RTP801/REDD1 was shown to be induced by hypoxia (Shoshani, 2002). This prompted an investigation of the effects of hypoxia on the scylla charybdis double mutants. scylla charybdis mutant larvae were raised on normal food at room temperature in a hypoxia chamber containing 9% oxygen during their entire development. In general, control flies as well as scylla charybdis homozygotes were 3-4 d delayed in development under these hypoxic conditions. However, whereas adult homozygous scylla charybdis double mutants could readily be recovered under standard culture conditions in normoxia, homozygous mutants of three independent scylla charybdis allelic combinations (char180 in combination with scy31, scy113, or scyEP9.85) were strongly underrepresented compared to normoxic conditions. The appearance of an increased number of dead pupae in the vials was observed. The scylla113 char180 double-mutant combination had the strongest effect, and only two escapers (out of 326 scored flies) hatched, whereas under normoxia flies with this genotype were recovered with nearly the expected Mendelian ratio. The eclosed homozygotes raised under hypoxia did not show obvious morphological defects. Thus, whereas simultaneous loss of Scylla and Charybdis under normoxic conditions results in a slight increase in growth, their absence under reduced oxygen concentrations severely compromises larval development. This indicates that Scylla and Charybdis have a critical function for survival under hypoxic conditions (Reiling, 2004).
The transcription factor HIF-1 is the key regulator of changes in gene expression in response to hypoxia. It consists of two bHLH-PAS domain protein subunits (HIF-1alpha and HIF-1beta). Under conditions of low oxygen, the HIF-1 protein complex is stabilized and binds to Hypoxia Response Elements (HRE), short regulatory DNA sequences (core recognition sequence 5'-TACGTG-3') located in the genomic region of target genes. Both the scylla and charybdis loci possess several HREs. Since RTP801/REDD1, a mammalian homolog of scylla and charybdis, is a direct target gene of HIF-1 and is induced under hypoxic conditions, it was enquired whether this function is evolutionarily conserved. Wild-type larvae were subjected to hypoxia (between 2% and 5% O2) and checked for the induction of scylla and charybdis expression. It is mainly the endoreplicative tissue such as fat body, gut, salivary glands, and tracheae that respond to changes in oxygen concentrations. scylla mRNA expression was up-regulated in the larval fat body and in the gut after hypoxia. charybdis, in contrast, is mildly induced in the midgut but not in the fat body. Whether hypoxia had an effect on Scylla protein levels and distribution was also tested. To detect the endogenous Scylla protein, advantage was taken of a transgenic Scylla-reporter line (a so-called protein trap line). This protein trap line bears a promoter-less green fluorescent protein (GFP)-reporter transgene in the scylla locus generating a Scylla-GFP fusion protein. Scylla-GFP is expressed in most larval tissues. Under normoxic conditions, nuclear accumulation of Scylla protein is observed in some cells of the endoreplicative tissue. Consistent with the mRNA data, upon exposure of third instar larvae to various hypoxia conditions, an up-regulation and nuclear localization of Scylla protein was observed in the fat body and in the gut (Reiling, 2004).
In Drosopohila, the bHLH-PAS family comprises Period (Per), Trachealess (Trh), Single-minded (Sim), Spineless (Ss), Dysfusion (Dys), Tango (Tgo), and Similar (Sima). Tgo is the ubiquitously expressed HIF-1beta ortholog, which dimerizes with any of the alpha-subunits. Sima has been shown to fulfill analogous functions to its mammalian homolog HIF-1 (Reiling, 2004).
To test whether scylla/charybdis transcription is regulated by bHLH-PAS proteins that recognize the same DNA stretch, sim, trh or sima were overexpressed together with tgo using the Lsp2-Gal4-driver that is active specifically in the fat body during the third larval stage. For Sima, a form lacking the oxygen-dependent degradation domain (ODD) was used, rendering it refractory to proteolytic destruction under normoxic conditions. Only the coexpression of tgo and sima induced scylla but not charybdis expression as assessed by mRNA in situ hybridization. This does not preclude, however, the possibility that charybdis is a target of Tgo-Sima, since its endogenous induction was observed upon hypoxia, but only in the gut and not in the fat body. Since neither expression of trh with tgo nor sim induced scylla or charybdis expression, the regulation of scylla by the Tgo-Sima heterodimer is specific. Thus, scylla and charybdis, like their mammalian homolog RTP801/REDD1, are induced by hypoxia, and at least scylla appears to be a direct target gene of the HIF-1 homolog Tgo-Sima (Reiling, 2004).
