PKA regulatory subunit 1A inactivating mutation induces serotonin signaling in primary pigmented nodular adrenal disease
Zakariae Bram, … , Jérôme Bertherat, Hervé Lefebvre
Zakariae Bram, … , Jérôme Bertherat, Hervé Lefebvre
Published September 22, 2016
Citation Information: JCI Insight. 2016;1(15):e87958. https://doi.org/10.1172/jci.insight.87958.
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Research Article Endocrinology

PKA regulatory subunit 1A inactivating mutation induces serotonin signaling in primary pigmented nodular adrenal disease

Abstract

Primary pigmented nodular adrenocortical disease (PPNAD) is a rare cause of ACTH-independent hypercortisolism. The disease is primarily caused by germline mutations of the protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene, which induces constitutive activation of PKA in adrenocortical cells. Hypercortisolism is thought to result from PKA hyperactivity, but PPNAD tissues exhibit features of neuroendocrine differentiation, which may lead to stimulation of steroidogenesis by abnormally expressed neurotransmitters. We hypothesized that serotonin (5-HT) may participate in the pathophysiology of PPNAD-associated hypercortisolism. We show that PPNAD tissues overexpress the 5-HT synthesizing enzyme tryptophan hydroxylase type 2 (Tph2) and the serotonin receptors types 4, 6, and 7, leading to formation of an illicit stimulatory serotonergic loop whose pharmacological inhibition in vitro decreases cortisol production. In the human PPNAD cell line CAR47, the PKA inhibitor H-89 decreases 5-HT4 and 5-HT7 receptor expression. Moreover, in the human adrenocortical cell line H295R, inhibition of PRKAR1A expression increases the expression of Tph2 and 5-HT4/6/7 receptors, an effect that is blocked by H-89. These findings show that the serotonergic process observed in PPNAD tissues results from PKA activation by PRKAR1A mutations. They also suggest that Tph inhibitors may represent efficient treatments of hypercortisolism in patients with PPNAD.

Authors

Zakariae Bram, Estelle Louiset, Bruno Ragazzon, Sylvie Renouf, Julien Wils, Céline Duparc, Isabelle Boutelet, Marthe Rizk-Rabin, Rossella Libé, Jacques Young, Dennis Carson, Marie-Christine Vantyghem, Eva Szarek, Antoine Martinez, Constantine A. Stratakis, Jérôme Bertherat, Hervé Lefebvre

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Figure 5

Regulation of the expression of Tph2 and 5-HT4/6/7 receptors in the shPRKAR1A H295R cell line.

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Regulation of the expression of Tph2 and 5-HT4/6/7 receptors in the shPR...
(A) Expression levels of TPH2 and HTR4/6/7 mRNAs normalized to PPIA in transfected H295R cells with either the empty vector (EV) or the doxycyclin-sensitive shPRKAR1A–expressing vector in the absence or presence of doxycyclin (Dox). (B) Tph2 and 5-HT4/6/7 receptor immunoreactivity in shPRKAR1A H295R cells cultured in the absence (Dox-) or presence of doxycyclin (Dox+). Scale bars: 20 μm. (C) Western blot analysis of Tph2 and 5-HT4/6/7 receptors in shPRKAR1A H295R cells cultured in the absence (Dox-) or presence of doxycyclin (Dox+). Data illustrates results of 3 cell batches (1, 2, 3). (D) Expression levels of TPH2 and HTR4/6/7 mRNAs in basal conditions and after treatment of shPRKAR1A H295R cells with forskolin (Fk; 10–5 M), forskolin and H-89 (Fk+H-89; 10–5 M), doxycyclin (0.5 μg/ml), and doxycyclin and H‑89 (Dox+H-89) for 24h. (E) Cortisol secretion by shPRKAR1A H295R cells incubated for 24h with 5-HT (10–8 M) and H-89 (10–5 M) in the absence or presence of doxycyclin. BL, basal level. Data of at least 4 determinations are presented as box plot. (F) Schematic representation of the abnormal serotonergic regulatory loop controlling cortisol secretion in patients with PRKAR1A mutation. Activation of PKA due to PRKAR1A inactive mutation stimulates abnormal expression of the limiting enzyme for 5-HT synthesis tryptophan hydroxylase type 2 (Tph2), as well as functional 5-HT receptors — i.e., the 5-HT4, 5-HT6, and 5-HT7 receptors — in adrenocortical cells. Release of locally produced 5-HT stimulates cortisol secretion through autocrine and paracrine mechanisms involving the 3 receptor types positively coupled to the cAMP/PKA pathway. Data were analyzed by using Mann-Whitney U test and Bonferroni’s test after one-way ANOVA. *P < 0.05; **P < 0.01; or ***P < 0.001.