Role of Nuclear Angiotensin-II Receptor Mediated Signalling in Cardiovascular Remodelling

Le remodelage cardiaque est le processus par lequel la structure ou la fonction cardiaque change en réponse à un déséquilibre pathophysiologique tel qu'une maladie cardiaque, un contexte d'arythmie prolongée ou une modification de l'équilibre hormonal. Le système rénine-angiotensine (SRA) est un système hormonal largement étudié et il est impliqué dans de nombreuses activités associées au remodelage cardiovasculaire. L’existence d'un système circulatoire couplé à un système de tissus locaux est une représentation classique, cependant de nouvelles données suggèrent un SRA indépendant et fonctionnellement actif à l'échelle cellulaire. La compréhension de l'activité intracellulaire du SRA pourrait mener à de nouvelles pistes thérapeutiques qui pourraient prévenir un remodelage cardiovasculaire défavorable. L'objectif de cette thèse était d'élucider le rôle du SRA intracellulaire dans les cellules cardiaques. Récemment, les récepteurs couplés aux protéines G (RCPG), les protéines G et leurs effecteurs ont été détectés sur des membranes intracellulaires, y compris sur la membrane nucléaire, et les concepts de RCPG intracellulaires fonctionnels sont en voie d'être acceptés comme une réalité. Nous avons dès lors fait l'hypothèse que la signalisation du SRA délimitant le noyau était impliquée dans le contrôle de l'expression des gènes cardiaques. Nous avons démontré la présence de récepteurs d'angiotensine de type-1 (AT1R) et de type-2 (AT2R) nucléaires dans les cardiomyocytes ventriculaires adultes et dans une fraction nucléaire purifiée de tissu cardiaque. Des quantités d'Ang II ont été détectées dans du lysat de cardiomyocytes et des microinjections d'Ang-II-FITC ont donné lieu à des liaisons préférentielles aux sites nucléaires. L'analyse transcriptionnelle prouve que la synthèse d'ARN de novo dans des noyaux isolés stimulés à l'Ang-II, et l'expression des ARNm de NF-κB étaient beaucoup plus importants lorsque les noyaux étaient exposés à de l'Ang II par rapport aux cardiomyocytes intacts. La stimulation des AT1R nucléaires a engendré une mobilisation de Ca2+ via les récepteurs de l'inositol trisphosphate (IP3R), et le blocage des IP3R a diminué la réponse transcriptionnelle. Les méthodes disponibles actuellement pour l'étude de la signalisation intracrine sont limitées aux méthodes indirectes. L'un des objectifs de cette thèse était de synthétiser et caractériser des analogues d'Ang-II cellule-perméants afin d’étudier spécifiquement dans les cellules intactes l'activité intracellulaire du SRA. Nous avons synthétisé et caractérisé pharmacologiquement des analogues photosensibles Ang-II encapsulée en incorporant un groupement 4,5-diméthoxy-2-nitrobenzyl (DMNB) photoclivable sur les sites actifs identifiés du peptide. Chacun des trois analogues d'Ang II encapsulée synthétisés et purifiés: [Tyr(DMNB)4]Ang-II, Ang-II-ODMNB et [Tyr(DMNB)4]Ang-II-ODMNB a montré une réduction par un facteur deux ou trois de l'affinité de liaison envers AT1R et AT2R dans les dosages par liaison compétitive et une activité réduite dans la contraction de l'aorte thoracique. La photostimulation de [Tyr(DMNB)4]Ang-II dans des cellules HEK a augmenté la phosphorylation d'ERK1/2 (via AT1R) et la production de cGMP (via AT2R) alors que dans les cardiomyocytes isolés elle générait une augmentation de Ca2+ nucléoplasmique et initiait la synthèse d'ARNr 18S et d'ARNm du NF-κB. Les fibroblastes sont les principaux générateurs de remodelage cardiaque structurel, et les fibroblastes auriculaires sont plus réactifs aux stimuli profibrotiques que les fibroblastes ventriculaires. Nous avons émis l'hypothèse que l’Ang-II intracellulaire et l'activation des AT1R et AT2R nucléaires associés contrôlaient les profils d'expression des gènes des fibroblastes via des systèmes de signalisation distincts et de ce fait jouaient un rôle majeur dans le développement de la fibrose cardiaque. Nous avons remarqué que les fibroblastes auriculaires expriment l’AT1R et l’AT2R nucléaire et l'Ang-II au niveau intracellulaire. L’expression d'AT1R nucléaire a été régulés positivement dans les cas d’insuffisance cardiaque (IC), tandis que l'AT2R nucléaire a été glycosylé post-traductionnellement. La machinerie protéique des protéines G, y compris Gαq/11, Gαi/3, et Gβ, a été observée dans des noyaux isolés de fibroblastes. AT1R et AT2R régulent l'initiation de la transcription du fibroblaste via les voies de transduction de signal d'IP3R et du NO. La photostimulation de [Tyr(DMNB)4]Ang-II dans une culture de fibroblastes auriculaire déclenche la libération de Ca2+ nucléoplasmique, la prolifération, et la synthèse et sécrétion de collagène qui ne sont pas inhibées par les bloqueurs d'AT1R et/ou AT2R extracellulaires.

