Toll-like receptor 4 promotes bladder cancer progression upon S100A8/A9 binding, which requires TIRAP-mediated TPL2 activation.

Bladder cancer is an often widely disseminated and deadly cancer. To block the malignant outgrowth of bladder cancer, we must elucidate the molecular-level characteristics of not only bladder cancer cells but also their surrounding milieu. As part of this effort, we have long been studying extracellular S100A8/A9, which is elevated by the inflammation associated with certain cancers. Extracellularly enriched S100A8/A9 can hasten a shift to metastatic transition in multiple types of cancer cells. Intriguingly, high-level S100A8/A9 has been detected in the urine of bladder-cancer patients, and the level increases with the stage of malignancy. Nonetheless, S100A8/A9 has been investigated mainly as a potential biomarker of bladder cancers, and there have been no investigations of its role in bladder-cancer growth and metastasis. We herein report that extracellular S100A8/A9 induces upregulation of growth, migration and invasion in bladder cancer cells through its binding with cell-surface Toll-like receptor 4 (TLR4). Our molecular analysis revealed the TLR4 downstream signal that accelerates such cancer cell events. Tumor progression locus 2 (TPL2) was a key factor facilitating the aggressiveness of cancer cells. Upon binding of S100A8/A9 with TLR4, TPL2 activation was enhanced by an action with a TLR4 adaptor molecule, TIR domain-containing adaptor protein (TIRAP), which in turn led to activation of the mitogen-activated protein kinase (MAPK) cascade of TPL2. Finally, we showed that sustained inhibition of TLR4 in cancer cells effectively dampened cancer survival in vivo. Collectively, our results indicate that the S100A8/A9-TLR4-TPL2 axis influences the growth, survival, and invasive motility of bladder cancer cells.

Histidine-Rich Glycoprotein Suppresses the S100A8/A9-Mediated Organotropic Metastasis of Melanoma Cells.

The dissection of the complex multistep process of metastasis exposes vulnerabilities that could be exploited to prevent metastasis. To search for possible factors that favor metastatic outgrowth, we have been focusing on secretory S100A8/A9. A heterodimer complex of the S100A8 and S100A9 proteins, S100A8/A9 functions as a strong chemoattractant, growth factor, and immune suppressor, both promoting the cancer milieu at the cancer-onset site and cultivating remote, premetastatic cancer sites. We previously reported that melanoma cells show lung-tropic metastasis owing to the abundant expression of S100A8/A9 in the lung. In the present study, we addressed the question of why melanoma cells are not metastasized into the brain at significant levels in mice despite the marked induction of S100A8/A9 in the brain. We discovered the presence of plasma histidine-rich glycoprotein (HRG), a brain-metastasis suppression factor against S100A8/A9. Using S100A8/A9 as an affinity ligand, we searched for and purified the binding plasma proteins of S100A8/A9 and identified HRG as the major protein on mass spectrometric analysis. HRG prevents the binding of S100A8/A9 to the B16-BL6 melanoma cell surface via the formation of the S100A8/A9 complex. HRG also inhibited the S100A8/A9-induced migration and invasion of A375 melanoma cells. When we knocked down HRG in mice bearing skin melanoma, metastasis to both the brain and lungs was significantly enhanced. The clinical examination of plasma S100A8/A9 and HRG levels showed that lung cancer patients with brain metastasis had higher S100A8/A9 and lower HRG levels than nonmetastatic patients. These results suggest that the plasma protein HRG strongly protects the brain and lungs from the threat of melanoma metastasis.

c-Jun N-terminal kinase (JNK)-mediated phosphorylation of SARM1 regulates NAD+ cleavage activity to inhibit mitochondrial respiration.

