S2P intramembrane protease RseP degrades small membrane proteins and suppresses the cytotoxicity of intrinsic toxin HokB.

The site2-protease (S2P) family of intramembrane proteases (IMPs) is conserved in all kingdoms of life and cleaves transmembrane proteins within the membrane to regulate and maintain various cellular activities. RseP, an Escherichia coli S2P peptidase, is involved in the regulation of gene expression through the regulated cleavage of the two target membrane proteins (RseA and FecR) and in membrane quality control through the proteolytic elimination of remnant signal peptides. RseP is expected to have additional substrates and to be involved in other cellular processes. Recent studies have shown that cells express small membrane proteins (SMPs; single-spanning membrane proteins of approximately 50-100 amino acid residues) with crucial cellular functions. However, little is known about their metabolism, which affects their functions. This study investigated the possible RseP-catalyzed cleavage of E. coli SMPs based on the apparent similarity of the sizes and structures of SMPs to those of remnant signal peptides. We screened SMPs cleaved by RseP in vivo and in vitro and identified 14 SMPs, including HokB, an endogenous toxin that induces persister formation, as potential substrates. We demonstrated that RseP suppresses the cytotoxicity and biological functions of HokB. The identification of several SMPs as novel potential substrates of RseP provides a clue to a comprehensive understanding of the cellular roles of RseP and other S2P peptidases and highlights a novel aspect of the regulation of SMPs. IMPORTANCE Membrane proteins play an important role in cell activity and survival. Thus, understanding their dynamics, including proteolytic degradation, is crucial. E. coli RseP, an S2P family intramembrane protease, cleaves membrane proteins to regulate gene expression in response to environmental changes and to maintain membrane quality. To identify novel substrates of RseP, we screened small membrane proteins (SMPs), a group of proteins that have recently been shown to have diverse cellular functions, and identified 14 potential substrates. We also showed that RseP suppresses the cytotoxicity of the intrinsic toxin, HokB, an SMP that has been reported to induce persister cell formation, by degrading it. These findings provide new insights into the cellular roles of S2P peptidases and the functional regulation of SMPs.

Experimental evidence on prevention of infection by the ectoparasitic protozoans Ichthyobodo salmonis and Trichodina truttae in juvenile chum salmon using ultraviolet disinfection of rearing water.

In northern Japan, juvenile chum salmon Oncorhynchus keta (Walbaum) are released from hatcheries to enhance the fishery resource. Infections with ectoparasitic protozoans, particularly the flagellate Ichthyobodo salmonis and the ciliate Trichodina truttae, occasionally cause severe mortality among hatchery-reared juveniles. This study examined the susceptibility of the two parasites to wide-ranging UV irradiation (experiment 1) and then investigated whether UV disinfection of the rearing water using a commercial device was useful for preventing infections among juveniles in a small-scale rearing system over a 28-day period (experiment 2). In experiment 1, parasite mortality reached 100% with UV irradiation doses of ≥9.60 × 105  µW s/cm2 for I. salmonis and ≥8.40 × 105  µW s/cm2 for T. truttae. In experiment 2, disinfection of the rearing water at a UV irradiation dose of 2.2 × 106  µW s/cm2 succeeded in complete prevention of both parasites in the juvenile salmon. These results elucidate the minimum dose of UV irradiation for inactivation of I. salmonis and T. truttae, and demonstrate the usefulness of water disinfection using a commercial UV irradiation device to prevent infections by these parasites in hatchery-reared juvenile chum salmon.

Effects of dietary supplementation with oregano essential oil on prevention of the ectoparasitic protozoans Ichthyobodo salmonis and Trichodina truttae in juvenile chum salmon Oncorhynchus keta.

The present study performed three experiments to establish a practical prevention strategy for the ectoparasitic flagellate Ichthyobodo salmonis and ciliate Trichodina truttae in hatchery-reared juvenile chum salmon Oncorhynchus keta using dietary supplementation with oregano essential oil. Experiment 1 showed that a diet supplemented for 3 weeks with 0.02% oregano essential oil significantly prevented infection with I. salmonis and T. truttae in juveniles reared in small tanks. Experiment 2, in outdoor hatchery ponds, demonstrated that the oregano treatment completely prevented I. salmonis infection for 52 days and T. truttae infection for 38 days. Oregano-treated juvenile mortality attributable to infection with these protozoans also decreased to 7.6% of control juvenile mortality, confirming the utility of this treatment in cultured O. keta. Physiological analyses of the oregano-treated juveniles elucidated the treatment's safety in relation to their metabolism, osmoregulation, natural immunity and olfactory responses and also detected carvacrol (a major component of oregano essential oil which shows antimicrobial activity) on the skin. In experiment 3, exposure of the two protozoans to oregano essential oil revealed a weak antiparasitic action on the body surface of the juvenile O. keta. The overall results demonstrate that dietary oregano supplementation is a practical prevention strategy for I. salmonis and T. truttae in hatchery-reared juvenile O. keta and suggest the possibility that its anti-parasitic action is attributable to a component of the oil that emerges onto the skin of the body of the fish.

