Protease Nexin-1 Research Articles

The Role of the P1 Residue of Protease Nexin 2 and Basic Pancreatic Trypsin Inhibitor in the Mechanism of Factor XIa Inhibition.

Factor XIa (FXIa), a plasma serine protease that activates FIX, is regulated by protease nexin 2 (PN2), a Kunitz-type protease inhibitor (KPI) secreted on platelet activation. The Kunitz protease inhibitor domain of protease nexin 2 (PN2KPI) is highly homologous (45% primary sequence identity) to basic pancreatic trypsin inhibitor (BPTI, another Kunitz-type protease inhibitor) and their backbone tertiary structures are nearly identical (J Biol Chem 280: 36165, 2005; J Mol Biol 230: 919, 2005). However, PN2KPI (Ki 1 nM) is 600-fold more potent as a FXIa inhibitor than bovine basic pancreatic trypsin inhibitor (BPTI; Ki 630 nM). The present study is aimed at examining why these two structurally similar inhibitors are so different in their affinities and specificities in FXIa inhibition and analyzing the mechanisms of FXIa inhibition by these two inhibitors. Reasoning that the P1 residue (Arg15 in PN2KPI and Lys15 in BPTI) might play a crucial role in determining the affinity and specificity for FXIa we expressed BPTI-K15R (mimicking PN2KPI at the P1 site) and PN2KPI-R15K (mimicking BPTI at the P1 site) and examined their inhibitory properties. BPTI-K15R was found to inhibit FXIa with a Ki (10 nM) ~60-fold tighter than BPTI (Ki 630 nM), whereas PN2KPI-R15K inhibited FXIa with a Ki (32 nM) that was 32- fold impaired compared with PN2KPI (Ki 1 nM). Progress curves of peptidyl fluorogenic substrate (Boc-Glu-Ala-Arg-AMC) hydrolysis by FXIa in the presence of all the inhibitors except BPTI clearly demonstrated higher initial rates that continuously decreased until reaching a steady state, typical of slow inhibition. Progress curves obtained using BPTI, on the other hand, were found to be linear at all concentrations of the inhibitor. Analysis using steady state kinetics clearly demonstrated that BPTI is a competitive inhibitor in FXIa hydrolysis of the peptidyl chromogenic substrate, pyro-Glu-Pro-Arg-pNA. Analysis of progress curves using kinetic equations for slow, tight-binding inhibitors confirmed that the formation of the FXIa:PN2KPI complex occurs in a single step that is slow, whereas formation of FXIa:PN2KPI-R15K, FXIa:BPTI and FXIa:BPTI-K15R complexes do not conform to the same kinetic mechanism. Thus PN2KPI and BPTI inhibit FXIa by distinctly different mechanisms and the Arg at the P1 site of PN2KPI confers specificity and high affinity for FXIa inhibition and in part determines the mechanism of PN2KPI as a slow, tight binding inhibitor.

Novel MMP-9 Substrates in Cancer Cells Revealed by a Label-free Quantitative Proteomics Approach

