ABSTRACT
We established a steroidogenic human ovarian granulosa-like tumor cell line, designated KGN, from a patient with invasive ovarian granulosa cell carcinoma. KGN had a relatively long population doubling time of about 46.4 h and had an abnormal karyotype of 45,XX, 7q-, -22. A steroid analysis of the cultured medium by RIA performed 5 yr after the initiation of culture showed that KGN was able to secrete pregnenolone and progesterone, and both dramatically increased after stimulation with (Bu)(2)cAMP. However, little or no secretion of 17alpha-hydroxylated steroids, dehydroepiandrosterone, androstenedione, or estradiol was observed. The aromatase activity of KGN was relatively high and was further stimulated by (Bu)(2)cAMP or FSH. These findings showed a pattern similar to that of steroidogenesis in human granulosa cells, thus allowing analysis of naturally occurring steroidogenesis in human granulosa cells. Fas-mediated apoptosis of KGN was also observed, which mimicked the physiological regulation of apoptosis in normal human granulosa cells. Based on these findings, this cell line is considered to be a very useful model for understanding the regulation of steroidogenesis, cell growth, and apoptosis in human granulosa cells.
Subject(s)
Granulosa Cell Tumor/pathology , Ovarian Neoplasms/pathology , Receptors, FSH/metabolism , Apoptosis/drug effects , Aromatase/metabolism , Bucladesine/pharmacology , Cell Culture Techniques/methods , Cell Division , Chromosome Aberrations , Cyclic AMP/metabolism , Female , Follicle Stimulating Hormone/metabolism , Granulosa Cell Tumor/genetics , Granulosa Cell Tumor/physiopathology , Humans , Interferon-gamma/pharmacology , Karyotyping , Middle Aged , Ovarian Neoplasms/genetics , Ovarian Neoplasms/physiopathology , Pregnenolone/metabolism , Progesterone/metabolism , Recombinant Proteins , Tetradecanoylphorbol Acetate/pharmacology , Tumor Cells, CulturedABSTRACT
Ad4BP, also known as SF-1, is a steroidogenic tissue-specific transcription factor that is also essential for adrenal and gonadal development. Two mechanisms for the transcriptional regulation of the mammalian FTZ-F1 gene encoding Ad4BP in adrenocortical cells have been proposed in the previous studies: the crucial role of a cis-element, an E box for the steroidogenic cell-specific expression of mouse and rat FTZ-F1 genes, and a possible autoregulatory mechanism of the rFTZ-F1 gene by Ad4BP itself through binding to the Ad4 (or SF-1) site in the first intron. In the present study, the transcriptional regulation of the human FTZ-F1 gene in adrenocortical cells was investigated from several angles, including the above two mechanisms. Using a series of deletion analyses of the 5'-flanking region of the hFTZ-F1 gene and site-directed mutagenesis for transient transfection studies, an E box element, CACGTG at -87/-82 from the transcriptional start site, was also found to be essential for the transcription of the hFTZ-F1 gene in mouse or human adrenocortical cell lines as well as in non-steroidogenic CV-1 cells. Despite the presence of a corresponding Ad4 site, CCAAGGCC at +163/+156 in the first intron of the hFTZ-F1 gene, an autoregulatory mechanism through the Ad4 site was found to be unlikely in the hFTZ-F1 gene mainly due to site-directed mutagenesis. In addition, the forced expression of Ad4BP had little effect on hFTZ-F1 gene transcription in non-steroidogenic CV-1 cells. Such Ad4BP-independent regulation of the hFTZ-F1 gene was in striking contrast to the regulation of steroidogenic CYP genes, such as the human CYP11A gene, in which the proximal promoter activity is Ad4BP-dependent and the transactivation by Ad4BP is silenced by DAX-1. Even though the Ad4BP-dependent transcriptional regulation of the DAX-1 gene has been reported, DAX-1 did not affect the transcriptional activity of the hFTZ-F1 gene in our study. Taken together, these observations suggest that the E box is indeed required for the expression of the FTZ-F1 gene, at least in mammalian species, but may not determine the tissue-specific expression of the hFTZ-F1 gene, and that, unlike the steroidogenic CYP gene, the regulation of the hFTZ-F1 gene appears to be independent of both Ad4BP and DAX-1.
