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ScienceDirect Cell cycle regulation of mitochondrial function Isabel C Lopez-Mejia and Lluis Fajas Specific cellular functions, such as proliferation, survival, growth, or senescence, require a particular adaptive metabolic response, which is fine tuned by members of the cell cycle regulators families. Currently, proteins such as cyclins, CDKs, or E2Fs are being studied in the context of cell proliferation and survival, cell signaling, cell cycle regulation, and cancer. We show in this review that cellular, animal and molecular studies provided enough evidence to prove that these factors play, in addition, crucial roles in the control of mitochondrial function; finally resulting in a dual proliferative and metabolic response. Addresses Department of Physiology, Universite´ de Lausanne, Lausanne CH-1005, Switzerland Corresponding author: Fajas, Lluis ([email protected])
Current Opinion in Cell Biology 2015, 33:19–25 This review comes from a themed issue on Cell regulation Edited by Johan Auwerx and Jodi Nunnari
http://dx.doi.org/10.1016/j.ceb.2014.10.006 0955-0674/# 2013 Elsevier Ltd. All rights reserved.
Introduction Mitochondria, which are believed to have evolved from the endosymbiosis of an alpha-proteobacterium [1], are cellular organelles that act as the engines of eukaryotic cells. Because of the energy and intermediate metaboliteproducing functions of mitochondria, their activity needs to be tightly regulated in response to cellular and organismal needs. Cells activate/trigger different cellular signaling pathways to fulfill their needs, and very often, these responses couple gene regulation to the dynamic regulation of organelle function. Numerous cellular signaling pathways drive cell cycle progression in response to specific stimuli. The regulation of the cell cycle has been a very productive area of research since the 1970s, after the discovery of the cdc2 gene by Paul Nurse [2]. In this review, we will focus on the regulation of the function of a specific organelle, the mitochondrion, by cell cycle regulators and on the reciprocal, subsequent regulation of the cell cycle by mitochondria. Cell cycle regulators control progression through the eukaryotic cell cycle, and major components of this group www.sciencedirect.com
of proteins are the family of cyclin-dependent kinases (CDKs) and their regulatory subunits, the cyclin proteins. The activity of CDKs is mainly regulated by the cyclic expression of their binding cyclins or by control of their interactions with these cyclins or the CDK inhibitory proteins (CDK inhibitors (CKIs) or Ink4 family members). Cyclin–CDK complexes catalyze the phosphorylation of members of the retinoblastoma (pRB) protein family (pRB, p107, and p130). Phosphorylation of pRB by cyclin–CDK releases the E2F-DP transcription factors, thereby ensuring the expression of genes required for cell cycle progression [3]. Conversely, the family of CDK inhibitors (INK and CIP/KIP) block CDK activity in response to quiescence stimuli. Numerous cellular signaling pathways, including those that drive cell division, interact tightly with the mechanisms that regulate mitochondrial function, namely mechanisms that regulate mitochondrial fission and fusion, mitochondrial biogenesis, mitochondrial activity, and finally, mitochondrial apoptosis (intrinsic pathway). In addition to archetypal cell cycle regulators, key transcription factors that also play roles in proliferation and cell cycle arrest, are also essential players in the regulation of mitochondrial function. This is the case of the tumor suppressor p53, which serves not only as a regulator of apoptosis but also as a direct regulator of mitochondrial DNA replication and integrity, and of autophagy. This has been recently reviewed in [4,5].
The role of cell cycle regulators in controlling the biogenesis and metabolic activity of mitochondria Mitochondrial biogenesis is controlled through coordinated transcriptional regulation of nuclear and mitochondrial genes. The key transcription factors ruling mitochondrial biogenesis are peroxisome proliferatoractivated receptors (PPARs), PGC1 coactivators (PGC1a and PGC1b), and nuclear respiratory factors 1 and 2 (NRF1 and NRF2). The activity of these transcription factors is controlled by the energetic demands of the cells (recently reviewed in [6]). Regulation of mitochondrial biogenesis sustains mitochondrial activity, which can be dependent on the tricarboxylic acid cycle (Krebs or TCA cycle) or electron transport chain (ETC). Cell cycle regulators have been recently linked to mitochondrial biogenesis. Indeed, cyclin D1 / hepatocytes exhibit increased mitochondrial size, increased mitochondrial activity, and increased expression of NRF1 [7]. On the other hand, the overexpression of cyclin D1 led to a Current Opinion in Cell Biology 2015, 33:19–25
20 Cell regulation
twofold decrease in mitochondrial activity that was mediated by CDK4 kinase activity but was independent of RB. Interestingly, the addition of serum to starved cells induced cyclin D1 expression and reduced NRF1 activity; more precisely, the NRF1 and cyclin D1 expression patterns were inversely correlated during cell cycle progression [7]. Furthermore, the deletion of pRB resulted in failure to induce the mitochondrial biogenesis transcription program in erythroid progenitors as a result of decreased PGC1b expression, thus decreasing the transcription of the PGC-1 regulated genes. Surprisingly, pRB / erythroid progenitors do not exhibit increased proliferation but fail to downregulate cell cycle genes and induce the mitochondrial biogenesis program [8]. The importance of the crosstalk of cell cycle regulators and mitochondrial function in red cell development was also highlighted in a recent study demonstrating that proper regulation of cyclin E levels is essential for limiting ROS accumulation, mitochondrial biogenesis and oxidative metabolism during erythrocyte maturation [9]. Insufficient mitochondrial content upon pRB deletion was also observed in a model of mouse muscle cells [10] and in mouse embryonic fibroblasts (MEFs) that also lacked the other two pocket protein family members, p107 and p130 [11]. In contrast, the loss of RBF1, the Drosophila pRB homolog, was recently shown to be associated with the opposite phenotype, that is, increased cellular oxidation by altered glutamine catabolism, using fly larvae and imaginal discs as models [12,13]. Consistent with this observation was the finding that increased CycD–Cdk4 in Drosophila led to increased mitochondrial biogenesis [14], which correlated with increased levels of mitochondrial DNA and elevated ATP synthase and cytochrome C expression. Interestingly, adult flies overexpressing CycD–Cdk4 were two times more active than control flies. On the other hand, flies deficient for CycD or Cdk4 exhibited mitochondrial biogenesis defects and were less active. The effects of CycD–Cdk4 on mitochondrial biogenesis were mediated by EWG (erect wing gene), the Drosophila NRF1 ortholog, TFAM and the Drosophila homolog of NRF2, Delg (Drosophila Ets-like gene) [15,16]. Because CDK4 and its partners, the type D cyclins, are negative regulators of pRB, the findings on the regulation of mitochondrial biogenesis in Drosophila contradict those using pRB-deficient and cyclin D1-deficient mouse-derived models. Distinct functions of the cyclin and CDK families in Drosophila and mammalian cells could underlie these differences. Our laboratory recently demonstrated that the cell cycle regulator E2F1, as a part of the CDK4-RB-E2F1 axis, acts upstream of the regulation of the expression of key oxidative metabolism genes that control energy expenditure in response to exercise or thermogenesic stimulation. E2f1, in association with pRB, represses mitochondrial genes under basal conditions, and this negative regulation of gene Current Opinion in Cell Biology 2015, 33:19–25
