Contributions of specificity protein-1 and steroidogenic factor 1 to Adcy4 expression in Y1 mouse adrenal cells.

The type 4 adenylyl cyclase, Adcy4, is the least abundant of five different adenylyl cyclase isoforms expressed in the Y1 mouse adrenocortical cell line and is deficient in a Y1 mutant with impaired steroidogenic factor 1 (SF1) activity. This study examines the contributions of SF1 and other DNA promoter/regulatory elements to Adcy4 expression in the Y1 cell line and its derivative Adcy4-deficient mutant. Primer extension and in silico analyses indicate that Adcy4 transcription initiates from multiple sites just downstream of a GC-rich sequence. Luciferase reporter gene assays identify a 124-bp sequence, situated 19 bp upstream of the major transcription start site and highly conserved among several mammalian species, as the major determinant of Adcy4 expression in Y1 cells and as a site of compromised activity in the Adcy4-deficient mutant. EMSAs using competitor nucleotides and specific antibodies indicate that this conserved region contains three specificity protein (Sp)-1/Sp3-binding sites and one SF1-binding site. As determined by site-specific mutagenesis, the 5'-most Sp1/Sp3-site enhances promoter activity, whereas the middle Sp1/Sp3 and SF1 sites each repress Adcy4 promoter activity. In the Adcy4-deficient mutant, mutating the SF1 site restores Adcy4 promoter activity and knocking down SF1 with small interfering RNAs increases Adcy4 expression, confirming the contribution of SF1 to the mutant phenotype. These studies demonstrate roles for Sp1/Sp3 and SF1 in Adcy4 expression in Y1 cells and establish a repressor function for SF1 in certain promoter contexts.

A genome-wide assessment of adrenocorticotropin action in the Y1 mouse adrenal tumor cell line.

This report summarizes the genome-wide effects of ACTH on transcript accumulation in mouse adrenal Y1 cells and the relative contributions of the cAMP-, protein kinase C- and Ca(2+)-dependent signaling pathways to these actions of the hormone. ACTH affected the accumulation of 1386 transcripts, a much larger number than previously appreciated. The cAMP signaling pathway accounted for approximately 56% of the ACTH effects whereas the protein kinase C- and Ca(2+)-dependent pathways made smaller contributions to ACTH action. Approximately 38% of the ACTH-affected transcripts could not be assigned to these signaling pathways and thus represent candidates for regulation via other mechanisms. The set of ACTH-regulated transcripts included clusters with functions in steroid metabolism, cell proliferation and alternative splicing. Collectively, our results suggest that Y1 adrenal cells undergo extensive remodeling upon prolonged stimulation with ACTH. The functional implications of ACTH on alternative splicing are explored.

Global profiles of gene expression induced by adrenocorticotropin in Y1 mouse adrenal cells.

ACTH regulates the steroidogenic capacity, size, and structural integrity of the adrenal cortex through a series of actions involving changes in gene expression; however, only a limited number of ACTH-regulated genes have been identified, and these only partly account for the global effects of ACTH on the adrenal cortex. In this study, a National Institute on Aging 15K mouse cDNA microarray was used to identify genome-wide changes in gene expression after treatment of Y1 mouse adrenocortical cells with ACTH. ACTH affected the levels of 1275 annotated transcripts, of which 46% were up-regulated. The up-regulated transcripts were enriched for functions associated with steroid biosynthesis and metabolism; the down- regulated transcripts were enriched for functions associated with cell proliferation, nuclear transport and RNA processing, including alternative splicing. A total of 133 different transcripts, i.e. only 10% of the ACTH-affected transcripts, were represented in the categories above; most of these had not been described as ACTH-regulated previously. The contributions of protein kinase A and protein kinase C to these genome-wide effects of ACTH were evaluated in microarray experiments after treatment of Y1 cells and derivative protein kinase A-defective mutants with pharmacological probes of each pathway. Protein kinase A-dependent signaling accounted for 56% of the ACTH effect; protein kinase C-dependent signaling accounted for an additional 6%. These results indicate that ACTH affects the expression profile of Y1 adrenal cells principally through cAMP- and protein kinase A- dependent signaling. The large number of transcripts affected by ACTH anticipates a broader range of actions than previously appreciated.

Minireview: transcriptional regulation of adrenocortical development.

