O carcinoma do córtex da supra-renal (ACC) é uma neoplasia rara e de prognóstico sombrio. Embora estudos moleculares tenham explorado diversos aspectos relacionados à tumorigênese destas neoplasias, o conhecimento das vias relacionadas à disseminação metastática é restrito. O objetivo do presente estudo é avaliar a expressão de genes relacionados a metástases em uma coorte de pacientes portadores de tumores do córtex da supra-renal metastáticos e não-metastáticos, a fim de identificar vias envolvidas na disseminação metastática destas neoplasias, novos marcadores prognósticos e eventuais alvos terapêuticos. Os perfis de expressão de 27 tumores do córtex da supra-renal de 15 pacientes adultos (8 ACC e 7 adenomas) e 12 pediátricos (5 metastáticos e 7 não-metastáticos) foram avaliados por um array de expressão contendo um painel de 113 genes que sabidamente estão envolvidos no processo de disseminação metastática de diversas neoplasias humanas. A análise de grupamentos mostrou que adenoma dos pacientes adultos forma um grupo distinto dos demais tumores (ACC de adultos e tumores pediátricos). Os genes MMP11e DENR foram identificados como diferencialmente expressos quando se compararam os adenomas e ACC de adultos. Na comparação dos tumores pediátricos nenhum gene foi diferencialmente expresso. Assim como a análise de grupamento, a PCA utilizando grupo selecionado de genes também não foi capaz partir os tumores pediátricos em subgrupos pela evolução. A expressão dos genes MMP2, TIMP3 e FN1 também foram avaliados por RT-PCR e foram concordantes com os dados gerados pelo array de expressão. O papel da LOH como causa da redução da expressão de TIMP3 foi estudado com tipagem de microssatélites. Em alguns casos, foi identificada LOH da região 22q13. Porém, em outros casos em que a expressão do TIMP3 foi bastante reduzida, não houve LOH. Em resumo, foram identificados aspectos moleculares importantes envolvidos na disseminação e metástases de neoplasias do córtex da supra-renal de adultos e crianças, bem como características biológicas deste processo. Diferentes padrões de expressão identificados em tumores metastáticos e não-metastáticos podem ajudar na predição do prognóstico / Adrenocortical carcinoma (ACC) is a rare neoplasm with a poor prognosis. Although molecular studies have uncovered many aspects of ACC tumorigenesis, little is known about molecular pathways involved in metastatic spread. The objective of our study is to analyze the expression profile of metastasis-related genes in a cohort of metastatic and nonmetastatic adrenocortical tumors in order to identify genes involved in the metastatic spread, as well as to find new prognostic markers. The expression profiles of 27 adrenocortical tumors from 15 adults (8 ACC and 7 adenomas) and 12 children (5 metastatic and 7 non-metastatic) were evaluated by an array of 113 known to be involved in human metastasis. Cluster analysis showed adult adrenocortical adenomas form a group distinct from other adrenocortical tumors (adult carcinomas and pediatric tumors). The comparison of adult adenoma and ACC revealed that MMP11 and DENR were differentially expressed between these two groups while no gene was differentially expressed among pediatric adrenocortical tumors. Similarly to cluster analysis, Principal component analysis failed to identify partition amongst pediatric tumors categorized by their evolution. The expression data of MMP2, TIMP3 and FN1 genes by RT-PCR agreed with those generated by the arrays. LOH of 22q12.3 region was detected in some cases in which TIMP3 down regulation was verified (but not in all cases). In conclusion, we have identified important aspects of molecular pathways and biological characteristics involved in metastatic spread of adrenocortical tumors. Distinctive patterns of gene expression between metastatic and nonmetastatic tumors may help in prognosis prediction

Sphingolipids modulate multiple cellular functions, including steroid hormone biosynthesis. Sphingosine is an antagonist ligand for the nuclear receptor steroidogenic factor 1 (SF-1), which is the primary transcriptional regulator of most steroidogenic genes. Furthermore, sphingosine-dependent repression of SF-1 function is dependent on the expression of acid ceramidase (ASAH1), an enzyme that forms sphingosine. Based on these data, I hypothesized that ACTH/cAMP signaling regulates ASAH1 function at both transcriptional and post-transcriptional levels. In addition, because SF-1 is predominantly a nuclear protein, I postulated that ASAH1 modulates SF-1 function and, therefore, steroidogenic