A PMAH é uma causa rara de síndrome de Cushing, cuja real prevalência parece subestimada. O processo fisiopatológico que culminaria com a PMAH ainda não foi totalmente elucidado. A produção de cortisol mediada por receptores acoplados a proteína G (ectópicos/tópicos da adrenal) geralmente hiperexpressos, bem como a regulação autócrina/parácrina do ACTH ectópico em clusters celulares dos macronódulos adrenais têm sido considerados como parte importante do processo fisiopatológico da PMAH. Além destes mecanismos, recentemente, foi demonstrado a associação de variantes patogênicas germinativas/somáticas no gene ARMC5 como uma causa frequente da PMAH. Os estudos funcionais que caracterizaram o ARMC5 como gene supressor de tumor e potencialmente envolvido na hiperplasia adrenal nodular e sua produção de cortisol foram realizados em células H295R, derivadas de carcinoma adrenocortical. Células estas que não representam em tese um modelo ideal para o estudo de uma doença absolutamente benigna. Objetivos: Obtenção e caracterização morfofuncional de culturas de células obtidas de nódulos adrenais de pacientes submetidos à adrenalectomia com diagnóstico histopatológico compatível com PMAH, como um modelo biológico para a análise funcional do gene ARMC5. Métodos: Foram utilizadas 13 culturas de células de PMAH caracterizadas do ponto de vista morfofuncional e molecular para as análises funcionais do gene ARMC5. Resultados: As culturas de PMAH apresentaram mutações germinativas no ARMC5 identificadas em 8 das 13 culturas analisadas, associadas ou não a segundos eventos moleculares. As culturas de células de PMAH apresentaram receptores eutópicos e ectópicos de uma maneira heterogênea e a presença de ACTH ectópico em clusters de células. O silenciamento do ARMC5 nas células de PMAH levou à diminuição da esteroidogênese, ao aumento de CCNE1 e ao número de células viáveis após 96h. Quando hiperexpresso, o gene ARMC5 induziu apoptose e necrose celular após 12h, diminuindo a viabilidade das células de células de PMAH. Conclusões: Legitimamos a importância do papel do ARMC5 na esteroidogênese relacionada à PMAH, bem como sua função favorecendo a apoptose celular; além disso, pela primeira vez, detectamos o envolvimento do ARMC5 na regulação do ciclo celular e proliferação, cuja importância será explorada em estudos futuros. / Background: PMAH is a rare cause of Cushings syndrome, with an apparent underestimated prevalence. The pathophysiology of PMAH is not yet fully understood, however the participation of aberrant receptors and intra-adrenal ACTH in the hyperplastic tissue are considered mechanisms that regulate hypercortisolism in PMAH. Additionally, germline ARMC5 mutations have been described as the most frequent genetic abnormality found in patients diagnosed with PMAH. Previous functional studies analyzed ARMC5 role using H295R cells, a cell line of adrenocortical carcinoma. Objectives: In this study we investigated the role of ARMC5 in cell cultures obtained from PMAH nodules. Results: We observed the presence of mutations in ARMC5 gene in 8 out of 13 PMAH cell cultures analyzed. We observed the presence of aberrant receptors and intra-adrenal ectopic ACTH, regardless the presence of mutations. ARMC5 silencing in non-mutated PMAH cell cultures decreased steroidogenesis-related genes and increased CCNE1 mRNA expression and the number of viable cells without affecting cell viability. Additionally, ARMC5 overexpression induced cell death in PMAH mutated cell cultures, thereby decreasing cell viability. Conclusions: We confirmed the role of ARMC5 as an important pro-apoptotic protein involved in PMAH-related steroidogenesis. We also report for the first time the possible involvement of ARMC5 in controlling proliferation and regulating cell cycle in PMAH cell cultures, which need to be further explored.

