Neurotropic influenza A virus infection causes prion protein misfolding into infectious prions in neuroblastoma cells.


Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice.

The cellular prion protein, PrPC, is a glycosylphosphatidylinositol anchored-membrane glycoprotein expressed most abundantly in neuronal and to a lesser extent in non-neuronal cells. Its conformational conversion into the amyloidogenic isoform in neurons is a key pathogenic event in prion diseases, including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals. However, the normal functions of PrPC remain largely unknown, particularly in non-neuronal cells. Here we show that stimulation of PrPC with anti-PrP monoclonal antibodies (mAbs) protected mice from lethal infection with influenza A viruses (IAVs), with abundant accumulation of anti-inflammatory M2 macrophages with activated Src family kinases (SFKs) in infected lungs. A SFK inhibitor dasatinib inhibited M2 macrophage accumulation in IAV-infected lungs after treatment with anti-PrP mAbs and abolished the anti-PrP mAb-induced protective activity against lethal influenza infection in mice. We also show that stimulation of PrPC with anti-PrP mAbs induced M2 polarization in peritoneal macrophages through SFK activation in vitro and in vivo. These results indicate that PrPC could activate SFK in macrophages and induce macrophage polarization to an anti-inflammatory M2 phenotype after stimulation with anti-PrP mAbs, thereby eliciting protective activity against lethal infection with IAVs in mice after treatment with anti-PrP mAbs. These results also highlight PrPC as a novel therapeutic target for IAV infection.


Prion protein protects mice from lethal infection with influenza A viruses.

The cellular prion protein, designated PrPC, is a membrane glycoprotein expressed abundantly in brains and to a lesser extent in other tissues. Conformational conversion of PrPC into the amyloidogenic isoform is a key pathogenic event in prion diseases. However, the physiological functions of PrPC remain largely unknown, particularly in non-neuronal tissues. Here, we show that PrPC is expressed in lung epithelial cells, including alveolar type 1 and 2 cells and bronchiolar Clara cells. Compared with wild-type (WT) mice, PrPC-null mice (Prnp0/0) were highly susceptible to influenza A viruses (IAVs), with higher mortality. Infected Prnp0/0 lungs were severely injured, with higher inflammation and higher apoptosis of epithelial cells, and contained higher reactive oxygen species (ROS) than control WT lungs. Treatment with a ROS scavenger or an inhibitor of xanthine oxidase (XO), a major ROS-generating enzyme in IAV-infected lungs, rescued Prnp0/0 mice from the lethal infection with IAV. Moreover, Prnp0/0 mice transgenic for PrP with a deletion of the Cu-binding octapeptide repeat (OR) region, Tg(PrPOR)/Prnp0/0 mice, were also highly susceptible to IAV infection. These results indicate that PrPC has a protective role against lethal infection with IAVs through the Cu-binding OR region by reducing ROS in infected lungs. Cu content and the activity of anti-oxidant enzyme Cu/Zn-dependent superoxide dismutase, SOD1, were lower in Prnp0/0 and Tg(PrPOR)/Prnp0/0 lungs than in WT lungs. It is thus conceivable that PrPC functions to maintain Cu content and regulate SOD1 through the OR region in lungs, thereby reducing ROS in IAV-infected lungs and eventually protecting them from lethal infection with IAVs. Our current results highlight the role of PrPC in protection against IAV infection, and suggest that PrPC might be a novel target molecule for anti-influenza therapeutics.


Anti-influenza virus activity of extracts from stems of Jatropha multifidi collected in Myanmar.