Matrix metalloproteinase-9 (MMP-9) is implicated in tumor metastasis as well as a variety of inflammatory and pathological processes. Although many substrates for MMP-9, including components of the extracellular matrix, soluble mediators such as chemokines, and cell surface molecules have been identified, we undertook a more comprehensive proteomics-based approach to identify new substrates to further understand how MMP-9 might contribute to tumor metastasis. Previous proteomics approaches to identify protease substrates have depended upon differential labeling of each sample. Instead we used a label-free quantitative proteomics approach based on ultraperformance LC-ESI-high/low collision energy MS. Conditioned medium from a human metastatic prostate cancer cell line, PC-3ML, in which MMP-9 had been down-regulated by RNA interference was compared with that from the parental cells. From more than 200 proteins identified, 69 showed significant alteration in levels after depletion of the protease (>+/-2-fold), suggesting that they might be candidate substrates. Levels of six of these (amyloid-beta precursor protein, collagen VI, leukemia inhibitory factor, neuropilin-1, prostate cancer cell-derived growth factor (PCDGF), and protease nexin-1 (PN-1)) were tested in the conditioned media by immunoblotting. There was a strong correlation between results by ultraperformance LC-ESI-high/low collision energy MS and by immunoblotting giving credence to the label-free approach. Further information about MMP-9 cleavage was obtained by comparison of the peptide coverage of collagen VI in the presence and absence of MMP-9 showing increased sensitivity of the C- and N-terminal globular regions over the helical regions. Susceptibility of PN-1 and leukemia inhibitory factor to MMP-9 degradation was confirmed by in vitro incubation of the recombinant proteins with recombinant MMP-9. The MMP-9 cleavage sites in PN-1 were sequenced. This study provides a new label-free method for degradomics cell-based screening leading to the identification of a series of proteins whose levels are affected by MMP-9, some of which are clearly direct substrates for MMP-9 and become candidates for involvement in metastasis.

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.

The expression and the role of protease nexin-1 on brain edema after intracerebral hemorrhage

Brain edema is one of the most frequent and serious complications of intracerebral hemorrhage (ICH), but how the ICH cause brain edema is unknown. Our studies were designed to investigate the regulation and distribution of protease nexin-1 (PN-1), thrombin and aquaporin-4 (AQP-4) in brain edema after ICH in rat and human brain in vivo. Our result showed that the severity of cerebral edema resulted from an acute stage of ICH. The PN-1-thrombin system modulated cerebral edema after ICH. Thrombin and AQP-4 increased to aggregate cerebral edema after ICH. In order to control the deleterious effect of thrombin's overexpression, PN-1 appeared quickly and abundantly to inhibit thrombin and lessen the cerebral edema. PN-1 was distributed in neurons and glial cells of cerebral cortex, hippocampus, thalamencephalon, basal ganglia, cerebellum and circum-encephalocoele in rat and human brain. The expression of AQP-4 is different between human and rat. Thus, we demonstrated that the animal experimental approach was, however, not sufficient by itself and needed to be corroborated by observations on human brains.

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.

Prostasin expression is regulated by airway surface liquid volume and is increased in cystic fibrosis

Airway surface liquid (ASL) absorption is initiated by Na+ entry via epithelial Na+ channels (ENaC), which establishes an osmotic gradient that drives fluid from the luminal to serosal airway surface. We and others have recently reported that a protease/anti-protease balance regulates ENaC in human airway epithelial cells (HAEC) and provides a mechanism for autoregulation of ASL volume. In cystic fibrosis (CF), this balance is disturbed, leading to constitutive proteolytic activation of ENaC and the pathological Na+ hyperabsorption characteristic of this airway disease. Prostasin is a glycosylphosphatidylinositol-anchored serine protease that activates ENaC and is expressed on the surface epithelium lining the airway. In this report we present evidence that prostasin expression is regulated by the ASL volume, allowing for increased proteolytic activation of ENaC when the ASL volume is high. Prostasin activity is further regulated by the cognate serpin protease nexin-1 (PN-1), which is expressed in HAEC and inhibits Na+ absorption by forming an inactive complex with prostasin and preventing the proteolytic processing of prostasin. Whereas these mechanisms regulate prostasin expression in response to ASL volume in non-CF epithelia, HAEC cultured from CF patients express >50% more prostasin on the epithelial surface. These findings suggest that a proteolytic cascade involving prostasin, an upstream prostasin-activating protease, and PN-1 regulate Na+ absorption in the airway and that abnormal prostasin expression contributes to excessive proteolytic activation of ENaC in CF patients.

