Metformin Protects Against Sunitinib-induced Cardiotoxicity: Investigating the Role of AMPK.

ABSTRACT: Sunitinib is associated with cardiotoxicity through inhibition of AMP-protein kinase (AMPK) signaling. By contrast, the common antidiabetic agent metformin has demonstrated cardioprotection through indirect AMPK activation. In this study, we investigate the effects of metformin during sunitinib-induced cytotoxicity. Left ventricular developed pressure, coronary flow, heart rate, and infarct size were measured in Langendorff-perfused rat hearts treated with 1 µM sunitinib ±50 µM metformin ±1 µM human equilibrative nucleoside transporter inhibitor S-(4-Nitrobenzyl)-6-thioinosine (NBTI). Western blot analysis was performed for p-AMPKα levels. Primary isolated cardiac myocytes from the left ventricular tissue were used to measure live cell population levels. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess adjunctive treatment of and metformin in human hepatoma G2 and promyelocytic leukemia (HL-60) cells treated with 0.1-100 µM sunitinib ±50 µM metformin. In the perfused hearts, coadministration of metformin attenuated the sunitinib-induced changes to left ventricular developed pressure, infarct size, and cardiac myocyte population. Western blot analysis revealed a significant decrease in p-AMPKα during sunitinib treatment, which was attenuated after coadministration with metformin. All metformin-induced effects were attenuated, and NBTI was coadministered. The MTT assay demonstrated an increase in the EC50 value during coadministration of metformin with sunitinib compared with sunitinib monotherapy in hepatoma G2 and HL-60 cell lines, demonstrating the impact and complexity of metformin coadministration and the possible role of AMPK signaling. This study highlights the novel cardioprotective properties of metformin and AMPK activation during sunitinib-induced cardiotoxicity when administered together in the Langendorff heart model.

Channel nuclear pore protein 54 directs sexual differentiation and neuronal wiring of female reproductive behaviors in Drosophila.

BACKGROUND: Female reproductive behaviors and physiology change profoundly after mating. The control of pregnancy-associated changes in physiology and behaviors are largely hard-wired into the brain to guarantee reproductive success, yet the gene expression programs that direct neuronal differentiation and circuit wiring at the end of the sex determination pathway in response to mating are largely unknown. In Drosophila, the post-mating response induced by male-derived sex-peptide in females is a well-established model to elucidate how complex innate behaviors are hard-wired into the brain. Here, we use a genetic approach to further characterize the molecular and cellular architecture of the sex-peptide response in Drosophila females. RESULTS: Screening for mutations that affect the sensitivity to sex-peptide, we identified the channel nuclear pore protein Nup54 gene as an essential component for mediating the sex-peptide response, with viable mutant alleles leading to the inability of laying eggs and reducing receptivity upon sex-peptide exposure. Nup54 directs correct wiring of eight adult brain neurons that express pickpocket and are required for egg-laying, while additional channel Nups also mediate sexual differentiation. Consistent with links of Nups to speciation, the Nup54 promoter is a hot spot for rapid evolution and promoter variants alter nucleo-cytoplasmic shuttling. CONCLUSIONS: These results implicate nuclear pore functionality to neuronal wiring underlying the sex-peptide response and sexual differentiation as a response to sexual conflict arising from male-derived sex-peptide to direct the female post-mating response.

Hakai is required for stabilization of core components of the m6A mRNA methylation machinery.

N6-methyladenosine (m6A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions. The E3 ubiquitin ligase Hakai was previously found in complex with components of the m6A methylation machinery in plants and mammalian cells but its precise function remained to be investigated. Here we show that Hakai is a conserved component of the methyltransferase complex in Drosophila and human cells. In Drosophila, its depletion results in reduced m6A levels and altered m6A-dependent functions including sex determination. We show that its ubiquitination domain is required for dimerization and interaction with other members of the m6A machinery, while its catalytic activity is dispensable. Finally, we demonstrate that the loss of Hakai destabilizes several subunits of the methyltransferase complex, resulting in impaired m6A deposition. Our work adds functional and molecular insights into the mechanism of the m6A mRNA writer complex.

