Protease Inhibitor-1 Research Articles

Alpha-secretase inhibition impairs Group I metabotropic glutamate receptor-mediated protein synthesis, long-term potentiation and long-term depression.

Group I metabotropic glutamate receptors (Gp1-mGluRs) exert a host of effects on cellular functions, including enhancement of protein synthesis and the associated facilitation of long-term potentiation (LTP) and induction of long-term depression (LTD). However, the complete cascades of events mediating these events are not fully understood. Gp1-mGluRs trigger α-secretase cleavage of amyloid precursor protein, producing soluble amyloid precursor protein-α (sAPPα), a known regulator of LTP. However, the α-cleavage of APP has not previously been linked to Gp1-mGluR's actions. Using rat hippocampal slices, we found that the α-secretase inhibitor tumour necrosis factor-alpha protease inhibitor-1, which inhibits both disintegrin and metalloprotease 10 (ADAM10) and 17 (ADAM17) activity, blocked or reduced the ability of the Gp1-mGluR agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) to stimulate protein synthesis, metaplastically prime future LTP and elicit sub-maximal LTD. In contrast, the specific ADAM10 antagonist GI254023X did not affect the regulation of plasticity, suggesting that ADAM17 but not ADAM10 is involved in mediating these effects of DHPG. However, neither drug affected LTD that was strongly induced by either high-concentration DHPG or paired-pulse synaptic stimulation. Our data suggest that moderate Gp1-mGluR activation triggers α-secretase sheddase activity targeting APP or other membrane-bound proteins as part of a more complex signalling cascade than previously envisioned. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

The susceptibility of SERPINE1 rs1799889 SNP in diabetic vascular complications: a meta-analysis of fifty-one case-control studies

BackgroundThe serine protease inhibitor-1 (SERPINE1) rs1799889 single nucleotide polymorphism (SNP) has been constantly associated with diabetes mellitus (DM) and its vascular complications. The aim of this meta-analysis was to evaluate this association with combined evidences.MethodsThe systematic search was performed for studies published up to March 2021 which assess the associations between SERPINE1 rs1799889 SNP and the risks of DM, diabetic retinopathy (DR), diabetic cardiovascular disease (CVD) and diabetic nephropathy (DN). Only case-control studies were identified, and the linkage between SERPINE1 rs1799889 polymorphism and diabetic vascular risks were evaluated using genetic models.Results51 comparisons were enrolled. The results revealed a significant association with diabetes risk in overall population (allelic: OR = 1.34, 95 % CI = 1.14–1.57, homozygous: OR = 1.66, 95 % CI = 1.23–2.14, heterozygous: OR = 1.35, 95 % CI = 1.08–1.69, dominant: OR = 1.49, 95 % CI = 1.18–1.88, recessive: OR = 1.30, 95 % CI = 1.06–1.59) as well as in Asian descents (allelic: OR = 1.45, 95 % CI = 1.16–1.82, homozygous: OR = 1.88, 95 % CI = 1.29–2.75, heterozygous: OR = 1.47, 95 % CI = 1.08-2.00, dominant: OR = 1.64, 95 % CI = 1.21–2.24, recessive: OR = 1.46, 95 % CI = 1.09–1.96). A significant association was observed with DR risk (homozygous: OR = 1.25, 95 % CI = 1.01–1.56, recessive: OR = 1.20, 95 % CI = 1.01–1.43) for overall population, as for the European subgroup (homozygous: OR = 1.32, 95 % CI = 1.02–1.72, recessive: OR = 1.38, 95 % CI = 1.11–1.71). A significant association were shown with DN risk for overall population (allelic: OR = 1.48, 95 % CI = 1.15–1.90, homozygous: OR = 1.92, 95 % CI = 1.26–2.95, dominant: OR = 1.41, 95 % CI = 1.01–1.97, recessive: OR = 1.78, 95 % CI = 1.27–2.51) and for Asian subgroup (allelic: OR = 1.70, 95 % CI = 1.17–2.47, homozygous: OR = 2.46, 95 % CI = 1.30–4.66, recessive: OR = 2.24, 95 % CI = 1.40–3.59) after ethnicity stratification. No obvious association was implied with overall diabetic CVD risk in any genetic models, or after ethnicity stratification.ConclusionsSERPINE1 rs1799889 4G polymorphism may outstand for serving as a genetic synergistic factor in overall DM and DN populations, positively for individuals with Asian descent. The association of SERPINE1 rs1799889 SNP and DR or diabetic CVD risks was not revealed.