<b>Protease Nexin-1 in Reproductive Tissues: a Review</b>

The Serpins comprise a family of proteins that bind irreversibly to and inactivate serine proteases such as thrombin and plasminogen activators. This review summarizes the current knowledge of one Serpin, SerpinE2 (also known as protease nexin-1, PN-1) in the reproductive system. By virtue of the ability to regulate the activity of secreted proteases, PN-1 is most likely involved in tissue remodeling and extracellular matrix formation and degradation. Accordingly, PN-1 is expressed in the developing hypothalamus and testis, both sites of significant cell migration. PN-1 is also upregulated during tissue remodeling in the ovarian follicle (at ovulation) and in the placenta during implantation, and downregulated in the mammary gland during pregnancy and lactation. PN-1 has been shown to be essential for formation of the copulatory plug in rodents. The physiological role of PN-1 is not clear, but given the fairly widespread expression of this protein in reproductive tissues, this protein clearly deserves greater attention from reproductive biologists.

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.

Structural Determinants for Heparin Binding in Human Coagulation Factor XI.

Previous studies from our laboratory and others have demonstrated that zymogen factor XI (FXI) binds to heparin with moderate (KD ∼110 nM) affinity via residues (K252, K253 and K255) located in the Apple 3 (A3) domain. In contrast, the enzyme, FXIa, was shown to bind to heparin (Biochemistry 40: 7569–7580, 2001) with significantly higher affinity (KD ∼9 nM by SPR and ∼1.5 nM by ELISA) via residues (K529, R530 and R532) within the catalytic domain (CD). This interaction potentiates by ∼10-fold the inhibition of FXIa by protease nexin-2. Also, polyanions heparin and dextran sulfate inhibit the catalytic activity of the enzyme factor XIa. The present study was designed to determine the relative contributions of positively charged residues as well as the dimeric structure of FXI to heparin binding. Mutational analysis of full-length FXI expressed in HEK293 cells was based on the following criteria:1.Conservation of the positively charged residues in FXI among various species;2.Surface exposure of the residues based on the X-ray crystal structure of FXI (Papagrigoriou E, McEwan P, Walsh PN, Emsley J, Nat. Struct. & Mol. Biol . 2006; 13:557–558); and3.comparison with human plasma prekallikrein (PK), which does not bind heparin.Two positively charged residues Arg507 (147, chymotrypsin numbering) and Arg532 (173) are conserved in FXI genes of all species for which sequences are available. In human PK, Arg507 is replaced by lysine, while Arg532 is replaced by a neutral glutamine. We have expressed and purified wtFXI, R507A, R532A as well as monomeric C321S/K331A and C321A/I290A. While wtFXI, R507A and R532A demonstrated normal activity in APTT assays; monomeric FXI mutants retained 60-70% activity. The R532A and R507A mutants demonstrated ∼75% decrease in total number of heparin binding sites based on the solid phase ELISA assay using 5F7 monoclonal antibody. Also, the apparent dissociation constants for R507 (11 nM) and R532A (22 nM) were 7 and 11-fold increased respectively compared with 1.6 nM for the wtFXI. We also characterized monomeric FXI C321S/K331A and C321A/I290A proteins for their ability to bind to heparin compared with wtFXI using surface plasmon resonance (SPR). Surprisingly, the monomeric FXI mutants, C321S/K331A and C321A/I290A, which had no mutations in any heparin-binding regions, displayed major defects in binding to heparin by SPR. Although kinetic analysis is challenging due to complex binding kinetics, while Rmax is about 10-fold lower, the off-rate for the binding of the monomeric FXI mutants is drastically increased when compared to that of wtFXI. These results suggest the possibility that the unique dimeric structure of FXI is required for cooperative binding to heparin. Thus, the dimeric structure of FXI and basic residues R507 and R532 in the catalytic domain of factor XI are both necessary for high-affinity heparin binding.

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.