Efeito das prostaglandinas ciclopentenônicas sobre a expressão gênica do fator nuclear PPAR-γ, do receptor de scavenger CD36 e proteínas de apoptose em macrófagos : implicações para a fisiopatologia da aterosclerose

Considerando que as doenças cardiovasculares representam a maior causa de mortalidade e morbidade em países ocidentais, a aterosclerose se destaca pelo fato de predispor os pacientes ao infarto do miocárdio, a acidentes vasculares cerebrais e a doenças vasculares periféricas. Neste contexto, a oxidação de lipoproteínas do plasma, particularmente LDL, é um dos fatores de risco para eventos cardiovasculares, pois é reconhecida e internalizada por macrófagos, ocasionando a sua diferenciação em foam cells. Diversos fatores participam deste processo de diferenciação, como a expressão de receptores de scavenger CD 36, proporcionando aumento na captação de LDL oxidada, aumento na síntese endógena de colesterol e ativação de fatores nucleares que iniciam a transcrição de proteínas específicas e fatores de crescimento que disparam a aterogênese. Os fenômenos celulares relacionados à apoptose também são de especial importância, tanto no desenvolvimento da lesão aterosclerótica como na estabilidade da placa e formação de trombos. As prostaglandinas (PGs) ciclopentenônicas (CP-PGs), em particular a PGA2 e a 15-desóxi-∆12,14-PGJ2 são uma classe especial de PGs que, em diminutas concentrações, disparam a expressão das proteínas de choque térmico (hsp), que são citoprotetoras. Além disso, CP-PGs bloqueiam a ativação do fator nuclear pró-inflamatório NF-κB tornando-as potentes agentes antiinflamatórios. Embora as PGs das famílias A e J guardem uma série de características em comum, a 15-desóxi-∆12,14- PGJ2 é o ligante fisiológico do fator nuclear pró-aterogênico PPAR-γ, enquanto as PGs da família A ativam apenas a via citoprotetora das hsp. Este trabalho teve como objetivo avaliar os efeitos das CP-PGs sobre a expressão gênica de fatores relacionados à diferenciação de macrófagos em foam cells, bem como proteínas reguladoras do processo de apoptose, em células da linhagem pró-monocítica humana U937. Para tal, as células foram tratadas com CPPGs em presença e/ou ausência de LDL nat e LDL ox, o RNA foi extraído para a realização de RT-PCR para PPAR-γ, CD 36, HMG-CoA redutase e proteínas de apoptose Caspase 3, p53 e Bcl-xL. O tratamento estatístico utilizado foi análise de variância (ANOVA one-way) e teste “t” de student, com resultados expressos como médias + desvios-padrão da média, com P<0,05. Os resultados obtidos demontraram que as CP-PGs PGA2 (20µM-24h) e PGJ2 (1,5µM-24h) inibiram a expressão gênica do fator nuclear PPAR- γ (64 % (PGA2), 88 % (15- d-PGJ2)) nas células U937, em presença de LDL oxidada, quando comparado ao controle. PGA2 inibiu a expressão de HMG-CoA redutase (33 %), enzima chave da síntese de colesterol intracelular, e o tratamento com as CP-PGs também inibiu a apoptose nas células tratadas em presença de LDL oxidada. Os dados sugerem que as CP-PGs apresentam grande potencial para o tratamento da aterosclerose, já que, além de apresentarem efeito antiinflamatório, inibem a expressão do fator nuclear pró-aterogênico PPAR-γ, do receptor de scavenger CD36 (apenas a 15-desóxi-∆12,14-PGJ2) e da enzima HMG-CoA redutase. O bloqueio da apoptose nas células estudadas pode estar relacionado à citoproteção oferecida por estas PGs. Embora investigações in vivo deste laboratório tenham mostrado a eficácia do tratamento com CP-PGs em camundongos portadores de aterosclerose, estudos adicionais são necessários para esclarecer-se o efeito antiaterogênico das mesmas.