To contribute to the development of novel anti-influenza drugs, we investigated the anti-influenza activity of crude extracts from 118 medicinal plants collected in Myanmar. We discovered that extract from the stems of Jatropha multifida Linn. showed anti-influenza activity. J. multifida has been used in traditional medicine for the treatment of various diseases, and the stem has been reported to possess antimicrobial, antimalarial, and antitumor activities. However, the anti-influenza activity of this extract has not yet been investigated. We prepared water (HO), ethyl acetate (EtOAc), n-hexane (Hex), and chloroform (CHCl) extracts from the stems of J. multifida collected in Myanmar, and examined the survival of Madin-Darby canine kidney (MDCK) cells infected with the influenza A (H1N1) virus, and the inhibitory effects of these crude extracts on influenza A viral infection and growth in MDCK cells. The HO extracts from the stems of J. multifida promoted the survival of MDCK cells infected with the influenza A H1N1 virus. The EtOAc and CHCl extracts resulted in similar, but weaker, effects. The HO, EtOAc, and CHCl extracts from the stems of J. multifida inhibited influenza A virus H1N1 infection; the HO extract possessed the strongest inhibitory effect on influenza infection in MDCK cells. The EtOAc, Hex, and CHCl extracts all inhibited the growth of influenza A H1N1 virus, and the CHCl extract demonstrated the strongest activity in MDCK cells. The HO or CHCl extracts from the stems of J. multifida collected in Myanmar demonstrated the strongest inhibition of influenza A H1N1 viral infection or growth in MDCK cells, respectively. These results indicated that the stems of J. multifida could be regarded as an anti-influenza herbal medicine as well as a potential crude drug source for the development of anti-influenza compounds.

BMC Complementary and Alternative Medicine


Energy metabolic disorder is a major risk factor in severe influenza virus infection: Proposals for new therapeutic options based on animal model experiments

Severe influenza is characterized by cytokine storm and multiorgan failure. Influenza patients with underlying diseases show a rapid progression in disease severity. The major mechanism that underlies multiorgan failure during the progressive stage of infection, particularly in patients with underlying risk factors, is mitochondrial energy crisis. The relationship between the factors that determine infection severity, such as influenza virus, cytokines, cellular trypsin as a hemagglutinin processing protease for viral multiplication, accumulation of metabolic intermediates and ATP crisis in mitochondria, is termed the "influenza virus-cytokine-trypsin" cycle. This occurs during the initial stages of infection, and is interconnected with the "metabolic disorders-cytokine" cycle in the middle to late phase of infection. Experiments using animal models have highlighted the complex relationship between these two cycles. New treatment options have been proposed that target the ATP crisis and multiorgan failure during the late phase of infection, rather than antiviral treatments with neuraminidase inhibitors that work during the initial phase. These options are (i) restoration of glucose oxidation in mitochondria by diisopropylamine dichloroacetate, which inhibits infection-induced pyruvate dehydrogenase kinase 4 activity, and (ii) restoration of long-chain fatty acid oxidation in mitochondria by l-carnitine and bezafibrate, an agonist of peroxisome proliferation-activated receptors-β/δ, which transcriptionally upregulates carnitine palmitoyltransferase II. The latter is particularly effective in patients with influenza-associated encephalopathy who have thermolabile and short half-life compound variants of carnitine palmitoyltransferase II.


Induction of systemic and mucosal immunity and maintenance of its memory against influenza A virus by nasal vaccination using a new mucosal adjuvant SF-10 derived from pulmonary surfactant in young cynomolgus monkeys

Induction of systemic and mucosal immunity and maintenance of its memory was investigated in 12 young male cynomolgus monkeys after intranasal instillation of flu vaccine using a new mucosal adjuvant SF-10 derived from pulmonary surfactant constituents. Split-product of influenza virus A/California/7/2009(H1N1)pdm hemagglutinin vaccine (HAv) at 15μg with or without SF-10 and the adjuvant alone were instilled intranasally three times every 2 weeks. SF-10-adjuvanted HAv (SF-10-HAv) elicited significantly higher HAv-specific IgG and hemagglutinin inhibition (HI) titers in serum and HAv-specific secretory IgA and its neutralizing activities in nasal washes compared with HAv antigen and SF-10 alone. Significant cross-neutralizing activities of nasal washes after the third vaccination to several other H1N1 and H3N2 strains were observed. HI titers in serum and neutralizing activities in nasal washes reached peak levels at 6 weeks after initial vaccination, then gradually decreased after 10 weeks and returned to the baseline levels at 36 weeks. A single intranasal revaccination of SF-10-HAv at 36 weeks rapidly and significantly increased both immunity in serum and nasal washes compared with naïve monkeys. Revaccination by one or two doses achieved almost maximal immunity at 2 or 4 weeks after instillation. Statistically significant adverse effects (e.g., body weight loss, elevated body temperature, nasal discharge, change in peripheral blood leukocyte and platelet counts) were not observed for 2 weeks after vaccination of SF-10-HAv, HAv or SF-10 and also during the experimental period. These results in young monkey model suggest the potential of clinical use SF-10 for intranasal flu vaccine.