Mitochondrial dysfunction is a key pathological feature of many different types of neurodegenerative disease. Sterile alpha and Toll/interleukin receptor motif-containing protein 1 (SARM1) has been attracting much attention as an important molecule for inducing axonal degeneration and neuronal cell death by causing loss of NAD (NADH). However, it has remained unclear what exactly regulates the SARM1 activity. Here, we report that NAD+ cleavage activity of SARM1 is regulated by its own phosphorylation at serine 548. The phosphorylation of SARM1 was mediated by c-jun N-terminal kinase (JNK) under oxidative stress conditions, resulting in inhibition of mitochondrial respiration concomitant with enhanced activity of NAD+ cleavage. Nonphosphorylatable mutation of Ser-548 or treatment with a JNK inhibitor decreased SARM1 activity. Furthermore, neuronal cells derived from a familial Parkinson's disease (PD) patient showed a congenitally increased level of SARM1 phosphorylation compared with that in neuronal cells from a healthy person and were highly sensitive to oxidative stress. These results indicate that JNK-mediated phosphorylation of SARM1 at Ser-548 is a regulator of SARM1 leading to inhibition of mitochondrial respiration. These findings suggest that an abnormal regulation of SARM1 phosphorylation is involved in the pathogenesis of Parkinson's disease and possibly other neurodegenerative diseases.

Newly developed anti-S100A8/A9 monoclonal antibody efficiently prevents lung tropic cancer metastasis.

The metastatic dissemination of cancer cells to remote areas of the body is the most problematic aspect in cancer patients. Among cancers, melanomas are notoriously difficult to treat due to their significantly high metastatic potential even during early stages. Hence, the establishment of advanced therapeutic approaches to regulate metastasis is required to overcome the melanoma disease. An accumulating mass of evidence has indicated a critical role of extracellular S100A8/A9 in melanoma distant metastasis. Lung S100A8/A9 is induced by melanoma cells from distant organs and it attracts these cells to its enriched lung environment since melanoma cells possess several receptors that sense the S100A8/A9 ligand. We hence aimed to develop a neutralizing antibody against S100A8/A9 that would efficiently block melanoma lung metastasis. Our protocol provided us with one prominent antibody, Ab45 that efficiently suppressed not only S100A8/A9-mediated melanoma mobility but also lung tropic melanoma metastasis in a mouse model. This prompted us to make chimeric Ab45, a chimera antibody consisting of mouse Ab45-Fab and human IgG2-Fc. Chimeric Ab45 also showed significant inhibition of the lung metastasis of melanoma. From these results, we have high hopes that the newly produced antibody will become a potential biological tool to block melanoma metastasis in future clinical settings.

exSSSRs (extracellular S100 soil sensor receptors)-Fc fusion proteins work as prominent decoys to S100A8/A9-induced lung tropic cancer metastasis.

Within the "seed and soil" theory of organ tropic cancer metastasis is a growing compilation of evidence that S100A8/A9 functions as a soil signal that attracts cancer cells to certain organs, which prove beneficial to their growth. S100A8/A9-sensing receptors including Toll-like receptor 4 (TLR4), advanced glycation end products (RAGE), and also important receptors we recently succeeded in identifying (EMMPRIN, NPTNß, MCAM, and ALCAM) have the potential to become promising therapeutic targets. In our study, we prepared extracellular regions of these novel molecules and fused them to human IgG2-Fc to extend half-life expectancy, and we evaluated the anti-metastatic effects of the purified decoy proteins on metastatic cancer cells. The purified proteins markedly suppressed S100A8/A9-mediated lung tropic cancer metastasis. We hence expect that our novel biologics may become a prominent medicine to prevent cancer metastasis in clinical settings through cutting the linkage between "seed and soil".

Neuroplastin-ß mediates S100A8/A9-induced lung cancer disseminative progression.

Compiling evidence indicates an unusual role of extracellular S100A8/A9 in cancer metastasis. S100A8/A9 secreted from either cancer cells or normal cells including epithelial and inflammatory cells stimulates cancer cells through S100A8/A9 sensor receptors in an autocrine or paracrine manner, leading to cancer cell metastatic progression. We previously reported a novel S100A8/A9 receptor, neuroplastin-ß (NPTNß), which plays a critical role in atopic dermatitis when it is highly activated in keratinocytes by an excess amount of extracellular S100A8/A9 in the inflammatory skin lesion. Interestingly, our expression profiling of NPTNß showed significantly high expression levels in lung cancer cell lines in a consistent manner. We hence aimed to determine the significance of NPTNß as an S100A8/A9 receptor in lung cancer. Our results showed that NPTNß has strong ability to induce cancer-related cellular events, including anchorage-independent growth, motility and invasiveness, in lung cancer cells in response to extracellular S100A8/A9, eventually leading to the expression of a cancer disseminative phenotype in lung tissue in vivo. Mechanistic investigation revealed that binding of S100A8/A9 to NPTNß mediates activation of NFIA and NFIB and following SPDEF transcription factors through orchestrated upstream signals from TRAF2 and RAS, which is linked to anchorage-independent growth, motility and invasiveness. Overall, our results indicate the importance of the S100A8/A9-NPTNß axis in lung cancer disseminative progression and reveal a pivotal role of its newly identified downstream signaling, TRAF2/RAS-NFIA/NFIB-SPDEF, in linking to the aggressive development of lung cancers.

