An operon of three transcriptional regulators controls horizontal gene transfer of the integrative and conjugative element ICEclc in Pseudomonas knackmussii B13.

The integrative and conjugative element ICEclc is a mobile genetic element in Pseudomonas knackmussii B13, and an experimental model for a widely distributed group of elements in Proteobacteria. ICEclc is transferred from specialized transfer competent cells, which arise at a frequency of 3-5% in a population at stationary phase. Very little is known about the different factors that control the transfer frequency of this ICE family. Here we report the discovery of a three-gene operon encoded by ICEclc, which exerts global control on transfer initiation. The operon consists of three consecutive regulatory genes, encoding a TetR-type repressor MfsR, a MarR-type regulator and a LysR-type activator TciR. We show that MfsR autoregulates expression of the operon, whereas TciR is a global activator of ICEclc gene expression, but no clear role was yet found for MarR. Deletion of mfsR increases expression of tciR and marR, causing the proportion of transfer competent cells to reach almost 100% and transfer frequencies to approach 1 per donor. mfsR deletion also caused a two orders of magnitude loss in population viability, individual cell growth arrest and loss of ICEclc. This indicates that autoregulation is an important feature maintaining ICE transfer but avoiding fitness loss. Bioinformatic analysis showed that the mfsR-marR-tciR operon is unique for ICEclc and a few highly related ICE, whereas tciR orthologues occur more widely in a large variety of suspected ICE among Proteobacteria.

cis- and trans-acting elements of the estrogen-regulated vitellogenin gene B1 of Xenopus laevis.

Vitellogenin genes are expressed under strict estrogen control in the liver of female oviparous vertebrates. Gene transfer experiments using estrogen-responsive cells have shown that the 13 bp perfect palindromic element GGTCACTGTGACC found upstream of the Xenopus laevis vitellogenin gene A2 promoter mediates hormonal stimulation and thus, was called the estrogen-responsive element (ERE). In the Xenopus vitellogenin genes B1 and B2 there are two closely adjacent EREs with one or more base substitutions when compared to the consensus ERE GGTCANNNTGACC. On their own, these degenerated elements have only a low or no regulatory capacity at all but act together synergistically to form an estrogen-responsive unit (ERU) with the same strength as the perfect palindromic 13 bp element. Analysis of estrogen receptor binding to the gene B1 ERU revealed a cooperative interaction of receptor dimers to the two adjacent imperfect EREs which most likely explains the synergistic stimulation observed in vivo. Furthermore, a promoter activator element located between positions --113 and --42 of the gene B1 and functional in the human MCF-7 and the Xenopus B3.2 cells has been identified and shown to be involved in the high level of induced transcription activity when the ERE is placed at a distance from the promoter. Finally, a hormone-controlled in vitro transcription system derived from Xenopus liver nuclear extracts was exploited to characterize two additional novel cis-acting elements within the vitellogenin gene B1 promoter. One of them, a negative regulatory element (NRE), is responsible for repression of promoter activity in the absence of hormone. The second is related to the NF-I binding site and is required, together with the ERE, to mediate hormonal induction. Moreover, we detected three trans-acting activities in Xenopus liver nuclear extracts that interact with these regions and demonstrated that they participate in the regulation of the expression of the vitellogenin promoter in vitro.

Short communication. SNP analyses of the 5HT1R and STAT genes in Pacific white shrimp, Litopenaeus vannamei

La industria de la producción de camarón es una de las industrias acuícolas que se encuentra en más crecimiento en la actualidad. Los estudios para encontrar marcadores genéticos son muy efectivos para la mejora de sus propiedades y de gran interés para los productores de camarón. En este trabajo se utilizaron seis individuos de una población de Litopenaeus vannamei, donde se encontraron cuatro polimorfismos de nucleótido único (SNPs) en el gen 5HT1R (5-hidroxitriptamina receptor1) y un SNP en el gen STAT (transductor de señal y activador de la transcripción). Sin embargo, el polimorfismo en el gen STAT resultó ser homocigoto en una población diferente utilizada para análisis de asociación. Los presentes análisis revelaron que el alelo C, en dos polimorfismos SNP (C109T y C395G) del gen 5HT1R, tiende a estar asociado con el aumento del peso corporal. Consideramos que hay necesidad de hacer nuevos estudios utilizando una muestra más amplia y diversa de la población en cuestión.