expression was abrogated upon CDK4 activation. Indeed E2f1 / mice showed increased expression of mitochondrial ETC, fatty acid oxidation and uncoupling genes as well as of genes involved in mitochondrial biogenesis in muscle and brown adipose tissue. Increased mitochondrial gene expression resulted in an increased number of mitochondria and in a markedly oxidative phenotype. Mechanistic studies revealed that the E2f1–pRB complex bound to the promoters of numerous oxidative genes. Upon appropriate stimulation, pRB was phosphorylated, thus facilitating gene transcription [17]. These results are in agreement with those of previous studies by other laboratories, which demonstrated that siRNAs against E2F1 in human cells prompted the induction of the TOP1MT (mitochondrial DNA Topoisomerase 1) mRNA as well as increased the transcription and replication of the mitochondrial genome [18]. Surprisingly, again in the Drosophila model, dDP mutants exhibited decreased ETC and OXPHOS gene expression, resulting in diminished ATP levels, decreased mitochondrial activity, and fragmented mitochondria. Moreover, using transmission electron microscopy (TEM), mitochondria from dDP mutant eye discs exhibited a more round morphology. Moreover, dDP, dE2f1, dE2f2 and RBF1 were detected at the promoters of the downregulated genes [19]. The same group confirmed their observations in a human osteosarcoma cell line in which E2F activity was blunted using DP1 and DP2 siRNAs, a dominant-negative form of DP1, or a mutant E2F that does not bind DNA. They observed similar ChIP occupancy, a punctuate mitochondrial pattern, reduced Mitotracker staining (thus reduced mitochondrial activity) and altered mitochondrial morphology at the ultrastructural level [19]. It is difficult to explain these paradoxical results other than that they are a result of differences in cell types or possibly due to the formation of distinct E2F complexes with specific activities. Other CDK family members have been linked to mitochondrial activity. CDK1, along with B1 cyclin, was demonstrated to couple the G2 > M transition with mitochondrial respiration by extensive phosphorylation of electron transport chain complex I subunits. Phosphorylation by CDK1 is necessary to activate the complex and the production of sufficient ATP for rapid cell cycle progression [20]. Overall, regulation of mitochondrial function by the CDK–RB–E2F pathway is conserved from flies to mammals, but this mechanism of control has most likely gained complexity due to the emergence of several paralogs of each of the genes (Table 1). Taken together, the current knowledge from both Drosophila and mouse models suggest that E2F1, similar to the regulation of cell cycle genes, can either repress or stimulate the expression of OXPHOS target genes in a pRB-dependent www.sciencedirect.com
Cell cycle regulation of mitochondrial function Lopez-Mejia and Fajas 21
Table 1 Cell cycle regulators in mitochondria. Roles/functions that implicate cell cycle regulators in the control of mitochondrial functions. The cellular/animal models used are also included in the table for the reader’s convenience Cell cycle regulator pRB pRB pRB, p107, p130
Model Mouse erythroid progenitors Rb deficient mouse muscle cells Triple RB KO MEFs
RBF1
Drosophila wing discs and 3rd instar larvae
RB
Several human cancer cell lines, as well as RPE human cells Mouse Hepatocytes and MEFs Mouse muscle and brown adipose tissue
Cyclin D1 E2F1
E2F1
Human HeLa cells
dE2F1, dDP
Drosophila eye discs and whole larvae
DP
CDK1
Human osteosarcoma cell lines (SAOS-2) Drosophila Fat body from third instar larvae Drosophila adult flies, fat body, salivary glands and wing discs Drosophila larvae fat bodies Human MCF10A cells
CDK1
Human HeLa cells
CycD–CDK4 CDK4
CDK4
Phenotype pRB KO erythroid progenitors fail to activate the mitochondrial biogenesis program necessary for their complete differentiation Rb deficiency causes mitochondrial loss via autophagy upon muscle differentiation Rb-Family deletion causes elevated anaplerosis of glutamine carbon and increased mitochondrial function Rb TKO cells contain significantly less mitochondria relative to WT cells RBF1 depleted cells show global metabolic changes, specially in glutamine metabolism, and increased cellular oxidation. This leads to increased sensitivity to DNA damage. RB depleted cells show global metabolic changes, specially in glutamine metabolism, and increased cellular oxidation. This leads to increased sensitivity to DNA damage Cyclin D1 represses NRF1 thus mitochondrial activity. This Cyclin D1 function is RB independent but CDK dependent E2F1 KO mice have increased oxidative phenotype and increased OXPHOS genes expression. They also lack thermogenic/fasting induction of oxidative metabolism Knockdown of E2F1 leads to the induction of the TOP1MT mRNA; as well as to increased transcription and replication of the mitochondrial genome dDP mutants have reduced mitochondrial activity, reduced ATP production, and fragmented and round mitochondria. They are resistant to irradiation induced apoptosis Knockdown of DP1 or DP2 leads to punctuate mitochondria, decreased expression of OXPHOS genes and abnormal mitochondrial ultrastructure Increased CycD–Cdk4 in Drosophila leads to increased mitochondrial biogenesis The effects of CycD–Cdk4 on mitochondrial biogenesis are mediated by NRF1 and TFAM. They lead to ‘whole’ fly activity changes The effects of CDK4 on mitochondrial biogenesis are also mediated by NRF2 CDK1 phosphorylates mitochondrial complex I proteins to increase their activity and ensure sufficient ATP production for rapid cell cycle progression Phosphorylation of DRP1 by CDK1 promotes mitochondrial fission
manner. We can speculate that, in E2f1 / mice, other members of the family, such as E2f2 or E2f3, can compensate for the activator function of E2F1 but cannot compensate for the repressor role. The ‘occupation’ of both the activator dE2F1 and the repressor dE2F2 on Drosophila mitochondrial gene promoters remains, in our opinion, unclear.
