ELSEVIER
MCE
Molecular and Cellular Endocrinology
Review
GATA transcription factors in adrenal development and tumors
Helka Parviainen ª, Sanne Kiiveri ª, Malgorzata Bielinskab, Nafis Rahman “, Ilpo T. Huhtaniemi c,d, David B. Wilson b,e, Markku Heikinheimo a,b,*
a Children’s Hospital and Program for Developmental and Reproductive Biology, Biomedicum Helsinki, University of Helsinki, 00014 Helsinki, Finland
b Department of Pediatrics, Washington University School of Medicine, St. Louis Children’s Hospital, St. Louis, MO 63110, USA ” Department of Physiology, University of Turku, 20520 Turku, Finland
d Imperial College, London, UK
e Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis Children’s Hospital, St. Louis, MO 63110, USA
Abstract
Of the six GATA transcription factors, GATA-4 and GATA-6 are expressed in the mouse and human adrenal with distinct developmental profiles. GATA-4 is confined to the fetal cortex, i.e. to the less differentiated proliferating cells, while GATA-6 is expressed both in the fetal and adult adrenal. In vitro, GATA-4 regulates inhibin-a and steroidogenic factor-1 implicated in normal adrenal function. GATA-6 probably has roles in the development and differentiation of adrenocortical cells, and in the regulation of steroidogenesis. GATA-4 expression is dramatically upregulated and GATA-6 downregulated in gonadotropin dependent mouse adrenocortical tumors. This is accompanied by the appearance of luteinizing hormone receptor (LHR). In vitro, GATA-4 transactivates LHR promoter, and gonadotropins upregulate GATA-4 levels. Human adrenal tumors occasionally express GATA-4, whereas GATA-6 levels are usually lower than normal. @ 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Gonadectomy; Luteinizing hormone; Inhibin; Steroidogenesis; Metaplasia
Contents
1. Introduction 00
1.1. Overview of mouse adrenal development 00
1.2. GATA transcription factors 00
1.3. GATA factors and their target genes in the adrenal 00
1.4. GATA factors in gonadal cells 00
2. Mouse models for adrenocortical tumors 00
2.1. GATA expression in inbred mouse strains 00
2.2. GATA expression in genetically modified strains 00
3. Clinical significance: GATA transcription factors in human adrenal tumors 00
4. Concluding remarks 00
Acknowledgements 00
References
00
1. Introduction
The adrenal cortex is a major source of steroid hormones that are synthesized from cholesterol through the sequential activity of a series of cytochrome P450 (CYP) enzymes. The adrenal cortex is divided into distinct functional and morphological lay- ers, which vary somewhat among species. In most mammals,
* Corresponding author at: Program for Developmental and Reproductive Biology, Biomedicum Helsinki, Room B530b, P.O. Box 63 (Haartmaninkatu 8), 00014 University of Helsinki, Finland. Tel .: +358 9 4717 1975; fax: +358 9 4717 1947.
E-mail address: markku.heikinheimo@helsinki.fi (M. Heikinheimo).
including humans, the cortex is composed of the outer zona glomerulosa, middle zona fasciculata, and inner zona reticu- laris. In addition, a thin stem cell zone is believed to be situated outermost, adjacent to the adrenal capsule, enabling continu- ous self-renewal of the adrenal cortex by centripetal migration (Bland et al., 2003; Vinson, 2003). The same stem cells seem, in many instances, to give rise to adrenocortical neoplasia.
1.1. Overview of mouse adrenal development
During embryonic mouse development, the gonads and the adrenals arise from common progenitor cells in the urogeni- tal ridge (Hatano et al., 1996). This adrenogonadal primordium expresses Wilm’s tumor-1, Wnt4, and steroidogenic factor-1 (SF-1), which, based on mutation studies, participate in the dif- ferentiation of both adrenocortical and gonadal stromal cells (Keegan and Hammer, 2002). A separate adrenocortical pri- mordium is distinguishable from embryonal day 11.5 onwards (Hatano et al., 1996).
