A soluble derivative of PrPC activates cell-signaling and regulates cell physiology through LRP1 and the NMDA receptor.

Cellular prion protein (PrPC) is a widely expressed glycosylphosphatidylinositol-anchored membrane protein. Scrapie prion protein is a misfolded and aggregated form of PrPC responsible for prion-induced neurodegenerative diseases. Understanding the function of the nonpathogenic PrPC monomer is an important objective. PrPC may be shed from the cell surface to generate soluble derivatives. Herein, we studied a recombinant derivative of PrPC (soluble cellular prion protein, S-PrP) that corresponds closely in sequence to a soluble form of PrPC shed from the cell surface by proteases in the A Disintegrin And Metalloprotease (ADAM) family. S-PrP activated cell-signaling in PC12 and N2a cells. TrkA was transactivated by Src family kinases and extracellular signal-regulated kinase 1/2 was activated downstream of Trk receptors. These cell-signaling events were dependent on the N-methyl-d-aspartate receptor (NMDA-R) and low-density lipoprotein receptor-related protein-1 (LRP1), which functioned as a cell-signaling receptor system in lipid rafts. Membrane-anchored PrPC and neural cell adhesion molecule were not required for S-PrP-initiated cell-signaling. S-PrP promoted PC12 cell neurite outgrowth. This response required the NMDA-R, LRP1, Src family kinases, and Trk receptors. In Schwann cells, S-PrP interacted with the LRP1/NMDA-R system to activate extracellular signal-regulated kinase 1/2 and promote cell migration. The effects of S-PrP on PC12 cell neurite outgrowth and Schwann cell migration were similar to those caused by other proteins that engage the LRP1/NMDA-R system, including activated α2-macroglobulin and tissue-type plasminogen activator. Collectively, these results demonstrate that shed forms of PrPC may exhibit important biological activities in the central nervous system and the peripheral nervous system by serving as ligands for the LRP1/NMDA-R system.

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.

The activities of LDL Receptor-related Protein-1 (LRP1) compartmentalize into distinct plasma membrane microdomains.

LDL Receptor-related Protein-1 (LRP1) is an endocytic receptor for diverse ligands. In neurons and neuron-like cells, ligand-binding to LRP1 initiates cell-signaling. Herein, we show that in PC12 and N2a neuron-like cells, LRP1 distributes into lipid rafts and non-raft plasma membrane fractions. When lipid rafts were disrupted, using methyl-ß-cyclodextrin or fumonisin B1, activation of Src family kinases and ERK1/2 by the LRP1 ligands, tissue-type plasminogen activator and activated α2-macroglobulin, was blocked. Biological consequences of activated LRP1 signaling, including neurite outgrowth and cell growth, also were blocked. The effects of lipid raft disruption on ERK1/2 activation and neurite outgrowth, in response to LRP1 ligands, were reproduced in experiments with cerebellar granule neurons in primary culture. Because the reagents used to disrupt lipid rafts may have effects on the composition of the plasma membrane outside lipid rafts, we studied the effects of these reagents on LRP1 activities unrelated to cell-signaling. Lipid raft disruption did not affect the total ligand binding capacity of LRP1, the affinity of LRP1 for its ligands, or its endocytic activity. These results demonstrate that well described activities of LRP1 require localization of this receptor to distinct plasma membrane microdomains.

LDL receptor-related protein-1 regulates NFκB and microRNA-155 in macrophages to control the inflammatory response.

LDL receptor-related protein-1 (LRP1) is an endocytic and cell-signaling receptor. In mice in which LRP1 is deleted in myeloid cells, the response to lipopolysaccharide (LPS) was greatly exacerbated. LRP1 deletion in macrophages in vitro, under the control of tamoxifen-activated Cre-ER(T) fusion protein, robustly increased expression of proinflammatory cytokines and chemokines. In LRP1-expressing macrophages, proinflammatory mediator expression was regulated by LRP1 ligands in a ligand-specific manner. The LRP1 agonists, α2-macroglobulin and tissue-type plasminogen activator, attenuated expression of inflammatory mediators, even in the presence of LPS. The antagonists, receptor-associated protein (RAP) and lactoferrin (LF), and LRP1-specific antibody had the entirely opposite effect, promoting inflammatory mediator expression and mimicking LRP1 deletion. NFκB was rapidly activated in response to RAP and LF and responsible for the initial increase in expression of proinflammatory mediators. RAP and LF also significantly increased expression of microRNA-155 (miR-155) after a lag phase of about 4 h. miR-155 expression reflected, at least in part, activation of secondary cell-signaling pathways downstream of TNFα. Although miR-155 was not involved in the initial induction of cytokine expression in response to LRP1 antagonists, miR-155 was essential for sustaining the proinflammatory response. We conclude that LRP1, NFκB, and miR-155 function as members of a previously unidentified system that has the potential to inhibit or sustain inflammation, depending on the continuum of LRP1 ligands present in the macrophage microenvironment.

Selective coexpression of VEGF receptor 2 in EGFRvIII-positive glioblastoma cells prevents cellular senescence and contributes to their aggressive nature.