A dominant function of p38 mitogen-activated protein kinase signaling in receptor activator of nuclear factor-kappa B ligand expression and osteoclastogenesis induction by Aggregatibacter actinomycetemcomitans and Escherichia coli lipopolysaccharide

Background and Objective: Lipopolysaccharide from gram-negative bacteria is one of the microbial-associated molecular patterns that initiate the immune/inflammatory response, leading to the tissue destruction observed in periodontitis. The aim of this study was to evaluate the role of the p38 mitogen-activated protein kinase (MAPK) signaling pathway in lipopolysaccharide-induced receptor activator of nuclear factor-kappa B ligand (RANKL) expression by murine periodontal ligament cells.Material and Methods: Expression of RANKL and osteoprotegerin mRNA was studied by reverse transcription-polymerase chain reaction upon stimulation with lipopolysaccharide from Escherichia coli and Aggregatibacter actinomycetemcomitans. The biochemical inhibitor SB203580 was used to evaluate the contribution of the p38 MAPK signaling pathway to lipopolysaccharide-induced RANKL and osteoprotegerin expression. Stable cell lines expressing dominant-negative forms of MAPK kinase (MKK)-3 and MKK6 were generated to confirm the role of the p38 MAPK pathway. An osteoclastogenesis assay using a coculture model of the murine monocytic cell line RAW 264.7 was used to determine if osteoclast differentiation induced by lipopolysaccharide-stimulated periodontal ligament was correlated with RANKL expression.Results: Inhibiting p38 MAPK prior to lipopolysaccharide stimulation resulted in a significant decrease of RANKL mRNA expression. Osteoprotegerin mRNA expression was not affected by lipopolysaccharide or p38 MAPK. Lipopolysaccharide-stimulated periodontal ligament cells increased osteoclast differentiation, an effect that was completely blocked by osteoprotegerin and significantly decreased by inhibition of MKK3 and MKK6, upstream activators of p38 MAPK. Conditioned medium from murine periodontal ligament cultures did not increase osteoclast differentiation, indicating that periodontal ligament cells produced membrane-bound RANKL.Conclusion: Lipopolysaccharide resulted in a significant increase of RANKL in periodontal ligament cells. The p38 MAPK pathway is required for lipopolysaccharide-induced membrane-bound RANKL expression in these cells.

The proliferating cell nuclear antigen index in breast carcinomas does not correlate with mitotic index and estrogen receptor immunoreactivity

To investigate the expression of a marker of cell proliferation (PCNA/Cyclin) and its putative relationship with histological grading, mitotic index and estrogen receptor immunoreactivity, we studied twenty-seven cases of invasive breast carcinoma in formalin-fixed, paraffin-embedded tissue sections. The PCNA and estrogen receptor were detected by the PC10 and H222 monoclonal antibodies respectively, using an avidin-biotin-pernxidase method. The median value of PCNA index was 20.9% with a range from 1.4 to 84.2%. We did not find any significant relationship between PCNA index anti the histological grading, mitotic index and estrogen receptor immunoreactivity. We conclude that PCNA detected by the monoclonal antibody PC10 in formalin-fixed material looks at present unrealiable as a proliferation marker in breast carcinoma.

