Cell Cycle 11:3, 554-568; February 1, 2012; © 2012 Landes Bioscience

RESEARCH PAPER

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*Correspondence to: Bruce A. Edgar; Email: b.edgar@dkfz.deSubmitted: 12/09/11; Accepted: 12/14/11http://dx.doi.org/10.4161/cc.11.3.19062

Introduction

CyclinD (CycD) and cyclin-dependent kinase 4 (Cdk4) form a growth-factor and mitogen-regulated complex acting at the interface between extracellular signaling and the nucleus to control progression through the cell cycle.1 Extracellular growth factors, oncogenes (Ras, Myc) and serum induce the activation of CycD/Cdk4 during G

1 phase of the cell cycle.1-6 Thus, the

CycD/Cdk4 complex is a common regulatory node for many growth signals induced by mitogens and oncogenes to ensure that cells enter the cell cycle when extracellular conditions are energetically favorable.4 In this study we focus on Drosophila CycD, orthologous to the three paralogs in mammals, CycD1, CycD2 and CycD3. Mammalian CycD1 has been character-ized as a proto-oncogene, whose gene amplification and pro-tein overexpression frequently occurs in many cancers.1 CycD activity initiates the phosphorylation-dependent inactivation of the retinoblastoma tumor suppressor and confers cells with a growth advantage. Thus, the deregulation of CycD/Cdk4 activity can contribute to neoplasia.1 The hyperactivation of mammalian D-type cyclins in vivo leads to robust cellular pro-liferation, growth and tumorgenesis.7,8 Accordingly, mamma-lian cells lacking D-cyclins show greatly reduced susceptibility to the oncogenic action of both Ras or Myc.9 CycD1-deficient

Drosophila cyclin D (CycD) is the single fly ortholog of the mammalian cyclin D1 and promotes both cell cycle progression and cellular growth. However, little is known about how CycD promotes cell growth. We show here that CycD/Cdk4 hyperactivity leads to increased mitochondrial biogenesis (mitobiogenesis), mitochondrial mass, NRF-1 activity (Tfam transcript levels) and metabolic activity in Drosophila, whereas loss of CycD/Cdk4 activity has the opposite effects. Surprisingly, both CycD/Cdk4 addition and loss of function increase mitochondrial superoxide production and decrease lifespan, indicating that an imbalance in mitobiogenesis may lead to oxidative stress and aging. In addition, we provide multiple lines of evidence indicating that CycD/Cdk4 activity affects the hypoxic status of cells and sensitizes animals to hypoxia. Both mitochondrial and hypoxia-related effects can be detected at global transcriptional level. We propose that mitobiogenesis and the hypoxic stress response have an antagonistic relationship, and that CycD/Cdk4 levels regulate mitobiogenesis contemporaneous to the cell cycle, such that only when cells are sufficiently oxygenated can they proliferate.

Drosophila cyclin D/Cdk4 regulates mitochondrial biogenesis and aging and sensitizes animals

to hypoxic stressAmalia Icreverzi,1,2 Aida Flor de la Cruz,1 Wayne A. Van Voorhies3 and Bruce A. Edgar1,4,*

1Basic Science Division; Fred Hutchinson Cancer Research Center; 2Molecular and Cellular Biology Program; University of Washington; Seattle, WA USA; 3Molecular Biology Program; New Mexico State University; Las Cruces, NM USA; 4German Cancer Research Center (DKFZ)-Center for Molecular Biology Heidelberg (ZMBH) Alliance; Heidelberg,

Germany

Key words: CycD, mitobiogenesis, aging, hypoxia, superoxide, Drosophila

mice have reduced body size, reduced viability and neurological defects.10

In vivo, loss of Drosophila CycD/Cdk4 leads to defects in growth, proliferation and fertility,11 whereas the overexpression of CycD/Cdk4 in Drosophila led to faster cell cycle progression, ectopic proliferation and increased cellular growth.12,13 We have previously shown that CycD/Cdk4 overgrowth requires func-tional mitochondria, mRpL12 (mitochondrial ribosomal protein L12) and Hph (Hif-1α-Prolyl-Hydroxylase).14,15 These require-ments suggests that the growth function of CycD/Cdk4 requires proficient mitochondrial translation and efficient Hif-1α degra-dation, since the only essential target of Hph in Drosophila has been demonstrated to be Hif-1α.16 Additionally, excess CycD/Cdk4 increased levels of Hph protein and amplified mitochon-drial function.14,15 Here, we investigate CycD/Cdk4 coordination of mitochondrial function with growth and distinguish between increases in mitobiogenesis and mitochondrial function.

Mitochondria are eukaryotic organelles that contain proteins central to cellular metabolism, respiration, nucleotide biosynthe-sis, apoptosis as well as cell growth.17 Generally, increased mito-chondrial function is generated via increased mitobiogenesis, the addition of mitochondrial mass to existing organelles.18 In previ-ous studies, we demonstrated that excess CycD/Cdk4 increased Mitotracker staining indicated enhanced mitochondrial

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RESEARCH PAPER REPORT

and translation of mitochondrially encoded proteins.18 To mea-sure mitobiogenesis, we developed a sensitive assay to quantify mtDNA relative to nuclear DNA (nDNA) using quantitative real-time PCR (qRT-PCR); we measured the relative amount of two mitochondrial genes (CO1 and CO2) to two nuclear genes (PCNA and RNR-S).