A hiperplasia adrenocortical macronodular primária (PMAH) é uma causa rara de Síndrome de Cushing (SC). Apresenta como características macronódulos funcionantes em ambas as glândulas suprarrenais e uma produção variável de cortisol. Os nódulos apresentam uma produção ectópica de hormônio adrenocorticotrófico (ACTH) tornando a doença independente do ACTH hipofisário. Estudos independentes mostraram que mutações germinativas no gene armadillo repeat cointaining 5 (ARMC5) são uma causa frequente de PMAH. Além dessa mutação parece haver a participação de receptores hormonais ectópicos no córtex suprarrenal, que estimulariam a esteroidogênese e a hiperplasia da glândula. No entanto, os estudos sobre a relação entre o ARMC5 e a participação dos receptores aberrantes na produção de cortisol são incipientes. Portanto, temos como hipótese que as alterações no gene ARMC5 podem estar envolvidas no padrão celular e funcional das células que compõem os nódulos hiperplásicos na PMAH. Desta forma, nossos objetivos foram analisar: 1) a proporção de células espongiformes e compactas em cortes corados com hematoxilina e eosina; 2) a expressão gênica, nas células espongiformes e proteica de ambas as células, do ARMC5, das enzimas esteroidogênicas StAR, 3βHSD2 e CYP17A1, dos receptores da vasopressina (AVP1AR), serotonina (5HT4R) e do peptídeo inibidor gástrico (GIPR) e do ACTH ectópico e 3) a expressão do antígeno de proliferação celular, a proteína PCNA, para análise do padrão de proliferação em cortes histológicos de nódulos de hiperplasias de pacientes que apresentam mutações germinativas, mutações germinativas e somáticas ou não mutação no gene ARMC5. Os resultados mostraram que os nódulos hiperplásicos são compostos, na sua maioria, por células espongiformes, cujo padrão foi independente da presença de mutação. A expressão do ARMC5 nas células espongiformes foi maior em pacientes com mutação, e os receptores ectópicos apresentaram uma expressão maior no tecido de PMAH em relação à adrenal normal. A reação de imunoistoquimica revelou células positivas para a proteína ARMC5 e StAR, em ambos os tipos celulares, enquanto a enzima 3βHSD2 foi predominante nas células espongiformes e a enzima CYP17A1 nas células compactas. A expressão de AVP1AR, 5HT4R, e do ACTH ectópico foi positiva em ambos os tipos celulares e independente da presença ou não da mutação no gene ARMC5, bem como a expressão da proteína PCNA. Portanto, nossos resultados sugerem que a presença ou não de mutações no gene ARMC5 nas hiperplasias não está envolvida no padrão celular das células que compõem os nódulos. O mesmo para a presença dos receptores AVP1AR, 5HT4R e GIPR, do ACTH ectópico e da proteína PCNA. Como conclusão, os resultados dos parâmetros analisados sugerem que as alterações no gene ARMC5 não estão envolvidas no padrão celular e funcional das células que compõem os nódulos hiperplásicos na PMAH, e podem ter o mesmo grau de importância na formação desses nódulos. / The primary macronodular adrenal hyperplasia (PMAH) is a rare cause of Cushing\'s Syndrome (SC). It is characterized as macronodules in the adrenal gland and by variable production of cortisol. The nodules present an ectopic production of adrenocorticotrophic hormone (ACTH) pituitary-independent. Several studies showed that the germ mutations in armadillo repeat cointaining 5 (ARMC5) are a frequent cause of PMAH. In addition, there is a participation of ectopic hormonal receptors in the adrenal cortex, which promotes steroidogenesis and hyperplasia of the gland. However, studies on a relationship between ARMC5 and aberrant receptor involvement in cortisol production are incipient. Therefore, our hypothesis is that ARMC5 gene may be involved in the cellular and functional pattern of the cells in PMAH. Thus, the objectives were to analyzed: 1) the proportion of spongiform and compact cells in sections stained with hematoxylin and eosin; 2) the gene expression in the spongiform cells and the gene and protein expression in both cells types of the ARMC5, the steroidogenic enzymes StAR ,3βHSD2 e CYP17A1, and the ectopic receptors of vasopressin (AVP1AR), serotonin (5HT4R) and gastric inhibitory peptide (GIPR) also the ACTH ectopic and 3) the proliferating cell nuclear antigen (PCNA) that shows the proliferation pattern in patients PMAH that present germline, germinative and somatic mutations or no mutation in ARMC5 gene. We showed that the hyperplastic nodules are mainly composed by spongiform cells independent of the ARMC5 mutation. The expression of ARMC5 in spongiform cells was higher in patients with mutation, and PMAH tissue have a higher expression of the ectopic receptors when compared with normal adrenal. With the immunohistochemical labeling, we observed the ARMC5 and StAR protein expression in both cell types. The 3βHSD2 enzyme was predominant in spongiform cells and the CYP17A1 enzyme in the compact cells. The expression of AVP1AR, 5HT4R, and ectopic ACTH was positive in both cell types regardless of the presence of the ARMC5 mutation, as well as the expression of the PCNA protein. Therefore, the results suggest that the mutation in ARMC5 is not involved with spongiform and compact cell function. Moreover, the presence of the AVP1AR, 5HT4R and GIPR receptors, the ectopic ACTH and the PCNA protein was not related to the gene mutation. In conclusion, the alterations in the ARMC5 gene are not involved in the cell functional pattern in the PMAH and in the onset of the nodules.