Circadian clock orchestration of signaling pathways influences mouse metabolism

Circadian clocks, present in organisms leaving in a rhythmic environment, constitute the mechanisms allowing anticipation and adaptation of behavior and physiology in response to these environmental variations. As a consequence, most aspects of metabolism and behavior are under the control of this circadian clock. At a molecular level, in all the studied species, the rhythmic expression of the genes involved are generated by interconnected transcriptional and translational feedback loops. In mammals, the heterodimer composed of BMAL1 and its partners CLOCK or NPAS2 constitutes a transcriptional activator regulating transcription of Per and Cry genes. These genes encode for repressors of the activity of BMAL1:CLOCK or BMAL1: NPAS2 heterodimers, thus closing a negative feedback loop that generates rhythms of approximately 24 hours. The aim of my doctoral work consisted in the investigation of the role of circadian clock in the regulation of different aspects of mouse metabolism through the rhythmic activation of signaling pathways. First, we showed that one way how the circadian clock exerts its function as an oscillator is through the regulation of mRNA translation. Indeed, we present evidence showing that circadian clock influences the temporal translation of a subset of mRNAs involved in ribosome biogenesis by controlling the transcription of translation initiation factors as well as the clock-dependent rhythmic activation of signaling pathways involved in their regulation. Moreover, the circadian oscillator regulates the transcription of ribosomal protein mRNAs and ribosomal RNAs. Thus the circadian clock exerts a major role in coordinating transcription and translation steps underlying ribosome biogenesis. In the second part, we showed the involvement of the circadian clock in lipid metabolism. Indeed, the three PAR bZip transcription factors DBP, TEF and HLF, are regulated by the molecular clock and play key roles in the control of lipid metabolism. Here we present evidence concerning the circadian expression and activity of PPARα via the circadian transcription of genes involved in the release of fatty acids, natural ligands of PPARα. It leads to the rhythmic activation of PPARα itself which could then play its role in the transcription of genes encoding proteins involved in lipid, cholesterol and glucose metabolism. In addition, we considered the possible role of lipid transporters, here SCP2, in the modulation of circadian activation of signaling pathways such as TORC1, PPARα and SREBP, linked to metabolism, and its feedback on the circadian clock. In the last part of this work, we studied the effects of these circadian clock-orchestrated pathways in physiology, as clock disruptions have been shown to be linked to metabolic disorders. We performed in vivo experiments on genetically and high-fat induced obese mice devoid of functional circadian clock. The results obtained showed that clock disruption leads to impaired triglycerides and glucose homeostasis in addition to insulin secretion and sensitivity. -- Les rythmes circadiens, présents chez tout organisme vivant dans un environnement rythmique, constituent l'ensemble de mécanismes permettant des réponses comportementales et physiologiques anticipées et adaptées aux variations environnementales. De ce fait, la plupart des aspects liés au métabolisme et au comportement de ces organismes apparaissent être sous le contrôle de l'horloge circadienne contrôlant ces rythmes. Au niveau moléculaire, dans toutes les espèces étudiées, l'expression rythmique de gènes impliqués sont générés par l'interconnexion de boucles de contrôle transcriptionnelles et traductionnelles. Chez les mammifères, l'hétérodimère composé de BMAL1 et de ses partenaires CLOCK ou NPAS2 constitue un activateur transcriptionnel régulant la transcription des gènes Per et Cry. Ces gènes codent pour des répresseurs de l'activité des hétérodimères BMAL1:CLOCK ou BMAL1:NPAS2. Cela a pour effet de fermer la boucle négative, générant ainsi des rythmes d'environ 24 heures. Le but de mon travail de thèse a consisté en l'investigation du rôle de l'horloge circadienne dans la régulation de certains aspects du métabolisme chez la souris via la régulation de l'activation rythmique des voies de signalisation. Nous avons tout d'abord montré que l'horloge circadienne exerce sa fonction d'oscillateur notamment au niveau de la régulation de la traduction des ARNm. En effet, nous présentons des preuves montrant que l'horloge circadienne influence la traduction temporelle d'un groupe d'ARNm impliqués dans la biogénèse des ribosomes en contrôlant la transcription de facteurs d'initiation de la traduction ainsi que l'activation rythmique des voies de signalisation qui sont impliquées dans leur régulation. De plus, l'oscillateur circadien régule la transcription d'ARNm codant pour les protéines ribosomales et d'ARN ribosomaux. De cette façon, l'horloge circadienne exerce un rôle majeur dans la coordination des étapes de transcription et traduction permettant la biogénèse des ribosomes. Dans la deuxième partie, nous montrons les implications de l'horloge circadienne dans le métabolisme des lipides. En effet, DBP, TEF et HLF, trois facteurs de transcription de la famille des PAR bZip qui sont régulés par l'horloge circadienne, jouent un rôle clé dans le contrôle du métabolisme des lipides par l'horloge circadienne. Nous apportons ici des preuves concernant l'expression et l'activité rythmiques de PPARα via la transcription circadienne de gènes impliqués dans le relargage d'acides gras, ligands naturels de PPARα, conduisant à l'activation circadienne de PPARα lui-même, pouvant ainsi jouer son rôle de facteur de transcription de gènes codant pour des protéines impliquées dans le métabolisme des lipides, du cholestérol et du glucose. De plus, nous nous sommes penchés sur le rôle possible de transporteurs de lipides, ici SCP2, dans la modulation de l'activation circadienne de voies de signalisation, telles que TORC1, PPARα et SREBP, qui sont liées au métabolisme, ainsi que son impact sur l'horloge elle-même. Dans la dernière partie de ce travail, nous avons étudié les effets de l'activation de ces voies de signalisation régulées par l'horloge circadienne dans le contexte physiologique puisqu'il a été montré que la perturbation de l'horloge pouvait être associée à des désordres métaboliques. Pour ce faire, nous avons fait des expériences in vivo sur des souris déficientes pour l'horloge moléculaire pour lesquelles l'obésité est induite génétiquement ou induite par la nourriture riche en lipides. Les résultats que nous obtenons montrent des dérèglements au niveau de l'homéostasie des triglycérides et du glucose ainsi que sur l'expression et la réponse à l'insuline.

