Hypothalamic gonadotropin-releasing hormone (GnRH) neurons originate in the olfactory placode and migrate to the forebrain during embryonic development. We found that GnRH neurons migrated in two different modes in the chick medial telencephalon: they initially underwent axophilic migration in association with a subset of olfactory fibers in a dorsocaudal direction. This was followed by ventrally directed tangential migration to the basal forebrain. Since many of the ventrally migrating GnRH neurons did not follow distinct fiber fascicles, it is proposed that diffusible guidance molecules played a role in this migratory process. A long-range diffusible factor, netrin 1, was expressed in the lower part of the commissural plate and the subpallial septum, but not along the axophilic migratory route of GnRH neurons. Failure of ventrally directed migration of GnRH neurons and their misrouting to the dorsomedial forebrain was induced by misexpression of netrin 1 in the dorsocaudal part of the septum near the top of the commissural plate, which is where the migration of GnRH neurons changed to a ventral direction. In such cases, a subset of olfactory fibers also extended, but close contact between aberrant fibers and misrouted GnRH neurons did not exist. A coculture experiment demonstrated that netrin 1 exerts an attractive effect on migrating GnRH neurons. These results provide evidence that netrin 1 acts as chemoattractant to migrating GnRH neurons at the dorsocaudal part of the septum and has the potential to regulate the ventral migration of GnRH neurons to the ventral septum and the preoptic area.

Helicobacter cinaedi is the most common enterohepatic Helicobacter species that causes bacteremia in humans, but its pathogenicity is unclear. Here, we investigated the possible association of H. cinaedi with atherosclerosis in vivo and in vitro. We found that H. cinaedi infection significantly enhanced atherosclerosis in hyperlipidaemic mice. Aortic root lesions in infected mice showed increased accumulation of neutrophils and F4/80(+) foam cells, which was due, at least partly, to bacteria-mediated increased expression of proinflammatory genes. Although infection was asymptomatic, detection of cytolethal distending toxin RNA of H. cinaedi indicated aorta infection. H. cinaedi infection altered expression of cholesterol receptors and transporters in cultured macrophages and caused foam cell formation. Also, infection induced differentiation of THP-1 monocytes. These data provide the first evidence of a pathogenic role of H. cinaedi in atherosclerosis in experimental models, thereby justifying additional investigations of the possible role of enterohepatic Helicobacter spp. in atherosclerosis and cardiovascular disease.

The estrogen receptor (ER) functions as a dimer and is involved in several different biological functions. However ER dimeric proteins have not been identified by in situ methodologies. Structured illumination microscopy (SIM) has been recently developed, which enabled the localization of protein and protein interaction. Therefore, in this study, we firstly demonstrated that ERs formed both homodimers and heterodimers in breast carcinoma cell lines using Nikon's SIM (N-SIM). ERα/α homodimers were detected in the nuclei of both ERα-positive MCF-7 and T-47D cells; 23.0% and 13.4% of ERα proteins formed ERα/α homodimers, respectively. ERα/β heterodimers were also detected in MCF-7 and T-47D. Approximately 6.6% of both ERα and ERβ1 proteins formed ERα/β1 heterodimers in MCF-7. In addition, 18.1% and 22.4% of ERα and ERβ proteins formed ERα/β2 heterodimers and ERα/β5 heterodimers in MCF-7, respectively. In addition, by using proximity ligation assay (PLA) in MCF-7, estradiol-induced ERα/α homodimers and ERα/β1 heterodimers were both detected after 15 to 45 min of treatment and at 15 min, respectively. The percentage of total ER proteins could also be determined using N-SIM. By using both methods, it has become possible to evaluate precise localization and ratio of ER dimers among different cell types.

Cysteine persulfide and cysteine polysulfides are cysteine derivatives having sulfane sulfur atoms bound to cysteine thiol. Accumulating evidence has suggested that cysteine persulfides/polysulfides are abundant in prokaryotes and eukaryotes and play important roles in diverse biological processes such as antioxidant host defense and redox-dependent signal transduction. Here, we show that enhancement of cellular polysulfides by using polysulfide donors developed in this study resulted in marked inhibition of lipopolysaccharide (LPS)-initiated macrophage activation. Polysulfide donor treatment strongly suppressed LPS-induced pro-inflammatory responses in macrophages by inhibiting Toll-like receptor 4 (TLR4) signaling. Other TLR signaling stimulants-including zymosan A-TLR2 and poly(I:C)-TLR3-were also significantly suppressed by polysulfur donor treatment. Administration of polysulfide donors protected mice from lethal endotoxin shock. These data indicate that cellular polysulfides negatively regulate TLR4-mediated pro-inflammatory signaling and hence constitute a potential target for inflammatory disorders.