gene expression by controlling the nuclear concentrations of SPH. To test these hypotheses, I first examined the effect of chronic ACTH/cAMP signaling on the transcription of the ASAH1 gene. Next, the functional significance of ASAH1 expression in adrenocortical cells was probed by generating an ASAH1-knockdown cell line. I subsequently characterized the role of ASAH1 as a transcriptional nuclear receptor coregulator. Finally, I defined the role of sphingosine-1-phosphate, a bi-product of ASAH1 activity, in the acute phase of cortisol biosynthesis. Using a variety of experimental approaches, I identified cAMP response element binding protein as an essential transcriptional activator of the ASAH1 gene. Analysis of adrenocortical cells lacking ASAH1 revealed that ASAH1 is a global regulator of steroidogenic capacity. Furthermore, I identified ASAH1 as a nuclear protein and defined the molecular determinants of the interaction between ASAH1 and SF-1. Collectively, this body of work establishes the integral role of ASAH1 in the regulation of ACTH-dependent adrenocortical cortisol biosynthesis.

Diss. (sammanfattning) Umeå : Univ., 2007. / Härtill 4 uppsatser.

Primary aldosteronism is the most common secondary cause of hypertension with a prevalence of about 10%. About half of PA cases are caused by aldosterone-producing adenomas (APA). Two APA subtypes, ZG-like and ZF-like APAs, have been described, according to the histological resemblance to normal zona glomerulosa (ZG) and zona fasciculata (ZF), underlying somatic mutations (KCNJ5 commonly found in ZF-like, CACN1AD, ATP1A1, ATP2B3, CTNNB1 in ZG-like APAs), and transcriptome profile. It is unknown if the process of tumorigenesis differs between ZG- and ZF-like APAs. In order to define ZG specific genes, we have compared the transcriptome of APAs and their adjacent adrenal glands by microarray assay. RNA was isolated by laser capture microdissection (LCM) from adjacent ZG, ZF and APAs from 14 patients with Conn’s and 7 patients with phaeocromocytoma. Two top hit genes from the comparison of ZG vs ZF were functionally studied, ANO4 and NEFM. NEFM, encoding neurofilament medium, was the fourth most up-regulated gene in ZG vs ZF, showing 14.8-fold-fold higher expression levels (p=9.16-12) in ZG than ZF. NEFM was also one of the most down-regulated genes in ZF-like vs ZG-like APAs. Immunohistochemistry (IHC) confirmed selective high expression of NEFM in ZG and ZG-like APAs. Silencing NEFM in H295R cells increased aldosterone secretion and cell proliferation. In addition, it increased stimulation and inhibition, respectively, of aldosterone secretion from H295R cells by the dopamine receptor D1R agonist fenoldopam and antagonist SCH23390. IHC showed predominantly intracellular staining for D1R in NEFM-rich ZG-like APAs, but membranous staining in NEFM-poor ZF-like APAs. Aldosterone secretion in response to fenoldopam in primary cells from ZG-like APAs was lower than in cells from ZF-like APAs. NEFM expression levels directly correlate with KCNJ5 phenotype: KCNJ5 mutations down-regulate NEFM mRNA and protein levels in H295R cells and in primary cells from ZG-like APAs. ANO4,encoding a Ca2+-activated chloride channel family member, was the third most upregulated gene, showing 19.9-fold higher expression levels (p=6.6x10-24) in ZG than ZF. IHC confirmed ZG selectivity of ANO4 protein in the adrenal cortex. The staining was mainly cytoplasmic. Unlike NEFM, there was no difference in expression of ANO4 between ZG- and ZF-like APAs, the levels being mid-way between those of ZF and ZG. Overexpression of ANO4 in H295R cells caused an increase in CYP11B2 and NR4A2 gene expression levels but basal aldosterone secretion was unchanged. In the presence of calcium agonists, ANO4 reduced aldosterone secretion. ANO4 subcellular localisation was confirmed as cytoplasmic by immunofluorescence microscopy of transfected cells. When exposed to calcium ionophores, ANO4 generated small chloride currents as detected by YFP assay. In summary, the comparison of transcriptome of ZG with paired ZF found unexpected up-regulated genes. Most of the highly up regulated genes in human ZG, including NEFM and ANO4, inhibit either basal or stimulated aldosterone secretion, and this may reflect an adaptive response to high salt intake. No clear-cut correspondence was found between transcriptome of APAs and their resembling zone of adrenal cortex. The down-regulation of NEFM following transfection of mutant KCNJ5 suggests that ZF-like properties may be a consequence of mutation, rather than tissue of origin.

