Hormone signaling and fatty liver in females: analysis of estrogen receptor α mutant mice.

BACKGROUND: Treatment with estrogen in early menopausal women protects against development of hepatic steatosis and nonalcoholic fatty liver disease but estrogen has undesirable side effects, which negate its beneficial effects in premenopausal and postmenopausal women. Targeted therapies require better understanding of the target sites and mechanisms by which estrogen signaling exerts its protective effects in women. Estrogen receptor α (ERα) is thought to be the primary mediator for estrogen signaling to protect against hepatic steatosis. ERα has several mechanisms for signal transduction: (1) inducing gene transcription by direct binding to specific DNA sequences, (2) inducing tethered transcription with other DNA-binding factors, and (3) stimulating nongenomic action through membrane-associated ERα. However, it is still unclear which mechanisms mediate ERα-dependent protection against hepatic steatosis. METHODS: To understand the mechanisms of estrogen signaling for protection against hepatic steatosis in females, we analyzed the global ERα knockout mouse (αERKO), ERα DNA-binding domain mutant mouse (KIKO) and liver-specific ERα knockout mouse (LERKO) fed high-fat diets (HFD). The KIKO mouse disrupts the direct DNA-binding transcription activity but retains tethered transcription regulation and nongenomic action. Hepatic steatosis was evaluated by scoring the macrovesicular and microvesicular steatosis as well as serum alanine aminotransferase (ALT) levels. We analyzed serum testosterone to assess its correlation with hepatic steatosis. RESULTS: Liver fat accumulation was far greater in HFD-fed αERKO and KIKO females than in HFD-fed wild-type (WT) controls. Conversely, HFD-fed LERKO females did not accumulate excess liver fat. HFD-fed αERKO and KIKO females showed higher microvesicular steatosis and ALT levels than WT controls that correlated with increased serum testosterone levels. CONCLUSIONS: ERα-mediated direct transcription in non-hepatic tissues is essential for estrogen-mediated protection against hepatic steatosis in HFD-fed females. The balance between non-hepatic estrogen signaling and hepatic or non-hepatic testosterone action may control hepatic steatosis.

Identification of equol producers in a Japanese population by high-performance liquid chromatography with coulometric array for determining serum isoflavones.

Using a method of high-performance liquid chromatography (HPLC) with coulometric array, we measured isoflavone levels in sera from seven volunteers before and after three days of ingesting Soyaflavone E (an isoflavones powder) and from 129 female farmers (Japanese Multiple Environmental Toxicants Study; JMETS). Results showed that the serum isoflavone concentrations rose dramatically after three days of ingesting Soyaflavone E in all subjects except for the serum equol concentrations in two subjects. The geometric mean concentrations of daidzein, genistein, and equol in the serum of 129 Japanese women were 25.0 ng/ml of daidzein, 94.1 ng/ml of genistein, and 9.6 ng/ml of equol. Interestingly, there existed two dominant groups in terms of serum equol concentrations in an independent manner of soy-derived product intake among the study participants.

An endoplasmic reticulum protein, p180, is highly expressed in human cytomegalovirus-permissive cells and interacts with the tegument protein encoded by UL48.

We have used a virus overlay assay to detect cellular proteins associated with human cytomegalovirus (HCMV) particles. The radiolabeled HCMV particles specifically bound to two host proteins with molecular sizes of 150 and 180 kDa. By a micro-amino-acid sequencing technique, the 180-kDa protein was identified as a human homologue of the ES130/p180 ribosome receptor (p180), which is an integral endoplasmic reticulum (ER) membrane protein possessing a very unique tandem repeat domain at its N-terminal region. The virus overlay assay using truncated p180 polypeptides revealed that HCMV binding to human p180 occurred through the N-terminal region. In HCMV-permissive cells the high level of expression of the human p180 protein was clearly observed regardless of cell type. Furthermore, we showed that p180 binds to the UL48 gene product, which is one of the predominant tegument proteins of HCMV and which is considered to be tightly associated with the capsid. The interaction between the two proteins was assumed to be specific and was observed both in vitro and in vivo. During the late phase of infection, the unique relocation of human p180 was observed, that is, to the juxtanuclear region, which appeared to be in the vicinity of the area where naked virions were frequently observed in an electron-microscopic study. Thus our data suggest that p180 interacts with the HCMV tegument, at least through pUL48, during the HCMV replication process. We discuss the possible role of the interaction between p180 and pUL48 in the intracellular transport of HCMV virions.