Using the heat inducible hs-Flp Act-Gal4 system to induce transgenes systemically, UAS genes were induced after animals eclosed, and whole males were analyzed 96 h later. We found that induction of either Drosophila CycD/Cdk4 or mammalian CycD1/Cdk4 complexes increased mtDNA ratios by more than 26% (Fig. 1a), indicating increased mtDNA synthesis. As a positive control of mitobiogenesis, we overexpressed Erect Wing (EWG), the Drosophila ortholog of NRF-1, a key regulator of mitobiogenesis.18 Ectopic EWG increased total adult mtDNA ratios by 18%. The depletion of CycD by RNAi decreased mtDNA content by 20%. We saw no significant reduction of mtDNA following induction of EWG RNAi. To ascertain whether CycD/Cdk4 requires EWG to induce mtDNA increases, we coexpressed CycD/Cdk4 and EWG RNAi. Knocking-down EWG with RNAi inhibited CycD/Cdk4-induced mtDNA increases, indicating a requirement for EWG (Fig. 1a). We assayed CycD and Cdk4 mRNA levels with Act-Gal4 driver, and found that CycD and Cdk4 mRNA levels were not decreased upon the addition of EWG RNAi, suggesting EWG is necessary for CycD/Cdk4 to increase mtDNA (Fig. S1). To confirm the results obtained using CycD RNAi, we assessed mtDNA ratios in cycD,1- and cdk4,3-null mutant males. In this case we discovered that their mtDNA ratios were similarly reduced, with 25% and 22% reductions relative to controls (Fig. 1b). To determine the tissue-specific effects of CycD/Cdk4 on mtDNA ratios, we inves-tigated the mtDNA contents of unfertilized eggs laid by mutant cycD or cdk4 females. We found that mutant females generated eggs with nearly 10% less mtDNA (Fig. 1C). The decreased mtDNA in mutants’ eggs suggests that mutant cycD or cdk4 females generate less mtDNA. The oocyte observation is consis-tent with whole-animal mtDNA content outcomes, suggesting that loss of cycD or cdk4 results in decreased mitochondrial DNA synthesis throughout development.

Cycd/Cdk4 regulates mitochondrial proteins. We next examined levels of ATP-synthase α (ATP-α) protein in cells overexpressing CycD/Cdk4 to further confirm mitobiogenesis. ATP-α is the nuclearly encoded, mitochondrially localized α subunit of complex 5 in the electron transport chain (ETC). Excess CycD/Cdk4 increased the ATP-α protein levels in both fat body and wing disc cells via Act or En drivers for 96 h (Fig. 2a and E). Fat body cells had decreased ATP-α levels with CycD RNAi (Fig. 2b). Surprisingly, EWG RNAi alone had no effect on ATP-α levels (Fig. 2C and G). However, additional EWG RNAi restricted CycD/Cdk4’s effects on ATP-α levels, leading to limited increases in ATP-α levels in fat body and wing cells when compared with CycD/Cdk4 alone. We saw similar ATP-α limitation when we combined CycD/Cdk4 and Tfam RNAi (Fig. S2). This restriction in ATP-α levels indicates that CycD/Cdk4 requires both EWG and Tfam to increase mitochondrial protein levels (Fig. 2d and h).

function.14 From this observation, we propose two possibilities for enhanced mitochondrial function: either increases in mito-biogenesis (mass) or mitochondrial efficiency (performance) could be responsible. We sought to distinguish between these two possibilities by quantifying mitobiogenesis using a novel mtDNA ratio assay (MRA).

Mitobiogenesis has been reported to be upregulated by growth factors and oncogenes.19-21 Mammalian studies have determined that both mitogens and growth factors are synergistically required to promote mitobiogenesis.21 Since CycD/Cdk4 has been shown to promote both cell cycle and cell growth in Drosophila on its own, perhaps it is also sufficient to promote mitochondrial bio-genesis.12 A key regulator of mitobiogenesis, nuclear respiratory factor-1 (NRF-1), is phosphorylated in vivo upon serum stimula-tion.22 That phosphorylation of NRF-1 increases its activity and is driven by the same stimuli that induce cell proliferation suggests that NRF-1 phosophorylation may be involved in coordinat-ing cell division with mitobiogenesis.21,23 Previous studies con-firmed that the CycD1/Cdk4 complex phosphorylated NRF-1 at residues identified as significant for NRF-1 function.24,25 Many mitochondrially localized proteins require NRF-1 to activate their transcription, including genes required for mitochondrial function and Tfam, the key mitochondrial transcription factor.18 Mutated mitochondrial proteins often have cell proliferation, cell growth and fertility defects. These include, for instance, tech-nical knockout (tko), encoding mRpS12 and bonsai, encoding mRpS15 in Drosophila.26,27

Recent studies demonstrate that reduced oxygen tension, hypoxia, interferes with mitochondrial function and cellular metabolism.28,29 These effects are mediated by Hif-1α (hypoxia-inducible factor-1α).30 Hif-1α is post-translationally marked for degradation in normoxia by Hph. If Hif-1α is stabilized, it translocates to the nucleus and transactivates genes adaptive for surviving hypoxic stress.31 Reports on Hif-1α link its activity to suppressing mitochondrial function and mass.28,29,32 Past studies described the detrimental effects hypoxia has on mitochondrial function and biogenesis, but the reciprocal relationship between mitochondrial function and hypoxia survival has not been well-defined.28

In this study, we examine the impact of CycD/Cdk4 levels on mitochondrial content and hypoxia survival. Here, we show that Drosophila cycD or cdk4 mutants have defects in mitobio-genesis, as measured by mtDNA content, and reduced mitochon-drial areas, but, surprisingly, these mutants also have increased hypoxia resistance. Conversely, we observe that excess CycD/Cdk4 increased mitobiogenesis, ATP-α levels and mitochondrial areas but also increased hypoxia sensitivity and animals develop hypersensitivity to consecutive hypoxia.