Heavy-ion beam mutagenesis of the ectomycorrhizal agaricomycete Tricholoma matsutake that produces the prized mushroom "matsutake" in conifer forests.

Tricholoma matsutake is an ectomycorrhizal agaricomycete that produces the prized mushroom "matsutake" in Pinaceae forests. Currently, there are no available cultivars or cultivation methods that produce fruiting bodies. Heavy-ion beams, which induce mutations through double-stranded DNA breaks, have been used widely for plant breeding. In the present study, we examined whether heavy-ion beams could be useful in isolating T. matsutake mutants. An argon-ion beam gave a suitable lethality curve in relation to irradiation doses, accelerating killing at 100-150 Gy. Argon-ion beam irradiation of the agar plate cultures yielded several transient mutants whose colony morphologies differed from that of the wild-type strain at the first screening, but which did not persist following culture transfer. It also generated a mutant whose phenotype remained stable after repeated culture transfers. The stable pleiotropic mutant not only exhibited a different colony morphology to the wild type, but also showed increased degradation of dye-linked water-insoluble amylose and cellulose substrates. Thus, heavy-ion beams may be useful for isolating mutants of T. matsutake, although precautions may be required to maintain the mutants, without phenotypic reversion, during repetitive culture of their mycelia.

Signal Diversity of Receptor for Advanced Glycation End Products.

The receptor for advanced glycation end products (RAGE) is involved in inflammatory pathogenesis. It functions as a receptor to multiple ligands such as AGEs, HMGB1 and S100 proteins, activating multiple intracellular signaling pathways with each ligand binding. The molecular events by which ligand-activated RAGE controls diverse signaling are not well understood, but some progress was made recently. Accumulating evidence revealed that RAGE has multiple binding partners within the cytoplasm and on the plasma membrane. It was first pointed out in 2008 that RAGE's cytoplasmic tail is able to recruit Diaphanous-1 (Dia-1), resulting in the acquisition of increased cellular motility through Rac1/Cdc42 activation. We also observed that within the cytosol, RAGE's cytoplasmic tail behaves similarly to a Toll-like receptor (TLR4)-TIR domain, interacting with TIRAP and MyD88 adaptor molecules that in turn activate multiple downstream signals. Subsequent studies demonstrated the presence of an alternative adaptor molecule, DAP10, on the plasma membrane. The coupling of RAGE with DAP10 is critical for enhancing the RAGE-mediated survival signal. Interestingly, RAGE interaction on the membrane was not restricted to DAP10 alone. The chemotactic G-protein-coupled receptors (GPCRs) formyl peptide receptors1 and 2 (FPR1 and FPR2) also interacted with RAGE on the plasma membrane. Binding interaction between leukotriene B4 receptor 1 (BLT1) and RAGE was also demonstrated. All of the interactions affected the RAGE signal polarity. These findings indicate that functional interactions between RAGE and various molecules within the cytoplasmic area or on the membrane area coordinately regulate multiple ligand-mediated RAGE responses, leading to typical cellular phenotypes in several pathological settings. Here we review RAGE's signaling diversity, to contribute to the understanding of the elaborate functions of RAGE in physiological and pathological contexts.

Enzymatic activity of cell-free extracts from Burkholderia oxyphila OX-01 bio-converts (+)-catechin and (-)-epicatechin to (+)-taxifolin.

This study characterized the enzymatic ability of a cell-free extract from an acidophilic (+)-catechin degrader Burkholderia oxyphila (OX-01). The crude OX-01 extracts were able to transform (+)-catechin and (-)-epicatechin into (+)-taxifolin via a leucocyanidin intermediate in a two-step oxidation. Enzymatic oxidation at the C-4 position was carried out anaerobically using H2O as an oxygen donor. The C-4 oxidation occurred only in the presence of the 2R-catechin stereoisomer, with the C-3 stereoisomer not affecting the reaction. These results suggest that the OX-01 may have evolved to target both (+)-catechin and (-)-epicatechin, which are major structural units in plants.

