SERPINE2/Protease Nexin-1 in vivo multiple functions: Does the puzzle make sense?

Cultures of glial cells and fibroblasts allowed and lead to the identification SERPINE2/Protease Nexin-1 (SERPINE2/PN-1). Cellular, biochemical, immunological and molecular characterization substantiated its variable expression in many organs as a function of development, adult stages, pathological situations or following injury. It is not a circulating serpin, but as other members of the family, its target specificity is influenced by components of the extracellular matrix. The challenges are to identify where and when SERPINE2/PN-1 modulatory action becomes crucial or even possibly specific in a mosaic of feasible in vivo impacts. Data providing correlations are not sufficient to satisfy this aim. Genetically modified mice, or tissue derived thereof, provide interesting in vivo models to identify and study the relevance of this serpin. This review will highlight sometimes-intriguing results indicating a crucial impact of SERPINE2/PN-1, especially in the vasculature, the nervous system or the behavior of cancer cells in vivo. Data presently available will be discussed in an attempt to define general trends in the diversity of SERPINE2/PN-1 modes of action in vivo.

Endogenous protease nexin-1 protects against cerebral ischemia.

The serine protease thrombin plays a role in signalling ischemic neuronal death in the brain. Paradoxically, endogenous neuroprotective mechanisms can be triggered by preconditioning with thrombin (thrombin preconditioning, TPC), leading to tolerance to cerebral ischemia. Here we studied the role of thrombin's endogenous potent inhibitor, protease nexin-1 (PN-1), in ischemia and in tolerance to cerebral ischemia induced by TPC. Cerebral ischemia was modelled in vitro in organotypic hippocampal slice cultures from rats or genetically engineered mice lacking PN-1 or with the reporter gene lacZ knocked into the PN-1 locus PN-1HAPN-1-lacZ/HAPN-1-lacZ (PN-1 KI) exposed to oxygen and glucose deprivation (OGD). We observed increased thrombin enzyme activity in culture homogenates 24 h after OGD. Lack of PN-1 increased neuronal death in the CA1, suggesting that endogenous PN-1 inhibits thrombin-induced neuronal damage after ischemia. OGD enhanced ß-galactosidase activity, reflecting PN-1 expression, at one and 24 h, most strikingly in the stratum radiatum, a glial cell layer adjacent to the CA1 layer of ischemia sensitive neurons. TPC, 24 h before OGD, additionally increased PN-1 expression 1 h after OGD, compared to OGD alone. TPC failed to induce tolerance in cultures from PN-1(-/-) mice confirming PN-1 as an important TPC target. PN-1 upregulation after TPC was blocked by the c-Jun N-terminal kinase (JNK) inhibitor, L-JNKI1, known to block TPC. This work suggests that PN-1 is an endogenous neuroprotectant in cerebral ischemia and a potential target for neuroprotection.

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 gamma-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.

Newly generated cells are increased in hippocampus of adult mice lacking a serine protease inhibitor.

BACKGROUND: Neurogenesis in the hippocampal dentate gyrus and the subventricular zone occurs throughout the life of mammals and newly generated neurons can integrate functionally into established neuronal circuits. Neurogenesis levels in the dentate gyrus are modulated by changes in the environment (enrichment, exercise), hippocampal-dependent tasks, NMDA receptor (NMDAR) activity, sonic hedgehog (SHH) and/or other factors. RESULTS: previously, we showed that Protease Nexin-1 (PN-1), a potent serine protease inhibitor, regulates the NMDAR availability and activity as well as SHH signaling. Compared with wild-type (WT), we detected a significant increase in BrdU-labeled cells in the dentate gyrus of mice lacking PN-1 (PN-1 -/-) both in controls and after running exercise. Patched homologue 1 (Ptc1) and Gli1 mRNA levels were higher and Gli3 down-regulated in mutant mice under standard conditions and to a lesser extent after running exercise. However, the number of surviving BrdU-positive cells did not differ between WT and PN-1 -/- animals. NMDAR availability was altered in the hippocampus of mutant animals after exercise. CONCLUSION: All together our results indicate that PN-1 controls progenitors proliferation through an effect on the SHH pathway and suggest an influence of the serpin on the survival of newly generated neurons through modulation of NMDAR availability.