Bakuchiol is a phenolic isoprenoid with novel enantiomer-selective anti-influenza A virus activity involving Nrf2 activation

Influenza represents a substantial threat to human health and requires novel therapeutic approaches. Bakuchiol is a phenolic isoprenoid compound present in Babchi (Psoralea corylifolia L.) seeds. We examined the anti-influenza viral activity of synthetic bakuchiol using Madin-Darby canine kidney cells. We found that the naturally occurring form, (+)-(S)-bakuchiol, and its enantiomer, (-)-(R)-bakuchiol, inhibited influenza A viral infection and growth and reduced the expression of viral mRNAs and proteins in these cells. Furthermore, these compounds markedly reduced the mRNA expression of the host cell influenza A virus-induced immune response genes, interferon-β and myxovirus-resistant protein 1. Interestingly, (+)-(S)-bakuchiol had greater efficacy than (-)-(R)-bakuchiol, indicating that chirality influenced anti-influenza virus activity. In vitro studies indicated that bakuchiol did not strongly inhibit the activities of influenza surface proteins or the M2 ion channel, expressed in Chinese hamster ovary cells. Analysis of luciferase reporter assay data unexpectedly indicated that bakuchiol may induce some host cell factor(s) that inhibited firefly and Renilla luciferases. Next generation sequencing and KeyMolnet analysis of influenza A virus-infected and non-infected cells exposed to bakuchiol revealed activation of transcriptional regulation by nuclear factor erythroid 2-related factor (Nrf), and an Nrf2 reporter assay showed that (+)-(S)-bakuchiol activated Nrf2. Additionally, (+)-(S)-bakuchiol up-regulated the mRNA levels of two Nrf2-induced genes, NAD(P)H quinone oxidoreductase 1 and glutathione S-transferase A3. These findings demonstrated that bakuchiol had enantiomer-selective anti-influenza viral activity involving a novel effect on the host cell oxidative stress response.


Oral Administration of Lactobacillus pentosus Strain S-PT84 Enhances Anti-Influenza Virus-Specific IgG Production in Plasma after Limited Doses of Influenza Virus Vaccination in Mice


Influenza A virus infection of vascular endothelial cells induces GSK-3β-mediated β-catenin degradation in adherens junctions, with a resultant increase in membrane permeability.

Multiorgan failure with vascular hyperpermeability is the final outcome in the progression of seasonal influenza virus pneumonia and influenza-associated encephalopathy, and it is also common in infection with highly pathogenic avian influenza virus. However, the precise molecular mechanism by which influenza virus infection causes vascular endothelial cell hyperpermeability remains poorly defined. We investigated the mechanisms of hyperpermeability of human umbilical vein endothelial cells infected with influenza A virus (IAV)/Puerto Rico/8/34 (PR8) (H1N1). The levels of β-catenin, a key regulatory component of the vascular endothelial-cadherin cell adhesion complex, were markedly decreased during infection for 28 h, with increments of vascular hyperpermeability measured by transendothelial electrical resistance. Lactacystin (at 2 μM), a proteasome inhibitor, inhibited the decrease in β-catenin levels. Since the N-terminal phosphorylation of β-catenin by glycogen synthase kinase (GSK)-3β is the initiation step of proteasome-dependent degradation, we examined the effects of GSK-3β suppression by RNA interference in endothelial cells. IAV-infection-induced β-catenin degradation was significantly inhibited in GSK-3β-knockdown cells, and transfection of cells with recombinant β-catenin significantly suppressed IAV-induced hyperpermeability. These findings suggest that IAV infection induces GSK-3β-mediated β-catenin degradation in the adherens junctional complexes and induces vascular hyperpermeability. The in vitro findings of β-catenin degradation and activation of GSK-3β after IAV infection were confirmed in lungs of mice infected with IAV PR8 during the course of infection from day 0 to day 6. These results suggest that GSK-3β-mediated β-catenin degradation in adherens junctions is one of the key mechanisms of vascular hyperpermeability in severe influenza.