MATRIX METALLOPROTEINASES AS MARKERS OF RESPIRATORY TRACT REMODELING AND POTENTIAL THERAPEUTIC TARGET IN PATIENTS WITH BRONCHIAL ASTHMA

It is evidently known that chronic inflammatory process in the bronchi of patients with bronchial asthma is associated with the emergence and progression of airway remodeling, resulting in irreversible obstruction. However, the exact mechanisms of connection between inflammation and airway remodeling are not very well understood and that is the reason for delay of development new specific targeted drugs aimed to inhibit the process of inflammation and remodeling. Therefore, our goal was aimed to analyze and systematize data on the role of matrix metalloproteinases in the occurrence of airway remodeling in patients with bronchial asthma and the possibility of therapeutic effects on this process. Materials and methods: search for information on the role and mechanisms of influence of matrix metalloproteinases on airway remodeling processes in patients with bronchial asthma and the possibility of its pharmacological correction in electronic databases such as PubMed and Google Scholar over the past 25 years. Matrix metalloproteinase-9 and tissue proteinase-1 inhibitor have been shown to play the most important role in airway remodeling in the presence of bronchial asthma, supported by numerous experimental and clinical studies. Much attention is paid to the comparison of these indicators in bronchoalveolar lavage, induced sputum and blood on the background of exacerbation and in the presence of a stable course of the disease. The analysis of their content depending on the severity of the course, dysfunction of external respiration and the degree of reversibility of bronchial obstruction. Studies of the possibility of drug effects on the content of remodeling markers have shown low clinical efficacy. The results are contradictory, but most of them prove the important role of matrix metalloproteinase-9, tissue protease inhibitor-1 and their relationship in the occurrence and progression of airway remodeling and, consequently, the severity of the disease, which dictates the need to develop new additional treatments.

Transmembrane tumor necrosis factor alpha attenuates pressure-overload cardiac hypertrophy via tumor necrosis factor receptor 2.

Tumor necrosis factor-alpha (TNF-α) plays an important pathogenic role in cardiac hypertrophy and heart failure (HF); however, anti-TNF is paradoxically negative in clinical trials and even worsens HF, indicating a possible protective role of TNF-α in HF. TNF-α exists in transmembrane (tmTNF-α) and soluble (sTNF-α) forms. Herein, we found that TNF receptor 1 (TNFR1) knockout (KO) or knockdown (KD) by short hairpin RNA or small interfering RNA (siRNA) significantly alleviated cardiac hypertrophy, heart dysfunction, fibrosis, and inflammation with increased tmTNF-α expression, whereas TNFR2 KO or KD exacerbated the pathological phenomena with increased sTNF-α secretion in transverse aortic constriction (TAC)- and isoproterenol (ISO)-induced cardiac hypertrophy in vivo and in vitro, respectively, indicating the beneficial effects of TNFR2 associated with tmTNF-α. Suppressing TNF-α converting enzyme by TNF-α Protease Inhibitor-1 (TAPI-1) to increase endogenous tmTNF-α expression significantly alleviated TAC-induced cardiac hypertrophy. Importantly, direct addition of exogenous tmTNF-α into cardiomyocytes in vitro significantly reduced ISO-induced cardiac hypertrophy and transcription of the pro-inflammatory cytokines and induced proliferation. The beneficial effects of tmTNF-α were completely blocked by TNFR2 KD in H9C2 cells and TNFR2 KO in primary myocardial cells. Furthermore, we demonstrated that tmTNF-α displayed antihypertrophic and anti-inflammatory effects by activating the AKT pathway and inhibiting the nuclear factor (NF)-κB pathway via TNFR2. Our data suggest that tmTNF-α exerts cardioprotective effects via TNFR2. Specific targeting of tmTNF-α processing, rather than anti-TNF therapy, may be more useful for the treatment of hypertrophy and HF.

Tazarotene-Induced Gene 1 (TIG1) Interacts with Serine Protease Inhibitor Kazal-Type 2 (SPINK2) to Inhibit Cellular Invasion of Testicular Carcinoma Cells.

