Novel, highly sensitive, in vivo screening method detects estrogenic activity at low doses of bisphenol A.

Low doses of bisphenol A (BPA), a typical endocrine-disrupting chemical (EDC), have been reported to exhibit estrogenic action in animals; however, the effects have not been fully clarified because of their non-reproducibility. Here, we developed a novel, short-term screening test for estrogen-like chemicals using in vivo bioluminescence imaging of estrogen-responsive reporter (E-Rep) mice. Comparative studies using 17α-ethinylestradiol and selective estrogen receptor modulators demonstrated that the method provides higher detection sensitivity and requires less time than the uterotrophic bioassay, a well-established, in vivo screening method for estrogen-like chemicals. Our method could detect the estrogenic effects of BPA at doses below tolerable daily intakes, whereas the uterotrophic bioassay could not. Our results indicated that in vivo bioluminescence imaging using E-Rep mice was extremely useful for screening estrogenic chemicals and detecting estrogenic effects at low doses of EDCs, including BPA. Our method should help resolve the controversy about low-dose effects of EDCs.

Evaluation of the Estrogenic Action Potential of Royal Jelly by Genomic Signaling Pathway in Vitro and in Vivo.

Royal jelly (RJ) has beneficial effects on human health, and some of these effects are reported to be the result of its estrogenic activity; however, chemicals with estrogenic activities may disrupt physiological estrogen signaling leading to adverse effects on human health. Thus, clarification of the mode of action of RJ is needed. Here, we investigated whether the estrogen-like actions of RJ are induced via estrogen receptors (ERs)-mediated genomic actions by using an in vitro reporter assay in human choriocarcinoma JEG3 cells and an estrogen-responsive reporter (E-Rep) mouse line that can be used to sensitively detect transactivation of ERs in multiple organs simultaneously. In the in vitro reporter assay, ERs-dependent transcriptional activity was significantly increased by 17ß-estradiol (E2) treatment at concentrations of 1 nM and above, confirming that the assay was highly responsive to estrogen; however, RJ did not exhibit any agonist activity via either the α or ß form of ER. Similarly, in E-Rep mice, E2 showed significant ERs-dependent genomic action in 17 tissue types including uterus and mammary gland, whereas RJ did not. Thus, unlike endocrine-disrupting chemicals, the estrogen-like activity of RJ is unlikely to be due to genomic actions via ERs.

The KEAP1-NRF2 System in Healthy Aging and Longevity.

Aging is inevitable, but the inherently and genetically programmed aging process is markedly influenced by environmental factors. All organisms are constantly exposed to various stresses, either exogenous or endogenous, throughout their lives, and the quality and quantity of the stresses generate diverse impacts on the organismal aging process. In the current oxygenic atmosphere on earth, oxidative stress caused by reactive oxygen species is one of the most common and critical environmental factors for life. The Kelch-like ECH-associated protein 1-NFE2-related factor 2 (KEAP1-NRF2) system is a critical defense mechanism of cells and organisms in response to redox perturbations. In the presence of oxidative and electrophilic insults, the thiol moieties of cysteine in KEAP1 are modified, and consequently NRF2 activates its target genes for detoxification and cytoprotection. A number of studies have clarified the contributions of the KEAP1-NRF2 system to the prevention and attenuation of physiological aging and aging-related diseases. Accumulating knowledge to control stress-induced damage may provide a clue for extending healthspan and treating aging-related diseases. In this review, we focus on the relationships between oxidative stress and aging-related alterations in the sensory, glandular, muscular, and central nervous systems and the roles of the KEAP1-NRF2 system in aging processes.

Triphenyl phosphate delayed pubertal timing and induced decline of ovarian reserve in mice as an estrogen receptor antagonist.

Although concerns have been raised about the adverse effects of triphenyl phosphate (TPhP) on female fertility, its risk to ovarian functioning remains unknown. In this study, female C57BL/6 mice at postnatal day 21 were exposed on a daily basis to TPhP dose of 2, 10, and 50 mg/kg for 40 days. A significant delay in pubertal timing was observed in the mice exposed to 50 mg/kg of TPhP. An estrogen-responsive reporter transgenic mice assay demonstrated that TPhP significantly downregulated the estrogen receptor (ER) signaling by 45.1% in the whole body in the 50 mg/kg group, and by 14.7-43.7% in the uterus for all exposure groups compared with the control. This strong antagonistic activity of TPhP toward ER explained the delay in pubertal timing. A significant reduction in the number of follicles in all stages was observed in mice after being exposed to TPhP for 40 days at concentrations of 10 and 50 mg/kg, resulting in a decline of the ovarian reserve. The elevation of the follicle-stimulating hormone concentration may have contributed to this phenomenon, as controlled by the antagonistic activity of TPhP toward ER in the brain. The toxic effects of TPhP on ovarian functioning highlight this chemical as a potential risk factor for female fertility.

