Protease Nexin-1 Expression Research Articles

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.

Effects of Actr2 gene Variants on Mouse Viability, Gene Expression, and Cytoskeletal Dynamics

Venous thromboembolism (VTE) is a prevalent human disease that exhibits significant heritability. Factor V Leiden (F5L) is the most common known VTE risk factor, but modifier genes significantly influence VTE development. We recently identified a p.R258G missense mutation in the Actr2 gene (Actr2+/G, ARP2 protein) as a genetic suppressor of lethal thrombosis in mice homozygous for F5L (F5L/L) and hemizygous for tissue factor pathway inhibitor (Tfpi+/-). However, the antithrombotic mechanism of Actr2+/G is unknown. We used genetic and genomic techniques to investigate the phenotypic effects of the Actr2G variant on cells and mice. We previously analyzed progeny from Actr2+/G x Actr2+/G (N7 backcross generations to C57BL6/J) to investigate the effects of Actr2G on mouse survival. The progeny deviated from Mendelian frequencies, as only ~12% were Actr2G/G mice (N=303; p&lt;9x10-7) and these mice have a significantly reduced lifespan of ~six months (N=131; p&lt;0.0001). The Actr2+/G mice, when compared to wildtype mice, were morphologically indistinguishable and were born at the anticipated frequency. In the context of F5L, a F5L/LActr2+/G x F5L/LActr2+/G cross yielded only 3 F5L/LActr2G/G, which also significantly departed from expected frequencies (N=66; p&lt;0.01). The F5L/LActr2+/G mice were born at the anticipated frequency. To determine if the mouse embryos were produced at normal frequencies, we examined pups at embryonic day 14.5 of this F5L/LActr2+/G x F5L/LActr2+/G cross. We observed normal frequencies at embryonic day 14.5, with 5 F5L/LActr2G/G embryos out of 18 total. Taken together, our results suggest that Actr2G exhibits gene dosage lethality, with Actr2G/G homozygosity detrimental for late stage embryonic development/early adulthood. We next studied the mechanism(s) of Actr2G thrombosis suppression by gene expression studies. RT-qPCR for multiple coagulation genes was performed in liver samples from Actr2+/+ and Actr2+/G mice. Serpine2 mRNA (Protease Nexin-1 (PN-1)) was significantly increased in Actr2+/G mice (N=3; p&lt;0.0001). We also observed an increase in plasma PN-1 by quantitative Western Blot analysis. To investigate the Actr2G regulation of PN-1 expression, mouse N2a cells containing Actr2+/G or V5-tagged PN-1 were generated using CRISPR/Cas9. Genomic analysis revealed homology directed repair (HDR)-mediated knock-in frequencies of 24.3% (9/37) for Actr2+/G and 5.6% (1/18) for PN-1-V5. We then attempted to generate PN-1-V5 cells carrying mutant and/or Myc-tagged ARP2 (GMyc2). For the GMyc2 lines, the HDR efficiency for double knock-in was 15.6% (7/45). Analysis of all of our cell lines revealed however, that indels were also present within both the endogenous Actr2 and Serpine2 loci. Subsequent analysis of our GMyc2 cells revealed 5 Actr2 alleles. This suggests that cells avoid the loss of Actr2 function and highlights the difficulty with creating an Actr2 mutant cell model. To further investigate the effects of the Actr2G mutation, we isolated mouse embryonic fibroblasts (MEFs) from Actr2+/+ and Actr2G/G mice. The Actr2G/G MEFs grew poorly in culture, and displayed a significant reduction in cell surface area 20 minutes after plating on fibronectin when compared to Actr2+/+ (q=0.0003). Further analysis revealed F-actin aggregation in the root of cellular protrusions of Actr2G/G MEFs. These results indicate cytoskeletal remodeling defects in Actr2G/G cells. In summary, our studies indicate that mutations affecting ARP2 function can have profound effects on mouse and cell survival, as well as pronounced effects on specific physiological processes like blood coagulation and the transcriptional regulation of coagulation related genes. Disclosures No relevant conflicts of interest to declare.

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).