Immunomodulator clarithromycin enhances mucosal and systemic immune responses and reduces re-infection rate in pediatric patients with influenza treated with antiviral neuraminidase inhibitors: a retrospective analysis.

Treatment with antiviral neuraminidase inhibitors suppresses influenza viral replication and antigen production, resulting in marked attenuation of mucosal immunity and mild suppression of systemic immunity in mice. This study investigated the effects of immunomodulator clarithromycin (CAM) supplementation on mucosal and systemic immunity in pediatric patients with influenza treated with neuraminidase inhibitors. A retrospective, non-randomized case series study was conducted among five treatment groups of 195 children aged 5.9±3.3 years infected with influenza A in 2008/2009 season. The five treatment groups were oseltamivir (OSV), zanamivir (ZNV), OSV+CAM, ZNV+CAM and untreated groups. Anti-viral secretory IgA (S-IgA) levels in nasal washes and IgG levels in sera were measured. The re-infection rate was analyzed among the same five treatment groups in the 2009/2010 season. Treatment of influenza with OSV and ZNV for 5 days attenuated the induction of anti-viral S-IgA in nasal washes and anti-viral IgG in serum, compared with the untreated group. The combination of CAM plus OSV or ZNV boosted and restored the production of mucosal S-IgA and systemic IgG. The re-infection rates in the subsequent season were significantly higher in the OSV and ZNV groups than the untreated, while CAM+OSV and CAM+ZNV tended to reduce such rate. CAM restored the attenuated anti-viral mucosal and systemic immunity and reduced the re-infection rate in the subsequent year in pediatric patients with influenza treated with OSV and ZNV.


Anti-Influenza Activity of C60 Fullerene Derivatives.

The H1N1 influenza A virus, which originated in swine, caused a global pandemic in 2009, and the highly pathogenic H5N1 avian influenza virus has also caused epidemics in Southeast Asia in recent years. Thus, the threat from influenza A remains a serious global health issue, and novel drugs that target these viruses are highly desirable. Influenza A RNA polymerase consists of the PA, PB1, and PB2 subunits, and the N-terminal domain of the PA subunit demonstrates endonuclease activity. Fullerene (C60) is a unique carbon molecule that forms a sphere. To identify potential new anti-influenza compounds, we screened 12 fullerene derivatives using an in vitro PA endonuclease inhibition assay. We identified 8 fullerene derivatives that inhibited the endonuclease activity of the PA N-terminal domain or full-length PA protein in vitro. We also performed in silico docking simulation analysis of the C60 fullerene and PA endonuclease, which suggested that fullerenes can bind to the active pocket of PA endonuclease. In a cell culture system, we found that several fullerene derivatives inhibit influenza A viral infection and the expression of influenza A nucleoprotein and nonstructural protein 1. These results indicate that fullerene derivatives are possible candidates for the development of novel anti-influenza drugs.


Oral clarithromycin enhances airway IgA immunity through induction of IgA class switching recombination and B-cell activating factor of the tumor necrosis factor family molecule on mucosal dendritic cells in mice infected with influenza A virus

We previously reported that the macrolide antibiotic clarithromycin (CAM) enhanced the mucosal immune response in pediatric influenza, particularly in children treated with the antiviral neuraminidase inhibitor oseltamivir (OSV) with low production of mucosal antiviral secretory IgA (S-IgA). The aims of the present study were to confirm the effects of CAM on S-IgA immune responses, by using influenza A virus (IAV) H1N1-infected mice treated with or without OSV, and to determine the molecular mechanisms responsible for the induction of mucosal IgA class switching recombination in IAV-infected CAM-treated mice. The anti-IAV S-IgA responses and expression levels of IgA class switching recombination-associated molecules were examined in bronchus-lymphoid tissues and spleens of infected mice. We also assessed neutralization activities of S-IgA against IAV. Data show that CAM enhanced anti-IAV S-IgA induction in the airway of infected mice and restored the attenuated antiviral S-IgA levels in OSV-treated mice to the levels in the vehicle-treated mice. The expression levels of B-cell-activating factor of the tumor necrosis factor family (BAFF) molecule on mucosal dendritic cells as well as those of activation-induced cytidine deaminase and Iμ-Cα transcripts on B cells were enhanced by CAM, compared with the levels without CAM treatment, but CAM had no effect on the expression of the BAFF receptor on B cells. Enhancement by CAM of neutralization activities of airway S-IgA against IAV in vitro and reinfected mice was observed. This study identifies that CAM enhances S-IgA production and neutralizing activities through the induction of IgA class switching recombination and upregulation of BAFF molecules in mucosal dendritic cells in IAV-infected mice.