The role of cell cycle regulators in the control of mitochondrial dynamics Mitochondrial dynamics, namely fission and fusion, determine the architecture and thus activity of mitochondria. These two processes implicate an equilibrium between the activities of fission-promoting proteins, that is, dynaminrelated protein 1 (DRP1), mitochondrial fission 1 protein (hFis1), mitochondrial fission factor (MFF), and fusionpromoting proteins, that is, optic atrophy protein 1 (OPA1), mitofusin 1 (MFN1) and MFN2. Mitochondrial dynamics are essential for the maintenance of mitochondrial fitness www.sciencedirect.com
Reference [8] [10] [11]
[13]
[13]
[7] [17]
[18] [19]
[19] [14] [15]
[16] [20] [23]
and for the ‘health’ of the mitochondrial genome (recently reviewed in [21]). During cell cycle progression, the ‘structure’ of the mitochondrial network within the cell evolves from an interconnected network in G1, through a hyperpolarized, giant single tubular network at the G1/S transition, to a very fragmented network in mitosis, and [22]. The cell cycle regulators CDK1 and cyclin B1 mediate mitochondrial fragmentation early in M phase by phosphorylating DRP1 on S585 (in rats, equivalent to human S616) [23]. At the end of M phase and after cell division, the fused mitochondrial network is restored, at least in part, due to the proteosomal degradation of DRP1. This process was shown to be mediated/catalyzed by another cell cycle regulator, the anaphase promoting complex (APC) [24]. Interestingly, another CDK, CDK5, is also able to phosphorylate the same residue, thus disrupting the microtubule targeting of DRP1 during interphase, which suggests a more general role for CDKs in mitochondrial localization Current Opinion in Cell Biology 2015, 33:19–25
22 Cell regulation
[25]. Notably, the phosphorylation of DRP1 by CDK5 was shown to be important for neuronal maturation [26]. Apoptosis, or programmed cell death, is a crucial process for metazoan development, that is also tightly regulated by mitochondria. Indeed, mitochondrial fission takes place early in the intrinsic apoptotic program, well before caspase activation. CDK1 has been shown to play a role in the regulation of mitosis-induced apoptosis, and the current model suggests that CDK1/cyclin B1 phosphorylates key apoptotic regulators such as caspase 2, caspase 9, BCL-XL, BCL-2 and Mcl-1 during mitosis [27–30]. Upon improper mitotic spindle formation, cells with deficiencies will undergo cell death during anaphase. In these abnormal conditions, CDK1 activity is preserved due to the lack of APC-mediated cyclin B1 degradation, and extensive degradation of phosphorylated Mcl-1 protein drives apoptosis (recently reviewed in [31]).
Feedback control of the cell cycle by mitochondria Current research emphasizes the importance of proper bioenergetics, which mainly rely on mitochondrial activity, for cell cycle progression. Undeniably, proliferation generates high energy demands to proceed through all biosynthetic processes that are necessary for lipid, protein and nucleic acid synthesis. Insufficient mitochondrial activity might, in turn, decrease ATP levels and subsequently increase the AMP/ATP ratio, which is known to activate AMPK and lead to G1 > S arrest, at least in Drosophila [32] and MEFs [33]. Direct defects in mitochondrial complexes can also elicit such cell cycle defects. Indeed, mitochondrial dysfunction due to loss-of-function mutants in the ETC complexes I or IV induce G1 > S arrest [32]. Mitochondrial dynamics also influence cell cycle progression. Cell cycle-mediated mitochondrial hyperfusion is necessary for cell cycle progression because it is a prerequisite for cyclin E accumulation and for the consequent entry into S phase. Importantly, triggering mitochondrial hyperfusion in G0-arrested cells (serum-starved) triggered entry into S phase [22]. Accordingly, loss of DRP1 function in Drosophila, led to a decrease in mitochondrial division and to an immortalized-like cellular phenotype as cells failed to enter their differentiation program [34]. Moreover, the mitochondrial fission regulator hFis1 was recently shown to tightly interact with the components of the cell cycle machinery at the G2 > M transition. Indeed, siRNA-mediated downregulation of hFis1 levels facilitated an elongated mitochondrial morphology, led to a reduction in the levels of several key regulators such as cyclin A, cyclin B and CDK1, and subsequently blocked G2 > M cell cycle progression [35]. A more global coordination of mitochondrial function and cell cycle regulation was described 10 years ago when Current Opinion in Cell Biology 2015, 33:19–25
ChIP-on-Chip analyses demonstrated that NRF1 occupies numerous E2F target promoters and coregulates E2F transcriptional activity [36]. The expression of NRF2 is equally important for cell cycle progression because the overexpression of this factor drove cells out of quiescence. Moreover, acute disruption of NRF2 in MEFs caused cell cycle arrest and prevented entry into S phase [37]. These studies on key mitochondrial master genes and their effects on the cell cycle once again demonstrate the strong coupling between regulation of the cell cycle and mitochondrial activity.