Protease Nexin I is a feedback regulator of EGF/PKC/MAPK/EGR1 signaling in breast cancer cells metastasis and stemness

Breast cancer is the most prevalent cancer in women worldwide, which remains incurable once metastatic. Breast cancer stem cells (BCSCs) are a small subset of breast cancer cells, which are the radical cause of drug resistance, tumor relapse, and metastasis in breast cancer. The extracellular serine protease inhibitor serpinE2, also named protease nexin-1 (PN-1), contributes to enhanced metastasis of cancer cells mainly by remodeling the tumor matrix. In this study, we found that PN-1 was up-regulated in breast cancer, which promoted cell invasion, migration and stemness. Furthermore, by using specific inhibitors, we discovered that epidermal growth factor (EGF) up-regulated PN-1 in breast cancer cells through cascade activation of epidermal growth factor receptor (EGFR) to the activation of protein kinase Cδ (PKCδ), mitogen-activated protein kinase (MEK) and extracellular signal-related kinase (ERK), which finally led to the up-regulation of early growth response protein 1 (EGR1). Moreover, EGF signaling was further activated as a feedback of PN-1 up-regulation through PN-1 blocking HtrA1. Taken together, our findings revealed a novel signaling axis that up-regulated PN-1 expression in breast cancer cells, and the new mechanism of PN-1-promoted breast cancer metastasis, which may provide new insights into identifying novel therapeutic targets for breast cancer.

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.

Protease nexin 1: a novel inducer of prostate tumor cell apoptosis

Protease nexin 1 (PN1), a member of serine protease inhibitors (SERPINs) family, is known for its ability to bind and inhibit a wide range of proteases. Recently, we have found that PN1 is able to induce prostate cancer cell undergoing apoptosis through a distinct mechanism of engaging uPA-uPAR complexes and the regulation of two independent downstream signaling cascades, leading to the repression of X-linked inhibitor of apoptosis protein (XIAP). In this process, PN1 expression reduces the expression of NF-κB signalling component p65 and thereby lessens xiap transcription. Alternately, PN1 activity can prevent the stability of XIAP by reducing XIAP phosphorylation at serine 87 via a blockade of AKT signaling. A combination of exogenous PN1 and TRAIL leads to a substantial growth lag in prostate cancer xenografts, indicating the potential of PN1 as a promising target for improving prostate cancer therapy. Furthermore, human prostate tissue arrays show inverse levels of PN1 and XIAP in tumor and normal prostate. Hence, the PN1-uPA regulatory axis may serve as an inducer of tumor cell apoptosis by modulating survival pathways, and therefore delay the tumor growth of prostate cancer.

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.

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 β-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.

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.

Serpinin: A Novel Chromogranin A-Derived, Secreted Peptide Up-Regulates Protease Nexin-1 Expression and Granule Biogenesis in Endocrine Cells

Previously we demonstrated that chromogranin A (CgA) promoted secretory granule biogenesis in endocrine cells by stabilizing and preventing granule protein degradation in the Golgi, through up-regulation of expression of the protease inhibitor, protease nexin-1 (PN-1). However, the mechanism by which CgA signals the increase of PN-1 expression is unknown. Here we identified a 2.9-kDa CgA-C-terminus peptide, which we named serpinin, in conditioned media from AtT-20 cells, a corticotroph cell line, which up-regulated PN-1 mRNA expression. Serpinin was secreted from AtT-20 cells upon high potassium stimulation and increased PN-1 mRNA transcription in these cells, in an actinomycin D-inhibitable manner. CgA itself and other CgA-derived peptides, when added to AtT-20 cell media, had no effect on PN-1 expression. Treatment of AtT-20 cells with 10 nm serpinin elevated cAMP levels and PN-1 mRNA expression, and this effect was inhibited by a protein kinase A inhibitor, 6-22 amide. Serpinin and a cAMP analog, 8-bromo-cAMP, promoted the translocation of the transcription factor Sp1 into the nucleus, which is known to drive PN-1 expression. Additionally, an Sp1 inhibitor, mithramycin A inhibited the serpinin-induced PN-1 mRNA up-regulation. Furthermore, a luciferase reporter assay demonstrated serpinin-induced up-regulation of PN-1 promoter activity in an Sp1-dependent manner. When added to CgB-transfected 6T3 cells, a mutant AtT20 cell line, serpinin induced granule biogenesis as evidenced by the presence of CgB puncta accumulation in the processes and tips. Our findings taken together show that serpinin, a novel CgA-derived peptide, is secreted upon stimulation of corticotrophs and plays an important autocrine role in up-regulating PN-1-dependent granule biogenesis via a cAMP-protein kinase A-Sp1 pathway to replenish released granules.

COMMENTARY: Breaking down fear memory (Commentary on Meins et al.)

