👤 Koshi Hashimoto

Neural circuit formation through synaptogenesis plays a crucial role in learning, memory, and the recovery of neural function following brain dysfunction. We previously reported that administering the low-dose cardiac glycoside digoxin, which activates brain Na/K-ATPase, promotes dendritic spine formation and improves motor learning. On the other hand, brain-derived neurotrophic factor (BDNF) is also involved in axon elongation, branching, attraction, and the maturation of dendritic spines. Since trans-2-decenoic acid ethyl ester (DAEE), an ester of medium-chain fatty acid with ten carbons, activates the signaling pathway downstream of BDNF-TrkB, co-administration of digoxin and DAEE could further improve motor learning. This study compared the effects of digoxin, DAEE, or both on motor learning performance and locomotor activity in mice. Digoxin improved early performance in the rotarod test without changing locomotor activity, but did not affect final performance. DAEE increased activity in the open-field test but had no effect on the running wheel and did not influence motor learning in the rotarod test. On the other hand, the combination of digoxin and DAEE improved performance on the rotarod test later in the study. These data indicate that combining digoxin with DAEE delays the peak effects of motor learning compared to digoxin monotherapy, a temporal shift that may offer therapeutic advantages in rehabilitation outcomes. Show less

Perioperative neurocognitive disorders (PND), primarily including postoperative delirium (POD) and postoperative cognitive dysfunction (POCD), are common and serious complications in elderly surgical patients. However, the exact mechanisms underlying PND are not fully understood. The lung-brain axis has recently been recognized as an important pathway in neurodegenerative diseases such as Alzheimer's disease (AD). Given that PND shares pathological features with AD, such as amyloid-β (Aβ) accumulation, the lung-brain axis may also represent a plausible mechanistic contributor to PND. Furthermore, elderly surgical patients often receive inhalation anesthetics and undergo mechanical ventilation during general anesthesia, which directly affect the lungs and may alter the pulmonary microenvironment. Therefore, we hypothesize that the lung-brain axis plays a role in the development of PND. In this article, we discuss potential mechanisms by which surgery and anesthesia-especially inhalation anesthetics and mechanical ventilation-may influence cognitive function via the lung-brain axis. Potential mechanisms include changes in the pulmonary microbiota, secretion of brain-derived neurotrophic factor, and lung-derived inflammatory responses. These pathways may disrupt the blood-brain barrier, promote neuroinflammation, and exacerbate Aβ deposition, ultimately leading to cognitive impairment. Exploring the role of the lung-brain axis could provide new insights into PND pathophysiology and reveal potential targets for prevention and treatment of PND by targeting pulmonary-mediated cascades. Show less

Depression is a major global health burden, and current treatments are limited by delayed onset and incomplete efficacy, highlighting the need for novel, mechanism-based therapies. Chronic restraint stress (CRS) induces behavioral, hormonal, and synaptic changes relevant to depression, but the role of adiponectin signaling remains unclear. Here, we examined whether the adiponectin receptor agonist AdipoRon exerts antidepressant-like effects via brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB) signaling in mice subjected to 14 days of CRS. CRS produced anxiety- and depression-like behaviors, elevated plasma corticosterone, reduced circulating adiponectin, and selectively decreased hippocampal adiponectin and adiponectin receptor 2 (AdipoR2), accompanied by reduced PSD-95 and GluA1 in CA3 and the dentate gyrus (DG). AdipoRon treatment (20 mg/kg, days 8-14) prevented behavioral deficits, normalized corticosterone and adiponectin levels, and restored hippocampal AdipoR2, PSD-95, and GluA1 expression in CA3 and DG. AdipoRon also reversed CRS-induced decreases in hippocampal phosphorylated AMPK (p-AMPK), PPARα, BDNF, and phosphorylated TrkB (p-TrkB), with p-AMPK/AMPK and PPARα levels positively correlating with BDNF. Immunofluorescence confirmed BDNF recovery in CA3 and DG. Importantly, pretreatment with the TrkB antagonist ANA-12 abolished the behavioral, hormonal, and molecular effects of AdipoRon, indicating that its actions require BDNF-TrkB activation. These findings suggest that AdipoRon mitigates CRS-induced deficits via hippocampal AdipoR2-AMPK-PPARα-BDNF-TrkB signaling and highlight AdipoR2 as a promising target for depression therapy under chronic stress. Show less

