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(The FASEB Journal. 2006;20:2133-2135.)
© 2006 FASEB

Regulation of c-jun mRNA expression in adult cardiocytes by MAP kinase interacting kinase-1 (MNK1)

Laura S. Spruill and Paul J. McDermott1

Department of Medicine, The Gazes Cardiac Research Institute, Medical University of South Carolina, and The Ralph H. Johnson Department of Veterans Affairs Medical Center, Charleston, South Carolina, USA

1Correspondence: The Gazes Cardiac Research Institute, Strom Thurmond Biomedical Research Bldg., Rm. 303, 114 Doughty St., Charleston, SC 29403, USA. E-mail: mcdermp{at}musc.edu

ABSTRACT

Hypertrophic growth of adult myocardium is associated with increased expression of the early response gene c-jun. The purpose of this study was to determine whether eukaryotic initiation factor (elF) 4E (eIF4E) regulates translational efficiency of c-jun mRNA as measured by flux into polysomes. Adult feline cardiomyocytes in primary culture were treated with 0.2 µM 12-O-tetradecanoylphorbol 13-acetate (TPA), and c-jun mRNA was quantified in total, monosome, and polysome fractions by real-time polymerase chain reaction. After 1 h, TPA increased total c-jun mRNA by 10.5-fold. The corresponding flux into polysomes was significantly lower (5-fold). Adenoviral-mediated overexpression of either eIF4E or a nonphosphorylatable mutant (S209/A) did not affect total c-jun mRNA or its flux between monosomes and polysomes. Similar results were obtained following overexpression of the eIF4E kinase Mnk1. Thus, translational efficiency of c-jun mRNA was not affected by changes in activity or amount of eIF4E. In contrast, a kinase-deficient Mnk1 mutant significantly reduced total c-jun mRNA from 9.8-fold to 6.0-fold while flux between monosomes and polysomes remained constant. The decrease in total c-jun mRNA resulted from increased decay of c-jun mRNA incorporated into the polysomes. We conclude that Mnk1 activity stabilizes c-jun mRNA in polysomes independent of eIF4E phosphorylation.—Spruill, L. McDermott, P. Regulation of c-jun mRNA expression in adult cardiocytes by MAP kinase interacting kinase-1 (MNK1).


Key Words: cardiac hypertrophy • translation • eIF4E • protein synthesis • mRNA stability

HYPERTROPHY OF THE ADULT myocardium develops in response to increased myocardial wall stresses generated during pathophysiologic states such as systemic hypertension, valvular stenosis or insufficiency, and myocardial infarction (1) . Hypertrophic growth is characterized by an overall enlargement of individual cardiac muscle cells (cardiocytes) to produce an adaptive increase in cardiac mass (2) . The link between increased myocardial wall stress and hypertrophic growth is cardiac protein synthesis, which is regulated by mechanisms that increase the efficiency (activity) and the capacity (amount) of the translation machinery, principally eukaryotic initiation factors (eIFs) and ribosomes (3 , 4 ). Essentially, all of the main regulatory components of the translation machinery are endpoints for convergence of signaling pathways that are known to initiate cardiac hypertrophy and remodeling (4 , 5) . By regulating translation, both general and specific changes in cardiocyte protein synthesis are produced that are integral for hypertrophic growth. General changes involve accelerating the rate of total protein synthesis in excess of the rate of protein degradation to produce a net increase in cardiocyte protein content (6 , 7) . Specific changes in protein synthesis entail controlling the flux of individual mRNAs from monosomes (mRNPs) into polysomes to coordinate changes in gene expression produced by transcriptional and posttranscriptional mechanisms (8 , 9) .

The elF 4F complex (eIF4F) has a central role in translational initiation by regulating the binding of mRNA to the 40S ribosomal subunit (10) . The eIF4F complex has three primary components: eIF4E, which binds to the 7mGppp cap of mRNA; eIF4A, which is a RNA helicase; and eIF4G, which functions as a protein scaffold for coordinating assembly of eIF4F. The activity of eIF4F is regulated either by increasing the assembly of eIF4F complexes or by altering the affinity of eIF4E for the 7mGppp cap. The assembly of eIF4F complexes is regulated by a family of eIF4E binding proteins (4E-BP1, 2, and 3) that compete with eIF4G for a common binding site on eIF4E, thereby controlling the amount of eIF4E available for eIF4F complex formation (11) . Phosphorylation of 4E-BPs on an integrated set of sites reduces their binding affinity for eIF4E, thereby enabling eIF4F complex formation (12 , 13) . In adult cardiocytes, activation of the PI3 kinase/Akt/mTOR signaling pathway causes 4E-BP phosphorylation and a corresponding increase in eIF4F complex formation (14 , 15) . The affinity of eIF4E for the 7mGppp cap is regulated by two molecular mechanisms, both of which are dependent on the assembly of eIF4F complexes. One mechanism is binding of eIF4E to eIF4G itself, which induces conformational changes in eIF4E that markedly enhance its affinity for the cap (16) . The other mechanism is phosphorylation of eIF4E on Ser-209 by the MAP kinase interacting kinases (Mnk1 and Mnk2), which reduce cap-binding affinity of eIF4E as demonstrated by in vitro studies (17 , 18) . A large body of evidence has shown that eIF4E phosphorylation is positively correlated with protein synthesis and growth in a wide range of cell types, including adult cardiocytes (19 20 21) .