Epizootiology of the ectoparasitic protozoans Ichthyobodo salmonis and Trichodina truttae on wild chum salmon Oncorhynchus keta.

Infestations of the ectoparasitic flagellate Ichthyobodo salmonis and the ciliate Trichodina truttae have caused acute mortalities of hatchery-reared juvenile chum salmon Oncorhynchus keta in Hokkaido, northern Japan. This study examined the epizootiology of I. salmonis and T. truttae on wild chum salmon as a possible infection source of the 2 parasitic protozoans in hatcheries. Infestations by both ectoparasites were detected on freshwater-adapted adult and juvenile chum salmon in all 4 rivers examined. This is the first study of an anadromous Pacific salmonid to report infestation of I. salmonis and T. truttae in adults returning for spawning. Among the marine-inhabiting phase of chum salmon, infestation with I. salmonis, but not T. truttae, was observed on adults and juveniles. The 2 protozoans were experimentally transmitted at the same time from wild to hatchery-reared chum salmon juveniles, and caused a high rate of mortality in the hatchery fish. In freshwater, the proliferation rate of T. truttae was greater than that of I. salmonis. These observations show that the euryhaline ectoparasite I. salmonis can infest chum salmon throughout their life cycle, in both river and ocean habitats, whereas T. truttae is able to infest these salmonids only in freshwater. Furthermore, wild chum salmon were shown to be a potential infestation source for both T. truttae and I. salmonis in hatchery fish.

Do failures in non-technical skills contribute to fatal medical accidents in Japan? A review of the 2010-2013 national accident reports.

OBJECTIVES: We sought to clarify how large a proportion of fatal medical accidents can be considered to be caused by poor non-technical skills, and to support development of a policy to reduce number of such accidents by making recommendations about possible training requirements. DESIGN: Summaries of reports of fatal medical accidents, published by the Japan Medical Safety Research Organization, were reviewed individually. Three experienced clinicians and one patient safety expert conducted the reviews to determine the cause of death. Views of the patient safety expert were given additional weight in the overall determination. SETTING: A total of 73 summary reports of fatal medical accidents were reviewed. These reports had been submitted by healthcare organisations across Japan to the Japan Medical Safety Research Organization between April 2010 and March 2013. PRIMARY AND SECONDARY OUTCOME MEASURES: The cause of death in fatal medical accidents, categorised into technical skills, non-technical skills and inevitable progress of disease were evaluated. Non-technical skills were further subdivided into situation awareness, decision making, communication, team working, leadership, managing stress and coping with fatigue. RESULTS: Overall, the cause of death was identified as non-technical skills in 34 cases (46.6%), disease progression in 33 cases (45.2%) and technical skills in two cases (5.5%). In two cases, no consensual determination could be achieved. Further categorisation of cases of non-technical skills were identified as 14 cases (41.2%) of problems with situation awareness, eight (23.5%) with team working and three (8.8%) with decision making. These three subcategories, or combinations of them, were identified as the cause of death in 33 cases (97.1%). CONCLUSIONS: Poor non-technical skills were considered to be a significant cause of adverse events in nearly half of the fatal medical accidents examined. Improving non-technical skills may be effective for reducing accidents, and training in particular subcategories of non-technical skills may be especially relevant.

Nephron, Wilms' tumor-1 (WT1), and synaptopodin expression in developing podocytes of mice.