An ancestral nuclear receptor couple, PPAR-RXR, is exploited by organotins.

Environmental chemicals have been reported to greatly disturb the endocrine and metabolic systems of multiple animal species. A recent example involves the exploitation of the nuclear receptor (NR) heterodimeric pair composed by PPAR/RXR (peroxisome proliferator-activated receptor/retinoid X receptor), which shows lipid perturbation in mammalian species. While gene orthologues of both of these receptors have been described outside vertebrates, no functional characterization of PPAR has been carried in protostome lineages. We provide the first functional analysis of PPAR in Patella sp. (Mollusca), using model obesogens such as tributyltin (TBT), triphenyltin (TPT), and proposed natural ligands (fatty acid molecules). To gain further insights, we used site-directed mutagenesis to PPAR and replaced the tyrosine 277 by a cysteine (the human homologous amino acid and TBT anchor residue) and an alanine. Additionally, we explored the alterations in the fatty acid profiles after an exposure to the model obesogen TBT, in vivo. Our results show that TBT and TPT behave as an antagonist of Patella sp. PPAR/RXR and that the tyrosine 277 is important, but not essential in the response to TBT. Overall, these results suggest a relation between the response of the mollusc PPAR-RXR to TBT and the lipid profile alterations reported at environmentally relevant concentrations. Our findings highlight the importance of comparative analysis between protostome and deuterostome lineages to decipher the differential impact of environmental chemicals.

Enhancer remodeling promotes tumor-initiating activity in NRF2-activated non-small cell lung cancers.

Transcriptional dysregulation, which can be caused by genetic and epigenetic alterations, is a fundamental feature of many cancers. A key cytoprotective transcriptional activator, NRF2, is often aberrantly activated in non-small cell lung cancers (NSCLCs) and supports both aggressive tumorigenesis and therapeutic resistance. Herein, we find that persistently activated NRF2 in NSCLCs generates enhancers at gene loci that are not normally regulated by transiently activated NRF2 under physiological conditions. Elevated accumulation of CEBPB in NRF2-activated NSCLCs is found to be one of the prerequisites for establishment of the unique NRF2-dependent enhancers, among which the NOTCH3 enhancer is shown to be critical for promotion of tumor-initiating activity. Enhancer remodeling mediated by NRF2-CEBPB cooperativity promotes tumor-initiating activity and drives malignancy of NRF2-activated NSCLCs via establishment of the NRF2-NOTCH3 regulatory axis.

Evaluation of surgical procedures of mouse urethra by visualization and the formation of fistula.

Visualization of the surgically operated tissues is vital to improve surgical model animals including mouse. Urological surgeries for urethra include series of fine manipulations to treat the increasing number of birth defects such as hypospadias. Hence visualization of the urethral status is vital. Inappropriate urethral surgical procedure often leads to the incomplete wound healing and subsequent formation of urethro-cutaneous fistula or urethral stricture. Application of indocyanine green mediated visualization of the urethra was first performed in the current study. Indocyanine green revealed the bladder but not the urethral status in mouse. Antegrade injection of contrast agent into the bladder enabled to detect the urethral status in vivo. The visualization of the leakage of contrast agent from the operated region was shown as the state of urethral fistula in the current hypospadias mouse model and urethral stricture was also revealed. A second trial for contrast agent was performed after the initial operation and a tendency of accelerated urethral stricture was observed. Thus, assessment of post-surgical conditions of urogenital tissues can be improved by the current analyses on the urethral status.

Gli3 is a Key Factor in the Schwann Cells from Both Intact and Injured Peripheral Nerves.

Hedgehog (Hh) signaling has been shown to be involved in regulating both intact and injured peripheral nerves. Therefore, it is critical to understand how Hh signaling is regulated in the peripheral nerve. One of the transcription factors of the Hh signaling pathway, Gli3, functions as both a repressor and an activator of Hh signaling activity. However, it remains unclear whether Gli3 is involved in controlling the intact and/or injured peripheral nerves. We found that Gli3 act as a repressor in the Schwann cells (SCs) of intact sciatic nerves. Although Dhh and Ptch1 expression were present, Hh signaling was not activated in these SCs. Moreover, heterozygous Gli3 mutation (Gli3-/+) induced ectopic Hh signaling activity in SCs. Hh signaling was thus suppressed by Gli3 in the SCs of intact sciatic nerves. Minor morphological changes were observed in the intact nerves from Gli3-/+ mice. Gli3 expression was significantly decreased following injury and ligand expression switched from Dhh to Shh, which activated Hh signaling in SCs from wild-type mice. Changes of these ligands was found to be important for nerve regeneration in which the downregulation of Gli3 was also involved. In fact, Gli3-/+ mice exhibited accelerated ligand switching and subsequent nerve regeneration. Both suppression of Hh signaling with Gli3 in the intact nerves and activation of Hh signaling without Gli3 in the injured nerve were observed in the SCs in an autocrine manner. Thus, Gli3 is a key factor in the control of intact peripheral nerve homeostasis and nerve regeneration.