Increased Severity of Hemorrhage in Transgenic Mice Expressing Cerebral Protease Nexin-2/Amyloid β-Protein Precursor

Secreted isoforms of amyloid beta-protein precursor (AbetaPP) that contain the Kunitz proteinase inhibitor domain, also known as protease nexin-2 (PN2), are enriched in brain. Although little is known of its physiological function, the potent inhibition of certain prothrombotic proteinases by PN2/AbetaPP suggests that it may function to regulate cerebral thrombosis during vascular injury events. To examine the antithrombotic function of cerebral PN2/AbetaPP in vivo, we performed measurements of carotid artery thrombosis and experimental intracerebral hemorrhage in transgenic mice with specific and modest overexpression of PN2/AbetaPP in brain. Comparisons were made with wild-type mice and Tg-rPF4/APP mice, a model that possesses specific and modest overexpression of PN2/AbetaPP in platelets and exhibits reduced thrombosis in vivo. Modest overexpression of PN2/AbetaPP in transgenic mouse brain had no effect on intraluminal carotid arterial thrombosis but resulted in larger hematoma volumes and hemoglobin levels (23.1+/-2.7 mm(3) [n=6; P<0.01] and 1411+/-202 microg/hemisphere [n=12; P<0.01], respectively), compared with wild-type mice (15.9+/-2.2 mm(3) [n=6] and 935+/-418 microg/hemisphere [n=12], respectively). These findings indicate that cerebral PN2/AbetaPP plays a significant role in regulating thrombosis in brain and that modest age-related increases in the cerebral levels of this protein could markedly enhance the extent of cerebral hemorrhage.

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.

Overexpression of protease nexin-1 mRNA and protein in oral squamous cell carcinomas

Protease nexin-1 (PN-1) belongs to the serpin family of serine protease inhibitors. It is the phylogenetically closest relative of plasminogen activator inhibitor-1 (PAI-1). Whilst there are numerous studies of the occurrence and functions of PAI-1 in cancer, a possible tumour biological role of PN-1 has been almost totally neglected. We have now compared the level of PN-1 mRNA in 20 cases of oral squamous cell carcinomas and in matched samples of the corresponding normal oral tissues. We found that the average PN-1 mRNA level in tumours and normal tissues was significantly different, being increased up to 13 fold in tumour samples compared with the average level in normal tissues. The PN-1 mRNA level was significantly higher in tumours from patients with lymph node metastasis than in tumours from patients without. We could conclude that PN-1 is frequently overexpressed in oral squamous cell carcinomas and that its level may correlate with the occurrence of lymph node metastasis. We hypothesise that PN-1 may have a tumour biological function similar to that of PAI-1.

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.

Protease Nexin-1 Interacts With Thrombomodulin and Modulates Its Anticoagulant Effect

The endothelial cell membrane glycoprotein thrombomodulin (TM) plays a critical role in the regulation of coagulation. TM is an essential cofactor in protein C activation by thrombin, and a direct inhibitor of thrombin-induced platelet activation and fibrinogen clotting. Protease nexin-1 (PN-1) is a serpin synthesized and secreted by a variety of cells including endothelial cells. PN-1 bound to the cell surface through interactions with glycosaminoglycans, is an efficient inhibitor of thrombin and controls thrombin-induced cell responses. An investigation of the interaction of PN-1 with TM using purified proteins and cultured human aortic endothelial cells was performed. Purified PN-1 was observed to bind to purified TM in a concentration-dependent manner. Double immunofluorescence studies indicated that PN-1 and TM were colocalized at the endothelial cell surface from which they were coprecipitated. Pretreatment of the cells with chondroitinase ABC greatly decreased the amount of the PN-1 associated to TM at the cell surface demonstrating the involvement of the TM chondroitin-sulfate chain in the formation of complexes. The inhibitory activity of the PN-1/TM complexes on the catalytic activity of thrombin, and on thrombin-induced fibrinogen clotting, was markedly enhanced when compared with the inhibitory activity of each partner. PN-1-overexpressing human aortic endothelial cells and PN-1-underexpressing human aortic endothelial cells exhibited respectively a significantly reduced ability and enhanced capacity to activate protein C. Furthermore, PN-1 decreased the cofactor activity of TM on thrombin activable fibrinolysis inhibitor activation by thrombin. These data show for the first time that PN-1 forms complexes with TM and modulates its anticoagulant activity.