Newborn mouse glomeruli are still immature with a morphological feature of an early capillary loop stage, but infant mice do not manifest proteinuria. Little is known about the molecular mechanism whereby infant mice are resistant to proteinuria. Nephrin and synaptopodin are crucial for slit diaphragm and foot process (FP) formation for avoiding proteinuria. Nephrin tyrosine phosphorylation means a transient biological signaling required for FP repair or extension during nephrotic disease. Using an immunohistochemical technique, we examined the natural course of nephrin, Wilms' tumor-1 (WT1) and synaptopodin at 16.5 days of embryonic age (E16.5d) and E19.5d, 7 days of post-neonatal age (P7d) and P42d during renal development of mice. As a result, nephrin and synaptopodin were detected at E19.5d in S-shaped bodies. WT1, a transcriptional factor for nephrin, was detected in nucleus in podocyte-like cells in all stages. Nephrin tyrosine phosphorylation was evident in glomeruli at P7d, and this was associated with an early-stage of FP extension. Inversely, nephrin phosphorylation became faint at P42d, along with maturated FP. Based on the present results, we suggest the sequential molecular mechanism to protect growing mice from proteinuria: (i) WT1-induced nephrin production by podocytes in S-shaped bodies at E19.5d; (ii) Synchronized induction of synaptopodin at the same period; and (iii) FP extension is initiated at a milk-suckling stage under a nephrin tyrosine-phosphorylated condition, while it is arrested at an adult stage, associated with a loss of nephrin-based signaling.

Proposal for optimal placement platform of bikes using queueing networks.

In recent social experiments, rental motorbikes and rental bicycles have been arranged at nodes, and environments where users can ride these bikes have been improved. When people borrow bikes, they return them to nearby nodes. Some experiments have been conducted using the models of Hamachari of Yokohama, the Niigata Rental Cycle, and Bicing. However, from these experiments, the effectiveness of distributing bikes was unclear, and many models were discontinued midway. Thus, we need to consider whether these models are effectively designed to represent the distribution system. Therefore, we construct a model to arrange the nodes for distributing bikes using a queueing network. To adopt realistic values for our model, we use the Google Maps application program interface. Thus, we can easily obtain values of distance and transit time between nodes in various places in the world. Moreover, we apply the distribution of a population to a gravity model and we compute the effective transition probability for this queueing network. If the arrangement of the nodes and number of bikes at each node is known, we can precisely design the system. We illustrate our system using convenience stores as nodes and optimize the node configuration. As a result, we can optimize simultaneously the number of nodes, node places, and number of bikes for each node, and we can construct a base for a rental cycle business to use our system.

Roles of the membrane-reentrant ß-hairpin-like loop of RseP protease in selective substrate cleavage.

Molecular mechanisms underlying substrate recognition and cleavage by Escherichia coli RseP, which belongs to S2P family of intramembrane-cleaving proteases, remain unclear. We examined the function of a conserved region looped into the membrane domain of RseP to form a ß-hairpin-like structure near its active site in substrate recognition and cleavage. We observed that mutations disturbing the possible ß-strand conformation of the loop impaired RseP proteolytic activity and that some of these mutations resulted in the differential cleavage of different substrates. Co-immunoprecipitation and crosslinking experiments suggest that the loop directly interacts with the transmembrane segments of substrates. Helix-destabilising mutations in the transmembrane segments of substrates suppressed the effect of loop mutations in an allele-specific manner. These results suggest that the loop promotes substrate cleavage by selectively recognising the transmembrane segments of substrates in an extended conformation and by presenting them to the proteolytic active site, which contributes to substrate discrimination.

Identification of a flavin-containing S-oxygenating monooxygenase involved in alliin biosynthesis in garlic.

S-Alk(en)yl-l-cysteine sulfoxides are cysteine-derived secondary metabolites highly accumulated in the genus Allium. Despite pharmaceutical importance, the enzymes that contribute to the biosynthesis of S-alk-(en)yl-l-cysteine sulfoxides in Allium plants remain largely unknown. Here, we report the identification of a flavin-containing monooxygenase, AsFMO1, in garlic (Allium sativum), which is responsible for the S-oxygenation reaction in the biosynthesis of S-allyl-l-cysteine sulfoxide (alliin). Recombinant AsFMO1 protein catalyzed the stereoselective S-oxygenation of S-allyl-l-cysteine to nearly exclusively yield (RC SS )-S-allylcysteine sulfoxide, which has identical stereochemistry to the major natural form of alliin in garlic. The S-oxygenation reaction catalyzed by AsFMO1 was dependent on the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and flavin adenine dinucleotide (FAD), consistent with other known flavin-containing monooxygenases. AsFMO1 preferred S-allyl-l-cysteine to γ-glutamyl-S-allyl-l-cysteine as the S-oxygenation substrate, suggesting that in garlic, the S-oxygenation of alliin biosynthetic intermediates primarily occurs after deglutamylation. The transient expression of green fluorescent protein (GFP) fusion proteins indicated that AsFMO1 is localized in the cytosol. AsFMO1 mRNA was accumulated in storage leaves of pre-emergent nearly sprouting bulbs, and in various tissues of sprouted bulbs with green foliage leaves. Taken together, our results suggest that AsFMO1 functions as an S-allyl-l-cysteine S-oxygenase, and contributes to the production of alliin both through the conversion of stored γ-glutamyl-S-allyl-l-cysteine to alliin in storage leaves during sprouting and through the de novo biosynthesis of alliin in green foliage leaves.

