Ing-Cherng Guo

Transcriptional regulation of the CYP11A1 and ferredoxin genes.

The first step in the synthesis of all steroids is the cleavage of cholesterol side chain, catalyzed by an electron transport system located in mitochondria consisting of ferredoxin reductase, ferredoxin, and cytochrome P450scc. These proteins are present in adrenal, gonad, placenta, and some parts of the brain. In addition, ferredoxin and ferredoxin reductase are also found in the kidney and liver. Whereas ferredoxin reductase levels remain constant in the cell, ferredoxin and P450scc levels are stimulated by trophic hormones using cAMP as an intracellular messenger. The ferredoxin promoter is relatively simple, consisting of a TATA box and two Sp1-binding sites. This simple module is enough to direct cAMP-dependent transcription in a steroidogenic cell-specific fashion. The regulatory region for the P450scc gene is more complex, containing many protein binding sites for different regulation purposes. Its TATA box directs cAMP-dependent transcription in a cell-type-specific manner. A transcription factor, steroidogenic factor 1 (SF1), activates P450scc gene expression. The tissue-specific expression of the P450scc gene is probably accomplished through the interaction of SF1 with other protein factors located further upstream of the control region. SF1 may also be involved in the cAMP response. An upstream region binding to cAMP-Responsive Element Binding Protein CREB and AP1 can respond to cAMP for gene activation. These analyses of regulatory elements provide the structural architecture for transcriptional regulation of the ferredoxin and the CYP11A11 gene.

Function of Cyp11a1 in animal models.

CYP11A1 encodes the P450scc enzyme that catalyzes the first and rate-limiting step of steroid biosynthesis. It is expressed in the adrenals and gonads under the control of pituitary peptide hormones. Transcription factor SF-1, which binds to the CYP11A1 promoter, plays an important role in the tissue-specific and hormonally regulated expression. Transgenic mouse experiments linking the CYP11A1 promoter to a reporter gene demonstrate the importance of the SF-1-binding site in directing balanced CYP11A1 gene expression. In addition, targeted mutagenesis of Cyp11a1 also reveals the importance of this gene in steroid secretion, gene regulation, and development of genitalia. Zebrafish cyp11a1 is expressed in the gonads and interrenal glands in the adults, similar to mammals. In addition, it is expressed in yolk syncytial layer during early embryogenesis. These two animal models complement each other for the understanding of gene regulatory pathways in human diseases related to steroid imbalance.

Transcriptional Regulation of CYP11A1

Steroid hormones are important physiological regulators that control our glucose and salt balance, as well as sexual characteristics. The synthesis of steroid hormones is under tight control; disturbed secretion of steroids often leads to diseases. The mechanism controlling the secretion of steroids, namely steroidogenesis, has been the focus of intensive studies. CYP11A1 controls the first and rate-limiting step of steroid biosynthesis. It is expressed in the adrenal cortex and gonads, under the control of pituitary hormones, through the cAMP-signaling pathway. The promoter of the CYP11A1 gene contains sequences that bind to transcription factor SF-1, which plays an important role in the tissue-specific and hormonally regulated expression of steroidogenic genes. Detailed transcriptional analysis documents the importance of SF-1 in activating CYP11A1 in vitro and in vivo. Other factors like c-Jun are also involved. The assembly of various transcription factors forming protein-DNA complexes appears to be the key step in CYP11A1 transcription.

Differential inhibition of progesterone synthesis in bovine luteal cells by estrogens and androgens

We investigated the roles of estrogens and androgens in the progesterone biosynthesis of bovine luteal cells. The responsiveness of primary luteal cells to the stimulation of tropic agents was observed in a dose-dependent manner. Estrogens and androgens significantly inhibited tropic agent-induced progesterone secretions, but glucocorticoids did not, which indicated the inhibitions were specific. The failure of exogenous 8-Br-cAMP to prevent these inhibitions suggested that took place at the post-cAMP steps. The immunoblot showed that testosterone remarkably decreased the amount of induced P450scc protein after 6-hour treatment, yet 17beta-estradiol did not. The 3beta-HSD activity assays demonstrated that both 17beta-estradiol and testosterone efficiently blocked induced 3beta-HSD activities. Both inhibitory effects of E2 and T on progesterone synthesis were observed one hour after treatment and accompanied with suppressed 3beta-HSD activities. This study presents that estrogens and androgens specifically inhibit bovine luteal function through different mechanisms.

Cell-type specificity of human CYP11A1 TATA box.