Análise de variantes alélicas no gene Armadillo Repeat Containing 5 (ARMC5) em pacientes com incidentaloma da suprarrenal identificamos 16 pacientes carreadores da mutação, a partir de dois irmãos inicialmente investigados. Aproximadamente 30% dos membros afetados desta genealogia, apresentavam somente uma suprarrenal acometida e a suprarrenal contra-lateral absolutamente normal ou discretamente espessada. Esta observação nos infere que a hiperplasia macronodular da PMAH pode ocorrer de forma assincrônica e dificultar aprincipio o seu diagnostico. A incidência de nódulos unilaterais identificados em exame de imagens na pratica clinica é muito elevada (1,4 - 2,9%, podendo chegar a 10% em pacientes com idade a cima de 70 anos) os denominados incidentalomas suprarrenais. Além disso, a grande maioria dos pacientes com nódulos suprarrenais apresentam alguma autonomia da secreção do cortisol. Considerando estes aspectos, nos propomos a investigar se os incidentalomas suprarrenais unilaterais com alguma autonomia da secreção do cortisol poderiam fazer parte do espectro de apresentação da PMAH. Nosso trabalho tem como foco analisar a frequência das variantes alélicas do ARMC5 em uma coorte multicêntrica de pacientes com nódulos suprarrenais incidentais e autonomia da secreção do cortisol. O esclarecimento da frequência da PMAH pode nos direcionar a uma melhor pratica clinica e cuidados com os pacientes / Primary macronodular adrenal hyperplasia (PMAH) was described in 1964 by Kirschner et al, and is considered a rare cause of endogenous ACTH-independent Cushing\'s syndrome (CS), usually associated with bilateral adrenal nodules. The clinical presentation of the patients is heterogeneous, ranging from subclinical to overt Cushing\'s syndrome, with all its characteristics. The cortisol secretion pattern, after the 1 mg dexamethasone overnight suppression test, identifies the intensity of cortisol secretion autonomy, ie, serum cortisol concentrations above 1.8 ug / dL and up to 5 ug / dL , characterize a possible autonomous cortisol secretion and above this value (5 ug / dL) an autonomous cortisol secretion. Patients are generally diagnosed with PMAH when undergoing ACTH-independent endogenous hypercortisolism investigation or, more frequently, during the investigation of bilateral adrenal incidentalomas and less likely in patients with metabolic syndrome associated with meningiomas of the central nervous system, since these are very prevalence in patients with PMAH. Bilateral adrenal involvement, along with the description in the literature of several PMAH-affected family relatives, has already indicated a genetic cause involved in the pathophysiology of this intriguing disease. In 2013, two studies, one Brazilian and one Franch, using distinct methodologies, identified germline mutations in ARMC5 as the major genetic cause of PMAH. The largest genealogy published so far was that of a Brazilian family, with 5 generations in which we identified 16 patients, carriers of the mutation, from two siblings initially investigated. Approximately 30% of the affected members of this genealogy had only one adrenal afected with the contralateral gland absolutely normal or discretely thickened. This observation infers us that the macronodular hyperplasia of PMAH can occur in an asynchronous way and make it difficult to diagnose it. The incidence of unilateral nodules identified in clinical imaging is very high (1.4-2.9%, and may reach 10% in patients over the age of 70), the so-called adrenal incidentalomas. In addition, the vast majority of patients with adrenal nodules have some autonomy in cortisol secretion. Considering these aspects, we propose to investigate whether unilateral adrenal incidentalomas, with some autonomy of cortisol secretion, could be part of the presentation spectrum of PMAH. Our work focuses on the frequency of allelic variants of ARMC5 in a multicentric cohort of patients with incidental adrenal nodules and autonomy of cortisol secretion. Clarifying the frequency of PMAH may lead to better clinical practice and patient care

