Regulation of hedgehog Ligand Expression by the N-End Rule Ubiquitin-Protein Ligase Hyperplastic Discs and the Drosophila GSK3β Homologue, Shaggy

Hedgehog (Hh) morphogen signalling plays an essential role in tissue development and homeostasis. While much is known about the Hh signal transduction pathway, far less is known about the molecules that regulate the expression of the hedgehog (hh) ligand itself. Here we reveal that Shaggy (Sgg), the Drosophila melanogaster orthologue of GSK3β, and the N-end Rule Ubiquitin-protein ligase Hyperplastic Discs (Hyd) act together to co-ordinate Hedgehog signalling through regulating hh ligand expression and Cubitus interruptus (Ci) expression. Increased hh and Ci expression within hyd mutant clones was effectively suppressed by sgg RNAi, placing sgg downstream of hyd. Functionally, sgg RNAi also rescued the adult hyd mutant head phenotype. Consistent with the genetic interactions, we found Hyd to physically interact with Sgg and Ci. Taken together we propose that Hyd and Sgg function to co-ordinate hh ligand and Ci expression, which in turn influences important developmental signalling pathways during imaginal disc development. These findings are important as tight temporal/spatial regulation of hh ligand expression underlies its important roles in animal development and tissue homeostasis. When deregulated, hh ligand family misexpression underlies numerous human diseases (e.g., colorectal, lung, pancreatic and haematological cancers) and developmental defects (e.g., cyclopia and polydactyly). In summary, our Drosophila-based findings highlight an apical role for Hyd and Sgg in initiating Hedgehog signalling, which could also be evolutionarily conserved in mammals

