Expression Of Protease Nexin-1 Research Articles

Cancer-associated Fibroblasts (CAFs) Regulate Lung Cancer Malignant Progression by Transferring SERPINE2 (PN1) Via Exosomes.

Cancer-associated fibroblasts (CAFs), one of the most abundant stromal cell types in tumor microenvironment (TME), have been a potential target for cancer treatment such as lung cancer. However, the underlying mechanism by which CAFs promote lung cancer progression remains elusive. We obtained primary CAFs, normal fibroblasts (NFs) and their exosomes, constructed protease nexin-1 (PN1) stably silenced or over-expressed CAFs cells using lentivirus. Bioinformatics was used to obtain the expression of PN1 in lung cancer and normal tissues, the relationship with overall survival, and the enriched pathways. The MTT assays and Transwell assays were performed to detect the proliferation, migration, and invasion abilities of lung cancer cells after treatments. Western blotting, qRT-PCR, immunohistochemistry, and xenograft models were used to illustrate how CAFs functions in lung cancer progression via exosomes. CAFs-derived exosomes, in which PN1 was higher expressed compared with NFs-derived ones, promoted effectively the proliferation, migration, and invasion of lung cancer cells A549 and H1975. Meanwhile, the expression of PN1 expressed higher in lung cancer tissues compared with normal ones, and was negatively associated with the overall survival rate of lung cancer patients. More importantly, over-expressing or silencing PN1 in A549 and H1975 could also promote or inhibit cell proliferation, migration, and invasion correspondingly. Furthermore, treated with PN1 over-expressed CAFs-derived exosomes, the lung cancer cells proliferation, migration, and invasion varied positively, and accompanied by activation of Toll-like and NF-κB signaling pathways. However, this phenomenon can be reversed by AN-3485, an antagonist of Toll-like pathway. Finally, over-expressing PN1 leads to an accelerated tumor growth by increasing the expression of proliferation biomarker Ki67 and activation of NF-κB signaling pathway in vivo. CAFs promoted lung cancer progression by transferring PN1 and activating Toll-like/NF-κB signaling pathway via exosomes.

Vascular Smooth Muscle Cells Release Protease Nexin-1 in Cell Models of Atherosclerosis and Human Carotid Tissue

Vascular smooth muscle cells (VSMCs) express protease nexin-1 (PN-1) in atherosclerotic lesions, but factors responsible for its release and its role in atherosclerosis remain unclear. PN-1 has been shown to induce collagen expression, a feature of plaque tissue and the synthetic VSMC phenotype. HDAC9 affects VSMC cellular phenotypic stability and has been implicated in large vessel ischemic stroke. We hypothesize that HDAC9 may influence PN-1 expression and be a target in atherosclerosis and stroke. Human primary VSMCs and control, asymptomatic and symptomatic (stroke or transient ischemic attack <7 days) carotid tissue from patients were examined. VSMCs were treated with cholesterol and oxidized phospholipids to induce the synthetic phenotype. Functional assays, qPCR, proteomic analysis and transwell co-culture with THP-1 monocytes, were used to assess phenotype characteristics and quantify protein and gene expression. Human VSMCs in atherogenic media have increased expression of PN-1, macrophage markers (LGALS3, CD68), and decreased expression of contractile genes (ACTA2). There was more PN-1 released from VSMCs grown in atherogenic media compared to control media. Monocytes exposed to atherogenic supernatant had higher rates of migration compared to control. Inhibition of HDAC9 by siRNA transfection decreased expression of PN-1, macrophage markers and restored VSMC contractile gene expression. Observations were replicated in human tissues. VSMCs from symptomatic plaque had increased expression of HDAC9, PN-1, macrophage markers and increased histone methylation, consistent with decreased contractile gene expression, compared to control and asymptomatic carotid tissues. HDAC9 and PN-1 have a previously unknown direct correlation, which is also related to VSMC synthetic phenotype. The mechanisms of this relationship and the role of PN-1 on plaque progression remains to be elucidated.

