Progressive neuronal and motor dysfunction in mice overexpressing the serine protease inhibitor protease nexin-1 in postmitotic neurons.

Perturbation of the homeostasis between proteases and their inhibitors has been associated with lesion-induced or degenerative neuronal changes. Protease nexin-1 (PN-1), a secreted serine protease inhibitor, is constitutively expressed in distinct neuronal cell populations of the adult CNS. In an earlier study we showed that transgenic mice with ectopic or increased expression of PN-1 in postnatal neurons have altered synaptic transmission. Here these mice are used to examine the impact of an extracellular proteolytic imbalance on long-term neuronal function. These mice develop disturbances in motor behavior from 12 weeks on, with some of the histopathological changes described in early stages of human motor neuron disease, and neurogenic muscle atrophy in old age. In addition, sensorimotor integration, measured by epicranial multichannel recording of sensory evoked potentials, is impaired. Our results suggest that axonal dysfunction rather than cell death underlies these phenotypes. In particular, long projecting neurons, namely cortical layer V pyramidal and spinal motor neurons, show an age-dependent vulnerability to PN-1 overexpression. These mice can serve to study early stages of in vivo neuronal dysfunction not yet associated with cell loss.

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.

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-H(6) can be purified from the conditioned medium upon recombinant expression.

An assay for high-sensitivity detection of thrombin activity and determination of proteases activating or inactivating protease-activated receptors.

This paper describes the development of galactosidase protease-activated receptor (GPAR) as a recombinant protein obtained by fusion of beta-galactosidase, the extracellular domains of protease-activated receptors (PARs), and a biotin acceptor domain. Used as an immobilized substrate, this protein allows the detection of thrombin in the sub-picomolar range. A comparative analysis for proteolytic cleavage of murine PAR1, PAR2, and PAR3 and human PAR4 was performed, involving mutated and nonmutated GPAR fusion proteins. Thrombin cleaved GPAR1 (2.6 mol(beta-galactosidase)/(mol(thrombin) * min)), GPAR3 (410 mmol(beta-galactosidase)/(mol(thrombin) * min)), and GPAR4 (4.3 mmol(beta-galactosidase)/(mol(thrombin) * min)) specifically at the proteolytic activation site. A second possible cleavage site for thrombin is present in murine PAR1 and PAR3. Trypsin and plasmin cleaved all receptor fusion proteins with little specificity for the activation site, except for a marked preference of trypsin for cleavage at the activation site of GPAR2. Chymotrypsin cleaves GPAR1 at a rate (58 mmol(beta-galactosidase)/(mol(thrombin) * min)) that suggests the possibility of chymotryptic inactivation of PAR1. Elastase may inactivate PAR1 and PAR3, but probably not PAR2 and PAR4. Neither activated protein C nor the plasminogen activators cleave any GPAR fusion protein at considerable rates.

An octamer-binding site is crucial for the activity of an enhancer active at the embryonic met-/mesencephalic junction.

An enhancer sequence found in the Protease Nexin-1 (PN-1) gene was shown to drive lacZ expression specifically at the met-/mesencephalic junction in transgenic mouse embryos. A functional study of this enhancer has been performed to better understand the mechanisms regulating isthmic gene expression. An octamer-binding site for POU domain factors was found to be crucial for the activity of the enhancer in vivo. Comparative expression studies of POU domain factors, electrophoretic mobility shift assays and transient transfection experiments, strongly suggest that Brn-1/-2 regulate the enhancer activity in vivo. In addition, in vitro experiments indicated that FGF-8 was required for the maintenance of the enhancer activity, but not for the synthesis of Bn-1/-2. The data represents the first functional evidence for a role of POU factors in the regulation of met-/mesencephalic gene expression. It also implies that at least two regulatory pathways, namely the FGF-8 signaling and the octamer-binding site pathway, synergistically interact to control the PN-1 enhancer activity in vivo.

Ligand binding properties of the very low density lipoprotein receptor. Absence of the third complement-type repeat encoded by exon 4 is associated with reduced binding of Mr 40,000 receptor-associated protein.