Although loss of Scylla function does not produce a mutant phenotype on its own, whether it would alter the PKB/PDK1 overexpression eye phenotype was tested. Indeed, loss of Scylla function enhances the PKB/PDK1 overgrowth phenotype. Thus, Scylla is essential for attenuating the increased growth in response to hyperactivation of the Inr pathway. Furthermore, loss of Scylla partially suppresses the growth reduction associated with reduced PKB function as assessed by comparing weights of PKB3 single mutants to scy31 PKB3 double mutants. In contrast, complete loss of Scylla in a heteroallelic S6K combination does not rescue the S6K single mutant phenotype indicating that S6K is epistatic over scylla (Reiling, 2004).
Moreover, overexpression of scylla and charybdis not only suppresses the growth phenotype caused by over-activation of the Inr pathway in the eye but to a certain extent also rescues the lethality associated with the ubiquitous increase in Inr pathway activity due to either overexpression of PKB or loss of PTEN. scylla rescues the male-specific lethality caused by ubiquitous expression of PKB and organismal lethality associated with the partial but not complete loss of PTEN function. Similarly, PKB-associated male lethality is also rescued by charybdis overexpression. This indicates that scylla and charybdis have the capacity to act as potent negative regulators of insulin signaling downstream of PKB and PDK1 (Reiling, 2004).
Several lines of evidence suggest that Scylla and Charybdis act upstream of TSC and Rheb. Tsc1/2 mutant flies can be rescued to adulthood by reducing S6K signaling, and a mere reduction of one TOR copy in a Tsc1 mutant context results in a rescue to the pupal stage. Whether ubiquitous scylla overexpression could rescue the larval lethality of heteroallelic Tsc1/2 mutant combinations (Tsc12G3/Tsc1Q87X and Tsc256/Tsc2192) was examined using the da-Gal4 or Act5C-Gal4 drivers in combination with a UAS-scy transgene or EPscy at 18°C, 25°C, and 29°C. Ubiquitous overexpression of scylla/charybdis in a Tsc1/2 mutant background did in no case extend larval development beyond first/second instar, and these larvae died at the same time as Tsc1/2 mutants. Moreover, the big head phenotype of Tsc2192 (and Tsc256) induced by the eyflp/FRT system was not further enhanced in scyEP9.85 char180 Tsc2 triple-mutant heads. It has been shown that heads composed almost entirely of scylla charybdis double-mutant cells are enlarged. Conversely, GMRGal4-driven co-overexpression of Tsc1, Tsc2, and scylla or charybdis in the eye does not further reduce the small eye phenotype induced by coexpression of Tsc1 and Tsc2 on their own. The absence of an additive growth effect upon loss of Tsc2, scylla, and charybdis or overexpression of Tsc1/2 and scylla or charybdis suggests that they function in the same pathway. These results are consistent with the idea that Scylla and Charybdis act upstream of the TSC complex. This conclusion is further supported by the fact that neither a Rheb-dependent bulging eye phenotype nor organismal lethality could be suppressed by scylla/charybdis coexpression (Reiling, 2004).
Robotic methods and the whole-genome sequence of Drosophila melanogaster were used to facilitate a large-scale expression screen for spatially restricted transcripts in Drosophila embryos. In this screen, scylla (scyl) and charybde (chrb), which code for dorsal transcripts in early Drosophila embryos and are homologous to the human apoptotic gene RTP801, were identified. In Drosophila, both gene products are transcriptionally regulated targets of Dpp/Zen-mediated signal transduction and appear more generally to be downstream targets of homeobox regulation. Gene disruption studies revealed the functional redundancy of scyl and chrb, as well as their requirement for embryonic head involution. From the perspective of functional genomics, these studies demonstrate that global surveys of gene expression can complement traditional genetic screening methods for the identification of genes essential for development: beginning from their spatio-temporal expression profiles and extending to their downstream placement relative to dpp and zen, these studies reveal roles for the scyl and chrb gene products as links between patterning and cell death (Scuderi, 2006).
