Teruo Sugawara

The Pathophysiology and Genetics of Congenital Lipoid Adrenal Hyperplasia

BACKGROUND Congenital lipoid adrenal hyperplasia results in severe impairment of steroid biosynthesis in the adrenal glands and gonads that is manifested both in utero and postnatally. We recently found mutations in the gene for the steroidogenic acute regulatory protein in four patients with this syndrome, but it was not clear whether all patients have such mutations or why there is substantial clinical variation in these patients. METHODS We directly sequenced the gene for steroidogenic acute regulatory protein in 15 patients with congenital lipoid adrenal hyperplasia from 10 countries. Identified mutations were confirmed and recreated in expression vectors, transfected into cultured cells, and assayed for the presence and activity of steroidogenic acute regulatory protein. RESULTS Fifteen different mutations in the gene for steroidogenic acute regulatory protein were found in 14 patients; the mutation Gln258Stop was found in 80 percent of affected alleles from Japanese and Korean patients, and the mutation Arg182Leu was found in 78 percent of affected alleles from Palestinian patients. We developed diagnostic tests for these and eight other mutations. Thirteen of the 15 mutations were in exons 5, 6, or 7, and all rendered the steroidogenic acute regulatory protein inactive in functional assays. Some mutants with amino acid replacements were capable of normal mitochondrial processing, indicating that the activity of steroidogenic acute regulatory protein is not associated with its translocation into mitochondria. Steroidogenic cells lacking the protein retained low levels of steroidogenesis. This explains the secretion of some steroid hormones by the ovaries after puberty before affected cells accumulate large amounts of cholesterol esters. CONCLUSIONS The congenital lipoid adrenal hyperplasia phenotype is the result of two separate events, an initial genetic loss of steroidogenesis that is dependent on steroidogenic acute regulatory protein and a subsequent loss of steroidogenesis that is independent of the protein due to cellular damage from accumulated cholesterol esters.

Regulation of expression of the steroidogenic acute regulatory protein (StAR) gene : A central role for steroidogenic factor 1

Steroidogenic acute regulatory protein (StAR) plays a critical role in regulating the rate-limiting step in steroid hormone synthesis, cholesterol side-chain cleavage. StAR gene expression is transcriptionally controlled in the gonads by gonadotropic hormones via a cAMP second message. We have begun to analyze factors responsible for the transcriptional activation of the StAR gene. The human StAR gene promoter has at least two cis elements that govern basal and cAMP-regulated gene expression. One of these elements (the distal element) is a consensus binding sequence for the orphan nuclear receptor transcription factor, steroidogenic factor 1 (SF-1); the other (the proximal element) is a related motif. The human StAR promoter is not active in BeWo choriocarcinoma cells, but is functional and cAMP-responsive in murine Y1 adrenal cortical tumor cells. Cotransfection of a plasmid expressing SF-1 allows a StAR promoter construct to function in BeWo cells. Other orphan nuclear transcription factors do not support StAR promoter function in BeWo cell hosts. Deletion or mutation of the distal and proximal cis elements individually substantially reduces SF-1-supported StAR promoter activity. The distal site binds SF-1 with high affinity, whereas the proximal site binds SF-1 with lower affinities. These findings demonstrate a requirement for SF-1 for human StAR gene expression.

Characterization of the Protein Responsible for the Acute Regulation of Steroidogenesis in Mouse Leydig Tumor Cells

This chapter discusses the cellular events that rapidly occur in response to the trophic hormone stimulation of steroidogenic tissues and result in the synthesis and secretion of arguably their most important products, the steroid hormones. These rapid or acute effects of hormone stimulation can be distinguished from the slower chronic effects in that acute effects occur within minutes, whereas chronic effects are those that require a period of many hours. Further, as pointed out earlier, the acute effects result in the synthesis and secretion of steroids only, whereas chronic effects usually involve increased gene transcription and translation of the proteins involved in the biosynthesis of these steroids in addition to steroid production. While both areas have been intensely studied, the molecular events that regulate the rapid production of hormonal steroids in steroidogenic tissues in response to trophic hormone stimulation have been the subject of intense investigation for over three decades. This chapter focuses only on those studies designed to elucidate the factors and mechanisms involved in the acute regulation of steroid production in response to hormone stimulation. Overviews of the effects of chronic stimulation on steroidogenic enzymes have appeared in several excellent review articles (1–4).

Structure, Function, and Regulated Expression of the Steroidogenic Acute Regulatory (StAR) Protein

Steroid hormones produced in the adrenals, gonads, and placenta are important regulators of tissue differentiation, development, and homeostasis. The first committed step in the synthesis of these hormones in all tissue types is the conversion of cholesterol into pregnenolone. This reaction is catalyzed by the cytochrome P450 side-chain cleavage enzyme (P450 scc ), located on the matrix side of the inner mitochondrial membrane. This first step in steroidogenesis, which is highly regulated in a temporal and tissue-specific manner, is primarily controlled at the level of cholesterol availability to the inner-mitochondrial matrix rather than at the level of P450 scc enzyme activity. It has been known for decades that the acute regulation of steroid-hormone synthesis in the adrenals and gonads is dependent upon the synthesis of a protein(s) in response to trophic hormones and that this protein is fast-acting (minutes) and functionally short-lived. The recently cloned Steroidogenic Acute Regulatory (StAR) protein appears to be the “labile protein” essential for the acute steroidogenic response. Absence of functional StAR protein causes congenital lipoid adrenal hyperplasia (lipoid CAH), a disease characterized by marked impairment of adrenal and gonadal steroid hormone synthesis. Recent data implicate cAMP-mediated pathways in the regulation of StAR mRNA expression via a mechanism that depends upon the orphan nuclear hormone receptor, steroidogenic factor-1 (SF-1). Additional data suggest that cAMP-mediated pathways stimulate StAR function at the posttranslational level by initiating protein kinase A (PKA)-mediated phosphorylation of StAR. Although StAR has been shown to be imported and processed by mitochondria, this event is not essential to StAR function. It appears that StAR acts on the outer mitochondrial membrane, stimulating sterol desorption from the sterol-rich outer membrane and its transfer to the relatively sterol-poor inner membrane.