The role of the peptidyl prolyl isomerase Rrd1 in the transcriptional stress response

La régulation de la transcription est un processus complexe qui a évolué pendant des millions d’années permettant ainsi aux cellules de s’adapter aux changements environnementaux. Notre laboratoire étudie le rôle de la rapamycine, un agent immunosuppresseur et anticancéreux, qui mime la carence nutritionelle. Afin de comprendre les mécanismes impliqués dans la réponse a la rapamycine, nous recherchons des mutants de la levure Saccaromyces cerevisiae qui ont un phenotype altérée envers cette drogue. Nous avons identifié le gène RRD1, qui encode une peptidyl prolyl isomérase et dont la mutation rend les levures très résistantes à la rapamycine et il semble que se soit associé à une réponse transcriptionelle alterée. Mon projet de recherche de doctorat est d’identifier le rôle de Rrd1 dans la réponse à la rapamycine. Tout d’abord nous avons trouvé que Rrd1 interagit avec l’ARN polymérase II (RNAPII), plus spécifiquement avec son domaine C-terminal. En réponse à la rapamycine, Rrd1 induit un changement dans la conformation du domaine C-terminal in vivo permettant la régulation de l’association de RNAPII avec certains gènes. Des analyses in vitro ont également montré que cette action est directe et probablement liée à l’activité isomérase de Rrd1 suggérant un rôle pour Rrd1 dans la régulation de la transcription. Nous avons utilisé la technologie de ChIP sur micropuce pour localiser Rrd1 sur la majorité des gènes transcrits par RNAPII et montre que Rrd1 agit en tant que facteur d’élongation de RNAPII. Pour finir, des résultats suggèrent que Rrd1 n’est pas seulement impliqué dans la réponse à la rapamycine mais aussi à differents stress environnementaux, nous permettant ainsi d’établir que Rrd1 est un facteur d’élongation de la transcription requis pour la régulation de la transcription via RNAPII en réponse au stress.

Understanding the transcriptional control of EIF4E and its dysregulation in acute myeloid leukemia: role of NF-κB

EIF4E, le facteur d’initiation de la traduction chez les eucaryotes est un oncogène puissant et qui se trouve induit dans plusieurs types de cancers, parmi lesquels les sous-types M4 et M5 de la leucémie aiguë myéloblastique (LAM). EIF4E est régulé à plusieurs niveaux cependant, la régulation transcriptionnelle de ce gène est peu connue. Mes résultats montrent que EIF4E est une cible transcriptionnelle directe du facteur nucléaire « kappa-light- chain- enhancer of activated B cells » (NF-κB).Dans les cellules hématopoïétiques primaires et les lignées cellulaires, les niveaux de EIF4E sont induits par des inducteurs de NF-κB. En effet, l’inactivation pharmaceutique ou génétique de NF-κB réprime l’activation de EIF4E. En effet, suite à l’activation de NF-κB chez l’humain, le promoteur endogène de EIF4E recrute p65 (RelA) et c-Rel aux sites évolutionnaires conservés κB in vitro et in vivo en même temps que p300 ainsi que la forme phosphorylée de Pol II. De plus, p65 est sélectivement associé au promoteur de EIF4E dans les sous-types LAM M4/M5 mais non pas dans les autres sous-types LAM ou dans les cellules hématopoïétiques primaires normales. Ceci indique que ce processus représente un facteur essentiel qui détermine l’expression différentielle de EIF4E dans la LAM. Les analyses de données d’expressions par séquençage de l’ARN provenant du « Cancer Genome Atlas » (TCGA) suggèrent que les niveaux d’ARNm de EIF4E et RELA se trouvent augmentés dans les cas LAM à pronostic intermédiaire ou faible mais non pas dans les groupes cytogénétiquement favorables. De plus, des niveaux élevés d’ARNm de EIF4E et RELA sont significativement associés avec un taux de survie relativement bas chez les patients. En effet, les sites uniques κB se trouvant dans le promoteur de EIF4E recrutent le régulateur de transcription NF-κB p65 dans 47 nouvelles cibles prévues. Finalement, 6 nouveaux facteurs de transcription potentiellement impliqués dans la régulation du gène EIF4E ont été prédits par des analyses de données ChIP-Seq provenant de l’encyclopédie des éléments d’ADN (ENCODE). Collectivement, ces résultats fournissent de nouveaux aperçus sur le control transcriptionnel de EIF4E et offrent une nouvelle base moléculaire pour sa dérégulation dans au moins un sous-groupe de spécimens de LAM. L’étude et la compréhension de ce niveau de régulation dans le contexte de spécimens de patients s’avère important pour le développement de nouvelles stratégies thérapeutiques ciblant l’expression du gène EIF4E moyennant des inhibiteurs de NF-κB en combinaison avec la ribavirine.