The serine protease inhibitor protease nexin-1 controls mammary cancer metastasis through LRP-1-mediated MMP-9 expression.

Through their ability to degrade the extracellular matrix, proteases mediate cancer cell invasion and metastasis. Paradoxically, some serine protease inhibitors (serpins) are often overexpressed in human tumors. Using computational analysis, we found that the RNA level of protease nexin-1 (PN-1), a serpin that blocks numerous proteases activity, is significantly elevated in estrogen receptor-alpha-negative and in high-grade breast cancer. The in silico approach was complemented by mechanistic studies on two mammary cancer cell lines, the PN-1-negative 168FARN cells and the PN-1-positive 4T1 cells, both of which form primary mammary tumors, but only 4T1 tumors are able to metastasize to the lungs. We show that treatment of 168FARN cells with PN-1 stimulates extracellular signal-regulated kinase activation via low-density lipoprotein receptor-related protein-1 (LRP-1) binding, resulting in increased matrix metalloproteinase (MMP)-9 RNA, protein, and secreted activity. PN-1-silenced 4T1 cells express low MMP-9 levels. Moreover, injection of PN-1-silenced cells into mice did not affect 4T1 primary mammary tumor outgrowth; however, the tumors had impaired metastatic potential, which could be restored by reexpressing soluble MMP-9 in the PN-1-silenced 4T1 cells. Thus, using mammary tumor models, we describe a novel pathway whereby the serpin PN-1 by binding LRP-1 stimulates extracellular signal-regulated kinase signaling, MMP-9 expression, and metastatic spread of mammary tumors. Importantly, an analysis of 126 breast cancer patients revealed that those whose breast tumors had elevated PN-1 levels had a significantly higher probability to develop lung metastasis, but not metastasis to other sites, on relapse. These results suggest that PN-1 might become a prognostic marker in breast cancer.

SHH pathway and cerebellar development.

The morphogenetic factor Sonic hedgehog (SHH) has been discovered as one of the masterplayers in cerebellar patterning and was subjected to intensive investigation during the last decade. During early postnatal development, this continuously secreted cholesterol-modified protein drives the expansion of the largest neuronal population of the brain, the granular cells. Moreover, it acts on Bergmann glia differentiation and would potentially affect Purkinje cells homeostasis at adult age. The cerebellar cortex constituted an ideal developmental model to dissect out the upstream mechanisms and downstream targets of this complex pathway. Its deep understanding discloses some of the mechanistic disorders underlying pediatric tumorigenesis, congenital ataxia, and mental retardation. Therapeutical use of its regulators has been consolidated on murine transgenic models and is now considered as a realistic human clinical application. Here, we will review the most recent advances made in the comprehensive understanding of SHH involvement in cerebellar development and pathology.

Control of thrombin signaling through PI3K is a mechanism underlying plasticity between hair follicle dermal sheath and papilla cells.

In hair follicles, dermal papilla (DP) and dermal sheath (DS) cells exhibit striking levels of plasticity, as each can regenerate both cell types. Here, we show that thrombin induces a phosphoinositide 3-kinase (PI3K)-Akt pathway-dependent acquisition of DS-like properties by DP cells in vitro, involving increased proliferation rate, acquisition of ;myofibroblastic' contractile properties and a decreased capacity to sustain growth and survival of keratinocytes. The thrombin inhibitor protease nexin 1 [PN-1, also known as SERPINE2) regulates all those effects in vitro. Accordingly, the PI3K-Akt pathway is constitutively activated and expression of myofibroblastic marker smooth-muscle actin is enhanced in vivo in hair follicle dermal cells from PN-1(-/-) mice. Furthermore, physiological PN-1 disappearance and upregulation of the thrombin receptor PAR-1 (also known as F2R) during follicular regression in wild-type mice also correlate with such changes in DP cell characteristics. Our results indicate that control of thrombin signaling interferes with hair follicle dermal cells plasticity to regulate their function.

Protease nexin 1 and its receptor LRP modulate SHH signalling during cerebellar development.