Tazarotene-induced gene 1 (TIG1) encodes a protein that is a retinoid-regulated tumor suppressor. TIG1 is expressed in most normal tissues, and downregulation of TIG1 expression in multiple cancers is caused by promoter hypermethylation. Kazal-type serine protease inhibitor-2 (SPINK2) is a serine protease inhibitor, and the SPINK protein family has been shown to inhibit the expression of urokinase-type plasminogen activator (uPA). In addition, increased levels of uPA and the uPA receptor were observed in testicular cancer tissues. This study demonstrated that TIG1 interacts with SPINK2 in NT2/D1 testicular carcinoma cells. TIG1 and SPINK2 were highly expressed in normal testis tissues, while low expression levels of TIG1 and SPINK2 were found in testicular cancer tissues. TIG1 inhibited cell invasion, migration, and epithelial–mesenchymal transition (EMT) of NT2/D1 cells. SPINK2 enhanced TIG1-regulated uPA activity and EMT suppression, while silencing SPINK2 alleviated TIG1-mediated EMT regulation, cell migration, and invasion. Therefore, the results suggest that the interaction between TIG1 and SPINK2 plays an important role in the inhibition of testicular cancer cell EMT, and suppression is mediated through downregulation of the uPA/uPAR signaling pathway.

SERPINE2 rs16865421 polymorphism is associated with a lower risk of chronic obstructive pulmonary disease in the Uygur population: A case-control study.

The present study aimed to investigate the relationship between seven polymorphisms of the serine protease inhibitor-2 (SERPINE2) gene and the risk of chronic obstructive pulmonary disease (COPD) in the Uygur population via a case-control study. In total, 440 Uygur patients with COPD were included in the patient group and 384 healthy individuals were recruited in the matched control group. Data on demographic variables, smoking status, occupational dust exposure history and living conditions were collected. Polymorphism analysis was performed for seven loci of the SERPINE2 gene by mass spectrometry. The genotype distribution of rs16865421 showed a significant difference between the patient and control groups (p<0.05). Participants carrying the rs16865421-AG heterozygous mutant genotype had a lower risk of COPD compared to those with the rs16865421-A allele (odds ratio=0.68, 95% confidence interval=0.47-0.98, p = 0.041). However, no such association was found for rs1438831, rs6734100, rs6748795, rs7583463, rs840088 and rs975278. No significant interaction was observed between the genotypes and risk factors. Polymorphisms of rs16865421-AG carried by the Uygur population may be protective against COPD.

Vaccine potential of antigen cocktails composed of recombinant Toxoplasma gondii TgPI-1, ROP2 and GRA4 proteins against chronic toxoplasmosis in C3H mice

The development of an effective and safe vaccine to prevent Toxoplasma gondii infection is an important aim due to the great clinical and economic impact of this parasitosis. We have previously demonstrated that immunization with the serine protease inhibitor-1 (TgPI-1) confers partial protection to C3H/HeN and C57BL/6 mice. In order to improve the level of protection, in this work, we combined this novel antigen with ROP2 and/or GRA4 recombinant proteins (rTgPI-1+rROP2, rTgPI-1+rGRA4, rTgPI-1+rROP2+rGRA4) to explore the best combination against chronic toxoplasmosis in C3H/HeN mice. All tested vaccine formulations, administered following a homologous prime-boost protocol that combines intradermal and intranasal routes, conferred partial protection as measured by the reduction of brain cyst burden following oral challenge with tissue cysts of Me49 T. gondii strain. The highest level of protection was achieved by the mixture of rTgPI-1 and rROP2 proteins with an average parasite burden reduction of 50% compared to the unvaccinated control group. The vaccine-induced protective effect was related to the elicitation of systemic cellular and humoral immune responses that included antigen-specific spleen cell proliferation, the release of Th1/Th2 cytokines, and the generation of antigen-specific antibodies in serum. Additionally, mucosal immune responses were also induced, characterized by secretion of antigen-specific IgA antibodies in intestinal lavages and specific mesenteric lymph node cell proliferation. Our results demonstrate that rTgPI-1+rROP2 antigens seem a promising mixture to be combined with other immunogenic proteins in a multiantigenic vaccine formulation against toxoplasmosis.

Editorial: Control of Plant Pathogens by Biogenic Elicitors and Possible Mechanisms of Action.