Symmetry in biology: from genetic code to stochastic gene regulation

Mathematical models, as instruments for understanding the workings of nature, are a traditional tool of physics, but they also play an ever increasing role in biology - in the description of fundamental processes as well as that of complex systems. In this review, the authors discuss two examples of the application of group theoretical methods, which constitute the mathematical discipline for a quantitative description of the idea of symmetry, to genetics. The first one appears, in the form of a pseudo-orthogonal (Lorentz like) symmetry, in the stochastic modelling of what may be regarded as the simplest possible example of a genetic network and, hopefully, a building block for more complicated ones: a single self-interacting or externally regulated gene with only two possible states: ` on` and ` off`. The second is the algebraic approach to the evolution of the genetic code, according to which the current code results from a dynamical symmetry breaking process, starting out from an initial state of complete symmetry and ending in the presently observed final state of low symmetry. In both cases, symmetry plays a decisive role: in the first, it is a characteristic feature of the dynamics of the gene switch and its decay to equilibrium, whereas in the second, it provides the guidelines for the evolution of the coding rules.

Regulation of insulin signalling by exercise in skeletal muscle

Regular physical activity improves insulin action and is an effective therapy for the treatment and prevention of type 2 diabetes. However, little is known of the mechanisms by which exercise improves insulin action in muscle. These studies investigate the actions of a single bout of exercise and short-term endurance training on insulin signalling. Twenty-four hours following the completion of a single bout of endurance exercise insulin action improved, although greater enhancement of insulin action was demonstrated following the completion of endurance training, implying that cumulative bouts of exercise substantially increase insulin action above that seen from the residual effects of an acute bout of prior exercise. No alteration in the abundance and phosphorylation of proximal members of the insulin-signalling cascade in skeletal muscle, including the insulin receptor and IRS-1 were found. A major finding however, was the significant increase in the serine phosphorylation of a known downstream signalling protein, Akt (1.5 fold, p ≤0.05) following an acute bout of exercise and exercise training. This was matched by the observed increase in protein abundance of SHPTP2 (1.6 fold, p ≤0.05) a protein tyrosine phosphatase, in the cytosolic fraction of skeletal muscle following endurance exercise. These data suggest a small positive role for SHPTP2 on insulin stimulated glucose transport consistent with transgenic mice models. Further studies were aimed at examining the gene expression following a single bout of either resistance or endurance exercise. There were significant transient increases in IRS-2 mRNA concentration in the few hours following a single bout of both endurance and resistance exercise. IRS-2 protein abundance was also observed to significantly increase 24-hours following a single bout of endurance exercise indicating transcriptional regulation of IRS-2 following muscular contraction. One final component of this PhD project was to examine a second novel insulin-signalling pathway via c-Cbl tyrosine phosphorylation that has recently been shown to be essential for insulin stimulated glucose uptake in adipocytes. No evidence was found for the tyrosine phosphorylation of c-Cbl in the skeletal muscle of Zucker rats despite demonstrating significant phosphorylation of the insulin receptor and Akt by insulin treatment and successfully immunoprecipitating c-Cbl protein. Surprisingly, there was a small but significant increase in c-Cbl protein expression following insulin-stimulation, however c-Cbl tyrosine phosphorylation does not appear to be associated with insulin or exercise-mediated glucose transport in skeletal muscle.

