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Home医源资料库在线期刊循环研究杂志2005年第95卷第3期

E2F2 and E2F4 but not E2F1 and E2F3 Induce DNA Synthesis in Cardiomyocytes Without Activation of Apoptosis

来源:循环研究杂志
摘要:pRbbindsandinactivatesE2Ftranscriptionfactorsinitshypophosphorylatedform,whereasphosphorylatedpRbreleasesE2Fs,whichthenactivategenesrequiredfornucleotidemetabolismandDNAsynthesis。AlthoughE2F1toE2F5allhavebothaDNAbindingdomain,adimerizationdomain,andatra......

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    the Institute of Physiological Chemistry (H.E., N.H., P.N., H.N., P.G., T.B.) and Departments of Medicine III (H.E., U.M.-W., K.W.) and Cardio-Thoracic Surgery (A.S.), University of Halle-Wittenberg, Germany
    the Max-Planck-Institute for Heart and Lung Research (T.B.), Bad Nauheim, Germany.

    Abstract

    Proliferation of mammalian cardiomyocytes ceases around birth when a transition from hyperplastic to hypertrophic myocardial growth occurs. Previous studies demonstrated that directed expression of the transcription factor E2F1 induces S-phase entry in cardiomyocytes along with stimulation of programmed cell death. Here, we show that directed expression of E2F2 and E2F4 by adenovirus mediated gene transfer in neonatal cardiomyocytes induced S-phase entry but did not result in an onset of apoptosis whereas directed expression of E2F1 and E2F3 strongly evoked programmed cell death concomitant with cell cycle progression. Although both E2F2 and E2F4 induced S-phase entry only directed expression of E2F2 resulted in mitotic cell division of cardiomyocytes. Expression of E2F5 or a control LacZ-Adenovirus had no effects on cell cycle progression. Quantitative real time PCR revealed that E2F1, E2F2, E2F3, and E2F4 alleviate G0 arrest by induction of cyclinA and E cyclins. Furthermore, directed expression of E2F1, E2F3, and E2F5 led to a transcriptional activation of several proapoptotic genes, which were mitigated by E2F2 and E2F4. Our finding that expression of E2F2 induces cell division of cardiomyocytes along with a suppression of proapoptotic genes might open a new access to improve the regenerative capacity of cardiomyocytes.

    Key Words: E2F  cardiomyocyte  cell cycle  proliferation

    Introduction

    Differentiation of cardiomyocytes is inversely correlated with proliferation. During embryogenesis precursor cells of cardiomyocytes within the precardial mesoderm show a very high rate of cell proliferation of approximately 70%, which drops to 45% on onset of cardiomyocyte differentiation.1 Around birth, a transition from hyperplastic to hypertrophic myocardial growth occurs, and cytokinesis and cell proliferation, which are hallmarks of fetal development, are superseded by hypertrophy and binucleation of cardiomyocytes.2 The proliferation block of cardiomyocytes prevents efficient replacement of functional myocardial on tissue damage, although some reports demonstrated division of cardiomyocytes in the failing heart, indicating that cardiomyocytes retain at least some proliferative capacity3

    The mammalian cell cycle is tightly regulated by a complex network of factors that either promote cell cycle progression or arrest cells at a certain cycle position. Stimulation by growth factors in proliferating cells leads to formation of complexes of D-type cyclins and cdk2/4 and phosphorylation of pocket proteins such as the retinoblastoma protein (pRb). pRb binds and inactivates E2F transcription factors in its hypophosphorylated form, whereas phosphorylated pRb releases E2Fs, which then activate genes required for nucleotide metabolism and DNA synthesis.4 In addition to the established role in regulation of cell proliferation, some E2Fs, in particular E2F1 and E2F3, have been shown to induce apoptosis5 and cause, at least in part, the RbeC/eC phenotype in mice, which is characterized by excessive apoptosis.6

    The family of E2F transcription factors comprises seven individual members: E2F1 to E2F7. Although E2F1 to E2F5 all have both a DNA binding domain, a dimerization domain, and a transactivating domain (see review7), E2F6 and E2F7 have no transactivating properties. Based on sequence homologies, binding of pocket proteins, and the ability to induce S-phase entry in quiescent cells, E2F1 to 5 can be further distinguished into "activating" E2Fs (E2F1, E2F2, and E2F3) and "repressing" E2Fs although such a classification holds true only to specific experimental setups.7

    Previous studies have demonstrated that activation of E2F1 in cardiomyocytes stimulates DNA synthesis but in parallel increases apoptosis.8 Because individual members of the E2F family serve distinct roles in different cell types and proapoptotic effects are a special hallmark of E2F1,5,9 we decided to exploit discrete properties of other E2F family members in control of cell proliferation to overcome the cell cycle block of cardiomyocytes without induction of apoptosis. Furthermore, we began to analyze the regulatory network downstream of E2F, which controls whether a cardiomyocyte is prone to die or to proliferate.