Atypical cyclins and CDKs Atypical members of the CDK and cyclin families also play a role in the regulation of mitochondrial function. The phosphorylation of Drp1 by the atypical CDK5 was described above. The CDK activating kinase, CDK7, which is both a cell cycle regulator and a more general transcriptional regulator, has been shown to regulate mitochondrial activity with its partners cyclin H and MAT1. In the mouse heart, deletion of MAT1 led to a decrease in CDK7 activity and to a general defect in transcriptional coactivation by PGC-1 family members [38]. CDK7 was also found to be a positive regulator of apoptosis in a Drosophila model through controlling the localization of the proapoptotic tail anchor protein Hid [39]. As with mitosis, stress shifts the balance of mitochondrial dynamics towards fission. The role of the atypical cyclin C–Cdk8p complex in this process has been extensively studied in Saccharomyces cerevisiae. Cyclin C–Cdk8p represses stress-responsive genes; however, under oxidative stress, cyclin C first translocates from the nucleus to mitochondria to elicit the transcription of stress-responsive genes. Outside the nucleus, cyclin C–Cdk8p promotes mitochondrial fission [40]. Upon sustained stress the localization of Cdk8p remains nuclear [41], and the translocation and subsequent destruction of cyclin C requires the intervention of the MAPK pathway (Slt2p and Kdx1p in yeast) [42]. Under normal conditions, this ‘altered’ mitochondrial response is prevented via Med13p-mediated nuclear retention of cyclin C [43]. These tight regulations demonstrate how cell cycle-like regulators can participate in the crosstalk between the nucleus and mitochondria and regulate mitochondrial activity, the stress response and programmed cell death.
Concluding remarks Mitochondrial function and dynamics must be closely regulated to fulfill the metabolic needs of the cells, and cell cycle regulators are major sensors and effectors of these needs. In this review, we discussed how these families of proteins coordinately regulate cell proliferation and mitochondrial activity to achieve an adapted cellular response to the metabolic requirements for cell division (Figure 1). In the future, other questions, such as how do cell cycle www.sciencedirect.com
Cell cycle regulation of mitochondrial function Lopez-Mejia and Fajas 23
Figure 1
Mitogens
DRP1 Fissioned mitochondria
–
CDK4/6
Cyclin D
APC DRP1 +
RB E2F
G0
Complex I
OXPHOS GENES
Cell cycle X progression genes
+
Cyclin D
Cyclin C li B
M
CDK1
CDK4/6
G1 G2
Fused mitochondria PP P
RB P
S Cyclin E Cyclin A
CDK2
E2F
OXPHOS GENES Cell cycle progression genes
CDK2 Hyperfused giant mitochondria Current Opinion in Cell Biology
Coupling cell cycle regulation with mitochondrial function and dynamics. Mitogen-induced activation of the cyclin D–CDK4/6 complex early in G1 phase leads to the partial inactivation of the pocket proteins pRB, p107 and p130. Inactivated pocket proteins will release E2F transcription factors activity and thus, allow the expression of genes necessary for the G1 > S transition. Next, CDK2 is active, as a complex with cyclin E or A type cyclins, during S phase and G2 phase respectively. Finally, CDK1 with B type cyclins, will drive cells through mitosis. The cycle will terminate upon activation of the APC complex and the subsequent degradation of type B cyclins. When separated, the daughter cells enter into G1. In parallel, when complexed to pRB the E2F proteins repress mitochondrial function genes, and this repression is also released when G1 > S CDKs are activated, even in a non-proliferative context. Cell cycle progression mediated changes in cyclin–CDK activity are accompanied by changes in mitochondrial morphology. The mitochondrial meshwork transforms from an interconnected network in G1 to a very fragmented network in mitosis, going through a hyperpolarized giant single tubular network at the G1/S transition. This is at least partially mediated by the activation and ensuing degradation of DRP1 during M phase by CDK1 and the APC respectively. CDK1 also facilitates the G2 > M transition by phosphorylating and activating the complex I of the ETC. Mitochondrial activity is also regulated by cell cycle regulators at the level of nuclear transcription. Indeed the RB-E2F complexes have been shown in several studies to regulate transcription of mitochondrial genes, but these studies are somehow contradictory, suggesting supplemental levels of complexity that remain to be investigated. Positive regulators of cell cycle are in white. Negative regulators of cell cycle are in black.
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The Fajas lab is funded by the Swiss National Science Foundation (SNF) and by the Ligue Suisse contre le Cancer.
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regulators specifically synchronize mitochondrial functions with the regulation of mitosis and other phases of the cell cycle, should be raised. In addition, new functions in the regulation of mitochondrial activity by some members of these families should be considered.
Acknowledgements
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Cell cycle regulation of mitochondrial function Lopez-Mejia and Fajas 25
Strich and colleagues reveal that ‘atypical’ members of the cyclin and CDK families are key mediators of the rapid stress responses. Cyclin C emerges as a crucial mediator of oxidative stress response in yeast.
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Current Opinion in Cell Biology 2015, 33:19–25
ScienceDirect Cell cycle regulation of mitochondrial function Isabel C Lopez-Mejia and Lluis Fajas Specific cellular functions, such as proliferation, survival, growth, or senescence, require a particular adaptive metabolic response, which is fine tuned by members of the cell cycle regulators families. Currently, proteins such as cyclins, CDKs, or E2Fs are being studied in the context of cell proliferation and survival, cell signaling, cell cycle regulation, and cancer. We show in this review that cellular, animal and molecular studies provided enough evidence to prove that these factors play, in addition, crucial roles in the control of mitochondrial function; finally resulting in a dual proliferative and metabolic response. Addresses Department of Physiology, Universite´ de Lausanne, Lausanne CH-1005, Switzerland Corresponding author: Fajas, Lluis ([email protected])
Current Opinion in Cell Biology 2015, 33:19–25 This review comes from a themed issue on Cell regulation Edited by Johan Auwerx and Jodi Nunnari
http://dx.doi.org/10.1016/j.ceb.2014.10.006 0955-0674/# 2013 Elsevier Ltd. All rights reserved.