Editorial: Control of Plant Pathogens by Biogenic Elicitors and Possible Mechanisms of Action.

Homologous prime-boost strategy with TgPI-1 improves the immune response and protects highly susceptible mice against chronic Toxoplasma gondii infection

Subunit-based vaccines are safer than live or attenuated pathogen vaccines, although they are generally weak immunogens. Thus, proper combination of immunization strategies and adjuvants are needed to increase their efficacy. We have previously protected C3H/HeN mice from Toxoplasma gondii infection by immunization with the serine protease inhibitor-1 (TgPI-1) in combination with alum. In this work, we explore an original vaccination protocol that combines administration of recombinant TgPI-1 by intradermal and intranasal routes in order to enhance protection in the highly susceptible C57BL/6 strain. Mice primed intradermally with rTgPI-1 plus alum and boosted intranasally with rTgPI-1 plus CpG-ODN elicited a strong specific Th1/Th2 humoral response, along with a mucosal immune response characterized by specific-IgA in intestinal lavages. A positive cellular response of mesentheric lymph node cells and Th1/Th2 cytokine secretion in the ileon were also detected. When immunized mice were challenged with the cystogenic Me49 T. gondii strain, they displayed up to 62% reduction in brain parasite burden. Moreover, adoptive transfer of mesenteric lymph node cells from vaccinated to naïve mice induced significant protection against infection. These results demonstrate that this strategy that combines the administration of TgPI-1 by two different routes, intradermal priming and intranasal boost, improves protective immunity against T. gondii chronic infection in highly susceptible mice.

Involvement of nitric oxide in the jasmonate-dependent basal defense against root-knot nematode in tomato plants.

Jasmonic acid (JA) and nitric oxide (NO) are well-characterized signaling molecules in plant defense responses. However, their roles in plant defense against root-knot nematode (RKN, Meloidogyne incognita) infection are largely unknown. In this study, we found that the transcript levels of the JA- and NO-related biosynthetic and signaling component genes were induced after RKN infection. Application of exogenous JA and sodium nitroprusside (SNP; a NO donor) significantly decreased the number of egg masses in tomato roots after RKN infection and partially alleviated RKN-induced decreases in plant fresh weight and net photosynthetic rate. These molecules also alleviated RKN-induced increases in root electrolyte leakage and membrane peroxidation. Importantly, NO scavenger partially inhibited JA-induced RKN defense. The pharmacological inhibition of JA biosynthesis significantly increased the plants’ susceptibility to RKNs, which was effectively alleviated by SNP application, showing that NO may be involved in the JA-dependent RKN defense pathway. Furthermore, both JA and SNP induced increases in protease inhibitor 2 (PI2) gene expression after RKN infestation. Silencing of PI2 compromised both JA- and SNP-induced RKN defense responses, suggesting that the PI2 gene mediates JA- and NO-induced defense against RKNs. This work will be important for deepening the understanding of the mechanisms involved in basal defense against RKN attack in plants.

Diacetyl induces amphiregulin shedding in pulmonary epithelial cells and in experimental bronchiolitis obliterans.

Diacetyl (DA), a component of artificial butter flavoring, has been linked to the development of bronchiolitis obliterans (BO), a disease of airway epithelial injury and airway fibrosis. The epidermal growth factor receptor ligand, amphiregulin (AREG), has been implicated in other types of epithelial injury and lung fibrosis. We investigated the effects of DA directly on the pulmonary epithelium, and we hypothesized that DA exposure would result in epithelial cell shedding of AREG. Consistent with this hypothesis, we demonstrate that DA increases AREG by the pulmonary epithelial cell line NCI-H292 and by multiple independent primary human airway epithelial donors grown under physiologically relevant conditions at the air-liquid interface. Furthermore, we demonstrate that AREG shedding occurs through a TNF-α-converting enzyme (TACE)-dependent mechanism via inhibition of TACE activity in epithelial cells using the small molecule inhibitor, TNF-α protease inhibitor-1, as well as TACE-specific small inhibitor RNA. Finally, we demonstrate supportive in vivo results showing increased AREG transcript and protein levels in the lungs of rodents with DA-induced BO. In summary, our novel in vitro and in vivo observations suggest that further study of AREG is warranted in the pathogenesis of DA-induced BO.

