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Biomedical subjects

Guojun Bu

Publications and source records attributed to Guojun Bu.

At least 37 records · Page 2Linked to original sources
PubMed2005

Estrogen therapy fails to alter amyloid deposition in the PDAPP model of Alzheimer's disease.

Epidemiological studies implicate estrogen deprivation as a risk factor for Alzheimer's disease and postmenopausal estrogen replacement as protective factor. One potential mechanism involves estrogen attenuation of beta-amyloid (Abeta) peptide accumulation. We examined the effect of estrogen on amyloid accumulation in female PDAPP mice, which express human amyloid precursor protein (APP) with the V717F mutation. These animals deposit Abeta 1-42 in the hippocampus and neocortex and develop Alzheimer-like neuropathology. Mice were subjected to ovariectomy, ovariectomy with estrogen replacement, or sham surgery at 3 months of age, and levels of cerebral Abeta 1-40 and 1-42 were determined after 5 months of treatment. Neither estrogen deprivation nor estrogen replacement altered Abeta accumulation in the hippocampus or neocortex. Similarly, immunoreactivity for full-length human APP and secreted APPalpha was unchanged. Estrogen status of the animals was confirmed using a variety of techniques, including uterine and pituitary weight, vaginal cytology, and plasma estradiol concentrations. There was no correlation between plasma estradiol levels and accumulation of either Abeta 1-40 or Abeta 1-42 in the brain. Our observations indicate that long-term estrogen therapy does not alter amyloid pathology in PDAPP mice, an animal model of Alzheimer's disease, and question the role of estrogen in Abeta deposition in brain.

Alzheimer Disease↗
PubMed2005

Rapid endocytosis of the low density lipoprotein receptor-related protein modulates cell surface distribution and processing of the beta-amyloid precursor protein.

The low density lipoprotein receptor-related protein (LRP) is a approximately 600-kDa multifunctional endocytic receptor that is highly expressed in the brain. LRP and its ligands apolipoprotein E, alpha2-macroglobulin, and beta-amyloid precursor protein (APP), are genetically linked to Alzheimer disease and are found in characteristic plaque deposits in brains of patients with Alzheimer disease. To identify which extracellular domains of LRP interact with APP, we used minireceptors of each of the individual LRP ligand binding domains and assessed their ability to bind and degrade a soluble APP fragment. LRP minireceptors containing ligand binding domains II and IV, but not I or III, interacted with APP. To test whether APP trafficking is directly related to the rapid endocytosis of LRP, we generated stable Chinese hamster ovary cell lines expressing either a wild-type LRP minireceptor or its endocytosis mutants. Chinese hamster ovary cells stably expressing wild-type LRP minireceptor had less cell surface APP than pcDNA3 vector-transfected cells, whereas those stably expressing endocytosis-defective LRP minireceptors accumulated APP at the cell surface. We also found that the steady-state levels of the amyloid beta-peptides (Abeta) is dictated by the relative expression levels of APP and LRP, probably reflecting the dual roles of LRP in both Abeta production and clearance. Together, these data establish a relationship between LRP rapid endocytosis and APP trafficking and proteolytic processing to generate Abeta.

Amino Acid Sequence↗
PubMed2005

ApoER2 is endocytosed by a clathrin-mediated process involving the adaptor protein Dab2 independent of its Rafts' association.

The apolipoprotein E receptor 2 (apoER2) is a member of the low-density lipoprotein receptor family which binds ligands such as reelin, apolipoprotein E and apolipoprotein J/clusterin and has been shown to play roles in neuronal migration during development and in male fertility. The function of apoER2 mainly depends on cellular signaling triggered by ligand binding. Although the receptor is internalized, the mechanism and functional significance of its endocytic trafficking remain unclear. Apolipoprotein E receptor 2 partitions into lipid rafts and interacts with caveolin-1, a feature that could modulate its endocytic behavior. Recent evidence also suggested that apoER2 might be endocytosed by a pathway independent of clathrin. Here, we show that despite a raft association, apoER2 internalization depends on its cytoplasmic FxNPXY motif that is similar to canonical motifs for clathrin-mediated endocytosis. This motif mediates receptor binding to the adaptor protein Dab2, which can interact directly with clathrin. Several inhibitory conditions of clathrin-mediated endocytosis, including expression of the dominant negative forms of eps15 and Dab2, decreased apoER2 internalization. In contrast, treatment with the drug nystatin, which blocks the caveolar/raft internalization pathway, has no effect on the receptor's endocytosis. Neither the transmembrane nor the proline-rich insert of the cytoplasmic domain, which has been previously reported to exclude the receptor from the clathrin-mediated pathway, altered apoER2 endocytic activity. These studies indicate that apoER2 internalizes through a clathrin-mediated pathway and that its association with caveolar and noncaveolar rafts does not determine its endocytosis.