    Materials and Methods

    Adenoviruses, Cell Culture, and FACS

    Adenoviruses expressing E2F1, E2F2, E2F3, E2F4, or E2F5 (Ad-E2F1/-E2F5) were kindly provided by J.R. Nevins (Howard Hughes Medical Institute, Durham, NC); nuclear -galactosidase (Ad-nlsLacZ) was a gift from T. Eschenhagen (University of Hamburg, Germany). Adenoviruses were used as described previously.10

    Cardiomyocytes from newborn rats and mice were prepared using standard procedures. In all experiments, cells were transferred to serum-free DMEM containing 25 mg/L BSA, 2.5 mg/L transferring, and 25 e/L insulin 24 hours before virus administration and maintained in this medium until fixation. Animals were bred at the animal facility of the Martin-Luther-University Halle-Wittenberg (MLU) or purchased from Harlan Winkelman GmbH (Borchen, Germany). All animal experimentations were endorsed by the local government and performed according to guidelines of the MLU.

    DNA content measurements by FACS analysis were performed essentially as described.11 The cell cycle profile was calculated using MultiCycle software (Phoenix Flow Systems). Proliferation of cardiomyocytes was determined using the BrdU-Hoechst method,11 which allows enumeration of cells at different stages of three consecutive cell cycles including mitotic cell division.12 All FACS analyses were restricted to MF20-positive cells to exclude contaminating fibroblasts.

    Immunhistochemistry and TUNEL Analysis

    To detect DNA-synthesizing nuclei, cells were incubated with BrdU for 24 hours and stained according to the manufacturer’s instructions (BrdU Immunohistochemistry System; Oncogene). Finally, cells were stained for myosin heavy chain (MyHC) using the MF20 antibody as described previously.

    DNA double strand breaks were marked by fluorescein-conjugated dUTP using an in situ Cell Death Detection Kit (Roche). To calculate the percentage of TUNEL-positive cells, both the total area of nuclei (Areanuclei) from Hoechst fluorescence and the area of TUNEL-positive nuclei (AreaTUNEL) were measured using imaging software (Scion Image). Percentage of TUNEL-positive nuclei was calculated as [% TUNEL-pos.]=AreaTUNEL/Areanucleix100.

    Quantitative Real-Time PCR

    Total RNA was isolated using Trizol reagent according to the instructions of the manufacturer. cDNAs were synthesized using oligo-dT-primers as described.13 Real-time PCR was performed as described.13 Primer sequences are listed in the expanded Materials and Methods section in the online data supplement available at http://circres.ahajournals.org. Expression levels are shown as follows: expression level=(copy number geneA per e蘈 cDNA)/(copy number G3PDH per e蘈 cDNA). Relative expression levels in different experiments were compared based on the expression of a gene of interest ("geneX") with and without overexpression of E2F: relative expression level=(GeneXE2F/G3PDHE2F)/(GeneXcontrol/G3PDHcontrol)).

    Protein Isolation, Western Blots, EMSA, and ELISA

    Protein isolation, subcellular fractioning, and Western blot analysis were performed according to standard techniques. Antibodies against E2Fs (sc-22820, sc-22820, sc-22822, sc-866, and sc-999) and pocket proteins (sc-50-G, sc-7986, sc-250, and sc-317-G) were purchased from Santa Cruz Biotech. Interactions between E2Fs and pocket proteins were analyzed using the "Mercury TransFactor Profiling Kit - Oncogenesis 1" according to the instructions supplied by the manufacturer (Clontech). Band-shift assays were done as described14 using the E2F binding site TCCGTTTTCGCGCTTAAATTTGAGAAAGGGCGCGAAACTGGA.5

    Results

    E2F1, E2F2, E2F3, and E2F4 but not E2F5 Stimulate S-Phase Entry of Cardiomyocytes