Risk Factors for Incisional and Organ Space Surgical Site Infections After Liver Resection are Different.

BACKGROUND: Surgical site infection (SSI) is a common cause of major morbidity after liver resection. This study aimed to identify the risk factors for incisional and organ/space SSIs after liver resection. METHODS: Our liver surgery database was retrospectively analyzed for patients treated between January 2009 and November 2012 in a tertiary care Swiss hospital. Univariate and multivariate analyses were conducted on preoperative, intraoperative, and postoperative variables to identify risk factors for incisional and organ/space SSIs. RESULTS: In a total of 226 patients, SSI incidences were 12.8 % (incisional), 4.0 % (organ/space), and 1.8 % (both). Univariate analysis showed that incisional SSIs were associated with high American Society of Anesthesiologists (ASA) scores, preoperative anemia, hypoalbuminemia, low prothrombin time, viral or alcoholic chronic hepatitis, liver cirrhosis, and prolonged operation times. Organ/space SSIs were associated with high rates of red blood cell transfusions, concomitant bowel surgery, and prolonged operation times. Multivariate analysis revealed that risk factors for incisional SSIs were anemia [odds ratio (OR) 2.82], high ASA scores (OR 2.88), presence of hepatitis or cirrhosis (OR 5.07), and prolonged operation times (OR 9.61). The only risk factor for organ/space SSIs was concomitant bowel surgery (OR 5.53). Hospital stays were similar in organ/space and incisional SSI groups, but significantly longer for those with both organ/space and incisional SSIs. CONCLUSIONS: High ASA scores, anemia, chronic hepatitis or liver cirrhosis, and prolonged operations increased the risk of incisional SSIs; concomitant bowel surgery increased the risk of organ/space SSI. Specific precautions to prevent organ/space and incisional SSIs may shorten hospital stays.