SERPINE2 (Protease Nexin I) Is a Feedback Regulator of EGF/PKC/MAPK/EGR1 Signaling in Breast Cancer Cells Metastasis and Stemness

Background: Protease nexin-1 (PN-1) contributes to enhanced metastasis of cancer cells. In this study, we showed PN-1 levels were remarkably upregulated in breast cancer cells and tissues, we aimed to uncover an unidentified mechanism underlying the up-regulation of PN-1 in breast cancers. Methods: The up-regulation of PN-1 in MCF-7 spheroid cells and breast cancer tissues was detected by RNA sequencing and qRT-PCR. The functions of PN-1 on breast cancer cells were investigated. The signaling pathway involved in epidermal growth factor (EGF) up-regulated PN-1 in breast cancer cells was identified by specific inhibitors or siRNAs of the candidate downstream genes. The feedback regulation of EGF/MAPK/EGR1 signaling by PN-1 was analyzed by Enzyme-linked immunosorbent assay (ELISA) and western blotting. Findings: The up-regulation of PN-1 in breast cancer mainly promoted cell migration, invasion and stemness. We uncovered that EGF induced the expression of PN-1 via its transcriptional factor, early growth response protein 1 (EGR1), through cascade activation of epidermal growth factor receptor (EGFR) to the activation of protein kinase Cδ (PKCδ), mitogenactivated protein kinase (MEK) and extracellular signal-related kinase (ERK). We further demonstrated EGF-induced PN-1 up-regulation could promote breast tumor cell metastasis in the mouse model for human breast cancer lung and liver metastasis. We also proved there was a positive feedback towards the activation of EGF signals by overexpression of PN-1. Interpretation: Our findings reveal a novel signaling axis that up-regulated PN-1 expression in breast cancer cells, which may provide new insights into identifying novel therapeutic targets for breast cancer. Funding Statement: This project is funded by National Natural Science Foundation of China (Grant No. 81570696, No. 81702941). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: All the patients provided written consent, and the experiments were approved by the Ethics Committee of China Pharmaceutical University (Nanjing, China).

The expression and role of protease nexin-1 in intervertebral disc cells nucleus pulposus

Objective To investigate the expression of Protease nexin-1 (PN-1) in Intervertebral Disc nucleus pulposus (NP) cells and the related signal pathways. Methods Human primary NP cells were isolated and cultured, and divided into control group, interleukine-1β (IL-1β), TNF-α group. Histological examinations were undertaken to observe the morphologic changes of NP cells during the process of differentiation at day 3, 7, 14 and 21. MMPs expression in NP cells were accessed by Western blot. Then, NP cells were cultured in medium containing 10 ng/ml IL-1β and different concenration of PN-1 (0, 10, 50, 100 ng/ml) for 24h. Protein were extracted, and The expression level of Interleukin-1 receptor-associated kinase 1 (IRAK1) , p38 mitogen-activated protein kinase (p38 MAP Ki-nase), phosphos-P38 MAP Kinase (P-P38) wereanalyzed by using Western blot. Results Histologically, under induction with IL-1β, both morphological and biological properties of NP cells were changed, cell proliferation became slowly, cell density was sparse within disordered arrangement, and obvious cell necrosis appeared. The results also showed the degenerative process of NP cells, MMPs production increased and Agg and Col II protein decreased significantly. PN-1 administration could inhibit the activation of MMP3,MMP9 and MMP13 induced by IL-1β, and enhance the expression of AGG and Col2. IL-1β administration could stimulate Nuclear factor κB (NF-κB) signal pathway through the up-regulated expression of IRAK1 and P-P38, while PN-1 could inhibit the inductive effects by suppress the expression of IRAK1 and the phosphorylation of P-38, thereby, inhibit the degeneration of NP cells. Conclusion PN-1 is involved during the degeneration of NP cells. PN-1 administration could antagonize IL-1β-induced proteases and inhibit the activation of MMPs, and potentially preserve NP cells, and reverse its degeneration via IRAK1/NF-κB signaling pathway. Key words: Intervertebral disc degeneration; Interleukin-1 Receptor-Associated Kinases; NF-kappa B

Uptake of Plasmin-PN-1 Complexes in Early Human Atheroma.

