Cyclin D

Transcriptional Regulation

Little is known about how patterns of cell proliferation and arrest are generated during development, a time when tight regulation of the cell cycle is necessary. In this study, the mechanism by which the developmental signaling molecule Wingless generates G₁ arrest in the presumptive Drosophila wing margin is examined in detail. Wg signaling promotes activity of the Drosophila retinoblastoma family (Rbf) protein, which is required for G₁ arrest in the presumptive wing margin. Wg promotes Rbf function by repressing expression of the G₁-S regulator Drosophila myc (dmyc). Ectopic expression of dMyc induces expression of Cyclin E, Cyclin D, and Cdk4, which can inhibit Rbf and promote G₁-S progression. Thus, G₁ arrest in the presumptive wing margin depends on the presence of Rbf, which is maintained by the ability of Wg signaling to repress dmyc expression in these cells. In addition to advancing the understanding of how patterned cell-cycle arrest is generated by the Wg signaling molecule during development, this study indicates that components of the Rbf/E2f pathway are targets of dMyc in Drosophila. Although Rbf/E2f pathway components mediate the ability of dMyc to promote G₁ progression, dMyc appears to regulate growth independently of the RBF/E2f pathway (Duman-Scheel, 2004).

The results indicate why exclusion of dMyc from the ZNC is necessary for Rbf activity. Overexpression of dMyc leads to high levels of Cyclin E, Cyclin D, and Cdk4 transcripts. dMyc also regulates Cyclin E posttranscriptionally in Drosophila. G₁-S Cyclins/Cdks function to phosphorylate and inhibit Rbf, suggesting that dMyc blocks Rbf activity through activation of G₁-S Cyclins/Cdks. Thus, inhibition of dMyc by Wg helps to ensure that G₁-S Cyclins/Cdks do not activate S phase. This idea is supported by the results that indicate that only a combination of both Dap and constitutively active Rbf (that cannot be regulated by Cyclins/Cdks) can restore G₁ arrest when Wg signaling is blocked or when dMyc is expressed. These data suggest that either Cyclin D or Cyclin E activity can mediate the ability of dMyc to promote S phase in the ZNC. Coexpressing Dap alone with dMyc, which would block only Cyclin E/Cdk2 activity, does not restore G₁ arrest. Furthermore, overexpression of dMyc in a cdk4 mutant background still results in ectopic S phases, suggesting that Cyclin E/Cdk2 also are sufficient to mediate dMyc's ability to promote G₁ progression. Thus, either Cyclin D/Cdk4 or Cyclin E/Cdk2 is sufficient to mediate the ability of dMyc to promote G₁ progression. The ability of Wg to inhibit dMyc expression is thus critical for RBF activation and G₁ arrest in the ZNC. Still, it is possible that Wg promotes G₁ arrest through other mechanisms that have not yet been uncovered. The observation that overexpression of a dominant-negative form of dTCF (dTCFDeltaN) with C96>Gal4 can promote S phase, even in a dmyc mutant background, supports this idea (Duman-Scheel, 2004).

It is likely that dMyc/dMax directly up-regulate transcription of Cyclin D and cdk4 in Drosophila. Myc/Max heterodimers regulate transcription by binding to various consensus sequences, such as the E box. Previous studies indicated that cMyc induces Cyclin D2 expression in mice by binding to two consensus E boxes in the Cyclin D2 promoter. cdk4 also was identified as a transcriptional target of c-Myc. Furthermore, it has been suggested that cdk4 is a transcriptional target of dMyc and Cyclin D is a transcriptional target of dMax. Although future studies should analyze the Drosophila Cyclin D and Cdk 4 regulatory regions in more detail, these results suggest that the observed ability of dMyc to induce Cyclin D and Cdk4 expression in the ZNC most likely occurs through transcriptional regulation of these proteins by dMyc/dMax. In contrast, Cyclin E was not identified as a target of dMyc or dMax. It is more likely that the ability of dMyc to induce growth in the wing indirectly leads to increased Cyclin E transcript levels (Duman-Scheel, 2004).

A double-assurance mechanism controls cell cycle exit upon terminal differentiation in Drosophila

Terminal differentiation is often coupled with permanent exit from the cell cycle, yet it is unclear how cell proliferation is blocked in differentiated tissues. The process of cell cycle exit was examined in Drosophila wings and eyes; cell cycle exit can be prevented or even reversed in terminally differentiating cells by the simultaneous activation of E2F1 and either Cyclin E/Cdk2 or Cyclin D/Cdk4. Enforcing both E2F and Cyclin/Cdk activities is required to bypass exit because feedback between E2F and Cyclin E/Cdk2 is inhibited after cells differentiate, ensuring that cell cycle exit is robust. In some differentiating cell types (e.g., neurons), known inhibitors including the retinoblastoma homolog Rbf and the p27 homolog Dacapo contribute to parallel repression of E2F and Cyclin E/Cdk2. In other cell types, however (e.g., wing epithelial cells), unknown mechanisms inhibit E2F and Cyclin/Cdk activity in parallel to enforce permanent cell cycle exit upon terminal differentiation (Buttitta, 2007).

Current models for cell cycle exit invoke repression of Cyclin/Cdk activity by CKIs or repression of E2F-mediated transcription by RBs as the proximal mechanisms by which cell cycle progression is arrested. Since these models include the potential for positive feedback between E2F and CycE/Cdk2, they predict that the induction of either E2F or a G1 Cyclin/Cdk complex should be sufficient to maintain the activity of the other and thereby sustain the proliferative state. However, in differentiating Drosophila tissues, both E2F and G1 Cyclin/Cdk activities had to be simultaneously upregulated to bypass or reverse cell cycle exit. An explanation for this resides in two observations. First, the ability of Cyclin/Cdk activity to promote E2F-dependent transcription is lost or reduced in the wing and eye after terminal differentiation. Second, increased E2F cannot sustain functional levels of CycE/Cdk2 activity after terminal differentiation, despite an increase in cycE and cdk2 mRNA to levels higher than those observed in proliferative-stage wings. Thus, crosstalk between E2F and Cyclin/Cdk activity appears to be limited, in both directions, as a consequence of differentiation (Buttitta, 2007).

How are these two regulatory interactions altered? One possibility is that Rbf2- or E2F2-dependent repression prevents ectopic Cyclin/Cdk activity from promoting E2F-dependent transcription after prolonged exit. While mRNA expression data and the existing genetic data on E2F2 and Rbf2 do not support this possibility, the roles of Rbf2 or E2F2 have not been tested in the presence of continued Cyclin/Cdk activity. Therefore, transcriptional repression of E2F targets by Rbf2 or E2F2 remains an important issue to address in future experiments (Buttitta, 2007).

More enigmatic is the inability of the ectopic CycE/Cdk2 provided by overexpressed E2F to promote cell cycle progression. One plausible explanation for this is that novel inhibitors of CycE are expressed with the onset of differentiation, and that these raise the threshold of Cyclin/Cdk activity required to promote cell cycle progression. Such inhibitors might make the critical substrates of CycE/Cdk2, which reside on chromatin in DNA-replication and -transcription initiation complexes, less accessible or otherwise recalcitrant to activation. The notion of an increased Cdk threshold is consistent with the observation that the >10-fold increase in CycE/Cdk2 provided by direct overexpression of the kinase bypassed cell cycle exit in conjunction with E2F, while the ~4-fold increase provided indirectly by ectopic E2F is insufficient to drive the cell cycle. Although a >10-fold increase in Cdk activity as applied in these experiments is far above the normal physiological range, such dramatic deregulation of cell cycle genes may be physiologically relevant to cancers, in which gene expression can be greatly amplified (Buttitta, 2007).