The six-layered neocortex is a shared characteristic of all mammals, but not of non-mammalian species, and its formation requires an inside-out pattern of neuronal migration. The extant reptilian dorsal cortex is thought to represent an ancestral form of the neocortex, although how the reptilian three-layered cortex is formed is poorly understood. Here, we show unique patterns of lamination and neuronal migration in the developing reptilian cortex. While the multipolar-to-bipolar transition of migrating neurons is essential for mammalian cortical development, the reptilian cortex lacks bipolar-shaped migrating neurons, resulting in an outside-in pattern of cortical development. Furthermore, dynamic regulation of Wnt signal strengths contributes to neuronal morphological changes, which is conserved across species. Our data preclude the idea that the six-layered mammalian neocortex emerged by simple addition to the reptilian dorsal cortex but suggest that the acquisition of a novel neuronal morphology based on conserved developmental programs contributed to neocortical evolution.

Estrogen receptors promote target gene transcription when they form a dimer, in which two identical (homodimer) or different (heterodimer) proteins are bound to each other. In hormone-dependent cancers, hormone receptor dimerization plays pivotal roles, not only in the pathogenesis or development of the tumors, but also in the development of therapeutic resistance. Protein⁻protein interactions (PPIs), including dimerization and complex formation, have been also well-known to be required for proteins to exert their functions. The methods which could detect PPIs are genetic engineering (i.e., resonance energy transfer) and/or antibody technology (i.e., co-immunoprecipitation) using cultured cells. In addition, visualization of the target proteins in tissues can be performed using antigen⁻antibody reactions, as in immunohistochemistry. Furthermore, development of microscopic techniques (i.e., electron microscopy and confocal laser microscopy) has made it possible to visualize intracellular and/or intranuclear organelles. We have recently reported the visualization of estrogen receptor dimers in breast cancer tissues by using the in situ proximity ligation assay (PLA). PLA was developed along the lines of antibody technology development, and this assay has made it possible to visualize PPIs in archival tissue specimens. Localization of PPI in organelles has also become possible using super-resolution microscopes exceeding the resolution limit of conventional microscopes. Therefore, in this review, we summarize the methodologies used for studying PPIs in both cells and tissues, and review the recently reported studies on PPIs of hormones.

Sirtuin 7 (SIRT7) is an NAD+-dependent lysine deacetylase that regulates diverse biological processes. We recently observed that SIRT7 deficiency suppresses the nuclear accumulation of p65, which is a component of nuclear factor kappa B. However, the underlying molecular mechanism remains elusive. In this study, we demonstrated that SIRT7 interacts with a small GTPase, Ras-related nuclear antigen (Ran), and deacetylates Ran at K37. The nuclear export of p65 was facilitated in SIRT7-deficient fibroblast cells, while the nuclear export was inhibited in SIRT7-deficient cells expressing K37R-Ran (deacetylation-mimicking mutant). Additionally, the nuclear export of p65 in wild-type fibroblast cells was promoted by K37Q-Ran (acetylation-mimicking mutant). K37Q-Ran exhibited an increased ability to bind to chromosome region maintenance 1 (CRM1), which is a major nuclear receptor that mediates the export of cargo proteins, and enhanced the binding between p65 and CRM1. These data suggest that SIRT7 is a lysine deacetylase that targets the K37 residue of Ran to suppress the nuclear export of p65.

The NLRP3 inflammasome is a multiprotein complex responsible for the maturation of precursor forms of interleukin (IL)-1β and IL-18 into active proinflammatory cytokines. Increasing evidence suggests that modulation of redox homeostasis contributes to the activation of the NLRP3 inflammasome. However, specific mechanistic details remain unclear. We demonstrate here that ATP exposure evoked a sharp decrease in glutathione (GSH) levels in macrophages, which led to NLRP3 inflammasome activation. We detected an increase in GSH levels in culture supernatants that was comparable to the GSH decrease in macrophages, which suggests that exposure to ATP stimulated GSH efflux. Exogenous addition of P2X7 receptor antagonist, GSH, or the oxidized form GSSG attenuated this efflux. Also, exogenous GSH or GSSG strongly inhibited NLRP3 inflammasome activation in vitro and in vivo. These data suggest that GSH efflux controls NLRP3 inflammasome activation, which may lead to development of novel therapeutic strategies for NLRP3 inflammasome-associated disorders.