Role of host cellular proteases in the pathogenesis of influenza and influenza-induced multiple organ failure

Influenza A virus (IAV) is one of the most common infectious pathogens in humans. Since the IVA genome does not have the processing protease for the viral hemagglutinin (HA) envelope glycoprotein precursors, entry of this virus into cells and infectious organ tropism of IAV are primarily determined by host cellular trypsin-type HA processing proteases. Several secretion-type HA processing proteases for seasonal IAV in the airway, and ubiquitously expressed furin and pro-protein convertases for highly pathogenic avian influenza (HPAI) virus, have been reported. Recently, other HA-processing proteases for seasonal IAV and HPAI have been identified in the membrane fraction. These proteases proteolytically activate viral multiplication at the time of viral entry and budding. In addition to the role of host cellular proteases in IAV pathogenicity, IAV infection results in marked upregulation of cellular trypsins and matrix metalloproteinase-9 in various organs and cells, particularly endothelial cells, through induced pro-inflammatory cytokines. These host cellular factors interact with each other as the influenza virus-cytokine-protease cycle, which is the major mechanism that induces vascular hyperpermeability and multiorgan failure in severe influenza. This mini-review discusses the roles of cellular proteases in the pathogenesis of IAV and highlights the molecular mechanisms of upregulation of trypsins as effective targets for the control of IAV infection. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics


Surfactant protein C is an essential constituent for mucosal adjuvanticity of Surfacten, acting as an antigen delivery vehicle and inducing both local and systemic immunity

We have reported that Surfacten(®) (St), a bovine pulmonary surfactant free of antigenic c-type lectins, is a useful mucosal adjuvant for nasal vaccination. To prepare ample supplies a synthetic adjuvant that mimics St, we analyzed essential constituents of St for mucosal adjuvanticity. Intranasal inoculation of influenza virus hemagglutinin (HA) vaccine combined with St free of surfactant protein (SP)-C resulted in failure of HA vaccine delivery to dendritic cells and loss of local and systemic immune responses. Naïve bovine SP-C, synthetic human or bovine SP-C peptide reconstituted with three major St lipids restored delivery activity and local and systemic immune responses to levels similar to those of St and provided almost complete protection against lethal doses of influenza virus challenge in mice. The delivery of fluoresceinated HA vaccine to cultured dendritic cells was significantly enhanced by co-administration of St or synthetic adjuvant, and moderately stimulated the expression of MHC class II and CD86. In addition, both St and synthetic adjuvant markedly sustained HA vaccine and achieved a wide antigen distribution in murine nasal cavity. These results suggest that synthetic mucosal adjuvant reconstituted with SP-C peptide and major St lipids is useful for ample supply of the potent mucosal adjuvant as an antigen delivery vehicle for intranasal vaccination.


Attenuation of respiratory immune responses by antiviral neuraminidase inhibitor treatment and boost of mucosal immunoglobulin A response by co-administration of immuno- modulator clarithromycin in paediatric influenza.


Activation of the highly pathogenic avian influenza virus replication by membrane-bound proteases MSPL and TMPRSS13 and its inhibition by the protease inhibitors


Attenuation of inducible respiratory immune responses by oseltamivir treatment in mice infected with influenza A virus.

The antiviral neuraminidase inhibitor oseltamivir (OSV) is widely used to suppress viral replication in the treatment of influenza. Here, we report that OSV administration significantly suppressed respiratory mucosal secretory IgA responses with respect to antigen (Ag)-specific antibody (Ab) production and also the induction of Ag-specific IgA Ab-forming cells, but not systemic IgG responses, in weanling mice as a model of pediatric influenza. Neutralizing activities of the airway fluids in oral OSV-treated mice were significantly less than those of sham-treated mice. Our findings suggest the risk of re-infection in patients showing a low mucosal response following OSV treatment.