The very low density lipoprotein receptor (VLDLR) binds, among other ligands, the Mr 40,000 receptor-associated protein (RAP) and a variety of serine proteinase-serpin complexes, including complexes of the proteinase urokinase-type plasminogen activator (uPA) with the serpins plasminogen activator inhibitor-1 (PAI-1) and protease nexin-1 (PN-1). We have analyzed the binding of RAP, uPA.PAI-1, and uPA.PN-1 to two naturally occurring VLDLR variants, VLDLR-I, containing all eight complement-type repeats, and VLDLR-III, lacking the third complement-type repeat, encoded by exon 4. VLDLR-III displayed approximately 4-fold lower binding of RAP than VLDLR-I and approximately 10-fold lower binding of the most C-terminal one of the three domains of RAP. In contrast, the binding of uPA.PAI-1 and uPA.PN-1 to the two VLDLR variants was indistinguishable. Surprisingly, uPA.PN-1, but not uPA.PAI-1, competed RAP binding to both VLDLR variants. These observations show that the third complement-type repeat plays a crucial role in maintaining the contact sites needed for optimal recognition of RAP, but does not affect the proteinase-serpin complex contact sites, and that two ligands can show full cross-competition without sharing the same contacts with the receptor. These results elucidate the mechanisms of molecular recognition of ligands by receptors of the low density lipoprotein receptor family.

Role of thrombin anion-binding exosite-I in the formation of thrombin-serpin complexes.

Site-directed mutagenesis was used to investigate the role of basic residues in the thrombin anion-binding exosite-I during formation of thrombin-antithrombin III (ATIII), thrombin-protease nexin 1 (PN1), and thrombin-heparin cofactor II (HCII) inhibitor complexes, in the absence and presence of glycosaminoglycans. In the absence of glycosaminoglycan, association rate constant (kon) values for the inhibition of the mutant thrombins (R35Q, K36Q, R67Q, R73Q, R75Q, R77(a)Q, K81Q, K109Q, K110Q, and K149(e)Q) by ATIII and PN1 were similar to wild-type recombinant thrombin (rIIa), whereas kon values were decreased 2-3-fold for HCII against the majority of the exosite-I mutants. The exosite-I mutants did not have a significant effect on heparin-accelerated inhibition by ATIII with maximal kon values similar to rIIa. A small effect was seen for PN1/heparin inhibition of the exosite-I mutants R35Q, R67Q, R73Q, R75Q, and R77(a)Q, where kon values were decreased 2-4-fold, compared with rIIa. For HCII/heparin, kon values for inhibition of the exosite-I mutants (except R67Q, R73Q, and K149(e)Q) were 2-3-fold lower than rIIa. Larger decreases in kon values for HCII/heparin were found for R67Q and R73Q thrombins with 441- and 14-fold decreases, respectively, whereas K149(e)Q was unchanged. For HCII/dermatan sulfate, R67Q and R73Q had kon values reduced 720- and 48-fold, respectively, whereas the remaining mutants were decreased 3-7-fold relative to rIIa. The results suggest that ATIII has no major interaction with exosite-I of thrombin with or without heparin. PN1 bound to heparin uses exosite-I to some extent, possibly by utilizing the positive electrostatic field of exosite-I to enhance orientation and thrombin complex formation. The larger effects of the thrombin exosite-I mutants for HCII inhibition with heparin and dermatan sulfate indicate its need for exosite-I, presumably through contact of the "hirudin-like" domain of HCII with exosite-I of thrombin.

Changes in the expression of protease-activated receptor 1 and protease nexin-1 mRNA during rat nervous system development and after nerve lesion.

HDs racI Thrombin causes profound metabolic and morphological changes in cultured neural cells via activation of the thrombin receptor, also called protease-activated receptor 1 (PAR1). PAR1 mRNA is present in the rat brain, but the role of this receptor in the nervous system remains elusive. The expression of PAR1 and the potent thrombin inhibitor protease nexin-1 (PN-1) was investigated in the developing rat brain and spinal cord and after peripheral nerve lesion. As seen by in situ hybridization, the PAR1 mRNA signal in the late embryonic and early postnatal nervous system was widespread, but generally of low intensity whereas in the adult it was more pronounced and confined to particular neuronal cells. These include the mesencephalic dopaminergic neurons, several thalamic and brainstem nuclei, the mitral cells in the olfactory bulb and the Purkinje cells in the cerebellum. In the spinal cord, PAR1 mRNA was abundant in motoneurons and a particularly high expression was detected in the preganglionic neurons of the autonomic nervous system. High PAR1 mRNA expression was also found in the dorsal root ganglia. Interestingly, strong immunoreactivity for the protease inhibitor PN-1 was present in spinal motoneuron cell bodies, although its transcript was undetectable there. In response to sciatic nerve transection, the signal intensity of PAR1 mRNA as seen by Northern analysis increased in the proximal and the distal part of the lesioned nerve and in the denervated muscle, whereas the PN-1 mRNA signal strongly increased only in the distal part of the nerve but remained unchanged in the proximal part and in the muscle. After facial nerve transection, PAR1 mRNA expression substantially decreased in facial motoneurons. No PAR1 transcript was detected in reactive astrocytes. Similar to PAR1, PN-1 mRNA which was expressed in interneurons within the facial nucleus was also decreased following facial nerve transection.