A subfamily of RNA-binding DEAD-box proteins acts as an estrogen receptor alpha coactivator through the N-terminal activation domain (AF-1) with an RNA coactivator, SRA.

One class of the nuclear receptor AF-2 coactivator complexes contains the SRC-1/TIF2 family, CBP/p300 and an RNA coactivator, SRA. We identified a subfamily of RNA-binding DEAD-box proteins (p72/p68) as a human estrogen receptor alpha (hER alpha) coactivator in the complex containing these factors. p72/p68 interacted with both the AD2 of any SRC-1/TIF2 family protein and the hER alpha A/B domain, but not with any other nuclear receptor tested. p72/p68, TIF2 (SRC-1) and SRA were co-immunoprecipitated with estrogen-bound hER alpha in MCF7 cells and in partially purified complexes associated with hER alpha from HeLa nuclear extracts. Estrogen induced co-localization of p72 with hER alpha and TIF2 in the nucleus. The presence of p72/p68 potentiated the estrogen-induced expression of the endogenous pS2 gene in MCF7 cells. In a transient expression assay, a combination of p72/p68 with SRA and one TIF2 brought an ultimate synergism to the estrogen-induced transactivation of hER alpha. These findings indicate that p72/p68 acts as an ER subtype-selective coactivator through ER alpha AF-1 by associating with the coactivator complex to bind its AF-2 through direct binding with SRA and the SRC-1/TIF2 family proteins.

A nuclear matrix-associated factor, SAF-B, interacts with specific isoforms of AUF1/hnRNP D.

One class of heterogeneous nuclear ribonucleoproteins (hnRNPs), AUF1/hnRNP D, consists of four isoform proteins (p45, p42, p40, and p37) which are generated by alternative splicing. The present study was therefore undertaken to clarify any isoform-specific differences in terms of their functions and nucleocytoplasmic localization. All isoforms primarily localized in the nucleus. However, heterokaryon analysis and a study using RNA polymerase II inhibitor revealed that p40/p37 exhibited a continuous shuttling between the nucleus and cytoplasm. Constant nuclear retention activity was mapped to the p45/p42-specific sequence at the C-terminal region, which is retained by alternative splicing. Using this domain as a probe, we performed a yeast two-hybrid screening and we found that scaffold attachment factor B (SAF-B), a nuclear matrix-associated protein, exhibits protein-protein interaction to this region. Colocalization of p45/p42 and SAF-B was observed as a speckle in the nucleus. Interestingly, p45/p42 isoforms appeared to act as a negative regulator in gene expression by forming a complex with SAF-B. Thus, the present study revealed that the isoform-specific functions of AUF1/hnRNP D are defined by intracellular shuttling capacity.

Binding of human cytomegalovirus to sulfated glucuronyl glycosphingolipids and their inhibitory effects on the infection.

Interactions between human cytomegalovirus (HCMV) and various carbohydrate structures were analysed using sulfated glucuronyl glycosphingolipids (SGGLs) and the structurally related glycosphingolipids (GLs). A thin-layer chromatography-overlay assay and a solid-phase binding assay revealed that HCMV strongly bound to sulfated glucuronyl lactosaminylparagloboside, one of the SGGLs having the repeating lactosamine structure (3Gal beta1-4GlcNAc1-)2 in addition to the 3-O-sulfated glucuronyl moiety. The virus bound less strongly to other 3-O-sulfated GLs, which included sulfated glucuronyl paragloboside and cerebroside sulfate ester, and also to (3Gal beta1-4GlcNAc1-)2-containing GLs that included nLc6Cer. Thus, a (3Gal beta1-4GlcNAc1-)2 and a 3-O-sulfated saccharide seem to be important structures for the binding by HCMV. When virus particles were preincubated with these GLs, inhibitory effects were observed both on expression of the viral immediate-early gene and on plaque formation by HCMV. These effects were very well correlated with the abilities of the GLs to bind to the virus. Pretreatment of host cells with HNK-1 monoclonal antibody, which specifically recognizes SGGLs, resulted in partial inhibition of plaque formation by HCMV. These results clearly show that HCMV recognizes and binds to the sulfated carbohydrate structure in SGGL and also suggest that binding of HCMV to the specific sugar structure may play an important role in HCMV infection.

The BglII-N fragment of herpes simplex virus type 2 contains a region responsible for resistance to antiviral effects of interferon.