Novel type II transmembrane serine proteases, MSPL and TMPRSS13, proteolytically activate membrane fusion activity of hemagglutinin of highly pathogenic avian influenza viruses and induce their multicycle replication.

Host cellular proteases induce influenza virus entry into cells by cleaving the viral surface envelope glycoprotein hemagglutinin (HA). However, details on the cellular proteases involved in this event are not fully available. We report here that ubiquitous type II transmembrane serine proteases, MSPL and its splice variant TMPRSS13, are novel candidates for proteases processing HA proteins of highly pathogenic avian influenza (HPAI) viruses, apart from the previously identified furin and proprotein convertases 5 and 6. HAs from all HPAI virus H5 and H7 strains have one of two cleavage site motifs, the R-X-K/R-R motif with R at position P4 and the K-K/R-K/T-R motif with K at position P4. In studies of synthetic 14-residue HPAI virus HA peptides with these cleavage site motifs, furin preferentially cleaved only HA peptides with the R-K-K-R motif in the presence of calcium and not peptides with the other motif, whereas MSPL and TMPRSS13 cleaved both types of HA peptides (those with the R/K-K-K-R motif) efficiently in the absence of calcium. Full-length recombinant HPAI virus HA with the K-K-K-R cleavage motif exhibited poor susceptibility to cleavage in the absence of MSPL or TMPRSS13 and the presence of furin in infected cells, but it was converted to mature HA subunits in transfected cells expressing MSPL or TMPRSS13, with membrane-fused giant-cell formation. This conversion and membrane fusion were suppressed by inhibitors of MSPL and TMPRSS13. Furthermore, infection with and multiplication of genetically modified live HPAI virus A/Crow/Kyoto/53/2004 (H5N1) with the K-K-K-R cleavage site motif were detected only in MSPL- and TMPRSS13-expressing cells.


Host envelope glycoprotein processing proteases are indispensable for entry into human cells by seasonal and highly pathogenic avian influenza viruses.

Influenza A virus (IAV) is one of the most common infectious pathogens in humans and causes considerable morbidity and mortality. The recent spread of highly-pathogenic avian IAV H5N1 viruses has reinforced the importance of pandemic preparedness. In the pathogenesis of IAV infection, cellular proteases play critical roles in the process of viral entry into cells that subsequently leads to tissue damage in the infected organs. Since there are no processing protease for the viral membrane fusion glycoprotein hemagglutinin precursor (HA(0)) in IAV, entry of the virus into cells is determined primarily by the host cellular HA(0) processing proteases that proteolytically activate membrane fusion activity. HA(0) of seasonal human IAV has the consensus cleavage site motif Q(E)-T/X-R and is selectively processed by at least seven different trypsin-type processing proteases identified to-date in animal model experiments using mouse-adapted IAV or gene expression system in MDCK cells. As is the case for the highly pathogenic avian influenza (HPAI) A virus, endoproteolytic processing of the HA(0) occurs through ubiquitous cellular processing proteases, which selectively recognize the multi-basic consensus cleavage site motifs, such as R-X-K/R-R, and K-X-K/R-R. The cleavage enzymes for the R-X-K/R-R motif, but not K-X-K/R-R motif, have been reported to be furin and pro-protein convertase (PC)5/6 in the trans-Golgi network. Here we report new members of type II transmembrane serine proteases of the cell membrane, mosaic serine protease large form (MSPL) and its splice variant TMPRSS13, which recognize and cleave both R-X-K/R-R and K-X-K/R-R motifs without calcium. Furthermore, IAV infection significantly up-regulates a latent ectopic pancreatic trypsin, one of the potent HA processing proteases, and pro-matrix metalloprotease-9, in various organs. These proteases may synergistically damage the blood-brain barrier in the brain and basement membrane of blood vessels in various organs, resulting in severe edema and multiple organ failure. In this review, we discuss these proteases as new drug target molecules for IAV treatment acting by inhibition of IAV multiplication and prevention of multiple organ failure, other than anti-viral agents, viral neuraminidase inhibitors.