A qPCR assay that specifically quantifies Tricholoma matsutake biomass in natural soil.

Tricholoma matsutake is an ectomycorrhizal basidiomycete that produces prized, yet uncultivable, "matsutake" mushrooms along densely developed mycelia, called "shiro," in the rhizosphere of coniferous forests. Pinus densiflora is a major host of this fungus in Japan. Measuring T. matsutake biomass in soil allows us to determine the kinetics of fungal growth before and after fruiting, which is useful for analyzing the conditions of the shiro and its surrounding mycorrhizosphere, predicting fruiting timing, and managing forests to obtain better crop yields. Here, we document a novel method to quantify T. matsutake mycelia in soil by quantifying a single-copy DNA element that is uniquely conserved within T. matsutake but is absent from other fungal species, including close relatives and a wide range of ectomycorrhizal associates of P. densiflora. The targeted DNA region was amplified quantitatively in cultured mycelia that were mixed with other fungal species and soil, as well as in an in vitro co-culture system with P. densiflora seedlings. Using this method, we quantified T. matsutake mycelia not only from shiro in natural environments but also from the surrounding soil in which T. matsutake mycelia could not be observed by visual examination or distinguished by other means. It was demonstrated that the core of the shiro and its underlying area in the B horizon are predominantly composed of fungal mycelia. The fungal mass in the A or A0 horizon was much lower, although many white mycelia were observed at the A horizon. Additionally, the rhizospheric fungal biomass peaked during the fruiting season.

DNAX-activating protein 10 (DAP10) membrane adaptor associates with receptor for advanced glycation end products (RAGE) and modulates the RAGE-triggered signaling pathway in human keratinocytes.

The receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of many inflammatory, degenerative, and hyperproliferative diseases, including cancer. Previously, we revealed mechanisms of downstream signaling from ligand-activated RAGE, which recruits TIRAP/MyD88. Here, we showed that DNAX-activating protein 10 (DAP10), a transmembrane adaptor protein, also binds to RAGE. By artificial oligomerization of RAGE alone or RAGE-DAP10, we found that RAGE-DAP10 heterodimer formation resulted in a marked enhancement of Akt activation, whereas homomultimeric interaction of RAGE led to activation of caspase 8. Normal human epidermal keratinocytes exposed to S100A8/A9, a ligand for RAGE, at a nanomolar concentration mimicked the pro-survival response of RAGE-DAP10 interaction, although at a micromolar concentration, the cells mimicked the pro-apoptotic response of RAGE-RAGE. In transformed epithelial cell lines, A431 and HaCaT, in which endogenous DAP10 was overexpressed, and S100A8/A9, even at a micromolar concentration, led to cell growth and survival due to RAGE-DAP10 interaction. Functional blocking of DAP10 in the cell lines abrogated the Akt phosphorylation from S100A8/A9-activated RAGE, eventually leading to an increase in apoptosis. Finally, S100A8/A9, RAGE, and DAP10 were overexpressed in the psoriatic epidermis. Our findings indicate that the functional interaction between RAGE and DAP10 coordinately regulates S100A8/A9-mediated survival and/or apoptotic response of keratinocytes.

Ectomycorrhizas in vitro between Tricholoma matsutake, a basidiomycete that associates with Pinaceae, and Betula platyphylla var. japonica, an early-successional birch species, in cool-temperate forests.

Tricholoma matsutake is an ectomycorrhizal basidiomycete that associates with Pinaceae in the Northern Hemisphere and produces prized "matsutake" mushrooms. We questioned whether the symbiont could associate with a birch that is an early-successional species in boreal, cool-temperate, or subalpine forests. In the present study, we demonstrated that T. matsutake can form typical ectomycorrhizas with Betula platyphylla var. japonica; the associations included a Hartig net and a thin but distinct fungal sheath, as well as the rhizospheric mycelial aggregate "shiro" that is required for fruiting in nature. The in vitro shiro also emitted a characteristic aroma. This is the first report of an ectomycorrhizal formation between T. matsutake and a deciduous broad-leaved tree in the boreal or cool-temperate zones that T. matsutake naturally inhabits.

