The roles of the nuclear receptor steroidogenic factor 1 in endocrine differentiation and development

ELSEVIER

BRIEF REVIEWS The Roles of the Nuclear Receptor Steroidogenic Factor 1 in Endocrine Differentiation and Development Keith L. Parker and Bernard P. Schimmer The orphan nuclear receptor steroidogenic factor 1 (SF-l) has emerged as a critical determinant of adrenal and gonadal differentiation, development, and function. SF-1 was initially isolated as a positive regulator of the cytochrome P450 steroid hydroxylases in the adrenal glands and gonads; developmental analyses subsequently showed that SF-I was also expressed in the diencephalon and anterior pituitary, suggesting additional roles in endocrine function. Analyses of knockout mice deficient in SF-l revealed multiple abnormalities, including adrenal and gonadal agenesis, male to female sex reversal of the internal genitalia, impaired gonadotrope function, and absence of the ventromedial hypothalamic nucleus. Taken togethel; these results implicate SF-l as a global regulator within the hypothalamic-pituitary-gonadal axis and the adrenal cortex. (Trends Endocrinol Metab 1996;7:203207).

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Overview

Recent studies suggest that steroidogenic factor 1 (SF-l) has distinct and pivotal roles in the differentiation of the develop ing embryo and in the regulation of endocrine function in the adult. In the adult, SF-l is required for the expression of enzymes that mediate steroid hormone biosynthesis in the adrenal cortex and gonads; SF-l also plays a permissive role in the expression of pituitary gonadotropins. During development, SF-1 is an essential

determinant

of adrenal

and go-

Keith L. Parker is at the Departments of Medicine and Pharmacology and the Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710, USA. Bernard P Schimmer is at the Banting and Best Department of Medical Research, University of Toronto, M5GlL6 Toronto, Canada.

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nadal development, male sexual differentiation, and formation of the ventromedial hypothalamic nucleus. SF-1 belongs to the nuclear receptor family of transcription factors and is expressed in a cell-selective manner, suggesting that its diverse actions result from its ability to regulate the orderly activation of target genes within selective tissues. This review highlights experiments that have revealed the pivotal roles of SF-1 and identifies areas in which additional experiments are needed to expand understanding of the mechanisms by which SF-1 exerts its profound effects on endocrine differentiation and function.

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Isolation and Characterization of Steroidogenic Factor 1

SF-1 was independently identified by two groups as a cell-specific protein that

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interacted in gel mobility shift assays with conserved promoter elements upstream of the cytochrome P450 steroid hydroxylases (Rice et al. 1991, Honda et al. 1993). Further characterization of mouse and bovine SF-1 revealed that SF-1 acts as a cell-selective transcription factor that regulates genes encoding the enzymes that make steroid hormones, as well as genes encoding gonadotropin subunits (Barnhart and Mellon 1994, Ingraham et al. 1994) and Miillerian-inhibiting substance (Shen et al. 1994, Hatano et al. 1994). The cloning of SF-l revealed that this transcription factor is itself a member of the nuclear hormone receptor family of transcriptional regulators (Lala et al. 1992, Honda et al. 1993). This family of transcription factors also mediates gene regulation by steroid hormones, thyroid hormone, vitamin D, and retinoids [for review, see Tsai and O’Malley (1994)]. The SF-I gene most closely resembles the Drosophila FTZ-Fl nuclear receptor, which regulates expression of the fuski tczrazu (ftz) paired rule segmentation gene; on this basis the mouse gene was designated Ftz-FI. Although SF- 1 shares extensive regions of homology with all nuclear receptor family members, especially in the zinc-finger DNA-binding and ligand-binding domains, putative ligands for SF-1 have not been identified, and SF-I thus remains an orphan member of the nuclear receptor family The Ftz-Fl gene also encodes a second protein that is structurally related to SFl-the embryonal long terminal repeatbinding protein (ELP). ELP, originally identified as a repressor of retroviral expression in embryonal carcinoma cells (Tsukiyama et al. 1992), is derived from the Ftz-Fl gene via alternative promoter usage and 3’ splice selection (Ikeda et al. 1993). The roles of ELP in gene expression and embryonic differentiation remain enigmatic; ELP has not been detected in mouse embryos (Ikeda et al. 1994), and reports regarding its pres-