Journal of Molecular and Genetic Medicine


Involvement of autophagy in trypsinogen activation within the pancreatic acinar cells

Autophagy is mostly a nonselective bulk degradation system within cells. Recent reports indicate that autophagy can act both as a protector and killer of the cell depending on the stage of the disease or the surrounding cellular environment (for review see Cuervo, A.M. 2004. Trends Cell Biol. 14:70-77). We found that cytoplasmic vacuoles induced in pancreatic acinar cells by experimental pancreatitis were autophagic in origin, as demonstrated by microtubule-associated protein 1 light chain 3 expression and electron microscopy experiments. To analyze the role of macroautophagy in acute pancreatitis, we produced conditional knockout mice lacking the autophagy-related 5 gene in acinar cells. Acute pancreatitis was not observed, except for very mild edema in a restricted area, in conditional knockout mice. Unexpectedly, trypsinogen activation was greatly reduced in the absence of autophagy. These results suggest that autophagy exerts devastating effects in pancreatic acinar cells by activation of trypsinogen to trypsin in the early stage of acute pancreatitis through delivering trypsinogen to the lysosome.


Identification and analysis of the promoter region of the type II transmembrane serine protease polyserase-1 and its transcript variants

Polyserase-1/TMPRSS9 and its alternative transcripts, serase-1B and serase-2B, are novel type II transmembrane serine proteases that may regulate physiological and pathological phenomena on the cell surface. To understand the mechanisms of gene expression and regulation of these transcripts, we cloned and characterized the 5' promoter region of the mouse polyserase-1 (mpolyserase-1) gene. Using 5'-rapid amplification of cDNA ends, we located the transcription initiation site 272 nucleotides upstream of the translation initiation site. Luciferase reporter gene analysis revealed that the region from +186 to +272 bp in the 5'-untranslated region (UTR), containing the GATA motif (AGATAA), glucocorticoid responsible element (TGTTCT), and E-box sequence (CAGGTG), is required for maximal promoter activity. Mutations introduced into the E-box sequence but not elsewhere in the promoter region caused a selective decrease in transcriptional activity. Furthermore, a DNA probe (+229 to +255 bp) containing the E-box sequence formed a single nuclear protein complex in a sequence-specific manner. These data suggest that the expression of mpolyserase-1 and its transcript variants is positively regulated by the E-box in its 5'-UTR, which might be responsible for the binding of basic helix-loop-helix transcription factors involved in the development of various organelles.


Serase-1B, a new splice variant of polyserase-1/TMPRSS9, activates urokinase-type plasminogen activator and the proteolytic activation is negatively regulated by glycosaminoglycans.

Polyserase-1 (polyserine protease-1)/TMPRSS9 (transmembrane serine protease 9) is a type II transmembrane serine protease (TTSP) that possesses unique three tandem serine protease domains. However, the physiological function of each protease domain remains poorly understood. We discovered a new splice variant of polyserase-1, termed Serase-1B, which contains 34 extra amino acids consisting a SEA module (a domain found in sea urchin sperm protein, enterokinase and agrin) adjacent to the transmembrane domain and the first protease domain with a mucin-like box at the C-terminus. The tissue distribution of this enzyme by RT (reverse transcription)-PCR analysis revealed high expression in the liver, small intestine, pancreas, testis and peripheral blood CD14+ and CD8+ cells. To investigate the role of Serase-1B, a full-length form recombinant protein was produced. Interestingly, recombinant Serase-1B was partly secreted as a soluble inactive precursor and it was also activated by trypsin. This activated enzyme selectively cleaved synthetic peptides for trypsin and activated protein C, and it was inhibited by several natural serine protease inhibitors, such as aprotinin, alpha2-antiplasmin and plasminogen activator inhibitor 1. In addition, Serase-1B efficiently converted pro-uPA (urokinase-type plasminogen activator) into active uPA and this activation was strongly inhibited by these natural inhibitors. Furthermore, this activation was also negatively regulated by glycosaminoglycans. Our results indicate that Serase-1B is a novel member of TTSPs that might be involved in uPA/plasmin-mediated proteolysis and possibly implicated in biological events such as fibrinolysis and tumour progression.