Selective upregulation of p66-Shc gene expression in the liver and brain of aged rats.

The phosphotyrosine signaling followed by various receptor activations conforms a unique signaling platform during metazoan evolution, and is crucial for animal development, maturation, and aging. Shc is the most versatile bipartite phosphotyrosine signal adaptor harboring phosphotrosine-biding (PTB) and Src-homology2 (SH2) domains. Among the Shc adaptor family members, p66-Shc is of potential interest in aging studies, since its deletion in mice resulted in a longer lifespan and/or higher quality of life in later stages of life. However, a few studies have examined the gene expression profiles of p66-Shc in aging tissues. Here, we quantified the expression levels of transcripts of Shc-related isoforms in the liver and brain of young adult, middle-aged, and aged rats, and found that p66-Shc gene expression is specifically up-regulated in the aged liver and brain. In the aged liver tissue, p66-Shc expression was also evident at the protein level, and accumulated in the soluble fraction of the aged tissue. These results indicate that p66-Shc is not only related to animal longevity but also affected during aging, and thus the repression of p66-Shc could become a potential target for an anti-aging strategy.

Position-dependent effect of a neural-restrictive silencer-like element present in the promoter downstream of the SCG10-like protein gene.

Neural-restrictive silencer (NRS) has been well characterized in SCG10 and many other neuron-specific genes; it is, however, unknown whether the promoters of the SCLIP and RB3 genes (two other SCG10 family members) share basal transcriptional mechanisms with SCG10 or not. To explore how NRS-mediated neural-specific gene transcription has evolved, we determined the genomic and promoter structures of the SCLIP gene, and found that the gene retained an NRS-like element that functioned as a negative regulator in non-neuronal cells. However, unlike the NRS in the SCG10 gene, this NRS(SCLIP) was located downstream of the transcription start site, and showed a position-dependent repressing activity. Further gel-shift and NRS factor (NRSF) co-transfection experiments revealed that NRS(SCLIP) was bound and regulated by NRSF. Such an element was not found in the gene promoter of RB3, suggesting that the NRS-NRSF regulatory system evolved once SCLIP had diverged from RB3 or stathmin.

In vitro assessment of transcriptional activation of the estrogen and androgen receptors of mosquitofish, Gambusia affinis affinis.

Sex-steroid hormones are essential for normal reproductive activity in both sexes. Estrogens are necessary for ovarian differentiation during a critical developmental stage in many vertebrates and promote the growth and differentiation of the female reproductive system. Androgens play essential roles in the development and functioning of the vertebrate male reproductive system as well as actively supporting spermatogenesis. Importantly, recent studies suggest that androgens and estrogens have important reproductive roles in both males and females. To understand the molecular mechanisms of estrogen and androgen actions and to evaluate estrogen and androgen receptor-ligand interactions in the mosquitofish, Gambusia affinis affinis, we used degenerate primer sets and PCR techniques to isolated DNA fragments encoding estrogen receptor alpha (ERalpha; ESR1), ERbeta1 (ERbeta1) and ERbeta2 from the ovary. Full-length mosquitofish ER (mfER) cDNAs were obtained using cDNA library screening and RACE techniques. Amino acid sequences of mfERs showed over-all homology of 46% (alpha versus beta1), 43% (alpha versus beta2), and 52% (beta1 versus beta2). We applied the ERE-luciferase reporter assay system to characterize these receptors. In this transient transfection assay system using mammalian cells, the mfER proteins displayed estrogen-dependent activation of transcription. In addition to ERs, the transactivation of mosquitofish ARs (mfARs) previously isolated by our group, were examined using an androgen-responsive MMTV-luciferase assay system. Mosquitofish ARs showed androgen-dependent activation of transcription from the MMTV promoter. These data provide a basic tool allowing future studies examining the receptor-ligand interactions and endocrine disrupting mechanisms in mosquitofish and also expands our knowledge of estrogen and androgen receptor evolution.

