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1 Department of Pharmacology, University of Virginia, Charlottesville, VA 22908, USA
2 Digestive Health Center of Excellence, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA
3 Department of Genetics, Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan
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Abstract |
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Introduction |
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Only recently, MNK1 function has been shown in translation of cytokines (Nikolcheva et al. 2002; Buxade et al. 2005; Andersson & Sundler 2006). Down-regulation of MNK1 function by siRNA or by a cell permeable kinase inhibitor (CGP57380) inhibits production of the cytokine TNF
in T-cells (Buxade et al. 2005). The molecular mechanism for regulation of cytokine production by MNK1 is not fully understood, but may require MNK1 phosphorylation of hnRNP A1 (Buxade et al. 2005). In addition to hnRNP A1, MNK1 phosphorylates eIF4G (Raught & Gingras 1999).
Mice lacking both MNK genes are apparently normal, suggesting that MNKs function in adaptive responses to stress. The strongest genetic evidence for a MNK phenotype is from a study in Drosophila melanogaster where the Drosophila homologue of MNK (LK6 gene) was deleted (Arquier et al. 2005). Loss of LK6 function in Drosophila decreases viability, slows development and decreases adult size. These outcomes are due to eIF4E phosphorylation because over-expression of Drosophila LK6 rescues lethality of eIF4E hypomorphs-dependent on intact Ser251 (homologous to human Ser209) (Arquier et al. 2005). This is supported by homozygous knockin mutations of Ser251 in Drosophila-eIF4E (Lachance et al. 2002). Mutant Drosophila homozygous for S251A eIF4E have reduced viability (35% lethality), delayed development, and adults of both males and females are smaller in overall size with aberrant wings. The phenotypes are largely rescued to normalcy by homozygous knockin of S251D. The size and wing phenotypes are primarily due to smaller cells, and not due to reduced numbers of cells, similar to phenotypes of loss of Drosophila S6 kinase (Montagne et al. 1999).
The observed phenotypes for Drosophila-MNK and Drosophila-eIF4E made it surprising that no overt phenotype resulted from deletion of MNKs in mice (Ueda et al. 2004). Here, we report a new phenotype for MNKs using mouse embryo fibroblasts (MEFs) from wild-type and MNK1/2 gene knockout mice. Loss of MNK function sensitizes fibroblasts to undergo apoptosis in response to withdrawal of serum.
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Results |
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We studied immortalized MEFs from wild-type mice and mice deficient in one or both MNK genes. We discovered a difference in appearance and morphology of fibroblasts immortalized from mnk1 (–/–)/mnk2 (–/–) mice in comparison to fibroblasts immortalized from mnk1 (–/–), mnk2 (–/–) or wild-type mice after withdrawal of serum. We will refer to these cell lines as mnk1
/mnk2, mnk1, mnk2 and wild-type. Using phase light microscopy, we observed that compared to wild-type MEF's the mnk1/mnk2 cells appeared stressed and detached from the plate more rapidly and completely in response to serum withdrawal (Fig. 1a). The differences in response to serum withdrawal between the individual MNK knockout cell lines were more variable between each other (mnk1 vs. mnk2); however, both lines were consistently more sensitive than the wild-type cells and less sensitive than the double knockout cells (Fig. 1a, and data not shown). Consistently, a larger percentage of mnk1/mnk2 cells were rounded, suggesting more rapid induction of apoptosis due to loss of MNKs. This was supported by an increase in nuclear condensation in the mnk1/mnk2 cells observed with Hoechst 33342 staining (Fig. 1b). We pursued these findings with the goal of establishing whether loss of MNKs was causally related to the phenotype, and not to elucidate the apoptotic cascades operant after serum withdrawal, which have been extensively studied (Schamberger et al. 2004, 2005; Austin & Cook 2005). The apoptotic pathway of immortalized fibroblasts upon withdrawal of serum requires caspase-3 but not caspase-9 (Schamberger et al. 2004, 2005).