The JAK/STAT3 Pathway Is a Common Inducer of Astrocyte Reactivity in Alzheimer's and Huntington's Diseases.

Astrocyte reactivity is a hallmark of neurodegenerative diseases (ND), but its effects on disease outcomes remain highly debated. Elucidation of the signaling cascades inducing reactivity in astrocytes during ND would help characterize the function of these cells and identify novel molecular targets to modulate disease progression. The Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway is associated with reactive astrocytes in models of acute injury, but it is unknown whether this pathway is directly responsible for astrocyte reactivity in progressive pathological conditions such as ND. In this study, we examined whether the JAK/STAT3 pathway promotes astrocyte reactivity in several animal models of ND. The JAK/STAT3 pathway was activated in reactive astrocytes in two transgenic mouse models of Alzheimer's disease and in a mouse and a nonhuman primate lentiviral vector-based model of Huntington's disease (HD). To determine whether this cascade was instrumental for astrocyte reactivity, we used a lentiviral vector that specifically targets astrocytes in vivo to overexpress the endogenous inhibitor of the JAK/STAT3 pathway [suppressor of cytokine signaling 3 (SOCS3)]. SOCS3 significantly inhibited this pathway in astrocytes, prevented astrocyte reactivity, and decreased microglial activation in models of both diseases. Inhibition of the JAK/STAT3 pathway within reactive astrocytes also increased the number of huntingtin aggregates, a neuropathological hallmark of HD, but did not influence neuronal death. Our data demonstrate that the JAK/STAT3 pathway is a common mediator of astrocyte reactivity that is highly conserved between disease states, species, and brain regions. This universal signaling cascade represents a potent target to study the role of reactive astrocytes in ND.

Research resource: the dynamic transcriptional profile of sertoli cells during the progression of spermatogenesis.

Sertoli cells (SCs), the only somatic cells within seminiferous tubules, associate intimately with developing germ cells. They not only provide physical and nutritional support but also secrete factors essential to the complex developmental processes of germ cell proliferation and differentiation. The SC transcriptome must therefore adapt rapidly during the different stages of spermatogenesis. We report comprehensive genome-wide expression profiles of pure populations of SCs isolated at 5 distinct stages of the first wave of mouse spermatogenesis, using RNA sequencing technology. We were able to reconstruct about 13 901 high-confidence, nonredundant coding and noncoding transcripts, characterized by complex alternative splicing patterns with more than 45% comprising novel isoforms of known genes. Interestingly, roughly one-fifth (2939) of these genes exhibited a dynamic expression profile reflecting the evolving role of SCs during the progression of spermatogenesis, with stage-specific expression of genes involved in biological processes such as cell cycle regulation, metabolism and energy production, retinoic acid synthesis, and blood-testis barrier biogenesis. Finally, regulatory network analysis identified the transcription factors endothelial PAS domain-containing protein 1 (EPAS1/Hif2α), aryl hydrocarbon receptor nuclear translocator (ARNT/Hif1β), and signal transducer and activator of transcription 1 (STAT1) as potential master regulators driving the SC transcriptional program. Our results highlight the plastic transcriptional landscape of SCs during the progression of spermatogenesis and provide valuable resources to better understand SC function and spermatogenesis and its related disorders, such as male infertility.