Astrocyte spreading in response to thrombin and lysophosphatidic acid is dependent on the Rho GTPase.

Astrocytes are typically star shaped cells playing diverse roles in the function of the nervous system. In astrocyte cultures established from the cerebral hemispheres of newborn rats, the cells have generally a polygonal fibroblast-like morphology, but acquire a stellate shape upon serum removal. When the serine protease thrombin or the bioactive lipid lysophosphatidic acid is added, the stellate cells revert to the flat morphology. Here we show that the effect of these agents is mediated via activation of the small GTP-binding protein Rho. Neither thrombin nor lysophosphatidic acid induced spreading of astrocytes microinjected with C3 transferase, an exoenzyme which ADP-ribosylates and thereby inactivates Rho. In contrast, the response of cells injected with a dominant negative form of Rac was unaffected. In addition, the injection of active Rho into stellate astrocytes mimicked the effect of thrombin and lysophosphatidic acid and an injection of C3 into flat cells grown in serum induced stellation. The conversion from a stellate to a spread morphology upon activation of Rho resulted in the formation of stress fibers and focal adhesions which most probably are key events in establishing and stabilizing the altered cytoarchitecture. These results suggest that Rho plays a crucial role in determining the shape of astrocytes and thereby may modulate their interaction with neurons in vivo.

Endogenous serine protease inhibitor modulates epileptic activity and hippocampal long-term potentiation.

Protease nexin-1 (PN-1), a member of the serpin superfamily, controls the activity of extracellular serine proteases and is expressed in the brain. Mutant mice overexpressing PN-1 in brain under the control of the Thy-1 promoter (Thy 1/PN-1) or lacking PN-1 (PN-1-/-) were found to develop epileptic activity in vivo and in vitro. Theta burst-induced long-term potentiation (LTP) and NMDA receptor-mediated synaptic transmission in the CA1 field of hippocampal slices were augmented in Thy 1/PN-1 mice and reduced in PN-1-/- mice. Compensatory changes in GABA-mediated inhibition in Thy 1/PN-1 mice suggest that altered brain PN-1 levels lead to an imbalance between excitatory and inhibitory synaptic transmission.

Protease nexin-1 is expressed at the mouse met-/mesencephalic junction and FGF signaling regulates its promoter activity in primary met-/mesencephalic cells.

The expression pattern of the serine protease inhibitor gene Protease nexin-1 (PN-1) has been analyzed by in situ hybridization during embryonic nervous system development. PN-1 was found to be specifically expressed at the junction between the mes- and metencephalon (mid- and hindbrain). Transgenic embryos expressing the bacterial lacZ gene under the control of different fragments of the PN-1 upstream regulatory region were used to demarcate an enhancer sufficient for expression at this putative segmental border. Primary cell cultures derived from the embryonic neural tube at the level of the met-/mesencephalic junction were used to demonstrate a specific effect on transcriptional activity by basic fibroblast growth factor and Engrailed transcription factors, providing evidence that PN-1 is a target gene of these factors. The results of this study place PN-1 into the signaling cascade(s) considered to be important for the development of this junction and suggest a role for PN-1 in the establishment or maintenance of the differences between these adjacent neuromeres.

Protease nexin 1 in the murine kidney: glomerular localization and up-regulation in glomerulopathies.

