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Essentials Plg-RKT-/- female mice give birth, but no offspring of Plg-RKT-/- female mice survive to weaning. Causal mechanisms of potential lactational failure in Plg-RKT-/- mice are unknown. Plg-RKT regulates extracellular matrix remodeling, cell proliferation, apoptosis, fibrin surveillance. Plg-RKT is essential for lactogenesis and mammary lobuloalveolar development. Summary Background Lactational competence requires plasminogen, the zymogen of the serine protease, plasmin. Plg-RKT is a unique transmembrane plasminogen receptor that promotes plasminogen activation to plasmin on cell surfaces. Plg-RKT-/- mice are viable, but no offspring of Plg-RKT-/- female mice survive to weaning. Objectives We investigated potential lactational failure in Plg-RKT-/- mice and addressed causal mechanisms. Methods Fibrin accumulation, macrophage infiltration, processing of extracellular matrix components, effects of genetic deletion of fibrinogen, expression of fibrosis genes, and proliferation and apoptosis of epithelial cells were examined in lactating mammary glands of Plg-RKT-/- and Plg-RKT+/+ mice. Results Milk was not present in the stomachs of offspring of Plg-RKT-/- female mice and the pups were rescued by foster mothers. Although the mammary ductal tree developed normally in Plg-RKT-/- glands, lobuloalveolar development was blocked by a hypertrophic fibrotic stroma and infiltrating macrophages were present. A massive accumulation of fibrin was also present in Plg-RKT-/- alveoli and ducts. Although this accumulation was decreased when Plg-RKT-/- mice were made genetically heterozygous for fibrinogen, defects in lobuloalveolar development were not rescued by fibrinogen heterozygosity. Transcriptional profiling revealed that EGF was downregulated 12-fold in Plg-RKT-/- glands. Furthermore, proliferation of epithelial cells was not detectable. In addition, the pro-survival protein, Mcl-1, was markedly downregulated and apoptosis was observed in Plg-RKT-/- but not Plg-RKT+/+ glands. Conclusions Plg-RKT is essential for lactogenesis and functions to maintain the appropriate stromal extracellular matrix environment, regulate epithelial cell proliferation and apoptosis, and, by regulating fibrinolysis, preserve alveolar and ductal patency.

SummaryBackground Plg-RKT is a novel integral membrane plasminogen receptor that binds plasminogen via a C-terminal lysine exposed on the cell surface and promotes plasminogen activation on the cell surface by both tissue plasminogen activator and urokinase plasminogen activator. Objectives To evaluate the role of Plg-RKT in vivo we generated Plg-RKT-/- mice using a homologous recombination technique. Methods We characterized the effect of Plg-RKT deletion on reproduction, viability, health, and spontaneous thrombosis and inflammation. Results Plg-RKT-/- mice were viable and fertile. Survival of Plg-RKT-/- mice and Plg-RKT+/+ littermates was not significantly different. However, quite strikingly, all pups of Plg-RKT-/- females died within 2 days of birth, consistent with a lactation defect in Plg-RKT-/- mothers. Additionally, there was a significant effect of Plg-RKT deficiency on growth rates of female, but not male mice. In experimental peritonitis studies, Plg-RKT-/- mice exhibited a marked defect in macrophage recruitment. As a contributing mechanism, the capacity of Plg-RKT-/- macrophages for plasminogen binding was markedly decreased. Conclusions These studies demonstrate that Plg-RKT is required for plasminogen binding and macrophage migration in vivo. In addition, Plg-RKT deficiency is not compatible with survival of the species, due to death of all offspring of Plg-RKT-/- females. This new mouse model will be important for future studies aimed at delineating the role of cell surface plasminogen activation in challenge and disease models in vivo. This article is protected by copyright. All rights reserved.

Summary Background The mechanisms by which PAI-1 biosynthesis is altered during stress have not been fully elucidated. Studies suggest a major role for neuro-peptidergic modulation of the stress response by PACAP (pituitary adenylate cyclase-activating polypeptide), a member of the VIP/secretin/glucagon family. Objective We tested the hypothesis that PACAP regulates PAI-1 biosynthesis during stress in vivo. Methods PAI-1 gene expression was monitored by RT-PCR in adrenal glands harvested from C57BL/6J mice that were unstressed, or subjected to restraint stress for 2 h, or treated with PACAP. Results PAI-1 mRNA expression was markedly increased in adrenals from stressed mice. Restraint stress resulted in much smaller increments in adrenal tPA mRNA, suggesting that local adrenal tPA/PAI-1 biosynthetic balance is markedly altered by stress. The observed increases in PAI-1mRNA during stress were substantially blunted (55 ± 4%, P