Results

Cycd/Cdk4 regulates mtdna levels. Mitochondria are semi-autonomous organelles containing their own genome, encod-ing essential mitochondrial proteins required for respiration.18 A preliminary step in mitobiogenesis is the replication of mito-chondrial DNA (mtDNA), which allows for future transcription

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cellular ultrastructure via transmission eectron microscopy (TEM) and quantified the mitochondrial size, numbers and area in larval salivary gland cells, as described in Hoppeler et al.33 Using the Ptc-Gal4 driver, we found that excess CycD/Cdk4 robustly increased the mitochondrial fractional area by 2-fold (Fig. 3b), and that knocking-down CycD or Tfam with RNAi decreased the mitochondrial area to 55% of control cells (Fig. 3d and E). By measuring the organelle size of individual mito-chondria and numbers of organelles per 25 microns2 of cytosol, we discovered that excess CycD/Cdk4 and EWG both increased mitochondrial numbers and area, but that mitochondrion size was not modified (Fig. 3F). Conversely, the expression of CycD

We also examined CycD/Cdk4’s influence on other mito-chondrial protein levels as determined by cytochrome C (Cyt C) staining. Cyt C is a nuclear-encoded mitochondrial ETC pro-tein. Excess CycD/Cdk4 increased the Cyt C staining in wing disc cells, while CycD RNAi decreased cellular Cyt C staining (Fig. S2). We conclude that excess CycD/Cdk4 leads to increased mitochondrial protein levels, as suggested by increased ATP-α and Cyt C staining, and requires EWG and Tfam to amplify nuclearly encoded mitochondrial proteins.

Cycd/Cdk4 increases mitochondrial fractional area and number. In order to address whether CycD/Cdk4 influenced mitochondrial fraction (mitochondria per cytosol), we examined

Figure 1. CycD/Cdk4 regulates mtDNA levels. Real-time qPCR was performed to quantify mtDNA:nDNA ratio using primers for two genes from each genome in (A) adult males expressing UAS-GFP and transgenes under the control of Act-Gal4 driver, at 96 hpi (hours post-gene induction), (B) 5-d old adult males and (C) oocytes from unmated mutant females, 8AED (hours after egg deposition). * p < 0.05, ** p < 0.01.

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mutants’ decrease in mitochondrial area inversely mirrors CycD/Cdk4 or EWG overexpression increasing mitochondrial fraction, indicating that a similar mechanism may be at work.

To resolve whether cycD mutants have any mitochondrial fission/fusion defects, we examined the mutants’ onion-stage spermatids. Developing sperm require regulated mitochondrial fusion to condense mitochondria.34 cycD mutant animals exhib-ited competently condensed mitochondrial organelles (Fig. S3). Hence, we do not believe that mutant’s mitobiogenesis defects could be caused by fusion/fission defects.

mitochondria generated by ectopic Cycd/Cdk4-activity are functional and contribute to increased resting metabolic rates and locomotor activity. We employed both metabolic- and activ-ity-based assays to analyze whether the mitochondria generated by CycD/Cdk4 animals were functional and active. Measuring respiratory rates (the rate of CO

2 production) of individual adult

flies, we discovered that excess CycD/Cdk4 augmented the ani-mals’ resting metabolic rates by 10% compared with GFP con-trols (Fig. 4a). However, both cycD and cdk4 mutants had a

or Tfam RNAi decreased the size of mitochondria, but no signifi-cant changes were observed in the total number of mitochondria. These smaller mitochondria lead to a 45% decrease in mitochon-drial area in Tfam or CycD RNAi cells (Fig. 3d and E).

Moreover, to determine whether the cycD or cdk4 mutants had any major mitochondrial morphological defects, we uti-lized TEM to inspect cellular ultrastructure and mitochondrial areas. We found that cycD and cdk4 mutants do not differ from control mitochondria, although they were fewer in number (Fig. 3i–K). Evaluating mitochondrial area indicated that cycD and cdk4 mutants have a 50% decrease in mitochondrial area, due mainly to a reduction to mitochondrial numbers (to nearly 50% of controls), but not declining individual mitochondrion size (Fig. 3l–n). We conclude that increased CycD/Cdk4 activ-ity leads to increases in mitochondrial area and numbers but not significant mitochondrial size changes, and that loss of cycD, cdk4 or Tfam interferes with mitobiogenesis, causing fewer (in cycD or cdk4 mutants) or smaller mitochondria (in CycD or Tfam RNAi), resulting in reduced mitochondrial areas. cycD and cdk4

Figure 2. CycD/Cdk4 regulates ATP-Synthase protein levels in fat body and wing disc cells. Fat body cells expressing transgenes under Act-Gal4 driver marked with UAS-GFP (A) UAS-CycD/Cdk4, (B) UAS-CycD RNAi, (C) UAS-EWG RNAi and (D) UAS-CycD/Cdk4 and EWG RNAi. Wing disc cells express-ing transgenes under En-Gal4 driver marked with UAS-GFP (E) UAS-CycD/Cdk4, (F) UAS-CycD RNAi, (G) UAS-EWG RNAi and (H) UAS-CycD/Cdk4 and EWG RNAi. (I) En-Gal4 ATP synthase intensity ratio. ** p < 0.01, significantly different from CycD/Cdk4 alone.

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Figure 3. CycD/Cdk4 regulates mitochondrial areas. TEM micrographs of 96AED larvae salivary glands from Ptc-Gal4 animals, (A) Ptc-Gal4/+ (control), (B) +CycD/Cdk4, (C) +EWG (NRF-1), (D) +CycD RNAi and (E) +Tfam RNAi. Quantification of micrographs from genotypes (A–E) of (F) mitochondrial area, (G) number of mitochondria in 25 microns2 and (H) average mitochondrion size. TEM micrographs of WL3 salivary glands from (I) control, (J) cycD-/- and (K) cdk4-/-. Quantification of micrographs from genotypes I–K of (L) mitochondrial area, (M) number of mitochondria in 25 microns2 and (N) average mitochondrion size. * p < 0.05, ** p < 0.01.