Neisseria meningitidis and Neisseria gonorrhoeae are differently adapted in the regulation of denitrification: single nucleotide polymorphisms that enable species-specific tuning of the aerobic–anaerobic switch

The closely related pathogenic Neisseria species N. meningitidis and N. gonorrhoeae are able to respire in the absence of oxygen, using nitrite as an alternative electron acceptor. aniA (copper-containing nitrite reductase) is tightly regulated by four transcriptional regulators: FNR (fumarate and nitrate reductase), NarP, FUR (Ferric uptake regulator) and NsrR. The four regulators control expression of aniA in N. meningitidis by binding to specific and distinct regions of the promoter. We show in the present study that FUR and NarP are both required for the induction of expression of aniA in N. meningitidis, and that they bind adjacent to one another in a non-co-operative manner. Activation via FUR/NarP is dependent on their topological arrangement relative to the RNA polymerase-binding site. Analysis of the sequence of the aniA promoters from multiple N. meningitidis and N. gonorrhoeae strains indicates that there are species-specific single nucleotide polymorphisms, in regions predicted to be important for regulator binding. These sequence differences alter both the in vitro DNA binding and the promoter activation in intact cells by key activators FNR (oxygen sensor) and NarP (which is activated by nitrite in N. meningitidis). The weak relative binding of FNR to the N. gonorrhoeae aniA promoter (compared to N. meningitidis) is compensated for by a higher affinity of the gonococcal aniA promoter for NarP. Despite containing nearly identical genes for catalysing and regulating denitrification, variations in the promoter for the aniA gene appear to have been selected to enable the two pathogens to tune differentially their responses to environmental variables during the aerobic–anaerobic switch.

Balancing immunity and tolerance: genetic footprint of natural selection in the transcriptional regulatory region of HLA-G

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Transcriptional Alterations Related to Neuropathology and Clinical Manifestation of Alzheimer's Disease

Alzheimer's disease (AD) is the most common cause of dementia in the human population, characterized by a spectrum of neuropathological abnormalities that results in memory impairment and loss of other cognitive processes as well as the presence of non-cognitive symptoms. Transcriptomic analyses provide an important approach to elucidating the pathogenesis of complex diseases like AD, helping to figure out both pre-clinical markers to identify susceptible patients and the early pathogenic mechanisms to serve as therapeutic targets. This study provides the gene expression profile of postmortem brain tissue from subjects with clinic-pathological AD (Braak IV, V, or V and CERAD B or C; and CDR >= 1), preclinical AD (Braak IV, V, or VI and CERAD B or C; and CDR = 0), and healthy older individuals (Braak <= II and CERAD 0 or A; and CDR = 0) in order to establish genes related to both AD neuropathology and clinical emergence of dementia. Based on differential gene expression, hierarchical clustering and network analysis, genes involved in energy metabolism, oxidative stress, DNA damage/repair, senescence, and transcriptional regulation were implicated with the neuropathology of AD; a transcriptional profile related to clinical manifestation of AD could not be detected with reliability using differential gene expression analysis, although genes involved in synaptic plasticity, and cell cycle seems to have a role revealed by gene classifier. In conclusion, the present data suggest gene expression profile changes secondary to the development of AD-related pathology and some genes that appear to be related to the clinical manifestation of dementia in subjects with significant AD pathology, making necessary further investigations to better understand these transcriptional findings on the pathogenesis and clinical emergence of AD.