PPARβ/δ ameliorates fructose-induced insulin resistance in adipocytes by preventing Nrf2 activation

We studied whether PPARβ/δ deficiency modifies the effects of high fructose intake (30% fructose in drinking water) on glucose tolerance and adipose tissue dysfunction, focusing on the CD36-dependent pathway that enhances adipose tissue inflammation and impairs insulin signaling. Fructose intake for 8weeks significantly increased body and liver weight, and hepatic triglyceride accumulation in PPARβ/δ-deficient mice but not in wild-type mice. Feeding PPARβ/δ-deficient mice with fructose exacerbated glucose intolerance and led to macrophage infiltration, inflammation, enhanced mRNA and protein levels of CD36, and activation of the JNK pathway in white adipose tissue compared to those of water-fed PPARβ/δ-deficient mice. Cultured adipocytes exposed to fructose also exhibited increased CD36 protein levels and this increase was prevented by the PPARβ/δ activator GW501516. Interestingly, the levels of the nuclear factor E2-related factor 2 (Nrf2), a transcription factor reported to up-regulate Cd36 expression and to impair insulin signaling, were increased in fructose-exposed adipocytes whereas co-incubation with GW501516 abolished this increase. In agreement with Nrf2 playing a role in the fructose-induced CD36 protein level increases, the Nrf2 inhibitor trigonelline prevented the increase and the reduction in insulin-stimulated AKT phosphorylation caused by fructose in adipocytes. Protein levels of the well-known Nrf2 target gene NAD(P)H: quinone oxidoreductase 1 (Nqo1) were increased in water-fed PPARβ/δ-null mice, suggesting that PPARβ/δ deficiency increases Nrf2 activity; and this increase was exacerbated in fructose-fed PPARβ/δ-deficient mice. These findings indicate that the combination of high fructose intake and PPARβ/δ deficiency increases CD36 protein levels via Nrf2, a process that promotes chronic inflammation and insulin resistance in adipose tissue.

Competition between SOCS36E and Drk modulates Sevenless receptor tyrosine kinase activity

Modulation of signalling pathways can trigger different cellular responses, including differences in cell fate. This modulation can be achieved by controlling the pathway activity with great precision to ensure robustness and reproducibility of the specification of cell fate. The development of the photoreceptor R7 in the Drosophila melanogasterretina has become a model in which to investigate the control of cell signalling. During R7 specification, a burst of Ras small GTPase (Ras) and mitogen-activated protein kinase (MAPK) controlled by Sevenless receptor tyrosine kinase (Sev) is required. Several cells in each ommatidium express sev. However, the spatiotemporal expression of the boss ligand and the action of negative regulators of the Sev pathway will restrict the R7 fate to a single cell. The Drosophila suppressor of cytokine signalling 36E (SOCS36E) protein contains an SH2 domain and acts as a Sev signalling attenuator. By contrast, downstream of receptor kinase (Drk), the fly homolog of the mammalian Grb2 adaptor protein, which also contains an SH2 domain, acts as a positive activator of the pathway. Here, we apply the Förster resonance energy transfer (FRET) assay to transfected Drosophila S2 cells and demonstrate that Sev binds directly to either the suppressor protein SOCS36E or the adaptor protein Drk. We propose a mechanistic model in which the competition between these two proteins for binding to the same docking site results in either attenuation of the Sev transduction in cells that should not develop R7 photoreceptors or amplification of the Ras-MAPK signal only in the R7 precursor.

Inflammatory and coagulation biomarkers and mortality in patients with HIV infection.