Recent studies of cycle exit in larval Drosophila eyes have concluded that Rbf1 and Dap are required to inhibit E2F and CycE/Cdk2 in differentiating photoreceptors. Other studies document the roles of Ago/Fbw7 and components of the Hippo/Warts-signaling pathway in downregulating CycE for cell cycle exit in nonneural cells in the eye. Although the data are consistent with these studies in the eye, Ago and the Hippo/Warts pathway are dispensable for cell cycle exit in the wing. Furthermore, deletion of Rbf1 did not prevent cell cycle exit in the epithelial wing, even when high levels of CycE/Cdk2 were provided. Conversely, deletion of Dap was not sufficient to keep wing cells cycling, even when excessive E2F activity was provided. These observations suggest that unknown inhibitors of E2F and Cyclin/Cdk activity mediate cell cycle exit in specific contexts, such as the wing (Buttitta, 2007).

In attempts to identify upstream factors regulating cell cycle exit, a variety of growth and patterning signals were manipulated in the pupal wing and eye, and their effects on cell cycle exit were examined. Surprisingly, signals that act as potent inducers of proliferation in wings and eyes at earlier stages did not prevent or even delay cell cycle exit upon terminal differentiation. Thus, an important focus for future studies will be the nature of the signals upstream of E2F and CycE that mediate cell cycle exit. These could be novel signals, or combinations of known signals delivered in unappreciated ways (Buttitta, 2007).

How general is double assurance? Studies of cell cycle exit in mammals do not offer a consistent answer to this question. S phase re-entry can be achieved in differentiated cells by activating E2F, CycE/Cdk2, or CycD/Cdk4 alone, but this does not lead to cell division or continued proliferation. Several studies with mammalian cells in vivo have shown that neither increased E2F nor Cyclin/Cdk activity alone is sufficient to fully reverse differentiation-associated quiescence, consistent with the double-assurance model propose in this study. Also consistent with this model is the ability of proteins from DNA tumor viruses, such as adenovirus E1A, SV40 LargeT, and HPV E6 and E7, to fully reverse differentiation-associated cell cycle exit in many cell types. These viral onco-proteins stimulate cell cycle progression by targeting multiple cell cycle factors, which ultimately increase both E2F and G1 Cyclin/Cdk activities simultaneously. For example, LargeT and E1A inhibit both RBs and CKIs, such as p21^Cip1 and p27^Kip1 (Buttitta, 2007).

There are some instances, however, in which differentiation-associated cell cycle exit has been bypassed, not just delayed, by the deletion of CKIs or RBs. In one such case, p19^Ink4d and p27^Kip1 were knocked out in the mouse brain, and ectopic mitoses were documented in neuronal cells weeks after they normally become quiescent. Similar results have been obtained with hair and support cells in the mouse inner ear, where deletion of p19^Ink4d, p27^Kip1, or pRB can bypass developmentally programmed cell cycle exit. In light of these findings, it is interesting to speculate that certain differentiated tissues may retain some ability to repair or regenerate by maintaining the capacity for positive feedback between E2F and CycE/Cdk2 activity. Inner-ear hair cells may be such an example, since in many vertebrates they are capable of regeneration, although this ability has been lost in mammals. Although the mammalian brain has a very limited capacity for regeneration, the cell cycle can be reactivated in the brains of other vertebrates, such as fish, in response to injury. Thus, the retention of crosstalk between E2F and Cyclin/Cdk activities in the evolutionary descendents of regeneration-competent cells might explain some of the tissue-specific sensitivities to loss of CKIs or RBs observed in mammals (Buttitta, 2007).

Nutrition controls mitochondrial biogenesis in the Drosophila adipose tissue through Delg and Cyclin D/Cdk4

Mitochondria are cellular organelles that perform critical metabolic functions: they generate energy from nutrients but also provide metabolites for de novo synthesis of fatty acids and several amino acids. Thus mitochondrial mass and activity must be coordinated with nutrient availability, yet this remains poorly understood. This study demonstrated that Drosophila larvae grown in low yeast food have strong defects in mitochondrial abundance and respiration activity in the larval fat body. This correlates with reduced expression of genes encoding mitochondrial proteins, particularly genes involved in oxidative phosphorylation. Second, genes involved in glutamine metabolism are also expressed in a nutrient-dependent manner, suggesting a coordination of amino acid synthesis with mitochondrial abundance and activity. Moreover, this study shows that Delg (Ets97D, CG6338, Delg), the Drosophila homologue to the alpha subunit of mammalian transcription factor NRF-2/GABP, is required for proper expression of most genes encoding mitochondrial proteins. The data demonstrate that Delg is critical to adjust mitochondrial abundance in respect to Cyclin D/Cdk4, a growth-promoting complex and glutamine metabolism according to nutrient availability. However, in contrast to nutrients, Delg is not involved in the regulation of mitochondrial activity in the fat body. These findings are the first genetic evidence that the regulation of mitochondrial mass can be uncoupled from mitochondrial activity (Baltzer, 2009).

In eukaryotes, cellular organelles are separated from the cytoplasm through lipid membranes, creating compartments with unique biological properties. Rather than static, organelle size and function are often dynamic, and tightly regulated in response to various stimuli. One of the best-studied organelles are mitochondria, which show large cell-type specific variations in morphology and abundance, demonstrating that mitochondria are highly regulated. Mitochondrial dysfunction is linked to various diseases, including metabolic disorders and cellular aging, therefore these organelles are critical for cellular homeostasis. Mitochondria perform multiple metabolic functions, most notably the generation of energy from carbohydrates, fatty acids and amino acids. Equally important, mitochondria also provide metabolites for anabolic processes such as de novo synthesis of fatty acids and amino acids. Although the metabolic biochemical reactions are well established, how these processes are coordinated in vivo is just beginning to be understood. One interesting question is how nutrients control mitochondrial mass and activity, and how this regulation affects cellular metabolism (Baltzer, 2009).

During cellular growth, amino acids are used for protein synthesis. In higher eukaryotes, essential amino acids are taken up through the diet, whereas nonessential amino acids are synthesized de novo. For the latter, mitochondria are critical, since they provide oxaloacetate for aspartate and asparagine, as well as 2-oxoglutarate (α-ketoglutarate) for glutamate, glutamine, arginine and proline biosynthesis. Of these amino acids, glutamine is particularly interesting: First, many cell types take up large amounts of glutamine, which can be used to produce cytoplasmic NAD⁺, NAPDH and lactate in a process called glutaminolysis. Second, since the TCA cycle intermediate citrate can be used as a substrate for fatty acid synthesis, glutamine can be converted into 2-oxoglutarate, thus replenishing the TCA cycle. Third, efflux of cytoplasmic glutamine, either taken up or synthesized de novo, is directly linked to the uptake of essential amino acids, both in mammals and Drosophila. Interestingly, all three processes are highly active in cancer cells, under conditions of high metabolic activity. One would therefore expect tight coordination between nutrients, mitochondrial activity and amino acid synthesis, in particular glutamine, yet factors mediating such links have not been described (Baltzer, 2009).