Development of the postnatal cerebellum relies on the tight regulation of cell number by morphogens that control the balance between cell proliferation, survival and differentiation. Here, we analyze the role of the serine-protease inhibitor protease nexin 1 (PN-1; SERPINE2) in the proliferation and differentiation of cerebellar granular neuron precursors (CGNPs) via the modulation of their main mitogenic factor, sonic hedgehog (SHH). Our studies show that PN-1 interacts with low-density lipoprotein receptor-related proteins (LRPs) to antagonize SHH-induced CGNP proliferation and that it inhibits the activity of the SHH transcriptional target GLI1. The binding of PN-1 to LRPs interferes with SHH-induced cyclin D1 expression. CGNPs isolated from Pn-1-deficient mice exhibit enhanced basal proliferation rates due to overactivation of the SHH pathway and show higher sensitivity to exogenous SHH. In vivo, the Pn-1 deficiency alters the expression of SHH target genes. In addition, the onset of CGNP differentiation is delayed, which results in an enlarged outer external granular layer. Furthermore, the Pn-1 deficiency leads to an overproduction of CGNPs and to enlargement of the internal granular layer in a subset of cerebellar lobes during late development and adulthood. We propose that PN-1 contributes to shaping the cerebellum by promoting cell cycle exit.

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 (t(1/2) 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.

Reciprocal regulation of urokinase receptor (CD87)-mediated cell adhesion by plasminogen activator inhibitor-1 and protease nexin-1.

Protease nexin-1 (PN-1) and plasminogen activator inhibitor-1 (PAI-1) are serine protease inhibitors that bind to the extracellular matrix protein vitronectin (VN) with high affinity. PAI-1 is known to inhibit cell adhesion and migration by binding to VN and inhibiting the interaction with integrins or the urokinase receptor (uPAR). Unexpectedly, PN-1 was found to increase the association between VN and uPAR in the presence of enzymatically active uPA. Through this mechanism PN-1 also stimulated uPAR-dependent cell adhesion to immobilized VN. In contrast to PAI-1, PN-1 did not influence VN binding to integrins or integrin-mediated cell adhesion. Upon adhesion of monocytes to VN there was an accumulation of uPAR and PN-1 at the interface between the cell and the matrix, whereas on fibronectin (FN) both components were distributed evenly over the whole cell as visualized by confocal microscopy. Immunohistochemistry of atherosclerotic vessels indicated that PN-1 was found associated with smooth muscle cells, macrophages and platelets. In some regions of the diseased vessels PN-1 was in close proximity to VN and uPAR, but no PN-1 was present in normal vessels. These results indicate a novel function of PN-1 linked to complex formation with uPA that leads to the regulation of VN-dependent adhesion of leukocytes.

The role of serine proteases and serine protease inhibitors in the migration of gonadotropin-releasing hormone neurons.

BACKGROUND: Mechanisms regulating neuronal migration during development remain largely undefined. Extracellular matrix cues, target site released factors, and components of the migratory neurons themselves are likely all coordinated in time and space directing neurons to their appropriate locations. We have studied the effects of proteases and their inhibitors on the extracellular matrix and the consequences to the migration of gonadotropin releasing hormone (GnRH) neurons in the embryonic chick. Chick GnRH neurons differentiate in the olfactory epithelium, migrate along the olfactory nerve and enter the forebrain. The accessibility of this coherent cell group make it amenable for studying protease/inhibitor roles in migratory processes. RESULTS: Affigel blue beads were used to deliver a serine protease inhibitor, protease nexin-1 (PN-1), and a target protease, trypsin, to the olfactory epithelium coincident with initiation of GnRH neuronal migration. PN-1 inhibited neuronal migration while trypsin accelerated their transit into the CNS. Prior to initiation of migration, neither PN-1 nor trypsin altered the timing of neuronal exit. Trypsin did, however, accelerate the timing of neuronal crossing into the nerve-forebrain junction. CONCLUSIONS: These data support the hypothesis that protease activity modulates neuronal movements across barriers. Moreover, the data suggest, for the first time, that aspects of GnRH neuronal migration may be cell autonomous but modulated by ECM alterations.