Background In the Strategies for Management of Anti-Retroviral Therapy trial, all-cause mortality was higher for participants randomized to intermittent, CD4-guided antiretroviral treatment (ART) (drug conservation [DC]) than continuous ART (viral suppression [VS]). We hypothesized that increased HIV-RNA levels following ART interruption induced activation of tissue factor pathways, thrombosis, and fibrinolysis. Methods and Findings Stored samples were used to measure six biomarkers: high sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), amyloid A, amyloid P, D-dimer, and prothrombin fragment 1þ2. Two studies were conducted: (1) a nested case-control study for studying biomarker associations with mortality, and (2) a study to compare DC and VS participants for biomarker changes. For (1), markers were determined at study entry and before death (latest level) for 85 deaths and for two controls (n¼170) matched on country, age, sex, and date of randomization. Odds ratios (ORs) were estimated with logistic regression. For each biomarker, each of the three upper quartiles was compared to the lowest quartile. For (2), the biomarkers were assessed for 249 DC and 250 VS participants at study entry and 1 mo following randomization. Higher levels of hsCRP, IL-6, and D-dimer at study entry were significantly associated with an increased risk of all-cause mortality. Unadjusted ORs (highest versus lowest quartile) were 2.0 (95% confidence interval [CI], 1.0-4.1; p¼0.05), 8.3 (95% CI, 3.3-20.8; p , 0.0001), and 12.4 (95% CI, 4.2-37.0; p , 0.0001), respectively. Associations were significant after adjustment, when the DC and VS groups were analyzed separately, and when latest levels were assessed. IL-6 and D-dimer increased at 1 mo by 30% and 16% in the DC group and by 0% and 5% in the VS group (p , 0.0001 for treatment difference for both biomarkers); increases in the DC group were related to HIV-RNA levels at 1 mo (p , 0.0001). In an expanded case-control analysis (four controls per case), the OR (DC/VS) for mortality was reduced from 1.8 (95% CI, 1.1-3.1; p¼0.02) to 1.5 (95% CI, 0.8-2.8) and 1.4 (95% CI, 0.8-2.5) after adjustment for latest levels of IL-6 and D-dimer, respectively. Conclusions IL-6 and D-dimer were strongly related to all-cause mortality. Interrupting ART may further increase the risk of death by raising IL-6 and D-dimer levels. Therapies that reduce the inflammatory response to HIV and decrease IL-6 and D-dimer levels may warrant investigation.

Metabolic effects of insulin and IGFs on gilthead sea bream (Sparus aurata) muscle cells.

Primary cultures of gilthead sea bream myocytes were performed in order to examine the relative metabolic function of insulin compared with IGF-I and IGF-II (insulin-like growth factors, IGFs) at different stages in the cell culture. In these cells, the in vitro effects of insulin and IGFs on 2-deoxyglucose (2-DG) and L-alanine uptake were studied in both myocytes (day 4) and small myotubes (day 9). 2-DG uptake in gilthead sea bream muscle cells was increased in the presence of insulin and IGFs in a time dependent manner and along with muscle cell differentiation. On the contrary, L-alanine uptake was also stimulated by insulin and IGFs but showed an inverse pattern, being the uptake higher in small myocytes than in large myotubes. The results of preincubation with inhibitors (PD-98059, wortmannin, and cytochalasin B) on 2-DG uptake indicated that insulin and IGFs stimulate glucose uptake through the same mechanisms, and evidenced that mitogenesis activator protein kinase (MAPK) and PI3K-Akt transduction pathways mediate the metabolic function of these peptides. In the same way, we observed that GLUT4 protein synthesis was stimulated in the presence of insulin and IGFs in gilthead sea bream muscle cells in a different manner at days 4 or 9 of the culture. In summary we describe here, for the first time, the effects of insulin and IGFs on 2-DG and L-alanine uptake in primary culture of gilthead sea bream muscle cells. We show that both MAPK and PI3K-Akt transduction pathways are needed in order to control insulin and IGFs actions in these cells. Moreover, changes in glucose uptake can be explained by the action of the GLUT4 transporter, which is stimulated in the presence of insulin and IGFs throughout the cell culture.

Differential Dimerization of Variants Linked to Enhanced S-Cone Sensitivity Syndrome (ESCS) Located in the NR2E3 Ligand-Binding Domain.

NR2E3 encodes the photoreceptor-specific nuclear hormone receptor that acts as a repressor of cone-specific gene expression in rod photoreceptors, and as an activator of several rod-specific genes. Recessive variants located in the ligand-binding domain (LBD) of NR2E3 cause enhanced short wavelength sensitive- (S-) cone syndrome (ESCS), a retinal degeneration characterized by an excess of S-cones and non-functional rods. We analyzed the dimerization properties of NR2E3 and the effect of disease-causing LBD missense variants by bioluminescence resonance energy transfer (BRET(2) ) protein interaction assays. Homodimerization was not affected in presence of p.A256V, p.R039G, p.R311Q, and p.R334G variants, but abolished in presence of p.L263P, p.L336P, p.L353V, p.R385P, and p.M407K variants. Homology modeling predicted structural changes induced by NR2E3 LBD variants. NR2E3 LBD variants did not affect interaction with CRX, but with NRL and rev-erbα/NR1D1. CRX and NRL heterodimerized more efficiently together, than did either with NR2E3. NR2E3 did not heterodimerize with TLX/NR2E1 and RXRα/NR2C1. The identification of a new compound heterozygous patient with detectable rod function, who expressed solely the p.A256V variant protein, suggests a correlation between LBD variants able to form functional NR2E3 dimers and atypical mild forms of ESCS with residual rod function.

