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K Morohashi

Publications and source records attributed to K Morohashi.

At least 37 records · Page 2Linked to original sources
PubMed1997

The expression of sf-1/Ad4BP is related to the process of luteinization in the marmoset (Callithrix jacchus) ovary.

Expression of the nuclear receptor, steroidogenic factor-1 (SF-1/Ad4BP), was studied in a primate (marmoset) ovary using immunohistochemical, RT-PCR, and immunoblot techniques. The periovulatory phase was compared with the luteal phase. With PCR we found a marmoset homolog of SF-1/Ad4BP to be expressed in ovarian and other steroidogenic tissues. Characteristically, the periovulatory ovaries consisted of growing (non-luteinized) small follicles together with large luteinizing follicles and many corpora lutea accessoria (Clas), which had developed from atretic large follicles. During the luteal phase, true corpora lutea (Cls) were additionally found. In general, we found that small follicles were devoid of any immunoreactivity of SF-1/Ad4BP. In large follicles, the luteinizing theca and granulosa cells express SF-1/Ad4BP. All luteal cells of Clas showed a nuclear staining in both ovary types. In Cls, only a few luteal cells were positive. Large follicles of different sizes showed no differences in expression level, as evidenced by immunoblot analysis. Our results indicate that SF-1/ Ad4BP participates in the activation of genc transcription during the onset of luteinization and that Clas are essential for ovarian luteal function.

Animals↗
PubMed1997

Transcriptional regulation of the bovine CYP17 gene by cAMP.

The transcription of steroid hydroxylase genes is controlled by ACTH and cAMP in the adrenal cortex. In most instances the regulation appears to rely on transcription factors traditionally not associated with cAMP-dependent gene expression. For the non-traditional factors it remains necessary to elucidate the coupling of increases in intracellular cAMP and cAMP-dependent protein kinase (PKA) activity to the function of these proteins. The bovine CYP17 gene, which encodes the steroid 17 alpha-hydroxylase, contains two discrete DNA elements within its promoter and upstream region (CRS1 and CRS2) that individually can confer cAMP responsiveness. The CRS1 element is a target for PKA signalling and for negative regulation via the protein kinase C signal transduction pathway. The homeodomain protein Pbx1 enhances CRS1-dependent transcription, but additional CRS1-binding proteins remain to be identified. Furthermore it is not known how PKA regulates the activity of Pbx1 or its possible binding partners. Closer to the promoter, the nuclear orphan receptors SF-1 and COUP-TF have overlapping binding sites in CRS2 and they bind in a mutually exclusive manner with very similar affinities; 8 and 10 nM, respectively. SF-1 stimulates whereas COUP-TF inhibits transcription from the bovine CYP17 promoter. Together, the data suggest that cAMP-dependent control of the amounts of the activator SF-1 vs. the repressor COUP-TF could influence CRS2-dependent transcription. In addition, PKA may influence the phosphorylation of SF-1, thus increasing its activity. In vitro, PKA will elicit phosphorylation of SF-1. However, although SF-1 can be immunoprecipitated from adrenocortical cells as a phosphroprotein, we have not been able to show cAMP-dependent increase in net phosphorylation in intact cells. More careful examination of individual phosphorylation sites in SF-1 may still reveal hormone- and cAMP-induced phosphorylation of SF-1.

Animals↗
PubMed1997

The regulation of 3 beta-hydroxysteroid dehydrogenase expression.