Mitochondria contain their own genome (mtDNA), encoding a small number of proteins required for oxidative phosphorylation (OXPHOS), as well as tRNAs and rRNAs for mitochondrial translation. The majority of mitochondrial proteins are encoded by the nuclear genome, including factors for mitochondrial transcription and translation. These proteins are translated in the cytoplasm and imported into mitochondria. Accordingly, the transcription of these nuclear genes is believed to be rate limiting for mitochondrial mass and activity. To understand the nutrient-specific regulation of mitochondria, one has to characterize how these nuclear transcription factors are regulated in response to nutrients. In Drosophila, genes encoding mitochondrial proteins are highly expressed during the larval growth and feeding period. Subsequently, as the larvae stop feeding at the end of the last larval instar and prepare for metamorphosis, expression of these genes is strongly downregulated. Thus Drosophila larval growth is an ideal system to study how mitochondria are regulated in response nutrients in vivo. This has been exploited in recent microarray studies, where expression profiles of normal fed and starved larvae were compared: Indeed, starvation led to a strong downregulation of genes involved in mitochondrial translation, respiration, TCA cycle, fatty acid oxidation and mitochondrial transport. Similar findings have been published using microarrays from fed or starved adult flies. Comparing larval fat body and muscle tissues, Teleman et al. discovered that many of these genes respond in a cell-type specific manner. Therefore, factors must exist that mediate a tissue-specific transcriptional control in response to nutrients. One candidate for such a factor is dFoxo, the fly homologue to mammalian forkhead O-type transcription factors (FoxO family). Importantly, dFoxo does only mediate the nutrient responsiveness for a subset of genes encoding mitochondrial proteins. This implies that other transcription factors must exist, yet they have not been described in Drosophila (Baltzer, 2009 and references there).

This study used Drosophila to characterize mitochondria in a developing organism in vivo. Focus was placed on the larval fat body, the fly adipose/liver tissue. Fat body cells are specified during embryogenesis, and show an enormous increase in cell size during larval stages that is accompanied by endoreduplication of mitochondrial DNA to a C-value of ~256. Growth of these cells is directly regulated by nutrient uptake, making the larval fat body an ideal system to study mitochondria in response to nutrition and nutrient-sensitive growth-promoting pathways. It was show that low-yeast food conditions, and thus amino acid starvation, leads to strongly reduced mitochondrial abundance and respiration activity. This correlates with reduced expression of genes encoding mitochondrial proteins, including enzymes involved in glutamine metabolism. Moreover, Delg (Ets at 97D), the fly homologue to the alpha subunit of mammalian transcription factor NRF-2/GABP, functions as a key regulator for mitochondrial mass. Surprisingly, reduced mitochondrial mass in delg mutants does not translate into reduced OXPHOS activity. Rather, residual mitochondria compensate by being more active. More importantly, the data show that Delg is critical to adjust mitochondrial abundance and expression levels of enzymes required for glutamine metabolism in response to nutrient availability. Finally, it was observed that the nutrient-sensitive growth-promoting complex Cyclin D/Cdk4 requires Delg for its effect on mitochondria. Thus the data demonstrate how Cyclin D/Cdk4 and Delg coordinate mitochondrial abundance and glutamine metabolism with nutrient availability in vivo (Baltzer, 2009).

Drosophila Delg (Drosophila Ets like gene) is a close homologue to mammalian NRF-2α, being 39% identical in amino acids sequence. In particular, the ETS domain is highly conserved, and 12 of the 13 residues that bind NRF-2α are also conserved. Delg was first identified as one of several Drosophila proteins containing an ETS domain. Specific mutants have developmental defects, particularly during oogenesis. A null mutant background (delg⁶¹³/Df(3R)ro^80b) is lethal during pupal stages, whereas a hypomorphic allele (delg^tne) gives raise to viable but sterile adults (Schulz, 1993). To stain for mitochondria, MitoTracker, which gave an abundant staining in the cytoplasm of wild type fat body cells, was first used. In contrast, delg null mutant cells showed a strong decrease in staining. Although reduced, mutant cells still retained staining that localized in a perinuclear manner. When quantified, a 30% reduction in MitoTracker staining was observed in delg mutant cells. Since these stainings were done on fixed tissues, they reflect mitochondrial abundance, but not mitochondrial activity. To further assay mitochondrial mass, NAO was used; this specifically labels the mitochondrial phospholipid cardiolipin, and is commonly used as a good readout to estimate mitochondrial mass. delg mutant cells showed a strong reduction in NAO. Again, residual mitochondria were concentrated around the nucleus. Finally, using electron microscopy, it was noticed that delg mutants had similar numbers of mitochondria, but mitochondria were strongly reduced in size, being on average 50% smaller in area. To test whether this effect is cell-autonomous, delg homozygous mutant clones were induced using the Flp/FRT system. Mutant cells, recognized by the absence of GFP, showed a strong reduction in MitoTracker. Taken together, these data demonstrate that mitochondrial mass is reduced in delg mutant fat body cells in a cell-autonomous manner. In contrast, ectopic expression of Delg did not result in an increase in mitochondrial abundance, demonstrating that Delg is required but not sufficient to control mitochondrial mass in vivo.

In Drosophila as well as other insects, the fat body is the major organ for de novo biosynthesis of fatty acids, leading to the storage of lipids as triacylglycerols. Equally important, the fat body is known to release amino acids, such a glutamine and proline, which are synthesized from the mitochondrial metabolite 2-oxoglutarate. Therefore, one would expect that mitochondrial mass and activity are regulated in response to nutrients in the larval fat body. Indeed, this study shows that strong decreases in mitochondrial abundance, respiration activity as well as expression levels of enzymes involved in glutamine and proline metabolism occur under low-yeast food. Under these feeding conditions, amino acids and fatty acids, which are both provided by yeast, become limited. Delg mutants show very similar phenotypes compared to normal fed controls, and do not show additive phenotypes in respect to mitochondrial abundance and amino acid metabolism upon low-yeast food. It is therefore proposed that Delg functions as a transcription factor to coordinate mitochondrial functions according to nutrient availability. One of these aspects is to adjust the synthesis of non-essential amino acids to the uptake of essential amino acids. In this respect, de novo synthesis of L-glutamine is particularly interesting, as the efflux of its cytoplasmic pool is used, both in mammals and Drosophila, for import of essential amino acids. It is proposed that Delg either directly senses nutrients, most likely amino acids, or is controlled by upstream sensors. Since the nutrient-sensitive Cyclin D/Cdk4 pathway functions through Delg, the latter seems more likely. Given the key role of the fat body in metabolic homeostasis of the whole animal, one might expect that fat body mitochondria be regulated differently from mitochondria in other tissues. Indeed, the phenotypes were specific to the fat body, demonstrating that Delg functions primarily in this tissue to coordinate the different anabolic and catabolic functions of mitochondria (Baltzer, 2009).

Mammalian NRF-2 was identified through its binding to the promoters of cytochrome c oxidase (COX) subunits (Virbasius, 1993; Gugneja, 1995), and has been purified as GABP (LaMarco, 1989). Active NRF-2 is a heterotetramer consisting of two alpha and two beta subunits. The alpha subunits mediate DNA binding, which requires direct GGAA/T repeats in the promoters. Accordingly, electromobility assays as well as luciferase reporter assays have shown that these motifs are functionally important. This is particular well understood for genes encoding electron transport proteins, as well as for mitochondrial protein import. Furthermore, direct NRF-2 binding to several promoters was shown by chromatin immunoprecipitation. Thus biochemical evidence links NRF-2/GABP to the transcriptional control of nuclear genes encoding mitochondrial proteins. Accordingly, RNAi studies found reduced expression of several COX subunits in cells having reduced NRF-2α levels, leading to reduced COX activity. Surprisingly, genetic data have not supported the biochemical data: MEFs lacking NRF-2α/GABPα do not have reduced mRNA or protein levels of several putative NRF-2 targets, and mitochondrial phenotypes were not reported. Drosophila Delg is the closest fly homologue to mammalian NRF-2α. Two-hybrid data show that Delg can bind to the Drosophila NRF-2β homologue CG32343, and preliminary data show that CG32343 mutants have mitochondrial defects very similar to delg mutants. Taken together, these data show that Delg functions analogues to mammalian NRF-2α, and the data are the first genetic evidence that links any member of the NRF-2α family to mitochondrial biogenesis (Baltzer, 2009 and references therein).