Zymogens are delivered to the arterial wall by radial transmural convection. Plasminogen can be activated within the arterial wall to produce plasmin, which is involved in evolution of the atherosclerotic plaque. Vascular smooth muscle cells (vSMCs) protect the vessels from proteolytic injury due to atherosclerosis development by highly expressing endocytic LDL receptor-related protein-1 (LRP-1), and by producing anti-proteases, such as Protease Nexin-1 (PN-1). PN-1 is able to form covalent complexes with plasmin. We hypothesized that plasmin-PN-1 complexes could be internalized via LRP-1 by vSMCs during the early stages of human atheroma. LRP-1 is also responsible for the capture of aggregated LDL in human atheroma. Plasmin activity and immunohistochemical analyses of early human atheroma showed that the plasminergic system is activated within the arterial wall, where intimal foam cells, including vSMCs and platelets, are the major sites of PN-1 accumulation. Both PN-1 and LRP-1 are overexpressed in early atheroma at both messenger and protein levels. Cell biology studies demonstrated an increased expression of PN-1 and tissue plasminogen activator by vSMCs in response to LDL. Plasmin-PN-1 complexes are internalized via LRP-1 in vSMCs, whereas plasmin alone is not. Tissue PN-1 interacts with plasmin in early human atheroma via two complementary mechanisms: plasmin inhibition and tissue uptake of plasmin-PN-1 complexes via LRP-1 in vSMCs. Despite this potential protective effect, plasminogen activation by vSMCs remains abnormally elevated in the intima in early stages of human atheroma.

Protease nexin-1 regulates retinal vascular development.

We recently identified protease nexin-1 (PN-1) or serpinE2, as a possibly underestimated player in maintaining angiogenic balance. Here, we used the well-characterized postnatal vascular development of newborn mouse retina to further investigate the role and the mechanism of action of PN-1 in physiological angiogenesis. The development of retinal vasculature was analysed by endothelial cell staining with isolectin B4. PN-1-deficient (PN-1(-/-)) retina displayed increased vascularization in the postnatal period, with elevated capillary thickness and density, compared to their wild-type littermate (WT). Moreover, PN-1(-/-) retina presented more veins/arteries than WT retina. The kinetics of retinal vasculature development, retinal VEGF expression and overall retinal structure were similar in WT and PN-1(-/-) mice, but we observed a hyperproliferation of vascular cells in PN-1(-/-) retina. Expression of PN-1 was analysed by immunoblotting and X-Gal staining of retinas from mice expressing beta-galactosidase under a PN-1 promoter. PN-1 was highly expressed in the first week following birth and then progressively decreased to a low level in adult retina where it localized on the retinal arteries. PCR arrays performed on mouse retinal RNA identified two angiogenesis-related factors, midkine and Smad5, that were overexpressed in PN-1(-/-) newborn mice and this was confirmed by RT-PCR. Both the higher vascularization and the overexpression of midkine and Smad5 mRNA were also observed in gastrocnemius muscle of PN-1(-/-) mice, suggesting that PN-1 interferes with these pathways. Together, our results demonstrate that PN-1 strongly limits physiological angiogenesis and suggest that modulation of PN-1 expression could represent a new way to regulate angiogenesis.

0262 : Involvement of protease nexin-1 in the clearance of serine proteases in human thoracic aneurysms of ascending aorta

Tissue activation of proteolysis is involved in progressive dilation and dissection of human thoracic aneurysms of ascending aorta (TAA). Vascular smooth muscle cells (VSMC) limit possible proteolytic wall injury by producing antiproteases, among which is protease nexin-1 (PN-1). PN-1 is a tissue serpin expressed by VSMC, able to inhibit plasmin activities by forming complexes. The main objective was to test whether human VSMC can clear plasmin/PN-1 complex through an LRP1-dependent mechanism. We have explored the expression of PN-1, t-PA and LRP-1 in human tissue and cultured VSMC of TAA compared with healthy aorta (control). PN-1, t-PA and LRP-1 mRNA were overexpressed in medial extracts of TAA compared with control. Both PN-1 and LRP-1 protein were overexpressed in cultured VSMC from TAA as compared to control whereas for t-PA there was no difference in protein expression between TAA and control. We have studied the localisation of PN-1/plasmin and LRP-1 in TAA. Intracellular presence of plasmin, PN-1 and LRP-1 in VSMC in human TAA was detected. We used cultured VSMC to study their abilitiy to endocytose PN-1 or PN-1/ plasmin complexes. VSMC endocytose PN-1 and PN-1/plasmin complexes but not plasmin. Our results suggest that PN-1, t-PA and LRP-1 are increased in order to protect the arterial wall from protease injury in TAA.

Protease nexin 1 induces apoptosis of prostate tumor cells through inhibition of X-chromosome-linked inhibitor of apoptosis protein.