Root endophyte symbiosis in vitro between the ectomycorrhizal basidiomycete Tricholoma matsutake and the arbuscular mycorrhizal plant Prunus speciosa.

We previously reported that Tricholoma matsutake and Tricholoma fulvocastaneum, ectomycorrhizal basidiomycetes that associate with Pinaceae and Fagaceae, respectively, in the Northern Hemisphere, could interact in vitro as a root endophyte of somatic plants of Cedrela odorata (Meliaceae), which naturally harbors arbuscular mycorrhizal fungi in South America, to form a characteristic rhizospheric colony or "shiro". We questioned whether this phenomenon could have occurred because of plant-microbe interactions between geographically separated species that never encounter one another in nature. In the present study, we document that these fungi formed root endophyte interactions and shiro within 140 days of inoculation with somatic plants of Prunus speciosa (=Cerasus speciosa, Rosaceae), a wild cherry tree that naturally harbors arbuscular mycorrhizal fungi in Japan. Compared with C. odorata, infected P. speciosa plants had less mycelial sheath surrounding the exodermis, and the older the roots, especially main roots, the more hyphae penetrated. In addition, a large number of juvenile roots were not associated with hyphae. We concluded that such root endophyte interactions were not events isolated to the interactions between exotic plants and microbes but could occur generally in vitro. Our pure culture system with a somatic plant allowed these fungi to express symbiosis-related phenotypes that varied with the plant host; these traits are innately programmed but suppressed in nature and could be useful in genetic analyses of plant-fungal symbiosis.

S100A11 is involved in the progression of colorectal cancer through the desmosome-catenin-TCF signaling pathway.

Compiling evidence has indicated that S100A11 expression at high levels is closely associated with various cancer species. Consistent with the results reported elsewhere, we have also revealed that S100A11 is highly expressed in squamous cell carcinoma, mesothelioma, and pancreatic cancers and plays a crucial role in cancer progression when secreted into extracellular fluid. Those studies are all focused on the extracellular role of S100A11. However, most of S100A11 is still present within cancer cells, although the intracellular role of S100A11 in cancer cells has not been fully elucidated. Thus, we aimed to investigate S100A11 functions within cancer cells, primarily focusing on colorectal cancer cells, whose S100A11 is abundantly present in cells and still poorly studied cancer for the protein. Our efforts revealed that overexpression of S100A11 promotes proliferation and migration, and downregulation inversely dampens those cancer behaviors. To clarify how intracellular S100A11 aids cancer cell activation, we tried to identify S100A11 binding proteins, resulting in novel binding partners in the inner membrane, many of which are desmosome proteins. Our molecular approach defined that S100A11 regulates the expression level of DSG1, a component protein of desmosome, by which S100A11 activates the TCF pathway via promoting nuclear translocation of γ-catenin from the desmosome. The identified new pathway greatly helps to comprehend S100A11's nature in colorectal cancers and others.

Lysyl oxidase-like 4 promotes the invasiveness of triple-negative breast cancer cells by orchestrating the invasive machinery formed by annexin A2 and S100A11 on the cell surface.

Background: Our earlier research revealed that the secreted lysyl oxidase-like 4 (LOXL4) that is highly elevated in triple-negative breast cancer (TNBC) acts as a catalyst to lock annexin A2 on the cell membrane surface, which accelerates invasive outgrowth of the cancer through the binding of integrin-ß1 on the cell surface. However, whether this machinery is subject to the LOXL4-mediated intrusive regulation remains uncertain. Methods: Cell invasion was assessed using a transwell-based assay, protein-protein interactions by an immunoprecipitation-Western blotting technique and immunocytochemistry, and plasmin activity in the cell membrane by gelatin zymography. Results: We revealed that cell surface annexin A2 acts as a receptor of plasminogen via interaction with S100A10, a key cell surface annexin A2-binding factor, and S100A11. We found that the cell surface annexin A2/S100A11 complex leads to mature active plasmin from bound plasminogen, which actively stimulates gelatin digestion, followed by increased invasion. Conclusion: We have refined our understanding of the role of LOXL4 in TNBC cell invasion: namely, LOXL4 mediates the upregulation of annexin A2 at the cell surface, the upregulated annexin 2 binds S100A11 and S100A10, and the resulting annexin A2/S100A11 complex acts as a receptor of plasminogen, readily converting it into active-form plasmin and thereby enhancing invasion.

