Dynamics of ACTH-Mediated Regulation of Gene Transcription in ATC1 and ATC7 Adrenal Zona Fasciculata Cell Lines

Abstract

We tested the hypothesis that mouse ATC1 and ATC7 cells, the first adrenocortical cell lines to exhibit a complete zona fasciculata (ZF) cell phenotype, respond to dynamic ACTH stimulation in a similar manner as the adrenal gland in vivo . Exploiting our previous in vivo observations that gene transcription within the steroidogenic pathway is dynamically regulated in response to a pulse of ACTH, we exposed ATC1 and ATC7 cells to various patterns of ACTH, including pulsatile and constant, and measured the transcriptional activation of this pathway. We show that pulses of ACTH administered to ATC7 cells can reliably stimulate a pulsatile pattern of transcriptional activity that is comparable to that observed in adrenal ZF cells in vivo . Hourly pulses of ACTH stimulate dynamic increases in CREB phosphorylation (pCREB) and transcription of genes involved in critical steps of steroidogenesis including signal transduction ( e.g., MRAP), cholesterol delivery ( e.g., StAR), and steroid biosynthesis ( e.g., CYP11A1), as well as those relating to transcriptional regulation of steroidogenic factors ( e.g., SF-1 and Nur-77). In contrast, constant ACTH stimulation results in a prolonged and exaggerated pCREB and steroidogenic gene transcriptional response. We also show that when a large dose of ACTH (100 nM) is applied after these treatment regimens, a significant increase in steroidogenic transcriptional responsiveness is achieved only in cells that have been exposed to pulsatile, rather than constant, ACTH. Our data support our in vivo observations that pulsatile ACTH is important for the optimal transcriptional responsiveness of the adrenal. Importantly, our data suggest that ATC7 cells respond to dynamic ACTH stimulation.

Co-culture of monocytes and zona fasciculata adrenal cells: An in vitro model to study the immune-adrenal cross-talk

“…Interestingly, the effect of ACTH on IL-6 mRNA was not observed in ATC7 cells co-cultured with THP1 cells in the absence of LPS, suggesting that anti-inflammatory cytokines secreted by THP1 cells in basal conditions may protect the adrenal cells from a non-inflammatory immune activation mediated by ACTH. We have recently shown that ACTH treatment dynamically increases the expression of steroidogenic genes in ATC7 cells ( Hazell et al, 2019 ). Our present data confirmed our previous findings, but also show that the dynamic effect of ACTH is disrupted in ATC7 cells co-cultured with THP1 cells, with a smaller effect on StAR mRNA, which was further decreased by pre-treatment with LPS, and complete suppression of DAX-1 mRNA.…”

“…Murine adrenocortical tumour ATC7 cells (a kind gift from Dr Pierre Val, Université Clermont Auvergne, Clermont-Ferrand, France), were cultured as previously described ( Hazell et al, 2019 ). Human monocytic THP1 cells were purchased from Sigma (Sigma, Gillingham, UK).…”

“…For protein quantification cells were lysed in SDS lysis buffer (2% SDS; 50 mM Tris pH 6.8; 10% glycerol) and Western immunoblotting performed as described in ( Hazell et al, 2019 ). In brief, all membranes were blocked with 1% BSA in Tris-buffered saline/0.05% Tween 20 (TBS/T) and probed with primary rabbit antibodies directed to StAR (1:1000; Santa Cruz Biotechnology, USA), pCREB (1:1000; Cell Signalling Technology, Inc., USA), followed by horseradish peroxidase-conjugated donkey α-rabbit secondary antibody (1:5000; Santa Cruz Biotechnology).…”

“…In brief, all membranes were blocked with 1% BSA in Tris-buffered saline/0.05% Tween 20 (TBS/T) and probed with primary rabbit antibodies directed to StAR (1:1000; Santa Cruz Biotechnology, USA), pCREB (1:1000; Cell Signalling Technology, Inc., USA), followed by horseradish peroxidase-conjugated donkey α-rabbit secondary antibody (1:5000; Santa Cruz Biotechnology). Vinculin (Goat α-vinculin primary (1:5000) followed by a Donkey α-Goat secondary (1:5000) (both Santa Cruz Biotechnology) was used as a loading control as previously shown ( Hazell et al, 2019 ). Protein bands were visualized with Luminata Forte Western HRP substrate (Millipore Corporation, Billerica, MA, USA) using a G BOX (Syngene, Cambridge, UK) and densitometry was determined using Image J (developed at the National Institutes of Health and freely available at http://rsb.info.nih.gov ).…”

“…The current study describes a novel co-culture model of adrenocortical tumour cell lines murine ATC7 cells, with complete zona fasciculata (ZF) cell phenotype ( Ragazzon et al, 2006 ; Hazell et al, 2019 ) and macrophages derived from THP1 monocytes (a human monocytic cell line derived from an acute monocytic leukaemia patient) ( Tsuchiya et al, 1980 ). Using this model, we explored the effects of an immunological stimulus (lipopolysaccharide, LPS) on the expression of the pro-inflammatory cytokine IL-6 as well as the expression of genes involved in steroidogenesis in ATC7 cells, both in basal conditions and under ACTH stimulation.…”