Zc3h13/Flacc is required for adenosine methylation by bridging the mRNA-binding factor Rbm15/Spenito to the m6A machinery component Wtap/Fl(2)d.

N6-methyladenosine (m6A) is the most abundant mRNA modification in eukaryotes, playing crucial roles in multiple biological processes. m6A is catalyzed by the activity of methyltransferase-like 3 (Mettl3), which depends on additional proteins whose precise functions remain poorly understood. Here we identified Zc3h13 (zinc finger CCCH domain-containing protein 13)/Flacc [Fl(2)d-associated complex component] as a novel interactor of m6A methyltransferase complex components in Drosophila and mice. Like other components of this complex, Flacc controls m6A levels and is involved in sex determination in Drosophila We demonstrate that Flacc promotes m6A deposition by bridging Fl(2)d to the mRNA-binding factor Nito. Altogether, our work advances the molecular understanding of conservation and regulation of the m6A machinery.

m6A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination.

N6-methyladenosine (m6A) is the most common internal modification of eukaryotic messenger RNA (mRNA) and is decoded by YTH domain proteins. The mammalian mRNA m6A methylosome is a complex of nuclear proteins that includes METTL3 (methyltransferase-like 3), METTL14, WTAP (Wilms tumour 1-associated protein) and KIAA1429. Drosophila has corresponding homologues named Ime4 and KAR4 (Inducer of meiosis 4 and Karyogamy protein 4), and Female-lethal (2)d (Fl(2)d) and Virilizer (Vir). In Drosophila, fl(2)d and vir are required for sex-dependent regulation of alternative splicing of the sex determination factor Sex lethal (Sxl). However, the functions of m6A in introns in the regulation of alternative splicing remain uncertain. Here we show that m6A is absent in the mRNA of Drosophila lacking Ime4. In contrast to mouse and plant knockout models, Drosophila Ime4-null mutants remain viable, though flightless, and show a sex bias towards maleness. This is because m6A is required for female-specific alternative splicing of Sxl, which determines female physiognomy, but also translationally represses male-specific lethal 2 (msl-2) to prevent dosage compensation in females. We further show that the m6A reader protein YT521-B decodes m6A in the sex-specifically spliced intron of Sxl, as its absence phenocopies Ime4 mutants. Loss of m6A also affects alternative splicing of additional genes, predominantly in the 5' untranslated region, and has global effects on the expression of metabolic genes. The requirement of m6A and its reader YT521-B for female-specific Sxl alternative splicing reveals that this hitherto enigmatic mRNA modification constitutes an ancient and specific mechanism to adjust levels of gene expression.

Concentration and Localization of Coexpressed ELAV/Hu Proteins Control Specificity of mRNA Processing.

Neuronally coexpressed ELAV/Hu proteins comprise a family of highly related RNA binding proteins which bind to very similar cognate sequences. How this redundancy is linked to in vivo function and how gene-specific regulation is achieved have not been clear. Analysis of mutants in Drosophila ELAV/Hu family proteins ELAV, FNE, and RBP9 and of genetic interactions among them indicates that they have mostly independent roles in neuronal development and function but have converging roles in the regulation of synaptic plasticity. Conversely, ELAV, FNE, RBP9, and human HuR bind ELAV target RNA in vitro with similar affinities. Likewise, all can regulate alternative splicing of ELAV target genes in nonneuronal wing disc cells and substitute for ELAV in eye development upon artificially increased expression; they can also substantially restore ELAV's biological functions when expressed under the control of the elav gene. Furthermore, ELAV-related Sex-lethal can regulate ELAV targets, and ELAV/Hu proteins can interfere with sexual differentiation. An ancient relationship to Sex-lethal is revealed by gonadal expression of RBP9, providing a maternal fail-safe for dosage compensation. Our results indicate that highly related ELAV/Hu RNA binding proteins select targets for mRNA processing through alteration of their expression levels and subcellular localization but only minimally by altered RNA binding specificity.

