Sanne Kiiveri

Transcription factors GATA-6, SF-1, and cell proliferation in human adrenocortical tumors.

Transcription factor GATA-6 is expressed in fetal and adult human adrenal cortex and has been suggested to have a role in adrenal androgen synthesis. In other tissues GATA-6 has been linked to the cell cycle regulation and the dedifferentiation of carcinoma cells. GATA-6 has been shown to be downregulated in mouse adrenocortical tumors, but has not been studied in human adrenocortical tumors in detail. We have now analyzed GATA-6 expression in 20 human adrenocortical adenomas and 16 carcinomas using Northern blot analysis and immunohistochemistry. GATA-6 mRNA and protein expression was remarkably diminished in adrenocortical carcinomas as compared to normal adrenal cortex and adenomas (p<0.05). In opposite to other tumor types GATA-6 expression was, however, high in virilizing carcinomas. Steroidogenic factor 1 (SF-1) has been functionally linked to GATA-6, and the expression of these two factors correlated in the adrenal tumors. Furthermore, GATA-6 immunoreactivity was linked to P450c17 expression. In contrast to GATA-6, we found upregulated cyclin-dependent kinase inhibitor p21 and proliferation marker Ki67 in adrenocortical carcinomas indicating that GATA-6 is not linked to cell proliferation in human adrenal tumors. Taken together, the present and earlier results link GATA-6 to adrenocortical steroidogenesis and to the benign adrenocortical phenotype.

Adrenocortical tumorigenesis, luteinizing hormone receptor and transcription factors GATA-4 and GATA-6

Luteinizing hormone (LH/hCG) responsiveness of normal and pathological human adrenal glands as well as the possibility of constitutive expressions of luteinizing hormone receptor (LHR) in adrenal cortex has been reported. Some recent studies showed a correlation between the LHR and abundant GATA-4 expression in both metastasizing and non-metastasizing human adrenocortical tumors, but not in normal adrenals, implicating the putative relevance of LHR and GATA-4 for adrenocortical pathophysiology. However, the physio- and pathophysiological significance of LHR and GATA-4 in the mechanism of adrenocortical tumorigenesis remains unclear. The paucity of suitable models for adrenal tumorigenesis makes the establishment of proper animal models highly important. LHR expression in the murine adrenal gland is an exception and not found in wild-type (WT) animal. We have previously shown that ectopic LHR expression in the murine adrenal gland can be induced by chronically elevated LH levels. We have generated a gonadotropin-responsive adrenal tumor model in gonadectomized transgenic (TG) mice expressing the inhibin alpha promoter/Simian Virus 40 T antigen transgene (inhalpha/Tag). Given the induction of expression and regulation of GATA-4 and GATA-6 zinc finger transcription factors in the gonads by gonadotropins, this review will explore their relationship to LHR expression and their role in adrenocortical tumorigenesis. A functional link between LHR and GATA-4 actions in the adrenal pathophysiology is proposed.

Transcription factors GATA-4 and GATA-6 in human adrenocortical tumors.

Transcription factors GATA‐4 and GATA‐6 are expressed during normal adrenocortical 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 be observed in human adrenocortical tumors, but the pathophysiological significance of these findings remains to be elucidated.

Transcription factors GATA-4 and GATA-6 during mouse and human adrenocortical development.

Our earlier work implicates transcription factors GATA-4 and GATA-6 in the murine adrenal. We have now studied their expression during mouse and human adrenal development in detail. GATA-4 and GATA-6 mRNAs are readily detectable from embryonic day 15 in mouse and gestational week 19 in human adrenal cortex. In postnatal adrenal, GATA-4 expression is down-regulated, whereas GATA-6 mRNA and protein continue to be abundantly present. In a human adrenocortical cell line NCI-H295R, GATA-6 mRNA is up-regulated by cAMP. This cell line does not express GATA-4. Our findings suggest that GATA-6 expression is hormonally controlled, and required throughout adrenal development from fetal to adult age. GATA-4, on the other hand, may serve a role in fetal adrenal gene regulation.

GATA transcription factors in adrenal development and tumors

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-alpha 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.

The regulated expression of c-IAP1 and c-IAP2 during the rat seminiferous epithelial cycle plays a role in the protection of germ cells from Fas-mediated apoptosis.

The inhibitor of apoptosis proteins, c-IAP1 and c-IAP2, are highly expressed in rat testis and potentially play a regulatory role in testicular apoptosis. To better understand their functions during spermatogenesis, we have analyzed their spatio-temporal distribution in rat testis, how their expression is controlled by the paracrine stem-cell factor (SCF) and how they affect Fas-mediated apoptosis. Both c-IAP1 and c-IAP2 showed cycles of transcriptional expression, throughout the seminiferous epithelial cycle. c-IAP1 protein showed a diffuse nuclear distribution in type B spermatogonia, preleptotene, leptotene, and zygotene spermatocytes. In pachytene spermatocytes, c-IAP1 colocalized with SUMO-1 in the XY-body. c-IAP2 protein was cytoplasmic in spermatocytes, from stage VI pachytene onwards, round spermatids, elongated spermatids and Leydig cells. Its expression was upregulated by SCF. Inhibition of IAP activity resulted in a greater sensitivity of germ cells to Fas-mediated apoptosis. These results suggest an important role for IAPs in the regulation of spermatogenic apoptosis.

Nude Mice as a Model for Gonadotropin‐Induced Adrenocortical Neoplasia

Certain inbred mice (e.g., DBA/2J, CE) develop sex steroid producing adrenocortical tumors following gonadectomy. This adrenal response is thought to result from an unopposed increase in circulating gonadotropins and/or a decrease in factor(s) of gonadal origin. To differentiate between these two possibilities, we utilized the NU/J strain of nude mice, which are immunologically compromised and therefore permissive to xenografts. One group of female nude mice was gonadectomized, while another group of females received xenografts of CHO cells stably transfected with human chorionic gonadotropin (hCG). After 1–2 months, subcapsular adrenocortical neoplasms containing sex steroid‐producing cells were observed in both groups. We conclude that high levels of circulating gonadotropins are sufficient to induce adrenocortical tumorigenesis, even in the presence of intact gonads.