Development of the adrenal cortex is dependent upon the specific regulation of cellular proliferation and differentiation. Although both intra-adrenal transcription factors and extra-adrenal peptide ...hormones have been demonstrated as indispensable for this regulatory process, the resulting distribution of proliferating and steroidogenic cell populations in the developing adrenal cortex has not been defined. Thus, we assessed expression and colocalization of a differentiation marker (3-β-hydroxysteroid dehydrogenase, 3β-HSD) and a proliferation marker (5-bromo-2′-deoxyuridine (BrdU) incorporation) at the various time points (embryonic day (E) 9.5 until 2 weeks post partum) during mouse adrenal development. In addition, adrenocorticotropin-hormone (ACTH) receptor (melanocortin-2-receptor (MC2-R)) expression was examined by in situ hybridization (ISH) and co-localized with 3β-HSD. As demonstrated by immunohistochemistry (IHC) the number of BrdU positive cells within the adrenal cortex decreased during development, whereas the number of 3β-HSD positive cells increased. While BrdU incorporation was evident in a scattered pattern throughout the adrenal gland up to day E13.5, at later time points BrdU positive cells assembled in a discrete subcapsular compartment possibly representing the stem cell layer of the adult adrenal cortex. Interestingly, only a small percentage of proliferating cells expressed 3β-HSD, while the majority of 3β-HSD positive cells co-stained for MC2-R expression by means of ISH. As demonstrated by semiquantitative RT-PCR, MC2-R mRNA levels increased from E11.5 until birth, while the highest adrenal secretory protease (AsP) expression was detected at E13.5 with a decrease thereafter. Taken together, these findings are in accordance with the concept of distinct cell populations present during adrenocortical development with a highly proliferative phenotype or differentiated steroidogenic properties.
A variety of transcription factors including Wilms tumor gene (Wt-1), steroidogenic factor 1 (Sf-1), dosage-sensitive sex reversal, adrenal hypoplasia congenita on the X-chromosome, Gene 1 (Dax-1), ...and pre-B-cell transcription factor 1 (Pbx1) have been defined as necessary for regular adrenocortical development. However, the role of Pbx1 for adrenal growth and function in the adult organism together with the molecular relationship between Pbx1 and these other transcription factors have not been characterized. We demonstrate that Pbx haploinsufficiency (Pbx1+/−) in mice is accompanied by a significant lower adrenal weight in adult animals compared with wild-type controls. Accordingly, baseline proliferating cell nuclear antigen levels are lower in Pbx1+/− mice, and unilateral adrenalectomy results in impaired contralateral compensatory adrenal growth, indicating a lower proliferative potential in the context of Pbx1 haploinsufficiency. In accordance with the key role of IGFs in adrenocortical proliferation and development, real-time RT-PCR demonstrates significant lower expression levels of the IGF-I receptor, and up-regulation of IGF binding protein-2. Functionally, Pbx1+/− mice display a blunted corticosterone response after ACTH stimulation coincident with lower adrenal expression of the ACTH receptor (melanocortin 2 receptor, Mc2-r). Mechanistically, in vitro studies reveal that Pbx1 and Sf-1 synergistically stimulates Mc2-r promoter activity. Moreover, Sf-1 directly activates the Pbx1 promoter activity in vitro and in vivo. Taken together, these studies provide evidence for a role of Pbx1 in the maintenance of a functional adrenal cortex mediated by synergistic actions of Pbx1 and Sf-1 in the transcriptional regulation of the critical effector of adrenocortical differentiation, the ACTH receptor.
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.
Pro-opiomelanocortin (POMC) is a polypeptide precursor that undergoes extensive processing to yield a range of peptides with biologically diverse functions. POMC-derived ACTH is vital for normal ...adrenal function and the melanocortin α-MSH plays a key role in appetite control and energy homeostasis. However, the roles of peptide fragments derived from the highly conserved N-terminal region of POMC are less well characterized. We have used mice with a null mutation in the Pomc gene (Pomc−/−) to determine the in vivo effects of synthetic N-terminal 1–28 POMC, which has been shown previously to possess adrenal mitogenic activity. 1–28 POMC (20 μg) given s.c. for 10 days had no effect on the adrenal cortex of Pomc−/− mice, with resultant cortical morphology and plasma corticosterone levels being indistinguishable from sham treatment. Concurrent administration of 1–28 POMC and 1–24 ACTH (30 μg/day) resulted in changes identical to 1–24 ACTH treatment alone, which consisted of upregulation of steroidogenic enzymes, elevation of corticosterone levels, hypertrophy of the zona fasciculate, and regression of the X-zone. However, treatment of corticosterone-depleted Pomc−/− mice with 1–28 POMC reduced cumulative food intake and total body weight. These anorexigenic effects were ameliorated when the peptide was administered to Pomc−/− mice with circulating corticosterone restored either to a low physiological level by corticosterone-supplemented drinking water (CORT) or to a supraphysiological level by concurrent 1–24 ACTH administration. Further, i.c.v. administration of 1–28 POMC to CORT-treated Pomc−/− mice had no effect on food intake or body weight. In wild-type mice, the effects of 1–28 POMC upon food intake and body weight were identical to sham treatment, but 1–28 POMC was able to ameliorate the hyperphagia induced by concurrent 1–24 ACTH treatment. In a mouse model which lacks all endogenous POMC peptides, s.c. treatment with synthetic 1–28 POMC alone can reduce food intake and body weight, but has no impact upon adrenal growth or steroidogenesis.