Neurotransmitter release by catecholaminergic cells is negatively regulated by prohormone cleavage products formed from plasmin-mediated proteolysis. Here, we investigated the expression and subcellular localization of Plg-R(KT), a novel plasminogen receptor, and its role in catecholaminergic cell plasminogen activation and regulation of catecholamine release. Prominent staining with anti-Plg-R(KT) mAb was observed in adrenal medullary chromaffin cells in murine and human tissue. In Western blotting, Plg-R(KT) was highly expressed in bovine adrenomedullary chromaffin cells, human pheochromocytoma tissue, PC12 pheochromocytoma cells, and murine hippocampus. Expression of Plg-R(KT) fused in-frame to GFP resulted in targeting of the GFP signal to the cell membrane. Phase partitioning, co-immunoprecipitation with urokinase-type plasminogen activator receptor (uPAR), and FACS analysis with antibody directed against the C terminus of Plg-R(KT) were consistent with Plg-R(KT) being an integral plasma membrane protein on the surface of catecholaminergic cells. Cells stably overexpressing Plg-R(KT) exhibited substantial enhancement of plasminogen activation, and antibody blockade of non-transfected PC12 cells suppressed plasminogen activation. In functional secretion assays, nicotine-evoked [(3)H]norepinephrine release from cells overexpressing Plg-R(KT) was markedly decreased (by 51 ± 2%, p0.001) when compared with control transfected cells, and antibody blockade increased [(3)H]norepinephrine release from non-transfected PC12 cells. In summary, Plg-R(KT) is present on the surface of catecholaminergic cells and functions to stimulate plasminogen activation and modulate catecholamine release. Plg-R(KT) thus represents a new mechanism and novel control point for regulating the interface between plasminogen activation and neurosecretory cell function.

Proteins of the plasminogen activation system are broadly expressed throughout the nervous system, and key roles for these proteins in neuronal function have been demonstrated. Recent reports have established that plasminogen is synthesized in neuroendocrine tissues, making this protein and the proteolytic activity of the product of its activation, plasmin, available at sites separated anatomically from circulating, hepatocyte-derived plasminogen. Results with plasminogen-deficient humans and mice suggest a role for plasminogen in neuritogenesis. To elucidate the role of the plasminogen activation system in these processes, the function of plasminogen during neuritogenesis and neurite outgrowth was studied. It is shown here that plasminogen participates in neuritogenesis, as plasmin inhibitors reduced both neurite outgrowth and neurite length in PC-12 cells. The addition of exogenous plasminogen enhanced neurite outgrowth and neurite length in both PC-12 cells and primary cortical neurons. The proteolytic activity of plasmin was required, since mutation of the catalytic serine residue completely abolished the stimulatory activity. Furthermore, mutation of the lysine binding site within kringle 5 of the plasminogen molecule also reduced the neuritogenic activity of plasminogen. Additionally, we demonstrate that plasminogen specifically bound to laminin-1, the interaction resulted in increased plasminogen activation by tissue-type plasminogen activator, and was dependent on a functional lysine binding site within plasminogen kringle 5. Moreover, during NGF-induced neuritogenesis, laminin-1 was degraded, and this cleavage was catalyzed by plasmin. This study provides the first direct evidence that plasminogen participates in neurite outgrowth and also suggests that laminin-1 degradation by plasmin contributes to the process of neuritogenesis.

Ectromelia virus (ECTV), the causative agent of mousepox, expresses an extracellular interferon-gamma binding protein (IFN-gammaBP) with homology to the ligand binding domains of the IFN-gamma high affinity receptor (IFN-gammaR1). Unlike the cellular receptor, the IFN-gammaBP binds IFN-gamma from several species. The IFN-gammaBP is synthesized early after infection, accumulating in the extracellular milieu as dimers composed of two protein species with Mr of 34.6 or 33.0 kDa. Homodimers are covalently linked by an interchain disulphide bond at position 216. The IFN-gammaBP has complex N-linked oligosaccharides at positions 41 and 149 as determined by site-directed mutagenesis and glycosidase treatment. Glycosylation at position 41 is required for secretion from mammalian cells and may play a role in the activity of the IFN-gammaBP. Glycosylation at position 149 is not required for secretion, and the lack of glycosylation at this site does not diminish ligand binding as measured by surface plasmon resonance (SPR) and ELISA.

