Transcription Factors GATA-4 and GATA-6 in Human Adrenocortical Tumors
S. Kiiveri,1,2 J. Liu,3 P. Heikkila,3,4 J. Arola,3,4 E. Lehtonen,3,4 R. Voutilainen,5 and M. Heikinheimo1,2,*
‘Children’s Hospital, 2Program for Developmental and Reproductive Biology, Biomedicum Helsinki, and 3Department of Pathology, University of Helsinki, Helsinki, Finland
4Department of Pathology, Helsinki University Central Hospital, Helsinki, Finland 5Department of Pediatrics, Kuopio University Hospital, Kuopio, Finland
ABSTRACT
Transcription factors GATA-4 and GATA-6 are expressed during normal adrenocor- tical development in mice and humans, and in vitro studies have linked them to adrenal steroidogenesis. GATA-4 is highly expressed in the adrenocortical tumors of gonadectomized mice, whereas GATA-6 is down-regulated in the tumor area. Based on these findings we studied GATA-4 and GATA-6 expression in 39 human adrenocortical tumors using RT-PCR, Northern analysis and immunohistochemistry. 6/18 adenomas and 4/11 carcinomas were positive for GATA-4 mRNA. GATA-6 mRNA was expressed in 19/19 adenomas and 9/10 carcinomas, and GATA-6 immunoreactivity was remarkably lower in adrenocortical carcinomas than in adenomas (p < 0.05). Some of the steroidogenically active human adrenocortical cells (NCI-H295R) were weakly positive for GATA-4, whereas steroidogenically inactive cells (ACT-1) were totally GATA-4 negative. In contrast, both cell lines expressed GATA-6. GATA expression patterns similar to the animal models can thus
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be observed in human adrenocortical tumors, but the pathophysiological significance of these findings remains to be elucidated.
Key Words: Transcription factor; GATA-4; GATA-6; Adrenal tumor.
INTRODUCTION
The GATA family of transcription factors consists of six conserved zinc finger proteins expressed with distinct developmental and tissue-specific profiles. Transcrip- tion factors GATA-4 and GATA-6 are essential during early embryogenesis, and they have roles in the development and function of several organs including the heart, gastrointestinal tract, gonads, and lung (1,2). They are both expressed in the adult testis and ovary, where they have been functionally linked to the regulation of several endocrine genes (3-5). GATA-4 and GATA-6 have also been related to cell prolifer- ation, apoptosis, and cancer (6,7).
We have previously shown that GATA-4 is expressed predominantly in the fetal and GATA-6 in both fetal and adult adrenal during normal development in mice and humans (8,9). Inhibin a/SV40 T-antigen transgenic mice subjected to gonadectomy develop adrenocortical carcinomas in a gonadotropin-dependent fashion (10). The adrenocortical tumors in these mice express GATA-4 and GATA-6 in the tumor area is reciprocally down-regulated similarly to other animal models (8,11,12). The first study on GATA transcription factor expression in human adrenocortical tumors has recently been published (13), but the role of these transcription factors and their targets during tumorigenesis remain unknown.
MATERIALS AND METHODS
A total of 36 adrenocortical tumors were studied and their characteristics are shown in Table 1. Steroidogenic NCI-H295R (ATCC, Rockville, MD) (14) and non- steroidogenic ACT-1 (kindly provided by Dr. Munehisa Ueno, Saitama Medical School, Japan) (15) human adrenocortical carcinoma cell lines were also used. GATA-4 and GATA-6 protein expression in the tissues and cells were studied with previously de- scribed methods (8,9).
RNA isolation and GATA-4 RT-PCR were performed as previously described (9). For Northern blot analysis, 10-20 µg of denatured RNA was subjected to electro- phoresis on a 1% denaturing agarose gel and then transferred onto nylon membranes. The membranes were hybridized with 32P-labelled (> 6000 Ci/mmol, Amersham) synthetic oligonucleotide probes for human GATA-4 (one probe) and GATA-6 (two probes) (Institute of Biotechnology, University of Helsinki, Finland). The sequences were 5’-GGC TGT TCC AAG AGT CCT GCT TGG AGC TGG-3’ corresponding to nucleotides 1551-1580 of human GATA-4 cDNA (GenBank no. NM-002052) and sequences 5’- CGT CTG GAT GGA GCC GCA GTT CAC GCA CTC -3’ and 5’-AAG CCG CCG TGA TGA AGG CAC GCG CTT CTG -3’ corresponding to nucleotides 1074-1103 and 1200-1229 of human GATA-6 cDNA (GenBank no. NM-005257). A 28S ribosomal RNA cDNA probe was used as a loading control.
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| Histology | Patient | IHC | PCR | |
|---|---|---|---|---|
| Sex (F/M) | Age | G6 (%) | G4 (+/total) | |
| Adenoma | ||||
| Nonfunctional | 4/1 | 65 | 60 | 1/4 |
| Conn's | 4/4 | 49 | 54 | 1/6 |
| Cushing's | 5/1 | 53 | 44 | 3/5 |
| Virilizing | 3/0 | 32 | 50 | 1/3 |
| Carcinoma | ||||
| Nonfunctional | 1/5 | 59 | 14 | 1/3 |
| Conn's | 2/1 | 66 | 27 | 1/1 |
| Cushing's | 3/2 | 41 | 23 | 1/4 |
| Virilizing | 2/1 | 29 | 77 | 1/3 |
Note: The number of samples is shown as total number of female (F) and male (M) patients. The mean age and results of immunohistochemistry (IHC) are shown. PCR results are shown as the number of positive samples per the total number of analyzed specimen. Abbreviations used are G6, GATA-6, G4, GATA-4.