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ence in adult steroidogenic tissues are conflicting (Morohashi et al. 1994, Sadovsky et al. 1995). At best, ELP transcripts are present in adult tissues in very small amounts, and the ELP protein apparently lacks the ability to activate transcription (Morohashi et al. 1994).

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The Developmental Profile of Steroidogenic Factor 1 Expression Suggests Important Roles in Endocrine Differentiation

The proposed roles of SF-l in steroid hydroxylase expression--coupled with the known actions of androgens in male sexual differentiation-suggested that SF-l might also play important roles in endocrine differentiation and sexual development. In situ hybridization analyses with SF- l-specific probes defined intriguing profiles of SF-1 expression, some of which were unanticipated. SF-l transcripts were detected throughout the adrenal primordium from very early stages of adrenal development [approximately embryonic day 10.5 (ElO.S)]. When the chromaffin cell precursors subsequently migrated into the adrenal primordium at -E12.5-E13.5, SF-I expression became restricted to the steroidogenic cortical cells. This early onset of SF-l expression, which precedes the acquisition of steroidogenic competence, is consistent with a key role in adrenal development. Within the gonads of both male and female embryos, SF-1 again was expressed at very early stages of gonadogenesis (-E9), when the intermediate mesoderm first condenses into the urogenital ridge that ultimately contributes cell lineages to both the gonad and kidney. Later in gonadogenesis, with the onset of morphological sexual differentiation at -E12.5, SF-l expression increased in the testes but decreased in the ovaries (Ikeda et al. 1994, Shen et al. 1994, Hatano et al. 1994). As discussed below, this sex-specific decrease in ovarian SF-l expression suggests that SF-1 regulates target genes whose expression would be deleterious to normal female sexual differentiation. Although SF-l expression in the adult testis is largely restricted to the steroidogeuic Leydig cells, SF-1 transcripts were detected in both compartments of the fetal testes: the interstitial region, where Leydig cells produce steroid hor-

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1. Steroidogenic factor 1 (SF-l) knockout mice lack adrenal glands and gonads and have female internal genitalia. The dissected genitourinary tracts of wild-type female (B) and male (D) and SF-1 knockout female (A) and male (C) mice are shown. Note the absence of adrenal glands and gonads in SF-l-deficient mice, and the presence of oviducts in both males and females. a, adrenal gland; e, epididymis; k, kidney: o, ovary; od, oviduct; and t, testis. Reprinted with permission from Luo et al. (1994).

Figure

mones, and the testicular cords, where fetal Sertoli cells produce Miillerian-inhibiting substance (MIS). The expression of SF- 1 by Sertoli cells hinted that SF- l’s role in endocrine development extended beyond regulating the expression of steroidogenic enzymes (Ikeda et al. 1994). Finally, SF-1 expression was also detected in the embryonic diencephalon (which ultimately contributes to the endocrine hypothalamus) and the anterior pituitary gland. These findings again suggested that SF-1 functions at other levels of the hypothalamic-pituitarysteroidogenic organ axis to regulate the development and function of the endocrine system (Ikeda et al. 1994).

