Benutzerspezifische Werkzeuge

Progress Report 2013

KFO252_long

Progress Report 2013: Project 6 - Mechanisms of adrenal dysregulation in triple A syndrome


Achievements in the first funding period:

Aim I: Expression and proteomic analysis after overexpression and downregulation of AAAS in the human adrenal cell line NCI-H295R: impact for adrenocortical cell function and steroidogenesis.

We investigated the role of ALADIN on cell function following stable inducible over-expression and sh-RNA knock-down of AAAS in the human adrenocortical carcinoma cell line NCI-H295R. Real-time quantitative PCR was used to monitor expression of key enzymes involved in adrenal steroidogenesis. Our findings indicate a down-regulation of these enzymes after AAAS knock-down. To assess functional consequences, steroids were quantified by a Waters® Xevo tandem mass-spectrometer with ACQUITY ultra performance liquid chromatography. Our investigations revealed that ALADIN knock-down cells show an accumulation of precursor metabolites which feed the androgen pathway. Cells over-expressing ALADIN show an increased production of glucocorticoids and mineralocorticoids. These experiments were conducted with and without oxidative stress. The knock-down leads to decreased cell viability whereas over-expressing cells show increased viability. Using both the AAAS knock-down and over-expression model we currently investigate cell viability under oxidative stress and the impairment of the nuclear import of proteins protecting the nucleus against oxidative stress using YFP-tagged vectors.

Aim II: Investigation of the role of ALADIN for the interplay between adrenocortical and medullary function.

We investigated the role of ALADIN on cellular mechanisms in primary bovine cells. To study the interaction between the adrenocortex and the medulla we established a co-culture system in which the Aaas gene was transient downregulated by different siRNAs. In this system we used the following combinations of knockdown and non-silencing control siRNA treated co-cultures: 1) adrenocortical cells AaasKD and chromaffin cells AaasKD 2) adrenocortical cells AaasKD and chromaffin cells AaasWT 3) adrenocortical cells AaasWT and chromaffin cells AaasKD 4) adrenocortical cells AaasWT and chromaffin cells AaasWT. The mRNAs of both cell types were prepared and the expression of genes coding for steroidogenic enzymes (Cyp11B1, Cyp11A1, Cyp17A1, Cyp21A2) in cortical cells and the expression of phenylethanolamine N-methyltransferase (PNMT) and tyrosine hydroxylase (TH) in chromaffin cells were measured by qPCR using Taqman. Our findings indicate that there is an ALADIN-dependent crosstalk between the adrenocortex and the medulla. We observed an increase in PNMT and TH expression due to the Aaas knockdown in both cell types but not in combination 2) or 3). We currently measure the steroids produced by the co-cultures and investigate the behaviour in downregulated co-cultures under enhanced oxidative stress conditions induced by H2O2 and paraquat.

Furthermore we investigated the influence of Aaas in bovine adrenal single-cultures and showed that an Aaas knockdown in chromaffin and adrenocortical cells leads to an increased proliferation time. We also could show that this downregulated cells.

Aim III: Investigation of the role of ALADIN for adrenocortical function under oxidative stress.

We established the oxidative stress conditioned Aaas-/-/Sod2+/- mice and currently investigate the steroidogenic enzyme expression in 30 weeks old animals. At the same time we assess the adrenal hormonal secretory function (steroides, catecholamines) and the histology of the adrenals. We will start soon with the exogenous application of oxidative stress in mice.

Collaboration within the KFO:

  • Dr. Monika Ehrhart-Bornstein (bovine cell culture model)
  • Prof. Eisenhofer (measurement of steroids and catecholamines)
  • Prof. Morawietz (oxidative stress model)