The foundation for the current study was a survey of RNA expression patterns by automated whole-mount RNA hybridization in situ. This screening protocol led to the identification of scyl as a dorsally restricted transcript in blastoderm stage embryos. Based on its spatial and temporal expression properties, which represent a subset of the dpp expression pattern, it was postulated that scyl is a transcriptionally regulated target of the Dpp signaling cascade that specifies early embryonic dorsal fates. To test this hypothesis, scyl transcript distributions were compared in wild-type and dorsoventral patterning mutant embryos. Fate determining genes functioning downstream of the ventral morphogen Dorsal (Dl) and/or the dorsal morphogen Dpp are expected to exhibit altered expression patterns in Dl-deficient/Dpp-constitutive and Dl-constitutive/Dpp-deficient mutant embryos. Indeed, this was found to be the case for the scyl transcript. Transcription of scyl in wild-type and mutant embryos was similar to that of zen, the best characterized target of Dpp, indicating that Dpp is both necessary and sufficient for scyl transcription. In blastoderm stage embryos, zen is expressed at the posterior pole and in the dorsal-most 40% of the developing embryo. In like fashion, scyl is expressed at the posterior pole and is dorsally restricted. scyl transcripts, however, are confined to the subset of zen-expressing cells that correspond to the dorsal-most 10% of the developing embryo. Both scyl and zen are ubiquitously expressed in dorsalized embryos derived from dl mutant females and in which the dorsal morphogen Dpp is ubiquitously expressed. Neither scyl nor zen is expressed in the abdominal regions of ventralized embryos, which are derived from cactus (cact) mutant females and which lack zygotic Dpp (Scuderi, 2006).
Two observations that led to an examination of the regulation of scyl and chrb by downstream components of the Dpp signaling cascade are: (1) scyl, like zen, is a transcriptionally regulated target of Dpp-mediated signaling and (2) scyl, chrb and zen are expressed in overlapping dorsal fields. The gene encoding the divergent homeobox transcription factor Zen is itself activated by Dpp-mediated signaling in dorsal domains of the blastoderm stage embryo. In zen mutant embryos, dorsally restricted scyl and chrb transcripts are lost, placing both genes downstream of the Zen transcriptional effector of Dpp-mediated signal transduction (Scuderi, 2006).
In addition to the dorsal field of scyl and chrb expression in early embryos, segmental expression along the anteroposterior axis suggests that scyl and chrb may also be sensitive to regulation by homeobox genes other than zen. Both scyl and chrb transcripts are localized in anteroposteriorly segmented patterns in ventral regions of the blastoderm and in the three thoracic segments of stage 13 embryos. In stage 13 embryos, genes of the bithorax complex (BX-C) repress expression of target genes in abdominal segments, restricting their expression to the thorax. The expression of scyl and chrb was examined in BX-C mutant embryos: expansion was observed of thoracic expression into abdominal segments of mutant embryos, thereby placing scyl and chrb downstream of the BX-C homeobox transcription factors, Ubx, Abd-A and/or Abd-B. Consistent with this observation is the finding (Chauvet, 2000) that Ubx binds to regulatory regions of both scyl and chrb (Scuderi, 2006).