Pathophysiology of genetic defects in pediatric Systemic Lupus Erythematosus

Notre laboratoire a découvert chez une famille de patients atteints de Lupus une mutation hétérozygote du gène ERN1 codant pour la protéine IRE1a : le principal inducteur de la réponse aux protéines mal conformées (UPR). L'UPR est un programme transcriptionnel ayant pour but de restaurer la fonction du réticulum endoplasmique (RE) lorsque celui-ci est surchargé de protéines mal conformées, un phénomène également appelé stress du RE. Le stress du RE va induire l'UPR par l'activation d'IRE1a, provoquant une réponse pro-survie dédiée au retour à l'homéostasie du RE dans le cas d'un stress aigu ou pro-apoptotique dans le cas d'un stress chronique. Par expression de la version humaine sauvage et mutée de la protéine dans la levure à l'état homozygote nous avons montrés que la mutation entraine une perte de fonction totale de son activité RNAse et partielle de son activité kinase. Avec un système de surexpression de IRE1a dans une lignée HEK293, nous avons confirmé que la protéine mutée était bien exprimée par des cellules humaines et cela malgré un fort impact de la mutation sur la protéine révélée par nos analyses in silico. Ces résultats nous ont orienté vers un modèle de mutation dominante négative, IRE1a fonctionnant sous la forme de dimères ou d'oligomères. Les analyses moléculaires de l'UPR réalisées sur des lymphocytes B immortalisés par le virus Epstein-Barr (B-EBV) issues du patient index montrent un épissage retardé d'XBP1, la cible principale de l'activité RNAse d'IRE1a, observable par l'expression diminuée de sa forme épissée (XBP1s) et augmentée de sa forme totale (XBP1u). Le ratio XBP1s/XBP1u confirme ce changement dans la régulation temporelle de la voie IRE1a-XBP1 qui montre des signes croissants d'activité jusqu'à 48h alors que la voie retourne à l'homéostasie chez les contrôles. Cette dérégulation ne semble pas modifier l'expression des cibles observées de ce facteur transcriptionnel : EDEM1, DNAJB9 et DNAJC3. En revanche, l'expression d'IL6 est fortement augmentée à l'état basal ainsi qu'après stimulation à la tunicamycine. Les analyses fonctionnelles ont montré une résistance à l'apoptose extrinsèque des B-EBV du patient index et cela sans diminution de l'expression des récepteurs FAS et TRAIL à la membrane. Cette résistance pourrait être à l'origine du phénotype d'auto-immunité présenté par les divers membres de la famille porteurs de la mutation. Une analyse par Western Blot a montré une expression diminuée de Caspase-3 comparée à un contrôle. Caspase-3 étant une cible du RIDD (Regulated IRE1a-dependent decay of mRNA), une activité de dégradation des ARNm issue de l'activité RNAse d'IRE1a, notre hypothèse est que l'activation augmentée dans le temps d'IRE1a va provoquer une perturbation dans l'induction de l'apoptose par l'UPR, la voie IRE1a devant être désactivée pour induire la mort cellulaire. La voie IRE1a-XBP1 possède des liens directs avec la voie PI3K : via l'association entre XBP1 et p85a/b pour augmenter sa translocation nucléaire d'une part et via l'inhibition induite d'IRE1a par la réactivation de la voie PI3K après induction d'un stress du RE d'autre part. Or, deux familles présentant un lupus ou un tableau clinique proche de nos patients ERN1, sont également porteurs de mutation dans PIK3R1 et PIK3R2 codant pour p85a/b, deux protéines essentielles à la signalisation de la voie PI3K/AKT. La voie PI3K/AKT étant importante pour la prolifération et la survie cellulaire et la phosphorylation d'AKT étant fortement diminuée dans notre modèle B-EBV ERN1 KO, nous supposons que des défauts de cette voie peuvent également converger vers la voie IRE1a-XBP1 et induire une auto-immunité par résistance à l'apoptose extrinsèque. Ces travaux renforcent la notion d'un lien entre le stress du RE et l'auto-immunité, apportant un premier élément de réponse par la description de ces cas de lupus monogénique.Our laboratory discovered in a familial case of lupus an heterozygous mutation or the ERN1 gene coding for the IRE1a protein : the major inducer of the Unfolded Protein Response (UPR). The UPR is a transcriptional program which can restore the function of the endoplasmic reticulum (ER) when it is overloaded with unfolded proteins, a process also called ER stress. The ER stress will induce the UPR by the activation of IRE1a, activating a pro-survival response to return to homeostasis in case of a acute stress or a pro-apoptotic response in case of a chronic stress. By expression the wild type and mutated human version of the protein in the yeast at the homozygous state, we showed that the mutation induces a complete RNAse and a partial kinase loss of function. With a cellular system overexpressiong IRE1a in a HEK293 cell line, we confirmed that the mutated protein can be expressed in human cells despite a strong impact of the mutation on the protein, as showed by our in silico analysis. These results have oriented us towards a dominant negative mutation model as IRE1a works as a dimere or an oligomere. Molecular analysis of the UPR on B lymphocytes immortalized by the Epstein-Barr virus (B-EBV) coming from our patient of interest showed a splicing delay in XBP1, the main target of IRE1a RNAse activity, as we could see it with a diminished XBP1 splice form expression (XBP1s) and an increased unspliced form expression (XBP1u). The XBP1s/XBP1u ratio confirms this temporal dysregulation of the IRE1a-XBP1 pathway which shows increasing activity until 48h in the patient, while the controls already returned to homeostasis. This dysregulation didn't modify the expression of XBP1 transcriptional targets : EDEM1, DNAJB9, DNAJC3. However, the expression of IL6 was greatly increase both at basal state and after tunicamycin treatment. The functional analysis demonstrated a resistance of B-EBV to extrinsic apoptosis in the patient of interest, without any diminution of FAS and TRAIL receptors at the membrane. This resistance could be at the origin of the autoimmune phenotype observable on the family members carrying the mutation. A Western Blot analysis showed a diminished expression of Caspase-3 compared to a control. As Caspase-3 is a target of the RIDD (Regulated IRE1a-dependant decay of mRNA), a mRNA degradation activity linked to the RNAse activity of IRE1a, our hypothesis is that the longer activation of IRE1a will induce a dysregulation of apoptosis induction in the UPR, as IRE1a needs to be desactivated to induce cell death. The IRE1a-XBP1 pathway also presents direct links with the PI3K/Akt pathway : first through the association of XBP1 and p85a/b to increase XBP1 nuclear translocation, second through the inhibition of IRE1a induced by the reactivation of the PI3K/Akt pathway after induction of ER stress. Moreover, two families presenting lupus cases or a clinical table close to our ERN1 patients are also carriers of PIK3R1 and PIK3R2 mutations, coding for p85a and p85b, two proteins essential for the signalisation of the PI3K/Akt pathway. Since this pathway is important for proliferation and cell survival and the fact that our B-EBV ERN1 KO cell line shows a strongly decreased phosphorylation of AKT, we suspect that defects of this pathway could converge to the IRE1a-XBP1 pathway and induce an autoimmunity with a resistance to extrinsic apoptosis. These data underlined the notion of a link between ER stress and autoimmunity, providing a first insight to this field by the description of these monogenic cases of lupus