Double infection with two interferon (IFN)-sensitive strains of herpes simplex virus (HSV), HSV-1(17syn) and HSV-2(UW268), showed reduced inhibition of virus growth by IFN. Intertypic recombinants with IFN resistance were obtained from the doubly infected cultures. These results indicate that HSV IFN resistance is controlled by at least two genetic regions. Restriction endonuclease analysis demonstrated that the recombinants were similar to HSV-2 in their genomic structure but the BamHI-A, BglII-I and BglII-N fragments of HSV-2 were commonly lost in the recombinants, suggesting that any of these fragments could be associated with HSV-2 IFN resistance. We cloned these fragments and BamHI-E, which overlaps BglII-N, from an IFN-resistant HSV-2 strain, HSV-2(G), and examined each fragment for its ability to rescue IFN resistance of HSV-2(UW268) by co-transfecting with the HSV-2(UW268) genome. Of the HSV-2(G) fragments, only BglII-N increased plating efficiency of progeny viruses in IFN-treated cells. An IFN-resistant HSV-2 clone was obtained from the BglII-N of HSV-2(G) and HSV-2(UW268) genome co-transfected culture, and a part of BglII-N of HSV-2(UW268) was replaced with that of HSV-2(G) in the HSV-2 clone. Thus, it was concluded that one of the HSV regions encoding IFN resistance is located on the BglII-N fragment of HSV-2.

Unexpected correlation in the sensitivity of 19 herpes simplex virus strains to types I and II interferons.

We compared the sensitivity of 19 herpes simplex virus (HSV) strains to type I (IFN-alpha and IFN-beta) and type II (IFN-gamma) human interferons in cultures of human retinal epithelial (K-1034) and lung (HEL) cells. Their sensitivities proved to be well correlated, even though type I and type II IFN have been reported to have different antiviral actions. The correlation was not because IFN-gamma stimulated the formation of IFN-beta, for an antibody that neutralized IFN-beta did not reduce its inhibitory effects. Our results show that each HSV strain has a characteristic and similar sensitivity to type I and type II IFN and suggest some common pathway in the mechanism of their antiviral actions.

Infection of a human retinal pigment epithelial cell line with human herpesvirus 6 variant A.

A retinal pigment epithelial (RPE) cell line (K-1034) was examined for its susceptibility to human herpesvirus 6 variant A (HHV-6A). Exposure of K-1034 cells to HHV-6A induced the formation of multinucleated giant cells, which was suppressed by an inhibitor of viral DNA synthesis. In the giant cells, herpesvirus nucleocapsids were demonstrated by electron microscopy and the viral glycoprotein B was detected by immunofluorescence assay. These results indicate that K-1034 cells are susceptible to HHV-6A and suggest that HHV-6A has an ability to directly destroy epithelial cells.

Enhanced cytopathic effect of human cytomegalovirus on a retinal pigment epithelium cell line, K-1034, by serum-free medium.

Although human cytomegalovirus (HCMV) predominantly infects epithelial cells in vivo, the majority of studies of HCMV gene expression and replication have been conducted using non-epithelial cell lines in part because of the absence of a good experimental system using epithelial cells. To address the nature of epithelial cell infection, we investigated the susceptibility of an epithelial cell line (K-1034) established from the retinal pigment epithelium to HCMV infection. This cell line exhibited high susceptibility to HCMV, as evidenced by detection of one of the immediate early antigens, IE2, in the nuclei of more than 80% of K-1034 cells at 24 h following inoculation at a multiplicity of infection of 3 plaque forming units per cell. However, the yield after one-step growth of HCMV in K-1034 cells was about twenty-fold less than that in human embryonic lung fibroblast cells. Cytopathic effect (CPE) on K-1034 cells was not prominent in medium supplemented with 10% fetal bovine serum and viral late antigens were detected in less than 5% of K-1034 cells. Interestingly, infected cells expressing late antigens and exhibiting CPE were markedly increased in serum-free medium, even though the yield of infectious HCMV and viral genome copy numbers were almost the same in the different serum concentrations, due to viral instability in the absence of serum. Thus, the progression of late antigens expression and the induction of CPE in infected epithelial cells is influenced by physiological conditions, and are negatively regulated by some serum factor.

In vitro selection of variants of herpes simplex virus type 1 which differ in cytopathic changes.