Effects of an androgenic growth promoter 17beta-trenbolone on masculinization of Mosquitofish (Gambusia affinis affinis).

Endocrine disrupting chemicals can affect normal hormone dependent processes through numerous mechanisms, including ligand mimicky. 17beta-Trenbolone (TB), a pharmaceutical, androgenic, anabolic steroid, is a potent agonist of androgen receptors, and has been extensively used as a growth promoter for beef cattle in the US. The effects of TB on adult and newborn mosquitofish (Gambusia affinis affinis) were examined. Two forms of mosquitofish androgen receptor (AR), ARalpha and ARbeta, were cloned. The mRNA expression levels of ARalpha and ARbeta were transiently increased in the anal fin of adult females at day 3 following exposure to TB (1-10 microg/L) or methyltestosterone (MT) (0.1-10 microg/L), a pharmaceutical androgen used as a positive control. Gonopodium differentiation from the adult female anal fin was induced after 28 days of exposure to TB (1-10 microg/L) or MT (0.1-10 microg/L). Gonopodium differentiation also was induced in all mosquitofish fry exposed for 28 days to 0.3, 1 or 10 microg/L TB. Furthermore, spermatozoa were observed histologically in the testes of male fry exposed for 28 days to 1 or 10 microg/L TB; spermatozoa are normally observed only in the testes of mature males. Surprisingly, all female fry exposed for 28 days to 1 or 10 microg/L TB displayed the formation of an ovotestis, as spermatozoa were found in the ovary. Thus, TB, like MT, induced masculinization of the anal fin accompanied by a transient up-regulation of ARalpha and ARbeta in adult females. TB also induced differentiation of the anal fin into a gonopodium in fry of both sexes, stimulated precocious spermatogenesis in the testes of males and the formation of ovotestes in females.

Effects of 17beta-estradiol, nonylphenol, and bisphenol-A on developing Xenopus laevis embryos.

Many chemicals released into the environment have the capacity to disrupt the normal development of aquatic animals. We investigated the influence of nonylphenol (NP), bisphenol-A (BPA), and 17beta-estradiol (E2) on developing Xenopus laevis embryos, as a model animal in the aquatic environment. Embryos were exposed to eight different concentrations of NP, BPA or E2 between 3 and 96 h post-fertilization (p.f.). Short body length, microcephaly, flexure, edema, and abnormal gut coiling were induced by 20 microM NP, BPA or 10 microM E2 by 96 h p.f. To clarify sensitive stages to these compounds, embryos were exposed to chemicals for 45 or 48 h starting at different developmental stages and experiments were terminated 96 h p.f. BPA and NP induced abnormalities in developing X. laevis, though the sensitive stages of embryos to these chemicals are different, BPA affecting earlier stages and NP affecting at later stages. To analyze the functional mechanisms of BPA and NP in induction of morphological changes, we adapted a DNA array technology and identified 6 X. laevis genes, XIRG, alpha skeletal tropomyosin, cyclin G1, HGF, troponin C2, and ribosomal protein L9. These findings may provide important clues to elucidate common mechanisms underlying teratogenic effects of these chemicals.

Abnormal angiogenesis in Foxo1 (Fkhr)-deficient mice.

Members of the Foxo family, Foxo1 (Fkhr), Foxo3 (Fkhrl1), and Foxo4 (Afx), are mammalian homologs of daf-16, which influences life span and energy metabolism in Caenorhabditis elegans. Mammalian FOXO proteins also play important roles in cell cycle arrest, apoptosis, stress resistance, and energy metabolism. In this study, we generated Foxo1-deficient mice to investigate the physiological role of FOXO1. The Foxo1-deficient mice died around embryonic day 11 because of defects in the branchial arches and remarkably impaired vascular development of embryos and yolk sacs. In vitro differentiation of embryonic stem cells demonstrated that endothelial cells derived from wild-type and Foxo1-deficient embryonic stem cells were able to produce comparable numbers of colonies supported by a layer of OP9 stromal cells. Although the morphology of the endothelial cell colonies was identical in both genotypes in the absence of exogenous vascular endothelial growth factor (VEGF), Foxo1-deficient endothelial cells showed a markedly different morphological response compared with wild-type endothelial cells in the presence of exogenous VEGF. These results suggest that Foxo1 is essential to the ability of endothelial cells to respond properly to a high dose of VEGF, thereby playing a critical role in normal vascular development.