Post-transkriptionelle Regulation der Expression der humanen induzierbaren NO-Synthase

Die Expression der humanen induzierbaren NO-Synthase (iNOS) wird sowohl über transkriptionelle als auch über post-transkriptionelle Mechanismen reguliert. Dabei spielt die Modulation der iNOS-mRNA-Stabilität durch RNA-bindende Proteine eine bedeutende Rolle. In dieser Arbeit konnte eine Beteiligung des p38-MAPK-Signaltransduktionsweges sowie der RNA-bindenden Proteine TTP, KSRP, HuR und PTB an der Regulation der iNOS-Expression dargestellt werden. Hemmung der p38-MAPK führte zu einer Reduktion der iNOS-mRNA-Expression, hatte aber keinen Effekt auf die iNOS-Promotoraktivität. Das RNA-bindende Protein Tristetraprolin (TTP) erhöhte die Stabilität der iNOS-mRNA nach Zytokin-Stimulation, ohne jedoch mit ihr zu interagieren. Die Proteinexpression von TTP war unter dem Einfluss von Zytokinen erhöht; Inhibition der p38-MAPK verursachte eine Verminderung der Zytokin-stimulierten TTP-Expression. Das „KH-type splicing regulatory protein" (KSRP) übte einen destabilisierenden Effekt auf die iNOS-mRNA aus. Der Abbau der mRNA wird dabei wahrscheinlich durch eine Zytokin-unabhängige Interaktion von KSRP mit dem Exosom vermittelt. Ebenso konnte zwischen KSRP und TTP eine Wechselwirkung beobachtet werden, die nach Induktion der iNOS-Expression mit Zytokinen verstärkt und durch p38-MAPK-Inhibitoren hemmbar war. Des Weiteren konnte gezeigt werden, dass die Bindung von KSRP an die iNOS-mRNA-3’-UTR für die Vermittlung des destabilisierenden Effekts essentiell ist. Eine genaue Lokalisierung der KSRP-Bindungsstelle ergab, dass KSRP ebenso wie HuR mit dem AU-reichen Element am 3’-Ende der 3’-UTR interagiert. KSRP und HuR sind in der Lage, um diese Bindungsstelle zu konkurrieren. Nach Zytokin-Stimulation war dementsprechend die endogene Bindung von KSRP an die iNOS-mRNA vermindert, während die endogene Bindung von HuR an die iNOS-mRNA verstärkt war. Die Stabilisierung der iNOS-mRNA nach Zytokin-Stimulation ergibt sich demnach aus einer Verminderung der Bindung des KSRP-Exosom-Komplexes an die iNOS-mRNA als Folge der verstärkten Interaktion von TTP und KSRP. Dies ermöglicht parallel eine vermehrte Bindung von HuR an die iNOS-3’-UTR und führt damit zu einer Stabilisierung der iNOS-mRNA und so letztendlich auch zu einer Erhöhung der iNOS-Expression. Außerdem konnte eine Beteiligung des Polypyrimidin-Trakt-bindenden Proteins (PTB) an der Regulation der humanen iNOS-Expression gezeigt werden. PTB erhöhte die Expression der iNOS und interagierte Zytokin-unabhängig mit KSRP. Zusammenfassend lässt sich schließen, dass ein Zusammenspiel verschiedener Proteine in einem komplexen Netzwerk für die fein abgestimmte Regulation der humanen iNOS-Expression auf post-transkriptioneller Ebene verantwortlich.