Advanced esophageal squamous cell carcinoma (ESCC) has a poor prognosis, and current treatments provide limited survival benefits. This study aimed to identify prognostic biomarkers and therapeutic targets by genomic profiling of advanced ESCC using circulating tumor DNA (ctDNA). The SCRUM-MONSTAR GOZILA study is a nationwide, plasma-based molecular profiling project using Guardant360, involving 31 core cancer institutions in Japan. We evaluated the genomic landscape of advanced ESCC and investigated associations between specific alterations and overall survival (OS). The correlation between blood tumor mutation burden (bTMB) and clinical outcomes in patients with PD-1 inhibitors was also assessed using multiple cutoff values (2, 4, 6, 8, and 10 mutations/Mb). Among 313 patients, alterations predominantly consisted of single nucleotide variants (SNVs, 68.9%) and copy number alterations (20.7%). ctDNA analysis identified key genomic alterations linked to poor outcomes in advanced ESCC, revealing potential prognostic biomarkers and therapeutic targets. In contrast, bTMB did not show predictive value for the efficacy of PD-1 inhibitors in this study. Show less

Frontotemporal dementia (FTD) is driven by progranulin haploinsufficiency, in which age-dependent microglial activation promotes neurodegeneration through TDP-43 proteinopathy. Cyclic phosphatidic acid (cPA) is a natural phospholipid mediator characterized by a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. A pharmacologically active derivative of cPA has been shown to suppress microglial activation. Based on this, we aimed to investigate the potential of cPA derivatives to prevent the onset of FTD. Specifically, we administered metabolically stabilized cPA derivatives, 2-carba-cPA (2ccPA) and its degradation product, 2-carba-LPA (2cLPA), to presymptomatic progranulin-deficient (Grn Show less

Flavobacterium oreochromis has been associated with elevated mortality rates during the early stages of tambaqui (Colossoma macropomum) aquaculture. This study investigated genetic responses to bacterial infection in juvenile fish by comparing gene expression profiles between symptomatic (IS) and asymptomatic (IA) individuals. Skin samples from both IA and IS individuals were collected for transcriptome sequencing. Approximately 21 million reads per library were aligned to the tambaqui genome. Differential expression analysis revealed 2,176 upregulated and 1,219 downregulated genes in IS individuals, whereas 1,358 genes were upregulated and 488 downregulated in IA individuals. Notably, genes upregulated in both groups were associated with autophagy (e.g., atg4b and ulk2) and oxidative stress responses (e.g., klf9 and txnip). In contrast, genes related to tissue integrity, such as unc45b and akap6, were consistently downregulated during infection. These results suggest a dual host response to F. oreochromis infection, characterized by activation of cellular stress pathways and the suppression of genes involved in maintaining structural integrity. The upregulation of autophagy-related (atg4b, ulk2) and oxidative stress-regulating (klf9 and txnip) genes likely reflects an attempt by the host to counteract bacterial invasion through enhanced intracellular degradation and redox homeostasis. Conversely, the downregulation of unc45b and akap6 may indicate a compromise in structural defense mechanisms. These findings offer valuable insights into the immunogenetics of tambaqui and have direct implications for enhancing disease resistance in aquaculture. Moreover, they contribute to a better understanding of the complex interplay between molecular pathways involved in F. oreochromis infection. Show less

To identify cytokines associated with insufficient response to aflibercept against neovascular age-related macular degeneration. This prospective, comparative control study enrolled 40 eyes of 40 patients with nAMD. Aqueous humor (AH) samples were collected at the baseline before the intravitreal administration of aflibercept. The patients were further classified into responder and non-responder groups based on the clinical course. Patients were classified as "responders" if they required three or fewer additional injections after the three initial monthly loading doses within one year, and as non-responders, if they required four or more injections after the initial three-monthly loading doses or were switched to alternative anti-VEGF agents or treatments such as photodynamic therapy. The concentration of Angiopoietin 1, angiopoietin like 4 (ANGPTL4), interferon gamma-induced protein 10, hepatocyte growth factor, interleukin 10, platelet derived growth factor BB, plasminogen activator inhibitor 1 (PAI1), vascular endothelial growth factor A, angiopoietin 2, monocyte chemotactic protein 1, IL8, IL12, platelet-derived growth factor (PlGF), and vascular cell adhesion molecule 1 in AH samples were analyzed using a multiplex immunoassay, in order to compare between responders and non-responders. 21 eyes were defined as responders, and 19 eyes were defined as non-responders. There were no significant differences in baseline characteristics. Multiple variate analysis using logistic regression analysis found that PAI1 (p = 0.023, coefficient = 0.025), PlGF (p = 0.016, coefficient = - 1.4), and ANGPTL4 (p = 0.032, coefficient = - 0.00070) at the baseline were significantly associated with the resistance to aflibercept. Baseline higher PAI1 and lower PlGF and ANGPTL4 were associated with insufficient response to aflibercept in 1 year. These cytokines can potentially predict the treatment effect against nAMD. Show less