Inhibition of cannabinoid CB1 receptor upregulates Slc2a4 expression via nuclear factor-kappa B and sterol regulatory element-binding protein-1 in adipocytes

Evidences have suggested that the endocannabinoid system is overactive in obesity, resulting in enhanced endocannabinoid levels in both circulation and visceral adipose tissue. The blockade of cannabinoid receptor type 1 (CB1) has been proposed for the treatment of obesity. Besides loss of body weight, CB1 antagonism improves insulin sensitivity, in which the glucose transporter type 4 (GLUT4) plays a key role. The aim of this study was to investigate the modulation of GLUT4-encoded gene (Slc2a4 gene) expression by CB1 receptor. For this, 3T3-L1 adipocytes were incubated in the presence of a highly selective CB1 receptor agonist (1 mu M arachidonyl-2'-chloroethylamide) and/or a CB1 receptor antagonist/inverse agonist (0.1, 0.5, or 1 mu M AM251, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide). After acute (2 and 4 h) and chronic (24 h) treatments, cells were harvested to evaluate: i) Slc2a4, Cnr1 (CB1 receptor-encoded gene), and Srebf1 type a (SREBP-1a type-encoded gene) mRNAs (real-time PCR); ii) GLUT4 protein (western blotting); and iii) binding activity of nuclear factor (NF)-kappa B and sterol regulatory element-binding protein (SREBP)-1 specifically in the promoter of Slc2a4 gene (electrophoretic mobility shift assay). Results revealed that both acute and chronic CB1 receptor antagonism greatly increased (similar to 2.5-fold) Slc2a4 mRNA and protein content. Additionally, CB1-induced upregulation of Slc2a4 was accompanied by decreased binding activity of NF-kappa B at 2 and 24 h, and by increased binding activity of the SREBP-1 at 24 h. In conclusion, these findings reveal that the blockade of CB1 receptor markedly increases Slc2a4/GLUT4 expression in adipocytes, a feature that involves NF-kappa B and SREBP-1 transcriptional regulation. Journal of Molecular Endocrinology (2012) 49, 97-106

Bombesin antagonist-based radioligands for translational nuclear imaging of gastrin-releasing peptide receptor-positive tumors

Bombesin receptors are overexpressed on a variety of human tumors. In particular, the gastrin-releasing peptide receptor (GRPr) has been identified on prostate and breast cancers and on gastrointestinal stromal tumors. The current study aims at developing clinically translatable bombesin antagonist-based radioligands for SPECT and PET of GRPr-positive tumors.

The nuclear mRNA export receptor Mex67-Mtr2 of Trypanosoma brucei contains a unique and essential zinc finger motif.

Trypanosoma brucei is the causative agent of Human African Trypanosomiasis. Trypanosomes are early diverged protozoan parasites and show significant differences in their gene expression compared with higher eukaryotes. Due to a lack of individual gene promoters, large polycistronic transcripts are produced and individual mRNAs mature by trans-splicing and polyadenylation. In the absence of transcriptional control, regulation of gene expression occurs post-transcriptionally mainly by control of transcript stability and translation. Regulation of mRNA export from the nucleus to the cytoplasm might be an additional post-transcriptional event involved in gene regulation. However, our knowledge about mRNA export in trypanosomes is very limited. Although export factors of higher eukaryotes are reported to be conserved, only a few orthologues can be readily identified in the genome of T. brucei. Hence, biochemical approaches are needed to identify the export machinery of trypanosomes. Here, we report the functional characterization of the essential mRNA export factor TbMex67. TbMex67 contains a unique and essential N-terminal zinc finger motif. Furthermore, we could identify two interacting export factors namely TbMtr2 and the karyopherin TbIMP1. Our data show that the general heterodimeric export receptor Mex67-Mtr2 is conserved throughout the eukaryotic kingdom albeit exhibiting parasite-specific features.