The adrenal glands are comprised of two distinct endocrine organs: the outer cortex, which is derived from mesoderm and synthesizes steroid hormones, and the inner medulla, which contains neuroectodermal cells derived from the neural crest and produces the catecholamine hormones norepinephrine and epinephrine. The developmental program that gives rise to the adrenal gland begins early during embryogenesis and continues throughout gestation and well after birth. In this article, we review the molecular mechanisms of adrenal differentiation and development, focusing on the contributions of genes responsible for the development of the adrenal cortex as identified from studies of experimental animal models and human subjects with clinical diseases. These studies identify a hierarchical network of transcription factors, including Wilms' tumor-1, steroidogenic factor-1, dosage-sensitive sex reversal, adrenal hypoplasia congenita, X-linked-1, PBX1, and CITED2, that both give rise to the adrenal cortex and subsequently determine its subsequent function in steroidogenesis.

Corticotropin (ACTH) regulates alternative RNA splicing in Y1 mouse adrenocortical tumor cells.

The stimulatory effect of ACTH on gene expression is well documented and is thought to be a major mechanism by which ACTH maintains the functional and structural integrity of the gland. Previously, we showed that ACTH regulates the accumulation of over 1200 transcripts in Y1 adrenal cells, including a cluster with functions in alternative splicing of RNA. On this basis, we postulated that some of the effects of ACTH on the transcription landscape of Y1 cells are mediated by alternative splicing. In this study, we demonstrate that ACTH regulates the alternative splicing of four transcripts - Gnas, Cd151, Dab2 and Tia1. Inasmuch as alternative splicing potentially affects transcripts from more than two-thirds of the mouse genome, we suggest that these findings are representative of a genome-wide effect of ACTH that impacts on the mRNA and protein composition of the adrenal cortex.

A polymorphic form of steroidogenic factor-1 is associated with adrenocorticotropin resistance in y1 mouse adrenocortical tumor cell mutants.

ACTH resistance in mutant derivatives of the Y1 mouse adrenocortical tumor cell line results from a defect that affects the activity of steroidogenic factor-1 (SF1), thereby preventing the expression of the melanocortin-2 receptor. In this report, we show that the SF1 genes in ACTH-resistant mutants differ from the gene in ACTH-responsive Y1 cells by two base changes-one that changes an Ala to Ser at codon 172, and one in the third position of codon 3 that does not affect the protein sequence. Furthermore, several of the mutants contain multiple copies of this alternate SF1 gene (SF1(S172)) on acentric chromosome fragments. The SF1(S172) allele represents a polymorphism rather than a spontaneous mutation because the two SF1 alleles can be traced to the hybrid mouse strain (C57L/J x A/HeJ) from which the original adrenal tumor was derived. The SF1(A172) allele also is found in C57Bl/6J and C57Bl/10J mice, whereas the SF1(S172) allele also is found in C3H/HeJ and DBA/2J mice. The two forms of SF1 had only modest differences in activity suggesting that the SF1 polymorphism per se is not directly responsible for ACTH resistance. Our results indicate that the SF1(S172) allele is a marker of ACTH resistance in this family of adrenocortical tumor cells.

Adrenocortical cell lines.

The human adrenal cortex is a complex endocrine organ that secretes mineralocorticoids, glucocorticoids and adrenal androgens. These steroids arise from morphologically and biochemically distinct zones of the adrenal gland. Studying secretion of these distinct steroid hormones can make use of cells isolated from the adrenal gland but this requires animal sacrifice and the need for continued isolation for long-term studies. In addition primary cultures of adrenal cells have a limited life-span in culture and the cultured cells are often contaminated by the presence of non-steroidogenic cells. For that reason in vitro cell culture models have several benefits for research on adrenocortical function. Herein we discuss the available adrenocortical cell lines and their uses as model systems for adrenal studies. Focus is placed on the human NCI-H295 and mouse Y-1 adrenal cell lines, which have been used extensively as adrenocortical model systems. These cell lines have proven to be of considerable value in studying the molecular and biochemical mechanisms controlling adrenal steroidogenesis. The current review will discuss the attributes and limitations of the currently available adrenocortical cell lines as models for adrenal studies.

Forskolin-resistant Y1 adrenal cell mutants are deficient in adenylyl cyclase type 4.