Stoichiometry of Heteromeric BAFF and APRIL Cytokines Dictates Their Receptor Binding and Signaling Properties.

The closely related TNF family ligands B cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL) serve in the generation and maintenance of mature B-lymphocytes. Both BAFF and APRIL assemble as homotrimers that bind and activate several receptors that they partially share. However, heteromers of BAFF and APRIL that occur in patients with autoimmune diseases are incompletely characterized. The N and C termini of adjacent BAFF or APRIL monomers are spatially close and can be linked to create single-chain homo- or hetero-ligands of defined stoichiometry. Similar to APRIL, heteromers consisting of one BAFF and two APRILs (BAA) bind to the receptors B cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI) but not to the BAFF receptor (BAFFR). Heteromers consisting of one APRIL and two BAFF (ABB) bind to TACI and BCMA and weakly to BAFFR in accordance with the analysis of the receptor interaction sites in the crystallographic structure of ABB. Receptor binding correlated with activity in reporter cell line assays specific for BAFFR, TACI, or BCMA. Single-chain BAFF (BBB) and to a lesser extent single-chain ABB, but not APRIL or single-chain BAA, rescued BAFFR-dependent B cell maturation in BAFF-deficient mice. In conclusion, BAFF-APRIL heteromers of different stoichiometries have distinct receptor-binding properties and activities. Based on the observation that heteromers are less active than BAFF, we speculate that their physiological role might be to down-regulate BAFF activity.

Combined loss of the BH3-only proteins Bim and Bmf restores B-cell development and function in TACI-Ig transgenic mice.

Terminal differentiation of B cells depends on two interconnected survival pathways, elicited by the B-cell receptor (BCR) and the BAFF receptor (BAFF-R), respectively. Loss of either signaling pathway arrests B-cell development. Although BCR-dependent survival depends mainly on the activation of the v-AKT murine thymoma viral oncogene homolog 1 (AKT)/PI3-kinase network, BAFF/BAFF-R-mediated survival engages non-canonical NF-κB signaling as well as MAPK/extracellular-signal regulated kinase and AKT/PI3-kinase modules to allow proper B-cell development. Plasma cell survival, however, is independent of BAFF-R and regulated by APRIL that signals NF-κB activation via alternative receptors, that is, transmembrane activator and CAML interactor (TACI) or B-cell maturation (BCMA). All these complex signaling events are believed to secure survival by increased expression of anti-apoptotic B-cell lymphoma 2 (Bcl2) family proteins in developing and mature B cells. Curiously, how lack of BAFF- or APRIL-mediated signaling triggers B-cell apoptosis remains largely unexplored. Here, we show that two pro-apoptotic members of the 'Bcl2 homology domain 3-only' subgroup of the Bcl2 family, Bcl2 interacting mediator of cell death (Bim) and Bcl2 modifying factor (Bmf), mediate apoptosis in the context of TACI-Ig overexpression that effectively neutralizes BAFF as well as APRIL. Surprisingly, although Bcl2 overexpression triggers B-cell hyperplasia exceeding the one observed in Bim(-/-)Bmf(-/-) mice, Bcl2 transgenic B cells remain susceptible to the effects of TACI-Ig expression in vivo, leading to ameliorated pathology in Vav-Bcl2 transgenic mice. Together, our findings shed new light on the molecular machinery restricting B-cell survival during development, normal homeostasis and under pathological conditions. Our data further suggest that Bcl2 antagonists might improve the potency of BAFF/APRIL-depletion strategies in B-cell-driven pathologies.