Adaptor Proteins, Signal Transducing↗
PubMed2005

LRP and Alzheimer's disease.

The low-density lipoprotein receptor (LDLR)-related protein, LRP, is a unique member of the LDLR family. Frequently referred to as a scavenger receptor, LRP is a large transmembrane endocytic receptor that can bind and internalize many functionally distinct ligands. Besides its role as a cargo-receptor, LRP has also been implicated in many signaling pathways. LRP knockout mice die at early embryonic age, which strongly suggests that LRP's functions are essential for normal development. Within the CNS, LRP is highly expressed in neuronal cell bodies and dendritic processes. In vitro, neurite outgrowth is stimulated by apolipoprotein E (apoE)-containing lipoprotein particles via binding to LRP. ApoE is the major cholesterol transporter in the brain and human carriers of one or two copies of the e4 allele of apoE are at a higher risk of developing Alzheimer's disease (AD). LRP also binds the amyloid precursor protein (APP) and its proteolytic fragment, the amyloid-beta peptide (Abeta), which are major players in the pathogenesis of AD. Finally, LRP has been linked to AD by genetic evidence. In this review we discuss the potential mechanisms by which LRP can affect APP and Abeta metabolism, and therefore contribute to the pathogenesis of AD.

Alzheimer Disease↗
PubMed2005

LRP5/6 in Wnt signaling and tumorigenesis.

The canonical Wnt signaling pathway is frequently overactivated in several types of human cancer. Defects in different components of the Wnt signaling pathway promote tumorigenesis and tumor progression. Accordingly, the pathway has been intensely studied to understand its importance in cancer biology and as therapeutic target. Recent studies have found that the low-density lipoprotein receptor-related protein (LRP)5 and 6 are essential Wnt coreceptors and interact with several key components of the Wnt signaling pathway. Furthermore, it has been demonstrated that LRP5 and 6 are potential oncogenic proteins. Thus, blockade of LRP56 function may lead to new strategies in cancer treatment.

Animals↗
PubMed2004

LRP6 expression promotes cancer cell proliferation and tumorigenesis by altering beta-catenin subcellular distribution.

The Wnt signaling pathway plays key roles in both embryogenesis and tumorigenesis. The low-density lipoprotein (LDL) receptor-related protein-6 (LRP6), a novel member of the expanding LDL receptor family, functions as an indispensable co-receptor for the Wnt signaling pathway. Although the role of LRP6 in embryonic development is now well established, its role in tumorigenesis is unclear. We report that LRP6 is readily expressed at the transcript level in several human cancer cell lines and human malignant tissues. Furthermore, using a retroviral gene transfer system, we find that stable expression of LRP6 in human fibrosarcoma HT1080 cells alters subcellular beta-catenin distribution such that the cytosolic beta-catenin level is significantly increased. This is accompanied by a significant increase in Wnt/beta-catenin signaling and cell proliferation. Finally, we demonstrate that LRP6 expression promotes tumorigenesis in vivo. These results thus indicate that LRP6 may function as a potential oncogenic protein by modulating Wnt/beta-catenin signaling.

Animals↗
PubMed2004

Efficient transfer of receptor-associated protein (RAP) across the blood-brain barrier.

We have sought to identify a high-capacity transport system that mediates transcytosis of proteins from the blood to the brain. The 39 kDa receptor-associated protein (RAP) functions as a specialized endoplasmic reticulum chaperone assisting in the folding and trafficking of members of the low-density lipoprotein (LDL) receptor family. RAP efficiently binds to these receptors and antagonizes binding of other ligands. Previous studies have shown that two large members of the LDL receptor family, LDL receptor-related protein 1 (LRP1) and LDL receptor-related protein 2 (LRP2 or megalin), possess the ability to mediate transcytosis of ligands across the brain capillary endothelium. Here, we tested whether blood-borne RAP crosses the blood-brain barrier (BBB) by LRP1- or megalin-mediated transport by studying the pharmacokinetics of [125I]-RAP transport into the brain in intact mice and across cell monolayers in vitro. Our results show that [125I]-RAP is relatively stable in blood for 30 minutes and has a mean influx constant of 0.62+/-0.08 microl/g-minute from blood to brain. In situ brain perfusion in blood-free buffer shows that transport of [125I]-RAP across the BBB is a saturable process. Capillary depletion of brain homogenates indicates that 70% of [125I]-RAP is localized in the parenchyma rather than in the vasculature of the brain. Results of transport in stably transfected MDCK cells are consistent with the hypothesis that megalin mediates most of the apical-to-basolateral transport across polarized epithelial cells. The inhibition of [125I]-RAP influx by excess RAP and the involvement of megalin indicate the presence of a saturable transport system at the BBB. The higher permeability of RAP compared with that of melanotransferrin and transferrin show that the LRP receptor is a high capacity transport system. These studies suggest that RAP may provide a novel means of protein-based drug delivery to the brain.