Department of Psychology and Neuroscience Program, University of Michigan, Ann Arbor, MI, USAIt is essential to rapidly learn and unfailingly remember threats in the environment. It is equally important to learn when those threats have passed,as well as the unique contexts in which one is safe from threat. In recent years, considerable progress has been made in understanding the neuralcircuits and molecular mechanisms that underlie the acquisition of fear memory in the mammalian brain (LeDoux, 2000; Maren, 2001). However,much less is known concerning the mechanisms for fear extinction, the learning process that suppresses fear when past threats no longer yieldaversive outcomes.Early work on the neural mechanisms of fear extinction revealed an essential role for N-methyl-d-aspartate (NMDA) receptors in the basolateralamygdala in fear extinction (Falls et al., 1992). More recently, the extinction circuit has expanded to include a network of inhibitory neurons in theamygdala, the intercalated cell masses (ITC), which play an important role in limiting the activity of neurons in the central nucleus (CE) that drivefear responses (Likhtik et al., 2008; Amano et al., 2010). In the current issue, Meins et al. (2010) shed new light on the molecular mechanismswithin these inhibitory amygdala circuits that are involved in the extinction of fear. Using a molecular genetic approach in mice, they first showthat inhibitory interneurons in the CE and ITC express a serine protease inhibitor, protease-nexin 1 (PN-1), which has previously been shown toregulate NMDA receptor function (Kvajo et al., 2004). Much weaker PN-1 expression was found in the basolateral nucleus of the amygdala (BA).Given the localization of PN-1 to inhibitory neurons in ITC and CE, Meins and colleagues next examined fear conditioning and extinction in PN-1knockout mice. Interestingly, PN-1 knockouts exhibited normal fear conditioning, but had marked impairments in the extinction of conditionalfear. Coupled with these behavioral deficits in extinction, PN-1 knockout mice exhibited reduced Fos expression in BA, as well as a reduction inphosphorylated alpha-calcium-calmodulin protein kinase II (aCamKII) in ITC neurons after extinction training. Hence, these data reveal animportant and novel role for PN-1 activity in extinction learning, and reinforce the important role for inhibitory interneurons in the amygdala inthis process.It has been proposed that NMDA receptor-dependent plasticity in the ITC is a mechanism for extinction learning (Amano et al., 2010). Insofaras PN-1 knockout mice exhibit impaired NMDA receptor function, the reduction of ITC c-Fos expression and aCamKII phosphorylation isconsistent with this possibility. Nonetheless, recent data indicate that NMDA receptor antagonism in the CE (and presumably ITC) does not affectthe acquisition of extinction in rats (Zimmerman & Maren, 2010). Further work is clearly required to understand the precise role for amygdaloidNMDA receptors and PN-1 regulation of NMDA receptor function in fear extinction. Nonetheless, the work by Meins and colleagues reveals anew player in the molecular organization of extinction learning within inhibitory interneurons of the amygdala, a finding that yields exciting newavenues for research in this rapidly moving field.

Abstract 3380: The serine protease inhibitor PN-1 induces activation of FGFR-mediated signaling