Cytokines are pivotal to a balanced innate or cell-mediated immune response, can be indicative of disease progression and/or resolution, and are being evaluated as therapeutics. There is a need to purify and/or to measure key cytokines rapidly with accuracy, precision, and sensitivity. The current assay technologies, which are based on RT-PCR, immunoassays, or bioassays, are limited in their use in the clinic, in particular because of the long time (1—3 h) required to carry out the assays. An alternative approach explored here is the use of pathogen-encoded cytokine- binding proteins, which have Kd in the nanomolar range. It is anticipated that pathogens have evolved binding proteins, antagonists, and/or specific neutralizing phenotypes directed against key signaling and effector molecules involved in the multifaceted host defense system. Thus, by screening the genomes of a wide range of microbial agents, we would expect to find coding sequences for binding proteins for the most important cytokines. Consistent with this view is the identification of poxvirus genes encoding binding activities for TNF, type I and type II interferons, interleukin (IL)-1β, IL-18, and β-chemokines. These high-affinity receptors have the potential to act as surrogate antibodies in a number of applications in cytokine quantification and purification and could be potentially useful reagents to complement the existing panel of anti-cytokine, monoclonal, polyclonal, or engineered antibodies that are currently available.

The local environment of neurosecretory cells contains the major components of the plasminogen activation system, including the plasminogen activators, tissue plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), as well as binding sites for t-PA, the receptor for u-PA (uPAR), and also the plasminogen activator inhibitor, PAI-1. Furthermore, these cells express specific binding sites for plasminogen, which is available in the circulation and in interstitial fluid. Colocalization of plasminogen and its activators on cell surfaces provides a mechanism for promoting local plasminogen activation. Plasmin is retained on the cell surface where it is protected from its inhibitor,α2-antiplasmin. In neurosecretory cells, localized plasmin activity provides a mechanism for extracellular processing of secreted hormones. Neurotransmitter release from catecholaminergic cells is negatively regulated by cleavage products formed by plasmin-mediated proteolysis. Recently, we have identified a major plasminogen receptor, Plg-RKT. We have found that Plg-RKTis highly expressed in chromaffin cells of the adrenal medulla as well as in other catecholaminergic cells and tissues. Plg-RKT-dependent plasminogen activation plays a key role in regulating catecholaminergic neurosecretory cell function.

Activation of plasminogen, the zymogen of the primary thrombolytic enzyme, plasmin, is markedly promoted when plasminogen is bound to cell surfaces, arming cells with the broad spectrum proteolytic activity of plasmin. In addition to its role in thrombolysis, cell surface plasmin facilitates a wide array of physiologic and pathologic processes. Carboxypeptidase B-sensitive plasminogen binding sites promote plasminogen activation on eukaryotic cells. However, no integral membrane plasminogen receptors exposing carboxyl terminal basic residues on cell surfaces have been identified. Here we use the exquisite sensitivity of multidimensional protein identification technology and an inducible progenitor cell line to identify a novel differentiation-induced integral membrane plasminogen receptor that exposes a C-terminal lysine on the cell surface, Plg-RKT (C9orf46 homolog). Plg-RKT was highly colocalized on the cell surface with the urokinase receptor, uPAR. Our data suggest that Plg-RKT also interacts directly with tissue plasminogen activator. Furthermore, Plg-RKT markedly promoted cell surface plasminogen activation. Database searching revealed that Plg-RKT mRNA is broadly expressed by migratory cell types, including leukocytes, and breast cancer, leukemic, and neuronal cells. This structurally unique plasminogen receptor represents a novel control point for regulating cell surface proteolysis.

Ectromelia virus (ECTV), a natural mouse pathogen and the causative agent of mousepox, is closely related to variola virus (VARV), which causes smallpox in humans. Mousepox is an excellent surrogate small-animal model for smallpox. Both ECTV and VARV encode a multitude of host response modifiers that target components of the immune system and that are thought to contribute to the high mortality rates associated with infection. Like VARV, ECTV encodes a protein homologous to the ectodomain of the host gamma interferon (IFN-γ) receptor 1. We generated an IFN-γ binding protein (IFN-γbp) deletion mutant of ECTV to study the role of viral IFN-γbp (vIFN-γbp) in host-virus interaction and also to elucidate the contribution of this molecule to the outcome of infection. Our data show that the absence of vIFN-γbp does not affect virus replication per se but does have a profound effect on virus replication and pathogenesis in mice. BALB/c mice, which are normally susceptible to infection with ECTV, were able to control replication of the mutant virus and survive infection. Absence of vIFN-γbp from ECTV allowed the generation of an effective host immune response that was otherwise diminished by this viral protein. Mice infected with a vIFN-γbp deletion mutant virus, designated ECTV-IFN-γbpΔ, produced increased levels of IFN-γ and generated robust cell-mediated and antibody responses. Using several strains of mice that exhibit differential degrees of resistance to mousepox, we show that recovery or death from ECTV infection is determined by a balance between the host's ability to produce IFN-γ and the virus' ability to dampen its effects.