Nrf2 Suppresses Oxidative Stress and Inflammation in App Knock-In Alzheimer's Disease Model Mice.

Nrf2 (NF-E2-related-factor 2) is a stress-responsive transcription factor that protects cells against oxidative stresses. To clarify whether Nrf2 prevents Alzheimer's disease (AD), AD model AppNL-G-F/NL-G-F knock-in (AppNLGF ) mice were studied in combination with genetic Nrf2 induction model Keap1FA/FA mice. While AppNLGF mice displayed shorter latency to escape than wild-type mice in the passive-avoidance task, the impairment was improved in AppNLGF ::Keap1FA/FA mice. Matrix-assisted laser desorption ionization-mass spectrometry imaging revealed that reduced glutathione levels were elevated by Nrf2 induction in AppNLGF ::Keap1FA/FA mouse brains compared to AppNLGF mouse brains. Genetic Nrf2 induction in AppNLGF mice markedly suppressed the elevation of the oxidative stress marker 8-OHdG and Iba1-positive microglial cell number. We also determined the plasmalogen-phosphatidylethanolamine (PlsPE) level as an AD biomarker. PlsPE containing polyunsaturated fatty acids was decreased in the AppNLGF mouse brain, but Nrf2 induction attenuated this decline. To evaluate whether pharmacological induction of Nrf2 elicits beneficial effects for AD treatment, we tested the natural compound 6-MSITC [6-(methylsulfinyl)hexyl isothiocyanate]. Administration of 6-MSITC improved the impaired cognition of AppNLGF mice in the passive-avoidance task. These results demonstrate that the induction of Nrf2 ameliorates cognitive impairment in the AD model mouse by suppressing oxidative stress and neuroinflammation, suggesting that Nrf2 is an important therapeutic target of AD.

From germ cells to neonates: the beginning of life and the KEAP1-NRF2 system.

The Kelch-like ECH-associated protein 1(KEAP1)-NF-E2-related factor 2 (NRF2) system is one of the most studied environmental stress response systems. In the presence of oxidative and electrophilic insults, the thiols of cysteine residues in KEAP1 are modified, and subsequently stabilized NRF2 activates its target genes that are involved in detoxification and cytoprotection. A myriad of recent studies has revealed the broad range of contributions of the KEAP1-NRF2 system to physiological and pathological processes. However, its functions during gametic and embryonic development are still open for investigation. Although oxidative stress is harmful for embryos, Nrf2-/- mice do not show any apparent morphological abnormalities during development, probably because of the compensatory antioxidant functions of NF-E2-related factor 1 (NRF1). It can also be considered that the antioxidant system is essential for protecting germ cells during reproduction. The maturation processes of germ cells in both sexes are affected by Nrf2 mutation. Hence, in this review, we focus on the stress response system related to reproduction and embryonic development through the functions of the KEAP1-NRF2 system.

T396I mutation of mouse Sufu reduces the stability and activity of Gli3 repressor.

Hedgehog signaling is primarily transduced by two transcription factors: Gli2, which mainly acts as a full-length activator, and Gli3, which tends to be proteolytically processed from a full-length form (Gli3FL) to an N-terminal repressor (Gli3REP). Recent studies using a Sufu knockout mouse have indicated that Sufu is involved in regulating Gli2 and Gli3 activator and repressor activity at multiple steps of the signaling cascade; however, the mechanism of specific Gli2 and Gli3 regulation remains to be elucidated. In this study, we established an allelic series of ENU-induced mouse strains. Analysis of one of the missense alleles, SufuT396I, showed that Thr396 residue of Sufu played a key role in regulation of Gli3 activity. SufuT396I/T396I embryos exhibited severe polydactyly, which is indicative of compromised Gli3 activity. Concomitantly, significant quantitative reductions of unprocessed Gli3 (Gli3FL) and processed Gli3 (Gli3REP) were observed in vivo as well as in vitro. Genetic experiments showed that patterning defects in the limb buds of SufuT396I/T396I were rescued by a constitutive Gli3REP allele (Gli3∆699), strongly suggesting that SufuT396I reduced the truncated Gli3 repressor. In contrast, SufuT396I qualitatively exhibited no mutational effects on Gli2 regulation. Taken together, the results of this study show that the Thr396 residue of Sufu is specifically required for regulation of Gli3 but not Gli2. This implies a novel Sufu-mediated mechanism in which Gli2 activator and Gli3 repressor are differentially regulated.