Grit, a GTPase-activating protein for the Rho family, regulates neurite extension through association with the TrkA receptor and N-Shc and CrkL/Crk adapter molecules.

Neurotrophins are key regulators of the fate and shape of neuronal cells and act as guidance cues for growth cones by remodeling the actin cytoskeleton. Actin dynamics is controlled by Rho GTPases. We identified a novel Rho GTPase-activating protein (Grit) for Rho/Rac/Cdc42 small GTPases. Grit was abundant in neuronal cells and directly interacted with TrkA, a high-affinity receptor for nerve growth factor (NGF). Another pool of Grit was recruited to the activated receptor tyrosine kinase through its binding to N-Shc and CrkL/Crk, adapter molecules downstream of activated receptor tyrosine kinases. Overexpression of the TrkA-binding region of Grit inhibited NGF-induced neurite elongation. Further, we found some tendency for neurite promotion in full-length Grit-overexpressing PC12 cells upon NGF stimulation. These results suggest that Grit, a novel TrkA-interacting protein, regulates neurite outgrowth by modulating the Rho family of small GTPases.

Developmental toxicity of estrogenic chemicals on rodents and other species.

Antenatal sex-hormone exposure induces lesions in mouse reproductive organs, which are similar to those in humans exposed in utero to a synthetic estrogen, diethylstilbestrol. The developing organisms including rodents, fish and amphibians are particularly sensitive to exposure to estrogenic chemicals during a critical window. Exposure to estrogens during the critical period induces long-term changes in reproductive as well as non-reproductive organs, including persistent molecular alterations. The antenatal mouse model can be utilized as an indicator of possible long-term consequences of exposure to exogenous estrogenic compounds including possible environmental endocrine disruptors. Many chemicals released into the environment potentially disrupt the endocrine system in wildlife and humans, some of which exhibit estrogenic activity by binding to the estrogen receptors. Estrogen responsive genes, therefore, need to be identified to understand the molecular basis of estrogenic actions. In order to understand molecular mechanisms of estrogenic chemicals on developing organisms, we are identifying estrogen responsive genes using cDNA microarray, quantitative RT-PCR, and differential display methods, and genes related to the estrogen-independent vaginal changes in mice induced by estrogens during the critical window. In this review, discussion of our own findings related to endocrine distuptor issue will be provided.

Temporal alteration of dose-dependent response to activin in newt animal-cap explants.

In this study, we examined the dose-dependent responses of animal-pole cells of the Japanese newt, Cynops pyrrhogaster, to activin. Cynops has a slower developmental rate and a simpler animal cap structure than Xenopus. These features enable temporal differences in animal-cap competence to be identified more easily and relatively sharp dose-response profiles can be obtained without cell dissociation. When Cynops caps were excised at the mid-blastula stage and transcript levels of marker genes were examined at the early gastrula stage, the strongest induction of brachyury occurred at a low activin dose, suggesting that cells can recognize changing concentrations of an inducing signal in the embryo. Furthermore, the time course of brachyury expression revealed that caps from the mid-blastula stage exposed to a high dose of activin maintained a low expression level after induction. This suggests that Cynops animal-pole cells can assess activin concentration in a simple and direct manner. In addition, we found that animal-cap competence significantly changes during the blastula stage. The data presented here suggest that this change does not autonomously occur within animal-pole cells but requires signals that emanate from other germ layers.