Pathophysiologie des défauts génétiques du Lupus Erythémateux disséminé pédiatrique : implications du stress du réticulum endoplasmique et de la voie PI3K dans le développement de l’auto-immunité

Our laboratory discovered in a familial case of lupus an heterozygous mutation or the ERN1 gene coding for the IRE1a protein : the major inducer of the Unfolded Protein Response (UPR). The UPR is a transcriptional program which can restore the function of the endoplasmic reticulum (ER) when it is overloaded with unfolded proteins, a process also called ER stress. The ER stress will induce the UPR by the activation of IRE1a, activating a pro-survival response to return to homeostasis in case of a acute stress or a pro-apoptotic response in case of a chronic stress. By expression the wild type and mutated human version of the protein in the yeast at the homozygous state, we showed that the mutation induces a complete RNAse and a partial kinase loss of function. With a cellular system overexpressiong IRE1a in a HEK293 cell line, we confirmed that the mutated protein can be expressed in human cells despite a strong impact of the mutation on the protein, as showed by our in silico analysis. These results have oriented us towards a dominant negative mutation model as IRE1a works as a dimere or an oligomere. Molecular analysis of the UPR on B lymphocytes immortalized by the Epstein-Barr virus (B-EBV) coming from our patient of interest showed a splicing delay in XBP1, the main target of IRE1a RNAse activity, as we could see it with a diminished XBP1 splice form expression (XBP1s) and an increased unspliced form expression (XBP1u). The XBP1s/XBP1u ratio confirms this temporal dysregulation of the IRE1a-XBP1 pathway which shows increasing activity until 48h in the patient, while the controls already returned to homeostasis. This dysregulation didn't modify the expression of XBP1 transcriptional targets : EDEM1, DNAJB9, DNAJC3. However, the expression of IL6 was greatly increase both at basal state and after tunicamycin treatment. The functional analysis demonstrated a resistance of B-EBV to extrinsic apoptosis in the patient of interest, without any diminution of FAS and TRAIL receptors at the membrane. This resistance could be at the origin of the autoimmune phenotype observable on the family members carrying the mutation. A Western Blot analysis showed a diminished expression of Caspase-3 compared to a control. As Caspase-3 is a target of the RIDD (Regulated IRE1a-dependant decay of mRNA), a mRNA degradation activity linked to the RNAse activity of IRE1a, our hypothesis is that the longer activation of IRE1a will induce a dysregulation of apoptosis induction in the UPR, as IRE1a needs to be desactivated to induce cell death. The IRE1a-XBP1 pathway also presents direct links with the PI3K/Akt pathway : first through the association of XBP1 and p85a/b to increase XBP1 nuclear translocation, second through the inhibition of IRE1a induced by the reactivation of the PI3K/Akt pathway after induction of ER stress. Moreover, two families presenting lupus cases or a clinical table close to our ERN1 patients are also carriers of PIK3R1 and PIK3R2 mutations, coding for p85a and p85b, two proteins essential for the signalisation of the PI3K/Akt pathway. Since this pathway is important for proliferation and cell survival and the fact that our B-EBV ERN1 KO cell line shows a strongly decreased phosphorylation of AKT, we suspect that defects of this pathway could converge to the IRE1a-XBP1 pathway and induce an autoimmunity with a resistance to extrinsic apoptosis. These data underlined the notion of a link between ER stress and autoimmunity, providing a first insight to this field by the description of these monogenic cases of lupus.Notre laboratoire a découvert chez une famille de patients atteints de Lupus une mutation hétérozygote du