Introduction Mitochondria, which are believed to have evolved from the endosymbiosis of an alpha-proteobacterium [1], are cellular organelles that act as the engines of eukaryotic cells. Because of the energy and intermediate metaboliteproducing functions of mitochondria, their activity needs to be tightly regulated in response to cellular and organismal needs. Cells activate/trigger different cellular signaling pathways to fulfill their needs, and very often, these responses couple gene regulation to the dynamic regulation of organelle function. Numerous cellular signaling pathways drive cell cycle progression in response to specific stimuli. The regulation of the cell cycle has been a very productive area of research since the 1970s, after the discovery of the cdc2 gene by Paul Nurse [2]. In this review, we will focus on the regulation of the function of a specific organelle, the mitochondrion, by cell cycle regulators and on the reciprocal, subsequent regulation of the cell cycle by mitochondria. Cell cycle regulators control progression through the eukaryotic cell cycle, and major components of this group www.sciencedirect.com
of proteins are the family of cyclin-dependent kinases (CDKs) and their regulatory subunits, the cyclin proteins. The activity of CDKs is mainly regulated by the cyclic expression of their binding cyclins or by control of their interactions with these cyclins or the CDK inhibitory proteins (CDK inhibitors (CKIs) or Ink4 family members). Cyclin–CDK complexes catalyze the phosphorylation of members of the retinoblastoma (pRB) protein family (pRB, p107, and p130). Phosphorylation of pRB by cyclin–CDK releases the E2F-DP transcription factors, thereby ensuring the expression of genes required for cell cycle progression [3]. Conversely, the family of CDK inhibitors (INK and CIP/KIP) block CDK activity in response to quiescence stimuli. Numerous cellular signaling pathways, including those that drive cell division, interact tightly with the mechanisms that regulate mitochondrial function, namely mechanisms that regulate mitochondrial fission and fusion, mitochondrial biogenesis, mitochondrial activity, and finally, mitochondrial apoptosis (intrinsic pathway). In addition to archetypal cell cycle regulators, key transcription factors that also play roles in proliferation and cell cycle arrest, are also essential players in the regulation of mitochondrial function. This is the case of the tumor suppressor p53, which serves not only as a regulator of apoptosis but also as a direct regulator of mitochondrial DNA replication and integrity, and of autophagy. This has been recently reviewed in [4,5].
The role of cell cycle regulators in controlling the biogenesis and metabolic activity of mitochondria Mitochondrial biogenesis is controlled through coordinated transcriptional regulation of nuclear and mitochondrial genes. The key transcription factors ruling mitochondrial biogenesis are peroxisome proliferatoractivated receptors (PPARs), PGC1 coactivators (PGC1a and PGC1b), and nuclear respiratory factors 1 and 2 (NRF1 and NRF2). The activity of these transcription factors is controlled by the energetic demands of the cells (recently reviewed in [6]). Regulation of mitochondrial biogenesis sustains mitochondrial activity, which can be dependent on the tricarboxylic acid cycle (Krebs or TCA cycle) or electron transport chain (ETC). Cell cycle regulators have been recently linked to mitochondrial biogenesis. Indeed, cyclin D1 / hepatocytes exhibit increased mitochondrial size, increased mitochondrial activity, and increased expression of NRF1 [7]. On the other hand, the overexpression of cyclin D1 led to a Current Opinion in Cell Biology 2015, 33:19–25
20 Cell regulation
twofold decrease in mitochondrial activity that was mediated by CDK4 kinase activity but was independent of RB. Interestingly, the addition of serum to starved cells induced cyclin D1 expression and reduced NRF1 activity; more precisely, the NRF1 and cyclin D1 expression patterns were inversely correlated during cell cycle progression [7]. Furthermore, the deletion of pRB resulted in failure to induce the mitochondrial biogenesis transcription program in erythroid progenitors as a result of decreased PGC1b expression, thus decreasing the transcription of the PGC-1 regulated genes. Surprisingly, pRB / erythroid progenitors do not exhibit increased proliferation but fail to downregulate cell cycle genes and induce the mitochondrial biogenesis program [8]. The importance of the crosstalk of cell cycle regulators and mitochondrial function in red cell development was also highlighted in a recent study demonstrating that proper regulation of cyclin E levels is essential for limiting ROS accumulation, mitochondrial biogenesis and oxidative metabolism during erythrocyte maturation [9]. Insufficient mitochondrial content upon pRB deletion was also observed in a model of mouse muscle cells [10] and in mouse embryonic fibroblasts (MEFs) that also lacked the other two pocket protein family members, p107 and p130 [11]. In contrast, the loss of RBF1, the Drosophila pRB homolog, was recently shown to be associated with the opposite phenotype, that is, increased cellular oxidation by altered glutamine catabolism, using fly larvae and imaginal discs as models [12,13]. Consistent with this observation was the finding that increased CycD–Cdk4 in Drosophila led to increased mitochondrial biogenesis [14], which correlated with increased levels of mitochondrial DNA and elevated ATP synthase and cytochrome C expression. Interestingly, adult flies overexpressing CycD–Cdk4 were two times more active than control flies. On the other hand, flies deficient for CycD or Cdk4 exhibited mitochondrial biogenesis defects and were less active. The effects of CycD–Cdk4 on mitochondrial biogenesis were mediated by EWG (erect wing gene), the Drosophila NRF1 ortholog, TFAM and the Drosophila homolog of NRF2, Delg (Drosophila Ets-like gene) [15,16]. Because CDK4 and its partners, the type D cyclins, are negative regulators of pRB, the findings on the regulation of mitochondrial biogenesis in Drosophila contradict those using pRB-deficient and cyclin D1-deficient mouse-derived models. Distinct functions of the cyclin and CDK families in Drosophila and mammalian cells could underlie these differences. Our laboratory recently demonstrated that the cell cycle regulator E2F1, as a part of the CDK4-RB-E2F1 axis, acts upstream of the regulation of the expression of key oxidative metabolism genes that control energy expenditure in response to exercise or thermogenesic stimulation. E2f1, in association with pRB, represses mitochondrial genes under basal conditions, and this negative regulation of gene Current Opinion in Cell Biology 2015, 33:19–25