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levels. Utilizing a Drosophila activity monitor (DAM), we discov-ered that CycD/Cdk4-overexpressing animals were nearly twice as active as GFP-expressing controls, and that excess EWG led to hyperactivity (Fig. 4C). Conversely, the cycD and cdk4 mutants (null for their entire life) had a 30% and 16% reduction in physi-cal activity (Fig. 4d). The different degree of loss of function and the time of onset thereof likely explain the differences between the mutants and RNAi-treated animals. We conclude that mito-chondria generated by excess CycD/Cdk4 are functional not in contributing to resting metabolism, but in increasing physical activity. In contrast, loss of cycD or cdk4 led to decreased loco-motor activity. The positive correlation between mtDNA copy number and physical activity is consistent with earlier reports of other Drosophila species, signifying a conserved mechanism.35

loss or gain of Cycd/Cdk4 reduces lifespan and increases roS. Previous studies of Drosophila metabolic rates demonstrated

~25% reduction in resting metabolic rates (Fig. 4a). Surprisingly, when we included flies’ body mass as a covariate in the analyses of metabolic rate data, we found that while excess CycD/Cdk4 led to larger animals, the rates of CO

2 production per gram of

mass remained stable (Fig. 4b). Accordingly, although cycD and cdk4 mutants are 25% smaller, their metabolic rates were reduced by nearly 25%, thus their metabolic rates per gram of mass remained unchanged (Fig. 4b). We conclude that the changes seen in mtDNA ratios, ATP-α protein levels and mitochondrial areas lead to increased organismal respiration rates (Fig. 4a) but do not change the animals’ resting metabolic rate per mass, when the majority of metabolically active muscles are idle (Fig. 4b).

Earlier reports indicated that mtDNA copy numbers were posi-tively correlated with physical activity, suggesting that mitochon-drial function is proportional to mtDNA synthesis.35 To assess the animals’ overall physical activity, we monitored the flies’ locomotor

Figure 4. CycD/Cdk4 regulates resting metabolic rates and locomoter activity in Drosophila. (A) Metabolic rates of adult males, resting respiration represented by produced CO2 uL/h. (B) Metabolic rates of adults per fly mass, respiration represented by produced CO2 uL/h/mg. (C) Average motion of adult males expressing UAS-GFP and other transgenes under the control of Act-Gal4 driver. CycD/Cdk4 and EWG increase males’ physical activity, while in (D) cycD and cdk4 mutants have decreased locomotion. * p < 0.05, ** p < 0.01.

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Cycd/Cdk4 transcriptionally upregulates tfam and mito-chondrial translation. To further investigate how CycD/Cdk4 overexpression drives increased mitobiogenesis, we interrogated CycD/Cdk4-upregulated transcripts employing Drosophila spotted 13,000-cDNA microarrays. Using the hs-Flp Act-Gal4 construct, we induced CycD/Cdk4 in 72AED larvae and iden-tified altered transcripts 16 h later. Using SAM (significance analysis of microarray data) software, we determined which transcripts were significantly induced by 1.5-fold or more in whole larvae. All 369 significantly upregulated transcripts were compared with E2F1-induced transcripts; we found significant similarities between the CycD/Cdk4- and E2F1-upregulated transcripts, although there were many CycD/Cdk4 targets that were not found in the E2F data; such as many transcripts cod-ing for mitochondrial and metabolic proteins (Fig. 6a). We also investigated which CycD/Cdk4-upregulated transcripts were mitochondrially localized by comparing to Flybase’s Drosophila-specific mitochondrial protein database and queried in silico for mitochondrial localization sequences (MLS) with iPSORT.39 Sixty-four genes (17.3% of total transcripts) upregulated by CycD/Cdk4 encode proteins predicted to be mitochondrially localized. These include 14 genes involved in mitochondrial translation (Fig. 6b and table S1). Using the gene ontology enrichment analysis and visualization tool (GORILLA) to search for enriched GO terms in the mitochondrial data set, we found that the “mitochondrial translation” set was significantly upregulated (table S2).40

We next inquired whether CycD/Cdk4 upregulates mito-chondrial proteins via known mechanisms, such as increased NRF-1 activity. Comparing CycD/Cdk4-induced transcripts to genes induced by NRF-1 activity, we found significant over-lap (table S3).41 We also selected the transcription of Tfam as a reporter of NRF-1 activity.18 We found that CycD/Cdk4 was capable of inducing Tfam mRNA 13-fold. Expression of EWG (NRF-1) itself only increased Tfam transcript levels 6-fold rela-tive to GFP controls. The co-expression of CycD/Cdk4 with EWG RNAi increased Tfam transcript levels slightly but not sig-nificantly, suggesting that CycD required EWG to increase Tfam mRNA (Fig. 6C).

We evaluated transcripts downregulated by excess CycD/Cdk4 with those upregulated by loss of tko (mRpS12, mito-chondrial ribosomal subunit S12) to determine whether CycD/Cdk4’s upregulation of mitochondrial translation was reflected in the transcripts repressed.27 We found significant overlap of tran-scripts repressed in CycD/Cdk4-expressing flies and transcripts upregulated by tko mutants (Fig. 6d and table S4). We con-clude that excess CycD/Cdk4 activity significantly upregulates mitochondrial translation and Tfam mRNA, indicating that CycD/Cdk4 increases mitobiogenesis via known avenues.

To ascertain whether loss of cycD was associated with defects in mitochondrial translation, we compared the transcriptional profiles of cycD mutants with tko (mRpS12) mutants.27 We observed that the expression profiles of both cycD and tko mutant animals were significantly similar, with many genes being upreg-ulated to the same degree in both cycD and tko mutants (Fig. 6E and table S5).27

that flies’ metabolic rates increased as the flies aged.35 In view of our results, this suggests that excess CycD/Cdk4 may lead to an “aged” phenotype. To assess whether excess CycD/Cdk4 con-tributed to an “aged” phenotype by increasing the flies’ physical activity, we examined the lifespan of adult animals overexpress-ing CycD/Cdk4, CycD RNAi and Tfam RNAi. We found that the mean lifespan of adult animals expressing excess CycD/Cdk4 or CycD RNAi was decreased by more than 36% or 17%, respec-tively (Fig. 5a). The mean lifespan of Tfam RNAi animals was increased by 27%. The additional expression of Tfam RNAi in animals with CycD/Cdk4 restored their survival rate for the first month, but then the slope matched the survival rates of CycD/Cdk4 animals, decreasing mean lifespan by 21% (Fig. 5a). The cycD and cdk4 mutants also demonstrated reduced mean lifes-pans by 33% and 36%, respectively (Fig. 5b). We found simi-lar lifespans in both unmated females and males and concluded that the influence of gain or loss of CycD/Cdk4 is conserved in both genders (Fig. 5a and b). We propose that excess CycD/Cdk4 leads to increased mitochondrial oxidative phosphoryla-tion (OXPHOS) and a concomitant increase in reactive oxygen species (ROS), which may decrease lifespan.