3 beta-Hydroxysteroid dehydrogenasel delta 5-->4-isomerase (3 beta-HSD) catalyzes the formation of delta 4-3-ketosteroids from delta 5-3 beta-hydroxysteroids, an obligate step in the biosynthesis not only of androgens and estrogens but also of mineralocorticoids and glucocorticoids. The enzyme is expressed in the adrenal cortex and in steroidogenic cells of the gonads, consistent with this role. However, 3 beta-HSD is also expressed in many other tissues, such as the liver and kidney, where its function is not entirely clear. It is established that a family of closely related genes encode for 3 beta-HSD. The various 3 beta-HSD isoforms are expressed in a tissue-specific manner involving separate mechanisms of regulation. The human type I 3 beta-HSD is expressed at high levels in syncytial trophoblast and in sebaceous glands, and the type II isoform is almost exclusively expressed in the adrenal cortex and gonads. An important feature in liver and kidney (at least of hamster, mouse, rabbit, and rat) is the sexual dimorphic nature of 3 beta-HSD expression. We briefly review studies on the regulation of the human 3 beta-HSD I and II genes in human trophoblast and adrenal cortex and extend this to discuss the rat 3 beta-HSD I gene expressed in adrenals and gonads. The complexity of 3 beta-HSD expression through multiple signaling pathways acting on a multigene family of enzymes may contribute importantly to the diverse patterns and locations of steroid hormone biosynthesis.

3-Hydroxysteroid Dehydrogenases↗
PubMed1997

The ontogenesis of the steroidogenic tissues.

Based on the common function of steroid hormone-producing tissues and homologous regulation by the hypothalamo-pituitary axis, the adrenal cortex and the gonads have been suggested to have an intimate ontogenic relationship. This assumption is also supported by the findings of common transcription factors implicated in the differentiation of both types of tissue and further supported by concomitant defects in such tissues due to the disruption of a single gene. Similarly, simultaneous anomalies in those tissues are also observed in some diseases caused by mutations of the genes encoding those transcription factors. A recent immunohistochemical study with one of the transcription factors, Ad4BP/SF-1, definitely demonstrated the presence of a particular cell population designated the 'adreno-genital primordium (AGP)' which gives rise to both the adrenal cortex and the gonads. In the process of differentiation from the AGP to the mature adrenal cortex and the gonads of the two sexes, several interesting issues can be raised as to the next targets for the study. To address these issues it is important to elucidate the upstream regulatory mechanisms and downstream target genes of such transcription factors as WT1, SRY, SOX9 and DAX1, in addition to Ad4BP/SF-1, all of which are implicated in steroidogenic tissue differentiation.

Adrenal Cortex↗
PubMed1997

In vivo gene transfer into the blastoderm of early developmental stage of chicken.

An attempt was made to improve gene transfer into chick embryos in order to produce transgenic chickens. The beta-actin-lacZ/MiwZ, a marker gene in transfection reagent, was injected into the blastodisc of either unincubated fertilized eggs (stage X) or eggs induced from the shell gland by treating the hens intravenously with oxytocin or arginine vasotocin (stages IV-VI). All the manipulated embryos were incubated to reach stage XIV, the period at which primordial germ cells (PGCs) migrate from the germinal crescent to the gonadal anlage via the blood stream. MiwZ was detected in the embryos, extraembryonic tissues and blood by the histochemical staining method of beta-galactosidase. The MiwZ DNA was detected in 57% (127/221) of the survival embryos and in 9% (12/127) of the embryonic tissues. The expression was observed mosaically in the epidermis, heart and neural tube. The PGCs in the blood collected from the vitelline artery or dorsal aorta also showed a positive histochemical staining. However, the expression of MiwZ using the soft shelled eggs was more intense in the extraembryonic tissues, although it did not emerge in the embryos. Thus, it is possible to introduce an exogenous gene into the embryonic tissues using incubated fertilized eggs without sacrificing the hens. This technique for successive genetic operations should facilitate the production of transgenic chickens.

Actins↗
PubMed1997

Expression of aldosterone synthase cytochrome P450 (P450aldo) mRNA in rat adrenal glomerulosa cells by angiotensin II type 1 receptor.