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Targeted Gene Disruption Defines Multiple Roles of Steroidogenic Factor 1

Although the ontogeny of SF- 1 expression strongly suggested that SF-1 was important for the development of steroidogenic tissues, these studies did not permit definitive conclusions about tbe role of SF-1 in vivo. To address this question, targeted gene disruption in embryonic stem cells was used to make SF-1 knockout mice. The initial targeting strategy inserted the neomycin resistance selectable marker into an exon that encodes the zinc-finger DNA-binding domain of SF-l, thereby inactivating protein function. Through this approach, viable mice were produced that

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Future

Directions

in Defining

the Roles of Steroidogenic 1 in Endocrine Function

Factor

Although the analyses of knockout have dramatically tial roles for SF-l

mice

demonstrated essenat multiple levels, a

number of unresolved questions need to be addressed. Perhaps it is most important to define the precise roles that SF-l plays within hierarchical cascades of endocrine development. For example, what genes activate SF-l expression at early stages of adrenal, gonadal, and hyand what pothalamic development, mechanisms extinguish in the ovary coincident

Figure 2. Steroidogenic factor 1 (SF-l)

knockout mice lack the ventromedial hypothalamic nucleus (VMH). Serial coronal sections from wild-type (lower left) and -/- male (upper right) and female (lower right) mice were stained and analyzed histologically. Shown at the upper left is a schematic diagram of anatomical regions found within these sections. Arc, arcuate nucleus; DMH, dorsomedial hypothalamic nucleus; Do, dorsal hypothalamic nucleus; ME, median eminence; mt, mammillothalamic tract; and 3V, 3rd ventricle. Modified with

permission

from Ikeda et al. (1995).

were homozygously deficient in all proteins encoded by the Ftz-Fl gene (Luo et al. 1994, Sadovsky et al. 1995). The F&-F1 knockout mice had female external genitalia irrespective of genetic sex, and died shortly after birth secondary to corticosteroid deficiency; identical phenotypes were produced in knockout mice with specific disruption of SF-l (Luo et al. 1995), indicating an essential role for SF-1 in

lamic nucleus (VMH) ablated in the knockout al. 1995, Shinoda et al. ing additional roles for hypothalamic-pituitary

was specifically mice (Ikeda et 1995), implicatSF-l within the axis.

Most recent studies have sought to refine our understanding of the phenotype of SF-l knockout mice. Given that hypothalamic abnormalities are also present, impaired gonadotrope function could re-

androgen and corticosteroid biosynthesis. What was not anticipated, as shown in Figure 1, was the complete absence of ad-

flect intrinsic defects in the gonadotropes or effects secondary to the ablation of the VMH. Although GnRH is present in

renal glands and gonads in the knockout mice-findings that revealed obligatory

GnRH neurons of the medial hypothalamus in apparently normal amounts, treat-

roles for SF-l

in the development

of the

ment

of the SF-l

knockout

mice with

primary steroidogenic tissues. The expression of SF-l in the anterior

GnRH restored pituitary expression of LH and FSH (Ikeda et al. 1995). These results

pituitary and hypothalamus suggested that the Ftz-Fl knockout mice might ex-

suggest that SF-1 is not absolutely essen-

hibit abnormalities

1994, Shinoda et al. 1995). Moreover, as shown in Figure 2, the region corre-

that, in contrast to the adrenals and gonads and the VMH, gonadotropes are not totally ablated in the absence of SF-l. These studies further suggest that the VMH, either directly or indirectly, interacts with the GnRH neurons to facilitate

sponding

GnRH release.

at additional

sites.

Consistent with this, the knockout mice lacked LH and FSH, two separate markers of gonadotropes (Ingraham et al.

to the ventromedial

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Inc.,

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and

SF-l expression with sexual dif-

ferentiation? The difficulties in working with mouse embryos at early stages of development and the lack of cell culture models that recapitulate critical developmental events have hampered efforts to analyze these issues. It is, nonetheless, clear that a complete understanding of SF-l’s role in endocrine development cannot come until these mechanisms that determine its expression are defined. Conversely, it will be important to define the full spectrum of target genes that SF-I regulates in the various endocrine tissues. Table 1 lists the genes that are known to be regulated by SF-l. Although this list includes a number of genes with disparate functions (for example, membrane receptors, secreted microsomal and mitoglycoproteins, chondrial steroid hydroxylases, and proteins implicated in cholesterol delivery), these genes alone do not account for the profound consequences of SF-1 knockout that include adrenal and gonadal agenesis and loss of the VMH. Presumably, SF-l, therefore, must either activate or inhibit the expression of target genes that are directly related to processes of programmed cell death. The identification of these genes will provide key insights into SF’s role in endocrine development. It should also be noted that the studies linking SF-l to expression of a number of these genes, including the mitochondrial steroid hydroxylases, have relied on promoter analyses performed in cultured cells. Inasmuch as SF-l knockout mice undergo regression of the glands that make steroid hormones, it ultimately will be necessary to generate animal models in which SF-1 inactivation is timed to occur