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.

Evidence for a Matriptase-Prostasin Proteolytic Cascade Regulating Terminal Epidermal Differentiation

Recent gene ablation studies in mice have shown that matriptase, a type II transmembrane serine protease, and prostasin, a glycosylphosphatidylinositol-anchored membrane serine protease, are both required for processing of the epidermis-specific polyprotein, profilaggrin, stratum corneum formation, and acquisition of epidermal barrier function. Here we present evidence that matriptase acts upstream of prostasin in a zymogen activation cascade that regulates terminal epidermal differentiation and is required for prostasin zymogen activation. Enzymatic gene trapping of matriptase combined with prostasin immunohistochemistry revealed that matriptase was co-localized with prostasin in transitional layer cells of the epidermis and that the developmental onset of expression of the two membrane proteases was coordinated and correlated with acquisition of epidermal barrier function. Purified soluble matriptase efficiently converted soluble prostasin zymogen to an active two-chain form that formed SDS-stable complexes with the serpin protease nexin-1. Whereas two forms of prostasin with molecular weights corresponding to the prostasin zymogen and active prostasin were present in wild type epidermis, prostasin was exclusively found in the zymogen form in matriptase-deficient epidermis. These data suggest that matriptase, an autoactivating protease, acts upstream from prostasin to initiate a zymogen cascade that is essential for epidermal differentiation.

Plasminogen Activator Inhibitor-1 in Chronic Kidney Disease

In 1984, Loskutoff et al. ([1][1]) purified plasminogen activator inhibitor-1 (PAI-1) from conditioned media of cultured endothelial cells. This 50-kd glycoprotein is the primary physiologic inhibitor of the serine proteases tissue-type and urokinase-type plasminogen activators (tPA and uPA,

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.

A Role for Plasminogen Activator Inhibitor-1 in Obesity: From Pie to PAI?

The regulation of fibrinolysis depends on the interaction of plasma fibrinolytic and anti-fibrinolytic proteins. Fibrinolysis depends on the enzymatic conversion of plasminogen to plasmin. This process is mediated by tissue-type and urokinase-type plasminogen activators (t & uPAs, respectively). See page 2209 Plasma antifibrinolytic activity is primarily regulated by plasminogen-activator inhibitors (PAIs).1 Four PAIs have been described: PAIs-1 through 3 and protease nexin. Importantly, PAI-1 is the main antagonist of t & uPAs, accounting for approximately 60% of the inhibition of plasminogen activators. PAI-2 mostly inhibits urokinase plasminogen activator (uPA), whereas protease nexin antagonizes plasmin, thrombin, and uPA. The antifibrinolytic activity of PAI-3 is uncertain. These molecules and others are also collectively known as serine protease inhibitors or “serpins”. PAI-1 is a single chain 45-kDa glycoprotein that contains from 379 to 381 amino acids. Endothelial and vascular smooth muscle cells are presumably the main sources of PAI-1 but other cells, such as platelets, hepatocytes, mesangial cells, fibroblasts, monocytes, macrophages, adipocytes, and stromal cells permeating the adipose tissue, have also been shown to secrete the serpin. The gene for PAI-1 is located on chromosome 7q21.3-q22, and its expression is mainly regulated at the transcriptional level through the action of several hormones [eg, renin-angiotensin-aldostrerone system (RAAS)], cytokines [eg, tumor necrosis factor (TNF) α], lipoproteins (eg, VLDL), glucose, and endotoxin.1 Polymorphisms that influence PAI-1 gene expression have been described. The single 4/5 guanine polymorphism (4G/5G) in the promoter region of PAI-1 gene is widespread and has been associated with variable plasma PAI-1 activity and antigen levels.1 Indeed, plasma expression of PAI-1 antigen and activity are augmented in homozygous subjects for the 4G allele but decreased in 5G allele homozygous. This polymorphism may have important implications for human cardiovascular disease as suggested by case–control studies showing a higher prevalence of the 4G …