Changes in the mRNA levels for aldosterone synthase cytochrome P450 (P450aldo or CYP11B2) in rat adrenal glands were studied in response to angiotensin II type 1 (AT1) and type 2 (AT2) receptor antagonists. Since 11 beta hydroxylase P450 (P45011 beta or CYP11B1), which shows high homology (88.5%) with P450aldo in their nucleotide sequences of the amino acid coding regions, is also expressed in the adrenal gland, RT-PCR was performed with specific primers for each P450. Upon sodium restriction (5 mmol Na+/kg of diet) of the rats for 14 days, the amount of the P450aldo mRNA in the adrenal glands increased 8.5 fold above from the rats fed on a normal diet (225 mmol Na+/kg of diet), whereas no significant change of the P45011 beta mRNA was observed after the dietary sodium restriction. As shown by an immunoblot analysis, the adrenal capsule portions (mainly zona glomerulosa) of the rats kept on the low Na diet for 14 days expressed significantly higher level of P450aldo than those from the rats fed the normal diet. In concert with the alteration, plasma aldosterone concentration increased. However, when a specific AT1 antagonist E4177 was given to the rats kept on a low Na diet, the amount and activity of P450aldo as well as the plasma aldosterone concentration was suppressed. On the other hand, the increase of P450aldo induced by the low Na diet was not affected by an AT2-specific antagonist, PD123177.

Aldosterone↗
PubMed1997

Regulation of aldosterone synthase cytochrome P450 (CYP11B2) and 11 beta-hydroxylase cytochrome P450 (CYP11B1) expression in rat adrenal zona glomerulosa cells by low sodium diet and angiotensin II receptor antagonists.

Changes in the mRNA levels for aldosterone synthase cytochrome P450 (CYP11B2 or P450aldo) and 11 beta hydroxylase cytochrome P450 (CYP11B1 or P45011 beta) in rat adrenal glands were studied in response to angiotensin II type 1 (AT1) and type 2 (AT2) receptor antagonists. CYP11B1 and CYP11B2 genes were highly homologous (88.5%) in their nucleotide sequences of the amino acid coding regions. Reverse transcription-polymerase chain reactions (RT-PCR) which are capable of discriminating between rat CYP11B1 and CYP11B2, were performed with specific primers for each P450. Upon sodium restriction (5 mmol Na(+)/kg of diet) of rats for 14d, the amount of the CYP11B2 mRNA in the adrenal glands was increased 8.5-fold compared to that from the rats fed a normal diet (225 mmol Na(+)/kg of diet), whereas no significant change in the CYP11B1 mRNA was observed after the dietary sodium restriction. As shown by an immunoblot analysis, the adrenal capsule portions (mainly zona glomerulosa) of the rats kept on the low Na diet for 14d expressed significantly higher levels of both CYP11B2 and CYP11B1, and contained a significantly higher amount of CYP11B2 than those from the rats fed by normal diet. The activities of the CYP11B2 enzyme were also found to be increased by about 8-fold on day 14. In concert with these alterations, the plasma aldosterone concentration (PAC) increased. However, when the specific AT1 antagonist E4177 was given to rats maintained on the low Na diet, the amount and activity of CYP11B2, as well as the PAC, were suppressed. In contrast, the increase in CYP11B2 induced by the low Na diet was not affected by the AT2-specific antagonist PD123177. These results indicate that the aldosterone synthase cytochrome P450 (CYP11B2) is an ultimate target of the regulation of aldosterone biosynthesis by an AT1 receptor antagonist.

Aldosterone↗
PubMed1996

Structural characterization of human Ad4bp (SF-1) gene.

Ad4BP (or steroidogenic factor 1, SF-1) has been implicated to be an essential transcriptional factor for steroidogenesis as well as for the development of the reproductive axis. We elucidated the structure of the human Ad4BP gene. The gene is about 30 kb long and is split into 7 exons including a non-coding exon 1. The deduced amino acid sequence of the human Ad4BP consists of 461 amino acid residues and was highly homologous to those of other mammalian species.