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Apoptosis was assessed by immunoblotting total cell lysates with an antibody that recognizes both the full length and the cleaved (17 kDa) active form of caspase-3 (Fig. 2). Caspase-3 activation in mnk1/mnk2, mnk1 and mnk2 cells was evident after 4 h of serum withdrawal, whereas caspase-3 cleavage was delayed in wild-type cells (Fig. 2). In repetitions and in independent experiments (not shown), this gross difference in sensitivity of the mnk1/mnk2 cells compared to wild-type was highly reproducible. The kinetics for detection of active caspase-3 in individual experiments was variable, with a delay of ~6 h for wild-type cells and ~0–1.5 h for mnk1/mnk2 cells. Hydrogen peroxide or sodium arsenite addition to mnk1/mnk2 cells in serum was a less potent stress than withdrawal of serum (data not shown), and the wild-type and mnk1/mnk2 cells did not respond differently to these stresses. This suggests a deficiency of mnk1/mnk2 cells in response to nutrient stress. Conditions for the comparison were as similar as possible; for these experiments this included plating equal amounts of the three cell lines and half as much of the mnk2 cells. This is because the mnk2 cells were larger in size than the other three lines, visualized after trypsinization using a hemocytometer (Fig. 3). We did not investigate if this was an effect of mnk2 loss and are not suggesting that it is related. However, in an effort to make the conditions equivalent, the experiments presented throughout this paper were performed using mnk2 cells plated at half the density of the other cell lines. Using fewer mnk2 cells, the signals for parent caspase-3 (uncleaved) and actin were similar for the four cell lines prior to serum withdrawal.
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Addition of fresh serum to Rat1 fibroblasts potently activates ERK2/p42MAPK (Catling et al. 1995) whereas withdrawal of serum from Rat1 fibroblasts has been reported to decrease activation of ERK2/p42MAPK (Catling et al. 1995) and activate p38 MAP kinases (Kummer et al. 1997). Since loss of MNK function sensitizes MEFs to withdrawal of serum, we next asked whether serum withdrawal alters MNK activity. Total cell lysates from wild-type cells removed from serum or left in serum were immunoblotted for anti-pS209 eIF4E, a sensitive measure for MNK activity, since it is the only kinase phosphorylating the protein at this site (Ueda et al. 2004) (Fig. 2). The results show that eIF4E is phosphorylated in the wild-type cells and that the phosphorylation is not altered by serum starvation when both MNKs are present; interestingly, mnk1 cells had increased eIF4E phosphorylation consistent with MNK2 being the more constitutively active of the two kinases and eIF4E was phosphorylated in response to serum starvation (Fig. 2). This is not fully understood since p38 MAP kinase is not essential for MNK2 activation. MNK2 is reported to have high constitutive activity (Scheper et al. 2001). These results dissociate lack of eIF4E phosphorylation with sensitization of apoptosis since mnk1 cells have increased eIF4E phosphorylation and increased caspase-3 activation in response to serum withdrawal.
The MNK inhibitor CGP57380 sensitizes wild-type cells to serum withdrawal
To determine if sensitization to serum withdrawal was due to genomic deletions of MNKs and not due to secondary effects, we used CGP57380 to inhibit the MNKs in wild-type cells (Fig. 4). The specificity of CGP57380 for MNKs is established [(Buxade et al. 2005), also see Supplementary Data therein]. Addition of CGP57380 (50 µM, final), but not the vehicle, sensitized the wild-type cells to serum withdrawal (Fig. 4). These data strongly support our conclusion that loss of MNK function is causally related to this phenotype.
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/mnk2 cells from sensitivity to serum withdrawal
MNK1, MNK2a, the kinase defective variants and the "empty" vector were introduced back into mnk1/mnk2 cells and cell lines stably expressing these proteins were created by retroviral transduction (see Experimental procedures). The protein levels of the transduced MNKs were comparable (Supplementary Fig. S1, upper panel), but were much higher than the endogenous MNKs (data not shown). The over-expressed MNK1 and MNK2a caused inducible and constitutive phosphorylation of eIF4E, respectively, in similar fashions as the endogenous MNKs, whereas neither the kinase-defective variants phosphorylated eIF4E at all (Supplementary Fig. S1, lower panel). Wild-type MNK1 was the only protein that significantly rescued sensitivity to caspase-3 activation (Fig. 5), suggesting selectivity for MNK1 in this process and a need for kinase activity. The relatively lower expression of MNK2a vs. MNK1 could have an impact and so a role for MNK2a is not ruled out by these results, but the level of eIF4E phosphorylation is similar between the two transduced cell lines (Supplementary Fig. S1) furthering support for dissociation between the loss of eIF4E phosphorylation and the induction of caspase-3 activity by serum starvation.