Transcriptional responses of the Helicobacter pylori cag pathogenicity island

The severity of Helicobacter pylori infections largely depends on the genetic diversity of the infecting strain, and particularly on the presence of the cag pathogenicity island (cag-PAI). This virulence locus encodes a type-IV secretion system able to translocate in the host cell at least the cag-encoded toxin CagA and peptidoglycan fragments, that together are responsible for the pathogenic phenotype in the host. Little is known about the bacterial regulators that underlie the coordinated expression of cag gene products, needed to assemble a functional secretion system apparatus. To fill this gap, a comprehensive analysis of the transcriptional regulation of the cag-PAI operons was undertaken. To pursue this goal, a robust tool for the analysis of gene expression in H. pylori was first implemented. A bioluminescent reporter system based on the P. luminescens luxCDABE operon was constructed and validated by comparisons with transcriptional analyses, then it was systematically used for the comprehensive study and mapping of the cag promoters. The identification of bona fide cag promoters had permitted to pinpoint the set of cag transcriptional units of the PAI. The responses of these cag transcriptional units to metabolic stress signals were analyzed in detail, and integrated with transcription studies in deletion mutants of important H. pylori virulence regulators and protein-DNA interaction analyses to map the binding sites of the regulators. Finally, a small regulatory RNA cncR1 encoded by the cag-PAI was identified, and the 5’- and 3’-ends of the molecule were mapped by primer extension analyses, northern blot and studies with lux reporter constructs. To identify regulatory effects exerted by cncR1 on the H. pylori gene expression, the cncR1 knock out strain was derived and compared to the parental wild type strain by a macroarray approach. Results suggest a negative effect exerted by cncR1 on the regulome of the alternative sigma54 factor.

Transcriptional dynamics of the human DMD locus

The human DMD locus encodes dystrophin protein. Absence or reduced levels of dystrophin (DMD or BMD phenotype, respectively) lead to progressive muscle wasting. Little is known about the complex coordination of dystrophin expression and its transcriptional regulation is a field of intense interest. In this work we found that DMD locus harbours multiple long non coding RNAs which orchestrate and control transcription of muscle dystrophin mRNA isoforms. These lncRNAs are tissue-specific and highly expressed during myogenesis, suggesting a possible role in tissue-specific expression of DMD gene isoforms. Their forced ectopic expression in human muscle and neuronal cells leads to a specific and negative regulation of endogenous dystrophin full lenght isoforms. An intriguing aspect regarding the transcription of the DMD locus is the gene size (2.4Mb). The mechanism that ensures the complete synthesis of the primary transcript and the coordinated splicing of 79 exons is still completely unknown. By ChIP-on-chip analyses, we discovered novel regions never been involved before in the transcription regulation of the DMD locus. Specifically, we observed enrichments for Pol II, P-Ser2, P-Ser5, Ac-H3 and 2Me-H3K4 in an intronic region of 3Kb (approximately 21Kb) downstream of the end of DMD exon 52 and in a region of 4Kb spanning the DMD exon 62. Interestingly, this latter region and the TSS of Dp71 are strongly marked by 3Me-H3K36, an histone modification associated with the regulation of splicing process. Furthermore, we also observed strong presence of open chromatin marks (Ac-H3 and 2Me-H3K4) around intron 34 and the exon 45 without presence of RNA pol II. We speculate that these two regions may exert an enhancer-like function on Dp427m promoter, although further investigations are necessary. Finally, we investigated the nuclear-cytoplasmic compartmentalization of the muscular dystrophin mRNA and, specifically, we verified whether the exon skipping therapy could influence its cellular distribution.

