Denis Monard

The Phosphatidylethanolamine-binding Protein Is the Prototype of a Novel Family of Serine Protease Inhibitors

Serine proteases are involved in many processes in the nervous system and specific inhibitors tightly control their proteolytic activity. Thrombin is thought to play a role in tissue development and homeostasis. To date, protease nexin-1 is the only known endogenous protease inhibitor that specifically interferes with thrombotic activity and is expressed in the brain. In this study, we report the detection of a novel thrombin inhibitory activity in the brain of protease nexin-1(− /− ) mice. Purification and subsequent analysis by tandem mass spectrometry identified this protein as the phosphatidylethanolamine-binding protein (PEBP). We demonstrate that PEBP exerts inhibitory activity against several serine proteases including thrombin, neuropsin, and chymotrypsin, whereas trypsin, tissue type plasminogen activator, and elastase are not affected. Since PEBP does not share significant homology with other serine protease inhibitors, our results define it as the prototype of a novel class of serine protease inhibitors. PEBP immunoreactivity is found on the surface of Rat-1 fibroblast cells and although its sequence contains no secretion signal, PEBP-H6can be purified from the conditioned medium upon recombinant expression.

Tissue‐type plasminogen activator requires a co‐receptor to enhance NMDA receptor function

Glutamate is the main excitatory neurotransmitter of the CNS. Tissue‐type plasminogen activator (tPA) is recognized as a modulator of glutamatergic neurotransmission. This attribute is exemplified by its ability to potentiate calcium signaling following activation of the glutamate‐binding NMDA receptor (NMDAR). It has been hypothesized that tPA can directly cleave the NR1 subunit of the NMDAR and thereby potentiate NMDA‐induced calcium influx. In contrast, here we show that this increase in NMDAR signaling requires tPA to be proteolytically active, but does not involve cleavage of the NR1 subunit or plasminogen. Rather, we demonstrate that enhancement of NMDAR function by tPA is mediated by a member of the low‐density lipoprotein receptor (LDLR) family. Hence, this study proposes a novel functional relationship between tPA, the NMDAR, a LDLR and an unknown substrate which we suspect to be a serpin. Interestingly, whilst tPA alone failed to cleave NR1, cell‐surface NMDARs did serve as an efficient and discrete proteolytic target for plasmin. Hence, plasmin and tPA can affect the NMDAR via distinct avenues. Altogether, we find that plasmin directly proteolyses the NMDAR whilst tPA functions as an indirect modulator of NMDA‐induced events via LDLR engagement.

Male fertility defects in mice lacking the serine protease inhibitor protease nexin-1

Understanding infertility and sterility requires knowledge of the molecular mechanisms underlying sexual reproduction. We have found that male mice deficient for the gene encoding the protease inhibitor protease nexin-1 (PN-1) show a marked impairment in fertility from the onset of sexual maturity. Absence of PN-1 results in altered semen protein composition, which leads to inadequate semen coagulation and deficient vaginal plug formation upon copulation. Progressive morphological changes of the seminal vesicles also are observed. Consistent with these findings, abnormal PN-1 expression was found in the semen of men displaying seminal dysfunction. The data demonstrate that the level of extracellular proteolytic activity is a critical element in controlling male fertility.

Thrombin in ischemic neuronal death.

Thrombin plays a role in cerebral ischemia as rats subjected to focal cerebral ischemia were protected by the intracerebral injection of hirudin, a selective thrombin inhibitor. To separate the roles of thrombin in cell death and in coagulation, we have used an in vitro approach to test the effect of hirudin and of protease nexin-1 (PN-1), a cerebral thrombin inhibitor, on neuronal ischemia. Rat organotypic hippocampal slice cultures were subjected to oxygen (5%) and glucose (1 mmol/L) deprivation (OGD) during 30 min. Hirudin or PN-1 administered after OGD significantly prevented neuronal death in the CA1 region. After 24 h, there was a marked increase in thrombin immunoreactivity on Western blots. Thrombin therefore contributes to ischemic damage in neural tissue in vitro.

Stabilization of colchicine-binding activity of neuroblastoma

Abstract High concentrations of glycerol or sucrose increase the half-life of colchicinebinding activity in extracts from neuroblastoma cells to about 220 hours. This is the greatest stabilization by non-specific reagents yet observed, and the effect is not dependent upon added guanosine triphosphate. Both compounds block vinblastine precipitation of the binding activity. Although thiol groups are shown to be essential for colchicine binding, their oxidation is not the critical step in inactivation.

Inhibition of PDGF-BB by Factor VII-activating protease (FSAP) is neutralized by protease nexin-1, and the FSAP–inhibitor complexes are internalized via LRP

FSAP (Factor VII-activating protease) can inhibit neointima formation and VSMC (vascular smooth-muscle cell) proliferation by cleavage of PDGF-BB (platelet-derived growth factor-BB). Negatively charged polyanions lead to autoactivation of the FSAP, but no information is available concerning the potential regulation of FSAP activity and its metabolism in the vessel wall. In the present study, we demonstrate that the enzymatic activity of FSAP can be inhibited by the serine protease inhibitor, PN-1 (protease nexin-1), that is found in the vasculature. This leads to the loss of the inhibitory effect of FSAP on PDGF-BB-mediated DNA synthesis and mitogen-activated protein kinase phosphorylation in VSMCs. The FSAP-PN-1 complexes bind to the LRP (low-density lipoprotein receptor-related protein) and are subsequently internalized. This binding is inhibited by receptor-associated protein, an antagonist of LRP, as well as heparin. While PDGFbetaR (PDGFbeta receptor) is internalized by an LRP-dependent mechanism after stimulation of cells by PDGF-BB, the FSAP-PN-1 complex neither influenced PDGF-BB-mediated phosphorylation of PDGFbetaR nor its internalization via LRP. Hence, PN-1 inhibits the enzymatic activity of FSAP and neutralizes its effect on PDGF-BB-mediated VSMC proliferation. The FSAP-inhibitor complexes are internalized via LRP without influencing the PDGF-BB signal transduction pathway.