Stromal cell-derived factor-1 (SDF1)-dependent recruitment of bone marrow-derived renal endothelium-like cells in a mouse model of acute kidney injury.

Ischemic acute kidney injury (AKI) is the most key pathological event for accelerating progression to chronic kidney disease through vascular endothelial injury or dysfunction. Thus, it is critical to elucidate the molecular mechanism of endothelial protection and regeneration. Emerging evidence indicates that bone marrow-derived cells (BMCs) contribute to tissue reconstitution in several types of organs post-injury, but little is known whether and how BMCs contribute to renal endothelial reconstitution, especially in an early-stage of AKI. Using a mouse model of ischemic AKI, we provide evidence that incorporation of BMCs in vascular components (such as endothelial and smooth muscle cells) becomes evident within four days after renal ischemia and reperfusion, associated with an increase in stromal cell-derived factor-1 (SDF1) in endothelium and that in CXCR4/SDF1-receptor in BMCs. Notably, anti-CXCR4 antibody decreased the numbers of infiltrated BMCs and BMC-derived endothelium-like cells, but not of BMC-derived smooth muscle cell-like cells. These results suggest that reconstitution of renal endothelium post-ischemia partially depends on a paracrine loop of SDF1-CXCR4 between resident endothelium and BMCs. Such a chemokine ligand-receptor system may be attributable for selecting a cellular lineage (s), required for renal vascular protection, repair and homeostasis, even in an earlier phase of AKI.

A Decrease in Glomerular Endothelial Cells and Endothelial-mesenchymal Transition during Glomerulosclerosis in the Tensin2-deficient Mice (ICGN strain).

The ICR-derived glomerulonephritis (ICGN) mouse is a unique model of nephrotic syndrome, and albuminuria becomes evident in a neonatal stage, due to a genetic mutation of tensin2. We previously provided evidence that an apparent decrease in nephrin, caused by tensin2-deficiencient states, leads to podocytopathy, albuminuria and eventually, chronic renal failure. In general, glomerular endothelial cells (ECs) function as a barrier through tight attachment of glomerular basement membrane to podocytes, while decreased ECs can worsen renal failure. Nevertheless, it is still unknown whether glomerular ECs are altered under the tensin-2-deficient states during the manifestation of chronic renal failure. Herein, we examined the changes of glomerular ECs, with focus on the expression of PECAM-1 and VE-cadherin (EC-specific markers), or of α-SMA (myofibroblast marker) in this mouse model by histological methods. Compared with the non-nephrotic (+/nep) mice, the nephrotic (nep/nep) mice exhibited the reduced expression of PECAM-1, or of VE-cadherin, in glomerular area. Notably, some glomerular ECs showed the positive stainings for both PECAM-1 and α-SMA, suggesting endothelial-to-mesenchymal transition (EndoMT) during progression of glomerular sclerosis. This is the first report showing that a decrease in glomerular ECs, at least in part, via EndoMT is involved in tensin2-deficient pathological conditions.

Up-regulation of cyclin-E(1) via proline-mTOR pathway is responsible for HGF-mediated G(1)/S progression in the primary culture of rat hepatocytes.

