Nai-Chi Hsu

Tissue-Specific, Hormonal, and Developmental Regulation of SCC-LacZ Expression in Transgenic Mice Leads to Adrenocortical Zone Characterization*

We report here the study of the human CYP11A1 promoter in driving tissue-specific, developmentally and hormonally regulated reporter gene expression. A 4.4-kb fragment containing all known regulatory elements is more efficient than a short basal promoter fused to an upstream adrenal enhancer in driving reporter LacZ gene expression both in cell culture and in transgenic mice. The LacZ gene controlled by the 4.4- and 2.3-kb promoters was expressed in the adrenal cortex, testicular Leydig cells, ovarian corpora lutea, and granulosa cells. Transgene expression in the adrenals was stimulated by ACTH, indicating the presence of ACTH-responsive sequence. β-Galactosidase activity was first detected in the adrenal primordia at 11.5 days postcoitum. Its expression continued throughout all stages of adrenal development in a pattern similar to that of the endogenous CYP11A1, which was expressed in all zones of the adrenal cortex, but was strongest in the X zone. The X zone grew before puberty but regressed afterward...

Regulation of steroidogenesis in transgenic mice and zebrafish.

Steroid hormones are important physiological regulators in the body. Steroid hormones are mainly synthesized in the adrenal and gonads. Their synthesis is stimulated by pituitary hormones through cAMP as an intracellular mediator. The first and rate-limiting step for steroid biosynthesis is catalyzed by CYP11A1. Important regulatory elements for the control of the CYP11A1 gene expression have been characterized both in vitro and in vivo. The SF-1-binding sites are cis-acting elements controlling the basal and cAMP-stimulated gene expression. Our transgenic mouse studies showed that the 2.3kb promoter contains information controlling developmentally regulated gene expression. Finally, we present our results on the cloning of steroidogenic genes in zebrafish, a new model organism for genetic studies.

Steroidogenesis in zebrafish and mouse models

Steroid hormones regulate physiological homeostasis for salt, sugar, and sex differentiation. All steroids are synthesized from a common precursor, cholesterol, in a step that converts cholesterol to pregnenolone. The enzyme carrying out this first conversion step is CYP11A1. To further investigate the importance of steroid biosynthesis, animal models with defects in the Cyp11a1 gene are used. Mice with targeted disruption of the Cyp11a1 gene produce no steroids with severe adrenal defects. These mice survive during embryogenesis, but die after birth. Zebrafish with a block in cyp11a1 gene function has an earlier defect, presumably because it lacks adequate maternal steroid supply. When cyp11a1 activity was compensated by the injection of antisense morpholino oligos, the embryos have shortened axis and a defect of epibolic cell movement during early embryogenesis. The discovery of steroid function in cell movement is novel, and should provide new insights into our understanding of diverse functions of steroids.

Mutation of Mouse Cyp11a1 Promoter Caused Tissue-Specific Reduction of Gene Expression and Blunted Stress Response without Affecting Reproduction

Steroids are synthesized mainly from the adrenal glands catalyzed by steroidogenic enzymes; the expression of these enzymes is controlled by transcription factor steroidogenic factor-1 (SF-1; NR5A1). To understand the physiological effect of genetic changes on steroid secretion, we used Cre-LoxP and gene targeting technology to mutate the binding sequence for SF-1 (SF-1 response element) on the promoter of the mouse Cyp11a1 gene, which encodes a critical enzyme for steroid biosynthesis. The resulting Cyp11a1 L/L mice expressed about 7-fold less cytochrome P450 side-chain cleavage enzyme (CYP11A1) in the adrenal and testis but expressed normal amounts of CYP11A1 in the placenta and ovary. This tissue-specific reduction of gene expression did not affect basal steroid secretion but attenuated the circadian rhythm of glucocorticoid secretion. These mice also failed to induce glucocorticoid secretion in response to stress, leading to retention of CD4+CD8+ double-positive thymocytes. Unlike complete Cyp11a1 disruption, which causes neonatal death, promoter mutation did not decrease life span and caused no defect in reproduction. Thus, CYP11A1 appears in normal mice to be expressed above the minimal required level, providing a large capacity for use in response to stress. Mutation of the SF-1 response element of Cyp11a1 results in reduced stress response due to decreased adrenal CYP11A1 expression and insufficient stress-induced glucocorticoids secretion.

Study of the function of proximal SF-1 binding sites on Cyp11A1 promoter.

The orphan nuclear receptor steroidogenic factor 1 (SF-1) is a key regulator for the expression of steroidogenic enzymes. Among the SF-1 target genes, Cyp11a1 encodes the steroidogenic enzyme cytochrome P450scc which catalyzes the first and rate-limiting step in the steroidogenesis. From our previous studies, one functional proximal SF-1 binding site on the promoter region of human CYP11A1 was identified to be essential for transcription. To investigate the regulatory function of proximal SF-1 binding site in vivo, we generated mice with mutation on the proximal SF-1 binding site of Cyp11a1 by gene targeting method. Mutating the proximal SF-1 binding sites (mtP) reduced Cyp11a1 expression (RNA or protein) for 80% in the adrenals, but mutant mice grow normally as wild-type mice. The mutation also diminished Cyp11a1 expression in the testis, however, not in the ovaries. No reproductive abnormalities were observed in both genders of mtP mice. Although the basal hormone levels were normal in mtP mice, abnormal lower corticosterone, and higher ACTH responses were seen in mtP mice when suffering the stress of 10-m restrain or 24-h fasting. The histology of the mtP adrenals showed well developed cortex and medulla but with vacuole structure in the cortex, which was proven to be oil droplets.