To identify the susceptibility loci for myopic macular neovascularization (mMNV) in patients with high myopia. A genome-wide association study (GWAS) meta-analysis (meta-GWAS). We included 2783 highly myopic individuals, including 608 patients with mMNV and 2175 control participants without mMNV. We performed a meta-analysis of 3 independent GWASs conducted according to the genotyping platform (Illumina Asian Screening Array [ASA] data set, Illumina Human610 BeadChip [610K] data set, and whole genome sequencing [WGS] data set), adjusted for age, sex, axial length, and the first to third principal components. We used DeltaSVM to evaluate the binding affinity of transcription factors (TFs) to DNA sequences around the susceptibility of single nucleotide polymorphisms (SNPs). In addition, we evaluated the contribution of previously reported age-related macular degeneration (AMD) susceptibility loci. The association between SNPs and mMNV in patients with high myopia. The meta-GWAS identified rs56257842 at TEX29- LINC02337 as a novel susceptibility SNP for mMNV (odds ratio [OR] Our study identified a novel locus associated with mMNV in high myopia. Subsequent analyses offered important insights into the molecular biology of mMNV, providing the potential therapeutic targets for mMNV. Furthermore, our findings imply shared genetic susceptibility between mMNV and AMD. Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article. Show less

This study aims to elucidate the clinical and molecular characteristics, treatment outcomes and prognostic factors of patients with histone H3 K27-mutant diffuse midline glioma. We retrospectively analyzed 93 patients with diffuse midline glioma (47 thalamus, 24 brainstem, 12 spinal cord and 10 other midline locations) treated at 24 affiliated hospitals in the Kansai Molecular Diagnosis Network for CNS Tumors. Considering the term "midline" areas, which had been confused in previous reports, we classified four midline locations based on previous reports and anatomical findings. Clinical and molecular characteristics of the study cohort included: age 4-78 years, female sex (41%), lower-grade histology (56%), preoperative Karnofsky performance status (KPS) scores ≥ 80 (49%), resection (36%), adjuvant radiation plus chemotherapy (83%), temozolomide therapy (76%), bevacizumab therapy (42%), HIST1H3B p.K27M mutation (2%), TERT promoter mutation (3%), MGMT promoter methylation (9%), BRAF p.V600E mutation (1%), FGFR1 mutation (14%) and EGFR mutation (3%). Median progression-free and overall survival time was 9.9 ± 1.0 (7.9-11.9, 95% CI) and 16.6 ± 1.4 (13.9-19.3, 95% CI) months, respectively. Female sex, preoperative KPS score ≥ 80, adjuvant radiation + temozolomide and radiation ≥ 50 Gy were associated with favorable prognosis. Female sex and preoperative KPS score ≥ 80 were identified as independent good prognostic factors. This study demonstrated the current state of clinical practice for patients with diffuse midline glioma and molecular analyses of diffuse midline glioma in real-world settings. Further investigation in a larger population would contribute to better understanding of the pathology of diffuse midline glioma. Show less

Myeloid and lymphoid neoplasms associated with FGFR1 abnormalities (MLN-FGFR1 abnormalities) are rare hematologic malignancies associated with chromosome 8p11.2 abnormalities. Translocations of 8p11.2 were detected in 10 of 17,039 (0.06%) unique patient cytogenetic studies performed at nine institutions in Japan. No inversions or insertions of 8p11.2 were detected. Among the 10 patients with 8p11.2 translocations, three patients were diagnosed with MLN-FGFR1 abnormalities, which were confirmed by FISH analysis. Peripheral blood eosinophilia was observed in all three patients, and all progressed to AML or T-lymphoblastic lymphoma/leukemia. The prevalence of 8p11.2 translocations in clinical practice and the proportion of MLN-FGFR1 abnormalities in patients with 8p11.2 translocations in Japan were consistent with those in previous reports from Western countries. Show less