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after the adrenal glands and gonads have developed, thereby permitting a direct analysis of the role of SF-l in steroid hydroxylase gene expression in vivo. It is also noteworthy, in this regard, that one of the steroid hydroxylases-that is, the cholesterol side-chain cleavage enzym+is expressed in the embryonic primitive gut of SF-l knockout mice (Keeney et al. 1995) as well as in placental cells originating from SF-l- deficient animals (Sadovsky et al. 1995). These latter results demonstrate that SF-1 is not always required for sidechain cleavage enzyme gene expression, suggesting that other regulatory mechanisms also control its expression. Another area that merits further investigation is regulation of SF-l function, including the identification of possible ligands for SF-l, the effects of posttranslational modifications such as phosphorylation, and the possible interactions between SF-l and other transcription factors. With respect to potential posttranslational modifications, several reports have noted that CAMP and CAMPdependent protein kinase modulate SFl-dependent transcriptional activation (Morohashi et al. 1994, Parrisenti et al. 1993). A recent report further showed that recombinantly expressed SF-l was phosphorylated in vitro by purified CAMP-dependent protein kinase at a threonine residue (Zhang and Mellon 1996). These studies provide a framework for further investigation of the role of phosphorylation in modulating SF-l activity. With respect to potential interactions of SF-l with other transcription factors, one attractive candidate is the recently described nuclear receptor DAX- 1. DAX-1 was isolated with the use of positional cloning of the gene responsible for X-linked congenital adrenal hypoplasia (Zanaria et al. 1994). This disorder is characterized by congenital adrenal hypoplasia; a subset of patients also have hypogonadotrophic hypogonadism (Letter et al. 1991. Muscatelli et al. 1994). This compound endocrine disorder thus resembles, in several respects, the phenotype seen in Ftz-Ff-disrupted mice. Like SF-l, DAX-1 encodes an orphan nuclear receptor; the DAX-1 structure is unusual in that the cDNA includes amino acids corresponding to the ligand-binding domain of nuclear receptors but lacks the zinc-finger DNA-binding domain typical of these transcrip-

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tional regulators. Perhaps most intriguing, preliminary studies indicate that DAX-1 is expressed in essentially all of the sites at which SF-l is expressed, including the adrenal cortex, gonads, gonadotropes, and VMH (Swain et al. 1996). This striking colocalization supports an intimate relationship between these two nuclear receptors, and suggests that protein-protein interactions between SF-l and DAX-1 may mediate the activation of downstream genes. Studies that define the mechanisms of their proposed interactions will undoubtedly provide new insights into the complex events in endocrine development and differentiation. It will also be of interest to determine whether SF-l, like DAX-1, is associated with clinical disorders affecting endocrine development. Although most cases of congenital adrenal hypoplasia are X-linked and result from DAX- 1 mutations, a small subset of patients appear to have autosomal recessive inheritance. These patients are candidates for mutations in SF-l. The human gene encoding SF-l has recently been cloned and mapped to chromosome 9q33 (Taketo et al. 1995), and efforts to determine the sequence of the human F&-F1 gene are in progress (M. Wong and M. Ramaya unpublished observation). A final area that merits additional study is the precise role of the different Ftz-FI-encoded transcripts in endocrine function. A recent report defined four distinct transcripts that are encoded by the mouse Ftz-Ff gene, two of which encode identical proteins despite arising from distinct promoters that are differentially expressed in adrenal glands and gonads versus pituitary and hypothalamus (Ninomiya et al. 1995). To date, none of the strategies used to disrupt the Ftz-Fl locus have selectively inactivated only one transcript; even the “SF-l-selective” knockout (Luo et al. 1995) would be predicted to inactivate three isoforms that utilize the same initiator methionine. Thus, the precise role that each transcript plays in endocrine development remains an important area for future investigation.