To identify structures that determine the 90 degree orientation of thin espalier dendritic trees of Purkinje cells with respect to parallel fibers (axonal neurite bundles of granule cells) in the cerebellar cortex, we designed five types of two-dimensional and three-dimensional cell and tissue cultures of cerebella from postnatal mice and analyzed the orientation of Purkinje cell dendrites with respect to neurite bundles and astrocyte fibers by immunofluorescence double or triple staining. We cultured dissociated cerebellar cells on micropatterned substrates and preformed neurite bundles of a microexplant culture two-dimensionally and in matrix gels three-dimensionally. Dendrites, but not axons, of Purkinje cells extended toward the neurites of granule cells and oriented at right angles two-dimensionally to aligned neurite bundles in the three cultures. In a more organized explant proper of the microexplant culture, Purkinje cell dendrites extended toward thin aligned neurite bundles not only consistently at right angles but also two-dimensionally. However, in the "organotypic microexplant culture," in which three-dimensionally aligned thick neurite bundles mimicking parallel fibers were produced, Purkinje cell dendrites often oriented perpendicular to the thick bundles three-dimensionally. Astrocytes were abundant in all cultures, and there was no definite correlation between the presence of and orientation to Purkinje cell dendrites, although their fibers were frequently associated in parallel with dendrites in the organotypic microexplant culture. Therefore, Purkinje cells may grow their dendrites to the newly produced neurite bundles of parallel fibers in the cerebellar cortex and be oriented at right angles three-dimensionally mainly via "perpendicular contact guidance."

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and its expression is influenced by environmental compounds, such as 3-methylcholanthrene (3-MC) and β-naphthoflavone (β-NF). AhR and its downstream genes, such as CYP1A1, are considered to play a pivotal role in xenobiotic responses. AhR signaling has also been proposed to mediate osteogenesis in experimental animals, but its details have remained unclear. Therefore, in this study, we examined the possible roles of AhR in human bone. Immunohistochemical analysis revealed that AhR was detected in both osteoblasts and osteoclasts. We then screened AhR-target genes using a microarray analysis in human osteoblastic hFOB cells. Results of microarray and subsequent PCR analysis did reveal that estrogen metabolizing and synthesizing enzymes, such as CYP1B1 and aromatase, were increased by 3-MC in hFOB and osteosarcoma cell line, MG-63. The subsequent antibody cytokine analysis also demonstrated that interleukin-1β and -6 expression was increased by 3-MC and β-NF in hFOB cells and these interleukins were well known to induce aromatase. We then examined the cell proliferation rate of hFOB and MG-63 cells co-treated with 3-MC and testosterone as an aromatase substrate. The status of cell proliferation in both hFOB and MG-63 cells was stimulated by 3-MC and testosterone treatment, which was also inhibited by an estrogen blocker, aromatase inhibitor, or AhR antagonist. These findings indicated that AhR could regulate estrogen synthesis and metabolism in bone tissues through cytokine/aromatase signaling.

SP7/Osterix (OSX) is a master regulatory transcription factor that activates a variety of genes during differentiation of osteoblasts. However, the influence of post-translational modifications on the regulation of its transactivation activity is largely unknown. Here, we report that sirtuins, which are NAD(+)-dependent deacylases, regulate lysine deacylation-mediated transactivation of OSX. Germline Sirt7 knockout mice develop severe osteopenia characterized by decreased bone formation and an increase of osteoclasts. Similarly, osteoblast-specific Sirt7 knockout mice showed attenuated bone formation. Interaction of SIRT7 with OSX leads to the activation of transactivation by OSX without altering its protein expression. Deacylation of lysine (K) 368 in the C-terminal region of OSX by SIRT7 promote its N-terminal transactivation activity. In addition, SIRT7-mediated deacylation of K368 also facilitates depropionylation of OSX by SIRT1, thereby increasing OSX transactivation activity. In conclusion, our findings suggest that SIRT7 has a critical role in bone formation by regulating acylation of OSX.