L’hyperplasie bilatérale macronodulaire des surrénales (HBMS) conduit au développement de nodules corticosurrénaliens bilatéraux entraînant un syndrome de Cushing diagnostiqué souvent au cours de la cinquième décennie. L’étiologie de cette maladie n’était que partiellement connue mais le caractère bilatéral de l’atteinte surrénalienne et l’observation de cas familiaux suggéraient, au début de ce travail, une origine génétique. L’analyse de l’ADN tumoral et leucocytaire d’une série de 33 patients opérés a permis de mettre en évidence chez 25% des patients une perte d’hétérozygotie neutre en nombres de copies (LOH) de tout le bras court du chromosome 16 (16p) au niveau du tissu surrénalien. Un séquençage complet du génome de 5 patients (ADN germinal et tumoral) a permis de mettre en évidence une mutation du gène ARMC5 localisé en 16p pour 4 patients. Le séquençage direct des parties codantes d’ARMC5 à partir de l’ADN somatique et germinal de l’ensemble des patients opérés de la cohorte a montré qu’au total, 55% des patients avaient une mutation d’ARMC5. L’inactivation du gène se fait selon la théorie de Knudson (un événement germinal inactivant le premier allèle associé à un autre événement somatique inactivant le second allèle) ce qui laisse supposer qu’ARMC5 est un gène suppresseur de tumeur. L’analyse de la cohorte d’HBMS de nos collaborateurs américains au National Institute Health (laboratoire de CA. Stratakis, Bethesda, USA) a permis de confirmer que les mutations d’ARMC5 étaient un événement fréquent. Des variants de ce gène sont aussi associés à l’hypertension à rénine basse chez les patients noirs-américains. Afin de déterminer des corrélations génotype-phénotype, notre cohorte initiale a été élargie pour constituer une série consécutive de 98 cas index de patients présentant des formes légères à sévères de la maladie, opérées ou non. Vingt-quatre patients (25%) présentaient une altération d’ARMC5. Par ailleurs, 31 nodules surrénaliens de 19 patients ont été analysés en somatique. Le second événement était une mutation dans 68% des cas et une LOH du locus pour les 32% restant. Chez un même patient, le second événement était différent dans chaque nodule présenté. Les patients mutés avaient un syndrome de Cushing plus sévère cliniquement et biologiquement par rapport aux patients non mutés. La taille de leurs surrénales étaient plus grandes avec un plus grand nombre de nodules. Les patients mutés étaient aussi plus jeunes au diagnostic et plus souvent hypertendus. Ces patients étaient ainsi plus souvent opérés. La fonction de la protéine ARMC5 n’était pas connue lors de son identification comme gène de l’HBMS. In vitro, la surexpression du gène sauvage induit l’apoptose. La surexpression des mutants faux-sens et du mutant p.F700del retrouvés chez les patients entraîne moins d’apoptose qu’ARMC5 sauvage. La protéine ARMC5 contient des domaines Armadillo et BTB, connus pour être impliqués dans l’interaction protéine-protéine. En physiologie, l’ACTH stimule la production d’AMPc et la voie de la protéine kinase A (PKA) est impliquée dans différentes pathologies corticosurrénaliennes. Nous avons pu montrer qu’ARMC5 interagissait avec la sous-unité catalytique alpha de la PKA. L’invalidation d’ARMC5 conduit in vitro à une diminution de l’expression d’enzymes de la stéroïdogénèse, de la production de cortisol et une diminution de l’activité PKA. Ainsi, l’hypothèse pour expliquer les HBMS liées à une inactivation d’ARMC5 est que la perte d’apoptose conduit à une hyperplasie nodulaire du tissu corticosurrénalien et que, même si la production de cortisol est diminuée à l’échelle unicellulaire, l’effet