Role of ALADIN in human adrenocortical cells for oxidative stress response and steroidogenesis.

Ramona Jühlen,Katrin Koehler,Angela E Taylor,Wiebke Arlt,Angela Huebner,Barbara Kind,Jan Idkowiak,Yoshiaki Tsuji

doi:10.1371/journal.pone.0124582

Ramona Jühlen, Katrin Koehler + Show 6 more

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https://doi.org/10.1371/journal.pone.0124582

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Abstract

Triple A syndrome is caused by mutations in AAAS encoding the protein ALADIN. We investigated the role of ALADIN in the human adrenocortical cell line NCI-H295R1 by either over-expression or down-regulation of ALADIN. Our findings indicate that AAAS knock-down induces a down-regulation of genes coding for type II microsomal cytochrome P450 hydroxylases CYP17A1 and CYP21A2 and their electron donor enzyme cytochrome P450 oxidoreductase, thereby decreasing biosynthesis of precursor metabolites required for glucocorticoid and androgen production. Furthermore we demonstrate that ALADIN deficiency leads to increased susceptibility to oxidative stress and alteration in redox homeostasis after paraquat treatment. Finally, we show significantly impaired nuclear import of DNA ligase 1, aprataxin and ferritin heavy chain 1 in ALADIN knock-down cells. We conclude that down-regulating ALADIN results in decreased oxidative stress response leading to alteration in steroidogenesis, highlighting our knock-down cell model as an important in-vitro tool for studying the adrenal phenotype in triple A syndrome.

Highlights

Triple A syndrome (MIMÃ231550) is an autosomal-recessive disease manifesting with the triad of ACTH-resistant adrenal insufficiency, achalasia of the cardia and alacrima (Triple A) in combination with progressive neurological impairment [1]
We show that the ALADIN-dependent impairment of the glucocorticoid and androgenic pathways is accompanied by a downregulation of the gene coding for P450 oxidoreductase (POR), an enzyme managing electron transfer from NADPH to CYP17A1 and CYP21A2 microsomal P450 hydroxylases (Fig 2)
The nuclear import of aprataxin, DNA ligase 1 and ferritin heavy chain 1 is disturbed by ALADIN. Using both the human adrenal NCI-H295R1-TR AAAS knock-down and over-expression models we investigated the potential impairment of the nuclear import of aprataxin, DNA ligase 1 and ferritin heavy chain 1 employing YFP-specific vectors transiently transfected into the cell lines

Summary

Introduction

Triple A syndrome (MIMÃ231550) is an autosomal-recessive disease manifesting with the triad of ACTH-resistant adrenal insufficiency, achalasia of the cardia and alacrima (Triple A) in combination with progressive neurological impairment [1]. The disease is caused by mutations in the AAAS (achalasia—adrenocortical insufficiency—alacrima syndrome) gene, which encodes the protein ALADIN (alacrima-achalasia-adrenal insufficiency neurologic disorder) [2,3]. AAAS is ubiquitously expressed, but shows an enhanced expression in the adrenal gland, gastrointestinal tract and pituitary gland [3]. In 2002, ALADIN was identified as a component of the nuclear pore complex (NPC) [4]. Human NPC is a large protein complex composed of approximately 30 different proteins, known as nucleoporins, which mediate the transport of macromolecules between the cytoplasm and the nucleoplasm [4].

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