To test this idea, and to determine whether excess CycD/Cdk4 leads to increased generation of ROS, we utilized Molecular Probes’ MitoSOX reagent. MitoSOX is a mitochondrial-targeted superoxide indicator and is a selective detector of the superox-ide anion generated as a byproduct of OXPHOS. Approximately 3% of mitochondrially consumed oxygen is not reduced; these “leaky” electrons interact with molecular oxygen (O

2) to form the

superoxide anion (O2

-), which is the predominant reactive oxy-gen species (ROS) generated in mitochondria.36 We found that excess CycD/Cdk4 increased MitoSOX intensity, as did CycD RNAi, by 34% and 24% respectively (Fig. 5C–G). We do not believe that the major defect in CycD RNAi cells is solely loss of Tfam activity, since Tfam RNAi efficiently decreased MitoSOX by 32% (Fig. 5E–G). The addition of Tfam RNAi to CycD/Cdk4-expressing cells slightly reduced their MitoSOX levels, but the signal remained 20% above control intensity (Fig. 5G). To further determine the source of the superoxide, we utilized the cytoplasmic superoxide indicator dihydroethidium (DHE), which, upon reaction with superoxide anions, forms a red fluores-cent product.37 CycD/Cdk4 expression increased DHE staining in wing discs slightly but not to the same degree as MitoSOX, indicating that mitochondria was the main source of superoxide in those cells (Fig. 3d and E). The knockdown of CycD with RNAi had no consequence on the cytoplasmic levels of DHE, again indicating exclusive mitochondrial localization of superox-ide generated in those cells (Fig. 5i and l). These results suggest that both loss and gain of CycD/Cdk4 activity lead to an imbal-ance in mitobiogenesis, resulting in inefficient OXPHOS cou-pling and increased mitochondrial superoxide production, and that Tfam RNAi ameliorates these detrimental effects (Fig. 5F and l). The resultant mitochondrial ROS may not be the cause of early mortality, but we found that is was definitely inversely correlated with mean lifespan (Fig. 5a). Indeed, other studies in Drosophila have demonstrated a convincing relationship between oxidative stress and lifespan.38

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hypoxia respond by reducing their CycD mRNA by nearly 50% (Fig. S1). Wen et al. have indicated a conserved mechanism by showing downregulation of CycD1 mRNA by Hif-1α in mam-malian cells.42

To clarify whether CycD/Cdk4-overexpressing animals could be habituated, we exposed them to consecutive hypoxia treat-ments. Excess CycD/Cdk4 severely impaired the animals’ ability to precondition to hypoxia. On a second exposure to hypoxia, consecutively treated CycD/Cdk4 animals were 4-fold more sen-sitive than after their first hypoxic treatment and 14-fold more vulnerable when compared with control GFP pretreated animals. The addition of Tfam RNAi to CycD/Cdk4 slightly rescued, by 5-fold, this sensitivity to consecutive hypoxia (Fig. 7). Not only did we see a quantitative variation in CycD/Cdk4 animals’ ability to habituate to hypoxia, we also observed qualitative dif-ferences. CycD/Cdk4 animals’ recovery to consecutive hypoxia treatments was less complete than controls’: they were unable to ambulate (videos S1 and S2). We propose that cycD or cdk4 mutants are more resistant to hypoxia due to their hypoxia-like expression signature and are thus chronically “transcriptionally poised” for hypoxia (Fig. 7).

loss of Cycd or extended Cycd/Cdk4 increases ldh protein in normoxia. To determine whether the cycD and cdk4 mutants’ hypoxia resistance was due to increased Hif-1α activity, we assayed the levels of lactate dehydrogenase (LDH) protein, one of Hif-1α’s best-characterized targets.30 This enzyme converts pyruvate, the final product of glycolysis, to lactate when oxygen is in short supply.30 We stained wing disc cells overexpressing Sima (Hif-1α) for 48 h, CycD/Cdk4 for 48 h, CycD RNAi for 96 h or CycD/Cdk4 for 96 h with LDH antibody. Cells overexpress-ing CycD RNAi or Sima (Hif-1α) had increased LDH protein in normoxia by 24 or 29% (Fig. 7). To assess acute vs. chronic consequences of CycD/Cdk4 activity, we expressed the complex for either 48 h or 96 h (Fig. 7). We found that the acute expres-sion of CycD/Cdk4 decreased cellular levels of LDH protein by 11% (Fig. 7). Remarkably, long-term expression of CycD/Cdk4 increased levels of LDH by 15% (Fig. 7). Chronic CycD RNAi or CycD/Cdk4 expression led to increased LDH protein, possibly due to the increased superoxide production inhibiting efficient hydroxylation of Hif-1α (Fig. 5C and d).43 This suggests that acute CycD/Cdk4 activity initially transcriptionally repressed Hif-1α and its targets, but that prolonged exposure to CycD/Cdk4 activity, and the associated increase in oxidative stress may lead to increased Hif-1α stability in normoxia (Figs. 5 and 7).