de masse global conduit au total à un hypercortisolisme. Nos travaux ont donc identifié et caractérisé un premier gène causal, ARMC5, fréquemment impliqué dans l’HBMS et associé à des formes plus sévères de la maladie. Cette découverte ouvre des perspectives pour le diagnostic familial et la prise en charge des patients. (...) / Primary bilateral macronodular adrenal hyperplasia (PBMAH) is a rare cause of adrenal Cushing’s syndrome and bilateral adrenal tumors. We suspected a genetic origin of the disease on the basis of the report of some familial cases and the involvement of both adrenal glands. The aim of this study was to find a genetic cause of non syndromic PBMAH. To look at chromosomal abnormalities, we use single-nucleotide polymorphism (SNP) arrays and microsatellite markers analysis in a first series of 33 patients all operated for PBMAH. We realize whole genome sequencing of 5 patients (blood and tumor DNAs matched). Then we genotyped by Sanger sequencing the gene Armadillo Repeat Containing 5 (ARMC5) in this first series and 66 additional patients. Clinical data were collected to establish genotype-phenotype correlation. In addition, the cohort of patients of our collaborators at the National Institute Health (Dr. Stratakis, Bethesda, USA) was studied. The effects of ARMC5 inactivation and overexpression and the partners of the protein were sought in cell-culture models. The most frequent somatic alteration was a loss of heterozygosity at 16p observed in tumors of 25% of the patients. The gene ARMC5, located at 16p11.2, was the most frequently mutated by whole genome sequencing: a mutation was found in 4/5 patients. 55% of the patients of the first cohort (33 patients treated by adrenalectomy for PBMAH) had ARMC5 alteration. One patient presented with germline microdeletion of the locus identified by SNP array. Every patient had two events: either a mutation or a deletion at the germline level, either a second mutation or a LOH at the somatic level. We showed that the two events were present on different alleles suggesting that ARMC5 is a tumor suppressor gene. In addition, we showed for several patients that the second hit was different in each adrenal nodules of a same patient. This first cohort included only operated patients with serious forms of the disease. The study of the American cohort and the analysis of the total cohort of our lab including non-operated patients and milder forms showed an alteration of ARMC5 in about 25% of the patients. Genotype-phenotype correlation showed that ARMC5 defects are associated with younger age at the diagnosis, higher hypercortisolism, bigger adrenals and higher number of nodules. In addition, a mutation of ARMC5 was shown in a patient with a PBMAH secreting both aldosterone and cortisol. Analysis of a series of patient affected by primary hyperaldosteronism suggested that ARMC5 may be associated with hypertension especially in African-American subjet. Overexpression of ARMC5 leads in vitro to cell apoptosis. We showed that this apoptosis was reduced when transfecting vector harboring missense mutations or single amino-acid deletion found in our cohort. Invalidation of ARMC5 leads to a decreased steroidogenic enzymes expression, cortisol production and reduced protein kinase A (PKA) activity. We showed that ARMC5 interacts with the calaytic subunit alpha of the PKA dissociated from the cAMP-bound regulatory subunits. More than one quarter of sporadic PBMAH patients present a pathogenic germline ARMC5 defect and these index cases present a more severe disease. Systematic genotyping of ARMC5 may help for early diagnosis of PBMAH, familial counseling, and patients’ management. ARMC5 appears to be a new regulator of PKA and might represent a new target for the development of pharmacological agents controlling PKA function and cortisol production.