Dissection of the signal transduction machinery responsible for the lysyl oxidase-like 4-mediated increase in invasive motility in triple-negative breast cancer cells: mechanistic insight into the integrin-ß1-NF-κB-MMP9 axis.

Background: Triple-negative breast cancer (TNBC) cells are a highly formidable cancer to treat. Nonetheless, by continued investigation into the molecular biology underlying the complex regulation of TNBC cell activity, vulnerabilities can be exposed as potential therapeutic targets at the molecular level. We previously revealed that lysyl oxidase-like 4 (LOXL4) promotes the invasiveness of TNBC cells via cell surface annexin A2 as a novel binding substrate of LOXL4, which promotes the abundant localization of integrin-ß1 at the cancer plasma membrane. However, it has yet to be uncovered how the LOXL4-mediated abundance of integrin-ß1 hastens the invasive outgrowth of TNBC cells at the molecular level. Methods: LOXL4-overexpressing stable clones were established from MDA-MB-231 cells and subjected to molecular analyses, real-time qPCR and zymography to clarify their invasiveness, signal transduction, and matrix metalloprotease (MMP) activity, respectively. Results: Our results show that LOXL4 potently promotes the induction of matrix metalloprotease 9 (MMP9) via activation of nuclear factor-κB (NF-κB). Our molecular analysis revealed that TNF receptor-associated factor 4 (TRAF4) and TGF-ß activated kinase 1 (TAK1) were required for the activation of NF-κB through Iκß kinase kinase (IKKα/ß) phosphorylation. Conclusion: Our results demonstrate that the newly identified LOXL4-mediated axis, integrin-ß1-TRAF4-TAK1-IKKα/ß-Iκßα-NF-κB-MMP9, is crucial for TNBC cell invasiveness.

Mobile DNA distributions refine the phylogeny of "matsutake" mushrooms, Tricholoma sect. Caligata.

"Matsutake" mushrooms are formed by several species of Tricholoma sect. Caligata distributed across the northern hemisphere. A phylogenetic analysis of matsutake based on virtually neutral mutations in DNA sequences resolved robust relationships among Tricholoma anatolicum, Tricholoma bakamatsutake, Tricholoma magnivelare, Tricholoma matsutake, and Tricholoma sp. from Mexico (=Tricholoma sp. Mex). However, relationships among these matsutake and other species, such as Tricholoma caligatum and Tricholoma fulvocastaneum, were ambiguous. We, therefore, analyzed genomic copy numbers of σ marY1 , marY1, and marY2N retrotransposons by comparing them with the single-copy mobile DNA megB1 using real-time polymerase chain reaction (PCR) to clarify matsutake phylogeny. We also examined types of megB1-associated domains, composed of a number of poly (A) and poly (T) reminiscent of RNA-derived DNA elements among these species. Both datasets resolved two distinct groups, one composed of T. bakamatsutake, T. fulvocastaneum, and T. caligatum that could have diverged earlier and the other comprising T. magnivelare, Tricholoma sp. Mex, T. anatolicum, and T. matsutake that could have evolved later. In the first group, T. caligatum was the closest to the second group, followed by T. fulvocastaneum and T. bakamatsutake. Within the second group, T. magnivelare was clearly differentiated from the other species. The data suggest that matsutake underwent substantial evolution between the first group, mostly composed of Fagaceae symbionts, and the second group, comprised only of Pinaceae symbionts, but diverged little within each groups. Mobile DNA markers could be useful in resolving difficult phylogenies due to, for example, closely spaced speciation events.

Root endophyte interaction between ectomycorrhizal basidiomycete Tricholoma matsutake and arbuscular mycorrhizal tree Cedrela odorata, allowing in vitro synthesis of rhizospheric "shiro".