Abstract We are studying the role of PN-1 (Protease Nexin-1) in breast cancer. PN-1 is a serine protease inhibitor (serpin) that blocks a broad spectrum of proteases including among others tPA, uPA and thrombin. Once bound to a target protease, the PN-1/protease complexes bind with high affinity to Low Density Lipoprotein Receptor related Protein (LRP-1). A bioinformatic analysis revealed that PN-1 is overexpressed in a significant proportion of human tumors from different origins. Moreover, PN-1 expression levels correlate with markers of poor prognosis in breast cancer. PN-1 is also expressed in breast tumor cell lines and we recently showed that PN-1 is required by the malignant 4T1 mammary cancer cell line to metastasize to the lungs. Indeed, when mammary fat pads of Balb/c mice were injected with PN-1-knock down (KD)-4T1 cells, primary mammary tumors developed and these grew similarly as control 4T1 cells. However, the PN-1 KD tumors displayed a significant decrease in their potential to form lung metastasis, in comparison to control tumors. The mechanism by which complexes of serpin/protease mediate their effects in cancer cells is still largely unknown. Thus, our current studies are aimed at deciphering how the protease/PN-1 complex impacts on intracellular signaling in order to promote metastasis. Using the PN-1 negative 168FARN mammary tumor cell line, we have found that PN-1/protease complexes bind LRP-1 receptor and induce MMP-9 expression via Erk1/2 pathway activation. Importantly, the PN-1-driven regulation of MMP-9 expression was also demonstrated to be central to PN-1's prometastatic effect in 4T1 cells. Here by the use of Western Blot and specific inhibitors, we show that PN-1/protease complex treatment of fibroblast growth factor receptor (FGFR)-expressing tumor cells induces Erk signaling. Furthermore, fibroblast growth factor receptor substrate 2 (FRS2), a lipid anchored docking protein that plays a crucial role in mediating FGFR and tropomyosin receptor kinase (Trk)-induced signaling, was also found to be phosphorylated upon PN-1/protease complex addition. Preincubation of the cells with the FGFR inhibitor, TKI 258, abrogated phosphorylation of FRS2, Erk and Shc upon PN-1/protease complex treatment. In contrast, inhibitors of EGFR or PDGR did not have any effect on PN-1-induced phosphorylation of these proteins. This suggests that the PN-1/protease complex via binding to LRP-1 may transactivate FGFR and potentially other receptors known to activate mitogenic signaling in cancer cells. Our results are in agreement with a recent study showing that other ligands for LRP-1, (e.g, tPA and α2-macroglobulin), could transactivate TrkA receptor. Thus, PN-1 may also transactivate tyrosine kinase receptors. Hence, these results suggest that PN-1/protease complexes might be considered as a new target in cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3380.

Macrophages and Platelets Are the Major Source of Protease Nexin-1 in Human Atherosclerotic Plaque

Protease nexin-1 (PN-1), a serpin constitutively expressed by vascular smooth muscle cells and endothelial cells, inhibits thrombin, plasminogen activators, and plasmin and can thus be expected to play a role in vascular biology. The present study addressed the question of PN-1 expression in human atherothrombosis. Immunohistochemistry and biochemical studies confirmed that PN-1 was expressed at a moderate level in the medial layer of normal human arteries and showed that PN-1 expression was increased in atherothrombotic lesions. In early noncomplicated plaques, PN-1 was associated with infiltrating mononuclear cells. A strong PN-1 signal was observed in advanced lesions, principally in intraplaque hemorrhage-related structures. Monocytes/macrophages and platelets were identified as the main sources of PN-1 within atherothrombotic material. Isolated human monocytes and platelets both expressed high levels of active PN-1, and monocyte PN-1 expression was upregulated, at both messenger and protein levels, in response to stimulation by lipopolysaccharides. In contrast, PN-1 expression was downregulated during their differentiation into macrophages which were shown to produce degraded forms of PN-1. Platelets and monocytes/macrophages are a major source of PN-1 in human atherothrombotic plaques. PN-1 could thus represent a new actor in the evolution of atherosclerotic lesions.

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.

Expression of Prostasin Serine Protease and Protease Nexin-1 (PN-1) in Rhesus Monkey Ovary During Menstrual Cycle and Early Pregnancy

Serine proteases and their cognate serpin-class inhibitors are involved in the controlled proteolytic events during follicular development, ovulation, formation, and maintenance of the corpus luteum (CL). In this study, we investigated the expression patterns of prostasin serine protease and protease nexin-1 (PN-1), a serine protease inhibitor also called serpin-E2, in rhesus monkey ovaries during the menstrual cycle and early pregnancy, by using in situ hybridization and immunohistochemistry. Expression of prostasin was localized in oocyte, granulosa cells, and/or theca cells of early antral follicles and antral follicles, with high levels observed in preovulatory follicles. Prostasin was also localized at high levels of abundance in the CL during the menstrual cycle and early pregnancy. During the menstrual cycle, PN-1 was coordinately localized with prostasin in oocytes, granulosa cells, and theca cells of antral follicles and preovulatory follicles and in the CL. In addition, the PN-1 expression level in macaque CL during early pregnancy increased as pregnancy proceeded. We propose that prostasin may be involved in follicular development, ovulation, and CL formation, whereas PN-1 may be present to regulate the proteolysis in these processes.