To analyze the mechanisms for in vitro emergence of the syncytial variants of herpes simplex virus type 1 (HSV-1), several cell lines were infected with a mixture of equal amounts of two HSV-1 variants, one syncytial and the other non-syncytial, and changes in their relative abundance were monitored during passage. With a combination of two variants of the Miyama strain of HSV-1, the syncytial variant became dominant during passage in Vero, RK-13 and FL cells. On the other hand, the ratios of the two variants remained around 1:1 during the passage in HEp-2, MGC and HEL cells. In another set of variants of the SKO strain of HSV-1, the outcomes were different from those of the Miyama strain in the FL, MGC and HEp-2 cells. The ratios of the two variants remained around 1:1 during passage in FL cells, while the non-syncytial variant became dominant during passage in MGC and HEp-2 cells. In addition, we examined the effects of a complement and interferon-beta (IFN-beta) on the outcome of the selection. As a result, the complement slowed the selection of a syncytial variant, whereas IFN-beta facilitated it.

Steroid hormones differentially induce transcription of the chicken ovalbumin gene, but stabilize the mRNA with the same half-life.

The stabilization of chicken ovalbumin (OVA) mRNA by different classes of steroid hormones (estrogen, progesterone, glucocorticoid, and androgen) was studied in the oviducts of chicks treated with combinations of four steroids. The combination of estrogen with progesterone, glucocorticoid, or androgen enhanced the induction of the OVA gene more than did estrogen alone. Run-on analysis of the isolated oviduct nuclei to measure the transcription rate of the OVA gene showed that the enhanced induction of the OVA gene by the combined hormone treatments was partly caused by an increased rate of transcription. The half-life of OVA mRNA as determined using a transcription inhibitor (actinomycin D) was estimated to be about 24 h irrespective of the hormone treatment, though the half-life was about 6 h in the absence of hormones. These results suggested that the prolongation of the half-life of OVA mRNA by steroid hormones is constant irrespective of differential transcription rates of the OVA gene.

Steroid hormone-induced expression of the chicken ovalbumin gene and the levels of nuclear steroid hormone receptors in chick oviduct.

The induction of the chicken ovalbumin (OVA) gene by different classes of steroid hormones and the mRNA levels of estrogen (ER), progesterone (PR), and glucocorticoid (GR) receptors were studied in chick oviducts. Combined treatment with two hormones increased the induction of the OVA gene more than single treatment, when the levels of OVA mRNA were measured with Slot blot analysis. To discover the role of nuclear steroid hormone receptors as transcriptional factors in the OVA gene induction, we analyzed the levels of ER (with RT-PCR), PR, and GR mRNAs (with Northern blotting). The level of PR mRNA was increased only by estrogen, while no steroid hormone affected the levels of ER and GR mRNAs. Thus, these findings show that the levels of nuclear receptors do not reflect the OVA mRNA level in the oviduct of steroid hormone-treated chicks.

Vitamin A is involved in estrogen-induced cell proliferation but not in cytodifferentiation of the chicken oviduct.

We examined vitamin A-deficient chicks to determine whether vitamin A affects the estrogen-induced development of the chick oviduct. When oviduct development was stimulated for 5 days with the synthetic estrogen, diethylstilbestrol, the wet weight of the oviduct in vitamin A-deficient chicks was only half that in control chicks. The DNA content in this tissue showed that the decreased oviduct weight in the vitamin A-deficient chicks was caused by the decreased proliferation of oviduct cells. However, the estrogen-induced expression of the ovalbumin gene was not affected by the vitamin A deficiency, suggesting that estrogen-induced cytodifferentiation is not affected by vitamin A. To clarify the vitamin A action on estrogen-induced development in the oviduct, transcripts of nuclear estrogen receptor (ER) and all-trans-retinoic acid (RAR alpha, beta and gamma) receptors, which exert the effects of estrogen and vitamin A, were measured. The ER, RAR alpha and RAR beta genes, but not that of RAR gamma, were expressed during oviduct development, indicating that estrogen and vitamin A may control the expression of target genes through their cognate receptors. Thus, we have shown that vitamin A is involved in estrogen-induced cell proliferation but not in cytodifferentiation of the chicken oviduct.

A synthetic oestrogen antagonist, tamoxifen, inhibits oestrogen-induced transcriptional, but not post-transcriptional, regulation of gene expression.