Field evaluation of the bacterial volatile derivative 3-pentanol in priming for induced resistance in pepper.

Plants are defended from attack by emission of volatile organic compounds (VOCs) that can act directly against pathogens and herbivores or indirectly by recruiting natural enemies of herbivores. However, microbial VOC have been less investigated as potential triggers of plant systemic defense responses against pathogens in the field. Bacillus amyloliquefaciens strain IN937a, a plant growth-promoting rhizobacterium that colonizes plant tissues, stimulates induced systemic resistance (ISR) via its emission of VOCs. We investigated the ISR capacity of VOCs and derivatives collected from strain IN937a against bacterial spot disease caused by Xanthomonas axonopodis pv. vesicatoria in pepper. Of 15 bacterial VOCs and their derivatives, 3-pentanol, which is a C8 amyl alcohol reported to be a component of sex pheromones in insects, was selected for further investigation. Pathogens were infiltrated into pepper leaves 10, 20, 30, and 40 days after treatment and transplantation to the field. Disease severity was assessed 7 days after transplantation. Treatment with 3-pentanol significantly reduced disease severity caused by X. axonopodis and naturally occurring Cucumber mosaic virus in field trials over 2 years. We used quantitative real-time polymerase chain analysis to examine Pathogenesis-Related genes associated with salicylic acid (SA), jasmonic acid (JA), and ethylene defense signaling. The expression of Capsicum annuum Pathogenesis-Related protein 1 (CaPR1), CaPR2, and Ca protease inhibitor2 (CaPIN2) increased in field-grown pepper plants treated with 3-pentanol. Taken together, our results show that 3-pentanol triggers induced resistance by priming SA and JA signaling in pepper under field conditions.

Identification of potential general markers of disease resistance in American oysters, Crassostrea virginica through gene expression studies

Several diseases have a significant impact on American oyster populations in the Atlantic coasts of North America. Knowledge about the responses of oysters to pathogenic challenge could help in identifying potential markers of disease resistance and biomarkers of the health status of an oyster population. A previous analysis of the transcriptome of resistant and susceptible American oysters in response to challenge with the bacterial pathogen Roseovarius crassostreae, as well as sequencing of suppression subtractive hybridization libraries from oysters challenged with the protozoan parasite Perkinsus marinus, provided a list of genes potentially involved in disease resistance or susceptibility. We investigated the patterns of inducible gene expression of several of these genes in response to experimental challenge with the oyster pathogens R. crassostreae, Vibrio tubiashii, and P. marinus. Oysters showing differential susceptibility to R. crassostreae demonstrated differential patterns of expression of genes coding for immune (serine protease inhibitor-1, SPI1) and stress-related (heat shock protein 70, HSP70; arginine kinase) proteins 30 days after challenge with this bacterial pathogen. Differential patterns of expression of immune (spi1, galectin and a matrix metalloproteinase) and stress-related (hsp70, histone H4, and arginine kinase) genes was observed in hemocytes from adult oysters challenged with P. marinus, but not with V. tubiashii. While levels of spi1 expression in hemocytes collected 8 and 21 days after P. marinus challenge were negatively correlated with parasite load in oysters tissues at the end of the challenge (62 days), levels of expression of hsp70 in hemocytes collected 1-day after challenge were positively correlated with oyster parasite load at 62 days. Our results confirm previous research on the role of serine protease inhibitor-1 in immunity and disease resistance in oysters. They also suggest that HSP70 and histone H4 could be used as a markers of health status or disease susceptibility in oysters.

Vaccination with a genetically modified Brugia malayi cysteine protease inhibitor-2 reduces adult parasite numbers and affects the fertility of female worms following a subcutaneous challenge of Mongolian gerbils (Meriones unguiculatus) with B. malayi infective larvae

Vaccination of Mongolian gerbils with Brugia malayi cysteine protease inhibitor-2 in which the amino acid Asn66 was mutated to Lys66 (Bm-CPI-2M) resulted in reduced parasite numbers of 48.6% and 48.0% at 42 and 90days p.i. with B. malayi L3s. Fertility of female worms was also affected at 90days p.i. In vitro killing of L3s observed in the presence of gerbil peritoneal exudate cells and anti-Bm-CPI-2M sera suggests antibody-dependent cell-mediated cytotoxicity as a putative protective mechanism. These observations suggest that Bm-CPI-2M is a promising prophylactic and anti-fecundity vaccine candidate.

