Control of the Hypoxic Response in Drosophila melanogaster by the Basic Helix-Loop-Helix PAS Protein Similar

Instituto de Investigaciones Bioquímicas Fundación Campomar, Buenos Aires 1405, Argentina.
Molecular and Cellular Biology (Impact Factor: 4.78). 11/2002; 22(19):6842-53. DOI: 10.1128/MCB.22.19.6842-6853.2002
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In mammalian systems, the heterodimeric basic helix-loop-helix (bHLH)-PAS transcription hypoxia-inducible factor (HIF) has emerged as the key regulator of responses to hypoxia. Here we define a homologous system in Drosophila melanogaster, and we characterize its activity in vivo during development. By using transcriptional reporters in developing transgenic flies, we show that hypoxia-inducible activity rises to a peak in late embryogenesis and is most pronounced in tracheal cells. We show that the bHLH-PAS proteins Similar (Sima) and Tango (Tgo) function as HIF-alpha and HIF-beta homologues, respectively, and demonstrate a conserved mode of regulation for Sima by oxygen. Sima protein, but not its mRNA, was upregulated in hypoxia. Time course experiments following pulsed ectopic expression demonstrated that Sima is stabilized in hypoxia and that degradation relies on a central domain encompassing amino acids 692 to 863. Continuous ectopic expression overrode Sima degradation, which remained cytoplasmic in normoxia, and translocated to the nucleus only in hypoxia, revealing a second oxygen-regulated activation step. Abrogation of the Drosophila Egl-9 prolyl hydroxylase homologue, CG1114, caused both stabilization and nuclear localization of Sima, indicating a central involvement in both processes. Tight conservation of the HIF/prolyl hydroxylase system in Drosophila provides a new focus for understanding oxygen homeostasis in intact multicellular organisms.

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Available from: Sofía Lavista Llanos,
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    • "Due to the variability among the different genes observed in the qRT-PCR results, we utilized hypoxia-sensitive LDH reporter lines as an additional test of whether drdlwf flies are hypoxic. These lines contain multiple hypoxia response and cyclic AMP response elements (HRE and CRE, respectively) from the murine LDH promoter upstream of a LacZ or Gal4 (UAS-GFP) reporter gene and both have previously been shown to be activated by hypoxia in Drosophila [18]. We crossed the LDH-Gal4 UAS-GFP chromosome onto a drdlwf background and screened for GFP expression in 3 day old flies. "
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    ABSTRACT: Mutations in the gene drop-dead (drd) cause diverse phenotypes in adult Drosophila melanogaster including early lethality, neurodegeneration, tracheal defects, gut dysfunction, reduced body mass, and female sterility. Despite the identification of the drd gene itself, the causes of early lethality and neurodegeneration in the mutant flies remain unknown. To determine the pattern of drd expression associated with the neurodegenerative phenotype, knockdown of drd with various Gal4 drivers was performed. Early adult lethality and neurodegeneration were observed upon knockdown of drd in the tracheal system with two independent insertions of the breathless-Gal4 driver and upon knockdown in the tracheal system and elsewhere with the DJ717-Gal4 driver. Surprisingly, rescue of drd expression exclusively in the tracheae in otherwise mutant flies rescued the neurodegenerative phenotype but not adult lethality. Gut dysfunction, as measured by defecation rate, was not rescued in these flies, and gut function appeared normal upon tracheal-specific knockdown of drd. Finally, the hypothesis that tracheal dysfunction in drd mutants results in hypoxia was tested. Hypoxia-sensitive reporter transgenes (LDH-Gal4 and LDH-LacZ) were placed on a drd mutant background, but enhanced expression of these reporters was not observed. In addition, manipulation of drd expression in the tracheae did not affect expression of the hypoxia-induced genes LDH, tango, and similar. Overall, these results indicate that there are at least two causes of adult lethality in drd mutants, that gut dysfunction and neurodegeneration are independent phenotypes, and that neurodegeneration is associated with tracheal expression of drd but not with hypoxia.