Protease nexin 1 (PN-1), a potent serpin-class antiprotease, is thought to be synthesized in the murine kidney. However, neither the cellular localization of PN-1 synthesis nor its role has yet been defined. To address these questions, we determined by in situ hybridizations RNase protection assay and immunoblotting, the sites of PN-1 mRNA accumulation in normal mouse kidneys and the modulation of PN-1 expression in several pathological conditions. In normal kidneys, PN-1 mRNA was detected primarily in glomeruli, most likely in mesangial cells. The glomerular expression of PN-1 was substantially enhanced not only in lupus-like glomerulonephritis (induced by IgG3 monoclonal rheumatoid factors or occurring spontaneously in lupus-prone mice), but also in mild glomerular lesions associated with intracapillary thrombi induced by IgG3 anti-trinitrophenyl monoclonal antibodies. In contrast, no modulation of PN-1 mRNA levels was observed during the course of lipopolysaccharide-induced acute tubular necrosis. A constitutive PN-1 gene expression and its up-regulation during glomerular injury suggest a possible role for PN-1 in glomerular biology. In view of its high inhibitory activity towards thrombin, mesangial PN-1 may be involved in the control of glomerular coagulation following initial glomerular injuries.

A Krox binding site regulates protease nexin-1 promoter activity in embryonic heart, cartilage and parts of the nervous system.

The rat protease nexin-1 (PN-1) promoter contains a GCGGGGGCG binding site for the transcription factors Krox-24, Krox-20 and NGFI-C. Mutations of this site abolished binding of Krox-24 in vitro. The wildtype protease nexin-1 promoter expressed beta-galactosidase similarity to the expression of protease nexin-1 mRNA. When the function of this Krox site was tested in vivo using transgenic F0 embryos, mutation had two opposite effects. beta-Galactosidase expression increased in cartilage and heart at both stages E11.5 and E13.5, but was abolished in nerves of the central and peripheral nervous system at stage E13.5. These results suggest that Krox factors are among the important transcription factors regulating protease nexin-1 expression and thereby intracellular proteolytic activity in embryonic heart, cartilage and parts of the nervous system.

The thrombin receptor is present in myoblasts and its expression is repressed upon fusion.

Cultured myoblasts derived from limb muscle of newborn rats express thrombin receptor immunoreactivity on their surface. Receptor expression is repressed upon myoblast fusion. This is due at least in part to a decrease in the amount of the thrombin receptor mRNA. Addition of thrombin triggers calcium transients only in mono- but not multinucleated muscle cells. Furthermore, thrombin increases the rate of myoblast proliferation that coincides with an activation of mitogen-activated protein kinase. Northern analysis of thrombin receptor mRNA expression in skeletal muscle showed that the transcript is present at a relatively high level at birth, but is almost undetectable in the adult. By in situ hybridization, the mRNA at birth appeared to be present mostly in mononucleated cells grouped in clusters, but not in muscle fibers. Very few nuclei surrounded by a mRNA signal were present on muscle sections of rats 24 days postnatally. These results suggest that the thrombin receptor plays a role in muscle development.

The serine protease granzyme A does not induce platelet aggregation but inhibits responses triggered by thrombin.

Granzyme A is a serine protease stored in cytoplasmic granules of cytotoxic and helper T lymphocytes. This protease seems to elicit thrombin receptor-mediated responses in neural cells, thereby triggering neurite retraction and reversal of astrocyte stellation. Here we report that granzyme A does not cause platelet aggregation even at concentrations that are more than two orders of magnitude higher than the EC50 for granzyme A in causing morphological changes in neural cells. However, granzyme A blocks thrombin-induced platelet aggregation in a dose-dependent manner without affecting the response to either ADP or to the peptide agonist of the thrombin receptor SFLLRN that corresponds in sequence to the tethered ligand domain. The inability of granzyme A to cause aggregation and its inhibition of thrombin-induced aggregation were seen in platelets from man, rat and mouse. Granzyme A does not affect the catalytic activity of thrombin in cleaving a chromogenic substrate or the macromolecular substrate fibrinogen. However, granzyme A does seem to cleave the thrombin receptor on platelets to produce a weak Ca2+ signal and reduce the response to subsequent challenge with thrombin, but does not induce a signal in thrombin-stimulated platelets. It is proposed that granzyme A interacts with the thrombin receptor found on platelets in a manner that is insufficient to cause aggregation, but sufficient to compete with thrombin for the receptor. These results suggest that granzyme A cleaves the thrombin receptor at a rate that is insufficient to cause platelet aggregation but is sufficient to cause morphological changes in neural cells. Furthermore, these observations demonstrate that granzyme A release occurring during immune responses within blood vessels would not directly cause platelet aggregation.

The thrombin receptor in the nervous system.