Amino Acid Sequence↗
PubMed1996

Autoregulatory loop in the regulation of the mammalian ftz-f1 gene.

The mammalian ftz-f1 (mftz-f1) gene encoding Ad4BP/SF-1 has been demonstrated to be essential for the development of adrenal and gonadal glands. In a previous study, we identified an E box as the transcriptional element in the 5'-upstream region of the rat mftz-f1 gene. In the present study, we found a steroidogenic cell-specific transcriptional element in the first intron of the gene. Gel mobility shift and DNase I footprint analyses clearly revealed that Ad4BP itself binds to the element (Ad4 site). This finding was further supported by the positive effect of an Ad4BP expression vector on the transcription and by the significant decrease in the transcription caused by nucleotide substitutions within the Ad4 site. Similar loss was also caused by substitutions in the E box, indicating that the two elements are essential for the full transcriptional activity of the gene. DNase I hypersensitivity assays revealed that the chromatin structure around the Ad4 site and the E box was "open up" in the adrenal glands and Y-1 cells, whereas "closed down" in the liver. These observations indicated that the mftz-f1 gene is controlled by the autoregulatory loop in the steroidogenic tissues. The autoregulatory mechanism seems to be necessary to keep the mftz-f1 gene activated and thus maintain the tissues differentiated.

Animals↗
PubMed1996

Follicle stimulating hormone-beta subunit gene is expressed in parallel with a transcription factor Ad4BP/SF-1 in human pituitary adenomas.

OBJECTIVES: A transcription factor Ad4BP/SF-1 is implicated in the differentiation of gonadotrophs in the pituitary gland, but it is not known whether human pituitary cells express this factor. The present study aimed to disclose (1) whether human pituitary adenomas express Ad4BP/SF-1, and (2) if this is the case, what kinds of adenoma express the factor. MATERIAL: Total RNA was extracted from 23 pituitary adenomas obtained by transsphenoidal surgery, and subjected to Northern blot analyses using cDNAs of bovine Ad4BP/SF-1, porcine FSH-beta, LH-beta and glycoprotein hormone-alpha (GPH-alpha) subunts as radiolabelled probes. These adenomas included 13 clinically non-functioning adenomas, 1 GH/PRL-producing adenoma, 6 GH-producing adenomas, 2 PRL-producing adenomas and 1 ACTH-producing adenoma. RESULTS: The expression of Ad4BP/SF-1 exactly coincided with the expression of FSH-beta. Thus 5 out of 13 clinically non-functioning adenomas expressed Ad4BP/SF-1 and only these 5 adenomas expressed FSH-beta. Interestingly, only one of the GH-producing adenomas also expressed Ad4BP/SF-1 as well as FSH-beta. GPH-alpha was expressed in 4 non-functioning adenomas and 2 hormonally functioning adenomas, but did not necessarily coincide with Ad4BP/SF-1 expression. None of the 23 adenomas we tested expressed LH-beta, probably because LH-beta-producing adenomas are rather rare. CONCLUSIONS: The expression of FSH-beta was parallel with Ad4BP/SF-1 expression, indicating that the expression of Ad4BP/SF-1 is restricted to cells derived from gonadotroph lineages in human pituitary adenomas.

Adenoma↗
PubMed1996

Identical origin of adrenal cortex and gonad revealed by expression profiles of Ad4BP/SF-1.