Of particular interest is the strong reduction in mitochondrial size in the delg mutants. This implies that mitochondrial fusion might be defective, and/or that fission occurs at an increased rate. Indeed, based on microarray data, expression of Opa1-like (CG8479), the fly homologue to mammalian fusion protein OPA1, showed a significant, 2-fold reduction in expression in delg mutant fat body samples. In contrast, fly homologues to mammalian Mitofusins, which are well-established fusion factors (fly homologues Marf/CG3869 and Fuzzy onions/CG4568), were not differently expressed. More work is required to test whether delg mutant have defects in mitochondrial fusion. In addition, when delg homozygous mutant clones were induced, a strong reduction was noted in cell size, yet the nuclear size, shown by the DAPI staining, was not changed, demonstrating growth defects, This is surprising, since endoreplication, and thus nuclear size, normally correlates with cell size in this tissue. Since mitochondrial biogenesis has been shown to correlate with nuclear DNA synthesis, the data suggest that Delg might be involved to link S-phase and potentially cell size to mitochondrial mass. Future work will be required to address this hypothesis (Baltzer, 2009).

Genes involved in mitochondrial OXPHOS activity, including RFeSP and Blw, showed similar reduced expression in the delg mutant or under low yeast nutrition. Importantly, additive defects were detected when delg mutant were grown under low yeast. Moreover, when oxygen consumption was measured in permeabilized fat body tissues, state 3 respirations were strongly affected by low yeast nutrition, yet this was independent of Delg. This demonstrates that factors other than Delg must regulate mitochondrial OXPHOS activity in response to nutrients. One candidate is Spargel/CG9809, the fly homologue to mammalian PGC-1 proteins, which are transcriptional coactivators that control mitochondrial mass and activity in response to external stimuli. Indeed, Spargel functions in parallel to Delg, and mediates a link between insulin-signalling and the expression of genes encoding mitochondrial proteins. Therefore, Delg and Spargel mediate two parallel pathways that control mitochondrial mass and OXPHOS activity in response to nutrients (Baltzer, 2009).

Drosophila Cyclin D/Cdk4 is a cyclin-dependent protein kinase complex, and controls cellular growth levels in addition to regulating cell cycle progression. Importantly for this study, overgrowth induced by ectopic expression of Cyclin D/Cdk4 is insensitive to nutrient conditions, demonstrating that the Cyclin D/Cdk4 pathway is nutrient-responsive. This study shows that ectopic expression of Cyclin D/Cdk4 in the larval fat body is sufficient to drive mitochondrial abundance in a Delg-dependent manner, suggesting a mechanism where Cyclin D/Cdk4 coordinate growth levels and mitochondrial mass. Furthermore, one would expect that the transcriptional activity associated with Delg is regulated in response to Cyclin D/Cdk4. Indeed, using lacZ insertions into the loci of several genes encoding mitochondrial proteins, including Blw, clonal expression of Cyclin D/Cdk4 was sufficient to stimulate expression of these genes. However, this effect was restricted to wandering third instar larvae, but was not seen in feeding L1, L2 or L3 larvae. Moreover, when larvae were grown under low-yeast conditions, ectopic expression of Cyclin D/Cdk4 led to reduced expression of these genes, again based on lacZ insertions. It is concluded that Cyclin D/Cdk4 is not a general activator of Delg function, but might mediate a nutrient and/or developmental-dependent control (Baltzer, 2009).

In mammals, D-type cyclins bound to Cdk4 or Cdk6 are best characterized for their functions during cell cycle progression, but several reports have shown additional roles, in particular the regulation of multiple transcription factors by direct binding. Importantly for this study, mitochondrial size and activity are regulated in response to mammalian Cyclin D1: Knockdown or knockout of Cyclin D1 leads to larger mitochondria that are more active. Conversely, ectopic expression of Cyclin D1 inhibits mitochondrial activity, a function that requires binding to Cdk4. Moreover, Cyclin D1 and NRF-1 are functional linked: Cyclin D1 stimulates NRF-1-dependent transcription, the two proteins interact, and a protein kinase associated to immunoprecipitated vCyclin D1 can phosphorylate NRF-1. This suggests a mechanism where Cyclin D1/Cdk4 inhibit mitochondrial mass and activity through inhibition of NRF-1 function. Thus D-type cyclins bound to Cdk4 have opposite effects on mitochondrial mass in mammals compared to flies, suggesting that different mechanisms have been adopted during evolution to control mitochondria (Baltzer, 2009).

Protein Interactions

Yeast two hybrid experiments indicate that CycD interacts with both cdc2 and cdc2c, the two cyclin dependent kinases of Drosophila (Finley, 1996).

During Drosophila eye development, cell proliferation is coordinated with differentiation. Immediately posterior to the morphogenetic furrow, cells enter a synchronous round of S phase called second mitotic wave. This study examines the role of RBF, the Drosophila RB family homolog, in cell cycle progression in the second mitotic wave. RBF-280, a mutant form of RBF that has four putative cdk phosphorylation sites mutated, can no longer be regulated by Cyclin D or Cyclin E. RBF-280 retains the wild-type RBF ability to inhibit transactivation by E2F1. Expression of RBF-280 in the developing eye reveals that RBF-280 does not inhibit G1/S transition in the second mitotic wave; rather, it delays the completion of S phase and leads to abnormal eye development. These observations suggest that RB and E2F control the rate of S-phase progression instead of G1/S transition in the second mitotic wave. Characterization of the role of RBF in Cyclin D/Cdk4-mediated cellular growth shows that RBF-280 blocks Cyclin D/Cdk4 induced cellular growth in the proliferating wing disc cells but not in the non-dividing eye disc cells. By contrast, RBF-280 does not block activated Ras-induced cellular growth. These results suggest that the ability of Cyclin D/Cdk4 to drive growth in the proliferating wing cells is distinct from that in the non-dividing eye cells or the ability of activated Ras to induce growth, and that RBF may have a role in regulating growth in the proliferating wing discs (Xin, 2002).

RBF-280 cannot be regulated by Cyclin D- and Cyclin E-dependent kinases. RBF-280 was used to test the role of RBF in developmentally regulated cell proliferation and in Cyclin D/Cdk4-induced cellular growth. Inhibiting the E2F target gene expression in the second mitotic wave of the developing eye, via expression of RBF-280, mainly delays S-phase completion instead of inhibiting S phase entry. These results suggest that cells in the second mitotic wave are driven into S phase through an RB/E2F-independent mechanism. In addition, while RBF-280 completely inhibits cellular growth induced by Cyclin D/Cdk4 in the proliferating wing discs, RBF-280 cannot block cellular growth induced by activated Ras in the wing disc cells or Cyclin D/Cdk4 in the non-dividing eye cells. These observations indicate that the ability of Cyclin D/Cdk4 to induce growth in the proliferating wing discs is distinct from the ability of activated Ras to induce growth or the ability of Cyclin D/Cdk4 to induce growth in the non-dividing eye cells. In addition, these results suggest that RBF may have a role in inhibiting growth (Xin, 2002).