Finally, as an extension of the observation that scyl and chrb are downstream targets of homeobox genes acting in distinct signaling pathways, bioinformatic tools were used to identify conserved elements of the scyl and chrb promoters in D. melanogaster and D. pseudoobscura. In a computational cross-genome comparison utilizing algorithms based on both Gibbs sampling and Artificial Neural Networks, one 24-nucleotide motif and three 16-nucleotide motifs were identified that are conserved in the promoter regions of both genes in both species. Motif 1 was found much more frequently than expected for a random sequence, suggestive of its role as a generic transcription factor binding site or regulatory element. Motifs 2-4 were observed much less frequently, and this statistic is interpreted to be indicative of more specific roles for motifs 2-4 in the co-regulation of scyl and chrb. With respect to the identification of defined binding sites, neither motif 1 nor motif 2 corresponds to any canonical transcription factor binding sites listed in the TRANSFAC transcription factor database. Motif 3, however, is GC-rich and contains canonical binding sites for two widely used transcriptional activators: SP1 (GCCCGCCCCCC) and AP2 (GCCCGCGGC). More notable, however, is the characterization of motif 4, which was found only twice in each of the Drosophila genomes (in the promoter regions of scyl and chrb). In scans of the TRANSFAC database, it was found that motif 4 harbors a 10-bp canonical binding site for Zen (ATTTAAATGA) (Scuderi, 2006).
To test whether the placement of Scylla between PKB and TSC can be corroborated biochemically, the effect of scylla overexpression on PKB and S6K activity was examined. PKB activity of adult female heads overexpressing scylla or charybdis was tested in conjunction with PKB and PDK1 under control of the GMR-Gal4 enhancer. The same experimental setup has previously been used to demonstrate that PDK1 increases PKB activity. Total fly head protein was extracted and PKB activity was assayed by incorporation of 32P-labeled phosphate into a synthetic PKB substrate (Crosstide, CT). Although scylla/charybdis overexpression substantially suppresses the PKB/PDK1-induced bulging eye phenotype, PKB activity is not reduced in these eyes. Moreover, PKB activity is also unaffected in a scylla-/- background (Reiling, 2004).
These results are consistent with the placement of Scylla downstream of PKB. To test the effect of Scylla on S6K activity, second instar larvae expressing scylla under the control of Act5C-Gal4 were collected, and larval extracts were assayed for S6K activity. On average, S6K activity was down-regulated by >50%. Although there may also be a slight reduction in total S6K protein levels, this effect cannot account for the much stronger reduction in S6K activity. Taken together with the genetic evidence, these results strongly support the argument that Scylla acts between PKB and TSC to regulate S6K activity. Furthermore, Brugarolas (2004) provide direct biochemical evidence that a functional TSC complex is required for RTP801/REDD1 to affect S6 phosphorylation. Altogether, these data indicate that Scylla functions upstream of TSC (Reiling, 2004).
FOXO is thought to function as a repressor of growth that is, in turn, inhibited by insulin signaling. However, inactivating mutations in Drosophila melanogaster FOXO result in viable flies of normal size, which raises a question over the involvement of FOXO in growth regulation. Previously, a growth-suppressive role for FOXO under conditions of increased target of rapamycin (TOR) pathway activity was described. This study further characterizes this phenomenon. Tuberous sclerosis complex 1 mutations cause increased FOXO levels, resulting in elevated expression of FOXO-regulated genes, some of which are known to antagonize growth-promoting pathways. Analogous transcriptional changes are observed in mammalian cells, which implies that FOXO attenuates TOR-driven growth in diverse species (Harvey, 2008).
To investigate mechanisms by which the TOR pathway controls tissue growth, transcriptional profiles were analyzed of tissue lacking Tsc1, which leads to hyperactivation of the TOR pathway and excessive growth. Eye-antennal imaginal discs from third instar Drosophila larvae were generated that were composed almost entirely of tissue derived from one of two different genotypes: Tsc1 or wild-type isogenic control. Three biologically independent first strand cDNA samples from each genotype were hybridized to microarray chips. Expression levels of 157 genes were elevated 1.5-fold or more, whereas 211 genes were repressed 1.5-fold or more when compared with control tissue. These genes have been implicated in diverse cellular functions including metabolism, membrane transport, stress response, cell growth, and cell structure (Harvey, 2008).