Hepatocyte growth factor (HGF) is a key ligand that elicits G1/S progression of epithelial cells, including hepatocytes. Proline is also required for DNA synthesis that is induced by growth factors in primary culture of hepatocytes. However, it remains unknown how proline contributes to the G1/S progression of hepatocytes. The primary culture of rat hepatocytes using HGF plus proline can be a conceptual model for elucidating the molecular linkage of amino acids and growth factors during G1/S progression. Using this in vitro model, we provide evidence that not only induction of cyclin-D1 by HGF but also up-regulation of cyclin-E1 by proline is required for hepatocytes to enter the S-phase. Proline-enhanced cyclin-E1 induction, without changing its mRNA level, is associated with the activation of mammalian target of rapamycin (mTOR)-dependent pathways. Indeed, proline enhanced the ribosomal protein S6 phosphorylations (i.e., mTOR target), concomitantly with an increase in cyclin-E1. Inversely, mTOR-inhibitor, rapamycin suppressed the proline-mediated induction of cyclin-E1. As a result, DNA synthesis of hepatocytes, which was induced by HGF in the presence of proline, was largely abolished by mTOR-inhibitor treatment. Such a co-mitogenic effect of proline was also dependent on collagen synthesis: collagen synthesis inhibitors, such as cis-OH-proline, diminished the proline-induced cyclin-E1, and then the G1/S progression of hepatocytes was also suppressed. Overall, proline-mediated mTOR activation and collagen synthesis were found critical for HGF-induced DNA synthesis, partly via the sufficient accumulation of cyclin-E1. This is the first report to demonstrate the molecular bridge between amino acids and growth factors that drive mitogenic outcomes.

HGF-MET cascade, a key target for inhibiting cancer metastasis: the impact of NK4 discovery on cancer biology and therapeutics.

Hepatocyte growth factor (HGF) was discovered in 1984 as a mitogen of rat hepatocytes in a primary culture system. In the mid-1980s, MET was identified as an oncogenic mutant protein that induces malignant phenotypes in a human cell line. In the early 1990s, wild-type MET was shown to be a functional receptor of HGF. Indeed, HGF exerts multiple functions, such as proliferation, morphogenesis and anti-apoptosis, in various cells via MET tyrosine kinase phosphorylation. During the past 20 years, we have accumulated evidence that HGF is an essential conductor for embryogenesis and tissue regeneration in various types of organs. Furthermore, we found in the mid-1990s that stroma-derived HGF is a major contributor to cancer invasion at least in vitro. Based on this background, we prepared NK4 as an antagonist of HGF: NK4 inhibits HGF-mediated MET tyrosine phosphorylation by competing with HGF for binding to MET. In vivo, NK4 treatments produced the anti-tumor outcomes in mice bearing distinct types of malignant cancers, associated with the loss in MET activation. There are now numerous reports showing that HGF-antagonists and MET-inhibitors are logical for inhibiting tumor growth and metastasis. Additionally, NK4 exerts anti-angiogenic effects, partly through perlecan-dependent cascades. This paper focuses on the chronology and significance of HGF-antagonisms in anti-tumor researches, with an interest in NK4 discovery. Tumor HGF-MET axis is now critical for drug resistance and cancer stem cell maintenance. Thus, oncologists cannot ignore this cascade for the future success of anti-metastatic therapy.

Dissociation of c-Met phosphotyrosine sites in human cells in response to mouse hepatocyte growth factor but not human hepatocyte growth factor: the possible roles of different amino acids in different species.

Hepatocyte growth factor (HGF) is essential for embryogenesis, tissue regeneration and tumour malignancy through the activation of its receptor, c-Met. We previously demonstrated that HGF α-chain hairpin-loop, K1 domain and ß-chain are required for c-Met signalling. The sequential phosphorylation of tyrosine residues, from c-Met kinase domain to multidocking regions, is required for HGF-signalling transduction. Herein, we provide evidence that the disconcerted activation of c-Met tyrosine regions fails to induce biological functions. When human cells were incubated with 'mouse HGF', kinase domain activation (i.e. phospho-Tyr-1230/34/35) became evident, but the multidocking site (i.e. Tyr-1349) was not phosphorylated, resulting in unsuccessful induction of migration and mitogenesis. The binding ability of mouse HGF α-chain, or of ß-chain, to human c-Met was lower than that of human HGF, as evidenced by HGF-chimera assay. Notably, only four amino acid positions in HGF α-chain hairpin-loop and K1 domain and six positions in ß-chain differed between human HGF and mouse HGF. The human-specific amino acids (such as Gln-95 in hairpin-loop, Arg-134 in K1 domain and Cys-561 in ß-chain) may be important for accurate c-Met assembly and signalling transduction.

Relationships between gill Na⁺,K⁺-ATPase activity and endocrine and local insulin-like growth factor-I levels during smoltification of masu salmon (Oncorhynchus masou).