Alzheimer's disease (AD) is an important human disease that mainly causes cognitive impairments. Growing evidence has shown that amyloid-β (Aβ) peptide plays a key role in AD pathogenesis in what is known as the Aβ cascade hypothesis. This hypothesis suggests the importance of suppressing Aβ aggregation and Aβ production. The latter process is governed by β-site APP Cleaving Enzyme1 (BACE1) and γ-secretase. We, therefore, focused on Aβ aggregation inhibitory activity, initially assessing numerous extracts derived from our marine-derived fungus collections. One EtOAc extract derived from an Aspergillus sp. exhibited Aβ aggregation inhibitory activity. Eleven known compounds (1-11) were isolated from CHCl Show less

We aimed to determine the similarities and differences in the roles of genic and regulatory copy number variations (CNVs) in bipolar disorder (BD), schizophrenia (SCZ), and autism spectrum disorder (ASD). Based on high-resolution CNV data from 8708 Japanese samples, we performed to our knowledge the largest cross-disorder analysis of genic and regulatory CNVs in BD, SCZ, and ASD. In genic CNVs, we found an increased burden of smaller (<100 kb) exonic deletions in BD, which contrasted with the highest burden of larger (>500 kb) exonic CNVs in SCZ/ASD. Pathogenic CNVs linked to neurodevelopmental disorders were significantly associated with the risk for each disorder, but BD and SCZ/ASD differed in terms of the effect size (smaller in BD) and subtype distribution of CNVs linked to neurodevelopmental disorders. We identified 3 synaptic genes (DLG2, PCDH15, and ASTN2) as risk factors for BD. Whereas gene set analysis showed that BD-associated pathways were restricted to chromatin biology, SCZ and ASD involved more extensive and similar pathways. Nevertheless, a correlation analysis of gene set results indicated weak but significant pathway similarities between BD and SCZ or ASD (r = 0.25-0.31). In SCZ and ASD, but not BD, CNVs were significantly enriched in enhancers and promoters in brain tissue. BD and SCZ/ASD differ in terms of CNV burden, characteristics of CNVs linked to neurodevelopmental disorders, and regulatory CNVs. On the other hand, they have shared molecular mechanisms, including chromatin biology. The BD risk genes identified here could provide insight into the pathogenesis of BD. Show less

RGPR-p117 was originally discovered as a novel transcription factor, which specifically binds to a nuclear factor I (NFI) consensus motif TTGGC(N) The NRK-52E wild-type cells and RGPR-p117-overexpressing NRK-52E cells were cultured in DMEM containing fetal bovine serum. The overexpression of RGPR-p117 repressed colony formation and proliferation of NRK-52E cells. Interestingly, RGPR-p117 overexpression blocked cell proliferation promoted by culturing with Bay K 8644, a calcium-entry agonist, and phorbol 12-myristate 13-acetate, an activator of protein kinase C. The depressive effects of RGPR-p117 overexpression on cell proliferation were not occurred by culturing with various inhibitors of cell cycle and intracellular signaling processes. RGPR-p117 overexpression increased the translocation of RGPR-p117 into the nucleus of NRK-52E cells. Mechanistically, RGPR-p117 overexpression diminished the levels of Ras, PI3 kinase, Akt, mitogen-activated protein kinase, and mTOR, while it raised the levels of p53, Rb, p21, and regucalcin. Furthermore, RGPR-p117 overexpression protected cell death caused by apoptosis-inducing factors, suggesting that the suppressive effects of RGPR-p117 on cell growth are independent of cell death. The present study demonstrates that the overexpressed transcription factor RGPR-p117 suppresses cell proliferation via targeting diverse signaling processes, suggesting a role of RGPR-p117 in cell regulation. Show less

Epstein-Barr virus-induced gene 3 (EBI3) is a subunit common to IL-27, IL-35, and IL-39. Here, we explore an intracellular role of EBI3 that is independent of its function in cytokines. EBI3-deficient naive CD4+ T cells had reduced IFN-γ production and failed to induce T cell-dependent colitis in mice. Similarly reduced IFN-γ production was observed in vitro in EBI3-deficient CD4+ T cells differentiated under pathogenic Th17 polarizing conditions with IL-23. This is because the induction of expression of one of the IL-23 receptor (IL-23R) subunits, IL-23Rα, but not another IL-23R subunit, IL-12Rβ1, was selectively decreased at the protein level, but not the mRNA level. EBI3 augmented IL-23Rα expression via binding to the chaperone molecule calnexin and to IL-23Rα in a peptide-dependent manner, but not a glycan-dependent manner. Indeed, EBI3 failed to augment IL-23Rα expression in the absence of endogenous calnexin. Moreover, EBI3 poorly augmented the expression of G149R, an IL-23Rα variant that protects against the development of human colitis, because binding of EBI3 to the variant was reduced. Taken together with the result that EBI3 expression is inducible in T cells, the present results suggest that EBI3 plays a critical role in augmenting IL-23Rα protein expression via calnexin under inflammatory conditions. Show less

