Adrenocorticotropin-dependent changes in SF-1/DAX-1 ratio influence steroidogenic genes expression in a novel model of glucocorticoid-producing adrenocortical cell lines derived from targeted tumorigenesis.

We established the first adrenocortical tumor cell lines with complete zona fasciculata (ZF) cell phenotype from tumors induced in transgenic mice by large T-antigen of simian virus 40 under the control of the aldose reductase-like akr1b7 gene promoter. Adrenocortical tumor cell lines produced high amounts of corticosterone and were responsive to ACTH. All genes that are supportive for glucocorticoid synthesis including cyp21a1 and cyp11b1 were expressed, and most of them were transiently up-regulated by ACTH at transcriptional level: stimulation culminated after 3-6 h and returned to basal levels after 24 h. Taking advantage of these cells, we have examined the effect of ACTH on DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on X-chromosome, gene 1) and SF-1 (steroidogenic factor 1), two transcription factors known to respectively repress and activate adrenocortical steroidogenesis by acting on common target genes. According to their antagonistic activities, DAX-1 mRNA and protein levels were transiently down-regulated by ACTH, whereas those of SF-1 were stimulated, with kinetics paralleling those of steroidogenic genes expression, notably of two known SF-1 target genes, star and akr1b7. This suggests an essential role of SF-1/DAX-1 proteins ratio to achieve proper ACTH control of steroidogenic gene expression in cells derived from ZF. This was confirmed in mice adrenals, where repression of dax-1 gene and concomitant up-regulation of sf-1, star, and akr1b7 genes were observed in response to ACTH stimulation. In conclusion, using both unique differentiated cell lines and in vivo approaches, we provide the first evidence that hormonally induced changes in SF-1/DAX-1 ratio are part of the molecular arsenal of ZF cells to fine tune ACTH responsiveness.

Decreased expression of cyclic adenosine monophosphate-regulated aldose reductase (AKR1B1) is associated with malignancy in human sporadic adrenocortical tumors.

The human aldose reductase, AKR1B1, participates in glucose metabolism and osmoregulation and is supposed to play a protective role against toxic aldehydes derived from lipid peroxidation and steroidogenesis that could affect cell growth/differentiation when accumulated. Adrenal gland is a major site of expression of AKR1B1, and we asked whether changes in its expression could be associated with adrenal disorders. Therefore, we examined AKR1B1 gene expression in human fetal adrenals, adrenocortical cell line, and tumors and compared the results with the expression of steroidogenic genes (StAR and CYP11A) and regulators of adrenal cortex development [steroidogenic factor-1 (SF-1) and dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX1)]. Using specific antibodies, Northern blotting, and enzymatic assays, we present evidences that AKR1B1 detectable in 15-wk-old fetal glands is regulated by cAMP in NCI-H295 cells and thus that AKR1B1 is functionally related to the ACTH-responsive murine akr1b7/mvdp gene rather than to its direct ortholog, the mouse aldose reductase akr1b3 gene. Although low DAX1 expression in aldosterone-producing adenomas (n = 5) was confirmed (P < 0.05), no correlation was found between the expression of all other genes and the tumors endocrine activity. In contrast, relative abundance of AKR1B1 mRNA was decreased in adrenocortical carcinomas (n = 5; mean +/- sem, 0.95 +/- 0.2) when compared with adenomas (n = 12; 9.29 +/- 3.05; P < 0.001). Most (seven of eight) adrenocortical carcinomas (19.0 +/- 5.4) had very low relative AKR1B1 protein levels when compared with benign tumors (cortisol-producing adenomas, n = 5, 63.0 +/- 9.8; nonfunctional adenomas, n = 5, 58.0 +/- 10.4; aldosterone-producing adenomas, n = 4, 65.3 +/- 7.7; P < 0.001), Cushing's hyperplasia (n = 5, 54.6 +/- 5.3; P < 0.01), or normal adrenals (n = 4; 37.1 +/- 5.3; P < 0.001). These properties provide the first evidence that expression of cAMP-regulated AKR1B1 is decreased in adrenocortical cancer. This might take part in adrenal tumorigenesis and could be investigated as a marker of malignancy for the diagnosis of adrenal tumors.