    PLoS ONE 07/2013; 8(7):e68032. DOI:10.1371/journal.pone.0068032 · 3.23 Impact Factor
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    • "Hypoxia is a known trigger of autophagy in mammalian cells, but its role has not been characterized in Drosophila autophagy. The fly ortholog of the mammalian HIF-1α gene is sima (short for similar) [29]. Overexpression of sima, or depletion of Vhl led to the formation of LTR-positive autolysosomes in fat body cell clones of well-fed larvae (Figure 4A-C). "
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    ABSTRACT: Two pathways are responsible for the majority of regulated protein catabolism in eukaryotic cells: the ubiquitin-proteasome system (UPS) and lysosomal self-degradation through autophagy. Both processes are necessary for cellular homeostasis by ensuring continuous turnover and quality control of most intracellular proteins. Recent studies established that both UPS and autophagy are capable of selectively eliminating ubiquitinated proteins and that autophagy may partially compensate for the lack of proteasomal degradation, but the molecular links between these pathways are poorly characterized. Here we show that autophagy is enhanced by the silencing of genes encoding various proteasome subunits (alpha, beta or regulatory) in larval fat body cells. Proteasome inactivation induces canonical autophagy, as it depends on core autophagy genes Atg1, Vps34, Atg9, Atg4 and Atg12. Large-scale accumulation of aggregates containing p62 and ubiquitinated proteins is observed in proteasome RNAi cells. Importantly, overexpressed Atg8a reporters are captured into the cytoplasmic aggregates, but these do not represent autophagosomes. Loss of p62 does not block autophagy upregulation upon proteasome impairment, suggesting that compensatory autophagy is not simply due to the buildup of excess cargo. One of the best characterized substrates of UPS is the alpha subunit of hypoxia-inducible transcription factor 1 (HIF-1alpha), which is continuously degraded by the proteasome during normoxic conditions. Hypoxia is a known trigger of autophagy in mammalian cells, and we show that genetic activation of hypoxia signaling also induces autophagy in Drosophila. Moreover, we find that proteasome inactivation-induced autophagy requires sima, the Drosophila ortholog of HIF-1alpha. We have characterized proteasome inactivation- and hypoxia signaling-induced autophagy in the commonly used larval Drosophila fat body model. Activation of both autophagy and hypoxia signaling was implicated in various cancers, and mutations affecting genes encoding UPS enzymes have recently been suggested to cause renal cancer. Our studies identify a novel genetic link that may play an important role in that context, as HIF-1alpha/sima may contribute to upregulation of autophagy by impaired proteasomal activity.
    BMC Cell Biology 06/2013; 14(1):29. DOI:10.1186/1471-2121-14-29 · 2.34 Impact Factor
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    • "There are three different isozymes of PHD viz: PHD1, PHD2, PHD3, which are present in all mammals (Epstein et al. 2001) but in humans these PHDs are referred as HPH3, HPH2, HPH1 (Bruick et al. 2001) whereas, in case of C.elegans these are referred as EGLN2, EGLN1, EGLN3 (Taylor et al. 2001). These three orthologs appear to be product of gene duplication as there is only one gene (EGL-9) in C. elegans (Wang et al. 1995) and (CG1114) in D.melanogaster (Lavista-Llanos et al. 2002). The PHD isozymes differ in their distribution, regulation and ability to hydroxylate HIF-1α due to the variation in amino acid sequence of the N-terminal region, whereas the C terminal region shares homology in their sequences (Huang et al. 2002; Metzen et al. 2003). "
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    ABSTRACT: A divergence in the supply and consumption of oxygen in brain tissue initiates complex cycle of biochemical and molecular events resulting in neuronal death. To overcome such adverse situation, the tissue has to adopt some cellular mechanisms such as induction of various transcription factors, such as hypoxia inducible factor (HIF). It is a transcriptional regulator of oxygen homeostasis and key factor to generate the adaptive responses through upregulation of various target genes involved in the erythropoiesis, angiogenesis as well as glucose metabolism and transport. On the other hand, some studies do suggest that HIF also plays a detrimental role in ischemic reperfusion injury by inducing the pro apoptotic molecules, cytokines such as Nix, BNip3, and IL-20 which cause mitochondrial dysfunction leading to cell death. Hence, modulation of HIF-1 activity seems to provide an innovative therapeutic target to reduce the cellular damage, which arises from ischemic injury. Apart from traditional oxygen dependent HIF regulation, the focus has now shifted toward oxygen independent regulation in cell specific manner through reactive oxygen species involving hypoxia-associated factor, and heat shock protein 90, etc. Therefore, future development of such small molecule regulators for HIF-1 stability and signaling may prove useful to therapeutically target for enhancing recovery and repair in I/R injury.
    Cellular and Molecular Neurobiology 02/2012; 32(4):491-507. DOI:10.1007/s10571-012-9803-9 · 2.51 Impact Factor
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