Targeting dysregulated nuclear proteins androgen receptor and enhancer of zeste homolog 2: Clinical implication and mechanism underline

Cancer is the most devastating disease that has tremendous impacts on public health. Many efforts have been devoted to fighting cancer through either translational or basic researches for years. Nowadays, it emerges the importance to converge these two research directions and complement to each other for battling with cancer. Thus, our study aims at both translational and basic research directions. The first goal of our study is focus on translational research to search for new agents targeting prevention and therapy of advanced prostate cancer. Hormone refractory prostate cancer is incurable and lethal. Androgen receptor (AR) mediates androgen's effect not only on the tumor initiation but also plays the major role in the relapse transition of prostate cancer. Here we demonstrate that emodin, a natural compound, can directly target AR to suppress prostate cancer cell growth in vitro and prolong the survival of C3(1)/SV40 transgenic mice in vivo. Emodin treatment resulted in repressing androgen-dependent transactivation of AR by inhibiting AR nuclear translocation. Emodin decreased the association of AR and heat shock protein 90 and increased the association of AR and MDM2, which in turn, induces AR degradation through a proteasome-mediated pathway in a ligand independent manner. Our work indicates a new mechanism for the emodin-mediated anticancer effect and justifies further investigation of emodin as a therapeutic and preventive agent for prostate cancer. The second goal of our study is try to elucidate the fundamental tumor biology of cancer progression then provide the rationale to develop more efficient therapeutic strategy. Enhancer of zeste homologue 2 (EZH2) plays an important role in many biological processes through its intrinsic methyltransferase activity to trimethylate lysine 27 in histone H3. Although overexpression of EZH2 has been shown to be involved in cancer progression, the detailed mechanisms are elusive. Here, we show that Akt phosphorylates EZH2 at serine 21 and suppresses its methyltransferase activity by impeding the binding to its substrate histone H3, resulting in a decrease of lysine 27 trimethylation and derepression of silenced genes, thus promotes cell proliferation and tumorigenicity. Our results also show that histone methylation is not permanent but regulated in a dynamic manner and that the Akt signaling pathway is involved in the regulation of this epigenetic modification through phosphorylation of EZH2, thus contributing to oncogenic processes. ^

Tumor necrosis factor (TNF)-mediated kinase cascades: Bifurcation of Nuclear Factor-κB and c-jun N-terminal kinase (JNK/SAPK) pathways at TNF receptor-associated factor 2

TNF-induced activation of the transcription factor NF-κB and the c-jun N-terminal kinase (JNK/SAPK) requires TNF receptor-associated factor 2 (TRAF2). The NF-κB-inducing kinase (NIK) associates with TRAF2 and mediates TNF activation of NF-κB. Herein we show that NIK interacts with additional members of the TRAF family and that this interaction requires the conserved “WKI” motif within the TRAF domain. We also investigated the role of NIK in JNK activation by TNF. Whereas overexpression of NIK potently induced NF-κB activation, it failed to stimulate JNK activation. A kinase-inactive mutant of NIK was a dominant negative inhibitor of NF-κB activation but did not suppress TNF- or TRAF2-induced JNK activation. Thus, TRAF2 is the bifurcation point of two kinase cascades leading to activation of NF-κB and JNK, respectively.

Leukemia-associated retinoic acid receptor α fusion partners, PML and PLZF, heterodimerize and colocalize to nuclear bodies

In acute promyelocytic leukemia (APL), the typical t(15;17) and the rare t(11;17) translocations express, respectively, the PML/RARα and PLZF/RARα fusion proteins (where RARα is retinoic acid receptor α). Herein, we demonstrate that the PLZF and PML proteins interact with each other and colocalize onto nuclear bodies (NBs). Furthermore, induction of PML expression by interferons leads to a recruitment of PLZF onto NBs without increase in the levels of the PLZF protein. PML/RARα and PLZF/RARα localize to the same microspeckled nuclear domains that appear to be common targets for the two fusion proteins in APL. Although PLZF/RARα does not affect the localization of PML, PML/RARα delocalizes the endogenous PLZF protein in t(15;17)-positive NB4 cells, pointing to a hierarchy in the nuclear targeting of these proteins. Thus, our results unify the molecular pathogenesis of APL with at least two different RARα gene translocations and stress the importance of alterations of PLZF and RARα nuclear localizations in this disease.

