells with LDL or cholesterol and increased by the preincubation with lipoprotein-deficient serum. Heparin-binding protein 44, an antagonist of the LDL receptor family, also suppressed the cell-mediated LDL oxidation in a dose-dependent manner. The cell-mediated LDL oxidation was dose-dependently blocked by an anti-LRP antibody but not by an anti-LDL receptor antibody. Furthermore, antisense oligodeoxyribonucleotides against LRP reduced the cell-mediated LDL oxidation under the conditions in which the expression of LRP was decreased. The results taken together indicate that LRP was involved essentially for the cell-mediated LDL oxidation by 12/15-lipoxygenase expressed in J774A.1 cells, suggesting an important pathophysiological role of this receptor-enzyme system as the initial trigger of the progression of atherosclerosis.

Expression of functional lung resistance–related protein predicts poor outcome in adult T-cell leukemia

Chemotherapy of patients with adult T-cell leukemia (ATL) has been unsuccessful. The poor outcome is thought to be caused mainly by the drug resistance of ATL cells. Lung resistance-related protein (LRP) is a novel protein associated with drug resistance. The expression of LRP messenger RNA (mRNA) was evaluated by slot blot analysis in 55 patients with ATL. Of these patients, 36 had acute, 12 chronic, and 7 lymphoma-type ATL. The expression levels of LRP mRNA were significantly higher in chronic ATL than in lymphoma-type ATL (P =.007). The expression of LRP mRNA was higher in patients with white blood cell counts above 30,000/microL (P =.038) or with abnormal lymphocyte counts above 10,000/microL (P =.007) than in the remaining patients. The enhanced efflux of [(14)C]doxorubicin from nuclei isolated from ATL cells that expressed high levels of LRP was inhibited by a polyclonal antibody against LRP, and the accumulation of doxorubicin in the isolated nuclei was increased by the anti-LRP antibody. In acute and lymphoma-type ATL patients, high expression of LRP mRNA at diagnosis correlated with shorter survival, and a Cox proportional hazards model showed that LRP expression is an independent prognostic factor. These findings suggest that functionally active LRP is expressed in some ATL cells and that it is involved in drug resistance and poor prognosis in ATL. (Blood. 2001;98:1160-1165)

Expression of lung resistance protein in epithelioid sarcoma in vitro and in vivo.

The incidence of epithelioid sarcoma among patients with malignant soft tissue tumors is small, but the rates of recurrence and metastasis of this type of sarcoma are high. To date, effective chemotherapy for advanced epithelioid sarcoma has not been established and, furthermore, epithelioid sarcoma is known to exhibit multidrug resistance (MDR). The chemosensitivities to anticancer agents of two cell lines established from epithelioid sarcoma were examined in this study. The results showed that the ES-OMC-MN and SFT-8606 cell lines were resistant to vincristine (IC50 1190 nM and 872 nM, respectively) and Adriamycin (IC50 921 nM and 650 nM, respectively), but sensitive to actinomycin D (IC50 < 10 nM). P-glycoprotein (p-Gp) and MDR-associated protein (MRP) were not expressed in these cell lines, but a high expression level of lung resistance protein (LRP) was observed. The original tumor tissues from which the two cell lines were established were also found to be LRP-positive but not to express p-Gp or MRP. Their chemosensitivities to Adriamycin were not significantly altered in the presence of 2.5 microg/ml anti-LRP antibody (LRP-56), but the IC50 of vincristine was much less (IC50 128 nM and 27 nM, respectively) than that for an untreated cell line. It is thus suggested that the vincristine resistance in the two cell lines is LRP-mediated. Since cyclosporin A, known to be a modifier of p-Gp, also induced reversal of vincristine resistance in the ES-OMC-MN and SFT-8606 cell lines (IC50 6.2 nM and 17 nM, respectively), it is suggested that cyclosporin A acts as a modifier of MDR mediated by LRP.

The low-density lipoprotein receptor-related protein, a multifunctional apolipoprotein E receptor, modulates hippocampal neurite development.