Changes in genomic structures underlie phenotypic diversification in organisms. Amino acid-changing mutations affect pleiotropic functions of proteins, although little is known about how mutated proteins are adapted in existing developmental programs. Here we investigate the biological effects of a variant of the GLI3 transcription factor (GLI3R1537C) carried in Neanderthals and Denisovans, which are extinct hominins close to modern humans. R1537C does not compromise protein stability or GLI3 activator-dependent transcriptional activities. In contrast, R1537C affects the regulation of downstream target genes associated with developmental processes. Furthermore, genome-edited mice carrying the Neanderthal/Denisovan GLI3 mutation exhibited various alterations in skeletal morphology. Our data suggest that an extinct hominin-type GLI3 contributes to species-specific anatomical variations, which were tolerated by relaxed constraint in developmental programs during human evolution.

p62/SQSTM1 (p62) is a multifunctional protein implicated in several signal transduction pathways and selectively degraded by autophagy, a process for lysosomal degradation of both protein and organelle. p62 was also recently reported to be overexpressed in various malignancies and its inhibition to suppress carcinoma cell proliferation. However, its correlation with autophagy in carcinoma cells has remained largely unknown. Therefore, in this study, we examined the effects of p62 inhibition on the regulation of autophagy and cell survival in p62-positive carcinoma cells. p62-silencing dramatically suppressed cell proliferation and induced autophagy in p62 expressing PC9 and A549 cells. Electron microscopical analysis revealed the formation of autophagosomes with multilayer membranes caused by p62-silencing. p62 silencing-mediated reduced cell viability was restored by both genomic and pharmacological inhibition of autophagy but not that of apoptosis. These findings were also detected in several types of carcinoma cell lines including adenocarcinomas and squamous cell carcinomas. Results of our present study revealed that an inhibition of p62 resulted in the formation of mis-regulated autophagosomes with multilayer membranes and an autophagic cell death, and p62 can therefore be an attractive target for the development of anti-neoplastic agents.

The amplification of distinct neural stem/progenitor cell subtypes during embryogenesis is essential for the intricate brain structures present in various vertebrate species. For example, in both mammals and birds, proliferative neuronal progenitors transiently appear on the basal side of the ventricular zone of the telencephalon (basal progenitors), where they contribute to the enlargement of the neocortex and its homologous structures. In placental mammals, this proliferative cell population can be subdivided into several groups that include Tbr2(+) intermediate progenitors and basal radial glial cells (bRGs). Here, we report that basal progenitors in the developing avian pallium show unique morphological and molecular characteristics that resemble the characteristics of bRGs, a progenitor population that is abundant in gyrencephalic mammalian neocortex. Manipulation of LGN (Leu-Gly-Asn repeat-enriched protein) and Cdk4/cyclin D1, both essential regulators of neural progenitor dynamics, revealed that basal progenitors and Tbr2(+) cells are distinct cell lineages in the developing avian telencephalon. Furthermore, we identified a small population of subapical mitotic cells in the developing brains of a wide variety of amniotes and amphibians. Our results suggest that unique progenitor subtypes are amplified in mammalian and avian lineages by modifying common mechanisms of neural stem/progenitor regulation during amniote brain evolution.

Estrogen plays an important role in the development of estrogen-dependent breast carcinoma. Recently, several studies demonstrated a possible involvement of several micro RNAs (miRNAs) in the development of resistance to endocrine therapy in breast cancer patients, but the correlation between estrogen actions and miRNA expression in breast carcinoma still remains largely unknown. Therefore, in this study, we examined the in vitro effects of estrogen upon miRNA expression profiles in breast carcinoma.

Mitogen-activated protein kinases (MAPKs) are involved in the intracellular pathways that respond to various extracellular signals. Extracellular signal-regulated kinase (ERK) is a member of MAPKs and has various functions in neural development. However, the in vivo distribution of the activated form of ERK (p-ERK) in the developing nervous system is not well understood. Here, we investigated the expression of p-ERK in the spinal cord and dorsal root ganglion (DRG) of chick embryos. In the spinal cord, p-ERK-positive cells appeared in the ventral ventricular zone on embryonic day 4 (E4). From E6 onward, they appeared in the gray matter and in the white matter, suggesting migration from the ventricular zone. A double labeling method revealed that these p-ERK-positive cells included oligodendrocyte precursors. In the dorsal horn, p-ERK-positive small cells appeared on E6. Subsequently, the positive cells in the dorsal horn increased transiently in number and then decreased markedly by E10. Motoneurons also expressed p-ERK transiently on E7. In the DRG, weak p-ERK immunoreaction appeared in the ventrolateral region on E5. From E6, the immunoreactivity became stronger and by E9 intense p-ERK-positive cells were observed throughout the DRG. These data provide a neuroanatomical framework to begin to examine the in vivo role of ERK in neural development.