Direct Visualization of the Human Estrogen Receptor α Reveals a Role for Ligand in the Nuclear Distribution of the Receptor

The human estrogen receptor α (ER α) has been tagged at its amino terminus with the S65T variant of the green fluorescent protein (GFP), allowing subcellular trafficking and localization to be observed in living cells by fluorescence microscopy. The tagged receptor, GFP-ER, is functional as a ligand-dependent transcription factor, responds to both agonist and antagonist ligands, and can associate with the nuclear matrix. Its cellular localization was analyzed in four human breast cancer epithelial cell lines, two ER+ (MCF7 and T47D) and two ER− (MDA-MB-231 and MDA-MB-435A), under a variety of ligand conditions. In all cell lines, GFP-ER is observed only in the nucleus in the absence of ligand. Upon the addition of agonist or antagonist ligand, a dramatic redistribution of GFP-ER from a reticular to punctate pattern occurs within the nucleus. In addition, the full antagonist ICI 182780 alters the nucleocytoplasmic compartmentalization of the receptor and causes partial accumulation in the cytoplasm in a process requiring continued protein synthesis. GFP-ER localization varies between cells, despite being cultured and treated in a similar manner. Analysis of the nuclear fluorescence intensity for variation in its frequency distribution helped establish localization patterns characteristic of cell line and ligand. During the course of this study, localization of GFP-ER to the nucleolar region is observed for ER− but not ER+ human breast cancer epithelial cell lines. Finally, our work provides a visual description of the “unoccupied” and ligand-bound receptor and is discussed in the context of the role of ligand in modulating receptor activity.

Epidermal growth factor-induced nuclear factor κB activation: A major pathway of cell-cycle progression in estrogen-receptor negative breast cancer cells

The epidermal growth factor (EGF) family of receptors (EGFR) is overproduced in estrogen receptor (ER) negative (−) breast cancer cells. An inverse correlation of the level of EGFR and ER is observed between ER− and ER positive (+) breast cancer cells. A comparative study with EGFR-overproducing ER− and low-level producing ER+ breast cancer cells suggests that EGF is a major growth-stimulating factor for ER− cells. An outline of the pathway for the EGF-induced enhanced proliferation of ER− human breast cancer cells is proposed. The transmission of mitogenic signal induced by EGF–EGFR interaction is mediated via activation of nuclear factor κB (NF-κB). The basal level of active NF-κB in ER− cells is elevated by EGF and inhibited by anti-EGFR antibody (EGFR-Ab), thus qualifying EGF as a NF-κB activation factor. NF-κB transactivates the cell-cycle regulatory protein, cyclin D1, which causes increased phosphorylation of retinoblastoma protein, more strongly in ER− cells. An inhibitor of phosphatidylinositol 3 kinase, Ly294–002, blocked this event, suggesting a role of the former in the activation of NF-κB by EGF. Go6976, a well-characterized NF-κB inhibitor, blocked EGF-induced NF-κB activation and up-regulation of cell-cycle regulatory proteins. This low molecular weight compound also caused apoptotic death, predominantly more in ER− cells. Thus Go6976 and similar NF-κB inhibitors are potentially novel low molecular weight therapeutic agents for treatment of ER− breast cancer patients.