Activation of ERK signaling upon alternative protease nexin‐1 internalization mediated by syndecan‐1

Protease nexin‐1 (PN‐1), an inhibitor of serine proteases, contributes to tissue homeostasis and influences the behavior of some tumor cells. The internalization of PN‐1 protease complexes is considered to be mediated by the low‐density lipoprotein receptor related protein 1 (LRP1). In this study, both wild‐type and LRP1−/− mouse embryonic fibroblasts (MEF) were shown to internalize PN‐1. Receptor associated protein (RAP) interfered with PN‐1 uptake only in wild‐type MEF cells, indicating that another receptor mediates PN‐1 uptake in the absence of LRP1. In LRP1−/− MEF cells, inhibitor sensitivity and kinetic values (t1/2 at 45 min) of PN‐1 uptake showed a similarity to syndecan‐1‐mediated endocytosis. In these cells, PN‐1 uptake was increased by overexpression of full‐length syndecan‐1 and decreased by RNA interference targeting this proteoglycan. Most important, in contrast to PKA activation known to be triggered by LRP1‐mediated internalization, our study shows that syndecan‐1‐mediated internalization of PN‐1 stimulated the Ras‐ERK signaling pathway. J. Cell. Biochem. 99: 936–951, 2006.

Impaired fear extinction in mice lacking protease nexin‐1

The serine protease inhibitor protease‐nexin‐1 (PN‐1) has been shown to modulate N‐methyl‐d‐aspartate receptor (NMDAR)‐mediated synaptic currents and NMDAR‐dependent long‐term potentiation of synaptic transmission. Here, we analysed the role of PN‐1 in the acquisition and extinction of classical auditory fear conditioning, two distinct forms of learning that both depend on NMDAR activity in the amygdala. Immunostaining revealed that PN‐1 is expressed throughout the amygdala, primarily in γ‐aminobutyric acid containing neurons of the central amygdala and intercalated cell masses (ITCs) and in glia. Fear extinction was severely impaired in mice lacking PN‐1 (PN‐1 KO). Consistent with a role for the basal nucleus of the amygdala in fear extinction, we found that, compared with wild‐type (WT) littermate controls, PN‐1 KO mice exhibited decreased numbers of Fos‐positive neurons in the basal nucleus after extinction. Moreover, immunoblot analysis of laser‐microdissected amygdala sub‐nuclei revealed specific extinction‐induced increases in the level of phosphorylated alpha‐calcium/calmodulin protein kinase II in the medial ITCs and in the lateral subdivision of the central amygdala in WT mice. These responses were altered in PN‐1 KO mice. Together, these data indicate that lack of extinction in PN‐1 KO mice is associated with distinct changes in neuronal activity across the circuitry of the basal and central nuclei and the ITCs, supporting a differential impact on fear extinction of these amygdala substructures. They also suggest a new role for serine protease inhibitors such as PN‐1 in modulating fear conditioning and extinction.

Prothrombin overexpressed in post-natal neurones requires blood factors for activation in the mouse brain.

Thrombin is thought to mediate, through protease‐activated receptors, both protective as well as cytotoxic effects. As thrombin receptors are expressed in the CNS, an important question arises as to whether the intact nervous system is able to generate thrombin by activation of its precursor prothrombin, derived endogenously or only upon extravasation following brain injury. To address this question, transgenic mice that express C‐terminally haemagglutinin tagged human prothrombin in post‐mitotic neurones were generated. In situ hybridization and immunohistochemical analysis showed abundant and widespread cerebral expression of the transgene. Amidolytic assays of brain homogenates and hippocampal slice cultures demonstrated that activation of transgenic prothrombin required added factors, such as snake venom or blood components. This strongly suggests that any possible action of thrombin in the adult CNS depends on blood‐derived factors that activate prothrombin. Furthermore, the results are consistent with the idea that in the non‐pathological situation an as yet unidentified ligand activates thrombin receptors in the nervous system.

Purification of tubulin from neuroblastoma cells: Absence of covalently bound phosphate in tubulin from normal and morphologically differentiated cells

The state of assembly of cytoplasmic microtubules is believed to be an important factor in the regulation of a number of cell functions [1 ]. The microtubules are composed primarily of tubulin, a 120 000 molecular weight heterodimeric protein. Morphological differentiation of neuroblastoma cells [2] or Chinese hamster ovary cells [3], and the accompanying appearance of microtubules, does not require protein synthesis. The assembly of microtubules from a pool of preformed tubulin subunits could be controlled by a covalent modification of the tubulin to a form with a different ability to assemble. Since phosphate has been found associated with tubulin [4,5] and since appropriate phosphokinase activities have been found in brain extracts [6], phosphorylation has been considered to be a possible control mechanism. Alternatively, assembly could be controlled by a non-covalent interaction of the subunits with some other cellular component [7,8]. To attempt to distinguish between these possibilities, we have prepared tubulin from neuroblastoma treated with a glial cell conditioned medium. This medium induces a high per centage of the cells to extend processes, but unlike other techniques does not affect the growth rate of the cells [9], making