Les récepteurs tyrosine kinases sont un groupe de molécules clés de signalisation, qui ont 2 fonctions: la détection des stimuli de l'environnement extérieur des cellules et la transmission de ces signaux à l’intérieur des cellules. Dans les 20 dernières années, notre laboratoire a choisi d'étudier la fonction d’Ephb6 kinase, un récepteur tyrosine kinase fortement exprimé dans les lymphocytes T.Comme Efnb1 et Efnb2 sont tous des ligands pour Ephb6, nous avons ensuite procédé à étudier leur rôle dans la fonction des cellules T in vitro et in vivo. Des cellules T spécifiques mutants (KO) dans les gènes Efnb1 ou Efnb2 ainsi que les doubles mutants Efnb1/b2 (double KO) ont été générés, mais il n’y avait que les souris double KO qui ont démontré de la déficience dans le développement des thymocytes, fonction de Th1 et Th17, la signalisation du récepteur d’IL-6, et les réponses antivirales. Des preuves solides indiquent que la reconnaissance d’auto-antigène par les cellules T est un événement précoce dans la pathogenèse de la PR. Donc, nous avons postulé que les cellules T spécifique Efnb1 / b2 double KO chez la souris peuvent protéger les souris de l’arthrite induite par collagène (CIA), un modèle de souris de la PR humaine. Nous avons trouvé que Efnb1et Efnb2 dans les cellules T étaient essentielles pour la production d'anticorps pathogéniques et de la migration des lymphocytes T vers les pattes enflammées chez les souris CIA. Notre étude clinique suggère que l'expression de EFNB1 dans les cellules T pourrait être un paramètre utile pour surveiller l'activité de la maladie de RA et la réponse de traitement. Pour élucider les événements dans le programme d'activation des lymphocytes T, nous avons exploré par l'analyse des micropuces d'ADN pour identifier des molécules qui ont été exprimées de manière différente dans le WT par rapport aux cellules T Ephb6 KO dans le stade précoce de l’activation des cellules T. Environ 30 molécules étaient sur ou sous exprimées plus de 3 fois dans les cellules T WT par rapport aux cellules T KO pendant les 16 premières heures après stimulation par l'anti-CD3. Stra6 (stimulée par le gène de l'acide rétinoïque 6) et Armc5 (Armadillo répéter contenant 5) ont été parmi ceux qui ont été validées pour leur expression altérée. STRA6 est un récepteur de haute affinité pour le plasma rétinol-binding protéine (RBP) et un médiateur pour absorption cellulaire de vitamine A. Cellules T KO et WT étaient similaires en termes de prolifération et les réponses immunitaires anti-virales de virus de la chorioméningite lymphocytaire (LCMV). Ainsi, la sur-régulation de Stra6 est soit un événement parallèle qui ne soit pas essentiel pour le programme d'activation des lymphocytes T, ou il est très essentiel que la redondance existe, et sa suppression ne montre aucun effet apparent sur l'activation des cellules T. ARMC5 est une protéine intracellulaire contenant sept répétitions en tandem d’armadillo et un domaine BTB. Les fonctions du ARMC5 dans le système immunitaire ne sont pas encore connues. Nos résultats d'hybridation in situ ont montré une expression élevée de Armc5 dans le thymus, et une expression modérée dans les ganglions lymphatiques et la rate. Nous avons généré des souris KO Armc5. Fait interessant, les cellules T Armc5 KO présentaient de la prolifération diminuée et de la différenciation compromise vers Th1 et Th17 in vitro. Les souris KO étaient résistantes à l'induction expérimentale d’encéphalite auto-immune, et ont été compromises dans les réponses immunitaires anti-LCMV. En utilisant de la levure 2-hybride test, nous avons identifié 8 protéines ARMC5-associantes, qui sont connues pour les rôles dans l'activation de la cellule, le cycle cellulaire et l'apoptose. Une étude mécanique est en cours. Nos résultats montrent que Armc5 est essentiel dans la programme d'activation/de prolifération/de différenciation des lymphocytes T. Nos études ont augmenté nos connaissances sur EFNB1, EFNB2, STRA6 et ARMC5 en biologie des lymphocytes T et leur pertinence à des troubles immunitaires dans des modèles animaux ainsi que chez l'être humaine. / Receptor tyrosine kinases are a group of key signaling molecules, which have dual functions: sensing the environmental stimuli outside the cells and transmitting them into the cells. 