Endocrine Disrupting Chemicals (EDC) are exogenous agents that mimic endogenous hormone activity in the body. EDC exposure during the critical period of neonatal development can potentially cause life-long neurological, behavioural and physiological disease. This thesis focuses on the EDC Bisphenol A (BPA), a synthetic xenoestrogen widely prevalent in everyday materials that has significant environmental relevance given its ubiquitous presence in humans around the world. The central research question of my thesis is: Does perinatal exposure to BPA affect rat pup development? A rodent model was selected to study the effects of BPA on the adrenal component of the hypothalamic-pituitary-adrenal axis (HPA axis) stress pathway, which has not been extensively studied. Rat dams were divided into five groups (vehicle control (VEH), positive control diethylstilbestrol (DES), BPA 5, BPA 50 and BPA 500 μg/kg bw/day) and dosed daily throughout gestation and for four days of lactation. Rat pups were sacrificed at two time-points at the beginning and the end of the stress hyporesponsive period (SHRP), at postnatal day (PND) 5 and PND 15. Changes in three morphometric parameters (bodyweight, crown-rump (CR) length and anogenital distance (AGD) were assessed based on the factors of Treatment and Sex. Adrenal gland glucocorticoid receptor (GR) and 18SrRNA expression was determined by qPCR in male pups at PND 5 and PND 15. At PND 5, compared to the VEH group, the BPA 50 pups were significantly heavier (ANOVA, Dunnett’s post-hoc) and the DES and BPA 50 pups had significantly longer CR lengths (ANOVA, Dunnetts’ post-hoc). At PND 15, xenoestrogen treatment significantly influenced CR length (ANOVA). At both time-points, males had significantly longer AGD than females, as physiologically expected (ANOVA). Adrenal gland GR expression in male pups was not significantly affected by treatment, but there was an effect of treatment in18SrRNA gene expression at PND 5 (Kruskal-Wallis). Using the Ct method to determine GR and 18SrRNA fold changes, we cautiously suggest that our experimental doses resulted in a non-monotonic dose response to BPA in the PND 5 animals and a monotonic dose response to BPA exposure in the PND 15 animals. This study highly values the importance of investigating the effects of environmentally relevant, low dose BPA exposure during the critical window of development, given the little that is known about potentially permanent alterations to the stress pathway due to exposure during this delicate period of development.