The Nuclear Export Receptor Xpo1p Forms Distinct Complexes with NES Transport Substrates and the Yeast Ran Binding Protein 1 (Yrb1p)

Xpo1p (Crm1p) is the nuclear export receptor for proteins containing a leucine-rich nuclear export signal (NES). Xpo1p, the NES-containing protein, and GTP-bound Ran form a complex in the nucleus that translocates across the nuclear pore. We have identified Yrb1p as the major Xpo1p-binding protein in Saccharomyces cerevisiae extracts in the presence of GTP-bound Gsp1p (yeast Ran). Yrb1p is cytoplasmic at steady-state but shuttles continuously between the cytoplasm and the nucleus. Nuclear import of Yrb1p is mediated by two separate nuclear targeting signals. Export from the nucleus requires Xpo1p, but Yrb1p does not contain a leucine-rich NES. Instead, the interaction of Yrb1p with Xpo1p is mediated by Gsp1p-GTP. This novel type of export complex requires the acidic C-terminus of Gsp1p, which is dispensable for the binding to importin β-like transport receptors. A similar complex with Xpo1p and Gsp1p-GTP can be formed by Yrb2p, a relative of Yrb1p predominantly located in the nucleus. Yrb1p also functions as a disassembly factor for NES/Xpo1p/Gsp1p-GTP complexes by displacing the NES protein from Xpo1p/Gsp1p. This Yrb1p/Xpo1p/Gsp1p complex is then completely dissociated after GTP hydrolysis catalyzed by the cytoplasmic GTPase activating protein Rna1p.

Targeted mutation of the murine arylhydrocarbon receptor nuclear translocator 2 (Arnt2) gene reveals partial redundancy with Arnt

The ubiquitously expressed basic helix–loop–helix (bHLH)-PAS protein ARNT (arylhydrocarbon receptor nuclear transporter) forms transcriptionally active heterodimers with a variety of other bHLH-PAS proteins, including HIF-1α (hypoxia-inducible factor-1α) and AHR (arylhydrocarbon receptor). These complexes regulate gene expression in response to hypoxia and xenobiotics, respectively, and mutation of the murine Arnt locus results in embryonic death by day 10.5 associated with placental, vascular, and hematopoietic defects. The closely related protein ARNT2 is highly expressed in the central nervous system and kidney and also forms complexes with HIF-1α and AHR. To assess unique roles for ARNT2 in development, and reveal potential functional overlap with ARNT, we generated a targeted null mutation of the murine Arnt2 locus. Arnt2−/− embryos die perinatally and exhibit impaired hypothalamic development, phenotypes previously observed for a targeted mutation in the murine bHLH-PAS gene Sim1 (Single-minded 1), and consistent with the recent proposal that ARNT2 and SIM1 form an essential heterodimer in vivo [Michaud, J. L., DeRossi, C., May, N. R., Holdener, B. C. & Fan, C. (2000) Mech. Dev. 90, 253–261]. In addition, cultured Arnt2−/− neurons display decreased hypoxic induction of HIF-1 target genes, demonstrating formally that ARNT2/HIF-1α complexes regulate oxygen-responsive genes. Finally, a strong genetic interaction between Arnt and Arnt2 mutations was observed, indicating that either gene can fulfill essential functions in a dose-dependent manner before embryonic day 8.5. These results demonstrate that Arnt and Arnt2 have both unique and overlapping essential functions in embryonic development.

Nuclear hormone receptor CHR3 is a critical regulator of all four larval molts of the nematode Caenorhabditis elegans

CHR3 (nhr-23, NF1F4), the homologue of Drosophila DHR3 and mammalian ROR/RZR/RevErbA nuclear hormone receptors, is important for proper epidermal development and molting in the nematode Caenorhabditis elegans. Disruption of CHR3 (nhr-23) function leads to developmental changes, including incomplete molting and a short, fat (dumpy) phenotype. Here, we studied the role of CHR3 during larval development by using expression assays and RNA-mediated interference. We show that the levels of expression of CHR3 (nhr-23) cycle during larval development and reduction of CHR3 function during each intermolt period result in defects at all subsequent molts. Assaying candidate gene expression in populations of animals treated with CHR3 (nhr-23) RNA-mediated interference has identified dpy-7 as a potential gene acting downstream of CHR3. These results define CHR3 as a critical regulator of all C. elegans molts and begin to define the molecular pathway for its function.