Animals↗
PubMed2004

Lipoprotein receptor binding, cellular uptake, and lysosomal delivery of fusions between the receptor-associated protein (RAP) and alpha-L-iduronidase or acid alpha-glucosidase.

Enzyme replacement therapy for lysosomal storage disorders depends on efficient uptake of recombinant enzyme into the tissues of patients. This uptake is mediated by oligosaccharide receptors including the cation-independent mannose 6-phosphate receptor and the mannose receptor. We have sought to exploit alternative receptor systems that are independent of glycosylation but allow for efficient delivery to the lysosome. Fusions of the human lysosomal enzymes alpha-l-iduronidase or acid alpha-glucosidase with the receptor-associated protein were efficiently endocytosed by lysosomal storage disorder patient fibroblasts, rat C6 glioma cells, mouse C2C12 myoblasts, and recombinant Chinese hamster ovary cells expressing individual members of the low-density lipoprotein receptor family. Uptake of the fusions exceeded that of phosphorylated enzyme in all cases, often by an order of magnitude or greater. Uptake was specifically mediated by members of the low-density lipoprotein receptor protein family and was followed by delivery of the fusions to the lysosome. The advantages of the lipoprotein receptor system over oligosaccharide receptor systems include more efficient cellular delivery and the potential for transcytosis of ligands across tight endothelia, including the blood-brain barrier.

Animals↗
PubMed2004

The low density lipoprotein receptor-related protein 1B retains beta-amyloid precursor protein at the cell surface and reduces amyloid-beta peptide production.

The low density lipoprotein (LDL) receptor-related protein 1B (LRP1B) is a newly identified member of the LDL receptor family that shares high homology with the LDL receptor-related protein (LRP). LRP1B was originally described as a putative tumor suppressor in lung cancer cells; however, its expression profile in several regions of adult human brain suggests it may have additional functions in the central nervous system. Since LRP1B has overlapping ligand binding properties with LRP, we investigated whether LRP1B, like LRP, could interact with the beta-amyloid precursor protein (APP) and modulate its processing to amyloid-beta peptides (Abetas). Using an LRP1B minireceptor (mLRP1B4) generated to study the trafficking of LRP1B, we found that mLRP1B4 and APP form an immunoprecipitable complex. Furthermore mLRP1B4 bound and facilitated the degradation of a soluble isoform of APP containing a Kunitz proteinase inhibitor domain but not soluble APP lacking a Kunitz proteinase inhibitor domain. A functional consequence of mLRP1B4 expression was a significant accumulation of APP at the cell surface, which is likely related to the slow endocytosis rate of LRP1B. More importantly, mLRP1B4-expressing cells that accumulated cell surface APP produced less Abeta and secreted more soluble APP. These findings reveal that LRP1B is a novel binding partner of APP that functions to decrease APP processing to Abeta. Consequently LRP1B expression could function to protect against the pathogenesis of Alzheimer's disease.

Adult↗
PubMed2004

Degradation of the LDL receptor class 2 mutants is mediated by a proteasome-dependent pathway.

Familial hypercholesterolemia is a genetic disorder that results from various gene mutations, primarily within the LDL receptor (LDLR). Approximately 50% of the LDLR mutations are defined as class 2 mutations, with the mutant proteins partially or entirely retained in the endoplasmic reticulum. To determine the degradation pathway of the LDLR class 2 mutants, we examined the effects of inhibition of several potential pathways on the levels of the wild-type LDLR and its four representative class 2 mutants (S156L, C176Y, E207K, and C646Y) stably expressed in Chinese hamster ovary (CHO) cells. We found that proteasome inhibitors MG132 and lactacystin blocked the degradation of the LDLR mutants, but not that of the wild-type LDLR. Treatment of CHO cells with these proteasome inhibitors led to a significant accumulation of the mutants at steady state. Furthermore, cell surface levels of the LDLR mutants were significantly increased upon inhibition of the proteasome degradation pathway. In contrast to the proteasome inhibitors, inhibitors of trypsin-like proteases, chymotrypsin-like proteases, and lysosomal pathway inhibitors did not affect the levels of the LDLR mutants. Taken together, these data demonstrate that the proteasome is the principal degradation pathway for LDLR class 2 mutants.