Four mutant clones independently derived from the Y1 mouse adrenocortical tumor cell line have adenylyl cyclase (AC) activities that are resistant to forskolin, a direct activator of AC. In this study the AC isoform composition of the forskolin-resistant mutants was examined in order to explore the underlying basis for the resistance to forskolin. As determined by Western blot and RT-PCR analysis, the four forskolin-resistant mutants all were deficient in AC-4; the levels of other AC isoforms (AC-1, AC-3 and AC-5/6) were comparable to the levels in parent Y1 cells. Transfection of one of the mutant clones with an AC-4 expression vector increased forskolin-stimulated cAMP signaling, and restored forskolin-induced changes in cell morphology and growth. Taken together, these observations indicate that AC-4 deficiency is a hallmark of the forskolin-resistant phenotype of these mutants and suggest that AC-4 is an important target of forskolin action in the Y1 adrenal cell line.

Expression of adenylyl cyclase-4 (AC-4) in Y1 and forskolin-resistant adrenal cells.

Forskolin-resistant mutants of a mouse adrenocortical cell line present a complex phenotype in which adenylyl cyclase (AC) is resistant to activation by forskolin and by ACTH. ACTH-resistance results from a defect affecting transcription of the ACTH receptor and can be overcome by transfecting mutant cells with expression vectors encoding G beta/gamma. Forskolin-resistance results from an AC-4 deficiency. We now demonstrate that the AC-4 deficiency in forskolin-resistant mutants results from a transcription defect affecting the promoter activity of the AC-4 gene. Furthermore, the underlying defect leading to AC-4 deficiency and forskolin-resistance can be overcome by transfection of mutant clones with expression vectors encoding G beta/gamma. These data support our hypothesis that AC-4 is a preferred target of forskolin action in Y1 cells, demonstrate novel roles for G beta/gamma in gene expression and indicate that a common underlying defect, suppressible by G beta/gamma, accounts for both the resistance to ACTH and to forskolin.

A polymorphic form of steroidogenic factor 1 associated with ACTH receptor deficiency in mouse adrenal cell mutants.

We have described a family of adrenocortical tumor cell mutants (including clones OS3, Y6, and 10r9) that are resistant to ACTH because they fail to express the gene encoding the ACTH receptor (MC2R). The MC2R deficiency results from a mutation that impairs the activity of the nuclear receptor steroidogenic factor 1 (SF1) at the MC2R promoter. In this report, we show that ACTH resistance in the mutant clones is associated with a Sf1 gene that has Ser at codon 172 instead of Ala. In two of the three mutant clones, this Sf1 allele is amplified together with flanking DNA from chromosome 2 that includes the genes encoding germ cell nuclear factor and the beta-type proteosome subunit Psmb7. SF1(A172) and SF1(S172) exhibit little or no difference in transcriptional activity in SF1-dependent reporter gene assays, suggesting that SF1(S172) per se is not directly responsible for the loss of MC2R expression. Instead, the Sf1(S172) allele appears to be a marker of ACTH resistance in this family of adrenocortical tumor cell mutants, possibly reflecting the activity of a neighboring gene.

Contributions of steroidogenic factor 1 to the transcription landscape of Y1 mouse adrenocortical tumor cells.

The contribution of steroidogenic factor 1 (SF-1) to the gene expression profile of Y1 mouse adrenocortical cells was evaluated using short hairpin RNAs to knockdown SF-1. The reduced level of SF-1 RNA was associated with global changes that affected the accumulation of more than 2000 transcripts. Among the down-regulated transcripts were several with functions in steroidogenesis that were affected to different degrees--i.e., Mc2r>Scarb1>Star≥Hsd3b1>Cyp11b1. For Star and Cyp11b1, the different levels of expression correlated with the amount of residual SF-1 bound to the proximal promoter regions. The knockdown of SF-1 did not affect the accumulation of Cyp11a1 transcripts even though the amount of SF-1 bound to the proximal promoter of the gene was reduced to background levels. Our results indicate that transcripts with functions in steroidogenesis vary in their dependence on SF-1 for constitutive expression. On a more global scale, SF-1 knockdown affects the accumulation of a large number of transcripts, most of which are not recognizably involved in steroid hormone biosynthesis.

Minireview: steroidogenic factor 1: its roles in differentiation, development, and disease.