expression was abrogated upon CDK4 activation. Indeed E2f1 / mice showed increased expression of mitochondrial ETC, fatty acid oxidation and uncoupling genes as well as of genes involved in mitochondrial biogenesis in muscle and brown adipose tissue. Increased mitochondrial gene expression resulted in an increased number of mitochondria and in a markedly oxidative phenotype. Mechanistic studies revealed that the E2f1–pRB complex bound to the promoters of numerous oxidative genes. Upon appropriate stimulation, pRB was phosphorylated, thus facilitating gene transcription [17]. These results are in agreement with those of previous studies by other laboratories, which demonstrated that siRNAs against E2F1 in human cells prompted the induction of the TOP1MT (mitochondrial DNA Topoisomerase 1) mRNA as well as increased the transcription and replication of the mitochondrial genome [18]. Surprisingly, again in the Drosophila model, dDP mutants exhibited decreased ETC and OXPHOS gene expression, resulting in diminished ATP levels, decreased mitochondrial activity, and fragmented mitochondria. Moreover, using transmission electron microscopy (TEM), mitochondria from dDP mutant eye discs exhibited a more round morphology. Moreover, dDP, dE2f1, dE2f2 and RBF1 were detected at the promoters of the downregulated genes [19]. The same group confirmed their observations in a human osteosarcoma cell line in which E2F activity was blunted using DP1 and DP2 siRNAs, a dominant-negative form of DP1, or a mutant E2F that does not bind DNA. They observed similar ChIP occupancy, a punctuate mitochondrial pattern, reduced Mitotracker staining (thus reduced mitochondrial activity) and altered mitochondrial morphology at the ultrastructural level [19]. It is difficult to explain these paradoxical results other than that they are a result of differences in cell types or possibly due to the formation of distinct E2F complexes with specific activities. Other CDK family members have been linked to mitochondrial activity. CDK1, along with B1 cyclin, was demonstrated to couple the G2 > M transition with mitochondrial respiration by extensive phosphorylation of electron transport chain complex I subunits. Phosphorylation by CDK1 is necessary to activate the complex and the production of sufficient ATP for rapid cell cycle progression [20]. Overall, regulation of mitochondrial function by the CDK–RB–E2F pathway is conserved from flies to mammals, but this mechanism of control has most likely gained complexity due to the emergence of several paralogs of each of the genes (Table 1). Taken together, the current knowledge from both Drosophila and mouse models suggest that E2F1, similar to the regulation of cell cycle genes, can either repress or stimulate the expression of OXPHOS target genes in a pRB-dependent www.sciencedirect.com
Cell cycle regulation of mitochondrial function Lopez-Mejia and Fajas 21
Table 1 Cell cycle regulators in mitochondria. Roles/functions that implicate cell cycle regulators in the control of mitochondrial functions. The cellular/animal models used are also included in the table for the reader’s convenience Cell cycle regulator pRB pRB pRB, p107, p130
Model Mouse erythroid progenitors Rb deficient mouse muscle cells Triple RB KO MEFs
RBF1
Drosophila wing discs and 3rd instar larvae
RB
Several human cancer cell lines, as well as RPE human cells Mouse Hepatocytes and MEFs Mouse muscle and brown adipose tissue
Cyclin D1 E2F1
E2F1
Human HeLa cells
dE2F1, dDP
Drosophila eye discs and whole larvae
DP
CDK1
Human osteosarcoma cell lines (SAOS-2) Drosophila Fat body from third instar larvae Drosophila adult flies, fat body, salivary glands and wing discs Drosophila larvae fat bodies Human MCF10A cells
CDK1
Human HeLa cells
CycD–CDK4 CDK4
CDK4
Phenotype pRB KO erythroid progenitors fail to activate the mitochondrial biogenesis program necessary for their complete differentiation Rb deficiency causes mitochondrial loss via autophagy upon muscle differentiation Rb-Family deletion causes elevated anaplerosis of glutamine carbon and increased mitochondrial function Rb TKO cells contain significantly less mitochondria relative to WT cells RBF1 depleted cells show global metabolic changes, specially in glutamine metabolism, and increased cellular oxidation. This leads to increased sensitivity to DNA damage. RB depleted cells show global metabolic changes, specially in glutamine metabolism, and increased cellular oxidation. This leads to increased sensitivity to DNA damage Cyclin D1 represses NRF1 thus mitochondrial activity. This Cyclin D1 function is RB independent but CDK dependent E2F1 KO mice have increased oxidative phenotype and increased OXPHOS genes expression. They also lack thermogenic/fasting induction of oxidative metabolism Knockdown of E2F1 leads to the induction of the TOP1MT mRNA; as well as to increased transcription and replication of the mitochondrial genome dDP mutants have reduced mitochondrial activity, reduced ATP production, and fragmented and round mitochondria. They are resistant to irradiation induced apoptosis Knockdown of DP1 or DP2 leads to punctuate mitochondria, decreased expression of OXPHOS genes and abnormal mitochondrial ultrastructure Increased CycD–Cdk4 in Drosophila leads to increased mitochondrial biogenesis The effects of CycD–Cdk4 on mitochondrial biogenesis are mediated by NRF1 and TFAM. They lead to ‘whole’ fly activity changes The effects of CDK4 on mitochondrial biogenesis are also mediated by NRF2 CDK1 phosphorylates mitochondrial complex I proteins to increase their activity and ensure sufficient ATP production for rapid cell cycle progression Phosphorylation of DRP1 by CDK1 promotes mitochondrial fission
manner. We can speculate that, in E2f1 / mice, other members of the family, such as E2f2 or E2f3, can compensate for the activator function of E2F1 but cannot compensate for the repressor role. The ‘occupation’ of both the activator dE2F1 and the repressor dE2F2 on Drosophila mitochondrial gene promoters remains, in our opinion, unclear.