Mice Lacking Protease Nexin-1 Show Delayed Structural and Functional Recovery after Sciatic Nerve Crush

Multiple molecular mechanisms influence nerve regeneration. Because serine proteases were shown to affect peripheral nerve regeneration, we performed nerve crush experiments to study synapse reinnervation in adult mice lacking the serpin protease nexin-1 (PN-1). PN-1 is a potent endogenous inhibitor of thrombin, trypsin, tissue plasminogen activators (tPAs), and urokinase plasminogen activators. Compared with the wild type, a significant delay in synapse reinnervation was detected in PN-1 knock-out (KO) animals, which was associated with both reduced proliferation and increased apoptosis of Schwann cells. Various factors known to affect Schwann cells were also altered. Fibrin deposits, tPA activity, mature BDNF, and the low-affinity p75 neurotrophin receptor were increased in injured sciatic nerves of mutant mice. To test whether the absence of PN-1 in Schwann cells or in the axon caused delay in reinnervation, PN-1 was overexpressed exclusively in the nerves of PN-1 KO mice. Neuronal PN-1 expression did not rescue the delayed reinnervation. The results suggest that Schwann cell-derived PN-1 is crucial for proper reinnervation through its contribution to the autocrine control of proliferation and survival. Thus, the precise balance between distinct proteases and serpins such as PN-1 can modulate the overall impact on the kinetics of recovery.

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.

Protease nexin-1 expression is altered in human breast cancer

BackgroundUrokinase-type Plasminogen Activator (uPA), a serine protease, plays a pivotal role in human breast cancer metastasis by mediating the degradation of extracellular matrix proteins and promoting cell motility. In more advanced breast cancers, uPA activity is significantly up regulated and serves as a prognostic indicator of poor patient outcome. Classically, regulation of uPA activity, especially in breast cancers, is thought to be mediated by Type 1 Plasminogen Activator Inhibitor (PAI-1). However, we have recently found that a lesser known natural inhibitor of uPA, Protease Nexin 1 (PN-1), is expressed in normal human mammary tissue. Based on this observation, we investigated if PN-1 is also expressed in human breast cancers where it may contribute to the regulation of uPA and participate in the development of a metastatic phenotype.ResultsUsing quantitative real-time PCR analysis, we measured PN-1 mRNA expression in tissues obtained from 26 human breast tumor biopsies and compared these values with those obtained from 10 normal breast tissue samples. Since both PAI-1 and uPA expression levels are known to be elevated in metastatic breast cancer, we also measured their levels in our 26 tumor samples for direct comparison with PN-1 expression. We found that PN-1 expression was elevated over that found in normal mammary tissue; an increase of 1.5- to 3.5-fold in 21 of 26 human breast tumors examined. As anticipated, both PAI-1 and uPA mRNA levels were significantly higher in the majority of breast tumors; 19 of 26 tumors for PAI-1 and 22 of 26 tumors for uPA. A quantile box plot of these data demonstrates that the elevated PN-1 expression in breast tumor tissues directly correlates with the increased expression levels found for PAI-1 and uPA.ConclusionThe fact that PN-1 expression is elevated in human breast cancer, and that its increased expression is directly correlated with increases measured for PAI-1 and uPA, suggests that PN-1 may contribute to the regulation of uPA-mediate tumor cell motility and metastatic spread.

The serpin protease nexin-1 regulates vascular smooth muscle cell adhesion, spreading, migration and response to thrombin.

Protease nexin-1 (PN-1) is an important physiological regulator of thrombin in the brain. PN-1 is also present in aortic smooth muscle cells and may thus participate in vascular biology. However, little is known about its function in the vessel wall. In this study, we investigated the effect of PN-1 overexpression in smooth muscle cells (SMCs), on their sensitivity to thrombin, and their capacity for adhesion, spreading and migration. Two clones exhibiting a two- to threefold increase in PN-1 expression were selected and compared with untransfected and mock-transfected cells. Overexpression of PN-1 was observed to inhibit thrombin-induced cell responses as indicated by a twofold decrease in induction of PAI-1 expression, a decreased calcium mobilization in response to low thrombin concentrations and a twofold increase in the capacity to inhibit thrombin catalytic activity. Overexpression of PN-1 did not modify adhesion, spreading, and migration of SMCs on type I collagen. In contrast, SMCs overexpressing PN-1 exhibited a 40% reduction in adhesion, a 50% reduction in spreading and a complete absence of migration on vitronectin when compared with control SMCs. Our studies thus reveal that PN-1 is likely to play a critical role in regulating essential cell functions such as (i) thrombin-induced responses, which are dependent on its antiprotease activity, and (ii) adhesion, spreading, and migration, which are independent of its antiprotease activity and may be related to its interaction with other partners, such as vitronectin in the present case.