ABSTRACT The fibrinolysis proteinase tissue-type plasminogen activator (tPA, also known as PLAT) triggers cell signaling and regulates cell physiology. In PC12 cells, Schwann cells and macrophages, the N-methyl-D-aspartate receptor (NMDA-R) mediates tPA signaling. Plasminogen activator inhibitor-1 (PAI1, also known as SERPINE1) is a rapidly acting inhibitor of tPA enzyme activity. Although tPA-initiated cell signaling is not dependent on its enzyme active site, we show that tPA signaling is neutralized by PAI1. In PC12 cells, PAI1 blocked the ERK1/2 activation mediated by tPA as well as neurite outgrowth. In Schwann cells, PAI1 blocked tPA-mediated ERK1/2 activation and cell migration. In macrophages, PAI1 blocked the ability of tPA to inhibit IκBα phosphorylation and cytokine expression. The cell signaling activity of tPA–PAI1 complex was rescued when the complex was formed with PAI1R 76E , which binds to LRP1 with decreased affinity, by pre-treating cells with the LRP1 antagonist receptor-associated protein and upon LRP1 gene silencing. The inhibitory role of LRP1 in tPA–PAI1 complex-initiated cell signaling was unanticipated given the reported role of LRP1 as an NMDA-R co-receptor in signaling responses elicited by free tPA or α 2 -macroglobulin. We conclude that PAI1 functions as an in­hibitor not only of the enzyme activity of tPA but also of tPA receptor-mediated activities.

ABSTRACT 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.

Summary In the injured adult mammalian central nervous system (CNS), products are generated that inhibit neuronal sprouting and regeneration. In recent years, most attention has focused on the myelin-associated inhibitory proteins (MAIs) Nogo-A, OMgp, and myelin-associated glycoprotein (MAG). Binding of MAIs to neuronal cell-surface receptors leads to activation of RhoA, growth cone collapse, and neurite outgrowth inhibition. In the present study, we identify low-density lipoprotein (LDL) receptor-related protein-1 (LRP1) as a high-affinity, endocytic receptor for MAG. In contrast with previously identified MAG receptors, binding of MAG to LRP1 occurs independently of terminal sialic acids. In primary neurons, functional inactivation of LRP1 with receptor-associated protein, depletion by RNA interference (RNAi) knock-down, or LRP1 gene deletion is sufficient to significantly reverse MAG and myelin-mediated inhibition of neurite outgrowth. Similar results are observed when LRP1 is antagonized in PC12 and N2a cells. By contrast, inhibiting LRP1 does not attenuate inhibition of neurite outgrowth caused by chondroitin sulfate proteoglycans. Mechanistic studies in N2a cells showed that LRP1 and p75NTR associate in a MAG-dependent manner and that MAG-mediated activation of RhoA may involve both LRP1 and p75NTR. LRP1 derivatives that include the complement-like repeat clusters CII and CIV bind MAG and other MAIs. When CII and CIV were expressed as Fc-fusion proteins, these proteins, purified full-length LRP1 and shed LRP1 all attenuated the inhibition of neurite outgrowth caused by MAG and CNS myelin in primary neurons. Collectively, our studies identify LRP1 as a novel MAG receptor that functions in neurite outgrowth inhibition.

Multiple sclerosis (MS) is an autoimmune disease in which myelin is progressively degraded. Because degraded myelin may both initiate and accelerate disease progression, clearing degraded myelin from extracellular spaces may be critical. In this study, we prepared myelin vesicles (MV) from rat brains as a model of degraded myelin. Murine embryonic fibroblasts (MEFs) rapidly internalized MVs, which accumulated in lysosomes only when these cells expressed low-density lipoprotein receptor-related protein (LRP1). Receptor-associated protein (RAP), which binds LRP1 and inhibits interaction with other ligands, blocked MV uptake by LRP1-expressing MEFs. As a complementary approach, we prepared primary cultures of rat astrocytes, microglia and oligodendrocytes. All three cell types expressed LRP1 and mediated MV uptake, which was inhibited by RAP. LRP1 gene-silencing in oligodendrocytes also blocked MV uptake. Myelin basic protein (MBP), which was expressed as a recombinant protein, bound directly to LRP1. MBP-specific antibody inhibited MV uptake by oligodendrocytes. In experimental autoimmune encephalomyelitis in mice, LRP1 protein expression was substantially increased in the cerebellum and spinal cord. LRP1 colocalized with multiple CNS cell types. These studies establish LRP1 as a major receptor for phagocytosis of degraded myelin, which may function alone or in concert with co-receptors previously implicated in myelin phagocytosis.

Urokinase-type plasminogen activator (uPA) binds to the uPA receptor (uPAR) and activates the Ras-extracellular signal-regulated kinase (ERK) signaling pathway in many different cell types. In this study, we demonstrated that endogenously produced uPA functions as a major determinant of the basal level of activated ERK in MDA-MB-231 breast cancer cells. When these cells were cultured in the presence of antibodies that block the binding of uPA to uPAR, the level of phosphorylated ERK decreased substantially. Furthermore, conditioned medium from MDA-MB-231 cells activated ERK in MCF-7 cells and this response was blocked by uPA-specific antibody. The mitogen-activated protein kinase kinase inhibitor, PD098059, decreased expression of uPA and uPAR in MDA-MB-231 cells. Thus, uPA and the uPAR-ERK signaling pathway form a positive feedback loop in these cells. When this feedback loop was disrupted with uPA- or uPAR-specific antibody, uPA mRNA-specific antisense oligodeoxynucleotides or PD098059, cell growth was inhibited and apoptosis was promoted, as determined by the increase in cytoplasmic nucleosomes and caspase-3 activity. Treating the cells simultaneously with PD098059 and uPA- or uPAR-specific antibody did not further promote apoptosis, compared with either reagent added separately, supporting the hypothesis that uPAR and ERK are components of the same cell growth/survival-regulatory pathway. The ability of uPA to signal through uPAR, maintain an elevated basal level of activated ERK and inhibit apoptosis represents a novel mechanism whereby the uPA-uPAR system may affect breast cancer progression in vivo.