    To analyze distinct effects of individual members of the E2F-transcription factor family on cell cycle progression of cardiomyocytes, E2F1, E2F2, E2F3, E2F4, and E2F5 were expressed in serum-starved neonatal rat cardiomyocytes using adenoviral vectors (Figure 1E). After 48 hours, DNA content of cardiomyocytes was analyzed by FACS (Figure 1A through 1C). As shown in Figure 1D, E2F1, E2F2, E2F3, and E2F4 but not E2F5 or LacZ control virus significantly increased the number of cardiomyocytes in S- and G2-phase although all E2Fs were expressed at similar degrees in infected cardiomyocytes as indicated by Western blot analysis (Figure 1E). To confirm these results, we prepared primary cardiomyocytes from newborn mice, infected them with E2F1, E2F2, E2F4, or LacZ adenoviruses and counted the number of DNA-synthesizing cardiomyocytes that had incorporated BrdU. In agreement with our findings obtained by FACS analysis using neonatal rat cardiomyocytes E2F1, E2F2, and E2F4 but not LacZ clearly increased the number of DNA-synthesizing cardiomyocytes (Figure 2A and 2B). Additionally, histological examination revealed that the number of binucleated cardiomyocytes was elevated after directed expression of E2F1 and E2F2 (Figure 2C).

    E2F1, E2F3, and E2F5, but not E2F2 or E2F4 Exert Proapoptotic Effects

    Parallel to the analysis of cell cycle progression, we quantified the extent of apoptosis of neonatal rat cardiomyocytes using FACS analysis. With some but not all E2F-adenoviruses, we noted a clear increase of the number of cells with a DNA content lower that G1-phase cells, which is indicative for cells undergoing programmed cell death. Interestingly, expression of E2F2 and E2F4 did not result in induction of apoptosis of neonatal rat cardiomyocytes concomitant with stimulation of S-phase entry, whereas E2F1, E2F3, and E2F5 all lead to a significant increase of apoptotic cells. E2F1, which has a unique ability to induce apoptosis when accumulating in cells devoid of proliferative signals, evoked the strongest apoptotic response relative to other E2F family members (Figure 3A and 3B). Surprisingly, we also noted a modest increase of apoptosis with the LacZ control virus when compared with uninfected cardiomyocytes or cells infected with E2F2 and E2F4. Toxic effects of reporter genes including LacZ, which lead to apoptosis, have been reported before and, in the case of EGFP, have occasionally even resulted in dilatative cardiomyopathy in transgenic animals.15

    Similar results were obtained when we quantified cells undergoing apoptosis using the TUNEL technique. After E2F1 expression the number of primary cardiomyocytes from neonatal mice labeled by TUNEL staining was elevated up to 23%. In marked contrast, treatment of cardiomyocytes with E2F2 or E2F4 did not result in an elevated rate of TUNEL-positive cells and remained close to 5% of all cardiomyocytes in culture. We again detected a slight increase of apoptotic cells after LacZ expression using this alternative technique (Figure 3C).

    Expression of E2F1 and E2F2, but not of E2F4, Induces Mitosis of Cardiomyocytes

    The experiments described earlier clearly showed that both E2F2 and E2F4 induce DNA synthesis in cardiomyocytes without provoking apoptosis. S-Phase entry, however, does not prove that cells complete the cell cycle and divide. It remained possible that cardiomyocytes arrested either in G1-16 or in G2-phase and thereby mimicked a hypertrophic phenotype as described earlier for E2F1.17 We therefore used the flowcytometric BrdU-Hoechst assay, which allows retrospective assessment of G1, S, and G2/M cell cycle phases of synchronous and asynchronous cell populations11,12 (Figure 4). In the absence of BrdU, all cells within G1-phase (either growth arrested or after mitosis) showed the same intensity of fluorescence for PI and Hoechst 33258 (Figure 4B). BrdU, which is incorporated into the DNA, quenches selectively the Hoechst fluorescence but does not interfere with PI. Hence, cardiomyocytes that have passed the cell cycle completely (S-phase and mitosis), will have the same PI fluorescence but a reduced Hoechst fluorescence compared with G1 arrested cells, which did not proliferate (Figure 4A). As seen in Figure 4C through 4E, the number of dividing cardiomyocytes increased significantly after expression of E2F1 and E2F2 but remained unchanged after expression of E2F4. Thus, only E2F1 and E2F2, but not E2F4, stimulated mitotic cell division of cardiomyocytes although all three E2Fs induced S-phase entry.

