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

Abstract

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.

Inactivation of the Carney Complex Gene 1 (Protein Kinase A Regulatory Subunit 1A) Inhibits SMAD3 Expression and TGFβ-Stimulated Apoptosis in Adrenocortical Cells

“…Human embryonic kidney cells HEK293, human adrenocortical carcinoma cells H295R, and murine adrenocortical cells ATC1 were cultured as previously described (26,27). HEK293 cells are used for efficient transfection and to control the siR1A efficiency.…”

60 YEARS OF NEUROENDOCRINOLOGY: Glucocorticoid dynamics: insights from mathematical, experimental and clinical studies

“…Following ACTH stimulation and activation of CREB, StAR expression is upregulated by the CREB co-activator SF-1 and inhibited by the co-repressor DAX-1. Interestingly, ACTH can induce the transcription of SF-1 (Ragazzon et al 2006) and Nur77 (Davis & Lau 1994), while inhibiting the transcription of DAX-1 (Ragazzon et al 2006). Gummow et al (2006) have shown that dexamethasone can induce DAX-1 transcription by a mechanism that involves the formation of an SF-1/GR complex, thus leading to inhibition of StAR transcription and ultimately to inhibition of CORT synthesis.…”

Transcriptional Control of Adrenal Steroidogenesis

“…RT-PCR-Total RNA was isolated from ATC1 cells and rat adrenal primary culture with TRIzol (Invitrogen) according to the manufacturer's instructions and reverse transcribed by Moloney murine leukemia virus reverse transcriptase (Promega) and amplified using TaqDNA polymerase (Promega) as described previously (23). Nucleotide sequences of the specific primers used were: Prlr forward, 5Ј-GAGAAAAACA-CCTATGAATGTC-3Ј and reverse 5Ј-AGCAGTTCTTCAG-ACTTCC-3Ј; ␤-actin forward, 5Ј-CGTGGGCCGCCCTAGG-CACCA-3Ј and reverse, 5Ј-TTGGCCTTAGGGTTCAGGG-GGG-3Ј.…”

“…Western Blot Analysis-Western blotting was performed as described previously (23 (29), and ␤-actin (Sigma Aldrich, 1/3000 e ) or monoclonal phosphotyrosine Py (Upstate, 1/1000 e ). Primary antibodies were detected with a secondary antibody conjugated to peroxidase (PARIS).…”