Der Typ I Transmembran-Rezeptor SORLA gehört zur VPS10p-Rezeptor Familie in Säugern. Der Rezeptor mit starker Homologie zu Endozytose- und Sorting-Rezeptoren ist am stärksten im zentralen Nervensystem (CNS) exprimiert. Außerhalb des CNS ist SORLA in einer Vielzahl von Geweben zu finden, unter anderem in der Niere. Das klare Expressionsmuster des Rezeptors im distalen Nephron lässt eine Rolle des Rezeptors in transepithelialen Transportprozessen vermuten. Um genau festzustellen, welche Prozesse von SORLA beeinflusst werden, wurde die Nierenfunktion von Mäusen mit einer vollständigen Defizienz des Sorla-Gens (Sorla-/-) untersucht. Diese Tiere zeigen Defekte in der renalen Ionenhomöostase: sie verlieren Na+, Cl-, K+, und Ca2+ (im Normalzustand und/oder nach Trinkwasserentzug). Eine Erniedrigung von Blutdruck und Herzfrequenz sowie eine fehlregulierte Sekretion von Aldosteron gehen mit dem Salzverlust-Phänotyp einher. Passend zu dieser Beobachtung konnte eine Expression von SORLA in der Nebenniere – speziell in der Zona glomerulosa, dem Ort der Aldosteron-Synthese – gezeigt werden. Des weiteren wurde eine signifikant verminderte Expression mehrerer Gene des Adrenalin-Synthesewegs in Sorla-/--Mäusen festgestellt, welcher in einer verringerten Menge des Hormons in den Nebennieren der Tiere resultiert. In der Niere bewirkt das Fehlen von SORLA insbesondere eine veränderte Phosphorylierung der beiden Kation-Chlorid-Cotransporter NCC und NKCC2 hervor, deren Aktivität durch Phosphorylierung reguliert wird. Es ist bekannt, dass die Signalkinase SPAK die Aktivität von NKCC2 und NCC reguliert. Die anormale Phosphorylierung fällt mit einer untypischen Verteilung der Kinase im TAL der SORLA-defizienten Mäuse zusammen. Dies deutet auf eine Funktion des Rezeptors beim Trafficking von SPAK hin. Durch die Identifizierung von Transportproteinen als putative Interaktionspartner SORLAs konnte diese Hypothese bekräftigt werden. / The type I transmembrane receptor SORLA is a member of the mammalian VPS10p-receptor family. The receptor, which is mainly expressed in the central nervous system (CNS), is characterized by high structural homology to endocytosis- and sorting-receptors. Outside the CNS, expression of SORLA can be found in a variety of tissues, including kidney. This distinct expression pattern in the distal nephron suggests a role for SORLA in transepithelial transport processes. To determine which processes the receptor might be involved in, the kidney function of mice, wich carry a complete deficiency of the Sorla gene, was analyzed. These animals show defects in ion handling: they are wasting Na+, Cl-, K+, and Ca2+ (under normal conditions and/or after water deprivation). The salt loss phenotype is accompanied by decreased mean arterial pressure and heart rate as well as mis-regulated secretion of aldosterone. In line with this observation, SORLA is also expressed in the adrenal gland, particularly in the zona glomerulosa, the place of aldosterone synthesis. Additionally, a significant down-regulation of several genes of the epinephrine synthesis pathway in mice lacking SORLA was found. This defect results in lower adrenal levels of the hormone. In the kidney, the lack of SORLA results especially in altered phosphorylation of the two cation-chloride cotransporters NCC and NKCC2, as their activity is regulated by phosphorylation. The signaling kinase SPAK has been reported to regulate the activity of NKCC2 and NCC. The transporters’ abnormal phosphorylation coincides with the atypical distribution of the kinase in TAL of Sorla-/- mice, suggesting a role of the receptor in establishing the localization of SPAK. This hypothesis was further substantiated by the identification of putative SORLA-interacting proteins involved in trafficking.