    S-Phase Entry of Cardiomyocytes After E2F-Expression Does Not Seem to Depend on D-Type Cyclins but on A- and E-Type Cyclins Whereas Induction of Mitosis Coincides With Expression of Cyclins B1 and B2

    We next decided to identify cell cycleeCrelated genes, which might be indispensable for E2F-induced S-phase entry. Because not all E2Fs induced S-phase entry of cardiomyocytes, we were able to distinguish potential E2F target genes that correlated with cell cycle progression of cardiomyocytes from those, which were upregulated by certain E2Fs but apparently dispensable for cell cycle progression.

    mRNA expression levels of a panel of cell cycle-related genes were determined by quantitative real-time RT-PCR after infection with adenoviruses coding for individual E2F family members. As shown in Table 1, D-type cyclins, which are considered to be major players to overcome the G1 restriction point in various cell types, were not consistently upregulated in cardiomyocytes infected with S-phase promoting E2Fs. Clearly, D-type cyclins were not stimulated in cardiomyocytes infected with E2F2, which strongly induced S-phase entry in our experimental system. Along the same line, E2F5, which does not promote DNA-synthesis in cardiomyocytes, led to a robust induction of cyclinD2.

    In contrast to D-type cyclins, the induction of A-type and particularly of E-type cyclins clearly correlated with DNA-synthesis in cardiomyocytes. As shown in Table 1, only those E2Fs, which significantly stimulated DNA synthesis, led to a parallel increase of these S-phase related cyclins. Whereas E2F1, E2F2, and E2F3 elevated cyclinA mRNA level, induction of cyclin E was primarily directed by E2F2 and E2F4, which also lacked apoptosis-inducing activities. Progression through M-phase in dividing cells is known to depend on activation of B-type cyclins.18 The finding that both cyclin B1 and B2 are induced only after expression of E2F1 and -2 is in line with our data that only E2F1 and E2F2 but not E2F4 lead to a full completion of the cell cycle and a division of cardiomyocytes.

    Because cell cycle progression does not only require the presence of cell cycle inducers but also the absence of cell cycle inhibitors such as cyclin-dependent kinase inhibitors (CKIs), which might be activated in cardiomyocytes in response to untimed proliferation signals, we measured changes in transcriptional activation of p15INK, p16INK, and p19INK after directed E2F expression in primary cardiomyocytes (Table 1). Despite an upregulation of p15INK after E2F2, of p16INK after E2F1 and E2F5, and of p19INK after E2F1 expression, no striking correlation between E2F expression, INK-type CKI activation, and S-phase entry or induction of apoptosis was noted.

    Differential Induction of Apoptosis Promoting Genes by E2F Transcription Factors in Cardiomyocytes

    The striking differences between individual E2Fs in induction of apoptosis suggested that the activation of apoptosis promoting genes by distinct E2Fs differ significantly. We therefore investigated whether p21CIP/WAF and Apaf-1, which are known to be direct targets of E2F1 in some cell types are also activated in cardiomyocytes and whether other E2F family members evoked similar responses. As shown in Table 2, E2F1, but also its proapoptotic relatives E2F3 and E2F5, strongly stimulated p21CIP/WAF expression. The induction of p21CIP/WAF expression was even 3-times higher using E2F5 compared with E2F1 (Table 2). In addition, we noted a robust induction of bax and caspase6 after expression of E2F1 and E2F5. Interestingly, expression of the proapoptotic genes p21CIP/WAF and caspase6 were decreased after treatment with E2F2 and E2F4 reflecting selective induction of cell cycle progression without induction of apoptosis by these E2F family members.

    Previous studies demonstrated that E2F1 has a special ability to induce apoptosis and that stimulation of expression of p19ARF is an early step in this process in several cell types. Therefore, we analyzed activation of p19ARF in cardiomyocytes after overexpression of individual E2Fs to disclose whether induction of p19ARF is a common feature of E2F-induced cell death or occurs only in E2F1 expressing cells. Interestingly, p19ARF mRNA was significantly upregulated only after E2F1 expression (Table 2) highlighting the distinctive apoptotic potential of E2F1, which needs to be suppressed during normal proliferation. We also observed a strong stimulation of mdm2 by E2F1. Mdm2 is generally believed to inhibit apoptosis and hence might counteract the capacity of E2F1 to trigger apoptosis. Clearly, however, this mechanism did not suffice to balance the apoptotic potential of E2F1 in cardiomyocytes. Because our cultures did contain a minor population of noncardiomyogenic cells, it has to be kept in mind that such cells might also contribute to a small degree to the transcriptional changes measured by RT-PCR.