BACKGROUND: Ad4BP/SF-1 was originally identified as a steroidogenic tissue-specific transcription factor. Recent gene disruption studies with the mammalian Ftz-F1 gene encoding Ad4BP/SF-1 clearly revealed the essential function of the factor for adrenal and gonadal differentiation. RESULTS: In this study, we examined the early development of these tissues using Ad4BP/SF-1 as the marker. In rat foetuses of 11.5 days post-coitum (d.p.c.), a cell population designated adrenogenital primordium was firstly observed on symmetrical lines extending from the dorsal aorta to the dorsal coelomic epithelia of the primitive urogenital ridges. From 12.5 d.p.c., the rostral half of the adreno-genital primordium started to separate into two distinct cell populations. Judging from the distribution of primordial germ cells, the cell population on the dorsal aortal side is a primordium for the adrenal cortex whereas that on the coelomic epithelial side is for the gonads. At 13.5 d.p.c., these two primordia have separated completely. CONCLUSION: These observations clearly identified a novel adreno-genital primordium from which both the adrenal cortex and the gonads originate. An RT-PCR study conducted to detect adrenal- and gonad-specific mRNAs supported the above observations.

Adrenal Cortex↗
PubMed1995

Ad4BP/SF-1 regulates cyclic AMP-induced transcription from the proximal promoter (PII) of the human aromatase P450 (CYP19) gene in the ovary.

Aromatase P450, which is responsible for the metabolism of C19 steroids to estrogens, is expressed in the pre-ovulatory follicles and corpora lutea of ovulatory women by means of a promoter proximal to the start of translation (PII). To understand how this transcription is controlled by cAMP, we constructed chimeric constructs containing deletion mutations of the proximal promoter 5'-flanking DNA fused to the rabbit beta-globin reporter gene. Assay of reporter gene transcription in transfected bovine granulosa and luteal cells revealed that cAMP-stimulated transcription was lost upon deletion from -278 to -100 base pairs, indicating the presence of a functional cAMP-responsive element in this region; however, no classical cAMP-responsive element was found. Mutation of an AGGTCA motif located at -130 base pairs revealed that this element is crucial for cAMP-stimulated reporter gene transcription. When a single copy of this element was placed upstream of a heterologous promoter, it could act as a weak cAMP-response element. Supershift electrophoretic mobility shift assay and UV cross-linking established that Ad4BP/SF-1 binds to this hexameric element. Ad4BP/SF-1 mRNA and protein levels and DNA binding activity are increased in forskolin-treated luteal cells. We conclude that cAMP-stimulated transcription of human aromatase P450 in the ovary is due, at least in part, to increased levels and DNA binding activity of Ad4BP/SF-1.

Animals↗
PubMed1995

An E box element is required for the expression of the ad4bp gene, a mammalian homologue of ftz-f1 gene, which is essential for adrenal and gonadal development.

Ad4BP, also known as SF-1, is a cell type-specific transcription factor regulating all the steroidogenic P-450 genes. Recently, the targeted disruption of the mouse ftz-f1 gene encoding Ad4BP/SF-1 has established its essential function in both adrenal and gonadal development and sexual differentiation. As an initial step toward understanding its role in the cascade of gene activations necessary for the differentiation of the steroidogenic tissues and the sex differentiation of the gonads, we isolated and characterized the rat ad4bp gene. A sequence analysis of the ad4bp gene revealed that another nuclear factor ELP was also transcribed from the same gene by alternative promoter usage and splicing. The promoter of the ad4bp gene showed activities in the steroidogenic cells such as Y-1 adrenocortical cells and I-10 testicular Leydig cells when examined by transient transfection assays. Using deletion analysis and site-directed mutagenesis, we identified a cis-element at the position from -82 bp to -77 bp in the 5'-upstream region. The cis-element was identical to the consensus E box element, which is the binding site for the basic-helix-loop-helix proteins. Gel mobility shift analyses revealed the amount of a binding factor to this E box in the nuclear extract prepared from the rat testes attained a maximal level 1 week after birth and then decreased dramatically thereafter, and only trace amounts were detected in adult rats. In contrast, the binding factor in the ovaries attained a maximal level just after birth and kept its level thereafter. These dimorphic expressions of the binding factor to the E box correlated well with those of Ad4BP, and thus suggested that the expression of Ad4BP, and thus suggested that the expression of Ad4BP is transcriptionally regulated through this E box element.