Cyclin D/Cdk4 is able to drive cellular growth in addition to induce cell proliferation. Interestingly, the consequence of Cyclin D/Cdk4 expression is different in different cell types: Cyclin D/Cdk4 primarily induces growth and lead to larger cells in the non-dividing differentiated eye cells (hypertrophy); Cyclin D/Cdk4 induces increased DNA endoreplication and increased cell size (hypertrophy) in the salivary gland cells; Cyclin D/Cdk4 induces growth and division coordinately without affecting cell size in the proliferating wing discs, leading to more rapid cell cycle and more cells (hyperplasia) (Datar, 2000). To test if these biological effects are mediated by RBF, the effect of RBF-280, a form of RBF that cannot be regulated by Cyclin D, was tested on cellular growth and proliferation induced by Cyclin D and Cdk4 (Xin, 2002).

RBF is an important target of Cyclin D/Cdk4 in G1/S regulation. RBF-280 blocks the ability of Cyclin D to induce S phase in G1 arrest eye disc cells and the excessive DNA endoreplication in the salivary gland cells. In addition, RBF-280 also blocks the ability of Cyclin D/Cdk4 to increase the rate of cell proliferation in the proliferating wing discs. These results demonstrate that the ability of Cyclin D/Cdk4 to induce cell proliferation (G1/S transition) is mediated through inactivation of RBF. By contrast, the ability of RBF-280 to block Cyclin D/Cdk4 induced growth varies in different cell types. In the proliferating wing disc cells, Cyclin D/Cdk4 expression leads to more rapid cell cycle and more cells (hyperplasia). RBF-280 completely blocks the effect of Cyclin D/Cdk4. The average size of RBF-280 clones is not significantly different from the average size of Cyclin D+Cdk4+RBF-280 clones (P=0.68). In addition, the number of cells in the clone is also not significantly different (P=0.32). These observations indicate that RBF-280 blocks both cell growth and proliferation induced by Cyclin D/Cdk4 in the proliferating wing discs. The effect of RBF-280 on Cyclin D/Cdk4-induced growth is distinct from the effect of RBF-280 on activated Ras induced growth. Activated Ras induces cellular growth and leads to larger cells without affecting the rate of cell doubling in the wing discs (hypertrophy). The average area of Ras^V12+RBF-280 clones is significantly larger than the area of RBF-280 clones (P<0.0001). The observed increase in clone size is mainly due to increased cell size. Although it is not possible to get enough cells for Facs analysis to determine the cell size directly, because clones with RBF-280 expression are extremely small, cell size estimation using the average cell sizes derived from clone area/cell number has shown that RBF-280+Ras^V12 cells arenoticeably larger than RBF-280 cells, while the size of RBF-280+Cyclin D+Cdk4 cells is similar to the size of RBF-280 cells. These results are consistent with the reported cell size effect of Cyclin D/Cdk4 and activated Ras on RBF-WT. It was shown that the Cyclin D/Cdk4+RBF-WT cells are similar in size as the RBF-WT cells, while the Ras^V12+RBF-WT cells are much larger than the RBF-WT cells. Taken together, this evidence supports the notion that Ras^V12 can stimulate cellular growth in the presence of functional RBF, while Cyclin D/Cdk4 induces growth at least in part through inactivation of RBF in the developing wing discs (Xin, 2002).

Similar to activated Ras, Cyclin D/Cdk4 also induces growth and leads to large eyes as a result of increased cell size in the non-dividing eye cells (hypertrophy). Consistent with the idea that the large eye phenotypes are the consequence of cellular growth induced by Cyclin D/Cdk4, which is mediated through targets distinct from RBF, RBF-280 blocks Cyclin D/Cdk4-induced ectopic S phase in the eye discs but not Cyclin D/Cdk4 induced large eye phenotype. It is likely that Cyclin D/Cdk4 drives growth through distinct targets in the non-proliferating eye cells and in the proliferating wing disc cells. The target in the non-proliferating eye cells can drive growth in the presence of RBF-280, while the target in the proliferating wing disc cells are either RBF itself or a target that can drive growth only when RBF is inactivated. Further studies will be needed to identify the targets that mediate the ability of Cyclin D/Cdk4 to induce growth (Xin, 2002).

Inactivation of Cyclin E-Cdk2 is essential for a timely arrest of the epidermal cell proliferation program during Drosophila embryogenesis. E-type cyclin-cdk complexes are thought to be activated by D-types titrating away inhibitors and inducing cyclin E transcription by activating E2F transcription factors via Rb phosphorylation. Therefore, a study was performed to see whether the developmentally controlled inactivation of Cyclin E-Cdk2, required for the epidermal cell proliferation arrest, occurs as a consequence of Cyclin D-Cdk4 inactivation. However, preventing Cyclin D-Cdk4 inactivation by overexpression has a minimal effect on Cyclin E expression and does not interfere with the initial G1 arrest, while it readily induces the E2F target RnrS in arresting epidermal cells. Prolonged Cyclin D-Cdk4 overexpression eventually interferes with maintenance of quiescence in some cells. Moreover, in Cdk4 mutant embryos, some RnrS expression is still induced by Cyclin E overexpression, and endogenous Cyclin E expression as well as cell cycle progression is not affected, except for late aspects of the endoreduplication program. These findings argue against the proposed necessity of complete Rb inactivation by sequential phosphorylation by D- and E-type cyclin-cdk complexes. They demonstrate that Cyclin D-Cdk4 does not function as the master regulator of the embryonic cell proliferation program (Meyer, 2002).

In conclusion, while Cyclin D-Cdk4 overexpression interferes in some cells with maintenance of quiescence, it does not preclude an initial timely entry into proliferative quiescence. Moreover, the effects of Cyclin D-Cdk4 and Cyclin E overexpression in Cyclin E and Cdk4 mutant embryos, respectively, cannot be easily reconciled with an obligatory sequential action of D- and E-type cyclin-cdk complexes, which has been postulated for some classes of E2F target genes based on evidence obtained in vitro and in mammalian cell culture systems. Cyclin D-Cdk4 plays a minor role in the control of Cyclin E-Cdk2 activity and cell cycle progression during the embryonic cell division cycles. These cycles are characterized by a relaxed coupling to cell growth because of the abundant maternal stores that are stockpiled in the egg. However, Cyclin D-Cdk4 is partially required for progression through endoreduplication cycles late in embryogenesis, which might already be triggered by nutrients extracted from the yolk by the newly formed gut, similar to the larval endoreduplication cycles, which are clearly nutrient dependent. Moreover, consistent with the proposal that Cyclin D-Cdk4 complexes are primarily involved in the regulation of growth rates, oogenesis and imaginal development, which are tightly coupled to growth, are most severely affected in Cdk4 mutants (Meyer, 2002).

The JAK/STAT signal transduction pathway regulates many developmental processes in Drosophila. However, the functional mechanism of this pathway is poorly understood. The Drosophila cyclin-dependent kinase 4 (Cdk4) exhibits embryonic mutant phenotypes identical to those in the Hopscotch/JAK kinase and stat92E/STAT mutations. Specific genetic interactions between Cdk4 and hop mutations suggest that Cdk4 functions downstream of the HOP tyrosine kinase. Cyclin D-Cdk4 (as well as Cyclin E-Cdk2) binds and regulates STAT92E protein stability. STAT92E regulates gene expression for various biological processes, including the endocycle S phase. These data suggest that Cyclin D-Cdk4 and Cyclin E-Cdk2 play more versatile roles in Drosophila development (Chen, 2003).

In a large screen for autosomal P element-induced zygotic lethal mutations associated with specific maternal effect lethal phenotypes, a mutation, l(2)sh0671, located at 53C, was identified that showed a maternal effect segmentation phenotype. The phenotype is similar to the effect of loss of hop and stat92E gene activity during oogenesis. The P element, l(2)sh0671, was inserted into the second intron of the Cdk4 gene before the ATG translation initiation code (Chen, 2003).