Discussion

A growing body of research supports a connection between mito-chondrial funcion and hypoxia.28,32 We and others have shown

Cycd/Cdk4 transcriptionally represses hif-1α-regulated hypoxia targets. We have previously demonstrated that CycD/Cdk4 requires Hif1-Prolyl-Hydroxylase (Hph) in order to induce cellular growth.15 An essential function of Hph/Fatiga in Drosophila is targeting Hif-1α for degradation.16 Hence, we sought to ascertain whether the CycD/Cdk4-mediated increase in Hph stability influenced Hif-1α activity.15 We compared published reports of genes upregulated by hypoxia treatments (increased Hif-1α activity) to transcripts repressed by CycD/Cdk4 overexpression. In this case, we discovered significant overlap (Fig. 6F and table S6).30 Furthermore, published sets of gene classes induced by hypoxia in Drosophila correspond to the ontology classes repressed by CycD/Cdk4. These ontology classes included glycolysis, carbohydrate metabolic process, proteolysis, carboxylic acid metabolic process and chitin metabolic process (table S7).31 We concluded that CycD/Cdk4 activity transcrip-tionally represses Hif-1α and hypoxia-responsive genes.30,31

cycD and cdk4 mutants have a hypoxic transcriptional profile in normoxia. To address whether cycD and cdk4 mutant animals have increased hypoxia-responsive transcripts due to decreased Hph stability, we compared the genes reported to be upregulated by hypoxia treatments.30 We discovered that significant overlap of hypoxia-regulated transcripts were overrepresented in normoxic cycD and cdk4 mutants (Fig. 6G and table S8). We concluded that loss of cycD led to a similar suite of transcripts upregulated by defects in mitochondrial translation, as in loss of tko mutants, and moreover, that loss of either cycD or cdk4 upregulates hypoxia-responsive genes in normoxia (tables S6 and S8).27,30 These observations suggest that cycD and cdk4 mutants’ defects in impaired mitobiogenesis are due to diminished mitochondrial translation and increased Hif-1α activity.27,30 Indeed, Frei et al. (2004) showed that suppression of mitochondrial translation using a mutation in mRpL12 increased the level of a reporter of the hypoxic response, in a dose-dependent fashion.

loss of Cycd/Cdk4 confers resistance to hypoxia. In order to assess the functional consequences of repression or upregu-lation of hypoxia-targets by excess CycD/Cdk4 or loss of cycD, we exposed animals to hypoxia (0.5% O

2). The survival of cycD

and cdk4 mutant animals was 2-fold increased over controls (Fig. 7); therefore, we term these mutants “habituated to hypoxia.” To determine whether wild-type animals pretreated with hypoxia would recover as well as “habituated” cycD or cdk4 mutants, we exposed WT animals to hypoxia and then allowed recovery before retesting them. Animals pretreated with hypoxia survived as did cycD or cdk4 mutants naïve to hypoxia (Fig. 7). The knock-down of CycD or Tfam with RNAi also conferred animals with the “habituated” phenotype, such that they survived hypoxia exposure (Fig. S4). Forced expression of CycD/Cdk4 appears to be incompatible with hypoxia survival. WT animals exposed to

Figure 5 (See opposite page). Adult lifespan is decreased by loss or gain of CycD/Cdk4, and is strongly correlated with mitochondrial superoxide production. The average of male and female lifespans of (A) adult lifespan of Act-Gal4, UAS-GFP/UAS-transgene animals Control (GFP), CycD/Cdk4 (p = 2.24e-14), CycD/Cdk4 + Tfam RNAi (p = 9.6e-03), Tfam RNAi (p = 0.0129), CycD RNAi (p = 0.00103) (B) mutant adult lifespan of cycD (p = 5.12e-04) and cdk4 (p = 7.35e-04). MitoSOX reagent in wing disc cells expressing transgenes under En-Gal4 driver marked with UAS-GFP overexpressing (C) CycD/Cdk4, (D) CycD RNAi, (E) Tfam RNAi or (F) CycD/Cdk4 and Tfam RNAi. (G) Quantification of MitoSOX intensity of En-Gal4 vs. adjacent control cells. DHE stain in wing disc cells expressing transgenes under En-Gal4 driver marked with UAS-GFP overexpressing (H) CycD/Cdk4, (I) CycD RNAi, (J) Tfam RNAi or (K) CycD/Cdk4 and Tfam RNAi. (L) Quantification of DHE intensity of En-Gal4 vs. adjacent control cells. * p < 0.05, ** p < 0.01, *** p < 0.001.

www.landesbioscience.com Cell Cycle 563

that CycD/Cdk4 interacts with both mitobiogenesis (NRF-1) and hypoxia proteins (Hph).15,24 Our data suggest that CycD/Cdk4 plays a role in mitobiogen-esis and hypoxia recovery. We provide evidence here that both Drosophila and mammalian CycD/Cdk4 can increase mtDNA content in vivo, and that both EWG (NRF-1) and Tfam, a key mtDNA transcription factor, are required. Other studies established that mammalian CycD1/Cdk4 complexes are capable of phosophorylating NRF-1 at serine resi-dues required for NRF-1 activation but obtained results that contradict ours with respect to function.24 Specifically, one study found that CycD1 deficiency increased mitochondrial size and activ-ity.24 However, in mammalian cells, when one cyclin D is disrupted, the other remaining D-type cyclins are tran-scriptionally and proteomically upregu-lated.44,45 Furthermore, since each of the D-cyclin paralogs is sufficient to drive oncogenic proliferation, we believe that their ability to drive mitochondrial bio-genesis may be similarly conserved.9 Hence, we believe that the phenotype demonstrated by the Wang et al. study could be due to the compensatory action of the remaining paralogs being upregu-lated.46,47 Our results in Drosophila, with its single CycD and Cdk4 genes, clarify the relationship between excess CycD/Cdk4 and increased mitobiogenesis.

Figure 6. Increased CycD/Cdk4 activity increases transcripts of mitochondrially localized proteins, including Tfam. The overexpression of CycD/Cdk4 significantly upregulated many E2f targets. (B) Increased CycD/Cdk4 activity significantly upregu-lated of many mitochondrially localized transcripts along with well-known cell cycle transcripts. (C) CycD/Cdk4 activity upregulat-ed Tfam transcript 13-fold. (D) Venn diagram of the overlap of transcripts repressed by CycD/Cdk4 and transcripts upregulated by tko mutants. (E) Venn diagram of the significant overlap of transcripts induced in both cycD and tko mutants. (F) Venn diagram of the overlap of transcripts repressed by CycD/Cdk4, and transcripts upregulated by hypoxia. (G) Venn diagram of overlap of transcripts induced by cycD mutants and by hypoxia. All Venn diagrams include p-values denoting significance of overlap. * p < 0.05, ** p < 0.01.