Adrenal Cortex↗
PubMed1995

Transcription factor adrenal 4 binding protein as a marker of adrenocortical malignancy.

Adrenal 4 binding protein (Ad4BP) is a transcription factor that regulates the expression of the steroidogenic enzymes and is expressed primarily in steroidogenic cells. We immunolocalized Ad4BP in adrenocortical carcinoma (eight cases) and various malignancies that histologically simulate an adrenocortical carcinoma to evaluate the value of Ad4BP as an immunohistochemical marker of adrenocortical carcinoma. These malignancies examined were renal cell carcinoma (20 cases), hepatocellular carcinoma (10 cases), malignant melanoma (eight cases), ovarian (six cases) and uterine (three cases) clear cell carcinoma, large cell carcinoma of the lung (five cases), and pheochromocytoma (three cases). Nuclear Ad4BP immunoreactivity was observed only in adrenocortical carcinoma cases but not in other tumors examined. Almost all of the adrenocortical carcinoma cells were immunohistochemically positive for Ad4BP including cells associated with bizarre nuclei. These results show that application of Ad4BP immunostain can contribute greatly to the differential diagnosis of adrenocortical carcinoma.

Adrenal Cortex Neoplasms↗
PubMed1995

Gene regulation of steroidogenesis.

The biosynthesis of various steroid hormones in animal tissues are catalyzed by six forms of cytochrome P450 and two hydroxysteroid dehydrogenases. The tissue-specific expression of these enzymes, which are under the control of the pituitary gland and mainly regulated at the transcriptional level, determines the steroidogenesis of animal tissues. Analysis of the promoter regions of the steroidogenic P450 genes revealed various cis-acting elements, including cAMP-responsive sequences (CRS), Ad4, and GC-rich sequences, which were needed for the tissue-specific and cAMP-dependent expression of the genes. Some of the nuclear protein factors binding to the cis-acting elements were isolated and characterized. A zinc-finger protein binding to Ad4, which was termed Ad4BP or SF-1, seems to be of particular importance in steroidogenesis. Ad4BP was expressed in the cells of the steroidogenic tissues, adrenal gland and gonadal tissues, in the rat fetus prior to the expression of steroidogenic P450s, and remained expressed only in steroidogenic cells in adult animals. Close investigation of the temporal and spacial expression of Ad4BP in the fetal tissues suggested its role in the differentiation of the steroidogenic tissues and the sex determination of the gonadal tissues.

Animals↗
PubMed1995

Function and distribution of a steroidogenic cell-specific transcription factor, Ad4BP.

Ad4BP was identified as an essential transcription factor regulating steroidogenic cell-specific and cAMP-dependent transcription of the genes of steroidogenic P450s. The Ad4BP transcript was detected in steroidogenic tissues such as adrenal gland, testis, ovary, placenta and brain by RT-PCR, and showed good correlation with the expression of steroidogenic P450s. The genes of steroidogenic P450s, which are transcribed only in steroidogenic cells, were transcribed in non-steroidogenic cells when an Ad4BP expression vector was introduced into the cells. To study the function of Ad4BP in the differentiation of the steroidogenic tissues, immunochemical and immunohistochemical studies were performed with the tissues prepared from various developmental stages of rats. Adrenal cortex expressed Ad4BP since the tissue was detected in the dorsal wall of the fetus. Gonadal tissues expressed Ad4BP in a sex-dependent manner. High levels of Ad4BP expression were detected in fetal and prepubertal testes and in prepubertal and adult ovaries, whereas low level expressions were observed in the adult testes and in the fetal ovaries. The expression of Ad4BP in the gonads correlates well with the expression of the Müllerian inhibiting substance gene as well as the steroidogenic P450 gene for both sexes. These observations indicate that Ad4BP plays an important role in the development and differentiation of the steroidogenic tissues including sexual differentiation of the gonadal tissues through activation of the transcription of its target genes.

Adrenal Glands↗