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/mnk2 cells
To better characterize the sensitization to apoptosis observed in our MNK knockout MEFs, we utilized annexin V staining. Binding of annexin V to phosphatidylserine is indicative of apoptosis in almost all cell lines and it is considered to be an event associated with early commitment to apoptosis (van Engeland et al. 1998). Interestingly, we found that even without the stimulus of withdrawing serum, the mnk1/mnk2 cells already had significantly more staining with annexin V—12% more than the wild-type cells, quantified by more annexin V positive cells in the lower right quadrant (Fig. 6). The slightly displaced appearance of the mnk2 cells on the FACS profile is likely due to the overall larger size of these cells compared to the other three lines. Finding that the mnk1/mnk2 cells had significant staining with annexin V without any other "treatment" suggests that these cells (or a subset of them) may be more likely to initiate apoptosis in response to routine manipulations like washing and trypsinization that are used to test for annexin V binding, but since these cells are routinely passed using these methods they must have the ability to recuperate in the presence of normal growth medium. This is in keeping with our findings that loss of MNK1/2 sensitizes MEFs to serum-withdrawal induced apoptosis.
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Discussion |
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There were also differences in the ability of MNK1 vs. MNK2 to rescue the phenotype. Expression of wild-type MNK1, but not MNK2, was successful in rescuing a relatively normal response to serum withdrawal in mnk1/mnk2, assessed by caspase-3 activation. This difference may have to do with expression levels of the two enzymes, but it is unlikely that phosphorylation of eIF4E is responsible since the expression of either MNK in these cells resulted in robust eIF4E phosphorylation.
There is a reported connection between caspase-3 and MNK1 (Orton et al. 2004). MNK1 is a specific target for the active form of PAK2 generated by caspase-3 cleavage, but not for PAK2 activated by GTP-liganded CDC42 (Orton et al. 2004). Caspase 3-activated PAK2 phosphorylates mouse MNK1 in its N-terminus at one non-conserved, consensus site (Thr23) and at one conserved, non-consensus site (Ser27). The conserved site in human MNK1 is RRGRATDS39LPGK. Mouse MNK1 is phosphorylated at Ser27 in HEK293 cells, and under conditions promoting apoptosis (treatment with H2O2), but the physiologic kinases require more clarification. The relevance of this work here is that phosphorylation by PAK2 significantly inhibits binding of mouse MNK1 to eIF4G as well as MNK1-dependent phosphorylation of eIF4G, but not eIF4E. Thus, if confirmed as caused by the conserved site, apoptotic pathways may inhibit some MNK1 function for eIF4F-dependent translation in mammalian cells. This generally agrees with our finding that loss of MNK function sensitizes cells to undergo apoptosis in response to serum withdrawal.
Our recent work establishes that inhibition of MNKs with CGP57380 (3.2–50 µM) inhibits growth of AU565 cells in soft agar (Chrestensen et al. 2007). AU565 cells are a line of breast carcinoma that over-expresses the HER2 receptor (Yoo & Hamburger 1998). Metastatic cancer cells must respond to nutrient stress and growth factor deprivation to survive. Adaptation includes post-transcriptional steps, including mRNA routing to and from stress granules (Guil et al. 2006) and usage of internal ribosomal entry sites (IRES) (Holcik 2004). MNKs are required for translation of some cytokines (Buxade et al. 2005) as well as being implicated in IRES function (Ross et al. 2006). Loss of this adaptation supplied by MNKs may sensitize cancer cells to undergo apoptosis.
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Experimental procedures |
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Antibodies: Caspase-3 (#9662), phosphoS209 eIF4E (#9741)—Cell Signaling (Danvers, MA); Actin (#69 100)—MP Biochemicals (Solon, OH); MNK1 C20 (#6965), MNK2 S20 (#6964), Cox-2 (#1747)—Santa Cruz Biotechnology (Santa Cruz, CA). CGP57380 was a generous gift of Hermann Gram (Arthritis and Bone Metabolism, Novartis Pharma AG, CH-4002 Basel, Switzerland). CGP57380 was stored as stock solution in DMSO, and always compared to DMSO at equivalent dilution as vehicle control.