The initiation of adrenal development is similar in the mouse and human, but there are species-specific differences in the development and organization of the adrenal cortex. The adrenal cortex of the postnatal mouse contains adjacent to the medulla a layer termed the X zone, which does not have a counterpart in postnatal human adrenal cortex (Keegan and Hammer, 2002). However, the X zone develops from the recently recognized mouse fetal zone, which is analogous to that of the human fetal adrenal (Zubair et al., 2006). As the X zone regresses in puberty (males) or during first pregnancy (females), an adult mouse cor- tex forms. It, too, is distinct from its human counterpart, since CYP17 is expressed in the mouse adrenal only transiently during development and not in the adult (Keegan and Hammer, 2002; Keeney et al., 1995). Consequently, the mouse adrenal does not produce androgens, and the primary glucocorticoid produced is corticosterone, instead of cortisol as in humans.
1.2. GATA transcription factors
The GATA factors, GATA-1 to GATA-6, are a family of zinc finger transcription factors that bind to a consensus DNA sequence (A/T)GATA(A/G) in gene promoters and enhancers (Tremblay and Viger, 2003). Factors GATA-1 to GATA-3 are expressed principally in hematopoietic cells, whereas GATA-4 to GATA-6 regulate gene expression, differentiation, cellu- lar proliferation, and apoptosis in various mesoderm- and endoderm-derived tissues including heart, gut, gonads, and adrenal cortex. Null mutations for each of the Gata genes, with the exception of that for Gata5, are embryonic lethal, underscor- ing their pivotal role in early development.
1.3. GATA factors and their target genes in the adrenal
The two GATA factors expressed in the adrenal are GATA- 4 and GATA-6 (Kiiveri et al., 2002). GATA-4 is expressed in a considerable proportion of fetal mouse adrenocortical cells, with a clear downregulation soon after birth. Also in humans, GATA- 4 can be detected in the fetal but not adult adrenal. Studies on
mice chimeric for Gata4-/- and wild type cells indicate that GATA-4 is not essential for early adrenocortical differentiation (Kiiveri et al., 2002). It has, however, been shown to work in concert with SF-1, an important factor for adrenocortical devel- opment and function (Tremblay and Viger, 2003). Additionally, in vitro transactivation experiments demonstrate that GATA-4 upregulates inhibin-a, CYP17, and steroidogenic acute regu- latory protein (StAR) (Tremblay and Viger, 2003). GATA-6 is abundantly expressed throughout development in both mouse and human adrenal cortex (Kiiveri et al., 2002). Whether it has roles in adrenocortical development remains to be established, but in transactivation experiments it regulates several steroido- genic enzymes. Specifically, GATA-6 acts in synergy with SF-1, enhancing the transactivation of genes encoding StAR, CYP11A1 (P450scc), CYP17, and dehydroepiandrosterone- sulfotransferase (SULT2A1), all necessary for adrenal steroid biosynthesis (Jimenez et al., 2003; Tremblay and Viger, 2003). Of interest, StAR is expressed in mouse adrenal cortex with a pattern similar to that of GATA-6 (Clark et al., 1995).
1.4. GATA factors in gonadal cells
Although the gonads and adrenal cortex have a common developmental origin, the GATA factor expression profile dif- fers in these organs. The urogenital ridge expresses GATA-4 at its genital part, and the expression persists in gonadal somatic cells throughout development (La Voie, 2003). In the adult mouse gonad, GATA-4 expression is strongest during periods of active proliferation, in ovarian granulosa and testicular Sertoli cells. GATA-6 is also expressed during gonadal development and it is more abundant than GATA-4 in ovarian theca and testicu- lar Leydig cells (Heikinheimo et al., 1994; Ketola et al., 1999). These differences in the temporospatial expression patterns sug- gest specific functional roles for each of these factors also in the gonadal somatic cells.
The adrenals and the gonads also share several GATA tar- get genes, including SF-1, StAR, and inhibin-a. In addition, the GATA-4 target genes anti-Müllerian hormone (AMH) and aromatase (CYP19) are key regulators specifically of gonadal development and function (Tremblay and Viger, 2003). GATA- 6 has been proposed to also transactivate several of these gonadal regulators, albeit more weakly (Tremblay and Viger, 2003).