gène ERN1 codant pour la protéine IRE1a : le principal inducteur de la réponse aux protéines mal conformées (UPR). L'UPR est un programme transcriptionnel ayant pour but de restaurer la fonction du réticulum endoplasmique (RE) lorsque celui-ci est surchargé de protéines mal conformées, un phénomène également appelé stress du RE. Le stress du RE va induire l'UPR par l'activation d'IRE1a, provoquant une réponse pro-survie dédiée au retour à l'homéostasie du RE dans le cas d'un stress aigu ou pro-apoptotique dans le cas d'un stress chronique. Par expression de la version humaine sauvage et mutée de la protéine dans la levure à l'état homozygote nous avons montrés que la mutation entraine une perte de fonction totale de son activité RNAse et partielle de son activité kinase. Avec un système de surexpression de IRE1a dans une lignée HEK293, nous avons confirmé que la protéine mutée était bien exprimée par des cellules humaines et cela malgré un fort impact de la mutation sur la protéine révélée par nos analyses in silico. Ces résultats nous ont orienté vers un modèle de mutation dominante négative, IRE1a fonctionnant sous la forme de dimères ou d'oligomères. Les analyses moléculaires de l'UPR réalisées sur des lymphocytes B immortalisés par le virus Epstein-Barr (B-EBV) issues du patient index montrent un épissage retardé d'XBP1, la cible principale de l'activité RNAse d'IRE1a, observable par l'expression diminuée de sa forme épissée (XBP1s) et augmentée de sa forme totale (XBP1u). Le ratio XBP1s/XBP1u confirme ce changement dans la régulation temporelle de la voie IRE1a-XBP1 qui montre des signes croissants d'activité jusqu'à 48h alors que la voie retourne à l'homéostasie chez les contrôles. Cette dérégulation ne semble pas modifier l'expression des cibles observées de ce facteur transcriptionnel : EDEM1, DNAJB9 et DNAJC3. En revanche, l'expression d'IL6 est fortement augmentée à l'état basal ainsi qu'après stimulation à la tunicamycine. Les analyses fonctionnelles ont montré une résistance à l'apoptose extrinsèque des B-EBV du patient index et cela sans diminution de l'expression des récepteurs FAS et TRAIL à la membrane. Cette résistance pourrait être à l'origine du phénotype d'auto-immunité présenté par les divers membres de la famille porteurs de la mutation. Une analyse par Western Blot a montré une expression diminuée de Caspase-3 comparée à un contrôle. Caspase-3 étant une cible du RIDD (Regulated IRE1a-dependent decay of mRNA), une activité de dégradation des ARNm issue de l'activité RNAse d'IRE1a, notre hypothèse est que l'activation augmentée dans le temps d'IRE1a va provoquer une perturbation dans l'induction de l'apoptose par l'UPR, la voie IRE1a devant être désactivée pour induire la mort cellulaire. La voie IRE1a-XBP1 possède des liens directs avec la voie PI3K : via l'association entre XBP1 et p85a/b pour augmenter sa translocation nucléaire d'une part et via l'inhibition induite d'IRE1a par la réactivation de la voie PI3K après induction d'un stress du RE d'autre part. Or, deux familles présentant un lupus ou un tableau clinique proche de nos patients ERN1, sont également porteurs de mutation dans PIK3R1 et PIK3R2 codant pour p85a/b, deux protéines essentielles à la signalisation de la voie PI3K/AKT. La voie PI3K/AKT étant importante pour la prolifération et la survie cellulaire et la phosphorylation d'AKT étant fortement diminuée dans notre modèle B-EBV ERN1 KO, nous supposons que des défauts de cette voie peuvent également converger vers la voie IRE1a-XBP1 et induire une auto-immunité par résistance à l'apoptose extrinsèque. Ces travaux renforcent la notion d'un lien entre le stress du RE et l'auto-immunité, apportant un premier élément de réponse par la description de ces cas de lupus monogénique

Sgg regulates <i>hh</i>-<i>lacZ</i> expression in both the posterior and anterior compartments.