Protease nexin 1 (PN1) is an endogenous serine protease inhibitor (SERPIN), expressed at high levels in the prostate, and capable of inhibiting the proliferation of prostate cancer cells. We previously showed that PN1-uPA complexes inhibited Sonic Hedgehog (SHH) signalling through engagement of the LRP receptor. Here, we describe an alternative anti-proliferative mechanism through which PN1 expression leads to apoptosis. In prostate cancer cells, increased expression of PN1 led to substantial reduction of XIAP levels and apoptosis mediated through the uPAR, but not the LRP receptor. The alterations in XIAP were effected in two ways 1) via alteration in the NF-κB pathway, a pathway known to signal XIAP transcription and 2) by promoting XIAP instability. The AKT pathway is known to phosphorylate XIAP at serine 87 leading to protein stability and PN1 expression is shown to interfere with this process. As a result of both mechanisms, programmed cell death is substantially increased. Consistent with these observations, reduced PN1 protein correlated with elevated p65/XIAP expression and with higher Gleason scores in human prostate tissue arrays. Thus, PN1 expression appears to differentially down-regulate distinct oncogenic pathways depending upon the cell surface receptor engaged by its complexes and demonstrates a novel molecular mechanism by which the protein can promote tumor cell apoptosis.

Increased expression of protease nexin-1 in fibroblasts during idiopathic pulmonary fibrosis regulates thrombin activity and fibronectin expression

Idiopathic pulmonary fibrosis (IPF) is a chronic diffuse lung disease characterized by an accumulation of excess fibrous material in the lung. Protease nexin-1 (PN-1) is a tissue serpin produced by many cell types, including lung fibroblasts. PN-1 is capable of regulating proteases of both coagulation and fibrinolysis systems, by inhibiting, respectively, thrombin and plasminergic enzymes. PN-1 is thus a good candidate for regulating tissue remodeling occurring during IPF. We demonstrated a significant increase of PN-1 expression in lung tissue extracts, lung fibroblasts and bronchoalveolar lavage fluids of patients with IPF. The increase of PN-1 expression was reproduced after stimulation of control lung fibroblasts by transforming growth factor-β, a major pro-fibrotic cytokine involved in IPF. Another serpin, plasminogen activator inhibitor-1 (PAI-1) is also overexpressed in fibrotic fibroblasts. Unlike PAI-1, cell-bound PN-1 as well as secreted PN-1 from IPF and stimulated fibroblasts were shown to inhibit efficiently thrombin activity, indicating that both serpins should exhibit complementary roles in IPF pathogenesis, via their different preferential antiprotease activities. Moreover, we observed that overexpression of PN-1 induced by transfection of control fibroblasts led to increased fibronectin expression, whereas PN-1 silencing induced in fibrotic fibroblasts led to decreased fibronectin expression. Overexpression of PN-1 lacking either its antiprotease activity or its binding capacity to glycosaminoglycans had no effect on fibronectin expression. These novel findings suggest that modulation of PN-1 expression in lung fibroblasts may also have a role in the development of IPF by directly influencing the expression of extracellular matrix proteins. Our data provide new insights into the role of PN-1 in the poorly understood pathological processes involved in IPF and could therefore give rise to new therapeutic approaches.

Smad2-Dependent Protease Nexin-1 Overexpression Differentiates Chronic Aneurysms From Acute Dissections of Human Ascending Aorta

Tissue activation of proteolysis is involved in acute intramural rupture (dissections, acute ascending aortic dissection) and in progressive dilation (aneurysms, thoracic aneurysm of the ascending aorta) of human ascending aorta. The translational aim of this study was to characterize the regulation of antiproteolytic serpin expression in normal, aneurysmal, and dissecting aorta. We explored expression of protease nexin-1 (PN-1) and plasminogen activator inhibitor-1 and their regulation by the Smad2 signaling pathway in human tissue and cultured vascular smooth muscle cells (VSMCs) of aneurysms (thoracic aneurysm of the ascending aorta; n=46) and acute dissections (acute ascending aortic dissection; n=10) of the ascending aorta compared with healthy aortas (n=10). Both PN-1 and plasminogen activator inhibitor-1 mRNA and proteins were overexpressed in medial tissue extracts and primary VSMC cultures from thoracic aneurysm of the ascending aorta compared with acute ascending aortic dissection and controls. Transforming growth factor-β induced increased PN-1 expression in control but not in aneurysmal VSMCs. PN-1 and plasminogen activator inhibitor-1 overexpression by aneurysmal VSMCs was associated with increased Smad2 binding on their promoters and, functionally, resulted in VSMC self-protection from plasmin-induced detachment and death. This phenomenon was restricted to aneurysms and not observed in acute dissections. These results demonstrate that epigenetically regulated PN-1 overexpression promotes development of an antiproteolytic VSMC phenotype and might favor progressive aneurysmal dilation, whereas absence of this counter-regulation in dissections would lead to acute wall rupture.

