GATA transcription factors in testicular adrenal rest tumours

Abstract

Testicular adrenal rest tumours (TARTs) are benign adrenal-like testicular tumours that frequently occur in male patients with congenital adrenal hyperplasia. Recently, GATA transcription factors have been linked to the development of TARTs in mice. The aim of our study was to determine GATA expression in human TARTs and other steroidogenic tissues. We determined GATA expression in TARTs (n = 16), Leydig cell tumours (LCTs; n = 7), adrenal (foetal (n = 6) + adult (n = 10)) and testis (foetal (n = 13) + adult (n = 8)). We found testis-like GATA4, and adrenal-like GATA3 and GATA6 gene expressions by qPCR in human TARTs, indicating mixed testicular and adrenal characteristics of TARTs. Currently, no marker is available to discriminate TARTs from LCTs, leading to misdiagnosis and incorrect treatment. GATA3 and GATA6 mRNAs exhibited excellent discriminative power (area under the curve of 0.908 and 0.816, respectively), while immunohistochemistry did not. GATA genes contain several CREB-binding sites and incubation with 0.1 mM dibutyryl cAMP for 4 h stimulated GATA3, GATA4 and GATA6 expressions in a human foetal testis cell line (hs181.tes). Incubation of adrenocortical cells (H295RA) with ACTH, however, did not induce GATA expression in vitro Although ACTH did not dysregulate GATA expression in the only human ACTH-sensitive in vitro model available, our results do suggest that aberrant expression of GATA transcription factors in human TARTs might be involved in TART formation.

Keywords: GATA transcription factors; Leydig cell tumour; congenital adrenal hyperplasia; testicular adrenal rest tumour.

Figure 1

Gene expressions of GATA1 (A), GATA3 (B), GATA4 (C) and GATA6 (D) in human TARTs, adult testes, foetal testes, adult adrenals and foetal adrenals. mRNA expression was calculated using the delta Ct method. All values were normalized to corresponding HPRT expression. The symbols in the graph represent all samples used and the error bars indicate median and 25th and 75th percentiles. Significance was tested with the Mann–Whitney-U (MWU) test or Kruskal–Wallis and Dunn’s post hoc test comparing selected pairs of variables. Underlined significances are from the biomarker analysis comparing TARTs and LCTs (MWU). Non-underlined significances are from the aetiology analysis comparing TART, adult adrenal, adult testis, foetal adrenal and foetal testis tissues. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ct>40 means that mRNA was not detected in these samples. LCTs, Leydig cell tumours; TARTs, testicular adrenal rest tumours.

Figure 2

Gene expression of GATA transcription factors during gonadal and adrenal developments. The figure summarizes the results of our gene expression analysis in relation to adrenogonadal development. Cells from the adrenal primordium combined with neural crest cells give rise to the foetal adrenal, which will mature into the adult adrenal. The testis develops from the bipotential gonad. In this study, we measured gene expression levels of GATA1, GATA3, GATA4 and GATA6 in human TART, foetal and adult adrenal, and foetal and adult testis tissues. Of note, we are uncertain of cell-specific expression as we measured expression in total tissue. GATA3 and GATA6 were expressed in both foetal and adult adrenals, while GATA4 was expressed in the foetal as well as the adult testes. GATA3, GATA4 and GATA6 gene expressions were all found in TARTs. TARTs, testicular adrenal rest tumours. Reproduced from Viger RS, Guittot SM, Anttonen M, Wilson DB, Heikinheimo M, Role of the GATA family of transcription factors in endocrine development, function, and disease, Molecular Endocrinology, 2008, volume 22, issue 4, pages 781–798, by permission of Oxford University Press. Copyright 2008, The Endocrine Society (18).

Figure 3

Discriminative potential of GATA3 and GATA6 gene expressions in distinguishing TARTs from LCTs. Receiver operating characteristic analyses were performed. An area under the curve of 0.908 was observed for GATA3, while this was 0.816 for GATA6. LCTs, Leydig cell tumours; TARTs, testicular adrenal rest tumours.

Figure 4

GATA3 and GATA6 protein expressions in human TARTs, benign LCTs, metastases of malignant LCTs and control tissues. GATA3 and GATA6 expressions (positive staining) are indicated by brown nuclei and/or brown cytoplasm. Protein expression of GATA3 is present in the positive control tissue (kidney), but absent in TARTs, benign LCTs and metastases of malignant LCTs. Protein expression of GATA6 is present in the cytoplasm and nuclei of TARTs and benign LCTs, however absent in metastases of malignant LCTs and the negative control (tissue slides without primary antibody). Representative pictures are shown. Scale bar represents a distance of 50 µm. LCTs, Leydig cell tumours; TARTs, testicular adrenal rest tumours.

Figure 5

GATA transcription factors and their possible role in the aetiology of TARTs. Long-term exposure to elevated levels of ACTH is present in patients with congenital adrenal hyperplasia and this is associated with the development of TARTs. ACTH binds to its receptor (MC2R), using cAMP as a second messenger. CREB-binding sites are present within the gene body or up to 10 kb upstream of the transcription start site in GATA3, GATA4 and GATA6. cAMP could therefore be a GATA expression-inducing second messenger. Source: http://sabiosciences.com/chipqpcrsearch.php?app=TFBS. (A) Hs181.tes cells were incubated with 0.1 mM dibutyryl cAMP (Hs181.tes cells do not express ACTH receptor) for 0 min, 30 min or 4 h. Gene expression was calculated using the delta Ct method and corresponding HPRT expression was used to normalize. (B) H295RA cells were incubated with 10 nM ACTH for 0 min, 30 min, 4 h or 24 h. Delta Ct method and corresponding HPRT value were used to calculate normalized gene expression.