<p>(A-U) Confocal images of EA disc of the indicated genotypes imaged for GFP (A,D,G,J,M,P,S) fluorescence and β-Gal (B,E,H,K,N,Q,T) and Ci¹⁵⁵ (C,F,I,L,O,R,U) immunofluorescence. Dashed lines indicate the division between the anterior and posterior compartments, dotted lines indicate regions of high β-Gal and Ci¹⁵⁵ expression within and anterior to the DVS region (N,O, respectively). The boxed regions (P-R) indicate a region harbouring three clones overexpressing β-Gal and Ci¹⁵⁵ in the anterior compartment, which are enlarged in (S-U). (V) Boxplots of quantification of the average β-Gal pixel intensity of non-GFP masked off images (not shown). n = >5 for each genotype, s.e.m indicated. Statistical analysis by one-way ANOVA and Tukey’s multiple comparison tests, which revealed all comparisons to be statistically significant, except those indicated as non-significant (ns). (W) Potential model to explain the effects observed in the posterior EA disc. The double-headed arrow indicates a physical interaction, the single-headed arrow a positive regulatory action and the round-headed arrow a negative regulatory action. Scale bar = 50μm.</p

<i>hyd</i>^{<i>k7</i>.<i>19</i>} EA discs exhibit abnormal Ptc expression.

<p>(A-D) Deconvolved widefield and (F-K) confocal image sections of control FRT82B (A,B) and <i>hyd</i>^{<i>k7</i>.<i>19</i>} (C,D and F-K) EA discs imaged for GFP fluorescence and the indicated antigens for IF. “Union Jack” lookup table applied to Ptc images (A,C) to visualise low (blue), medium (white) and high (red) intensity levels. (E) Quantification of the area of medium and high Ptc signal. n = 3, s.e.m and indicated p value determined by Student’s t-test. (F-K) Overlapping expression of Ci¹⁵⁵ and Ptc immunofluorescence within a <i>hyd</i>^{<i>k7</i>.<i>19</i>} GFP-positive clone anterior to the Ci¹⁵⁵ DVS (F yellow dotted outline, which is overlaid onto G,H). The yellow dashed line indicates the Ci¹⁵⁵ DVS, which is overlaid onto H). Scale bars = 50μm (A-D) and 10μm (F-K).</p

<i>hyd</i>^{<i>K7</i>.<i>19</i>} EA discs exhibit aberrant Ci¹⁵⁵ expression patterns and morphogenetic furrow-associated features.

<p>(A-D) Deconvolved widefield and confocal image (F-Q) sections of control <i>FRT82B</i> (A,B, F-K)) and <i>hyd</i>^{<i>k7</i>.<i>19</i>} (C,D, L-Q) EA discs imaged for direct GFP fluorescence (A,C,G,K), Ci¹⁵⁵ immunofluorescence (B,D,H,I,K) and DAPI (A,B,C,E,F,G). (A-D) <i>hyd</i>^{<i>k7</i>.<i>19</i>} EA discs exhibit abnormal Ci¹⁵⁵ expression patterns. A “Union Jack” lookup table was applied to Ci¹⁵⁵ images to visualise low (blue), medium (white) and high (red) intensity levels and arrows marks the presumed Ci¹⁵⁵ DVS / morphogenetic furrow and an asterisk indicates increased Ci¹⁵⁵ staining as a result of the tissue folding over in itself (B). (D) <i>hyd</i>^{<i>k7</i>.<i>19</i>} EA discs exhibited ectopic Ci¹⁵⁵ expression in the posterior compartment (E, marked by a dashed yellow line, which is also overlaid onto C). (E) Quantification of the area of medium-to-high Ci¹⁵⁵ signal in control and <i>hyd</i>^{<i>k7</i>.<i>19</i>} EA discs. n = 5, s.e.m and indicated p value determined by Student’s t-test. (F-Q) <i>hyd</i>^{<i>k7</i>.<i>19</i>} EA discs exhibit abnormal markers of the morphogenetic furrow. Control FRT82B EA discs exhibited normal nuclei distribution (F) and DVS Ci¹⁵⁵ expression (H), while <i>hyd</i>^{<i>k7</i>.<i>19</i>} discs exhibited irregular patterns (L,N). (F-H) Dashed lines indicated the DVS’s associated high anterior and low posterior Ci¹⁵⁵ expression margins (H), which is overlaid onto (F,G). (L-N) A region of low Ci¹⁵⁵ expression flanked by two DVS-like regions of high Ci¹⁵⁵ expression is marked by a dashed outline (N), which is overlaid onto (L,M). Arrows mark high Ci¹⁵⁵ DVS (H), or DVS-like (N), signals. Scales bars (A-D) 50μm and (F-Q) 10μm.</p

