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Oliver Zwermann, Dominik M Schulte, Martin Reincke, and Felix Beuschlein

Objectives: Although several lines of evidence suggest that the overall effects of the ACTH receptor, melanocortin 2 receptor (MC2-R), mediated signal transduction on adrenocortical growth and tumorigenesis are anti-proliferative, activation of MC2-R induces mitogens like jun, fos, and myc and activates the MAPK pathway. In vivo, potential effects of endogenous ACTH on adrenal tumori-genesis can not be separated from effects of other POMC derived peptides.

Methods: Murine adrenocortical tumor cells that lack MC2-R expression (Y6pcDNA) and Y6 cells stablely transfected with MC2-R (Y6MC2-R) were generated. Presence of functional MC2-R was demonstrated by RT-PCR and Western blot using an antibody for phosphorylated CREB. As a syngenic tumor model, LaHeF1/J mice simultaneously received 107 Y6MC2-R and Y6pcDNA subcutaneously, giving rise to MC2-R positive and negative tumors within the same animal. Animals were treated for 3 weeks in groups of 12 according to the following schedule: group A, control animals receiving saline injection; group B, animals receiving 5.7 ng/injection of a slow release formula of ACTH 1-24 administered i.p. three times a week (aiming at a low physiologic dose); and group C, animals receiving 57 ng/injection of ACTH 1-24 (high physiological dose).

Results: Twenty days of ACTH 1-24 treatment did not significantly affect corticosterone levels, endogenous ACTH levels or adrenal and thymus weight compared with saline injection. However, ACTH 1-24 treatment of group B and C mice significantly reduced tumor weight in MC2-R positive tumors in a dose dependent manner (P = 0.03), while no significant difference in tumor mass was observed in MC2-R negative tumors. PCNA and TUNEL staining, together with morphological characterization, demonstrated that these in vivo effects were due to reduced proliferation, while apoptosis and cellular hypertrophy within the tumor remained unchanged.

Conclusion: MC2-R expression is associated with a less aggressive adrenal tumor phenotype and anti-proliferative effects can be amplified through stimulation with physiological doses of ACTH.

Free access

Nicole Reisch, Marc Slawik, Oliver Zwermann, Felix Beuschlein, and Martin Reincke

Objective: Adrenocorticotropic hormone (ACTH) is the primary secretagogue stimulating secretion of adrenal androgens (AA). Yet, genetic and environmental factors are assumed to play a determining role in the regulation of their biosynthesis and thus might explain the high variability of AA levels. Here we investigate the influence of an ACTH receptor promoter polymorphism affecting ACTH receptor gene transcription on ACTH-dependent dehydroepiandrosterone (DHEA) secretion.

Design: We recently reported a polymorphism within the transcription initiation site of the ACTH receptor gene promoter that alters the consensus sequence from CTC to CCC at −2 bp. This results in lower promoter activity in vitro and is associated with impaired cortisol response to ACTH stimulation in vivo. We now studied 14 normal, lean volunteers aged 20–35 years (eight CTC/CTC and six CCC/CCC carriers) in a 6-h ACTH stimulation test.

Methods: After overnight dexamethasone suppression, ACTH1-24 was administered continuously in each subject with hourly increasing doses (120–3840 ng/m2 body surface area/h) within a 6-h period. On a separate day, baseline DHEA samples were collected.

Results: In the 6-h ACTH stimulation test, CTC/CTC carriers showed a significantly higher DHEA response than CCC/CCC carriers (area under the curve: 19 367 ± 2919 vs 11 098 ± 1241 nmol/l per min; P < 0.04, Mann–Whitney U-test). In contrast, baseline DHEA concentrations did not differ between groups.

Conclusion: These data demonstrate that genetic variations within the ACTH receptor promoter result in decreased DHEA secretion. Thus, we might have identified one of the genetic factors responsible for variation in ACTH-dependent DHEA secretion.

Free access

Heinrich Kahles, Elizabeth Ramos-Lopez, Britta Lange, Oliver Zwermann, Martin Reincke, and Klaus Badenhoop

Background: Endocrine autoimmune disorders share genetic susceptibility loci, causing a disordered T-cell activation and homeostasis (HLA class II genes, CTLA-4). Recent studies showed a genetic variation within the PTPN22 gene to be an additional risk factor.

Materials and Methods: Patients with type 1 diabetes (n = 220), Hashimoto’s thyroiditis (n = 94), Addison’s disease (n = 121) and healthy controls (n = 239) were genotyped for the gene polymorphism PTPN22 1858 C/T.

Results: Our study confirms a significant association between allelic variation of the PTPN22 1858 C/T polymorphism and type 1 diabetes mellitus (T1D). 1858T was observed more frequently in T1D patients (19.3% vs 11.3%, P = 0.0009; odds ratio for allele T = 1.88, 95% confidence interval [1.3–2.7]). Furthermore, we found a strong association in female patients with T1D (P = 0.0003), whereas there was no significant difference between male patients with type 1 diabetes and male controls. No significant difference was observed between the distribution of PTPN22 C/T in patients with Hashimoto’s thyroiditis or Addison’s disease and healthy controls.

Conclusion: The PTPN22 polymorphism 1858 C/T may be involved in the pathogenesis of type 1 diabetes mellitus by a sex-specific mechanism that contributes to susceptibility in females.

Free access

Oliver Zwermann, Felix Beuschlein, Enzo Lalli, Albrecht Klink, Paolo Sassone-Corsi, and Martin Reincke

Background: The ACTH receptor (ACTH-R) is a member of the seven transmembrane domain receptor super-family. In non-functional adrenal adenomas and adrenocortical carcinomas, ACTH-R expression is low. However, no inhibitory factor for ACTH-R expression has been defined to date. DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene-1) is a general repressor of steroid production, inhibiting steroidogenic factor-1 (SF-1)-dependent expression of multiple steroidogenic enzymes. The aim of this study was to investigate whether ACTH-R gene transcription is affected by DAX-1 and whether this mechanism is involved in down-regulation of ACTH-R expression in adrenocortical tumors.

Methods: We screened 22 adrenocortical tumors for ACTH-R and DAX-1 mRNA expression by Northern blot. For in vitro analyses we co-transfected mouse Y1 adrenocortical carcinoma cells with the luciferase reporter gene vector pGL3 containing full-length constructs of human (h) or mouse (m) ACTH-R promoter together with a DAX-1 expression plasmid. These experiments were also performed using ACTH-R promoter 5′-deletion constructs and constructs mutated at the SF-1-binding sites.

Results: We found a negative correlation between DAX-1 and ACTH-R mRNA expression (R = −0.47, P < 0.02). Accordingly, in vitro expression of DAX-1 significantly reduced hACTH-R and mACTH-R promoter activity by 89 and 55% respectively. DAX-1 inhibition was also present in the shortest construct of a series of 5′-deletion constructs of the human promoter extending from −64 to +40 bp relative to the transcription start site. Mutation of the SF-1-binding sites within the hACTH-R promoter resulted in reduced or abolished DAX-1 inhibition, arguing for a mechanism that involves SF-1 for DAX-1 inhibition.

Conclusions: These data support the concept that DAX-1 is a major repressor of ACTH-R gene expression in vitro and in vivo.