The role of cell cycle regulators in the control of mitochondrial dynamics Mitochondrial dynamics, namely fission and fusion, determine the architecture and thus activity of mitochondria. These two processes implicate an equilibrium between the activities of fission-promoting proteins, that is, dynaminrelated protein 1 (DRP1), mitochondrial fission 1 protein (hFis1), mitochondrial fission factor (MFF), and fusionpromoting proteins, that is, optic atrophy protein 1 (OPA1), mitofusin 1 (MFN1) and MFN2. Mitochondrial dynamics are essential for the maintenance of mitochondrial fitness www.sciencedirect.com
Reference [8] [10] [11]
[13]
[13]
[7] [17]
[18] [19]
[19] [14] [15]
[16] [20] [23]
and for the ‘health’ of the mitochondrial genome (recently reviewed in [21]). During cell cycle progression, the ‘structure’ of the mitochondrial network within the cell evolves from an interconnected network in G1, through a hyperpolarized, giant single tubular network at the G1/S transition, to a very fragmented network in mitosis, and [22]. The cell cycle regulators CDK1 and cyclin B1 mediate mitochondrial fragmentation early in M phase by phosphorylating DRP1 on S585 (in rats, equivalent to human S616) [23]. At the end of M phase and after cell division, the fused mitochondrial network is restored, at least in part, due to the proteosomal degradation of DRP1. This process was shown to be mediated/catalyzed by another cell cycle regulator, the anaphase promoting complex (APC) [24]. Interestingly, another CDK, CDK5, is also able to phosphorylate the same residue, thus disrupting the microtubule targeting of DRP1 during interphase, which suggests a more general role for CDKs in mitochondrial localization Current Opinion in Cell Biology 2015, 33:19–25
22 Cell regulation
[25]. Notably, the phosphorylation of DRP1 by CDK5 was shown to be important for neuronal maturation [26]. Apoptosis, or programmed cell death, is a crucial process for metazoan development, that is also tightly regulated by mitochondria. Indeed, mitochondrial fission takes place early in the intrinsic apoptotic program, well before caspase activation. CDK1 has been shown to play a role in the regulation of mitosis-induced apoptosis, and the current model suggests that CDK1/cyclin B1 phosphorylates key apoptotic regulators such as caspase 2, caspase 9, BCL-XL, BCL-2 and Mcl-1 during mitosis [27–30]. Upon improper mitotic spindle formation, cells with deficiencies will undergo cell death during anaphase. In these abnormal conditions, CDK1 activity is preserved due to the lack of APC-mediated cyclin B1 degradation, and extensive degradation of phosphorylated Mcl-1 protein drives apoptosis (recently reviewed in [31]).
Feedback control of the cell cycle by mitochondria Current research emphasizes the importance of proper bioenergetics, which mainly rely on mitochondrial activity, for cell cycle progression. Undeniably, proliferation generates high energy demands to proceed through all biosynthetic processes that are necessary for lipid, protein and nucleic acid synthesis. Insufficient mitochondrial activity might, in turn, decrease ATP levels and subsequently increase the AMP/ATP ratio, which is known to activate AMPK and lead to G1 > S arrest, at least in Drosophila [32] and MEFs [33]. Direct defects in mitochondrial complexes can also elicit such cell cycle defects. Indeed, mitochondrial dysfunction due to loss-of-function mutants in the ETC complexes I or IV induce G1 > S arrest [32]. Mitochondrial dynamics also influence cell cycle progression. Cell cycle-mediated mitochondrial hyperfusion is necessary for cell cycle progression because it is a prerequisite for cyclin E accumulation and for the consequent entry into S phase. Importantly, triggering mitochondrial hyperfusion in G0-arrested cells (serum-starved) triggered entry into S phase [22]. Accordingly, loss of DRP1 function in Drosophila, led to a decrease in mitochondrial division and to an immortalized-like cellular phenotype as cells failed to enter their differentiation program [34]. Moreover, the mitochondrial fission regulator hFis1 was recently shown to tightly interact with the components of the cell cycle machinery at the G2 > M transition. Indeed, siRNA-mediated downregulation of hFis1 levels facilitated an elongated mitochondrial morphology, led to a reduction in the levels of several key regulators such as cyclin A, cyclin B and CDK1, and subsequently blocked G2 > M cell cycle progression [35]. A more global coordination of mitochondrial function and cell cycle regulation was described 10 years ago when Current Opinion in Cell Biology 2015, 33:19–25
ChIP-on-Chip analyses demonstrated that NRF1 occupies numerous E2F target promoters and coregulates E2F transcriptional activity [36]. The expression of NRF2 is equally important for cell cycle progression because the overexpression of this factor drove cells out of quiescence. Moreover, acute disruption of NRF2 in MEFs caused cell cycle arrest and prevented entry into S phase [37]. These studies on key mitochondrial master genes and their effects on the cell cycle once again demonstrate the strong coupling between regulation of the cell cycle and mitochondrial activity.
Atypical cyclins and CDKs Atypical members of the CDK and cyclin families also play a role in the regulation of mitochondrial function. The phosphorylation of Drp1 by the atypical CDK5 was described above. The CDK activating kinase, CDK7, which is both a cell cycle regulator and a more general transcriptional regulator, has been shown to regulate mitochondrial activity with its partners cyclin H and MAT1. In the mouse heart, deletion of MAT1 led to a decrease in CDK7 activity and to a general defect in transcriptional coactivation by PGC-1 family members [38]. CDK7 was also found to be a positive regulator of apoptosis in a Drosophila model through controlling the localization of the proapoptotic tail anchor protein Hid [39]. As with mitosis, stress shifts the balance of mitochondrial dynamics towards fission. The role of the atypical cyclin C–Cdk8p complex in this process has been extensively studied in Saccharomyces cerevisiae. Cyclin C–Cdk8p represses stress-responsive genes; however, under oxidative stress, cyclin C first translocates from the nucleus to mitochondria to elicit the transcription of stress-responsive genes. Outside the nucleus, cyclin C–Cdk8p promotes mitochondrial fission [40]. Upon sustained stress the localization of Cdk8p remains nuclear [41], and the translocation and subsequent destruction of cyclin C requires the intervention of the MAPK pathway (Slt2p and Kdx1p in yeast) [42]. Under normal conditions, this ‘altered’ mitochondrial response is prevented via Med13p-mediated nuclear retention of cyclin C [43]. These tight regulations demonstrate how cell cycle-like regulators can participate in the crosstalk between the nucleus and mitochondria and regulate mitochondrial activity, the stress response and programmed cell death.
