ESPE Abstracts (2016) 86 FC1.1

DNA Methylation of HSD3B2, NUR77 and RAR[beta] Promoter Genes is Not Involved in functional Differentiation of Human Androgen-Producing Adrenocortical Cells

Maria Cecilia Alonso Burgos, Javier Goni, Nora Saraco, Natalia Perez Garrido, Marco A. Rivarola, Alicia Belgorosky & Maria Sonia Baquedano

Garrahan National Pediatric Hospital, CABA, Argentina

Background: RARβ cooperates with Nur77 to in-vitro regulate HSD3B2 transcription. NUR77 expression parallels HSD3B2 expression with a much lower level in androgen-producing adrenocortical tissues (childhood virilizing adrenocortical tumours (VAT), fetal zone (FeZ) and zona reticularis, ZR). RARβ is down-regulated in starved, hyperandrogenic H295R cells. However, the mechanisms regulating this expression pattern and the relevance of RARβ to human adrenal physiology are unknown.

Objective: To evaluate developmental changes in DNA methylation of HSD3B2, NUR77 and RARβ genes in VAT and normal human adrenal tissues (HAT).

Method: VAT (n=11, age 0.75–4.5 year) and HAT collected from 3postnatal groups: Gr1:<3mo, n=9, FeZ involution; Gr2:3mo to 6 year, n=9, pre-adrenarche; and Gr3:>6 to 20 year, n=8, post-adrenarche, were evaluated. Total DNA and RNA from whole tissue and from laser capture microdissected zona fasciculata (ZF) and ZR in Gr3 were isolated. Total RARβ (RARβT) and RARβ2 expression were studied using qRT-PCR. Promoter methylation pattern were examined by bisulfite sequencing.

Results: RARβT and RARβ2 mRNAs were similar among HAT from the 3 groups. RARβT mRNA was lower in VAT compared to age-matched group HAT (P<0.05). RARβ2 tended to be lower in VAT compared to HAT without statistical significance. RARβT and RARβ2 mRNA expression showed no significant difference between micro-dissected ZR and ZF. HSD3B2 NUR77 mRNAs were much lower in ZR cells (P<0.05). In silico screened showed that NUR77 promoter was embedded within a CpG island but it remained completely unmethylated in HAT from the 3Grs and in VAT. No differences in adrenal zone-specific NUR77 methylation were observed.

Conclusion: RARβ was not associated with ZR-specific down-regulation of HSD3B2 in postnatal human adrenocotical zonation. DNA methylation would not be involved in zone-specific and VAT downregulation of adrenal NUR77. Lack of CpG Island in HSD3B2 suggested that the known downregulation of HSD3B2 gene expression in human ZR would not be directly mediated by DNA methylation.

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