Acetylcysteine↗
PubMed2004

Increased soluble amyloid-beta peptide and memory deficits in amyloid model mice overexpressing the low-density lipoprotein receptor-related protein.

Amyloid-beta peptide (Abeta) is central to the pathogenesis of Alzheimer's disease, and the low-density lipoprotein receptor-related protein (LRP) has been shown to alter Abeta metabolism in vitro. Here, we show that overexpression of a functional LRP minireceptor in the brain of PDAPP mice results in age-dependent increase of soluble brain Abeta, with no changes in Abeta plaque burden. Importantly, soluble brain Abeta was found to be primarily in the form of monomers/dimers and to be highly correlated with deficits in spatial learning and memory. These results provide in vivo evidence that LRP may contribute to memory deficits typical of Alzheimer's disease by modulating the pool of small soluble forms of Abeta.

Amyloid beta-Peptides↗
PubMed2004

Is tissue-type plasminogen activator a neuromodulator?

In the last few years, it has been evidenced that serine proteases play key roles in the mammalian brain, both in physiological and pathological conditions. It has been well established that among these serine proteases, the tissue-type plasminogen activator (t-PA) is critically involved in development, plasticity, and pathology of the nervous system. However, its mechanism of action remains to be further investigated. By using pharmacological and immunological approaches, we have evidenced in the present work that t-PA should be considered as a neuromodulator. Indeed, we have observed that: (i). neuronal depolarization induces a release of t-PA; (ii). this release of t-PA is sensitive to exocytosis inhibition and calcium chelation; (iii). released t-PA modulates NMDA receptor signaling and (iv). astrocytes are able to recapture extracellular t-PA through a low-density lipoprotein (LDL) receptor-related protein (LRP)-dependent mechanism.

Action Potentials↗
PubMed2003

Essential role of the low density lipoprotein receptor-related protein in vascular smooth muscle cell migration.

The low density lipoprotein receptor-related protein (LRP) is a multifunctional cell surface receptor highly expressed in human aortic smooth muscle cells. In the present study, we used the short interfering RNA (siRNA) technique to explore the role of LRP in smooth muscle cell migration. We identified an LRP-specific siRNA that selective silences LRP expression in human aortic smooth muscle cells. As a consequence, LRP-mediated ligand degradation was significantly reduced. More important, we found that platelet-derived growth factor-dependent cell migration was inhibited in cells transfected with LRP siRNA. These results demonstrate an important role of LRP in smooth muscle cell migration.

Aorta↗
PubMed2003

Differential distribution of low-density lipoprotein-receptor-related protein (LRP) and megalin in polarized epithelial cells is determined by their cytoplasmic domains.

Megalin and the low-density lipoprotein (LDL) receptor-related protein (LRP) are two large members of the LDL receptor family that bind and endocytose multiple ligands. The molecular and cellular determinants that dictate the sorting behavior of these receptors in polarized epithelial cells are largely unknown. Megalin is found apically distributed, whereas the limited information on LRP indicates its polarity. We show here that in Madin-Darby canine kidney cells, both endogenous LRP and a minireceptor containing the fourth ligand-binding, transmembrane and LRP cytosolic domains were basolaterally sorted. In contrast, minireceptors that either lacked the cytoplasmic domain or had the tyrosine in the NPTY motif mutated to alanine showed a preferential apical distribution. In LLC-PK1 cells, endogenous megalin was found exclusively in the apical membrane. Studies were also done using chimeric proteins harboring the cytosolic tail of megalin, one with the fourth ligand-binding domain of LRP and the other two containing the green fluorescent protein as the ectodomain and transmembrane domains of either megalin or LRP. Findings from these experiments showed that the cytosolic domain of megalin is sufficient for apical sorting, and that the megalin transmembrane domain promotes association with lipid rafts. In conclusion, we show that LRP and megalin both contain sorting information in their cytosolic domains that directs opposite polarity, basolateral for LRP and apical for megalin. Additionally, we show that the NPTY motif in LRP is important for basolateral sorting and the megalin transmembrane domain directs association with lipid rafts.

Amino Acid Sequence↗
PubMed2002

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.

Antigens, Differentiation↗