2. Mouse models for adrenocortical tumors
Several mouse models exhibit a pattern of adrenal gonadal- type metaplasia and/or neoplasia following gonadectomy (Bielinska et al., 2006; Matzuk et al., 1994). The phenomenon of gonadectomy-induced adrenal tumor formation was initially described in 1940s in inbred strains, and the abnormal cells were characterized as spindle-shaped “A” cells and lipid-laden “B” cells (Fig. 1). The tumors arise from subcapsular progeni- tors and invade to the deeper layers of the adrenal cortex. The inbred strains susceptible to tumor formation include DBA/2J, NU/J nude, C3H, and CE, of which CE mice develop carcino- mas and the other strains adenomas (Bielinska et al., 2006). The genetic basis for the strain susceptibility is still unclear, but it has
C
been linked to a polymorphism in the SF-1 gene (Frigeri et al., 2002).
Since gonadectomy disrupts the feedback inhibition at the hypothalamus-pituitary-gonadal axis, gonadectomized inbred mice exhibit high blood luteinizing hormone (LH) levels. This chronic gonadotropin elevation is needed for tumor formation, as hypophysectomy abrogates the tumorigenesis (Huseby and Bittner, 1951). Studies on NU/J nude mice reveal that xenografts of human chorionic gonadotropin (hCG) producing cells are able to cause adrenocortical tumor formation without gonadectomy (Bielinska et al., 2005). This suggests that it is indeed the ele- vated luteinizing hormone level, and not the abrogation of any gonadal factor in the gonadectomized mice, that is sufficient to drive tumor formation in the adrenal cortex of susceptible strains.
Some genetically modified mice, including inhibin-a null mice (Inh-/-) and transgenic mice expressing Simian virus T-antigen driven by inhibin-« promoter (Inha/Tag) also pro- duce gonadotropin-dependent adrenocortical tumors in response to gonadectomy (Kananen et al., 1996; Matzuk et al., 1994). However, if gonadectomy is not performed these knockout and transgenic mice develop gonadal tumors, a phenomenon dis- tinct from the inbred strains (Kananen et al., 1995; Matzuk et al., 1992). In these models, the adrenal tumors are situated juxtamedullarly at the X zone, but the origin of the tumors remains controversial. Based on the analogous expression pro- file of the tumors of the susceptible inbred strains, it has been proposed that following gonadectomy the Inh-/- and Inha/Tag adrenocortical tumors also arise from the subcapsular progenitor
CELL
Type A
Type B
Tumor
cells (Looyenga and Hammer, 2006). Since the Inha/Tag mice develop A and B cell-like neoplasia near the tumor area (Fig. 2) (Rahman et al., 2004), the subcapsular origin for the tumors is quite possible.
2.1. GATA expression in inbred mouse strains
Expression of GATA-4 mRNA and protein in both A and B type adrenocortical tumor cells is evident in post-gonadectomy adrenocortical tumors in several of the inbred mouse models, and it accompanies the presumed gonadal-type metaplasia of B cells (Fig. 1 and Table 1) (Bielinska et al., 2005, 2003). This process is characterized by expression of gonadal markers such as AMH, LH receptor (LHR), CYP17, and estrogen receptor a, and production of androgens, estrogens, or both. Type A cells, in contrast, express GATA-4 and the receptor for anti-Müllerian hormone but no other gonadal markers; the gonadal counter- part of A cells could be the stromal cells of the postmenopausal ovary. Neither A nor B cells express melanocortin-2 receptor or the CYP enzymes for the synthesis of corticosterone or aldos- terone, suggesting that the cells have lost the adrenocortical
| Normal cortex | DBA | NU/J nude | Inha/Tag | Inh-/- | |
|---|---|---|---|---|---|
| GATA-4 | − | + | + | + | + |
| GATA-6 | + | - | - | − | − |
| LHR | − | + | + | + | + |
| Inhibin-& | + | + | + | + | − |
| AMH | − | n/a | + | n/a | + |
| CYP17 | − | + | + | n/a | + |
| CYP19 | − | n/a | + | n/a | + |
LHR, luteinizing hormone receptor; AMH, anti-Müllerian hormone; CYP17, cytochrome P450c17; CYP19, aromatase; n/a, unknown.