Genetic analysis of the role of Alx4 in the coordination of lower body and external genitalia formation.

Although several syndromes include abnormalities of both the ventral body wall and external genitalia, the developmental bases of this correlation are largely unknown. Naturally occurring mutations in Aristaless-like 4 (Alx4, Strong's luxoid: Alx4Lst) have ventral body wall and pelvic girdle abnormalities. We sought to determine whether the development of the genital tubercle (GT) and its derivatives, the external genitalia, is affected by this mutation. We thus performed genetic and tissue labeling analyses in mutant mice. Alx4Lst/Lst mutants displayed hypoplasia of the dorsal GT and reduced expression of Fibronectin. We analyzed cell migration during GT formation by tissue labeling experiments and discovered that the cells located in the proximal segment of the umbilical cord (infra-umbilical mesenchyme) migrate toward the dorsal part of the GT. The Alx4Lst/Lst mutants also displayed augmented expression of Hh signal-related genes. Hence, we analyzed a series of combinatorial mutants for Alx4, Sonic hedgehog (Shh) and GLI-Kruppel family member 3 (Gli3). These phenotype-genotype analyses suggested a genetic interaction between Alx4 and Hh signaling during GT formation. Moreover, Hh gain-of-function mutants phenocopied some of these phenotypes. These observations reveal novel information regarding the pathogenic mechanisms of syndromic lower ventral body malformations, which are largely unknown.

The role of sonic hedgehog-Gli2 pathway in the masculinization of external genitalia.

During embryogenesis, sexually dimorphic organogenesis is achieved by hormones produced in the gonad. The external genitalia develop from a single primordium, the genital tubercle, and their masculinization processes depend on the androgen signaling. In addition to such hormonal signaling, the involvement of nongonadal and locally produced masculinization factors has been unclear. To elucidate the mechanisms of the sexually dimorphic development of the external genitalia, series of conditional mutant mouse analyses were performed using several mutant alleles, particularly focusing on the role of hedgehog signaling pathway in this manuscript. We demonstrate that hedgehog pathway is indispensable for the establishment of male external genitalia characteristics. Sonic hedgehog is expressed in the urethral plate epithelium, and its signal is mediated through glioblastoma 2 (Gli2) in the mesenchyme. The expression level of the sexually dimorphic genes is decreased in the glioblastoma 2 mutant embryos, suggesting that hedgehog signal is likely to facilitate the masculinization processes by affecting the androgen responsiveness. In addition, a conditional mutation of Sonic hedgehog at the sexual differentiation stage leads to abnormal male external genitalia development. The current study identified hedgehog signaling pathway as a key factor not only for initial development but also for sexually dimorphic development of the external genitalia in coordination with androgen signaling.

Genetic analysis of Hedgehog signaling in ventral body wall development and the onset of omphalocele formation.

BACKGROUND: An omphalocele is one of the major ventral body wall malformations and is characterized by abnormally herniated viscera from the body trunk. It has been frequently found to be associated with other structural malformations, such as genitourinary malformations and digit abnormalities. In spite of its clinical importance, the etiology of omphalocele formation is still controversial. Hedgehog (Hh) signaling is one of the essential growth factor signaling pathways involved in the formation of the limbs and urogenital system. However, the relationship between Hh signaling and ventral body wall formation remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: To gain insight into the roles of Hh signaling in ventral body wall formation and its malformation, we analyzed phenotypes of mouse mutants of Sonic hedgehog (Shh), GLI-Kruppel family member 3 (Gli3) and Aristaless-like homeobox 4 (Alx4). Introduction of additional Alx4(Lst) mutations into the Gli3(Xt/Xt) background resulted in various degrees of severe omphalocele and pubic diastasis. In addition, loss of a single Shh allele restored the omphalocele and pubic symphysis of Gli3(Xt/+); Alx4(Lst/Lst) embryos. We also observed ectopic Hh activity in the ventral body wall region of Gli3(Xt/Xt) embryos. Moreover, tamoxifen-inducible gain-of-function experiments to induce ectopic Hh signaling revealed Hh signal dose-dependent formation of omphaloceles. CONCLUSIONS/SIGNIFICANCE: We suggest that one of the possible causes of omphalocele and pubic diastasis is ectopically-induced Hh signaling. To our knowledge, this would be the first demonstration of the involvement of Hh signaling in ventral body wall malformation and the genetic rescue of omphalocele phenotypes.