Observed transcriptional changes were validated for several genes using Drosophila gene-enhancer trap lines. The UAS-Gal4 system was used to activate the TOR pathway in a specific tissue domain by driving expression of Rheb under the control of the glass multiple reporter (GMR) promoter. Induction of astray (aay) and 4E-BP (both of which were found to be elevated in Tsc1 tissue by microarray analysis) were observed in the GMR expression domain (posterior to the morphogenetic furrow) when Rheb was misexpressed but were not induced when the negative control Gal4 gene was misexpressed. QPCR was also used to confirm expression changes observed in Tsc1 tissue for charybdis (chrb), scylla (scy), phosphoenolpyruvate carboxy kinase, 4E-BP, and aay (Harvey, 2008).
Intriguingly, several gene products whose expression was elevated in Tsc1 tissue have been implicated in tissue growth controlled by the insulin and TOR pathways, including 4E-BP, Chrb, and Scy. 4E-BP is a repressor of cap-dependent translation. Upon phosphorylation by TOR, 4E-BP dissociates from eIF4e, allowing assembly of the initiation complex at the mRNA cap structure, ribosome recruitment, and subsequent translation. Scy and Chrb, and their mammalian orthologues REDD1 and REDD2, inhibit insulin and TOR signaling in response to hypoxia and energy stress and restrict growth during Drosophila development. The finding that inhibitors of growth are highly expressed in Tsc1 tissue led to the hypothesis that such genes are transcriptionally induced as part of a feedback loop that restricts tissue growth under conditions of excessive TOR activity. Feedback loops are an important activity-modulating feature of many signaling pathways, including the TOR and insulin pathways (Harvey, 2008).
To examine the mechanism whereby transcription of growth inhibitors is induced in response to TOR hyperactivation, attempts were made to determine which transcription factors were responsible for their expression. One obvious candidate was FOXO, a member of the forkhead transcription factor family, which has a well-established role as an effector of insulin signaling. If FOXO has a role in inducing expression of negative regulators of growth in Tsc tissue, then expression of some of those genes should be elevated under conditions of increased FOXO activity. To investigate this hypothesis, the expression profiles were examined of Tsc1 LOF tissue and Drosophila S2 cells expressing FOXOA3, a mutant version of FOXO that is insensitive to phosphorylation-dependent inhibition by Akt. This analysis revealed that 25 genes were up-regulated 1.5-fold or greater in both Tsc1 LOF and FOXO GOF expression profiles, which represents a highly significant degree of overlap as determined by calculation of the hypergeometric distribution. A highly statistically significant P value strongly suggests that there is a functional overlap between these two datasets that cannot be explained by random variation (Harvey, 2008).
Interestingly, two genes previously implicated in tissue growth regulated by the insulin and TOR pathways 4E-BP and scy were elevated in both microarray experiments, whereas the chrb growth-inhibiting gene was not. Thus, a subset of genes elevated in Tsc1 tissue appears to respond to FOXO activity and was investigated further (Harvey, 2008).
4E-BP is a well-characterized FOXO target gene. To determine whether FOXO could directly activate transcription of genes that were elevated in Tsc1 tissue other than 4E-BP, focus was placed on scy and the phosphoserine phosphatase aay (one of the most highly elevated transcripts in each microarray experiment). scy and aay both possess consensus FOXO recognition elements (FREs) in their promoters comparable to those found in dInR and 4E-BP promoters. Therefore, whether these genes are bona fide FOXO targets was examined by measuring their expression in Drosophila S2 cells misexpressing FOXOA3 in the presence of insulin. aay and scy mRNAs were up-regulated 19.4- and 4.3-fold, respectively, relative to a control gene, actin, as determined by QPCR (Harvey, 2008).
Luciferase reporter assays in S2 cells were used to determine whether the aay promoter region containing putative FREs was sensitive to FOXO activity. Luciferase activity dependent on the aay promoter was strongly induced by FOXOA3. In addition, using in vitro band shift assays, it was demonstrated that FOXO directly binds to the aay promoter, indicating that FOXO likely activates expression of aay by directly binding to the FRE. Surprisingly, in parallel luciferase reporter assays, activation of the scy promoter by FOXO could not be demonstrated, despite the fact that strong binding of FOXO to the putative scy FRE was observed using in vitro band-shift assays. A possible explanation is that the scy-promoter construct lacked the minimal promoter elements required for transcription of luciferase (Harvey, 2008).