Dysregulation of neuropeptides may play an important role in aging-induced impairments. Among them, pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent cytoprotective peptide that provides an endogenous control against a variety of tissue-damaging stimuli. We hypothesized that the progressive decline of PACAP throughout life and the well-known general cytoprotective effects of PACAP lead to age-related pathophysiological changes in PACAP deficiency, supported by the increased vulnerability to various stressors of animals partially or totally lacking PACAP. Using young and aging CD1 PACAP knockout (KO) and wild type (WT) mice, we demonstrated pre-senile amyloidosis in young PACAP KO animals and showed that senile amyloidosis appeared accelerated, more generalized, more severe, and affected more individuals. Histopathology showed age-related systemic amyloidosis with mainly kidney, spleen, liver, skin, thyroid, intestinal, tracheal, and esophageal involvement. Mass spectrometry-based proteomic analysis, reconfirmed with immunohistochemistry, revealed that apolipoprotein-AIV was the main amyloid protein in the deposits together with several accompanying proteins. Although the local amyloidogenic protein expression was disturbed in KO animals, no difference was found in laboratory lipid parameters, suggesting a complex pathway leading to increased age-related degeneration with amyloid deposits in the absence of PACAP. In spite of no marked inflammatory histological changes or blood test parameters, we detected a disturbed cytokine profile that possibly creates a pro-inflammatory milieu favoring amyloid deposition. In summary, here we describe accelerated systemic senile amyloidosis in PACAP gene-deficient mice, which might indicate an early aging phenomenon in this mouse strain. Thus, PACAP KO mice could serve as a model of accelerated aging with human relevance. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. Show less

The cytokine, interleukin-18 (IL-18), was originally identified as an interferon-γ-inducing proinflammatory factor; however, there is increasing evidence suggesting that it has non-immunological effects on physiological functions. We have previously investigated the potential pathophysiological relationship between IL-18 and dyslipidemia, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, which were mediated by lipid energy imbalance. Therefore, herein we focused on brown adipocytes (BAs) and brown adipose tissue (BAT) related to energy consumption as non-shivering thermogenesis. Il18 Compared with Il18 This study demonstrated the critical function of IL-18 in differentiation and lipid metabolism in BAs. Furthermore, IL-18 may contribute to novel treatments by improving the energy imbalance. Show less

Genome-wide association studies (GWASs) have identified several susceptibility loci for bipolar disorder (BD) and shown that the genetic architecture of BD can be explained by polygenicity, with numerous variants contributing to BD. In the present GWAS (Phase I/II), which included 2964 BD and 61 887 control subjects from the Japanese population, we detected a novel susceptibility locus at 11q12.2 (rs28456, P=6.4 × 10 Show less

Liver X Receptors (LXRs) are sterol-activated transcription factors that play major roles in cellular cholesterol homeostasis, HDL biogenesis and reverse cholesterol transport. The aim of the present study was to investigate the mechanisms that control the expression of the human LXRα gene in hepatic cells. A series of reporter plasmids containing consecutive 5' deletions of the hLXRα promoter upstream of the luciferase gene were constructed and the activity of each construct was measured in HepG2 cells. This analysis showed that the activity of the human LXRα promoter was significantly reduced by deleting the -111 to -42 region suggesting the presence of positive regulatory elements in this short proximal fragment. Bioinformatics data including motif search and ChIP-Seq revealed the presence of a potential binding motif for Hepatocyte Nuclear Factor 4 α (HNF-4α) in this area. Overexpression of HNF-4α in HEK 293T cells increased the expression of all LXRα promoter constructs except -42/+384. In line, silencing the expression of endogenous HNF-4α in HepG2 cells was associated with reduced LXRα protein levels and reduced activity of the -111/+384 LXRα promoter but not of the -42/+384 promoter. Using ChiP assays in HepG2 cells combined with DNAP assays we mapped the novel HNF-4α specific binding motif (H4-SBM) in the -50 to -40 region of the human LXRα promoter. A triple mutation in this H4-SBM abolished HNF-4α binding and reduced the activity of the promoter to 65% relative to the wild type. Furthermore, the mutant promoter could not be transactivated by HNF-4α. In conclusion, our data indicate that HNF-4α may have a wider role in cell and plasma cholesterol homeostasis by controlling the expression of LXRα in hepatic cells. Show less