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Acknowledgment

References Barnhart

KM, Mellon PM: 1994. The orphan

nuclear receptor, steroidogenic ulates the glycoprotein

factor 1, reg-

hormone

gene in pituitary gonadotropes.

u-subunit Mol Endo-

crinol9:878-885. Hatano 0, Takayama

K, Imai T, et al.: 1994.

Sex-dependent expression of a transcription factor, Ad4BP, regulating steroidogenic P-450 genes in the gonads during prenatal and postnatal rat development. Development 120: 2787-2797. Honda S-I, Morohashi K-I, Nomura M, Takeya H, Kitajima M, Omura T 1993. Ad4BP regulating steroidogenic

P-450 gene is a member

of steroid hormone receptor Biol Chem 268~7494-7502.

superfamily. J

Ikeda Y. Lala DS, Luo X, Kim E, Moisan M-P, Parker

KL:

1993.

mouse FTZ-Fl

Characterization

of the

gene, which encodes a key

regulator of steroid hydroxylase gene expression. Mol Endocrinol 7:852-860. Ikeda Y Shen W-H, Ingraham HA, Parker KL: 1994. Developmental expression of mouse steroidogenic factor 1, an essential regulator of the steroid hydroxylases. 8654-662.

Mol Endocrinol

Ikeda Y, Luo X, Abbud R, Nilson JH, Parker KL: 1995. The nuclear receptor steroidogenic factor 1 is essential for the formation of the ventromedial hypothalamic nucleus. Mol Endomino1 9478486. Ingraham

HA, Lala DS, Ikeda Y, et al.: 1994.

The nuclear receptor

steroidogenic

factor

1

acts at multiple levels of the reproductive axis. Genes Dev 8:2302-2312. Keeney DS, Ikeda Y, Waterman MR. and Parker KL: 1995. Cholesterol side-chain cleavage cytochrome P450 gene expression in primitive gut of the mouse embryo does not require steroidogenic 9:1091-1098.

factor

1.

Mol

Endocrinol

Kletter GB, Got&i JL, Kelch Rp: 1991. Congenital adrenal hypoplasia and isolated gonadotropin deficiency. Trends Endocrinol 2:123-128. Lala

The authors’ work is supported by the Howard Hughes Medical Institute (K.L.P.) and the Medical Research Council of Can-

Elsevier Science Inc., 1043.2760/96/$15.00

ada and the National Cancer Institute of Canada (BPS.). We thank Dr. Ken Morohashi for helpful discussions and communication of unpublished data, and Douglas Rice, Deepak Lala, Amadeo Parrisenti, Yayoi Ikeda, Xunrong Luo, and Beverly Koller for key contributions to some of the experiments reviewed here.

DS,

Rice

DA, Parker

KL:

Metab

1992.

Ste-

roidogenic factor I, a key regulator of steroidogenic enzyme expression, is the mouse homolog of fushi tarazu-factor I. Mol Endocrinol 6:1249-1258.

PI1 SlO43-2760(96)00105-l

TEM Vol.7,No. 6,1996

Lavorgna G, Ueda H, Clos J, Wu C: 1991. FTZFl,

a steroid hormone

implicated Science

receptor-like

in the activation

protein

of fishi

iarazu.