Regulation of renal sodium handling through the interaction between serine proteases and serine protease inhibitors

Sodium balance, extracellular fluid volume, and ultimately blood pressure are maintained by precise regulation of the activity of epithelial sodium channels (ENaC). Multiple mechanisms such as hormones, intracellular factors, and other regulatory factors contribute to regulation of ENaC activity. Prostasin, a glycosylphosphatidylinositol-anchored serine protease, has been identified as an activator of ENaC that increases its open probability. Furin cleaves αENaC at two sites and γENaC at one site at the Golgi. Prostasin cleaves γENaC at one site that is distinct from the furin site at the plasma membrane. Dual cleavage of α- and γ-subunit releases inhibitory segments from ENaC, leading to channel activation. Protease nexin-1 (PN-1), an endogenous prostasin inhibitor, inhibits ENaC activity through suppression of prostasin activity. Aldosterone and transforming growth factor-β1 reciprocally regulate expression of prostasin, PN-1, and ENaC in renal epithelial cell, resulting in sodium retention or natriuresis, respectively. These findings strongly suggest the possibility that coordinated regulation of serine protease, serpin, and ENaC expression plays a key role in sodium handling in the kidney.

Protease Nexin-1, tPA, and PAI-1 are Upregulated in Cryoglobulinemic Membranoproliferative Glomerulonephritis

Thymic stromal lymphopoietin (TSLP) transgenic mice develop cryoglobulin-associated membranoproliferative glomerulonephritis, characterized by renal monocyte/macrophage infiltration, marked expansion of extracellular matrix, and variable intraluminal and mesangial deposits of cryoglobulins. A microarray approach was used to study global gene expression in glomerular RNA obtained from these mice, as well as from combined TSLP transgenic and Fcγ receptor IIb null mice (TSLP/FcIIb−/−), which develop aggravated membranoproliferative glomerulonephritis. Protease nexin-1 (PN-1) and tissue plasminogen activator (tPA), two potential regulators of fibrosis that are involved in the fibrinolytic and coagulation pathways, were dramatically upregulated in TSLP mice compared with wild-type controls. In situ hybridization revealed minimal expression of PN-1 mRNA in the glomeruli of wild-type mice, increased expression in TSLP mice, and the greatest expression in the mesangial cells of TSLP/FcIIb−/− mice. Immunohistochemistry demonstrated greater expression of PN-1, tPA, and PAI-1 in the mesangial cells of TSLP mice compared with wild-type and the greatest in TSLP/FcIIb−/− mice. In cultured mesangial cells, incubation with cryoglobulins induced an upregulation of PN-1 mRNA; increased expression of PN-1, tPA, and PAI-1 proteins; and stimulated secretion of TGF-β1. It is concluded that PN-1, tPA, PAI-1, and TGF-β1 are likely important mediators of murine cryoglobulinemic glomerulonephritis and that the cryoglobulins may directly upregulate their expression.

Inhibition of prostasin-induced ENaC activities by PN-1 and regulation of PN-1 expression by TGF-β1 and aldosterone