Apolipoprotein A5 gene promoter region T-1131C polymorphism (APOA5 T-1131C) is known to be associated with elevated plasma TG levels, although little is known of the influence of the interaction between APOA5 T-1131C and lifestyle modification on TG levels. To investigate this matter, we studied APOA5 T-1131C and plasma TG levels of subjects participating in a three-month lifestyle modification program. A three-month lifestyle modification program was conducted with 297 participants (Age: 57 ± 8 years) in Izumo City, Japan, from 2001-2007. Changes in energy balance (the difference between energy intake and energy expenditure) and BMI were used to evaluate the participants' responses to the lifestyle modification. Even after adjusting for confounding factors, plasma TG levels were significantly different at baseline among three genotype subgroups: TT, 126 ± 68 mg/dl; TC, 134 ± 74 mg/dl; and CC, 172 ± 101 mg/dl. Lifestyle modification resulted in significant reductions in plasma TG levels in the TT, TC, and CC genotype subgroups: -21.9 ± 61.0 mg/dl, -20.9 ± 51.0 mg/dl, and -42.6 ± 78.5 mg/dl, respectively, with no significant differences between them. In a stepwise regression analysis, age, APOA5 T-1131C, body mass index (BMI), homeostasis model assessment-insulin resistance (HOMA-IR), and the 18:1/18:0 ratio showed independent association with plasma TG levels at baseline. In a general linear model analysis, APOA5 T-1131C C-allele carriers showed significantly greater TG reduction with decreased energy balance than wild type carriers after adjustment for age, gender, and baseline plasma TG levels. The genetic effects of APOA5 T-1131C independently affected plasma TG levels. However, lifestyle modification was effective in significantly reducing plasma TG levels despite the APOA5 T-1131C genotype background. Show less

The activation of liver X receptor (LXR) α or LXRβ negatively regulates the expression of pro-inflammatory genes in mammalian cells. We recently reported that 25-hydroxycholesterol, a representative LXR-activating oxysterol, suppresses IL-6 production in mouse mast cells (MCs) following its engagement of the high-affinity IgE receptor (FcεRI). This finding suggests that murine MCs express functional LXRs; however, the mechanisms underlying the LXR-dependent repression of the MC-mediated production of pro-inflammatory cytokines, including IL-6, are poorly understood. Therefore, we employed the synthetic LXR ligand GW3965 to examine the functions of LXRα and LXRβ in the production of pro-inflammatory cytokines by murine bone marrow-derived MCs (BMMCs). We prepared BMMCs from wild-type (WT), LXRα(-/-), and LXRα/β(-/-) mice. Each group of BMMCs was pretreated with GW3965 and then stimulated with IgE+antigen (Ag) or lipopolysaccharide (LPS). Cytokine production was then analyzed using specific ELISA kits. The activation of LXRs by GW3965 significantly attenuated the production of IL-1α and IL-1β, but not of IL-6, in the WT and LXRα(-/-) BMMCs stimulated with IgE+Ag. However, GW3965 treatment decreased the production of IL-1α, IL-1β, and IL-6 in WT and LXRα(-/-) BMMCs upon stimulation with LPS, while the GW3965-mediated suppression of cytokine production was nearly absent from the LXRα/β(-/-) BMMCs. These findings demonstrate, for the first time, that the activation of LXRs by GW3965 attenuates the antigen- or LPS-induced production of pro-inflammatory cytokines, such as IL-1α and IL-1β, in murine MCs and that LXRβ plays an important role in the LXR-mediated repression of cytokine production. Show less

The Hairy-related transcription factor family of Notch- and ALK1-downstream transcriptional repressors, called Hrt/Hey/Hesr/Chf/Herp/Gridlock, has complementary and indispensable functions for vascular development. While mouse embryos null for either Hrt1/Hey1 or Hrt2/Hey2 did not show early vascular phenotypes, Hrt1/Hey1; Hrt2/Hey2 double null mice (H1(ko) /H2(ko) ) showed embryonic lethality with severe impairment of vascular morphogenesis. It remained unclear, however, whether Hrt/Hey functions are required in endothelial cells or vascular smooth muscle cells. In this study, we demonstrate that mice with endothelial-specific deletion of Hrt2/Hey2 combined with global Hrt1/Hey1 deletion (H1(ko) /H2(eko) ) show abnormal vascular morphogenesis and embryonic lethality. Their defects were characterized by the failure of vascular network formation in the yolk sac, abnormalities of embryonic vascular structures and impaired smooth muscle cell recruitment, and were virtually identical to the H1(ko) /H2(ko) phenotypes. Among signaling molecules implicated in vascular development, Robo4 expression was significantly increased and activation of Src family kinases was suppressed in endothelial cells of H1(ko) /H2(eko) embryos. The present study indicates an important role of Hrt1/Hey1 and Hrt2/Hey2 in endothelial cells during early vascular development, and further suggests involvement of Robo4 and Src family kinases in the mechanisms of embryonic vascular defects caused by the Hrt/Hey deficiency. Show less