Deletion of Parasite Immune Modulatory Sequences Combined with Immune Activating Signals Enhances Vaccine Mediated Protection against Filarial Nematodes

BackgroundFilarial nematodes are tissue-dwelling parasites that can be killed by Th2-driven immune effectors, but that have evolved to withstand immune attack and establish chronic infections by suppressing host immunity. As a consequence, the efficacy of a vaccine against filariasis may depend on its capacity to counter parasite-driven immunomodulation.Methodology and Principal FindingsWe immunised mice with DNA plasmids expressing functionally-inactivated forms of two immunomodulatory molecules expressed by the filarial parasite Litomosoides sigmodontis: the abundant larval transcript-1 (LsALT) and cysteine protease inhibitor-2 (LsCPI). The mutant proteins enhanced antibody and cytokine responses to live parasite challenge, and led to more leukocyte recruitment to the site of infection than their native forms. The immune response was further enhanced when the antigens were targeted to dendritic cells using a single chain Fv-αDEC205 antibody and co-administered with plasmids that enhance T helper 2 immunity (IL-4) and antigen-presenting cell recruitment (Flt3L, MIP-1α). Mice immunised simultaneously against the mutated forms of LsALT and LsCPI eliminated adult parasites faster and consistently reduced peripheral microfilaraemia. A multifactorial analysis of the immune response revealed that protection was strongly correlated with the production of parasite-specific IgG1 and with the numbers of leukocytes present at the site of infection.ConclusionsWe have developed a successful strategy for DNA vaccination against a nematode infection that specifically targets parasite-driven immunosuppression while simultaneously enhancing Th2 immune responses and parasite antigen presentation by dendritic cells.

Kinetic Analysis, Expression Pattern, and Production of a Recombinant Fungal Protease Inhibitor of Tasar Silkworm Antheraea mylitta

Antheraea mylitta, a tasar silk-producing insect of Saturniidae family, expresses a fungal protease inhibitor named as A. mylitta fungal protease inhibitor-1 (AmFPI-1). AmFPI-1 inhibits alkaline protease of Aspergillus oryzae but its mechanism of action is not known. To understand the mode of inhibition of AmFPI-1 against the fungal protease, it was purified from the hemolymph of A. mylitta larvae and inhibitory activity against A. oryzae protease was studied. Kinetic analysis of purified AmFPI-1 on alkaline protease of A. oryzae showed that AmFPI-1 acts as a canonical-type competitive inhibitor with equilibrium dissociation constant (K ( i )) of 60nM. Expression of AmFPI-1 in different body tissues of fifth instar A. mylitta larvae was determined by real-time PCR, and the highest expression was observed in fat body followed by integument, silk gland, and gut, indicating that AmFPI-1 has pleiotropic functions including protection from invading fungi. The cDNA of AmFPI-1 was expressed in Escherichia coli, and recombinant His-tagged fusion protein was purified by Ni-NTA chromatography. Recombinant AmFPI-1 showed inhibitory activity against A. oryzae protease and suggested its use in various biological applications to prevent proteolysis.

Insect Eggs Can Enhance Wound Response in Plants: A Study System of Tomato Solanum lycopersicum L. and Helicoverpa zea Boddie

Insect oviposition on plants frequently precedes herbivory. Accumulating evidence indicates that plants recognize insect oviposition and elicit direct or indirect defenses to reduce the pressure of future herbivory. Most of the oviposition-triggered plant defenses described thus far remove eggs or keep them away from the host plant or their desirable feeding sites. Here, we report induction of antiherbivore defense by insect oviposition which targets newly hatched larvae, not the eggs, in the system of tomato Solanum lycopersicum L., and tomato fruitworm moth Helicoverpa zea Boddie. When tomato plants were oviposited by H. zea moths, pin2, a highly inducible gene encoding protease inhibitor2, which is a representative defense protein against herbivorous arthropods, was expressed at significantly higher level at the oviposition site than surrounding tissues, and expression decreased with distance away from the site of oviposition. Moreover, more eggs resulted in higher pin2 expression in leaves, and both fertilized and unfertilized eggs induced pin2 expression. Notably, when quantified daily following deposition of eggs, pin2 expression at the oviposition site was highest just before the emergence of larvae. Furthermore, H. zea oviposition primed the wound-induced increase of pin2 transcription and a burst of jasmonic acid (JA); tomato plants previously exposed to H. zea oviposition showed significantly stronger induction of pin2 and higher production of JA upon subsequent simulated herbivory than without oviposition. Our results suggest that tomato plants recognize H. zea oviposition as a signal of impending future herbivory and induce defenses to prepare for this herbivory by newly hatched neonate larvae.