A
B
C
D
m
m
E
F
m
phenotype (Bielinska et al., 2003). Also GATA-6, abundantly expressed in normal adrenocortical cells, is downregulated in the tumors. GATA-4 protein can also be detected in the neoplas- tic adrenocortical cells of the neutered ferret (Peterson et al., 2004).
2.2. GATA expression in genetically modified strains
In the adrenals of Inha/Tag mice, GATA-4 expression is first visible 3 months after gonadectomy. Thereafter, the whole tumor area abundantly expresses GATA-4 (Fig. 3) (Kiiveri et al., 1999; Rahman et al., 2004). Interestingly, GATA-6 is downregu- lated during the tumor formation so that tumor areas expressing GATA-4 display no significant GATA-6 expression. Also LHR is expressed in the tumors, with a similar temporospatial pattern to that of GATA-4, and in in vitro transactivation experiments GATA-4 was found to induce LHR expression (Rahman et al., 2004).
A recent report demonstrates that the tumors of the gonadec- tomized Inh-/- mice express GATA-4, have lost the expression
of GATA-6, and comprise two cell types with different expres- sion profiles resembling either theca cells or granulosa cells of the ovary (Looyenga and Hammer, 2006). The control wild- type gonadectomized mice did not develop adrenal neoplasms, but expression of gonad-restricted proteins, including GATA-4, could be detected. This suggests that inhibin action in the pre- vention of the neoplasms is preceded by gonadotropin action in cell fate decision-chronic gonadotropin elevation seems to be the earliest event in tumor induction.
3. Clinical significance: GATA transcription factors in human adrenal tumors
In humans, benign adrenal masses are often referred to as incidentalomas, paying respect to the most common route to diagnosis. The majority of these adenomas are steroidogeni- cally silent, but in some cases symptoms develop of hormone secretion-either excessive amounts of a substance secreted nor- mally by the adrenal (e.g. cortisol or androgens) or exogenous secretion of a hormone foreign to the adrenal (e.g. insulin).
Adrenocortical adenomas are fairly common in older popula- tion, but adrenocortical carcinoma is a rare disease.
GATA-4 mRNA and protein expression is detected in a small proportion of adrenocortical adenomas and carcinomas (Kiiveri et al., 2005), and has been linked to aggressive behavior (Barbosa et al., 2004). GATA-6 expression is usually diminished in adrenal tumors but its expression level in adenomas exceeds that of car- cinomas, except in virilizing tumors (Kiiveri et al., 2005). The latter finding may be related to the putative role for GATA-6 in regulating androgen synthesis (Jimenez et al., 2003).
There are reports of subcapsular adrenocortical neoplasms in humans with elevated gonadotropin levels, also implicating a possible role for LH in human adrenal adenomas (Bielinska et al., 2006). Given the linkage between GATA-4 and LHR in mice, we have studied the expression of these factors in a small series of human adrenocortical neoplasias, finding that LHR expression was present in 50% of benign and 18% of malignant adreno- cortical tumors (Kiiveri, Kero et al., unpublished data). There was no correlation between LHR and GATA-4 expression in our material, consistent with findings in a Brazilian series (Barbosa et al., 2004). This might suggest a distinct regulatory pathway for LHR signaling and GATA-4 in human adrenocortical tumors, although the human tumor series studied so far are small.
4. Concluding remarks
Changes in the expression of transcription factors GATA- 4 and GATA-6 during adrenocortical tumorigenesis have been detected in humans and in various animal models. There seems to be a link between upregulated gonadotropin secretion, LHR, GATA-4, and the onset of neoplasia. There are, however, likely to be multiple pathways contributing to the formation of the tumors and the maintenance of the neoplastic phenotype, and these pathways may vary between tumor types and species. Some pathways may well be connected to GATA proteins through their involvement in signaling cascades directing gene transcription and cellular functions in adrenocortical cells. Unravelling the role of GATA factors and associated molecules in adrenocorti- cal development and tumors can contribute to the development of diagnostic tools and future therapies for this cancer.
Acknowledgements
The Finnish Cultural Foundation, the Finnish Foundation for Pediatric Research, and the Sigrid Juselius Foundation finan- cially supported this work.
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