We established profiles of insulin-like growth factor (IGF)-I mRNA in the liver, gill and white muscle and circulating IGF-I during smoltification of hatchery-reared masu salmon, and compared with that of gill Na(+),K(+)-ATPase (NKA) activity. Gill NKA activity peaked in May and dropped in June. Liver igf1 mRNA was high in March and decreased to low levels thereafter. Gill igf1 increased from March, maintained its high levels during April and May and decreased in June. Muscle igf1 mRNA levels were relatively high during January and April when water temperature was low. Serum IGF-I continuously increased from March through June. Serum IGF-I during March and May showed a positive correlation with NKA activity, although both were also related to fish size. These parameters were standardized with fork length and re-analyzed. As a result, serum IGF-I and gill igf1 were correlated with NKA activity. On the other hand, samples from desmoltification period (June) that had high serum IGF-I levels and low NKA activity disrupted the relationship. Expression of two IGF-I receptor (igf1r) subtypes in the gill decreased in June, which could account for the disruption by preventing circulating IGF-I from acting on the gill and retaining it in the blood. The present study suggests that the increase in gill NKA activity in the course of smoltification of masu salmon was supported by both endocrine and local IGF-I, and the decrease during desmoltification in freshwater was due at least in part to the down-regulation of gill IGF-I receptors.

Improvement of sepsis by hepatocyte growth factor, an anti-inflammatory regulator: emerging insights and therapeutic potential.

Sepsis-induced multiple organ failure (MOF) is the most frequent lethal disease in intensive care units. Thus, it is important to elucidate the self-defensive mechanisms of sepsis-induced MOF. Hepatocyte growth factor (HGF) is now recognized as an organotrophic factor, which is essential for organogenesis during embryonic growth and regeneration in adulthood. HGF production is enhanced in response to infectious challenges, but the increase in endogenous HGF levels is transient and insufficient, with a time lag between tissue injuries and HGF upregulation, during progression of septic MOF. Thus, administration of active-formed HGF might be a new candidate for therapeutic development of MOF. HGF has an ability to target endotoxin-challenged macrophages and inhibits the upregulation of inflammatory cytokines through nuclear factor-κB-inactivated mechanisms. HGF also targets the endothelium and epithelium of various organs to suppress local inflammation, coagulation, and apoptotic death. This paper summarizes the novel mechanisms of HGF for attenuating sepsis-related pathological conditions with a focus on sepsis-induced MOF.

Hepatocyte growth factor overexpression in the nervous system enhances learning and memory performance in mice.

Hepatocyte growth factor (HGF) and its receptor, c-Met, play pivotal roles in the nervous system during development and in disease states. However, the physiological roles of HGF in the adult brain are not well understood. In the present study, to assess its role in learning and memory function, we used transgenic mice that overexpress HGF in a neuron-specific manner (HGF-Tg) to deliver HGF into the brain without injury. HGF-Tg mice displayed increased alternation rates in the Y-maze test compared with age-matched wild-type (WT) controls. In the Morris water maze (MWM) test, HGF-Tg mice took less time to find the platform on the first day, whereas the latency to escape to the hidden platform was decreased over training days compared with WT mice. A transfer test revealed that the incidence of arrival at the exact location of the platform was higher for HGF-Tg mice compared with WT mice. These results demonstrate that overexpression of HGF leads to an enhancement of both short- and long-term memory. Western blot analyses revealed that the levels of N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR2B, but not NR1, were increased in the hippocampus of HGF-Tg mice compared with WT controls, suggesting that an upregulation of NR2A and NR2B could represent one mechanism by which HGF enhances learning and memory performance. These results demonstrate that modulation of learning and memory performance is an important physiological function of HGF that contributes to normal CNS plasticity, and we propose HGF as a novel regulator of higher brain functions.

Identification of mannose receptor as receptor for hepatocyte growth factor ß-chain: novel ligand-receptor pathway for enhancing macrophage phagocytosis.