7-Dehydrocholesterol (7-DHC) is a common precursor of vitamin D3 and cholesterol. Although various oxysterols, oxygenated cholesterol derivatives, have been implicated in cellular signaling pathways, 7-DHC metabolism and potential functions of its metabolites remain poorly understood. We examined 7-DHC metabolism by various P450 enzymes and detected three metabolites produced by sterol 27-hydroxylase (CYP27A1) using high-performance liquid chromatography. Two were further identified as 25-hydroxy-7-DHC and 26/27-hydroxy-7-DHC. These 7-DHC metabolites were detected in serum of a patient with Smith-Lemli-Opitz syndrome. Luciferase reporter assays showed that 25-hydroxy-7-DHC activates liver X receptor (LXR) α, LXRβ and vitamin D receptor and that 26/27-hydroxy-7-DHC induces activation of LXRα and LXRβ, although the activities of both compounds on LXRs were weak. In a mammalian two-hybrid assay, 25-hydroxy-7-DHC and 26/27-hydroxy-7-DHC induced interaction between LXRα and a coactivator fragment less efficiently than a natural LXR agonist, 22(R)-hydroxycholesterol. These 7-DHC metabolites did not oppose agonist-induced LXR activation and interacted directly to LXRα in a manner distinct from a potent agonist. These findings indicate that the 7-DHC metabolites are partial LXR activators. Interestingly, 25-hydroxy-7-DHC and 26/27-hydroxy-7-DHC suppressed mRNA expression of sterol regulatory element-binding protein 1c, an LXR target gene, in HepG2 cells and HaCaT cells, while they weakly increased mRNA levels of ATP-binding cassette transporter A1, another LXR target, in HaCaT cells. Thus, 7-DHC is catabolized by CYP27A1 to metabolites that act as selective LXR modulators. Show less

Selective Alzheimer's disease (AD) indicator-1 (Seladin-1) gene has been identified as a gene, whose expression is down-regulated in the vulnerable region in the brain of AD patients. Thyroid hormone (TH) is important to maintain the function of central nervous system and TH receptor (TR) is known to crosstalk with liver X receptor (LXR) on the lipid metabolism-related gene promoter. Recently, we have demonstrated that TR-β up-regulates the mouse Seladin-1 gene promoter at the transcriptional levels and LXR-α compensates the promoter activation only when the thyroid function is insufficient. In the current study, we have identified that TH and an LXR artificial agonist, TO901317 (TO) activated the human Seladin-1 promoter (-1024/+57 base pair (bp)) including consensus TH response element (TRE) half site (site A: -381 to -375 bp), and the site A mutation deteriorated the activation by TH and TO. Both TR-β and LXR-α heterodimerize with retinoid X receptor (RXR)-α on the site A, and chromatin immunoprecipitation (ChIP) assay revealed that TR-β, LXR-α and RXR-α are recruited to the site A. Moreover, TR-β and LXR-α functionally compete for the promoter activation in CV1 cells. Taken together, we concluded that TR-β and LXR-α competitively up-regulate the human Seladin-1 promoter, sharing the same response element, site A. Show less