252:848-851.

and sexual differentia-

biosynthesis.

biology of steroid Endocr

Rev 9:295-

318. K, Lida H, Nomura M, et al.: 1994.

Functional

difference

between

ELP, and their distributions tissues. Mol Endocrinol

Ad4BP

and

in steroidogenic

X-linked

in the DAX-1 gene give rise to both adrenal

hypoplasia

hypogonadotrophic

congenita

hypogonadism.

and

Nature

372~672-676.

nization

N, Suzuki K,

T, Niwa 0: 1995. Genomic

and isoforms

gene. J Biochem

of the mouse

orgaELP

(Tokyo) 1 l&380-389.

Parisenti AM, Parker KL, Shimmer BP: 1993. Identification of promoter elements in the mouse 21-hydroxylase quire

a functional

BP:

1993.

(Cyp21) gene that recyclic

Tianscrip-

of the adrenal steroidogenic

A shared promoter

element regulates the exenzymes. Mol

Sadovsky Y, Crawford PA, Woodson KG, et al.: 1995. Mice deficient

in the orphan receptor

steroidogenic

1 lack adrenal

factor

cleavage

but

enzyme

express

P450

embryonic

serum

glands

sidechain-

in the placenta

and have

levels of cortico-

roidogenic

adenosine

3’,5’-

HA: 1994. Nuclear factor

1 regulates

substance

determination

receptor

ste-

the Mtillerian

gene: a link to the sex

cascade.

Cell 77:65 l-661.

(in

1 map to mouse chromosome

2 and human

chromosome

25:565-567.

9q33. Genomics

Tsai MJ, O’Malley BW

1994. Molecular

anisms of action of steroid/thyroid superfamily

members.

mech-

receptor

Annu Rev Biochem

63~451486. Tsukiyama T, Ueda H, Hirose S, Niwa 0: 1992. Embryo& protein

long

terminal

is a murine

repeat-binding

homolog

of FTZ-Fl,

a

defects of the ventromedial

hypo-

Swain A, Ikeda Y, Weber TJ, et al.: 1996. Stefactor

Zanaria E, Muscatelli

F, Bardoni B, et al.: 1994.

An unusual member of the nuclear hormone superfamily

receptor X-linked

adrenal

responsible

hypoplasia

congenita.

for Na-

ture 3721635441.

thalamic nucleus and pituitary gonadotroph in the Ftz-FIdisrupted mice. Dev Dyn 204: 22-29. roidogenic

Endocrinol

Mol Cell Biol 12:1286-291. CCD, Ikeda Y, Parker KL,

Shinoda K, Lei H, Yoshii H, et al.: 1995. Developmental

Mol

member of the steroid receptor superfamily.

10,943. Shen W-H, Moore Ingraham

development.

press). Homologs of Drosophila Fushi Tamzu Factor

Endocrinol5:1552-1561.

and gonads

multiple cell lineages: potential links in endo

Taketo M, Parker K L, Howard TA, et al.: 1995.

Metab 4:46-50.

Rice DA, Mouw AR, Bogerd A, Parker KL: 1991.

inhibiting

Ninomiya Y, Okada M, Kotomura Tsukiyama

Schimmer

steroids. Proc Natl Acad Sci USA 92:10,939-

Muscatelh F, Strom TM, Walker AP, et al.: 1994. Mutations

KL,

normal

8643-653.

kinase.

crine

pression of three steroidogenic

tion. Cell 77:481490. Miller WL: 1988. Molecular

Morohashi

protein

enzymes. Trends Endocrinol

is essential for adrenal and

gonadal development

hormone

Parker

tional regulation

Luo X, Ikeda Y, Parker KL: 1994. A cell-specific nuclear receptor

monophosphatedependent Mol Endocrinol7:283-290.

1 and Dax-1 co-localize

in

Zhang P, Mellon SH: 1996. The orphan nuclear receptor

steroidogenic

cyclic adenosine

3’,5’-

diated transcriptional chrome

factor-l

regulates the

monophosphate-meactivation

of rat cyto-

P45Oc 17

( 17cy-hydroxylase/cl7-20 TEM lyase). Mol Endocrinol 101147-158.

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