TOR pathway hyperactivation caused by Tsc deficiency has been shown to strongly repress activity of Akt. FOXO is normally inactivated by Akt-dependent phosphorylation, which restricts nuclear entry of FOXO and leads to its ubiquitin-dependent destruction. Therefore, in response to TOR pathway hyperactivation, it was predicted that reduced Akt activity would cause FOXO protein to accumulate. To examine this hypothesis, expression of FOXO protein was analyzed in mosaic Tsc1 imaginal discs. It was found that FOXO protein was markedly increased in Tsc1 clones when compared with neighboring wild-type tissue (Harvey, 2008).
In addition, FOXO protein appeared to be mostly nuclear in Tsc1 tissue and cytoplasmic in wild-type tissue. Consistent with this observation, nuclear localization of the mouse FOXO orthologue FOXO1 is observed in endothelial cells of Tsc2 mutant hemangiomas, whereas FOXO1 is mostly cytoplasmic in normal cells. FOXO mRNA levels are unchanged in Tsc1 tissue as determined by microarray analysis, which suggests that changes in translation or stability of FOXO protein account for its accumulation in Tsc1 tissue. The presence of increased FOXO protein in the nuclei of Tsc1 cells is consistent with the hypothesis that FOXO is responsible for increased expression of some of the growth inhibitors that are up-regulated in Tsc1 cells (Harvey, 2008).
To determine whether FOXO was necessary for transcriptional induction of genes that were elevated in Tsc1 tissue, QPCR analysis was used to measure 4E-BP, aay, and scy expression in Tsc1 and Tsc1-FOXO double mutant eye-antennal imaginal discs. Consistent with microarray analysis, increased expression of 4E-BP, aay, and scy was observed in Tsc1 tissue. In Tsc1-FOXO tissue, however, 4E-BP was expressed at approximately equivalent amounts as in wild-type tissue, whereas aay and scy expression was only partially reduced. This demonstrates that elevated expression of 4E-BP in Tsc1 tissue is dependent on the FOXO transcription factor and provides evidence that FOXO activity increases when the TOR pathway is hyperactivated. Expression of aay and scy appear to be partially dependent on FOXO but are likely stimulated by additional transcription factors in Tsc1 tissue (Harvey, 2008).
Next, attempts were made to determine whether FOXO is required to limit growth of tissues with increased TOR pathway activity. In addition, a potential role was examined for another transcription factor, HIF-1, for retardation of TOR-driven growth. HIF-1 is a dual-subunit transcription factor consisting of α and β subunits that functions in response to insulin/TOR signaling and drives transcription of the growth-inhibiting genes scy and chrb, both of which are elevated in Tsc1 tissue (Harvey, 2008).
Drosophila possesses several HIF-1α subunits and a sole HIF-1β subunit, tango (tgo), which partners with each HIF-1α subunit. If FOXO and/or HIF-1 are required to induce expression of genes that limit tissue growth when the TOR pathway is hyperactivated, one might predict that Tsc1-FOXO and/or Tsc1-tgo double mutant tissue would possess a greater capacity to grow than Tsc1 tissue alone. To test this hypothesis, the size was examined of Drosophila eyes comprised almost entirely of the following genotypes: control, tgo, FOXO, Tsc1, Tsc1-tgo, and Tsc1-FOXO. Mutant eyes were created by driving mitotic recombination of chromosomes bearing flipase recognition target (FRT) sites and the appropriate gene mutations, specifically in developing Drosophila eye-antennal imaginal discs. Eyes lacking either tgo or FOXO were approximately the same size as control eyes, whereas Tsc1 eyes were considerably larger. Tsc1-tgo double mutant eyes did not exhibit a further increase in size, which suggests that HIF-1 is not required to inhibit tissue growth in response to Tsc1 loss. In contrast, Tsc1-FOXO double mutant eyes were substantially larger than Tsc1 eyes. This finding is particularly significant in light of the finding that eyes lacking FOXO were indistinguishable in size from wild-type eyes. Thus, it appears that FOXO is normally dispensable for control of eye size, but when growth control is altered by virtue of increased TOR activity, FOXO partially offsets the increased tissue growth. These findings are consistent with observations that FOXO protein accumulates in Tsc1 tissue and that transcriptional profiles of FOXO GOF and Tsc1 LOF cells overlap significantly (Harvey, 2008).