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Figure 7. For figure legend, see page 565.

www.landesbioscience.com Cell Cycle 565

decreases we see in mutants’ mitochondrial mass phenocopies the declines seen in mammalian cells in hypoxic environments.32 In contrast, while Drosophila CycD/Cdk4 represses Hif-1α tran-scriptionally, we and others have demonstrated a reciprocal rela-tionship.49 Mammalian Hif-1α has been shown to downregulate CycD1 expression by binding to its promoter region directly.49 This further supports our hypothesis that hypoxia response is often incompatible with mitobiogenesis and may be mediated though CycD. CycD levels may not only be controlled by extra-cellular growth factors, but also by oxygen levels (or Hif-1α) in order to ensure that cells only enter the cell cycle when they are sufficiently oxygenated.4

We would like to propose a mechanism for how CycD/Cdk4 regulates mitobiogenesis and suppresses hypoxia response. Previously, CycD had been shown to phosphorylate NRF-1.24 We have demonstrated that CycD/Cdk4 upregulates transcription of NRF-1 targets, including Tfam and mitochondrial ribosomes, and represses Hif-1α targets and hypoxia-responsive transcripts. Hence we propose that CycD/Cdk4 activity regulates mitobio-genesis by the activation of NRF-1 and consequent induction of Tfam and mitochondrial ribosomal transcription. This would add both NRF-1 and Tfam to CycD’s transcription factor dance list.

Perhaps excess CycD1 contributes to tumorgenesis initially by driving rampant mitobiogenesis, which may also relax selec-tive pressures for efficient mitochondria and allow increased “ROS-producing” mitochondria. This oxidative stress, resem-bling hypoxia due to ROS inhibiting hydroxylation of Hif-1α, may contribute to increased tumor angiogenesis and/or lead to the Warburg effect by suppressing oxidative metabolism,48,50 Alternatively, the build-up of superoxide anions produced by CycD/Cdk4 cells may contribute to mitochondrial genome instability, a feature of many tumor cells.51

Materials and Methods

drosophila genetics. UAS-trangenes were driven with either hs-Flp Act-Gal4 for fat body/systemic or En-Gal4 driver for wing disc analyses. Complete genotypes are listed in supplement.

mtdna ratio assay. Total DNA was extracted from 10 males or 50 unfertilized eggs. Briefly, tissues were homogenized with Grinding Buffer and Proteinase K, incubated at 65°C for 30 min, and total DNA was ethanol extracted. Quantitative PCR was performed on BioRAD iQ5 SYBR Green (170–8,880) + 50 ug total DNA + primers for two nuclear or two mitochondrial genes. mtDNA ratios were determined using the log

2 of the Δ

between the Ct (cycle threshold) values between the mitochon-drial and nuclear genes; experiments were done in triplicate. Primer sequences are listed in the supplement.

We show here that excess CycD/Cdk4 leads to an increase in mitobiogenesis, as indicated by increases of mtDNA, mitochon-drial areas and numbers. The observations that either CycD/Cdk4 or EWG (NRF-1) overexpression result in increased mtDNA and mitochondrial areas indicate that perhaps CycD/Cdk4 and EWG may be operating via a similar mechanism. Conversely, loss of cycD or cdk4 appeared to slow mitobiogenesis, reducing mtDNA and mitochondrial mass, similar to the declines in Tfam RNAi-expressing animals.

Mitochondria generated by excess CycD/Cdk4 activity con-tributed to increased physical activity and mortality. This sug-gests that excess CycD/Cdk4 complexes trigger mitobiogenesis, initially contributing primarily to increased capacity for cellular growth, but also by increasing respiration rates and concomitant production of superoxide anions, which may damage mitochon-dria. This condition, when chronic, might yield even less efficient mitochondria. Increased ROS might explain the high suscepti-bility of CycD-overexpressing cells to transformation and why animals expressing ectopic CycD/Cdk4 had shorter lifespans.9 Additionally, the increased ROS levels we see in both cells with excess CycD/Cdk4 or CycD RNAi may indicate that any misreg-ulation of mitobiogenesis contributes to increased electron leak-age in the ETC and, ultimately, to shortened lifespan. Conversely, we found that cycD and cdk4 mutants have fewer mitochondria as measured by mtDNA and TEM. These mutant animals had near-normal metabolic and activity rates, overcoming their 42% deficit in mitochondrial areas. This suggests that while the mutants have reduced mtDNA and mitochondrial mass, they may maximize their mitochondrial function, perhaps creating a highly selective environment for only efficient mitochondria and thereby possibly protecting their cells from oncogene-induced transformation.

Excess CycD/Cdk4 appears to be incompatible with hypoxia survival. This is most likely due to ectopic CycD/Cdk4 leading to repression of transcripts upregulated by hypoxia treatment, including Hif-1α transcript directly. Another route to CycD/Cdk4’s hypoxia sensitivity could be mediated via superoxide’s interference with Hif-1α stabilization, which may hinder any habituation to hypoxia and thus contribute to increased suscepti-bility to consecutive hypoxia.48 WT animals exposed to hypoxia respond by reducing their CycD mRNA by nearly 50%, thus loss of cycD or cdk4 conferred resistance to hypoxic stress. This is most likely due to the transcriptional “hypoxic signature” found in these mutants even when they are normoxic. This hypoxic sig-nature in cycD and cdk4 mutants corresponds to published work30 and is best described as the “constant hypoxia” model proposed by Azad et al.31 These observations lead to our hypothesis that mutant animals are “habituated to hypoxia.” This “habituated” phenotype is, to our knowledge, the first demonstration that loss of cycD or cdk4 can provoke hypoxia resistance in vivo. The

Figure 7 (See opposite page). CycD/Cdk4 regulates hypoxia survival and lactate dehydrogenase levels in Drosophila. Hypoxia (0.5% O2) was adminis-tered for 8 h, and recovery measured was measured in (A) naïve cycD mutants, cdk4 mutants, WT animals or WT animals pretreated with hypoxia, and (B) animals with Act-Gal4 overexpressing GFP (control), CycD RNAi, Tfam RNAi, CycD/Cdk4, CycD/Cdk4 and Tfam RNAi, and Sima (Hif-1a). (C) Consecutive hypoxia treatments were administered to animals with Act-Gal4 overexpressing GFP (control), CycD/Cdk4, CycD/Cdk4 and Tfam RNAi. Lactate dehydro-genase (LDH) protein levels in wing disc cells expressing transgenes with En-Gal4 driver marked with GFP with (D) Sima (Hif-1a) 48 hpi, (E) CycD/Cdk4 48 hpi, (F) CycD RNAi 96 hpi or (G) CycD/Cdk4 96 hpi. (H) Quantification of LDH intensity of En-Gal4 vs. adjacent control cells. * p < 0.05, ** p < 0.01.