    Differential Association of Adenoviral Delivered E2F2s With Pocket Proteins in Cardiomyocytes

    E2F-dependent transcription is negatively regulated by three different pocket proteins (Rb, p107, and p130), which also serve as adaptors to recruit various histone modifying enzymes to E2F bindings sites. E2Fs bound to Rb/p107/p130 are not only incapable to stimulate cell cycle progression but also actively repress transcription of several genes containing E2F binding site.19 We therefore wanted to analyze to what extent adenoviral-delivered E2Fs interacted with different pocket proteins in cardiomyocytes using band-shift and ELISA based complex formation tests. As shown in Figure 5A, Rb complexed primarily with E2F1, E2F2, and E2F3, whereas p107 only showed a significant interaction with E2F2 and E2F4. Interestingly, the majority of E2F proteins were not bound to pocket proteins as indicated by the much higher concentration of extract needed to detect Rb and p107 compared with E2F1 and E2F2 in the ELISA assay (Figure 5B). Based on the different concentrations needed to yield similar signal strength, we calculated that the concentration of unbound E2F1 and E2F2 exceeded the bound fraction by 10-fold probably owing to the massive delivery of E2F by the adenoviral vectors. Similarly, we readily detected individual E2F-complexes in conventional band-shift experiments using a radioactively labeled E2F binding site with extracts from E2F-transfected cardiomyocytes (Figure 6) that were apparently devoid of bound pocket proteins. Correspondingly, we were unable to super-shift the E2F-complexes using antibodies against Rb, p107, and p130, respectively, most probably due to the limited sensitivity of this system (data not shown).

    Discussion

    Replacement of dead or dying cardiomyocytes would certainly herald a new era of chronic heart failure treatment. Although autologous cell transplantations using, for example, muscle progenitor cells or adult stem cells20 show some promise, it is currently unclear whether the benefit, mode of action, and potential side effects of stem cell transplantations justify a broad application. An alternative approach is the stimulation of proliferation of remaining cardiomyocytes. Such a strategy would be particularly useful in situations of localized myocardial damage as, eg, after myocardial infarction.

    The E2F-transcription factors are key regulators of S-phase entry in a wide variety of cell types. In the last years, several attempts were made to increase E2F-activity or to express different oncogenes to bypass the cardiomyocyte’s restriction point and stimulate proliferation. It has been shown that both overexpression of E2F18 and E1A-induced elimination of pRb, which in turn leads to increased E2F-activity,21 can reactivate DNA synthesis in cardiomyocytes. Unfortunately, however, E2F1-induced cell cycle reentry was always accompanied by a severe increase of apoptosis of cardiomyocytes. Surprisingly, no attempts have been made to use other members of the E2F transcription factor family, which also stimulate DNA synthesis in quiescent cells but show no (or less) effects on apoptosis, for induction of cell cycle entry of cardiomyocytes. Beside E2F1, especially E2F2 and E2F3 have been demonstrated to induce S-phase strongly5 and are therefore often classified as "activating" E2Fs. Unlike E2F1, which seems to have a special role to induce apoptosis, E2F2 and E2F3 have not been linked to increased apoptosis in the majority of experimental systems, although there are reports describing a role of E2F3 in induction of programmed cell death.5 Additionally, in a transgenic mouse model in which E2F4 was expressed by the keratin5 promoter in the epidermis Wang et al22 found an increase of proliferation similar to E2F1 but no significant increase of apoptosis. Interestingly, these in vivo results differ from findings in cell culture, which originally reported only very poor transactivating and S-phase promoting activities for E2F4.23

    In this study, we have identified E2F2 and E2F4 as factors that can induce S-phase entry of cardiomyocytes without provoking apoptosis. In particular, E2F2 is a promising candidate to stimulate proliferation of cardiomyocytes because E2F2 was most potent to simulate cell cycle progression including mitosis of cardiomyocytes but did not cause any signs of apoptosis. These findings overcome restrictions of a potential therapeutic use of E2Fs that are associated with a concomitant induction of apoptosis as seen for E2F1 and E2F3. Initially, our finding that directed expression of E2F5-induced apotosis in cardiomyocytes came as a surprise because several reports demonstrated that E2F5 expression correlates with the differentiated phenotype of growth arrested cells during development.24,25 However, none of the studies cited analyzed effects of E2F5-overexpression, which obviously is an artificial situation that might lead to effects different from the physiological role of E2F5 during development. Another observation, which also does not fit the role of E2F5 as a solitary repressor, was recently made by Ruutu and colleagues, who reported a strong correlation between elevated E2F5 levels and selective growth advantage in a HPV-33eCpositive cell line.26

    The differential induction of apoptosis by distinct E2Fs in cardiomyocytes argues against a model that proposes that E2F1, E2F2, and E2F3 indiscriminately provoke S-phase entry and apoptosis depending on expression levels.7 In contrast, directed expression of E2F2 in cardiomyocytes resulted in a reduced expression of various apoptosis-related genes including p21CIP/WAF and caspase 6 restraining the activity of proapoptotic pathways. According to our knowledge, this is the first example of suppression of proapoptotic genes by a member of the E2F-family.