Concluding remarks Mitochondrial function and dynamics must be closely regulated to fulfill the metabolic needs of the cells, and cell cycle regulators are major sensors and effectors of these needs. In this review, we discussed how these families of proteins coordinately regulate cell proliferation and mitochondrial activity to achieve an adapted cellular response to the metabolic requirements for cell division (Figure 1). In the future, other questions, such as how do cell cycle www.sciencedirect.com
Cell cycle regulation of mitochondrial function Lopez-Mejia and Fajas 23
Figure 1
Mitogens
DRP1 Fissioned mitochondria
–
CDK4/6
Cyclin D
APC DRP1 +
RB E2F
G0
Complex I
OXPHOS GENES
Cell cycle X progression genes
+
Cyclin D
Cyclin C li B
M
CDK1
CDK4/6
G1 G2
Fused mitochondria PP P
RB P
S Cyclin E Cyclin A
CDK2
E2F
OXPHOS GENES Cell cycle progression genes
CDK2 Hyperfused giant mitochondria Current Opinion in Cell Biology
Coupling cell cycle regulation with mitochondrial function and dynamics. Mitogen-induced activation of the cyclin D–CDK4/6 complex early in G1 phase leads to the partial inactivation of the pocket proteins pRB, p107 and p130. Inactivated pocket proteins will release E2F transcription factors activity and thus, allow the expression of genes necessary for the G1 > S transition. Next, CDK2 is active, as a complex with cyclin E or A type cyclins, during S phase and G2 phase respectively. Finally, CDK1 with B type cyclins, will drive cells through mitosis. The cycle will terminate upon activation of the APC complex and the subsequent degradation of type B cyclins. When separated, the daughter cells enter into G1. In parallel, when complexed to pRB the E2F proteins repress mitochondrial function genes, and this repression is also released when G1 > S CDKs are activated, even in a non-proliferative context. Cell cycle progression mediated changes in cyclin–CDK activity are accompanied by changes in mitochondrial morphology. The mitochondrial meshwork transforms from an interconnected network in G1 to a very fragmented network in mitosis, going through a hyperpolarized giant single tubular network at the G1/S transition. This is at least partially mediated by the activation and ensuing degradation of DRP1 during M phase by CDK1 and the APC respectively. CDK1 also facilitates the G2 > M transition by phosphorylating and activating the complex I of the ETC. Mitochondrial activity is also regulated by cell cycle regulators at the level of nuclear transcription. Indeed the RB-E2F complexes have been shown in several studies to regulate transcription of mitochondrial genes, but these studies are somehow contradictory, suggesting supplemental levels of complexity that remain to be investigated. Positive regulators of cell cycle are in white. Negative regulators of cell cycle are in black.
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The Fajas lab is funded by the Swiss National Science Foundation (SNF) and by the Ligue Suisse contre le Cancer.
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regulators specifically synchronize mitochondrial functions with the regulation of mitosis and other phases of the cell cycle, should be raised. In addition, new functions in the regulation of mitochondrial activity by some members of these families should be considered.
Acknowledgements
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32. Mandal S, Guptan P, Owusu-Ansah E, Banerjee U: Mitochondrial regulation of cell cycle progression during development as revealed by the tenured mutation in Drosophila. Dev Cell 2005, 9:843-854. 33. Jones RG, Plas DR, Kubek S, Buzzai M, Mu J, Xu Y, Birnbaum MJ, Thompson CB: AMP-activated protein kinase induces a p53-dependent metabolic checkpoint. Mol Cell 2005, 18:283-293. 34. Mitra K, Rikhy R, Lilly M, Lippincott-Schwartz J: DRP1-dependent mitochondrial fission initiates follicle cell differentiation during Drosophila oogenesis. J Cell Biol 2012, 197:487-497. 35. Lee S, Park YY, Kim SH, Nguyen OT, Yoo YS, Chan GK, Sun X, Cho H: Human mitochondrial Fis1 links to cell cycle regulators at G2/M transition. Cell Mol Life Sci 2014, 71:711-725. 36. Cam H, Balciunaite E, Blais A, Spektor A, Scarpulla RC, Young R, Kluger Y, Dynlacht BD: A common set of gene regulatory networks links metabolism and growth inhibition. Mol Cell 2004, 16:399-411. 37. Yang ZF, Mott S, Rosmarin AG: The Ets transcription factor GABP is required for cell-cycle progression. Nat Cell Biol 2007, 9:339-346. This study demonstrates that there is extensive ‘coordinated corregulation’ of the cell cycle and mitochondrial transcription programs. And that it is a genome-wide phenomenon. 38. Sano M, Izumi Y, Helenius K, Asakura M, Rossi DJ, Xie M, Taffet G, Hu L, Pautler RG, Wilson CR et al.: Menage-a-trois 1 is critical for the transcriptional function of PPARgamma coactivator 1. Cell Metab 2007, 5:129-142. 39. Morishita J, Kang MJ, Fidelin K, Ryoo HD: CDK7 regulates the mitochondrial localization of a tail-anchored proapoptotic protein, Hid. Cell Rep 2013, 5:1481-1488. 40. Cooper KF, Khakhina S, Kim SK, Strich R: Stress-induced nuclear-to-cytoplasmic translocation of cyclin C promotes mitochondrial fission in yeast. Dev Cell 2014, 28:161-173. www.sciencedirect.com
Cell cycle regulation of mitochondrial function Lopez-Mejia and Fajas 25
Strich and colleagues reveal that ‘atypical’ members of the cyclin and CDK families are key mediators of the rapid stress responses. Cyclin C emerges as a crucial mediator of oxidative stress response in yeast.
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