SF-1 e LRH-1 são fatores de transcrição que exercem um papel fundamental na produção de esteroides nas gônadas e na suprarrenal, além de estarem envolvidos no processo tumorigênico desses órgãos. Por outro lado, POD-1 apresenta menor expressão em carcinomas adrenocorticais, e parece regular Sf-1. Nesse trabalho foi analisado o papel de POD-1 na regulação de SF-1 e de LRH-1 em células de tumores adrenocorticais. A hiperexpressão de POD-1 resultou em redução da expressão SF-1/SF-1. Em contraste, houve um aumento da expressão gênica de LRH-1, devido à diminuição da expressão de SHP, um regulador negativo de LRH-1. Nas células transfectadas com siRNA-POD-1, os níveis de POD-1 foram reduzidos e de SF-1 aumentado, reforçando o mecanismo regulatório entre os fatores. No ChIP assay, POD-1 se ligou a sequência E-box do promotor de SF-1. Por outro lado, não foi caracterizado a ligação de POD-1 no promotor LRH-1, embora POD-1 tenha se ligado ao E-box do promotor SHP. A redução de SF-1 diminuiu a expressão de StAR, mas não modulou a proliferação das células tumorais. Em resumo, POD-1 pode ter um papel mais amplo como regulador da transcrição de fatores que controlam o processo tumorigênico, e é um candidato a gene supressor de tumor nas células adrenocorticais. / SF-1 and LRH-1 have played a critical role in steroid production, adrenal and gonads. Moreover, there are evidences that they have acted in tumorigenesis process in these organs. POD-1 is downregulated in adrenocortical carcinoma (ACC) it seems to regulate Sf-1. In this work, it has been to analyse the role of POD-1 in SF-1 and LRH-1 regulation in adrenocortical tumor cells. The POD-1 overexpression has reduced SF-1/SF-1 expression. However, there was an increase of LRH-1 gene expression due to SHP expression decrease which is negative regulate of LRH-1. The POD-1 and SF-1 gene expression in transfected cells with siRNA-POD-1 has shown POD-1 decrease and SF-1 increase emphasizing a regulatory mechanism between POD-1 and SF-1. By ChIP assay it was shown that POD-1 binded in SF-1 promoter E-box sequence. It was not characterized that POD-1 binded in LRH-1 promoter, although POD-1 can bind in SHP promoter E-box sequence. The reduction of SF-1 expression by POD-1 has decreased the StAR expression, however, it was not enough to change tumor cell proliferation. In summary, POD-1 must have a wider role as regulator of fator transcription which controls tumorigenese process being a possible candidate as tumor supressor gene in adrenocortical cells.

Existem evidências que o hormônio adrenocorticotrópico (ACTH) tem um papel importante no equilíbrio entre proliferação e morte celular na glândula supra-renal. As proteínas dos genes de resposta primária, proteínas da família Fos e Jun são componentes do fator de transcrição AP1, que dependendo de sua composição, pode estar relacionado com proliferação, diferenciação ou morte celular. Nesse trabalho utilizamos culturas de células primárias de adrenal de ratos para avaliar por imunocitoquímica e por imunoblotting, os efeitos do ACTH e do FGF2, na expressão das proteínas c-Fos, FosB, Fra1, Fra2, c-Jun, JunB e JunD. Os resultados mostram que tratamentos com ACTH e FGF2 modificam o padrão de expressão dessas proteínas. O ACTH induz aumento consistente da expressão de JunB, o que sugere uma composição de AP1 formada por JunB/c-Fos ou FosB. Tratamentos com FGF2, indicam a formação de um complexo c-Jun/c-Fos, FosB e Fra2. Esses resultados estão de acordo com os efeitos biológicos observados da ação do ACTH e do FGF2, como, inibição e proliferação celular nessas células. / There are evidences that in vivo the adrenocorticotropic hormone (ACTH) displays an important role in the balance of proliferation and cellular death in the adrenal gland. The early response gene proteins, Fos and Jun family, are components of the transcription factor AP-1 that, depending on its composition, could be related with proliferation, differentiation or cellular death. In this work we have been used adrenocortex cells of rat primary cultures, to evaluate, by immunocytochemistry and immunoblotting, the effects of ACTH and FGF2, in the expression of c-Fos, FosB, Fra1, Fra2, c-Jun, JunB and JunD proteins, and in such wise as to predict the composition of AP1 complex. The results showed that ACTH and FGF2 treatments modify the expression pattern of these proteins, inducing consistent and expressive increase of JunB expression in the ACTH-treated cells, suggesting an AP1 composition with JunB/c-Fos or FosB. FGF2 treatments indicate the composition of c-Jun/c-Fos or FosB or Fra-2 complexes. These results are in agreement with the biological effects observed in rat adrenal primary culture treated with ACTH and FGF2, with inhibition and cellular proliferation.