BACKGROUND: In glioblastoma (GBM), the gene for epidermal growth factor receptor (EGFR) is frequently amplified. EGFR mutations also are common, including a truncation mutation that yields a constitutively active variant called EGFR variant (v)III. EGFRvIII-positive GBM progresses rapidly; however, the reason for this is not clear because the activity of EGFRvIII is attenuated compared with EGF-ligated wild-type EGFR. We hypothesized that EGFRvIII-expressing GBM cells selectively express other oncogenic receptors that support tumor progression. METHODS: Mining of The Cancer Genome Atlas prompted us to test whether GBM cells in culture, which express EGFRvIII, selectively express vascular endothelial growth factor receptor (VEGFR)2. We also studied human GBM propagated as xenografts. We then applied multiple approaches to test the effects of VEGFR2 on GBM cell growth, apoptosis, and cellular senescence. RESULTS: In human GBM, EGFR overexpression and EGFRvIII positivity were associated with increased VEGFR2 expression. In GBM cells in culture, EGFRvIII-initiated cell signaling increased expression of VEGFR2, which prevented cellular senescence and promoted cell cycle progression. The VEGFR-selective tyrosine kinase inhibitor cediranib decreased tumor DNA synthesis, increased staining for senescence-associated ß-galactosidase, reduced retinoblastoma phosphorylation, and increased p27(Kip1), all markers of cellular senescence. Similar results were obtained when VEGFR2 was silenced. CONCLUSIONS: VEGFR2 expression by GBM cells supports cell cycle progression and prevents cellular senescence. Coexpression of VEGFR2 by GBM cells in which EGFR signaling is activated may contribute to the aggressive nature of these cells.

The NMDA receptor functions independently and as an LRP1 co-receptor to promote Schwann cell survival and migration.

NMDA receptors (NMDA-Rs) are ionotropic glutamate receptors, which associate with LDL-receptor-related protein-1 (LRP1) to trigger cell signaling in response to protein ligands in neurons. Here, we demonstrate for the first time that the NMDA-R is expressed by rat Schwann cells and functions independently and with LRP1 to regulate Schwann cell physiology. The NR1 (encoded by GRIN1) and NR2b (encoded by GRIN2B) NMDA-R subunits were expressed by cultured Schwann cells and upregulated in sciatic nerves following crush injury. The ability of LRP1 ligands to activate ERK1/2 (also known as MAPK3 and MAPK1, respectively) and promote Schwann cell migration required the NMDA-R. NR1 gene silencing compromised Schwann cell survival. Injection of the LRP1 ligands tissue-type plasminogen activator (tPA, also known as PLAT) or MMP9-PEX into crush-injured sciatic nerves activated ERK1/2 in Schwann cells in vivo, and the response was blocked by systemic treatment with the NMDA-R inhibitor MK801. tPA was unique among the LRP1 ligands examined because tPA activated cell signaling and promoted Schwann cell migration by interacting with the NMDA-R independently of LRP1, albeit with delayed kinetics. These results define the NMDA-R as a Schwann cell signaling receptor for protein ligands and a major regulator of Schwann cell physiology, which may be particularly important in peripheral nervous system (PNS) injury.

LRP1 assembles unique co-receptor systems to initiate cell signaling in response to tissue-type plasminogen activator and myelin-associated glycoprotein.

In addition to functioning as an activator of fibrinolysis, tissue-type plasminogen activator (tPA) interacts with neurons and regulates multiple aspects of neuronal cell physiology. In this study, we examined the mechanism by which tPA initiates cell signaling in PC12 and N2a neuron-like cells. We demonstrate that enzymatically active and inactive tPA (EI-tPA) activate ERK1/2 in a biphasic manner. Rapid ERK1/2 activation is dependent on LDL receptor-related protein-1 (LRP1). In the second phase, ERK1/2 is activated by tPA independently of LRP1. The length of the LRP1-dependent phase varied inversely with the tPA concentration. Rapid ERK1/2 activation in response to EI-tPA and activated α2-macroglobulin (α2M*) required the NMDA receptor and Trk receptors, which assemble with LRP1 into a single pathway. Assembly of this signaling system may have been facilitated by the bifunctional adapter protein, PSD-95, which associated with LRP1 selectively in cells treated with EI-tPA or α2M*. Myelin-associated glycoprotein binds to LRP1 with high affinity but failed to induce phosphorylation of TrkA or ERK1/2. Instead, myelin-associated glycoprotein recruited p75 neurotrophin receptor (p75NTR) into a complex with LRP1 and activated RhoA. p75NTR was not recruited by other LRP1 ligands, including EI-tPA and α2M*. Lactoferrin functioned as an LRP1 signaling antagonist, inhibiting Trk receptor phosphorylation and ERK1/2 activation in response to EI-tPA. These results demonstrate that LRP1-initiated cell signaling is ligand-dependent. Proteins that activate cell signaling by binding to LRP1 assemble different co-receptor systems. Ligand-specific co-receptor recruitment provides a mechanism by which one receptor, LRP1, may trigger different signaling responses.