Prostasin has been shown to regulate sodium handling in the kidney. Recently, a serine protease inhibitor, protease nexin-1 (PN-1), was identified as an endogenous inhibitor for prostasin. Therefore, we hypothesized that PN-1 may regulate sodium reabsorption by reducing prostasin activity, and that expression of PN-1 was regulated by transforming growth factor-beta1 (TGF-beta1) or aldosterone, like prostasin. cRNAs for epithelial sodium channel (ENaC), prostasin, and PN-1 were expressed in Xenopus oocytes, and the amiloride-sensitive sodium currents (I(Na)) were measured. The effect of TGF-beta1 and aldosterone on the mRNA and protein abundance of PN-1 and ENaC was detected by real-time polymerase chain reaction and immunoblotting in M-1 cells. Expression of PN-1 substantially decreased prostasin-induced I(Na) by approximately 68% in oocytes. Treatment of M-1 cells with 20 ng/ml TGF-beta1 significantly increased protein expression of PN-1 by 3.8+/-0.5-fold, whereas administration of 10(-6) M aldosterone markedly decreased protein expression of PN-1 to 53.7+/-6.7%. Basolateral, but not apical, application of TGF-beta1 significantly reduced I(eq). To elucidate the involvement of PN-1 in basal ENaC activity, we silenced the expression of PN-1 by using short-interfering RNA. This increased I(eq) by 1.6+/-0.1-fold. Our study indicates that PN-1 could have a natriuretic role by inhibiting prostasin activity and suggests the possibility that aldosterone and TGF-beta reciprocally regulate the expression of PN-1 in renal epithelial cells contributing to salt retention or natriuresis, respectively by an additional mechanism. PN-1 could represent a new factor that contributes to regulation of ENaC activity in the kidney.

Expression of protease nexin-1 and plasminogen activators during follicular growth and the periovulatory period in cattle

Extracellular matrix remodeling occurs during ovarian follicular development, mediated by plasminogen activators (PAs) and PA inhibitors including protease nexin-1 (PN-1). In the present study we measured expression/activity of the PA system in bovine follicles at different stages of development by timed collection of ovaries during the first follicular wave and during the periovulatory period, and in follicles collected from an abattoir. The abundance of mRNA encoding PN-1, tissue-type PA (tPA), urokinase (uPA) and PA inhibitor-1 (PAI-1) were initially upregulated by human chorionic gonadotropin (hCG) in bovine preovulatory follicular wall homogenates. PN-1, PAI-1 and tPA mRNA expression then decreased near the expected time of ovulation, whereas uPA mRNA levels remained high. PN-1 concentration in follicular fluid (FF) decreased and reached the lowest level at the time of ovulation, whereas plasmin activity in FF increased significantly after hCG. Follicles collected from the abattoir were classified as non-atretic, early-atretic or atretic based on FF estradiol and progesterone content: PN-1 protein levels in FF were significantly higher in non-atretic than in atretic follicles, and plasmin activity was correspondingly higher in the atretic follicles. No changes in PN-1 levels in FF were observed during the growth of pre-deviation follicles early in a follicular wave. These results indicate that PN-1 may be involved in the process of atresia in non-ovulatory dominant follicles and the prevention of precocious proteolysis in periovulatory follicles.

Brain Endothelial Hemostasis Regulation by Pericytes

Pericytes are known to regulate brain capillary endothelial functions. The purpose of this study was to define the hemostatic regulatory role of human brain pericytes. We used blood-brain barrier models consisting of human pericytes grown on transwell membrane inserts and cocultured with human brain microvascular endothelial cells (HBEC), or pericytes grown in direct contact with HBEC. When grown in cocultures in which pericytes were physically separated from endothelial cells, pericytes induced significant changes in endothelial tissue plasminogen activator (tPA) messenger ribonucleic acid (mRNA) and protein: tPA mRNA level was decreased in pericyte cocultures (52%+/-25% of monocultures, P < 0.05) and tPA protein level was decreased (66%+/-23% of monocultures, P < 0.05). Pericyte effects on endothelial fibrinolysis were enhanced when the two cell types were cocultured in direct contact, with tPA protein reduced in cocultures compared with monocultures (25%+/-15% of monocultures, P < 0.05). Endotoxin (lipopolysaccharide (LPS)), used as a standardized stimulus to define brain-specific inflammation-induced change, amplified pericyte-induced enhanced release of the tPA inhibitor plasminogen activator inhibitor-1 (PAI-1); the latter was released by endothelial cells first cocultured with pericytes and then incubated with LPS in the absence of pericytes. Pericytes (in contrast to endothelial cells and astrocytes) were found to be the principal in vitro source of the serpin protease nexin-1 (PN-1), known to have primarily antithrombin effects. These in vitro findings suggest that pericytes negatively regulate brain endothelial cell fibrinolysis, while pericyte expression of PN-1 may provide endogenous anticoagulant activity.