Existem evidências que o hormônio adrenocorticotrópico (ACTH) tem um papel importante no equilíbrio entre proliferação e morte celular na glândula supra-renal. As proteínas dos genes de resposta primária, proteínas da família Fos e Jun são componentes do fator de transcrição AP1, que dependendo de sua composição, pode estar relacionado com proliferação, diferenciação ou morte celular. Nesse trabalho utilizamos culturas de células primárias de adrenal de ratos para avaliar por imunocitoquímica e por imunoblotting, os efeitos do ACTH e do FGF2, na expressão das proteínas c-Fos, FosB, Fra1, Fra2, c-Jun, JunB e JunD. Os resultados mostram que tratamentos com ACTH e FGF2 modificam o padrão de expressão dessas proteínas. O ACTH induz aumento consistente da expressão de JunB, o que sugere uma composição de AP1 formada por JunB/c-Fos ou FosB. Tratamentos com FGF2, indicam a formação de um complexo c-Jun/c-Fos, FosB e Fra2. Esses resultados estão de acordo com os efeitos biológicos observados da ação do ACTH e do FGF2, como, inibição e proliferação celular nessas células. / There are evidences that in vivo the adrenocorticotropic hormone (ACTH) displays an important role in the balance of proliferation and cellular death in the adrenal gland. The early response gene proteins, Fos and Jun family, are components of the transcription factor AP-1 that, depending on its composition, could be related with proliferation, differentiation or cellular death. In this work we have been used adrenocortex cells of rat primary cultures, to evaluate, by immunocytochemistry and immunoblotting, the effects of ACTH and FGF2, in the expression of c-Fos, FosB, Fra1, Fra2, c-Jun, JunB and JunD proteins, and in such wise as to predict the composition of AP1 complex. The results showed that ACTH and FGF2 treatments modify the expression pattern of these proteins, inducing consistent and expressive increase of JunB expression in the ACTH-treated cells, suggesting an AP1 composition with JunB/c-Fos or FosB. FGF2 treatments indicate the composition of c-Jun/c-Fos or FosB or Fra-2 complexes. These results are in agreement with the biological effects observed in rat adrenal primary culture treated with ACTH and FGF2, with inhibition and cellular proliferation.

T cells, which constitute an essential arm in the adaptive immunity, complete their development in the thymus through a process called thymopoiesis. However, thymic involution can be induced by a couple of factors, which impairs T cell functions and is slow to recover. Therefore, understanding how thymopoiesis is regulated may lead effort to accelerate thymic recovery and improve immune functions in thymocyte-depleted patients. In this project, we identified scavenger receptor BI (SR-BI), a high density lipoprotein (HDL) receptor, as a novel modulator in thymopoiesis. In mice, absence of SR-BI causes a significant reduction in thymus size after puberty and a remarkable decrease in thymic output. Consequently, SR-BI-null mice show a narrowed naïve T cell pool in the periphery and blunted T cell responses, indicating that the impaired thymopoiesis due to SR-BI deficiency leads to compromised T cell homeostasis and functions. The impaired thymopoiesis of SR-BI-null mice is featured by a significant reduction in the percentage of earliest T progenitors (ETPs) but unchanged percentages of other thymocyte subtypes, suggesting that SR-BI deficiency causes a reduction in progenitor thymic entry. Further investigations reveal that SR-BI deficiency impairs thymopoiesis through affecting bone marrow progenitor thymic homing without influencing the lymphoid progenitor development in bone marrow. Importantly, SR-BI-null mice exhibit delayed thymic recovery after sublethal irradiation, indicating that SR-BI is also required for thymic regeneration. Using bone marrow transplantation models, we elucidate that it is non-hematopoietic rather than hematopoietic SR-BI deficiency that results in the defects in thymopoiesis. However, SR-BI deficiency-induced hypercholesterolemia is not responsible for the impaired thymopoiesis. Using adrenal transplantation models, we found that absence of adrenal SR-BI is responsible for the impaired thymopoiesis, as shown by that adrenalectomized mice transplanted with SR-BI-null adrenal gland display reduced thymus size, decreased percentage of ETPs and delayed thymic regeneration compared with those transplanted with wild-type adrenal. Altogether, results from this study elucidate a previously unrecognized role of SR-BI in thymopoiesis. We reveal that SR-BI expressed in adrenal gland is critical in maintaining normal T cell development and enhancing thymic regeneration, providing novel links between adrenal functions and T cell development.