The orphan nuclear receptor steroidogenic factor 1 (SF-1, also called Ad4BP, encoded by the NR5A1 gene) is an essential regulator of endocrine development and function. Initially identified as a tissue-specific transcriptional regulator of cytochrome P450 steroid hydroxylases, studies of both global and tissue-specific knockout mice have demonstrated that SF-1 is required for the development of the adrenal glands, gonads, and ventromedial hypothalamus and for the proper functioning of pituitary gonadotropes. Many genes are transcriptionally regulated by SF-1, and many proteins, in turn, interact with SF-1 and modulate its activity. Whereas mice with heterozygous mutations that disrupt SF-1 function have only subtle abnormalities, humans with heterozygous SF-1 mutations can present with XY sex reversal (i.e. testicular failure), ovarian failure, and occasionally adrenal insufficiency; dysregulation of SF-1 has been linked to diseases such as endometriosis and adrenocortical carcinoma. The current state of knowledge of this important transcription factor will be reviewed with a particular emphasis on the pioneering work on SF-1 by the late Keith Parker.

Genes essential for early events in gonadal development.

The acquisition of a sexually dimorphic phenotype is a key event in mammalian development. The underlying principle of this essential process is that genetic sex, determined by the presence or absence of a Y chromosome at fertilization, directs the embryonic gonads to differentiate into either testes or ovaries. Hormones produced by the testes then trigger the developmental program that leads to male phenotypic sexual differentiation. Without testes and their biochemical products, differentiation proceeds along the female pathway. Recent studies have identified several transcription factors that are required for gonadal development and sexual differentiation, i.e. Wilms' tumor related 1 (WT1), steroidogenic factor 1 (SF-1), SOX9, and GATA4, presumably because they activate the expression of essential target genes. Studies also have identified another transcriptional regulator, designated DAX-1, that inhibits target gene induction by all of these transcriptional activators. This chapter provides an overview of gonadal development and sexual differentiation, reviews the studies that have led to the isolation and characterization of these genes in the gonads, and then discusses how they interact to regulate critical events in sexual differentiation.

SF1 polymorphisms in the mouse and steroidogenic potential.

ACTH-resistance in four mutant derivatives of a mouse adrenocortical tumor cell line results from a defect that reduces the activity of steroidogenic factor-1 (SF1) thereby preventing expression of the ACTH receptor and other SF1-dependent genes. The SF1 genes from these mutants contain a sequence difference that changes an Ala to Ser at codon 172. Steroidogenic factor-1(S172) represents a polymorphism rather than a spontaneous mutation since the two forms of SF1, SF1(A172), and SF1(S172), can be traced to the hybrid mouse strain (C57L/J x A/HeJ) from which the original adrenal tumor was derived. The SF1(S172) allele is amplified in three of the four mutant clones together with the neighboring genes germ cell nuclear factor and LIM homeobox2. The two forms of SF1 had only modest differences in transcriptional activity in reporter gene assays, suggesting that the SF1 polymorphism per se is not directly responsible for the loss of mc2r expression. Rather, ACTH resistance in this family of adrenocortical tumor cell mutants may be due to a closely linked gene on the SF1(S172) allele. Mouse strains with reportedly high steroidogenic capacity (C57Bl/6J, C57Bl/10J) also have the SF1(A172) allele while mouse strains with low steroidogenic capacity (C3H/HeJ, DBA/2J) have the SF1(S172) allele. These latter observations suggest that the two SF1 alleles also may be markers of steroidogenic potential among mouse strains.

Steroidogenic factor 1: an essential mediator of endocrine development.

The orphan nuclear receptor steroidogenic factor 1 (SF-1, also called Ad4BP and officially designated NR5A1) has emerged as an essential regulator of endocrine development and function. Initially identified as a tissue-specific transcriptional regulator of the cytochrome P450 steroid hydroxylases, SF-1 has considerably broader roles, as evidenced from studies in knockout mice lacking SF-1. The SF-1-knockout mice lacked adrenal glands and gonads and therefore died from adrenal insufficiency within the first week after birth. In addition, SF-1 knockout mice exhibited male-to-female sex reversal of their internal and external genitalia, impaired expression of multiple markers of pituitary gonadotropes, and agenesis of the ventromedial hypothalamic nucleus (VMH). These studies delineated essential roles of SF-I in regulating endocrine differentiation and function at multiple levels, particularly with respect to reproduction. This chapter will review the experiments that established SF-1 as a pivotal, global determinant of endocrine differentiation and function. We next discuss recent insights into the mechanisms controlling the expression and function of SF-1 as well as the current status of research aimed at delineating its roles in specific tissues. Finally, we highlight areas where additional studies are needed to expand our understanding of SF-1 action.