In mammals and Drosophila, Cdk4 forms a protein complex that regulates the cell cycle progression. The Cyclin D and Cdk4 complex (CycD-Cdk4) phosphorylates and releases RB from RB/E2F; free E2F then activates gene expression, including Cyclin E (CycE). Cyclin E and Cdk2 form a complex (CycE-Cdk2) and regulate the cell cycle at the G1-S transition point. To further examine relations between the HOP/STAT92E signal transduction pathway and cell cycle regulation, the genetic interaction of hop with CycE was tested. Like HS-Cdk4, HS-CycE rescues hop^C111 embryo segmentation defects but has no effect on stat92E mutant embryos (Chen, 2003).

The viability and formation of melanotic tumors at 29°C were compared in females heterozygous for hop^Tum-l and CycE with females heterozygous only for hop^Tum-l. An improved survival rate was observed by removing a single copy of CycE in hop^Tum-l heterozygous females. As in the case of Cdk4, the formation of melanotic tumors is less affected by removing a single copy of CycE in hop^Tum-l heterozygous females. These results suggest that CycD-Cdk4 and CycE-Cdk2 complexes are members of the HOP/STAT92E signal transduction pathway and function downstream of the HOP tyrosine kinase and either upstream of or parallel to the STAT92E transcription factor (Chen, 2003).

Thus Cdk4 functions in the HOP/STAT92E pathway and regulates embryonic segmentation, tracheal formation, eye development, and melanotic tumor formation. Specific genetic interactions between Cdk4 and hop or stat92E mutations suggest that Cdk4 functions upstream of STAT and parallel to or downstream of the HOP tyrosine kinase. Furthermore, CycD-Cdk4 and CycE-Cdk2 bind and regulate STAT92E protein stability. These data demonstrate that, besides their role in regulating the cell cycle, CycD-Cdk4 and CycE-Cdk2 have a role in regulating cell fate determination and proliferation via STAT signaling (Chen, 2003).

Hif-1 prolyl hydroxylase is a regulator of cellular growth and that it is a key mediator for CycD/Cdk4

The Drosophila cyclin-dependent protein kinase complex Cyclin D/Cdk4 induces cell growth (accumulation of mass) as well as proliferation (cell cycle progression). To understand how CycD/Cdk4 promotes growth, a screen was performed for modifiers of CycD/Cdk4-driven overgrowth in the eye. Loss-of-function mutations in Hif-1 prolyl hydroxylase (Hph), an enzyme involved in the cellular response to hypoxic stress, dominantly suppress the growth but not the proliferation function of CycD/Cdk4. hph mutant cells are defective for growth, and, remarkably, ectopic expression of Hph is sufficient to increase cellular growth. Epistasis analysis places Hph downstream of CycD/Cdk4. Overexpressed CycD/Cdk4 causes an increase in Hph protein in tissues where Hph induces growth, suggesting a mechanism whereby Hph levels are regulated posttranscriptionally in response to CycD/Cdk4. These data suggest that Hph, in addition to its function in hypoxic response, is a regulator of cellular growth and that it is a key mediator for CycD/Cdk4. Nevertheless, observations weigh against an important role for Hif-1 in Hph-driven growth, suggesting a potential Hif-1-independent function of Hph (Frei, 2004).

The synthetic promoter element Glass multiple reporter (GMR) is expressed in the eye imaginal disc posterior to the morphogenetic furrow, where only cells in the second mitotic wave undergo one synchronized cell division. GMR can be used to drive expression of the yeast transcription factor Gal4. Gal4 can be used to direct transcription of transgenes placed next to the UAS binding site of Gal4. Therefore, UAS transgenes driven by Glass-activated GMR-Gal4 are expressed predominantly in postmitotic cells. Under these circumstances, expression of CycD/Cdk4 leads to an enlargement of the adult eye, bigger ommatidia and bristles, and a general rough appearance. Although some ommatidia have additional cells, the main cause of the enlargement is an increase in cell size leading to 61% larger ommatidia. In order to identify loss-of-function mutants that modify this phenotype, CycD/Cdk4 was expressed in a deficiency collection background and screened under the light microscope for modifiers. Out of 162 deficiencies that cover 60%-70% of the genome, four deficiencies were isolated that dominantly suppress CycD/Cdk4. Df(3R)6-7, which deletes polytene segments 82D3/8-F3/6, led to a decrease in the enlargement of the eye and bristle size. Most strikingly, under these conditions, CycD/Cdk4 led to an increase in ommatidia size of only 17. Subsequently, partial overlapping deficiencies were tested: Df(3R)3-4 showed the same suppression phenotype but not a third deficiency, Df(3R)110, demonstrating that the gene of interest is between 82F3 and F7. All available mutants in this region were tested, and two lethal P element insertions were identified that showed an identical suppression phenotype compared to the deficiencies: l(3)02255 is inserted 104 bp, and l(3)S030304 is inserted 1111 bp upstream of the putative transcription start site of Hph/dmHph/CG1114 (Frei, 2004).

Whether l(3)02255 and l(3)S030304 are alleles of hph and whether their loss of function causes the suppression of CycD/Cdk4 were addressed. Northern blot experiments revealed that both heterozygous mutants l(3)02255 and l(3)S030304 have reduced expression of hph compared to the endogenous levels and that l(3)02255/l(3)S030304 transheterozygotes lack all detectable hph expression. In addition to these loss-of-function mutants, the EP3200 line has an EP insertion 382 bp upstream of Hph. Expression of hph using this EP element, by the hs-Flp Act>CD2>Gal4 system, led to a weak expression of hph in the absence of a heat shock, due to leakage of the system. A further increase in hph expression occured upon heat shock (Frei, 2004).

A full-length Hph cDNA was cloned under the control of a UAS promoter and injected into flies. These Hph transgenes suppress at least partially the cell growth phenotype of homozygous hph mutants and completely suppress the reduced viability of l(3)02255/l(3)S030304 transheterozygotes. Furthermore, when UAS-Hph transgenes were coexpressed with CycD/Cdk4 using the GMR-Gal4 driver, the dominant suppression of CycD/Cdk4-driven overgrowth by l(3)02255 was inhibited and the overgrowth phenotype was restored. It is concluded that l(3)02255 and l(3)S030304 are alleles of Hph and that the reduction of Hph can suppress CycD/Cdk4-induced overgrowth (Frei, 2004).

Whether growth or proliferation would be suppressed by hph in the eye imaginal disc was tested. GFP was expressed either alone or together with CycD/Cdk4 in wild-type, hph⁰²²⁵⁵/+, or Df(3R)3-4/+ backgrounds using the GMR-Gal4 driver. Imaginal eye discs from wandering third instar larvae were dissected, trypsinized to single cells, and analyzed by FACS for their cell size by using the forward scatter (FCS). Expression of CycD/Cdk4 in a wild-type background led to an increase in the forward scatter of 20%-30%, which was reduced to 10%-15% in an hph⁰²²⁵⁵/+ or Df(3R)3-4/+ background. Acridine orange staining showed that the suppression phenotype was not due to an increase in cell death. Furthermore, the cell cycle distribution was analyzed of eye imaginal discs from wandering larvae or pupae 48 hr after prepupae formation. At both time points, the increase in cells entering S and G2/M phases of the cell cycle due to ectopic expression of CycD/Cdk4 was not altered in an hph⁰²²⁵⁵/+ background. Taken together, these data demonstrate that the cell size and proliferation functions of CycD/Cdk4 can be separated. Furthermore, Hph is required for the increase in cell size but not required for proliferation, suggesting that Hph functions downstream of CycD/Cdk4 in a growth-specific manner (Frei, 2004).