Sgg and Hyd genetically interact to govern animal viability and head and wing development.

<p>(A-J) Sgg perturbation modifies the <i>hyd</i>^{<i>k7</i>.<i>19</i>} head phenotype. (A-D) Brightfield images of adult <i>Drosophila</i> heads of the indicated genotypes shown either ‘head on’ (upper panels) or ‘side on’ (lower panels). Both gain (C) and loss (D) of <i>sgg</i> function appeared to rescue the <i>hyd</i>^{<i>k7</i>.<i>19</i>} phenotype. Boxplots indicating head width (E, n = ≥8 for each genotype) and counts of eye scars (F, n = ≥8 for each genotype) of the indicated genotypes, with statistical analysis by one-way ANOVA (E) and Fishers exact test (F) revealed statistical significance (asterisks). (G-J) Representative GFP fluorescent signals in adult <i>Drosophila</i> heads of the indicated genotypes revealed only <i>hyd</i>^{<i>k7</i>.<i>19</i>}<i>+sgg</i>^<i>S9A</i> animals lack a GFP signal (n = ≥4 for each genotype). Scale bars = 175μm. (K-P) <i>hyd</i>^<i>WT</i> overexpression promotes the <i>sgg</i>^<i>S9A</i>-mediated wing phenotype. (K-N) Brightfield images of adult <i>Drosophila</i> wings showing (K) normal, (L) mildly deformed, (M) severely deformed and (N) wing-to-notum phenotypes. (O) Percentage of adult wing phenotypes of <i>vg-GAL4</i> flies expressing the indicated transgenes, revealing that the <i>hyd</i>^<i>WT</i> transgene enhanced, and the <i>hyd</i>^<i>C>A</i> transgene suppressed the severity of the <i>sgg</i>^<i>S9A</i> wing defects (n ≥12 for each genotype). (P) Model showing the genetic interaction between <i>sgg</i>^<i>S9A</i> and <i>hyd</i> UAS-transgenes with respect to the wing-to-notum phenotype. Arrows indicate promotion and blockhead arrows inhibition. (Q-R) <i>hyd</i>^<i>WT</i> overexpression rescues <i>sgg</i>^<i>RNAi</i>-mediated embryonic lethality. Percentage viability of <i>sca-GAL4</i> flies expressing the indicated transgenes revealed a >95% rescue of embryonic lethality upon co-expression with the <i>UAS-hyd</i>^<i>WT</i>, but not <i>UAS</i>-<i>hyd</i>^<i>C>A</i>, transgene (16 individual crosses per genotype). (R) Model showing the genetic interaction between <i>sgg</i> and <i>hyd</i> UAS-transgenes. Arrows indicate promotion, blockhead arrows inhibition and dotted blockhead arrow weak inhibition. Scale bar = 250μm.</p

Hyd binds the Hedgehog pathway’s key transcriptional effector Ci¹⁵⁵ and the Ci-regulatory kinase Sgg.