Subject(s)
Adrenal Cortex/metabolism , DNA-Binding Proteins/genetics , Gene Expression Regulation , Repressor Proteins , Transcription Factors/genetics , Animals , Base Sequence , Cattle , Chloramphenicol O-Acetyltransferase/genetics , DAX-1 Orphan Nuclear Receptor , DNA Primers/chemistry , DNA-Binding Proteins/metabolism , Electrophoresis, Agar Gel , Fushi Tarazu Transcription Factors , Gene Expression Regulation/genetics , Genetic Vectors , Haplorhini , Homeodomain Proteins , Humans , Luciferases/metabolism , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Receptors, Cytoplasmic and Nuclear , Receptors, Retinoic Acid/metabolism , Regulatory Sequences, Nucleic Acid , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Nucleic Acid , Steroidogenic Factor 1 , Transcription Factors/metabolism , Transcription, Genetic , Transfection , Tumor Cells, Cultured/metabolismABSTRACT
Ad4BP (or SF-1) is 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 spliced variants of Ad4BP gene, ELP1 and ELP2 in mice, are unlikely to be present in humans since the analysis of the human gene revealed an in frame stop codon, 36-bp before the first ATG of Ad4BP. The promoter sequence of human Ad4BP, upstream of non-coding exon 1 was highly conserved, and E-box was also found to be essential for the transcription of human Ad4BP gene. During the process of the human Ad4BP gene cloning, we happened to obtain an Ad4BP-related gene, FTZ-F1beta which also belongs to the nuclear receptor family. We revealed cDNA structures of rat FTZ-F1beta, and found that rat has at least two types of FTZ-F1beta isoforms, which differ only by 21 amino acids length in the A/B domain. The tissue distributions of FTZ-F1beta in rat examined by RT-PCR, was found to be abundant in liver, pancreas, and gastrointestinal tracts. These results suggest that the physiological significance of FTZ-F1beta is different from that of Ad4BP.
Subject(s)
DNA-Binding Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Base Sequence , DNA Primers , DNA-Binding Proteins/genetics , Fushi Tarazu Transcription Factors , Homeodomain Proteins , Humans , Molecular Sequence Data , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Steroidogenic Factor 1 , Transcription Factors/geneticsABSTRACT
We cloned 537 basepairs (bp) of rat partial peroxisome proliferator-activated receptor gamma2 (PPARgamma2) cDNA and examined the effect of fasting or obesity on the expression of two isoforms of rat PPARgamma, gamma1 and gamma2, in either subcutaneous or mesenteric adipose tissue specimens using an RNase A protection assay. In Wistar rats, expression of both isoforms was dramatically reduced after 48 hours of fasting in the two fat tissue specimens. In comparing genetically obese (fa/fa) Zucker rats and lean control rats, no significant difference was observed in expression of the two isoforms in either type of adipose tissue. From these findings, we conclude that the adipose tissue level of rat PPARgamma depends on nutritional deprivation but is not closely associated with either obesity or insulin resistance in obese Zucker rats.
Subject(s)
Adipose Tissue/metabolism , Gene Expression Regulation/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Transcription Factors/genetics , Amino Acid Sequence , Animals , Base Sequence , Blood Glucose/analysis , Body Weight/genetics , Cloning, Molecular , Fasting , Insulin/blood , Insulin Resistance/genetics , Molecular Sequence Data , Obesity/genetics , RNA, Messenger/genetics , Rats , Rats, Wistar , Rats, Zucker , Ribonucleases/metabolism , Sequence Analysis, DNAABSTRACT
The sex-determining region Y chromosome (SRY) triggers testis determination. We report a 46,XX true hermaphrodite who had ambiguous genitalia at birth. A laparotomy at one year of age revealed this patient to have a testis on the right side and an ovotestis on the left side. By polymerase chain reaction analysis no SRY was detected in the DNA from the leukocytes but it was found in the DNA from the ovotestis. The hidden mosaicism with the Y-bearing cells in the gonads is likely the cause of the dual gonads in this patient.