In the experiments described above, CycD/Cdk4 was induced in mostly postmitotic cells of the eye imaginal disc. To test suppression by hph in mitotically dividing cells, CycD/Cdk4 was induced during larval development, and wing discs cells were analyzed. Ectopic expression of CycD/Cdk4 shows a distinctive induction of growth: cells divide at a faster rate but are otherwise indistinguishable from control cells from the same disc. Therefore, when single clones are measured, the clone area is increased, and the clone consists of more cells with no change in cell size or cell cycle phasing. Since columnar cells of wing discs form a single cell layer, measuring the clone area gives an accurate estimation of the amount of mass that was accumulated during the growth of the clone. CycD/Cdk4 was overexpressed together with GFP in random clones using the hs-Flp Act>CD2>Gal4 system and analyzed after a 48 hr growth period. Compared to external control clones expressing only GFP, expression of CycD/Cdk4 caused a 75% increase in the median clone size. This phenotype depends on Hph, since the median clone size was reduced to control level in a heterozygous hph⁰²²⁵⁵ mutant background. The suppression did not correlate with an increase in apoptosis, since coexpression of the cell death inhibitor p35 gave identical phenotypes. When cell size and cell cycle phasing were analyzed by FACS, there was no difference between cells expressing CycD/Cdk4 and internal control cells in either wild-type or hph/+ mutant backgrounds. These results demonstrate that the induction of growth by CycD/Cdk4 depends on normal levels of Hph. Furthermore, since hph suppressed growth but not proliferation in the eye imaginal discs, expression of CycD/Cdk4 in the wing should lead to a change in cell size if only growth but not proliferation were suppressed. However, no difference in cell size was detected, suggesting that the increase in proliferation caused by CycD/Cdk4 is secondary to the induction of growth (Frei, 2004).

Whether hph function was required for normal rates of cell growth was tested. Most hph⁰²²⁵/hph⁰²²⁵⁵ or hph⁰²²⁵⁵/Df(3R)3-4 animals die during embryogenesis, and only a few larvae hatch. These mutant larvae have severe growth defects and die within 2 to 3 days. Transheterozygotic hph^S030304/hph⁰²²⁵⁵ mutants develop normally until pupariation, but very few escaper adults eclose. These escapers are smaller than their heterozygous siblings but have normal body proportions. Weight measurements showed that hph^S030304/hph⁰²²⁵⁵ mutant flies are 18% lighter than heterozygotes. Therefore, hph mutant animals show a phenotype similar to homozygous cdk4³ flies (Meyer, 2000) or wild-type flies reared at low oxygen (Palos, 1979; Frazier, 2001; Frei, 2004).

To test whether hph mutant cells are autonomously defective for growth, homozygous mutant clones were induced in the fat body using ionizing radiation. hph⁰²²⁵⁵ was crossed to flies expressing GFP under the control of a constitutively active promoter inserted on the same arm of the chromosome as Hph (3R). The progeny were irradiated during embryogenesis, emerging larvae were grown in regular food, wandering third instar larvae were dissected, and their fat bodies were fixed and mounted. Homozygous hph⁰²²⁵⁵ mutant cells lacked GFP, whereas heterozygous mutant cells expressed GFP. hph⁰²²⁵⁵/hph⁰²²⁵⁵ cells were smaller and contain less DNA than heterozygous neighboring cells. Importantly, the presence of a UAS-Hph transgene partially suppresses this phenotype, indicating that loss of Hph is the cause of the growth defect (Frei, 2004).

Whether ectopic expression of Hph is sufficient to stimulate growth was tested. EP3200 or UAS-Hph transgenes were used to induce Hph expression. Cell clones expressing Hph were induced in wing imaginal discs, and the median clone size was measured. Expression of Hph led to an increase in clone area very similar to CycD/Cdk4. Surprisingly, expression of Hph together with CycD/Cdk4 stimulated clonal growth to the same extent as Hph alone. However, in the presence of the apoptosis inhibitor p35, an additive phenotype was detected when both growth drivers were coexpressed. As for CycD/Cdk4, overexpressed Hph did not change cell size or cell cycle phasing, as assayed by FACS (Frei, 2004).

To test whether Hph functions downstream of CycD/Cdk4 also in this tissue, clones expressing Hph were induced in a homozygous cdk4³ mutant background, and the median clone size was measured. Under these conditions, Hph led to a very similar induction of growth as in a wild-type background. FACS analysis indicates that there are no detectable changes in cell size or cell cycle phasing. These data show that Hph is sufficient to stimulate growth, and the finding that this stimulation is independent of Cdk4 suggests that Hph functions downstream of CycD/Cdk4 (Frei, 2004).

Ectopic expression of CycD/Cdk4 in the posterior compartment of the wing imaginal disc using the en-Gal4 promoter leads to an enlargement of the posterior compartment in adult wings with no change in trichome (hair) density. Since the trichome density is proportional to the number of cells per area, the increase in compartment size is due to more cells of the same size. When Hph was expressed under en-Gal4 control using EP3200 or UAS-Hph transgenes, a similar result was obtained: posterior compartments were bigger and contained more cells of the same size. Thus, Hph induces growth in a similar manner to CycD/Cdk4 in wing imaginal discs. However, when Hph was expressed in the eye imaginal disc using the GMR-Gal4 driver, no increase in cell size, as assayed by FACS, was observed. Furthermore, adult eyes were not enlarged. Further experiments are required to understand why Hph expression is not sufficient to increase growth in the eye imaginal disc (Frei, 2004).

Little is known about how Hph RNA or protein levels are regulated. In Drosophila embryos, hph is expressed uniformly and does not seem to be subject to patterning. Vertebrate cells have three Hph orthologs (Taylor, 2001): HPH-3 protein localizes to the nucleus, HPH-2 exclusively to the cytoplasm, and HPH-1 mainly in the cytoplasm with a little staining in the nucleus (Huang, 2002; Metzen, 2003; Frei, 2004).

To test whether the subcellular localization or levels of Hph are altered in response to CycD/Cdk4, polyclonal antibodies were raised to full-length Hph. To test the specificity of the antiserum, Hph was overexpressed in the posterior compartment of the wing using the en-Gal4 driver. Hph staining increased in posterior regions, both in the peripodial and columnar epithelium. Homozygous hph mutant cells, marked by the absence of GFP, lacked detectable Hph staining. Therefore, the serum is specific for Hph and is able to detect endogenous levels of Hph. Furthermore, in third instar imaginal wing discs, Hph staining was uniform throughout the disc and specific for the nucleoplasm of the cells. Very little staining was detectable in the cytoplasm or the nucleolus (Frei, 2004).

When Hph staining was analyzed in cells expressing ectopic CycD/Cdk4, increased Hph levels were observed. Antiserum staining in the peripodial epithelium shows that these cells had an increase in nucleoplasmic as well as cytoplasmic Hph. In contrast, when homozygous cdk4³ mutant cells, marked by the absence of GFP, were analyzed, only background levels were detected. Therefore, Hph protein levels are regulated in response to CycD/Cdk4 gain and loss of function. When hph expression was analyzed by RT-PCR from wing discs expressing ectopic CycD/Cdk4, no effect on Hph RNA levels was detected. Furthermore, when whole third instar larvae were analyzed by RT-PCR or microarray analysis, no change in Hph expression was observed. Taken together, these observations suggest that CycD/Cdk4 affects Hph levels posttranscriptionally (Frei, 2004).