RESULTS
Northern blot analysis and RT-PCR for GATA-4 mRNA expression was first performed for the tumor samples. We found that 6/18 adenomas and 4/11 carcinomas were GATA-4 positive (Table 1, Fig. 1). Most of the tumors (19/19 adenomas and 9/10 carcinomas) expressed GATA-6 mRNA to some degree (data not shown). By immuno- histochemistry 19/20 adenomas and 11/16 carcinomas were positive for GATA-6, and notably the immunoreactivity (% positive cells) was significantly diminished in carcinomas (p < 0.05) (Table 1). Only a little GATA-4 protein was detected in a small proportion of the NCI-H295R cells and none in the ACT-1 cells, whereas both cell lines were positive for GATA-6 (Fig. 2).
Ovarian tumor
Cushing’s ca
Conn’s ca
Virilizing ca
Virilizing ca
Cushing’s aden
Conn’s aden
Virilizing aden
GATA-4
-4.4 kb
28S
GATA-6
GATA-4
A
B
NCI-H295R
D
ACT-1
DISCUSSION
GATA-4 has been linked to carcinogenesis, for example, in the ovary (6). A recent study demonstrated an intense GATA-4 expression in human adrenocortical tumors with metastasing behavior (13). In the present series GATA-4 was expressed in both adenomas and carcinomas when studied with RT-PCR, but could be detected only in carcinomas by Northern blot analysis suggesting a more abundant expression in malignant than benign tumors. The expression of this transcription factor did not markedly associate with any specific hormonal profile. In the Brazilian work a significant GATA-6 mRNA down-regulation was observed in the malignant tumors (13), as shown now at the protein level. GATA-4 and GATA-6 regulate genes for steroidogenic enzymes involved in the adrenocortical androgen production (16,17). Supporting this view, GATA-6 expression remained high in the virilizing carcinomas when compared to the other tumor groups (this work). We also showed, that steroidogenically active (NCI- H295R) and inactive (ACT-1) human adrenocortical carcinoma cell lines expressed GATA-6, but not GATA-4. This suggests, that GATA-6 alone is not sufficient for driving steroidogenesis in the adrenocortical cells. Recent findings implicate functions for GATA-6, rather than GATA-4, in the normal adrenal. The expression of GATA transcription factors is altered during adrenocortical tumorigenesis, but the roles of GATA-4 and GATA-6 during tumor formation need further investigation.
ACKNOWLEDGMENTS
This work was supported by the Finnish Pediatric Foundation, Emil Aaltonen Foundation, Maud Kuistila Foundation, Finnish Medical Society Duodecim, Jalmari
Transcription Factors GATA-4 and GATA-6
and Rauha Ahokas Foundation, Sigrid Juselius Foundation, Finnish Cancer Foundation, Academy of Finland and Kuopio University Hospital. Mrs Merja Haukka, Ritva Löfman and Taru Jokinen are thanked for the expert technical assistance, and Dr Munehisa Ueno is thanked for providing the ACT-1 cells.
REFERENCES
1. Kuo CT, Morrisey EE, Anandappa R, Sigrist K, Lu MM, Parmacek MS, Soudais C, Leiden JM. Genes Dev 1997; 11:1048-1060.
2. Morrisey EE, Tang Z, Sigrist K, Lu MM, Jiang F, Ip HS, Parmacek MS. Genes Dev 1998; 12:3579-3590.
3. Heikinheimo M, Scandrett JM, Wilson DB. Dev Biol 1994; 164:361-373.
4. Ketola I, Rahman N, Toppari J, Bielinska M, Porter-Tinge SB, Tapanainen JS, Huhtaniemi IT, Wilson DB, Heikinheimo M. Endocrinology 1999; 140:1470-1480.
5. Tremblay JJ, Viger RS. Endocrinology 2001; 142:977-986.
6. Capo-chichi CD, Roland IH, Vanderveer L, Bao R, Yamagata T, Hirai H, Cohen C, Hamilton TC, Godwin AK, Xu XX. Cancer Res 2003; 63:4967-4977.
7. Aries A, Paradis P, Lefebvre C, Schwartz RJ, Nemer M. Proc Natl Acad Sci U S A 2004; 101:6975-6980.
8. Kiiveri S, Siltanen S, Rahman N, Bielinska M, Lehto VP, Huhtaniemi IT, Muglia LJ, Wilson DB, Heikinheimo M. Mol Med 1999; 5:490-501.
9. Kiiveri S, Liu J, Westerholm-Ormio M, Narita N, Wilson DB, Voutilainen R, Heikinheimo M. Endocrinology 2002; 143:3136-3143.
10. Kananen K, Markkula M, Mikola M, Rainio EM, McNeilly A, Huhtaniemi I. Mol Endocrinol 1996; 10:1667-1677.
11. Bielinska M, Parviainen H, Porter-Tinge SB, Kiiveri S, Genova E, Rahman N, Huhtaniemi IT, Muglia LJ, Heikinheimo M, Wilson DB. Endocrinology 2003; 144:4123-4133.
12. Peterson RA II, Kiupel M, Bielinska M, Kiiveri S, Heikinheimo M, Capen CC, Wilson DB. Vet Pathol 2004; 41:446-449.
13. Barbosa AS, Giacaglia LR, Martin RM, Mendonca BB, Lin CJ. BMC Endocr Disord 2004; 4.
14. Rainey WE, Bird IM, Mason JI. Mol Cell Endocrinol 1994; 100:45-50.
15. Ueno M, Nakashima J, Akita M, Ban S-I, Nakanoma T, Iida M, Deguchi N. Int J Urol 2001; 8:17-22.
16. Jimenez P, Saner K, Mayhew B, Rainey WE. Endocrinology 2003; 144:4285- 4288.
17. Flück CE, Miller WL. Mol Endocrinol 2004; 18:1144-1157.