Regulation of Brain Proteolytic Activity Is Necessary for theIn VivoFunction of NMDA Receptors

Serine proteases are considered to be involved in plasticity-related events in the nervous system, but their in vivo targets and the importance of their control by endogenous inhibitors are still not clarified. Here, we demonstrate the crucial role of a potent serine protease inhibitor, protease nexin-1 (PN-1), in the regulation of activity-dependent brain proteolytic activity and the functioning of sensory pathways. Neuronal activity regulates the expression of PN-1, which in turn controls brain proteolytic activity. In PN-1-/- mice, absence of PN-1 leads to increased brain proteolytic activity, which is correlated with an activity-dependent decrease in the NR1 subunit of the NMDA receptor. Correspondingly, reduced NMDA receptor signaling is detected in their barrel cortex. This is coupled to decreased sensory evoked potentials in the barrel cortex and impaired whisker-dependent sensory motor function. Thus, a tight control of serine protease activity is critical for the in vivo function of the NMDA receptors and the proper function of sensory pathways.

Protease nexin-1 inhibits plasminogen activation-induced apoptosis of adherent cells.

Degradation of adhesive glycoproteins by plasmin is implicated in cell migration. In this study, we further explored the role of plasminogen activation in cell adhesion and survival and show that uncontrolled plasminogen activation at the cell surface may induce cell detachment and apoptosis. We hypothesized that this process could be prevented in adherent cells by expression of protease nexin-1, a potent serpin able to inhibit thrombin, plasmin, and plasminogen activators. Using two- and three-dimensional culture systems, we demonstrate that Chinese hamster ovary fibroblasts constitutively express tissue-type plasminogen activator and efficiently activate exogenously added plasminogen in a specific and saturable manner (K(m) = 46 nm). The formation of plasmin results in proteolysis of fibronectin and laminin, which is followed by cell detachment and apoptosis. Protease nexin-1 expressed by transfected cells significantly inhibited the activity of plasmin and tissue-type plasminogen activator via the formation of inhibitory complexes and prevented cell detachment and apoptosis. In conclusion, protease nexin-1 may be an important anti-apoptotic factor for adherent cells. This cell model could be a useful tool to evaluate therapeutic agents such as serpins in vascular pathologies involving pericellular protease-protease inhibitor imbalance.

The serpin protease-nexin 1 is present in rat aortic smooth muscle cells and is upregulated in L-NAME hypertensive rats.

Protease-nexin 1 (PN-1) belongs to the serpin superfamily and behaves as a specific thrombin inhibitor in the pericellular environment. Little is known about PN-1 expression and its regulation in the vascular system. In this study, we examined the expression of functionally active PN-1 in vitro in rat aortic smooth muscle cells and in vivo in rat arterial media and its regulation in hypertensive rats. The vascular PN-1 formed specific covalent complexes with thrombin involving the catalytic site of the protease, and heparin increased the formation of these complexes. We also demonstrated PN-1 in rat arterial media by immunohistochemical staining. Moreover, we examined in vivo vascular expression of PN-1 in a model of chronic hypertension induced by long-term administration of N(G)-nitro-L-arginine methyl ester (L-NAME). Marked increases in PN-1 mRNA (3-fold) and protein (2-fold) were observed after 2 months of hypertension. Increased expression of PN-1 in the vascular wall was associated with an increase in the formation of complexes between radiolabeled-thrombin and PN-1, indicating that PN-1 was functional. PN-1 may thus participate in the mechanisms that regulate thrombin activity in the vessel wall.

Increase of prothrombin-mRNA after global cerebral ischemia in rats, with constant expression of protease nexin-1 and protease-activated receptors

Prothrombin, protease-activated receptors (PARs) and the specific thrombin inhibitor protease nexin-1 (PN-1) are expressed in the brain. Recent studies have shown that the serine protease thrombin, depending on its concentration, plays an important role in neuronal degeneration or protection after cerebral ischemia. However, it is still uncertain whether a change in prothrombin or alterations in the expression of specific PAR-subtypes or PN-1 are associated with postischemic thrombin effects. Using semi-quantitative reverse transcription-polymerase chain reaction analysis, we show that prothrombin was up-regulated in the hippocampal formation 24 h after transient global ischemia in rats (two-vessel occlusion with hypotension), whereas the expression of PN-1 and the expression of PAR-subtypes 1–3 did not change significantly. Thus, control of the balance between the expression of prothrombin and PN-1 may reflect an important mechanism that underlies postischemic thrombin effects.

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.