Multiple pathways mediate the sex-peptide-regulated switch in female Drosophila reproductive behaviours.

Male-derived sex-peptide (SP) induces profound changes in the behaviour of Drosophila females, resulting in decreased receptivity to further mating and increased egg laying. SP can mediate the switch in female reproductive behaviours via a G protein-coupled receptor, SPR, in neurons expressing fruitless, doublesex and pickpocket. Whether SPR is the sole receptor and whether SP induces the postmating switch in a single pathway has not, to our knowledge been tested. Here we report that the SP response can be induced in the absence of SPR when SP is ectopically expressed in neurons or when SP, transferred by mating, can access neurons through a leaky blood brain barrier. Membrane-tethered SP can induce oviposition via doublesex, but not fruitless and pickpocket neurons in SPR mutant females. Although pickpocket and doublesex neurons rely on G(o) signalling to reduce receptivity and induce oviposition, G(o) signalling in fruitless neurons is required only to induce oviposition, but not to reduce receptivity. Our results show that SP's action in reducing receptivity and inducing oviposition can be separated in fruitless and doublesex neurons. Hence, the SP-induced postmating switch incorporates shared, but also distinct circuitry of fruitless, doublesex and pickpocket neurons and additional receptors.

ELAV-mediated 3'-end processing of ewg transcripts is evolutionarily conserved despite sequence degeneration of the ELAV-binding site.

Regulation of alternative mRNA processing by ELAV (embryonic lethal abnormal visual system)/Hu proteins is mediated by binding to AU-rich elements of low complexity. Since such sequences diverge very rapidly during evolution, it has not been clear if ELAV regulation is maintained over extended phylogenetic distances. The transcription factor Erect wing (Ewg) is a major target of ELAV in Drosophila melanogaster and coordinates metabolic gene expression with regulation of synaptic plasticity. Here, we demonstrate evolutionary conservation of ELAV regulation of ewg despite massive degeneration of its binding site and of associated elements in the regulated intronic 3'-end processing site in distantly related Drosophila virilis. In this species, the RNA-binding part of ELAV protein is identical to D. melanogaster. ELAV expression as well as expression and regulation of ewg are also conserved. Using in vitro binding assays and in vivo transgene analysis, we demonstrate, however, that the ELAV-binding site of D. virilis is fully functional in regulating alternative splicing of ewg intron 6 in D. melanogaster. Known features of the ELAV-binding site, such as the requirement of multiple poly(U) motifs spread over an extended binding site of ∼150 nt and a higher affinity to the 3' part of the binding site, are conserved. We further show that the 135-bp ELAV-binding site from D. melanogaster is sufficient for ELAV recruitment in vivo. Hence, our data suggest that ELAV/Hu protein-regulated alternative RNA processing is more conserved than anticipated from the alignment of degenerate low-complexity sequences.

Determinants of ELAV gene-specific regulation.

How RNA-binding proteins recognize their complement of targets in a complex cellular environment remains poorly understood. Sequence degeneracy and redundancy of short motifs at genomic scales have mostly eluded predictions of specific target genes for gene-specific ELAV (embryonic lethal abnormal visual system)/Hu proteins that bind ubiquitous AU-rich motifs. Using the genetic tools of Drosophila, we have analysed binding properties of ELAV in vitro and ELAV-dependent regulation of its major target ewg (erect wing) in neurons. These studies reveal that an integral part of ELAV gene-specific regulation involves combinatorial binding to variably spaced short U-rich motifs on an extensive binding site.

Regulation of the ELAV target ewg: insights from an evolutionary perspective.