    In line with our findings, several studies demonstrated that E2F transcription factors are involved in cardiac hypertrophy.17 Hyperplasia and hypertrophy are relatively common reactions of myocardial cells to adverse conditions depending on the developmental stage of the cells.27 The overall effect of E2Fs might strongly depend on the current status of the cell, the concentration level of E2Fs, and their interaction partners and of pro- and antiapoptotic effectors, which thereby determine whether a cardiomyocyte undergoes hypertrophy, cell division, or programmed cell death.

    The divergent effects of individual E2Fs on cell cycle and apoptosis in cardiomyocytes were reflected by a differential stimulation of several cell cycle control genes because we observed induction of different cyclins and CKIs depending on the respective E2F. Interestingly, we found that mRNA levels of D-type cyclins in our experiments did not parallel S-phase entry. Although surprising, the finding that expression of E2F2 leads to S-phase entry of cardiomyocytes without induction of D-type cyclins confirms previous reports demonstrating that E2F1 can overcome growth arrest in the absence of D-type cyclin/cdk-activity28 and that alternative pathways are able to direct proliferation of cells in absence of cyclin-D activity.29

    On the other hand, transgenic overexpression of D-type cyclins was reported to induce DNA synthesis in cardiomyocytes,30 which seems to be in conflict with our data. However, it is not unlikely that a long-term exposure to increased cyclinD levels in transgenic animals results in stronger effects, compared with a short-term exposure, and to activation of further growth promoting pathways.

    In contrast to D-type cyclins, A- or E-type cyclins were always found elevated after infection with S-phase inducing E2Fs. We did not find evidence that induction of cyclin A participates in programmed cell death of cardiomyocytes despite a reported role of cyclin A in hypoxia-induced apoptosis of cardiomyocytes.31

    A favored model is that E2F triggers apoptosis via transcriptional activation of p19ARF32 preferably by E2F1, although E2F1 can provoke apoptosis in the absence of p19ARF, indicating the presence of additional pathways for E2F-induced apoptosis.33,34 In addition, it has been described that E2F2 and E2F3 also activate the ARF gene in fibroblasts without obligatory induction of programmed cell death.35,36 We only found a relevant induction of p19ARF in cardiomyocytes after expression of E2F1 but not of E2F2 and E2F3. This finding suggests (1) that E2F1 specifically activates the ARF gene as already proposed by DeGregori et al5 and (2) that alternative pathways mediate E2F3 and E2F5-induced apoptosis. Beside other effects that are caused by E2Fs such as induction of p73 and inhibition of TNFR-associated survival response,7 the induction of p21CIP/WAF, which has been shown to induce apoptosis in various cells,37 is a likely explanation.

    The specificity by which individual E2Fs activate putative downstream target genes remains an unsolved issue. In the course of our experiments, we found that proapoptotic E2Fs induced mdm2, p21CIP/WAF, caspase 6, and Apaf-1, whereas E2F2 and E2F4 had the opposite effect and repressed transcription of these genes. Likewise, it has been shown that E2F1 can bind directly and activate p21CIP/WAF and apaf-1 promoters38 while we also observed an induction by E2F3 and E2F5 but not by E2F2 or E2F4, respectively. Further experiments will reveal the cause for specific effects of individual E2Fs on target promoters.

    In summary, we found that the transcription factor E2F2 is an excellent candidate to stimulate proliferation of cardiomyocytes. Induction of cell cycle progression by E2F2 and E2F4 is not accompanied by increased death rates of cardiomyocytes. In contrast, both E2F2 and E2F4 seemed to confer increased resistance to apoptosis, further supporting a potential application in repair of damaged myocardium and treatment of heart failure.

    Acknowledgments

    This work was supported by the Deutsche Forschungsgemeinschaft, SFB 598, and the Wilhelm-Roux-Program for Research of the Martin-Luther-University.

    References

    Pasumarthi KB, Field LJ. Cardiomyocyte cell cycle regulation. Circ Res. 2002; 90: 1044eC1054.

    Ueno H, Perryman MB, Roberts R, Schneider MD. Differentiation of cardiac myocytes after mitogen withdrawal exhibits three sequential states of the ventricular growth response. J Cell Biol. 1988; 107: 1911eC1918.