For more than three decades, enhanced permeability and retention (EPR)-effect-based nanomedicines have received considerable attention for tumor-selective treatment of solid tumors. However, treatment of advanced cancers remains a huge challenge in clinical situations because of occluded or embolized tumor blood vessels, which lead to so-called heterogeneity of the EPR effect. We previously developed a method to restore impaired blood flow in blood vessels by using nitric oxide donors and other agents called EPR-effect enhancers. Here, we show that two novel EPR-effect enhancers-isosorbide dinitrate (ISDN, Nitrol®) and sildenafil citrate-strongly potentiated delivery of three macromolecular drugs to tumors: a complex of poly(styrene-co-maleic acid) (SMA) and cisplatin, named Smaplatin® (chemotherapy); poly(N-(2-hydroxypropyl)methacrylamide) polymer-conjugated zinc protoporphyrin (photodynamic therapy and imaging); and SMA glucosamine-conjugated boric acid complex (boron neutron capture therapy). We tested these nanodrugs in mice with advanced C26 tumors. When these nanomedicines were administered together with ISDN or sildenafil, tumor delivery and thus positive therapeutic results increased two- to four-fold in tumors with diameters of 15 mm or more. These results confirmed the rationale for using EPR-effect enhancers to restore tumor blood flow. In conclusion, all EPR-effect enhancers tested showed great potential for application in cancer therapy.

Programmed cell death ligand 1 (PD-L1) status has been reported to be different between metastatic and primary lesions in some cases. Therefore, the interaction between carcinoma and immune cells could influence their expression in the tumor microenvironment. PD-L1 is known to bind not only to Programmed cell death 1 (PD-1) but also to B7-1 (CD80). In this study, we examined the interaction between lung carcinoma cell lines and peripheral blood mononuclear cells (PBMCs) in vitro. We then examined the significance of B7-1 expression non-small cell lung cancer (NSCLC) microenvironment.

Olig2 is indispensable for motoneuron and oligodendrocyte fate-specification in the pMN domain of embryonic spinal cords, and also involved in the proliferation and differentiation of several cell types in the nervous system, including neural progenitor cells (NPCs) and oligodendrocytes. However, how Olig2 controls these diverse biological processes remains unclear. Here, we demonstrated that a novel Olig2-binding protein, DEAD-box helicase 20 (Ddx20), is indispensable for the survival of NPCs and oligodendrocyte progenitor cells (OPCs). A central nervous system (CNS)-specific Ddx20 conditional knockout (cKO) demonstrated apoptosis and cell cycle arrest in NPCs and OPCs, through the potentiation of the p53 pathway in DNA damage-dependent and independent manners, including SMN complex disruption and the abnormal splicing of Mdm2 mRNA. Analyzes of Olig2 null NPCs showed that Olig2 contributed to NPC proliferation through Ddx20 protein stabilization. Our findings provide novel mechanisms underlying the Olig2-mediated proliferation of NPCs, via the Ddx20-p53 axis, in the embryonic CNS.

Reactive persulfides such as cysteine persulfide and glutathione persulfide are produced by bacteria including Salmonella during sulfur metabolism. The biological significance of bacterial reactive persulfides in host-pathogen interactions still warrants investigation. We found that reactive persulfides produced by Salmonella Typhimurium LT2 regulate macrophage autophagy via metabolizing 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), an electrophilic product of reactive oxygen species and nitric oxide signaling. 8-Nitro-cGMP signaling was required for efficient autophagy-mediated clearance of Salmonella from infected macrophages. In the infected cells, 8-nitro-cGMP caused cGMP adduct formation (S-guanylation) of bacterial surface proteins, which triggered recruitment of autophagy-related proteins p62 and LC3-II to the intracellular bacteria. We also found that Salmonella-produced reactive persulfides downregulated this autophagy by decreasing cellular 8-nitro-cGMP content, thereby inhibiting electrophilic signaling. These data reveal a pathogenic role of bacteria-derived reactive persulfides via suppression of anti-bacterial autophagy.