Hepatocyte growth factor (HGF), a heterodimer composed of the α-chain and ß-chain, exerts multifunctional actions for tissue repair and homeostasis via its receptor, MET. HGF is cleaved by proteases secreted from inflammatory cells, and NK4 and ß-chain remnant (HGF-ß) are generated. Here, we provide evidence that HGF-ß binds to a new receptor other than MET for promoting a host cell clearance system. By an affinity cross-linking, radiolabeled HGF-ß was bound to liver non-parenchymal cells, particularly to Kupffer cells and sinusoidal endothelial cells, but not to parenchymal hepatocytes. The cross-linked complex was immunoprecipitated by anti-HGF antibody, but not anti-MET antibody, implying that HGF-ß binds to non-parenchymal cells at a site distinct from MET. Mass spectrometric detection of the ligand receptor complex revealed that the binding site of HGF-ß was the mannose receptor (MR). Actually, an ectopic expression of MR in COS-7 cells, which express no endogenous MR or MET, enabled HGF-ß to bind these cells at a K(D) of 89 nM, demonstrating that MR is the new receptor for HGF-ß. Interaction of HGF-ß and MR was diminished by EGTA, and by an enzymatic digestion of HGF-ß sugar chains, suggesting that MR may recognize the glycosylation site(s) of HGF-ß in a Ca(2+)-dependent fashion. Notably, HGF-ß, but not other MR ligands, enhanced the ingestion of latex beads, or of apoptotic neutrophils, by Kupffer cells, possibly via an F-actin-dependent pathway. Thus, the HGF-ß·MR complex may provide a new pathway for the enhancement of cell clearance systems, which is associated with resolution of inflammation.

Decreases in podocin, CD2-associated protein (CD2AP) and tensin2 may be involved in albuminuria during septic acute renal failure.

Podocytes have a peculiar structure constituting slit diaphragm (SD) and foot process (FP), and play essential roles in the glomerular filtration barrier. There is now ample evidence that SD- and FP-associated molecules, such as podocin and CD2-associated protein (CD2AP), are down-regulated during albuminuria of chronic kidney disease. However, it is still unclear whether these molecules are altered during acute renal failure (ARF) with albuminuria. Using lipopolysaccharide (LPS)-treated mice as a model of septic ARF, we provide evidence that the expression of SD- and FP-associated molecules becomes faint, along with albuminuria. In the LPS-treated mice, urinary albumin levels gradually increased, associated with the elevation of blood urea nitrogen levels, indicating the successful induction of albuminuria during septic ARF. In this pathological process, glomerular podocin expression became faint, especially at 36 hr post-LPS challenge (i.e., a peak of albuminuria). Likewise, LPS treatment led to a significant decrease in CD2AP, an anchorage between podocin and F-actin. With regard to this, tensin2 is a novel molecule that stabilizes F-actin extension. Interestingly, glomerular tensin2 expression levels were also decreased during the albuminuric phase, associated with losses of glomerular F-actin and synaptopodin under septic states. As a result, there were some lesions of podocytic FP effacement, as shown by electron microscopy. Based on these data, we emphasize the importance of concomitant decreases in podocin, CD2AP and tensin2 during septic ARF-associated proteinuria.

Preservations of nephrin and synaptopodin by recombinant hepatocyte growth factor in podocytes for the attenuations of foot process injury and albuminuria in nephritic mice.

AIM: Podocytes provide a slit diaphragm to inhibit proteinuria, and nephrin between podocytes functions as a barrier during glomerular filtration. Hepatocyte growth factor (HGF) can improve proteinuria in rodents with various renal injuries, but little is known about the role of HGF in podocyte-based events during glomerulonephritis. In the present study, we examined whether and how nephrin expression is sustained by podocytes during the HGF-mediated attenuation of albuminuria. METHODS: Lipopolysaccharide (LPS)-treated mice were used as an animal model of albuminuria. We evaluated the effect of HGF on slit proteins using immunohistochemistry, western blotting and real-time polymerase chain reaction. RESULTS: Albuminuria occurred 36 h after LPS treatment in mice. This albuminuria did not involve podocyte loss, but was associated with a decrease in nephrin and its key anchor, synaptopodin. In these processes, c-Met tyrosine phosphorylation, which represented HGF signal activation, occurred in glomerular cells including podocytes. When recombinant HGF was administrated to nephritic mice, c-Met tyrosine phosphorylation became evident in podocytes. The enhancement of the HGF-c-Met signal was associated with increases in nephrin and synaptopodin. An electron microscopic examination revealed that LPS induced the foot process effacement of podocytes, while HGF injections suppressed the foot process injury. Overall, albuminuria was attenuated in the LPS-treated mice after HGF administration. CONCLUSION: HGF protects podocytes from a loss of nephrin, at least in part, through maintaining synaptopodin. As a result, HGF was shown to sustain foot process structure, and albuminuria was attenuated under inflammation.