20 years ago, our laboratory started to study the function of Ephb6 kinase, a receptor tyrosine kinase highly expressed in T lymphocytes. As both Efnb1 and Efnb2 are the ligands for Ephb6, we then proceeded to study their roles in T cell function in vitro and in vivo. T cellspecific Efnb1, Efnb2 single gene knockout (KO), as well as Efnb1/b2 double KO mice were generated, but only the double KO mice showed compromised thymocyte development, Th1 and Th17 function, IL-6 receptor signaling, and anti-virus responses. Strong evidence indicates that T cells play a crucial role in the pathogenesis of rheumatoid arthritis (RA). Thus, we postulated that T cell-specific Efnb1/b2 double KO in mice may protect mice from collagen-induced arthritis (CIA), a mouse model for human RA. We found that Efnb1 and Efnb2 in T cells were essential for pathogenic antibody production and T cell migration to the inflamed paws in mice with CIA. Our clinical study suggests that the expression of EFNB1 in T cells might be a useful parameter for monitoring RA disease activity and treatment responses. Naïve T cells have the ability to expansion and differentiation into effector cells once they encounter foreign antigens, during which a large number of molecules are modulated. Some of these molecules play essential regulatory roles, while others exert house keeping functions and/or act as supporters to cope with increased or changed metabolic demands. To fully elucidate events in the T cell activation program, we undertook unbiased exploration with DNA microarray analysis to identify molecules that were differentially expressed in WT versus Ephb6 KO T cells in the early T-cell activation stage. About 30 molecules were up- or down-regulated more than three folds in WT T cells compared with KO T cells. Stra6 (stimulated by retinoic acid gene 6) and Armc5 (Armadillo repeat-containing 5) were among those that had been validated for their altered expression. We generated mice with these two genes deleted to study their roles in T cell function in vitro and in vivo. STRA6 is a high-affinity receptor for plasma retinol-binding protein (RBP) and mediates cellular vitamin A uptake. Stra6 KO mice manifest normal spleen and thymus in size, cellularity and lymphocyte subpopulations. KO and WT T cells were similar regarding proliferation, differentiation and anti-viral immune responses to lymphocytic choriomeningitis virus (LCMV). Thus, the up-regulation of Stra6 is either a parallel event which is not essential for the T cell activation program or it is so critical that heavy redundancy exists. ARMC5 is an intracellular protein containing seven tandem armadillo repeats and one BTB domain. Functions of ARMC5 in the immune system are not known previously. Our in situ hybridization results showed high expression of Armc5 in the thymus and moderate expression in the spleen and lymph nodes. A transient increase of Armc5 expression in T cells after TCR activation was found. To investigate its roles in T cell function, Armc5 KO mice were generated. The KO mice weighed 40% less than their WT counterparts. Lymphoid organs (the thymus, spleen and lymph nodes) of the KO mice appeared to be of normal size, weight, cellularity, and lymphocyte subpopulations. Intriguingly, Armc5 KO T cells presented decreased proliferation and compromised differentiation towards Th1 and Th17 in vitro. The KO mice were resistant to experimental autoimmune encephalitis induction and were compromised in anti-LCMV immune responses. Using yeast 2-hybrid assay, we have identified 8 ARMC5-assciating proteins, which have known functions in cell cycling and apoptosis. Further mechanistic study is underway. Our results reveal that Armc5 is vital in the T cell activation/proliferation /differentiation program. Our studies have augmented our knowledge about EFNB1, EFNB2, STRA6 and ARMC5 in T cell biology and their relevance to immune disorders in animal models as well as in humans.

ARMC5 est une protéine qui contient sept motifs Armadillo répétitifs organisés en tandem et un domaine BTB. Nous avons observé que cette protéine était fortement exprimée dans les organes lymphoïdes, les glandes surrénales et le cerveau. Les souris avec une délétion d’Armc5 (souris KO) étaient de petite taille, et présentaient une diminution de la prolifération et la différenciation des lymphocytes T. L’absence d’ARMC5 entraînait une déficience de la réponse immunitaire médiée par les lymphocytes CD4+ et CD8+ dans les modèles expérimentaux d’encéphalomyélite auto-immune et d’infection au virus de la chorioméningite lymphocytaire, respectivement. Par la suite, plusieurs études ont révélé que la mutation ARMC5 était associée à l’hyperplasie macronodulaire bilatérale primitive des surrénales (HMBPS), qui représente une cause rare du syndrome de Cushing. Nous avons ensuite confirmé que l’hyperplasie des glandes surrénales s’était développée chez les souris KO âgées, et qu’elle s’accompagnait d’une légère augmentation des taux sériques de glucocorticoïdes. Comme ARMC5 ne présentait pas d’activité enzymatique, il était probable qu’elle faisait appel à d’autres protéines pour exercer sa fonction. Nous avons identifié plusieurs protéines qui se liaient à ARMC5, et plus particulièrement le