    Beltrami AP, Urbanek K, Kajstura J, Yan SM, Finato N, Bussani R, Nadal-Ginard B, Silvestri F, Leri A, Beltrami CA, Anversa P. Evidence that human cardiac myocytes divide after myocardial infarction. N Engl J Med. 2001; 344: 1750eC1757.

    DeGregori J, Kowalik T, Nevins JR. Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes. Mol Cell Biol. 1995; 15: 4215eC4224.

    DeGregori J, Leone G, Miron A, Jakoi L, Nevins JR. Distinct roles for E2F proteins in cell growth control and apoptosis. Proc Natl Acad Sci U S A. 1997; 94: 7245eC7250.

    Yamasaki L, Bronson R, Williams BO, Dyson NJ, Harlow E, Jacks T. Loss of E2F-1 reduces tumorigenesis and extends the lifespan of Rb1(+/eC) mice. Nat Genet. 1998; 18: 360eC364.

    Trimarchi JM, Lees JA. Sibling rivalry in the E2F family. Nat Rev Mol Cell Biol. 2002; 3: 11eC20.

    Agah R, Kirshenbaum LA, Abdellatif M, Truong LD, Chakraborty S, Michael LH, Schneider MD. Adenoviral delivery of E2F-1 directs cell cycle reentry and p53-independent apoptosis in postmitotic adult myocardium in vivo. J Clin Invest. 1997; 100: 2722eC2728.

    Hallstrom TC, Nevins JR. Specificity in the activation and control of transcription factor E2F-dependent apoptosis. Proc Natl Acad Sci U S A. 2003; 100: 10848eC10853.

    Ebelt H, Braun T. Optimized, highly efficient transfer of foreign genes into newborn mouse hearts in vivo. Biochem Biophys Res Commun. 2003; 310: 1111eC1116.

    Rabinovitch PS. Regulation of human fibroblast growth rate by both noncycling cell fraction transition probability is shown by growth in 5-bromodeoxyuridine followed by Hoechst 33258 flow cytometry. Proc Natl Acad Sci U S A. 1983; 80: 2951eC2955.

    Ormerod MG. Cell-cycle analysis of asynchronous populations. Methods Mol Biol. 2004; 263: 345eC354.

    Schafer K, Neuhaus P, Kruse J, Braun T. The homeobox gene Lbx1 specifies a subpopulation of cardiac neural crest necessary for normal heart development. Circ Res. 2003; 92: 73eC80.

    Gunther S, Mielcarek M, Kruger M, Braun T. VITO-1 is an essential cofactor of TEF1-dependent muscle-specific gene regulation. Nucleic Acids Res. 2004; 32: 791eC802.

    Huang WY, Aramburu J, Douglas PS, Izumo S. Transgenic expression of green fluorescence protein can cause dilated cardiomyopathy. Nat Med. 2000; 6: 482eC483.

    Kirshenbaum LA, Abdellatif M, Chakraborty S, Schneider MD. Human E2F-1 reactivates cell cycle progression in ventricular myocytes and represses cardiac gene transcription. Dev Biol. 1996; 179: 402eC411.

    von Harsdorf R, Hauck L, Mehrhof F, Wegenka U, Cardoso MC, Dietz R. E2F-1 overexpression in cardiomyocytes induces downregulation of p21CIP1 and p27KIP1 and release of active cyclin-dependent kinases in the presence of insulin-like growth factor I. Circ Res. 1999; 85: 128eC136.

    Kang MJ, Kim JS, Chae SW, Koh KN, Koh GY. Cyclins and cyclin dependent kinases during cardiac development. Mol Cells. 1997; 7: 360eC366.

    DeGregori J. The Rb network. J Cell Sci. 2004; 117: 3411eC3413.

    Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, Pickel J, McKay R, Nadal-Ginard B, Bodine DM, Leri A, Anversa P. Bone marrow cells regenerate infarcted myocardium. Nature. 2001; 410: 701eC705.

    Akli S, Zhan S, Abdellatif M, Schneider MD. E1A can provoke G1 exit that is refractory to p21 and independent of activating cdk2. Circ Res. 1999; 85: 319eC328.

    Wang D, Russell JL, Johnson DG. E2F4 and E2F1 have similar proliferative properties but different apoptotic and oncogenic properties in vivo. Mol Cell Biol. 2000; 20: 3417eC3424.