ELAV (embryonic lethal abnormal visual system)/Hu family proteins are prototype RNA-binding proteins with binding preferences for AU-rich regions. Due to frequent occurrence of AU-rich motifs in introns and untranslated regions, it is poorly understood how gene-specific RNA-binding proteins, such as ELAV/Hu family members, recognize their complement of target RNAs in a complex cellular environment. The powerful genetic tools of Drosophila make the fruitfly an excellent model to study alternative mRNA processing in vivo in a developing organism. Recent sequencing of 12 Drosophila genomes will provide a novel resource to enhance our understanding of how gene-specific regulation of mRNA processing is achieved by ELAV/Hu family proteins.

Erect wing regulates synaptic growth in Drosophila by integration of multiple signaling pathways.

BACKGROUND: Formation of synaptic connections is a dynamic and highly regulated process. Little is known about the gene networks that regulate synaptic growth and how they balance stimulatory and restrictive signals. RESULTS: Here we show that the neuronally expressed transcription factor gene erect wing (ewg) is a major target of the RNA binding protein ELAV and that EWG restricts synaptic growth at neuromuscular junctions. Using a functional genomics approach we demonstrate that EWG acts primarily through increasing mRNA levels of genes involved in transcriptional and post-transcriptional regulation of gene expression, while genes at the end of the regulatory expression hierarchy (effector genes) represent only a minor portion, indicating an extensive regulatory network. Among EWG-regulated genes are components of Wingless and Notch signaling pathways. In a clonal analysis we demonstrate that EWG genetically interacts with Wingless and Notch, and also with TGF-beta and AP-1 pathways in the regulation of synaptic growth. CONCLUSION: Our results show that EWG restricts synaptic growth by integrating multiple cellular signaling pathways into an extensive regulatory gene expression network.

Sex-peptide-regulated female sexual behavior requires a subset of ascending ventral nerve cord neurons.

BACKGROUND: Male-derived Sex-peptide (SP) elicits egg laying and rejection of courting males in mated Drosophila females. Little is known about the genes that specify the underlying neuronal circuits and mediate this switch in female sexual behavior. RESULTS: Here we show that the egghead gene involved in glycosphingolipid biosynthesis provides an essential component to the SP response. We have isolated viable alleles of the vital egghead gene that abolish egghead expression from a distal promoter resulting in the absence of the largest transcript of this complex transcription unit. Temporally and spatially restricted expression of egghead revealed a requirement for egghead early in the development of apterous-expressing ventral nerve cord neurons to rescue the SP response. In viable egghead alleles, these ascending interneurons, three per abdominal and seven per thoracic hemisegment, fail to innervate the central brain. egghead expression in apterous neurons rescues neuronal targeting and the response to SP. Furthermore, neurotransmission in apterous neurons is required to elicit the SP response. CONCLUSION: Together with the former finding of SP binding to afferent nerves , these results suggest that SP-mediated modification of sensory input switches female sexual behavior from the virgin to the mated state.

Sex-peptides bind to two molecularly different targets in Drosophila melanogaster females.

Sex-Peptide (SP) and the peptide DUP99B elicit two postmating responses in Drosophila melanogaster females: receptivity is reduced and oviposition is increased. Both are synthesized in the male genital tract and transferred into the female during copulation. To elucidate their function, we characterized the binding properties of SP and DUP99B in females. Cryostat sections of adult females were incubated with alkaline phosphatase (AP)-tagged peptides. In virgin females, both peptides have specific target sites in the nervous system and in the genital tract. The binding pattern is almost identical for both peptides. Incubation of sections of mated females confirm that some of these target sites correspond to the in vivo targets of the two peptides. Neuronal binding is dependent on an intact C-terminal sequence of SP, binding in the genital tract is less demanding in terms of amino acid sequence requirement. On affinity blots the AP-SP probe binds to membrane proteins extracted from abdomen and head plus thorax, respectively. The binding proteins in the nervous system and the genital tract differ in their molecular properties. Calculation of dissociation constants (K(d)), and also determination of the minimal peptide concentrations necessary for binding, indicate that SP is the more important peptide inducing the postmating responses. Our results suggest that binding of SP in the nervous system is responsible for eliciting the postmating responses, whereas binding in the genital tract reflects the presence of a peptide transporter.