complexe ARMC5/Cullin3 qui formait une ubiquitine ligase (E3) spécifique de la sous-unité RPB1 de l’ARN polymérase II. ARMC5 contrôlait le processus d’ubiquitination de RPB1 qui, par conséquent, s’accumulait dans plusieurs organes majeurs : les glandes surrénales, les ganglions lymphatiques, le cerveau, les poumons, le foie, etc. chez la souris KO. Ces résultats démontrent un rôle clé de l’ubiquitine ligase dans la dégradation de la protéine RPB1. Une accumulation similaire a également été observée dans les tissus hyperplasiques des surrénales provenant de patients atteints d’HMBPS et porteurs de la mutation ARMC5, ce qui souligne la pertinence clinique de nos résultats de recherche fondamentale dans les maladies humaines. Un défaut de dégradation de RPB1 augmentait le pool d’ARN polymérase II. Par ailleurs, nous avons identifié un groupe de gènes fortement surexprimés dans les glandes surrénales déficientes en ARMC5, parmi lesquels figurent les gènes effecteurs qui seraient impliqués dans l’hyperplasie des surrénales chez les souris KO et l’HMBPS chez les patients porteurs de la mutation ARMC5. Finalement, nous avons montré que la délétion ou la mutation d’Armc5 augmentait considérablement le risque des anomalies du tube neural chez les souris et les humains. Chez les patients souffrant de myéloméningocèle, nous avons constaté neuf différentes mutations faux-sens délétères, dont une diminuait l’interaction entre ARMC5 et RPB1. L’augmentation du pool d’ARN polymérase II dans les cellules précurseurs neurales (CPN), causée par la délétion ARMC5, influençait un groupe particulier de gènes, dont certains (p. ex. Folh1) seraient susceptibles de participer au développement du tube neural. En résumé, l’association ARMC5 et Cullin3 forme un complexe E3 qui cible RPB1 provoquant son ubiquitination et sa dégradation. En absence d’un tel mécanisme, on observe une perturbation de l’homéostasie de l’ARN polymérase II, qui mène à une diminution de la réponse immunitaire médiée par lymphocytes T, le développement d’HMBPS et un risque accru d’anomalies du tube neural. / ARMC5 protein contains seven tandem Armadillo repeats and one BTB domain. We observed that Armc5 was highly expressed in the lymphatic organs, adrenal glands, and brain. Armc5 knockout (KO) mice were small in size and exhibited compromised T cell proliferation and differentiation. The absence of ARMC5 resulted in an impairment of the CD4 + cell- and CD8 + cell-mediated immune response in the experimental autoimmune encephalomyelitis model and lymphocytic choriomeningitis virus infection model, respectively. Subsequently, several studies revealed that ARMC5 mutations were related to primary bilateral macronodular adrenal hyperplasia (PBMAH), which is a rare cause of Cushing’s syndrome. We then confirmed that adrenal gland hyperplasia was indeed developed in aged Armc5 KO mice with mildly increased serum glucocorticoid levels. Since ARMC5 did not exhibit enzymatic activity, its function likely depends on the interaction with other proteins. We identified several proteins that binds to ARMC5, most notably ARMC5 binding to Cullin3, forming a ubiquitin ligase (E3) specific for RNA polymerase II subunit I (RPB1). ARMC5 regulated the ubiquitination of RPB1, and its deletion resulted in RPB1 accumulation in major organs (e.g., adrenal glands, lymph nodes, brain, lung, and liver), indicating the critical role of this E3 in RPB1 degradation. A similar accumulation was also found in hyperplasia tissues from adrenal glands of PBMAH patients carrying ARMC5 mutations, underscoring the clinical relevance of our basic research findings in human disease. Defective degradation of RPB1 led to an enlarged RNA polymerase II (Pol II) pool. In addition, we have identified a group of genes strongly upregulated in KO adrenal glands, including the effector genes which would be involved in adrenal gland hyperplasia in Armc5 KO mice and PBMAH patients carrying ARMC5 mutation. Finally, we have shown that deleting or mutating Armc5 significantly augments the risk of neural tube defects in mice and humans. In patients with myelomeningocele, we found nine deleterious missense mutations in ARMC5, one of which weakened the interaction between ARMC5 and RPB1. The enlarged Pol II pool in Armc5 KO neural precursor cells (NPCs) influenced a particular group of genes, some of which (e.g., Folh1) are thought to be involved in the development of the neural tube. In summary, ARMC5 and CUL3 form an E3 complex, which targets RPB1 causing its ubiquitination and degradation. In the absence of such a mechanism, there is a disturbance of RNA polymerase II homeostasis, which leads to a decrease in the T cell-mediated immune response, the development of PBMAH and an increased risk of neural tube defects.