cis-Regulation des Drosophila-melanogaster-Gens optomotor-blind

Die zeitliche und räumliche Expression von Genen trägt zu einem entscheidenden Ausmaß zu der Entwicklung eines Organismus bei. Unter vielen Faktoren spielt dabei die transkriptionelle Regulation eine wichtige Rolle. Diese basiert auf Anwesenheit und Binden von regulatorischen Proteinen an cis-regulatorischen Sequenzen (CRMs) und deren Einfluss auf die Transkriptionsmaschinerie am Promotor. Veränderungen der CRMs können zu Veränderungen der Genexpression führen, und somit einen Beitrag zur morphologischen Evolution leisten. rnIn dieser Arbeit wurde die transkriptionelle Regulation des Drosophila melanogaster Gens optomotor-blind insbesondere in den pupalen Tergiten untersucht. In einem Enhancer-Reporter screen wurde eine regulatorische Region in Intron IV, die Reportergen-Expression in den pupalen Tergiten treibt, identifiziert. Große Teile dieser Region (ombTU10 und ombTU11) trieben Reportergen-Expression in einem omb-ähnlichen Muster. Eine weitere Region (ombTU12) trieb Expression in einem für Hh-Zielgene typischen Expressionsmuster. Für ombTU12 konnte eine Hh-Abhängigkeit nachgewiesen werden. Die für Hh-Zielgene typische Enhanceraktivität konnte in dem Subfragment ombTU12Amin lokalisiert werden, welches zwei konservierte Bindestellen des Effektors der Hh-Signaltransduktionskaskase, Cubitus interruptus (Ci), enthält. Eine deutliche Abhängigkeit der Expression dieses Fragments von den Ci-Bindestellen konnte bisher aber noch nicht nachgewiesen werden.rnDeletionen verschiedener Bereiche dieser Tergitenenhancer-Region aus dem endogenen Gen sollten Aufschluss über deren Notwendigkeit in der Regulation von omb geben. Die Deletion des Fragments ombTU10 (ΔombTU10-2) führte zu einer Variabilität in der Pigmentierung der Abdominalsegmente A5 und A6 der Weibchen. Eine Deletion von Teilen des hh-responsiven Fragments ombTU12 (ΔombTU12A) zeigte keinen abdominalen Phänotyp. Dies deutet auf eine redundante Wirkung der Fragmente untereinander, oder mit einem weiteren bisher nicht identifizierten Tergitenenhancer im omb-Locus hin.rnFragmente, die in den pupalen Tergiten Reportergen-Expression trieben, waren zum Teil auch in Imaginalscheiben von Larven aktiv. Desweiteren wurde gezeigt, dass Fragmente, die in Isolation Reportergen-Expression trieben, als Fusionskonstrukt mit benachbarten genomischen Sequenzen keine Expression zeigten und somit im genomischen Kontext inaktiv sein können. Demzufolge sind nicht nur Aktivator- sondern auch Repressorregionen für die korrekte Expression eines Gens von Bedeutung.rnDie Analyse von omb Enhancer-Trap Insertionen zeigte, dass von drei untersuchten Typen (PlacW, PGalW und PGawB) nur Insertionen vom letzteren in den pupalen Tergiten aktiv waren. Von vier PGawB Insertionen waren nur drei aktiv. Es ist denkbar, dass die Orientierung der inaktiven Insertion für die mangelnde Responsivität verantwortlich ist.rn

CLEC5A (MDL-1) is a novel PU.1 transcriptional target during myeloid differentiation

C-type lectin domain family 5, member A (CLEC5A), also known as myeloid DNAX activation protein 12 (DAP12)-associating lectin-1 (MDL-1), is a cell surface receptor strongly associated with the activation and differentiation of myeloid cells. CLEC5A associates with its adaptor protein DAP12 to activate a signaling cascade resulting in activation of downstream kinases in inflammatory responses. Currently, little is known about the transcriptional regulation of CLEC5A. We identified CLEC5A as one of the most highly induced genes in a microarray gene profiling experiment of PU.1 restored myeloid PU.1-null cells. We further report that CLEC5A expression is significantly reduced in several myeloid differentiation models upon PU.1 inhibition during monocyte/macrophage or granulocyte differentiation. In addition, CLEC5A mRNA expression was significantly lower in primary acute myeloid leukemia (AML) patient samples than in macrophages and granulocytes from healthy donors. Moreover, we found activation of a CLEC5A promoter reporter by PU.1 as well as in vivo binding of PU.1 to the CLEC5A promoter. Our findings indicate that CLEC5A expression in monocyte/macrophage and granulocytes is regulated by PU.1.