Background The Nod-like receptor NACHT, LRR, and PYD domain–containing protein 3 (NLRP3) and Bruton tyrosine kinase (BTK) are protagonists in innate and adaptive immunity, respectively. NLRP3 senses ...exogenous and endogenous insults, leading to inflammasome activation, which occurs spontaneously in patients with Muckle-Wells syndrome; BTK mutations cause the genetic immunodeficiency X-linked agammaglobulinemia (XLA). However, to date, few proteins that regulate NLRP3 inflammasome activity in human primary immune cells have been identified, and clinically promising pharmacologic targeting strategies remain elusive. Objective We sought to identify novel regulators of the NLRP3 inflammasome in human cells with a view to exploring interference with inflammasome activity at the level of such regulators. Methods After proteome-wide phosphoproteomics, the identified novel regulator BTK was studied in human and murine cells by using pharmacologic and genetic BTK ablation. Results Here we show that BTK is a critical regulator of NLRP3 inflammasome activation: pharmacologic (using the US Food and Drug Administration–approved inhibitor ibrutinib) and genetic (in patients with XLA and Btk knockout mice) BTK ablation in primary immune cells led to reduced IL-1β processing and secretion in response to nigericin and the Staphylococcus aureus toxin leukocidin AB (LukAB). BTK affected apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and caspase-1 cleavage and interacted with NLRP3 and ASC. S aureus infection control in vivo and IL-1β release from cells of patients with Muckle-Wells syndrome were impaired by ibrutinib. Notably, IL-1β processing and release from immune cells isolated from patients with cancer receiving ibrutinib therapy were reduced. Conclusion Our data suggest that XLA might result in part from genetic inflammasome deficiency and that NLRP3 inflammasome–linked inflammation could potentially be targeted pharmacologically through BTK.
Neuroendocrine neoplasias (NEN) comprise a rare tumor entity with heterogeneous biology, prognosis and therapeutic options. Together with the recent publication of the first German guidelines on ...diagnostics and therapy of NEN, an analysis of the German NET-registry cohort of the German Society of Endocrinology (DGE) was performed. For this purpose, 2686 cases were extracted and their patient characteristics (e. g., age, sex, histopathological characterization, grading and staging) were displayed and outcomes were calculated. Additionally, the systemic treatment reality in the two largest subgroups, small intestinal and pancreatic NEN, was analyzed within metastatic patients.Distribution of primary tumor localization, histopathological classification, disease stage and overall survival was comparable with results from international registry studies. In concordance with current guidelines, somatostatin analogues (SSA) and peptide-receptor-radionuclide-therapy (PRRT) were the most common therapeutic modalities in small intestinal NEN. In pancreatic NEN, chemotherapy was used in first line as often as SSA. In second line, chemotherapy was used as often as PRRT. WHO-classification of 2010 and TNM staging proved to be of prognostic relevance.The current analysis of the German NET-registry characterizes a multicentric, interdisciplinary cohort of NEN patients throughout Germany and it describes the applied systemic treatment modalities and overall outcome as well as the prognostic value of the WHO classification of 2010 and TNM staging.
Inflammation and metabolism have been shown to be evolutionary linked and increasing evidence exists that pro-inflammatory factors are involved in the pathogenesis of obesity and type 2 diabetes. ...Until now, most data suggest that within adipose tissue these factors are secreted by cells of the innate immune system, e. g. macrophages. In the present study we demonstrate that B lymphocyte stimulator (BLyS) is increased in human obesity. In contrast to several pro-inflammatory factors, we found the source of BLyS in human adipose tissue to be the adipocytes rather than immune cells. In grade 3 obese human subjects, expression of BLyS in vivo in adipose tissue is significantly increased (p<0.001). Furthermore, BLyS serum levels are elevated in grade 3 human obesity (862.5+222.0 pg/ml vs. 543.7+60.7 pg/ml in lean controls, p<0.001) and are positively correlated to the BMI (r = 0.43, p<0.0002). In the present study, bariatric surgery significantly altered serum BLyS concentrations. In contrast, weight loss due to a very-low-calorie-formula-diet (800 kcal/d) had no such effect. To examine metabolic activity of BLyS, in a translational research approach, insulin sensitivity was measured in human subjects in vivo before and after treatment with the human recombinant anti-BLyS antibody belimumab. Since BLyS is known to promote B-cell proliferation and immunoglobulin secretion, the present data suggest that adipocytes of grade 3 obese human subjects are able to activate the adaptive immune system, suggesting that in metabolic inflammation in humans both, innate and adaptive immunity, are of pathophysiological relevance.
PDF
