The Inhibition of the Sonic Hedgehog Pathway Impairs Adrenal Steroidogenesis

Presentation Number: MON 393
Date of Presentation: April 3rd, 2017

Maíra F. Ribeiro*1, Ana Carolina Bueno2, Debora Cristiane Gomes3, Margaret de Castro2 and Sonir R. Antonini2
1Ribeirao Preto Medical School, University of Sao Paulo, 2Ribeirao Preto Medical School - University of Sao Paulo, Ribeirao Preto, Brazil, 3School of Medicine of Federal University of Uberlandia, Uberlandia MG, Brazil

Abstract

Background: the Sonic Hedgehog (SHH) pathway plays a key role in the adrenal cortex development and maintenance. The potential role of the SHH pathway in the regulation of the adrenal steroidogenesis in humans has not been evaluated yet. There is a differential expression of SHH pathway during fetal and postnatal life and this pathway seems to influence the steroidogenesis pattern in adrenocortical carcinoma cells.

Objectives: to investigate the role of the SHH pathway in adrenal steroidogenesis.

Methods: In NCI-H295 human adrenocortical cells, the SHH second receptor SMOOTHENED was inhibited by knocking down SMO gene (siRNA) or by Cyclopamine (Cyclo: 5-30μM) while adrenal steroidogenesis was stimulated by ACTH (10nM) and Forskolin (FSK; 10μM) up to 96h. Cell viability (MTS), gene expression (qPCR) of SHH pathway components (SHH, GLI1, GLI2, GLI3, SMO e PTCH), steroidogenic enzymes genes (CYP11B1, CYP11B2, CYP17, StAR), steroidogenic factor 1 (NR5A1), 17α-hydroxylase (immunofluorescence), as well as steroid secretion (cortisol, DHEA-S and testosterone secretion - RIA) were evaluated.

Results: Cyclo reduced the mRNA expression of SHH (30μM, 48h) and GLI1 (5 and 20μM, 48h), and increased the expression of SMO (5, 20 and 30μM, 96h). The association of Cyclo 20μM and ACTH reduced by 43% the increase of GLI1 expression induced by ACTH alone (48h; p<0.01). After 48h, Cyclo 20μM in association with ACTH reduced the expression of CYP11B1 (67%; p<0.05), CYP17A1 (60%; p<0.01) and NR5A1 (52%; p<0.05) as compared to the effects induced by ACTH alone. Similarly, Cyclo 20μM in association with FSK reduced the expression of CYP11B1 (48%; p<0.05), CYP17A1 (33%; p<0.05), NR5A1 (32%; p<0.05) and impaired 17α-hydroxylase fluorescence, as compared to increase induced by FSK alone. Conversely, SMO knockdown (-57%) increased the mRNA expression of GLI1 (p=0.006), CYP17A1 (p=0.01), CYP11B1 (p=0.001) and CYP11B2 (p=0.01). In line, Cyclo also impaired steroid secretion. Cyclo alone decreased DHEAS (50%) and Testosterone (20%). Moreover, the association of Cyclo partially prevented the increase of cortisol secretion induced by ACTH (25% and 95%, 12h and 48h, respectively) and by FSK (93% and 27%, 12h and 48h, respectively). SMO knockdown did not reduce cell viability until 96h whereas Cyclo 20μM slightly reduced cell viability only after 48h (14.5%). Thus, the effects observed on gene expression and hormonal secretion at 12h and 24h, and likely at 48h, cannot be ascribed to cell death.

Conclusion: in vitro, the inhibition of the SHH pathway decreased the expression of genes encoding steroidogenic enzymes and the secretion of adrenal steroids stimulated by ACTH and the FSK in NCI-H295 cells. These novel results indicate that, in addition to its role in the development and maintenance of the adrenal cortex cells, the SHH pathway may also play a role in the regulation of adrenal steroidogenesis.

 

Nothing to Disclose: MFR, ACB, DCG, MDC, SRA