Mouse embryo fibroblasts (MEFs)
The immortalized MEFs from mnk1 (–/–), mnk2 (–/–) and mnk1 (–/–)/mnk2 (–/–) mice were generated as described previously (Ueda et al. 2004). After primary MEFs stopped growing (by senescence after several passages), the medium continued to be changed every 5–7 days and cell populations that started growing again were expanded. At the University of Virginia, vials of expanded cell stocks have been stored in liquid nitrogen in 90% FBS, 10% DMSO. MEFs for the experiments described were maintained in DMEM media supplemented with 10% FBS in flasks at 37 °C with 5% CO2. Working stocks were replaced with newly thawed cells every 6–8 weeks. The double knockout cells stably expressing wild-type (WT) MNK1, kinase-defective (KD) MNK1, WT-MNK2a, KD-MNK2a, or vector control were established by infection of mnk1 (–/–)/mnk2 (–/–) cells with recombinant retroviruses that express a bicistronic mRNA consisting of one of the flag-epitope-tagged Mnk cDNAs and IRES-EGFP. After infection, transfectants expressing EGFP were enriched by using FACSAria cell sorter, and were passaged similarly to the mnk knockout lines (above).
Serum-withdrawal induced apoptosis
Modifications to assays in different experiments are described in the figure legends. Cells were usually studied in 6-well plates (35 mm2/well). After trypsinization and centrifugation, the cells were re-suspended in complete medium and seeded at ~2 x 105 cells per well, so that they would be ~85% confluent by the next day; later experiments used ~1 x 105 for mnk2 cells because they were larger (see Results). Serum starvation was done in DMEM containing high glucose after washing the cells 2 times with DMEM. Hoechst 33342 staining was done on live cells at 2 µg/mL for 30 min before visualization.
Cell harvesting and Western blot analysis
During the treatment cells were rounding and lifting from the plate (a finding consistent with an apoptotic response). To ensure we obtained the entire population of cells, media was first collected and centrifuged. Then the adherent cells were washed gently with cold PBS, lysed in 110 µL of lysis buffer (50 mM Tris, pH 7.4 at 25 °C, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1.5 mM MgCl2, 1% Triton-X100, 0.5% IpegalTM (Sigma-Aldrich) CA-360 (detergent replacing Nonidet P-40), 10 µg/mL each leupeptin and aprotinin, 1 µM PefaBlockTM (Boehringer), 100 µM Na3VO4, 1 µM microcystin LR) and then added to the pellet from the supernatant. These lysates were then incubated in ice for a minimum of 30 min, and then were either flash frozen for later analysis or made into samples for Western blot analysis. For analysis of actin, ~8% of the lysate was loaded; for caspase-3 and other antibodies the portion was ~30%.
Annexin V staining and FACS analysis
Cells were plated at ~4 x 105 cells per 60 cm dish (with the exception of mnk2 which were plated at ~2 x 105). After 24 h, the cells were harvested as follows: (i) The media was first collected and saved to isolate any "floaters" which may be either detached apoptotic cells or dividing cells; (ii) Remaining attached cells were washed with PBS, trypsinized and then gently suspended with an equal amount of fresh growth media; (iii) Collected cells (mixed floating and attached) for each experimental treatment (see legend for Fig. 6) were combined and centrifuged at low speed (1500 rpm, 5 min). The supernatant was aspirated, and the cell pellet was washed once in 1x annexin V binding buffer (Annexin V-FITC apoptosis detection kit 1, BD Pharmingen), then re-centrifuged and finally resuspended in 1X annexin V binding buffer containing annexin V-FITC and propidium iodide, as directed by the kit. An additional plate of wild-type cells was always used as a control for instrument calibration in each repetition of the experiments. The control cells were fixed by adding 35% ethanol and a portion was left unstained, stained with propidium iodide or stained with annexin V-FITC so that fluorescence overlap could be compensated prior to running experimental samples. Data were collected on a FACSCalibur flow cytometer (Becton Dickinson) and analyzed using CELL QUEST (BD) software.
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Acknowledgements |
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Footnotes |
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Communicated by: Eisuke Nishida * Correspondence: E-mail: thomas_sturgill{at}virginia.edu
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References |
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Received: 17 April 2007
Accepted: 1 July 2007
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