Differential regulation of glucocorticoid synthesis in murine intestinal epithelial versus adrenocortical cell lines

Glucocorticoids are steroid hormones with important functions in development, immune regulation, and glucose metabolism. The adrenal glands are the predominant source of glucocorticoids; however, there is increasing evidence for extraadrenal glucocorticoid synthesis in thymus, brain, skin, and vascular endothelium. We recently identified intestinal epithelial cells as an important source of glucocorticoids, which regulate the activation of local intestinal immune cells. The molecular regulation of intestinal glucocorticoid synthesis is currently unexplored. In this study we investigated the transcriptional regulation of the steroidogenic enzymes P450 side-chain cleavage enzyme and 11beta-hydroxylase, and the production of corticosterone in the murine intestinal epithelial cell line mICcl2 and compared it with that in the adrenocortical cell line Y1. Surprisingly, we observed a reciprocal stimulation pattern in these two cell lines. Elevation of intracellular cAMP induced the expression of steroidogenic enzymes in Y1 cells, whereas it inhibited steroidogenesis in mICcl2 cells. In contrast, phorbol ester induced steroidogenic enzymes in intestinal epithelial cells, which was synergistically enhanced upon transfection of cells with the nuclear receptors steroidogenic factor-1 (NR5A1) and liver receptor homolog-1 (NR5A2). Finally, we observed that basal and liver receptor homolog-1/phorbol ester-induced expression of steroidogenic enzymes in mICcl2 cells was inhibited by the antagonistic nuclear receptor small heterodimer partner. We conclude that the molecular basis of glucocorticoid synthesis in intestinal epithelial cells is distinct from that in adrenal cells, most likely representing an adaptation to the local environment and different requirements.

Regulation of phosphoinositide 3-kinase expression in health and disease

Both the biology and the therapeutic potential of the phosphoinositide 3-kinase (PI3K) signalling axis have been the subject of intense investigation; however, little is known about the regulation of PI3K expression. Emerging evidence indicates that PI3K levels change in response to cellular stimulation with insulin and nuclear receptor ligands, and during various physiological and pathological processes including differentiation, regeneration, hypertension and cancer. Recently identified mechanisms that control PI3K production include increased gene copy number in cancer, and transcriptional regulation of the p110alpha PI3K gene by FOXO3a, NF-kappaB and p53, and of the PI3K regulatory subunits by STAT3, EBNA-2 and SREBP. In most instances, however, the impact of alterations in PI3K expression on PI3K signalling and disease remains to be established.

Differential regulation of human 3β-hydroxysteroid dehydrogenase type 2 for steroid hormone biosynthesis by starvation and cyclic AMP stimulation: studies in the human adrenal NCI-H295R cell model

Human steroid biosynthesis depends on a specifically regulated cascade of enzymes including 3β-hydroxysteroid dehydrogenases (HSD3Bs). Type 2 HSD3B catalyzes the conversion of pregnenolone, 17α-hydroxypregnenolone and dehydroepiandrosterone to progesterone, 17α-hydroxyprogesterone and androstenedione in the human adrenal cortex and the gonads but the exact regulation of this enzyme is unknown. Therefore, specific downregulation of HSD3B2 at adrenarche around age 6-8 years and characteristic upregulation of HSD3B2 in the ovaries of women suffering from the polycystic ovary syndrome remain unexplained prompting us to study the regulation of HSD3B2 in adrenal NCI-H295R cells. Our studies confirm that the HSD3B2 promoter is regulated by transcription factors GATA, Nur77 and SF1/LRH1 in concert and that the NBRE/Nur77 site is crucial for hormonal stimulation with cAMP. In fact, these three transcription factors together were able to transactivate the HSD3B2 promoter in placental JEG3 cells which normally do not express HSD3B2. By contrast, epigenetic mechanisms such as methylation and acetylation seem not involved in controlling HSD3B2 expression. Cyclic AMP was found to exert differential effects on HSD3B2 when comparing short (acute) versus long-term (chronic) stimulation. Short cAMP stimulation inhibited HSD3B2 activity directly possibly due to regulation at co-factor or substrate level or posttranslational modification of the protein. Long cAMP stimulation attenuated HSD3B2 inhibition and increased HSD3B2 expression through transcriptional regulation. Although PKA and MAPK pathways are obvious candidates for possibly transmitting the cAMP signal to HSD3B2, our studies using PKA and MEK1/2 inhibitors revealed no such downstream signaling of cAMP. However, both signaling pathways were clearly regulating HSD3B2 expression.