<p>Co-immunoprecipitation (A,D) and affinity-purification (B,C) studies with the indicated affinity reagents were examined by SDS-PAGE and Western blotting with the indicated antibodies. (A) <i>Drosophila</i> CL8 cells were lysed and incubated with either Hyd or control IgG antibodies and affinity purified by Protein G beads. An arrow indicates the position of the expected size band and an asterisk indicates the presence of an uncharacterised faster migrating Hyd species. (B) Mammalian HEK293 cells were transfected with the indicated constructs and lysates underwent Streptactin-mediated purification (Strp) to purify Haemagglutinin-Streptactin-EDD (HS-EDD) and detect co-purified Myc-GLI2. (C) <i>Drosophila</i> S2 cells were transfected with either <i>HS-hyd or HS-vector</i> control, lysed and then incubated with Streptactin-affinity resin. Control and Hyd-coated beads were then incubated with Sgg-FLAG expressing S2 lysate and, following washing, analysed for bound Sgg-FLAG. Only the HS-Hyd beads purified FLAG-Sgg. (D) <i>Drosophila</i> S2 cells were co-transfected with the indicated <i>hyd</i> mutant and <i>sgg</i>-<i>FLAG</i> constructs and FLAG-affinity purified complexes were analysed with the indicated antibodies.</p

<i>hyd</i>^{<i>k7</i>.<i>19</i>} EA discs exhibit increased <i>hh-lacZ</i>-associated β-Gal expression within the posterior compartment and DVS-region.

<p>Confocal image sections of <i>FRT82B</i> control (A-E, L-N)) and <i>hyd</i>^{<i>k7</i>.<i>19</i>} (F-J, O-T) EA discs imaged for direct GFP fluorescence (A,F,N,Q,R), β-Gal (B,C,G,H,L-Q,S) and Ci¹⁵⁵ immunofluorescence (D,E,I,J,M,P,T). (A-K) <i>hyd</i>^{<i>k7</i>.<i>19</i>} EA discs exhibited increased β-Gal expression (H) relative to <i>FRT82B</i> controls (C). Non-clonal regions (GFP—ve regions) were ‘masked off’ to help visualise β-Gal and Ci¹⁵⁵ expression only within GFP-positive clones (C,H and E,J, respectively). Yellow dotted lines indicate the division between anterior and posterior compartment (B—E and G-J). Dashed yellow lines indicate regions of high <i>hh</i> expression (H) and corresponding low Ci¹⁵⁵ expression (E). (K) Quantification of the β-Gal average pixel intensity of the masked off images. n = 5, s.e.m and indicated p value determined by Student’s t-test. Scale bars = 50μm. (L-Q) <i>hyd</i>^{<i>k7</i>.<i>19</i>} DVS regions exhibited abnormal β-Gal (O) and Ci¹⁵⁵ (P) expression. Dashed lines indicate high Ci¹⁵⁵ DVS expression (M,P), which are overlaid onto the other panels. The dotted line marks the anterior front of high β-Gal expression (L), which is overlaid on (M,N). (R-T) Two GFP positive <i>hyd</i>^{<i>k7</i>.<i>19</i>} clones (R), located in the posterior compartment clearly overexpressed β-Gal (S). Of those clones, only one (R yellow dashed line, which is overlaid onto S,T) also harboured increased Ci¹⁵⁵ expression (T). A specific clonal subregion (T, dotted line) with the clone coincided with low β-Gal expression (S, dotted line). Scale bars = 10μm.</p

Regulation of lipid metabolism by the unfolded protein response.

The endoplasmic reticulum (ER) is the site of protein folding and secretion, Ca(2+) storage and lipid synthesis in eukaryotic cells. Disruption to protein folding or Ca(2+) homeostasis in the ER leads to the accumulation of unfolded proteins, a condition known as ER stress. This leads to activation of the unfolded protein response (UPR) pathway in order to restore protein homeostasis. Three ER membrane proteins, namely inositol-requiring enzyme 1 (IRE1), protein kinase RNA-like ER kinase (PERK) and activating transcription factor 6 (ATF6), sense the accumulation of unfolded/misfolded proteins and are activated, initiating an integrated transcriptional programme. Recent literature demonstrates that activation of these sensors can alter lipid enzymes, thus implicating the UPR in the regulation of lipid metabolism. Given the presence of ER stress and UPR activation in several diseases including cancer and neurodegenerative diseases, as well as the growing recognition of altered lipid metabolism in disease, it is timely to consider the role of the UPR in the regulation of lipid metabolism. This review provides an overview of the current knowledge on the impact of the three arms of the UPR on the synthesis, function and regulation of fatty acids, triglycerides, phospholipids and cholesterol