Expression of the CYP11A1 (SCC) genes, which encode the enzyme important for the first step of steroid biosynthesis, occurs in the adrenal gland and gonads, and is stimulated by cAMP. Transfection of serial deletions of the SCC promoter, which drives reporter gene expression, showed that a minimal promoter containing only the TATA box could direct cAMP-dependent transcription. Transcription factor SF1, which binds to a site next to the TATA box, can stimulate basal transcription but not cAMP response, either in adrenal cell lines or in COS-1 co-transfected with the SF1 expression plasmid. These data lead to the conclusion that the minimal promoter containing only the TATA box can drive cell type-specific, cAMP-dependent transcription. Additional experiments replacing the TATA sequence of SCC with other TATA sequences suggested that the TATA sequence itself is important for this cAMP-dependent transcription.

Effect of cAMP on protein binding activities of three elements in upstream promoter of human CYP11A1 gene

CYP11A1 gene encodes the P450scc enzyme. which catalyzes the first step of steroidogenesis under the modulation of cAMP signal. To understand the signaling pathway of cAMP, we analyzed three elements in upstream cAMP-responsive sequences of CYP11A1 gene. The sequence of -1617/-1609 fragment was identical to the conserved SF1 binding site (SF1/Ad4BP). Both sequences of -1637/-1626 and -1559/-1545 fragments are highly homologous to consensus TRE or CRE. The cAMP-treated adrenal Y1 nuclear extracts formed significantly higher intensities of complexes with these three elements than untreated Y1 nuclear extracts. The EMSA demonstrated potential binding of SF1 protein to -1617/-1609, and CREB like protein and AP1 family to -1637/-1626 and -1559/-1545. This is the first report to describe the enhancement of cAMP on protein binding activities of three elements of CYP11A1 promoter to SF1/Ad4BP or CREB/AP1-like proteins.

Protein Binding Activity and Cyclic Amp-Responsiveness of a Weak Sp1 Site in Proximal Promoter of Human CYP11A1 Gene

The cholesterol side-chain cleavage enzyme (P450scc) encoded by CYP11A1 gene catalyzes the conversion of cholesterol into pregneneolone, which is the first step in steroid biosynthesis and regulated by tropic hormones via cAMP-PKA pathway. The regulatory region mediating signal of cAMP on the transcription of human CYP11A1 gene contains upstream cAMP-responsive sequences (U-CRS) and proximal cAMP-responsive sequences (P-CRS). The P-CRS, located in -145 bp, harbors at least three highly conserved sequences including TATA box, SF1-binding site and Sp1-like sequence. By electrophoretic mobility shift assay, we showed oligonucleotide -117/-94 formed two specific DNA-protein complexes with nuclear extract from adrenocortical Y1, placental JEG-3, or cervical HeLa cells. The oligonucleotide containing Sp1 consensus sequence formed the same pattern as -117/-94 did. Although Sp1 was always stronger, -117/-94 and Sp1 oligonucleotides were effective competitors for each other. These data indicated a weak Sp1-binding site located in -117/-94 fragment. To test for its function, the -117/-94 fragment was fused to the TATA box of OVEC vector, before transfection into Y1 or JEG-3 cells. Its transcription activity was measured by RNA analysis after 8-Br-cAMP treatment. The results of primer extension showed -117/-94 directed cAMP-dependent transcription. Based on the observations of DNA-protein interaction assay and transcription activity assay, we conclude that a weak Sp1-binding site in the -117/-94 fragment is important for transcription of the CYP11A1 gene by interacting with basal transcription factors.

Transcriptional regulation ofCYP11A1

Steroid hormones are important physiological regulators that control our glucose and salt balance, as well as sexual characteristics. The synthesis of steroid hormones is under tight control; disturbed secretion of steroids often leads to diseases. The mechanism controlling the secretion of steroids, namely steroidogenesis, has been the focus of intensive studies.CYP11A1 controls the first and rate-limiting step of steroid biosynthesis. It is expressed in the adrenal cortex and gonads, under the control of pituitary hormones, through the cAMP-signaling pathway. The promoter of theCYP11A1 gene contains sequences that bind to transcription factor SF-1, which plays an important role in the tissue-specific and hormonally regulated expression of steroidogenic genes. Detailed transcriptional analysis documents the importance of SF-1 in activatingCYP11A1 in vitro and in vivo. Other factors like c-Jun are also involved. The assembly of various transcription factors forming protein-DNA complexes appears to be the key step inCYP11A1 transcription.