Because individual components of the insulin and TOR pathways are highly conserved among eukaryotes, important regulatory mechanisms that control tissue growth via these pathways are also likely to be conserved. To investigate this idea, transcriptional control was analyzed of mouse orthologues of genes that were elevated in D. melanogaster Tsc1 tissue. Initially, Northern blotting analysis was performed on Tsc2 primary mouse embryonic fibroblasts (MEFs; derived on a p53 background to overcome premature senescence induced by Tsc2 loss). It is reasonable to predict that transcriptional changes that occur because of loss of either Tsc1 or Tsc2 should be very similar because TSC1 and TSC2 function together in an obligate fashion, and mutation of either gene leads to almost indistinguishable phenotypes. It was found that several gene expression changes observed in Drosophila Tsc1 tissue are conserved in Tsc2 MEFs (Harvey, 2008).
The homologues of aay, heat shock protein (hsp) 23, scy, and chrb (PSPH, hsp 27, REDD1, and REDD2, respectively) were all significantly up-regulated in Tsc2 MEFs when compared with control MEFs and expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control. To demonstrate that these expression changes were a specific consequence of Tsc2 loss, Tsc2 expression was reconstituted in Tsc2 null cells, which substantially suppressed mammalian TOR activity and expression of these genes. Interestingly, expression of phosphoenolpyruvate carboxy kinase and 4E-BP1/2 was not altered between wild-type and Tsc2 cells, which might reflect tissue- or species-specific differences in the transcriptome of Drosophila epithelial cells and MEFs (Harvey, 2008).
To determine whether the mode of transcription of these genes was also conserved in mammals, expression was analyzed of the scy homologue REDD1. Like scy, mammalian REDD1 orthologues possess a putative consensus FRE within their proximal promoters. Cotransfection of a version of FOXO that is insensitive to phosphorylation-dependent inhibition by Akt (TM-FKHRL-1) induced robust activation of a mouse REDD1 reporter construct in primary MEFs. To determine whether induction was mediated through the identified FRE, a mutant reporter was created lacking this sequence. Deletion of the REDD1 FRE consistently reduced FOXO-mediated induction of the REDD1 promoter. Finally, to directly assess whether FOXO-dependent transcription was activated in mammalian cells lacking Tsc2, activity of the REDD1 promoter reporter or the corresponding mutant FRE reporter was examined in wild-type and Tsc2 MEFs. As predicted, the wild-type REDD1 promoter exhibited robust activation in Tsc2 cells compared with wild-type cells, and this activation was substantially reduced by deletion of the FRE. Together, these findings provide evidence that transcriptional changes resulting from Tsc1/Tsc2 deficiency are conserved in diverse species (Harvey, 2008).
This study has identified of an evolutionary conserved transcriptional program important for restricting tissue overgrowth driven by excessive activation of the TOR pathway. The FOXO transcription factor plays a key role in this transcriptional response, likely by stimulating expression of several growth inhibitory genes. Thus, although the requirement for FOXO in restricting growth under normal development conditions appears dispensable, this is no longer the case under conditions of excessive TOR activation. These findings have important implications for cancer syndromes that arise because of inappropriate TOR pathway activation, such as the human hamartomatous syndrome, tuberous sclerosis. TOR-dependent feedback inhibition is thought to contribute to the benign nature of Tsc1 and Tsc2 tumors (Ma, 2005; Manning, 2005). Conceivably, inactivating mutations in FOXO family transcription factors and/or FOXO target genes that possess growth-inhibiting properties could promote further growth in normally benign Tsc1 and Tsc2 tumors (Harvey, 2008).
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