    Verona R, Moberg K, Estes S, Starz M, Vernon JP, Lees JA. E2F activity is regulated by cell cycle-dependent changes in subcellular localization. Mol Cell Biol. 1997; 17: 7268eC7282.

    Gaubatz S, Lindeman GJ, Ishida S, Jakoi L, Nevins JR, Livingston DM, Rempel RE. E2F4 and E2F5 play an essential role in pocket protein-mediated G1 control. Mol Cell. 2000; 6: 729eC735.

    Ohtani N, Brennan P, Gaubatz S, Sanij E, Hertzog P, Wolvetang E, Ghysdael J, Rowe M, Hara E. Epstein-Barr virus LMP1 blocks p16INK4a-RB pathway by promoting nuclear export of E2F4/5. J Cell Biol. 2003; 162: 173eC183.

    Ruutu M, Peitsaro P, Johansson B, Syrjanen S. Transcriptional profiling of a human papillomavirus 33-positive squamous epithelial cell line which acquired a selective growth advantage after viral integration. Int J Cancer. 2002; 100: 318eC326.

    Oh H, Taffet GE, Youker KA, Entman ML, Overbeek PA, Michael LH, Schneider MD. Telomerase reverse transcriptase promotes cardiac muscle cell proliferation, hypertrophy, and survival. Proc Natl Acad Sci U S A. 2001; 98: 10308eC10313.

    DeGregori J, Leone G, Ohtani K, Miron A, Nevins JR. E2F-1 accumulation bypasses a G1 arrest resulting from the inhibition of G1 cyclin-dependent kinase activity. Genes Dev. 1995; 9: 2873eC2887.

    Kozar K, Ciemerych MA, Rebel VI, Shigematsu H, Zagozdzon A, Sicinska E, Geng Y, Yu Q, Bhattacharya S, Bronson RT, Akashi K, Sicinski P. Mouse development and cell proliferation in the absence of D-cyclins. Cell. 2004; 118: 477eC491.

    Soonpaa MH, Koh GY, Pajak L, Jing S, Wang H, Franklin MT, Kim KK, Field LJ. Cyclin D1 overexpression promotes cardiomyocyte DNA synthesis and multinucleation in transgenic mice. J Clin Invest. 1997; 99: 2644eC2654.

    Adachi S, Ito H, Tamamori-Adachi M, Ono Y, Nozato T, Abe S, Ikeda M, Marumo F, Hiroe M. Cyclin A/cdk2 activation is involved in hypoxia-induced apoptosis in cardiomyocytes. Circ Res. 2001; 88: 408eC414.

    Zhu JW, DeRyckere D, Li FX, Wan YY, DeGregori J. A role for E2F1 in the induction of ARF, p53, and apoptosis during thymic negative selection. Cell Growth Differ. 1999; 10: 829eC838.

    Tolbert D, Lu X, Yin C, Tantama M, Van Dyke T. p19(ARF) is dispensable for oncogenic stress-induced p53-mediated apoptosis and tumor suppression in vivo. Mol Cell Biol. 2002; 22: 370eC377.

    Lindstrom MS, Wiman KG. Myc and E2F1 induce p53 through p14ARF-independent mechanisms in human fibroblasts. Oncogene. 2003; 22: 4993eC5005.

    Parisi T, Pollice A, Di Cristofano A, Calabro V, La Mantia G. Transcriptional regulation of the human tumor suppressor p14(ARF) by E2F1, E2F2, E2F3, and Sp1-like factors. Biochem Biophys Res Commun. 2002; 291: 1138eC1145.

    Rogoff HA, Pickering MT, Debatis ME, Jones S, Kowalik TF. E2F1 induces phosphorylation of p53 that is coincident with p53 accumulation and apoptosis. Mol Cell Biol. 2002; 22: 5308eC5318.

    Wu Q, Kirschmeier P, Hockenberry T, Yang TY, Brassard DL, Wang L, McClanahan T, Black S, Rizzi G, Musco ML, Mirza A, Liu S. Transcriptional regulation during p21WAF1/CIP1-induced apoptosis in human ovarian cancer cells. J Biol Chem. 2002; 277: 36329eC36337.

    Radhakrishnan SK, Feliciano CS, Najmabadi F, Haegebarth A, Kandel ES, Tyner AL, Gartel AL. Constitutive expression of E2F-1 leads to p21-dependent cell cycle arrest in S phase of the cell cycle. Oncogene. 2004; 23: 4173eC4176.

作者: Henning Ebelt, Nadine Hufnagel, Petra Neuhaus, Her 2007-5-18
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