Neurite retraction and reversal of astrocyte stellation triggered by the serine protease thrombin are receptor-mediated events. This article summarizes the current knowledge about the cellular effects that are induced by thrombin and its receptor in neural cells. The data presented show that the thrombin receptor messenger RNA is expressed in cultured astrocytes and that the reversal of stellation caused by thrombin in these cells is prevented by the protein kinase inhibitor staurosporine. Peptides based in sequence on the tethered ligand domain of the thrombin receptor were shown to mimic the effect of thrombin in most systems investigated. Platelets of some species, however, aggregate only in response to thrombin but not to the peptides. This observation is confirmed here. Rodent receptor-activating peptides did not cause aggregation of rat or mouse platelets. In contrast, all peptides triggered reversal of stellation in rat astrocytes and neurite retraction in mouse neuroblastoma cells, supporting the proposed mechanism of cleavage-induced receptor activation in neural cells. Finally, evidence is presented that serum withdrawal causes a decrease in the amount of the thrombin receptor mRNA in different types of neuronal cells. The possible role played by the thrombin receptor in the nervous system is discussed.

Excitotoxic brain lesion modifies binding to a USF binding site acting as a negative regulatory element in the Protease nexin-1 promoter.

The expression of the serine protease inhibitor Protease nexin-1 (PN-1) is upregulated in glial cells following different types of lesion in the nervous system. A strong negative regulatory element has been shown by the missing nucleoside technique to be a CACGTG site (E-box) in the proximal part of the PN-1 promoter. The factor binding to this site is specifically recognized by antibodies directed against the human upstream stimulatory factor (USF). Point mutations in the E-box binding site which abolish USF binding in vitro increase the transcriptional activity of the PN-1 promoter. Cotransfection of a PN-1 promoter/reporter construct together with an expression vector for human USF1 confirms the negative regulatory function of this site. Finally, we show that the binding to this USF site changed after ibotenic acid-induced lesion of the caudate putamen in the rat brain.

Regulation of protease nexin-1 and angiotensin II receptor subtype 1 expression: inverse relationship in experimental models of nerve injury.

The up-regulation of PN-1 following nerve lesion has been investigated in vitro in cultures of dorsal root ganglion (DRG) explants, sciatic nerve segments, and isolated Schwann cells. In the first culture model, Schwann cells associated with neuronal processes synthesized small amounts of PN-1. Injury of the neurites emerging from the DRGs led to enhanced levels of PN-1 in Schwann cells located distal to the lesion site where degeneration of neuronal processes took place. In cultured sciatic nerve segments, PN-1 synthesis increased with a time-course comparable to that in ganglion explants following lesion. In the third model, PN-1 levels gradually rose in isolated Schwann cells during the first 3-8 days in culture. Dissociation of Schwann cells from the sciatic nerve therefore causes an effect similar to nerve damage. Impairment of Schwann cells-neuron interactions was followed by a reduction in the expression levels of the angiotensin II (Ang II) receptor subtype AT1 in all three systems studied. Since the neuropeptide Ang II is able to repress PN-1 synthesis in cultured Schwann cells, loss of neuronal contact might decrease their responsiveness to Ang II, thus resulting in PN-1 up-regulation by default.

Inactivation of protease nexin-1 by xanthine oxidase-derived free radicals.

Neuronal viability is affected by reactive oxygen species. Lipid peroxidation is often defined as a major reason for cellular breakdown. Additionally, certain indispensable proteins are possible targets for excessively formed reactive oxygen species. Evidence is given here that protease nexin-1 (PN-1), an endogenous thrombin inhibitor and neurite outgrowth promoter, is inactivated by xanthine oxidase-derived free radicals. Varying protection by superoxide dismutase and catalase was observed, depending on the reaction conditions. The water-soluble alpha-tocopherol analogues MDL 74,406 (R(+)-3,4-dihydro-6-hydroxy-N,N,N-2,5,7,8-heptamethyl-2H-1-benzopy ran-2- ethanaminium 4-methylbenzenesulfonate), MDL 74,180DA (2,3-dihydro-2,2,4,6,7-pentamethyl-3-(4-methyl-piperazino)-1-benzo furan-5-ol dihydro-chloride) and trolox also protected PN-1. Neurodegeneration may be triggered by oxidative inactivation of protease inhibitors such as PN-1. Protection of PN-1 in Alzheimer's or Parkinson's diseases, could be a possible target for a therapeutic function of antioxidants in these diseases.