Selective Alzheimer's disease (AD) indicator 1 (Seladin-1) has been identified as a gene down-regulated in the degenerated lesions of AD brain. Up-regulation of Seladin-1 reduces the accumulation of β-amyloid and neuronal death. Thyroid hormone (TH) exerts an important effect on the development and maintenance of central nervous systems. In the current study, we demonstrated that Seladin-1 gene and protein expression in the forebrain was increased in thyrotoxic mice compared with that of euthyroid mice. However, unexpectedly, no significant decrease in the gene and protein expression was observed in hypothyroid mice. Interestingly, an agonist of liver X receptor (LXR), TO901317 (TO) administration in vivo increased Seladin-1 gene and protein expression in the mouse forebrain only in a hypothyroid state and in the presence of mutant TR-β, suggesting that LXR-α would compensate for TR-β function to maintain Seladin-1 gene expression in hypothyroidism and resistance to TH. TH activated the mouse Seladin-1 gene promoter (-1936/+21 bp) and site 2 including canonical TH response element (TRE) half-site in the region between -159 and -154 bp is responsible for the positive regulation. RXR-α/TR-β heterodimerization was identified on site 2 by gel-shift assay, and chromatin immunoprecipitation assay revealed the recruitment of TR-β to site 2 and the recruitment was increased upon TH administration. On the other hand, LXR-α utilizes a distinct region from site 2 (-120 to -102 bp) to activate the mouse Seladin-1 gene promoter. Taking these findings together, we concluded that TH up-regulates Seladin-1 gene expression at the transcriptional level and LXR-α maintains the gene expression. Show less

Signaling by fibroblast growth factor (FGF) receptor (FGFR) 2IIIb regulates branching morphogenesis in the mammalian lung. FGFR2IIIb is primarily expressed in epithelial cells, whereas its ligands, FGF-10 and keratinocyte growth factor (KGF; FGF-7), are expressed in mesenchymal cells. FGF-10 null mice lack lungs, whereas KGF null animals have normal lung development, indicating that FGF-10 regulates lung branching morphogenesis. In this study, we determined the effects of FGF-10 on lung branching morphogenesis and accompanying gene expression in cultures of embryonic rat lungs. Embryonic day 14 rat lungs were cultured with FGF-10 (0-250 ng/ml) in the absence or presence of heparin (30 ng/ml) for 4 days. Gene expression profiles were analyzed by Affymetrix microchip array including pathway analysis. Some of these genes, functionally important in FGF-10 signaling, were further analyzed by Northern blot, real-time PCR, in situ hybridization and immunohistochemistry. Exogenous FGF-10 inhibited branching and induced cystic lung growth only in cultures containing heparin. In total, 252 upregulated genes and 164 downregulated genes were identified, and these included Spry1 (Sprouty-1), Spry2 (Sprouty-2), Spred-1, Bmp4 (bone morphogenetic protein-4, BMP-4), Shh (sonic hedgehog, SHH), Pthlh (parathyroid hormone-related protein, PTHrP), Dusp6 (MAP kinase phosphatase-3, MKP-3) and Clic4 (chloride intracellular channel-4, CLIC-4) among the upregulated genes and Igf1 (insulin-like growth factor-1, IGF-1), Tcf21 (POD), Gyg1 (glycogenin 1), Sparc (secreted protein acidic and rich in cysteine, SPARC), Pcolce (procollagen C-endopeptidase enhancer protein, Pro CEP) and Lox (lysyl oxidase) among the downregulated genes. Gsk3β and Wnt2, which are involved in canonical Wnt signaling, were up- and downregulated, respectively. Unlike FGF-7, FGF-10 effects on lung branching morphogenesis are heparin-dependent. Sprouty-2, BMP-4, SHH, IGF-1, SPARC and POD are known to regulate branching morphogenesis; however, potential roles of CLIC-4 and MKP-3 in lung branching morphogenesis remain to be investigated. FGF-10 may also function in regulating branching morphogenesis or inducing cystic lung growth by inhibiting Wnt2/β-catenin signaling. Show less

To obtain novel transrepression-selective liver X receptor (LXR) ligands, we adopted a strategy of reducing the transactivational agonistic activity of the 5,11-dihydro-5-methyl-11-methylene-6H-dibenz[b,e]azepin-6-one derivative 10, which exhibits LXR-mediated transrepressional and transactivational activity. Structural modification of 10 based on the reported X-ray crystal structure of the LXR ligand-binding domain led to a series of compounds, of which almost all exhibited transrepressional activity at 1 or 10 μM but showed no transactivational activity even at 30 μM. Among the compounds obtained, 18 and 22 were confirmed to have LXR-dependent transrepressional activity by using peritoneal macrophages from wild-type and LXR-null mice. A newly developed fluorescence polarization assay indicated that they bind directly to LXRα. Next, further structural modification was performed with the guidance of docking simulations with LXRα, focusing on enhancing the binding of the ligands with LXRα through the introduction of substituents or heteroatom(s). Among the compounds synthesized, compound 48, bearing a hydroxyl group, showed potent, selective, and dose-dependent transrepressional activity. Show less