Flagellin/TLR5 responses induce mucus hypersecretion by activating EGFR via an epithelial cell signaling cascades

Mucus hypersecretion is an important manifestation in patients with chronic inflammatory airway diseases. Excessive production of mucin leads to airway mucus obstruction and contributes to morbidity and mortality in these diseases. The molecular mechanisms underlying mucin overproduction, however, still remain largely unknown. Here, we report that the bacterium Pseudomonas aeruginosa (P. aeruginosa), an important human respiratory pathogen, induced MUC5AC mucin expression via an epithelial cell signaling cascade in human airway epithelial cells. The flagellin purified from P. aeruginosa up-regulated MUC5AC expression by activating its receptor Toll-like receptor 5 (TLR5) in 16HBE cells. This effect was inhibited by NADPH oxidase inhibitor (DPI), small interfering RNA of dual oxidase 2 (Duox2) and reactive oxygen species (ROS) scavengers (nPG and DMSO). Flagellin induced TGF-α release, and stimulated phosphorylated epidermal growth factor receptor (EGFR) and MUC5AC overproduction. These effects were prevented by EGFR and TGF-α neutralizing antibodies, metalloprotease inhibitors (GM6001 and TNF-α protease inhibitor-1) and specific knockdown of TNF-α-converting enzyme (TACE) with TACE siRNA. These findings may bring new insights into the molecular pathogenesis of P. aeruginosa infections and lead to novel therapeutic intervention for mucin overproduction in patients with P. aeruginosa infections.

Tumor suppressor cell adhesion molecule 1 (CADM1) is cleaved by a disintegrin and metalloprotease 10 (ADAM10) and subsequently cleaved by γ-secretase complex

Cell adhesion molecule 1 (CADM1) is a type I transmembrane glycoprotein expressed in various tissues. CADM1 is a cell adhesion molecule with many functions, including roles in tumor suppression, apoptosis, mast cell survival, synapse formation, and spermatogenesis. CADM1 undergoes membrane-proximal cleavage called shedding, but the sheddase and mechanisms of CADM1 proteolysis have not been reported. We determined the cleavage site involved in CADM1 shedding by LC/MS/MS and showed that CADM1 shedding occurred in the membrane fraction and was inhibited by tumor necrosis factor-α protease inhibitor-1 (TAPI-1). An siRNA experiment revealed that ADAM10 mediates endogenous CADM1 shedding. In addition, the membrane-bound fragment generated by shedding was further cleaved by γ-secretase and generated CADM1-intracellular domain (ICD) in a mechanism called regulated intramembrane proteolysis (RIP). These results clarify the detailed mechanism of membrane-proximal cleavage of CADM1, suggesting the possibility of RIP-mediated CADM1 signaling.

In Vivo Invasion of Head and Neck Squamous Cell Carcinoma Cells Does Not Require Macrophages

Invasion of tumor cells into the local stroma is an important component in cancer progression. Here we report studies of the in vivo invasion of head and neck squamous cell carcinoma (HNSCC) cells in response to applied gradients of a growth factor [epidermal growth factor (EGF)] and a chemokine (CXCL12), using orthotopic floor-of-mouth models. Analysis of the invading cells indicated that >75% of them were tumor cells, about 15% macrophages, and <10% were unidentified. Surprisingly, although macrophages invaded together with tumor cells, macrophage contributions were not required for HNSCC invasion. CXCL12-induced in vivo invasion of HNSCC cells was also observed and found to occur via a unidirectional transactivation of epidermal growth factor receptor (EGFR) through CXCR4. Inhibition of tumor necrosis factor-α-converting enzyme using TNF-α protease inhibitor-2 selectively inhibited CXCL12-induced invasion but not EGF-induced invasion, consistent with CXCL12 activation of EGFR via release of EGFR ligands.