The ectomycorrhizal basidiomycete Tricholoma matsutake associates with members of the Pinaceae such as Pinus densiflora (red pine), forming a rhizospheric colony or "shiro," which produces the prized "matsutake" mushroom. We investigated whether the host specificity of T. matsutake to conifers is innately determined using somatic plants of Cedrela odorata, a tropical broad-leaved tree (Meliaceae) that naturally harbors arbuscular mycorrhizal fungi. We found that T. matsutake could form in vitro shiro with C. odorata 140 days after inoculation, as with P. densiflora. The shiro was typically aromatic like that of P. densiflora. However, this was a root endophytic interaction unlike the mycorrhizal association with P. densiflora. Infected plants had epidermal tissues and thick exodermal tissues outside the inner cortex. The mycelial sheath surrounded the outside of the epidermis, and the hyphae penetrated into intra- and intercellular spaces, often forming hyphal bundles or a pseudoparenchymatous organization. However, the hyphae grew only in the direction of vascular bundles and did not form Hartig nets. Tricholoma fulvocastaneum or "false matsutake" naturally associates with Fagaceae and was also able to associate with C. odorata as a root endophyte. With T. matsutake, C. odorata generated a number of roots and showed greatly enhanced vigor, while with T. fulvocastaneum, it generated a smaller number of roots and showed somewhat lesser vigor. We argue that the host-plant specificity of ectomycorrhizal matsutake is not innately determined, and that somatic arbuscular mycorrhizal plants have a great potential to form mutualistic relationships with ectomycorrhizal fungi.

Near-complete de novo assembly of Tricholoma bakamatsutake chromosomes revealed the structural divergence and differentiation of Tricholoma genomes.

Tricholoma bakamatsutake, which is an edible ectomycorrhizal fungus associated with Fagaceae trees, may have diverged before the other species in Tricholoma section Caligata. We generated a highly contiguous whole-genome sequence for T. bakamatsutake SF-Tf05 isolated in an Oak (Quercus salicina) forest in Japan. The assembly of high-fidelity long reads, with a median read length of 12.3 kb, resulted in 13 chromosome-sized contigs comprising 142,068,211 bases with an average guanine and cytosine (GC) content of 43.94%. The 13 chromosomes were predicted to encode 11,060 genes. A contig (122,566 bases) presumably containing the whole circular mitochondrial genome was also recovered. The chromosome-wide comparison of T. bakamatsutake and Tricholoma matsutake (TMA_r1.0) indicated that the basic number of chromosomes (13) was conserved, but the structures of the corresponding chromosomes diverged, with multiple inversions and translocations. Gene conservation and cluster analyses revealed at least 3 phylogenetic clades in Tricholoma section Caligata. Specifically, all T. bakamatsutake strains belonged to the "bakamatsutake" clade, which is most proximal to the "caligatum" clade consisting of Tricholoma caligatum and Tricholoma fulvocastaneum. The constructed highly contiguous nearly telomere-to-telomere genome sequence of a T. bakamatsutake isolate will serve as a fundamental resource for future research on the evolution and differentiation of Tricholoma species.

STAT1/3 signaling suppresses axon degeneration and neuronal cell death through regulation of NAD+-biosynthetic and consuming enzymes.

Nicotinamide adenine dinucleotide (NAD)+-biosynthetic and consuming enzymes are involved in various intracellular events through the regulation of NAD+ metabolism. Recently, it has become clear that alterations in the expression of NAD+-biosynthetic and consuming enzymes contribute to the axonal stability of neurons. We explored soluble bioactive factor(s) that alter the expression of NAD+-metabolizing enzymes and found that cytokine interferon (IFN)-γ increased the expression of nicotinamide nucleotide adenylyltransferase 2 (NMNAT2), an NAD+-biosynthetic enzyme. IFN-γ activated signal transducers and activators of transcription 1 and 3 (STAT1/3) followed by c-Jun N-terminal kinase (JNK) suppression. As a result, STAT1/3 increased the expression of NMNAT2 at both mRNA and protein levels in a dose- and time-dependent manner and, at the same time, suppressed activation of sterile alpha and Toll/interleukin receptor motif-containing 1 (SARM1), an NAD+-consuming enzyme, and increased intracellular NAD+ levels. We examined the protective effect of STAT1/3 signaling against vincristine-mediated cell injury as a model of chemotherapy-induced peripheral neuropathy (CIPN), in which axonal degeneration is involved in disease progression. We found that IFN-γ-mediated STAT1/3 activation inhibited vincristine-induced downregulation of NMNAT2 and upregulation of SARM1 phosphorylation, resulting in modest suppression of subsequent neurite degradation and cell death. These results indicate that STAT1/3 signaling induces NMNAT2 expression while simultaneously suppressing SARM1 phosphorylation, and that both these actions contribute to suppression of axonal degeneration and cell death.