Endocrine Abstracts

Background: Triple A syndrome is a rare, autosomal recessive cause of adrenal insufficiency. Additional features include alacrima, achalasia of the oesophageal cardia, and neurodegenerative disease in 60%. The AAAS gene product is the nuclear pore complex protein ALADIN of unknown function. AAAS patient dermal fibroblasts have been described as hypersensitive to oxidative stress^1,2,3.

Objective: To establish a better disease model by knockdown of AAAS-gene expression in H295R human adrenocortical tumour cells.

Methods: AAAS-knockdown was achieved using synthetic shRNA lentiviral transduction. H₂O₂ was used as an inducer of oxidative stress.

Results: Cell viability, measured by MTS assay, was significantly reduced in AAAS-knockdown cells compared with controls at baseline (n=4, P<0.01) and following H₂O₂ treatment (n=3, P<0.05). Application of the anti-oxidant N-acetylcysteine resulted in a significant increase in cell viability compared with controls (n=4, P<0.01).

A baseline increase in oxidative stress was suggested by a significant decrease in the ratio of reduced to oxidised glutathione in AAAS-knockdown cells compared with controls (n=3, P<0.05). Cell cycle arrest is observed in ALADIN deficient cells (n=4, P<0.05). Following H₂O₂ treatment there is an increase in apoptosis, assessed by cleavage of poly-ADP ribose polymerase, of AAAS-knockdown cells compared with controls (n=3, P<0.05).

We observe a significant reduction in the protein expression of the steroidogenic acute regulatory protein (StAR) in AAAS-knockdown cells compared with controls at baseline (n=4, P<0.01) and following H₂O₂ treatment (n=4, P<0.001). An impact on function is seen with a significant decrease in cortisol production in ALADIN-deficient cells compared with controls (n=6, P<0.0001).

Conclusion: Steroidogenic activity in the adrenals induces a highly oxidative environment. A model for H₂O₂ mediated control of steroidogenesis has recently been proposed⁴. Our data suggests that an imbalance of redox homeostasis in ALADIN-deficient adrenal cells results in a reduction in StAR protein expression with subsequent impact on steroidogenesis.

References:

1. Hirano M et al. PNAS 103 2298–2303, 2006.

2. Kiriyama T et al. Biochemical and Biophysical Research Communications 374 631–634, 2008.

3. Kind B et al. Journal of Molecular Medicine 88 1233–1242, 2010.

4. Kil et al. Molecular Cell 46 584–594, 2012.