The epsilon 4 allele of apolipoprotein E (apoE) is an important risk factor for Alzheimer's disease. A major neuronal receptor for apoE within the brain is the low-density lipoprotein receptor-related protein (LRP). Using primary cultured hippocampal neurons, we examined the role of LRP in early neuronal development. LRP, as well as a 39-kDa protein that regulates its activity, is localized abundantly in developing neurons. Both the 39-kDa protein and an anti-LRP antibody inhibited neurite outgrowth of primary hippocampal neurons cultured in either serum-containing medium or on cortical astrocyte monolayers in serum-free medium. It is noteworthy that microtubule-associated protein-2 immunoreactive process outgrowth was decreased significantly in hippocampal neurons cultured on cortical astrocytes derived from apoE-deficient mice and was not diminished further following incubation with LRP inhibitors. Thus, these results suggest that LRP can influence aspects of neuronal process development and that apoE-containing lipoproteins may be one of the major LRP ligands that can contribute to this process.

Receptor-mediated endocytosis of tissue-type plasminogen activator by low density lipoprotein receptor-related protein on human hepatoma HepG2 cells

Hepatic parenchymal cells play an essential role in the clearance of circulating tissue-type plasminogen activator (t-PA) in vivo as a major pathway in the regulation of plasma fibrinolytic activity. Previous studies have identified plasminogen activator inhibitor type 1 (PAI-1)-dependent t-PA-binding sites in the human hepatoma cell line HepG2. In this study, we demonstrate that receptor-mediated binding and endocytosis of the t-PA-PAI-1 complex are largely mediated by a recently identified low density lipoprotein receptor-related protein (LRP). A 39-kDa LRP receptor-associated protein that modulates ligand binding to LRP was found to bind specifically to HepG2 cells and to inhibit approximately 70-80% of specific 125I-t-PA.PAI-1 binding. This inhibition by the 39-kDa protein was not due to inhibition of complex formation between 125I-t-PA and PAI-1; instead, the 39-kDa protein inhibited 125I-t-PA.PAI-1 binding to LRP. Polyclonal anti-LRP antibody raised against purified human LRP also inhibited 70-80% of specific 125I-t-PA.PAI-1 binding. A similar extent of inhibition by the 39-kDa protein was also observed for 125I-t-PA.PAI-1 endocytosis and degradation. Chemical cross-linking experiments demonstrated the direct interaction between 125I-t-PA.PAI-1 and LRP on HepG2 cells as anti-LRP antibody, in addition to anti-t-PA and anti-PAI-1 antibodies, was able to immunoprecipitate the 125I-t-PA.PAI-1 complex following binding of 125I-t-PA.PAI-1 to HepG2 cells and cross-linking. This interaction of the t-PA.PAI-1 complex with LRP on HepG2 cells was also observed when the unlabeled t-PA.PAI-1 complex was cross-linked to [35S]methionine-labeled HepG2 cells. In addition, the direct binding of the 39-kDa protein to LRP on HepG2 cells was demonstrated by similar cross-linking experiments. Thus, these data clearly show that LRP is the major cell-surface receptor responsible for t-PA.PAI-1 complex binding and endocytosis on human hepatoma HepG2 cells and extend the multifunctional nature of LRP as an endocytosis receptor for several structurally and functionally distinct ligands.

Determinants of binding and internalization of tissue-type plasminogen activator by human vascular smooth muscle and endothelial cells

Vascular injury induced by angioplasty is often followed by smooth muscle cell (SMC) proliferation, migration, and accumulation of extracellular matrix. Since plasminogen activators and their receptors may be important both in cell migration and the clearance of plasminogen activators, we studied the binding, internalization, and degradation of radiolabeled tissue-type plasminogen activator (t-PA) by explant cultures of human vascular SMC. Binding of t-PA to SMC at 4 degrees C was rapid, specific, saturable, and inhibitable by antibodies to plasminogen activator inhibitor type 1 (PAI-1). At 37 degrees C, labeled t-PA was internalized and degraded by SMC but not by human umbilical vein endothelial cells. Internalization and degradation was mediated by the low density lipoprotein receptor related protein/alpha 2-macroglobulin receptor (LRP) in that these processes were inhibited by an anti-LRP antibody, recombinant LRP-associated protein, urokinase-type plasminogen activator-PAI-1 complexes, and lactoferrin. The portion of t-PA most important for internalization after complexing with PAI-1 is likely to be in the finger and/or epidermal growth factor domains or in the carbohydrate at amino acid 117, in that the internalization of preformed t-PA.PAI-1 complexes or complexes formed on the cell surface was inhibited by an excess of active site-blocked wild type t-PA, but not by an active site blocked t-PA variant missing these domains. These studies are consistent with a model in which t-PA binds initially to SMC-associated PAI-1 with subsequent t-PA.PAI-1 internalization via LRP. SMC may play an important role in clearing t-PA-PAI-1 complexes from within the vessel wall.