Oestrogen (E2) regulates the expression of its target genes at transcriptional and post-transcriptional levels. To clarify the mechanism of E2-induced post-transcriptional regulation, with attention to the involvement of the oestrogen receptor (ER), we studied the effect of tamoxifen (TAM), a synthetic E2 antagonist that inhibits ER-mediated transcription, on E2-induced transcriptional and post-transcriptional regulation of the chicken ovalbumin (OVA) gene in chick oviducts. Run-on analysis with oviduct nuclei isolated from E2-treated chicks showed that TAM treatment completely blocked E2-induced transcription of the OVA gene within 24 h without affecting ER gene expression. Likewise, the rate of transcription fell to below the limit of detection after E2 withdrawal from the chicks. Reflecting the transcription rate, OVA mRNA accumulated linearly in E2-treated chicks, and E2 withdrawal caused a rapid loss of OVA mRNA. However, in the chicks treated with TAM and E2, OVA mRNA was degraded slowly over 48 h with a half-life of 24 h, suggesting that TAM does not inhibit E2-induced mRNA stabilization. Moreover, E2-induced mRNA stabilization was observed even when transcription of the OVA gene was blocked by a transcription inhibitor. Western-blot analysis showed that the remaining OVA mRNA was translatable. Thus the present study indicates that E2 regulates expression of the OVA gene via distinct pathways at transcriptional and post-transcriptional levels.

Positive and negative regulation of retinoid X receptor gene expression by thyroid hormone in the rat. Transcriptional and post-transcriptional controls by thyroid hormone.

The 9-cis-retinoic acid receptors (RXRs), belonging to the members of the steroid/thyroid hormone receptor superfamily, act as auxiliary proteins, heterodimerizing with other nuclear receptors such as retinoic acid receptors (RARs), vitamin D receptor, thyroid hormone receptors, and peroxisome-proliferator activated receptor, thereby transactivating target genes in a ligand-dependent manner. We have previously reported that in the rat, thyroid hormone (TH) positively and negatively regulates the hepatic mRNA levels of RXR beta and RXR gamma, respectively. In the present study, we have tried to elucidate the level at which TH regulates the gene expression of RXR beta and RXR gamma in the rat. A RNA synthesis inhibitor (actinomycin D), but not a protein synthesis inhibitor (cycloheximide), blocked the induction of RXR beta mRNA by TH. On the other hand, none of these drugs inhibited the decrease of RXR gamma mRNA levels caused by TH. Nuclear run-on assays showed that the transcription rate of the RXR beta gene was positively regulated by TH, whereas the transcription of RXR gamma gene was not controlled by TH. Taken together, these results indicate that the gene expression of RXR beta is positively regulated by TH at transcriptional level, while the negative regulation of the RXR gamma gene expression by TH may occur at a post-transcriptional level in intact rat. Thus, the RXR-mediated signal transductions may be modulated in part through TH control of the levels of RXR beta and RXR gamma.

Relationship between chromosomal alpha-specific suppressors of sir4-11 and polymorphism of the HMRa-bearing fragment.

In the course of studying sir4-11 which is responsible for the non-mating phenotype of asd-homothallism of Saccharomyces cerevisiae, we detected chromosomal alpha-specific suppressors of it. By examining the mating-type cassette constitution of two strains and the spore-clones derived from a diploid culture formed by a mass mating between these strains, we obtained the following results; (1) the HMRa-bearing HindIII-Bg/II restriction fragment was dimorphic, as judged by size, within each tetrad, (2) while one form was common to all tetrads, the other varied among tetrads, (3) spore-clones with the alpha-specific suppressor of sir4-11 had the variant forms while those without had the common form, and (4) while the two parents had the common form, each independent diploid clone had the common form plus a variant form. From these results, we conclude that the mating of cells in certain combinations induces a change of DNA structure at or near HMRa in a mating-pair specific manner and that the change makes HMRa non-derepressible or non-functional when derepressed.

Asd-homothallism of Saccharomyces cerevisiae: identification of asd1-1 as an allele of sir4 and detection of alpha-specific suppressors of it.

Asd-homothallism of Saccharomyces cerevisiae involves a life cycle characterized by a non-mating phenotype and endomitotic diploidization. The former trait is determined by a single mutation, asd1-1. This mutation was mapped between hom2 and lys4 on the right arm of chromosome IV and was complemented by the cloned SIR4 gene. Therefore, we conclude that asd1-1 is an allele of sir4-11 and renamed it sir4-11. Endomitotic diploidization of asd-homothallism is caused by the collaboration of three to four mutations including sir4-11. In the course of this study, we detected alpha-specific suppressors of sir4-11.