Abstract
Retinoids play a pivotal role in adrenal development and differentiation. Recent clinical trials revealed therapeutic potential of both all-trans and 9-cis retinoic acid in patients with cortisol excess due to a pituitary ACTH-secreting adenoma and indicated that retinoids might act also on the adrenal. Aim of the present study was to evaluate the effect of 9-cis retinoic acid on adrenals from patients with ACTH-dependent Cushing’s syndrome. Adrenal specimens from six patients with Cushing’s disease were incubated with 10 nM–1 µM 9-cis retinoic acid with and without 10 nM ACTH. Cortisol secretion was measured by immunoassay and expression of genes involved in steroidogenesis as well as retinoic acid action were evaluated by real-time RT-PCR. Incubation with 10–100 nM 9-cis retinoic acid increased spontaneous cortisol secretion and expression of STAR and CYP17A. On the other hand, in wells treated with ACTH, 9-cis retinoic acid markedly diminished ACTH receptor upregulation and no stimulatory effect on cortisol secretion or steroidogenic enzyme synthesis was observed. ACTH itself increased ligand-induced retinoic acid receptor expression, possibly enhancing sensitivity to retinoic acid. Our findings indicate that the effect of 9-cis retinoic acid in presence of ACTH is distinct from unchallenged wells and support the hypothesis of a direct adrenal action in patients with Cushing’s disease.
Highlights
Retinoids play a pivotal role in adrenal development and differentiation
Aim of the present study was to assess the effects of 9-cis retinoic acid on cortisol secretion and on genes involved in steroidogenesis and retinoid action in adrenal glands from patients with Cushing’s disease
We evaluated 17hydroxylase, StAR, hormone-sensitive lipase E (LIPE), the ACTH receptor (MC2R), as well as known retinoid target genes, such as retinoic acid receptors alpha and beta, liver X receptor (LXR), peroxisome proliferator activated receptor delta (PPARD), chicken ovoalbumin upstream promoter transcription factor 1 (COUP-TF1), sterol regulatory element binding transcription factor 1 (SREBP1), mitochondrial dehydrogenases mND1 and mND6, and genes involved in both pathways, e.g., dosage-sensitive sex-reversal adrenal hypoplasia critical region in the X chromosome (DAX-1) and steroidogenic factor 1 (SF-1)
Summary
9-cis Retinoic acid increased cortisol secretion in adrenal primary cultures from patients with ACTH-dependent Cushing’s syndrome. Incubation with ACTH increased cortisol secretion (335.79 ± 134.64% control, p < 0.05 vs unchallenged wells) and induced the expression of CYP17A1, LIPE , MC2R and StAR. 9-cis Retinoic acid blunted ACTH-stimulated MC2R expression by roughly 50% (Fig. 2b) and did not affect the ACTH-induced cortisol response (Fig. 2a) nor ACTH-induced changes in CYP17A and StAR (Fig. 2b). This was replicated at analysis of gene expression normalized to control wells, as the changes in steroidogenic gene expression during retinoic acid-ACTH co-incubated wells were comparable to wells incubated with ACTH alone (Table 1). We analysed cortisol secretion during ACTH/retinoid co-incubation by two approaches: the effect of 9-cis retinoic acid on the cortisol response to ACTH was compared to cortisol leves with 10 nM ACTH and the retinoid (10 nM: 92.2 ± 12.8% ACTH; 100 nM: 115.3 ± 16.8% ACTH; 1 μM: 123.6 ± 13.3% ACTH, all comparisons N.S., Fig. 2a) and to cortisol levels with each 9-cis retinoic acid concentration without ACTH (10 nM: 112.9 ± 13.7% RA; 100 nM: 144.6 ± 28.1% RA; 1 μM: 158.6 ± 32.5% RA, all comparisons N.S.); of note, the expected increase in cortisol levels with ACTH is over 300% of unchallenged wells (see above)
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