566 Cell Cycle Volume 11 Issue 3

(Molecular Probes) in DMSO for 10 min. Tissues were washed 2x in PBS, fixation was then performed in 8% PFA in PBS for 10 min with DNA-stain Hoechst 33,258. Tissues were washed 2x in PBS and mounted with VECTASHIELD (Vector Labs).

dhE. 96AED En-Gal4, UAS-GFP/UAS-X animals were dis-sected in PBS, incubated at RT with stained with 30 μM DHE (Sigma Aldrich D7008) solution for 10–15 min. Brains were washed 2 times in PBS buffer, fixation was then performed in 8% PFA in PBS for 10 min with DNA-stain Hoechst 33,258. Tissues were washed 2x in PBS and mounted with VECTASHIELD (Vector Labs).54

microarray analysis (Sam, iPSort and Flybase). Using hs-Flp Act-Gal4 driver to induce CycD/Cdk4 (or E2F1/Dp) complexes for 16 h in 72AED larvae, we extracted total RNA 16 h later, synthesized cDNA, labeled with Cy5 or Cy3 and hybridized to Drosophila-specific 13K-cDNA microarrays. Experiments were performed five times. All significantly regu-lated transcripts, as determined by SAM (significant analysis program), were compared with Flybase’s mitochondrial protein database, and queried in silico for MLS with iPSORT prediction program.

mrna quantitative rt-PCr. Total RNA was purified from four males using RNeasy (Qiagen). cDNA was synthesized using random hexamers and reverse transcribed with Superscript II (Invitrogen) according to the manufacturer’s protocol. RT-PCR was performed by monitoring in real time the increase in fluores-cence of the SYBR Green dye (BioRad). Transcript levels were determined using the log

2 of the Δ between the Ct (cycle thresh-

old) values between the Tfam and Dp genes; experiments were done in triplicate.

disclosure of Potential Conflicts of interest

No potential conflicts of interest were disclosed.

acknowledgements

This work was supported by NIH GM68105 and NIH NS058230 to B.A.E and US National Cancer Institute CA96286 to W.V.V.

author Statement

Amalia Icreverzi performed the bulk of the experiments, the microarray data analysis, and wrote the paper. Wayne Van Voorhies performed the respiration assays (Fig. 4a and b). Aida de la Cruz contributed the microarray data. Bruce Edgar super-vised the project and helped to write the manuscript.

note

Supplemental materials can be found at: www.landesbioscience.com/journals/cc/article/19062

atP-α/ldh antibody. Tissues from 96AED larvae were inverted, fixed in 8% paraformaldehyde (PFA) for 2 h or 4% PFA for 30 min for fat body and wing discs, respectively. Tissues were then washed 3x in PBS with 0.1% Triton (PBT) and then blocked in PBT and 5% w/vol BSA. Tissues were incubated with 1:1K ATP-α (Mito Sciences, MS507) or 1:10K LDH (Abcam, ab2101) o/n at 4°C. Tissues were washed in PBT for 2 h at RT. Tissues were incubated with Alexa 546 secondary antibodies 1:2K (Molecular Probes A11035), washed 2x in PBT; DNA was stained with 1:1K Hoechst 33258, and mounted with VECTASHIELD (Vector Labs, H-1000).

tEm. Salivary glands from 10 Ptc-Gal4 96AED larvae were fixed o/n at 4°C in 2% PFA plus 1% glutaraldehyde, postfixed in 1% osmium tetroxide (OsO

4) at RT, dehydrated in an ethanol

series and embedded in Epon 812. Ultrathin sections (80 nm) were examined with JEOL 1230 transmission electron micro-scope at 100 kV. 100 micrographs from each genotype were taken at 25,000x magnification.

quantification of Em micrographs. 100 micrographs per genotype of 25,000x images were analyzed to quantify mito-chondrial area, numbers and size. Mitochondrial area was quan-tified using ImageJ measurement tool similar to Hoppeler et al.33 By assessing the contribution of mitochondrial area per cell, we analyzed the mitochondrial proportion with ImageJ. Analysis was done blind.

metabolic rate. Metabolic rates were calculated by analyzing the CO

2 production from unmated adult males. CO

2 is produced

by oxidative metabolism and in Drosophila there is a near per-fect linear correlation between CO

2 production and oxygen con-

sumption as demonstrated by Van Voorhies et al.52 Supplemental methods contains details.

drosphila activity monitoring. Locomotor activity was analyzed using the Drosophila activity monitoring system (TriKinetics DAM5). Twenty adult males were monitored singly in 10 mm vials with food at one end and a cotton stopper at the other. The DAM system observes and quantifies each pass mid-vial; measurements were summed each 10 min, and data was col-lected mid-morning.

lifespan. Adult flies were collected within 8 h after eclosion, segregated by gender and heat shocked for 35 min to induce gene induction with the hs-Flp Act-Gal4 promoter. Flies were assayed for viability and transferred to fresh vials every 3 d, similar to Zid et al. Vials contained 25 adults all experiments were done in trip-licate for a total of 75 animals per gender; we averaged male and female survival for adult survival and performed log-rank test to determine p-values.

mitoSox. 96 AED En-Gal4, UAS-GFP/UAS-X animals were dissected in PBS, incubated at RT with 5 μM MitoSOX

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