The hydroxylation activity of HPHs depends of Fe²⁺ bound to the active site. Therefore, iron chelators like deferoxamine mesylate (DFO) are commonly used to experimentally mimic hypoxic conditions. When Drosophila larvae are raised on regular food supplemented with 2 mM DFO, they show an induction in Hif-1α/β, as assayed with a reporter construct, that is very similar to that seen under hypoxic conditions. When CycD/Cdl4 or Hph are expressed in the posterior compartment of the wing imaginal disc, the increase in compartment areas is suppressed by DFO. Moreover, when DFO is added to flies expressing CycD/Cdk4 in the postmitotic eye using the GMR-Gal4 driver, the enlargement of the adult eye as well as the rough appearance is suppressed, however, not to the same extent as in the heterozygous hph mutant backgrounds. This suggests that the hydroxylation activity of Hph is required for its growth function and that Hph is a major growth effector of CycD/Cdk4 (Frei, 2004).

The finding that Drosophila Hph functions downstream of CycD/Cdk4 and is sufficient to increase growth when overexpressed suggests that CycD/Cdk4 and Hph work in a common pathway. Consistent with this, heterozygous hph mutants do not suppress the extra growth induced by components of the insulin pathway or dMyc. Moreover, increases in cell size and changes in cell cycle phasing induced by the insulin signaling pathway, dMyc, or Ras in wing imaginal disc cells, do not depend on Cdk4. Taken together, these results suggest that the CycD/Cdk4-Hph pathway functions separately from these other growth regulatory pathways (Frei, 2004).

Since the kinase activity of Cdk4 is required for the induction of growth and proliferation, Hph could be a phosphorylation target of Cdk4. The consensus sequence of vertebrate pocket proteins, the only known targets of cyclin D1/Cdk4, can be different from the classical CDK sequence. In Drosophila Rbf1, two potential sites have been found that disrupt its regulation by CycD/Cdk4 and CycE/Cdk2: T³⁵⁶PLTR and S⁷²⁸PHPK. Both sites are different from the vertebrate consensus sequence. Therefore, a search for putative consensus sequences on Hph is difficult. However, there are three sites that have the minimal requirement of a serine or threonine residue followed by a proline: T⁹¹PDAP, T²⁰⁴PGTT, and T²⁸⁵PPAA. None of these resemble the consensus sequences recognized by either the vertebrate or Drosophila complex. Nevertheless, future experiments should address whether CycD/Cdk4 phosphorylates Hph on these or other sites and how this affects Hph function (Frei, 2004).

It is proposed that in wing discs, Hph protein levels are regulated in response to CycD/Cdk4. Although it cannot be excluded that growth is also induced in an Hph-independent manner, the findings that overgrowth driven by CycD/Cdk4 and Hph is suppressed nearly completely by the iron chelator DFO or by heterozygosity for hph suggest that this is a major mechanism. Moreover, it is proposed that the small size of flies reared at low oxygen concentrations is caused at least partially by a decrease in Hph activity due to the absence of oxygen (Frei, 2004).

How does Hph induce growth? Since the hydroxylation inhibitor DFO suppressed the increases in growth caused by CycD/Cdk4 or Hph, Hph's hydroxylation activity is probably required. The only characterized hydroxylation target of Hph is Hif-1α, a mediator of the transcriptional response to hypoxia. Although mutant alleles of the Drosophila Hif-1α ortholog sima are not available, a partial loss-of-function allele of the Hif-1β ortholog, tango¹, was available. To test the potential role of Hif1 in growth control, the ey-Flp/FRT method was used to generate flies in which the eyes were >80% homozygous mutant for tango¹. If Hph stimulates growth by hydroxylating Hif-1α and targeting it for degradation, then loss of Hif-1 activity might be expected to result in overgrowth phenotypes. Contrary to this expectation, overgrowth was not observed in tango¹/tango¹ eyes. Moreover, GMR-driven expression of CycD/Cdk4 led to the same degree of overgrowth in tango¹/tango¹ eyes as in wild-type controls. Although these observations weigh against an important role for Hif-1 in Hph-driven growth, it is important to note that tango¹ is not a null allele and that Tango is thought to be expressed in excess over its binding partner, Sima. Thus, further analysis using sima mutants and overexpression will be required to definitively test whether Hph drives cell growth via a Hif-1-dependent mechanism or through hydroxylation of novel targets. The finding that only one of the three vertebrate Hph orthologs is required for regulation of Hif-1α levels in vivo (Berra, 2003) further suggests that additional targets may be important (Frei, 2004).

There is little data that suggest a growth function for vertebrate HPH. Rat HPH-1/SM-20 was identified first as a gene upregulated by growth factors or serum. The induction is very fast and peaks at 60 min after stimulation. Remarkably, this induction does not require de novo protein synthesis, as it is not blocked by the translation inhibitor cyclohexamide. The effect on growth upon deregulation of mouse Falkor/HPH-3 is controversial: whereas expression of a C-terminal fragment induced cells to grow faster and to a higher density, expression of a wild-type construct had no effect. An antisense oligonucleotide specific for Falkor induced cells to grow faster. Thus, the function of vertebrate HPH family member in growth control is still ambiguous (Frei, 2004).

Drosophila Hph has at least two functions: response to hypoxia and regulation of growth. How are they linked? In response to hypoxia, Sima/Tango activity is strongly induced in endoreplicative tissues like trachea, gut and fat body, and to a much lesser extent, in imaginal discs (Lavista-Llanos, 2002). Although endopreplicative cells lacking Hph are impaired for growth, ectopic overexpression of Hph in these cells does not increase their size. In contrast, in imaginal discs, Hph can increase growth when overexpressed. It is speculated that in endoreplicative tissues, Hph's main function is to regulate the hypoxic response and, to a minor extent, growth, whereas in imaginal tissues, Hph's main function is to regulate growth. Taken to the environment of wild Drosophila, this suggests that hypoxic conditions, which are often found in fermenting fruit, may induce a strong hypoxic response in endoreplicative tissues. Since these tissues are metabolically highly active, this response may be required for the generation of sufficient ATP by the induction of glycolysis. In imaginal discs, cell cycle progression is not controlled primarily by extrinsic factors but by disc intrinsic growth cues. Therefore, even under hypoxic stress, growth and development of imaginal discs continues but may be slowed down, presumably by inactivation of Hph activity, in order to ensure the formation of adult animals (Frei, 2004).

In fat body cells, Hph is a nuclear protein, and homozygous Cdk4 mutant cells lack detectable Hph levels. Moreover, ectopic expression of CycD/Cdk4 leads to more Hph protein in the cytoplasm and/or the nucleus. Surprisingly, a reporter line showed an increase, rather than a decrease, in Sima activity upon expression of CycD/Cdk4. It is proposed that in the fat body, Hph induced by CycD/Cdk4 is not sufficient to hydroxylate Hif-1α. In addition to the cofactors oxygen and iron, hydroxylation activity requires the binding of 2-oxoglutarate to the active site of HPH (Epstein, 2001; Bruick, 2001). 2-oxoglutarate is an intermediate of the citrate cycle, and its levels might correlate with the metabolic activity of the cell. Therefore, Hph protein may be induced by CycD/Cdk4 but may require 2-oxoglutarate and oxygen for catalytic activity in the fat body. In this model, Hph would integrate the regulation of growth by CycD/Cdk4 and its upstream regulators, with the regulation of growth by the metabolic activity, mediated by oxygen and 2-oxoglutarate (Frei, 2004).

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