The precise role of eIF4E phosphorylation in regulating cellular growth has not been established. On the one hand, a loss of function mutation in Drosophila showed that eIF4E phosphorylation was essential for normal cellular enlargement, while the effect on cell numbers was minimal (22) . On the other hand, there is no evidence that eIF4E phosphorylation causes a general increase in translational initiation. In fact, neither overexpression of eIF4E nor increasing eIF4E phosphorylation was sufficient to accelerate the rate of total protein synthesis (21 , 23) . These findings reinforce the hypothesis that eIF4E phosphorylation selectively regulates translation of specific mRNAs, for example mRNAs that are relatively weak with respect to translational efficiency because of extensive secondary structure in their respective 5'-UTRs (24 , 25) . These mRNAs tend to encode for proteins involved in growth regulation such as transcription factors, proto-oncogenes, growth factors, and their receptors (24) . By increasing eIF4E phosphorylation, subsequent changes in the activity of the eIF4F complex could promote growth by optimizing translational efficiency of these specific types of mRNAs. Another possible function of eIF4E phosphorylation is to couple translation of a specific mRNA to its stability (26) . Changes in cap binding affinity of eIF4E could modify the activity of enzymes involved in deadenylation-dependent mRNA decay such as decapping enzymes and deadenylases (27) .

In adult cardiocytes, eIF4E phosphorylation and protein synthesis were increased concomitantly in response to stimuli that induce hypertrophic growth (20) . However, changes in eIF4E phosphorylation were not necessary to accelerate the rate of total protein synthesis (21) . The effects of eIF4E phosphorylation on translational efficiency of specific mRNAs were tested in adult cardiocytes using luciferase reporter mRNAs with incremental increases in the predicted amount of secondary structure in the corresponding 5'-UTRs (28) . These studies demonstrated an inverse relationship between the amount of secondary structure in the 5'-UTR and the translational efficiency of the reporter mRNA. It was shown further that increased eIF4E phosphorylation produced by overexpression of Mnk1 improved translational efficiency independent of the predicted amount of secondary structure in the 5'-UTR of the reporter mRNAs. The increases in translational efficiency occurred in association with corresponding reductions in reporter mRNA levels, which suggested that the positive effect of eIF4E phosphorylation on translational efficiency in adult cardiocytes is coupled to stability of mRNA.

In this study, we tested the hypothesis that translational efficiency of endogenous mRNAs is regulated in adult cardiocytes by activity of the eIF4F complex. Specifically, we assayed whether changes in eIF4E levels or eIF4E phosphorylation regulate the movement of c-jun mRNA between monosomes and the translationally active polysome pool, a process we refer to as "flux". We chose to examine c-jun mRNA for two primary reasons. First, its 5'-UTR has structural features capable of diminishing translational efficiency, specifically a length of 973 nucleotides, a G+C content of 65.3%, and a high degree of secondary structure as predicted by m-fold ({Delta}G=–380 kCal/mol) (25) . Second, c-jun functions during cardiac hypertrophy as an early response gene encoding for the activating protein (AP)-1 transcription factor, which in turn regulates transcription of target genes involved in growth (29 30 31) . Following transcriptional activation of c-jun, these studies demonstrate that the distribution of c-jun mRNA between the monosome- and polysome-bound pools is a function of both translational efficiency and mRNA stability and that the flux of c-jun mRNA into polysomes was not improved by increasing the amount or extent of eIF4E phosphorylation. Finally, Mnk1 activity stabilizes c-jun mRNA in polysomes by a mechanism that does not involve eIF4E phosphorylation.

MATERIALS AND METHODS

Cell culture
Adult feline cardiocytes were isolated by collagenase digestion as described previously (32) . Cardiocytes were plated onto laminin-coated tissue culture dishes at a concentration of 1.2 x 106 cells per 100 mm dish. The medium was changed after 4 h to remove nonadherent cells and was replaced with a serum-free medium (33) . The next day, cardiocytes were infected for 4 h with either cytomegalovirus (CMV)-ßGal adenovirus, or with one of the following adenoviruses: 4E-wild-type, 4E-209A, Mnk1, or the kinase-deficient mutant KD-Mnk1 as described previously (21 , 28 , 34) . Each protein contains a hemagglutinin (HA) epitope tag to distinguish between adenoviral-mediated and endogenous protein expression. The media were changed and the cardiocytes were incubated for an additional 48 h to allow for protein expression. Cardiocytes were challenged for 1 h with 0.2 mM phorbol 12-O-tetradecanoyl phorbol-13-acetate (TPA) prior to analysis of mRNA distribution.

Isolation of monosome and polysome fractions from adult cardiocytes
The distribution of mRNA was determined by fractionation of cardiocyte homogenates on 15 to 50% (w/v) linear sucrose gradients as described before (35) . RNA was extracted from the monosome fractions (mRNP) and polysome-bound fractions of the gradients using the Trizol method and stored in ethanol.

Measurement of RNA levels by real-time RT-PCR
RNA samples were pelleted and resuspended in 1 mM MgCl2. RQ1 DNase was added and the samples were incubated at 37°C for 20–30 min. Subsequently, the samples were heated for 5 min at 90°C, and relative levels of RNA were measured by a one-step SYBR green RT-polymerase chain reaction (RT-PCR) using the Quantitect SYBR Green RT-PCR kit (Qiagen). The primer sets are shown in Table 1 . Real-time RT-PCR was done using a Bio-Rad iCycler as follows: 45 min at 50°C for the reverse-transcription reaction, 15 min at 95°C, 35 cycles consisting of 15 s at 94°C, 30 s at 60°C, and 30 s at 72°C. At the end of each run, a temperature gradient step-down curve was run to produce a melt curve. In addition, reaction products were run on 2% agarose gels to verify the size and purity of the polymerase chain reaction (PCR) products.


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  		Table 1. Primers sets for RT-PCR

Standard curves for each run were generated by making a serial dilution of a control sample of RNA. The standard curves were used to extrapolate relative mRNA concentrations. The relative concentrations of GAPDH mRNA and c-jun mRNA were normalized to 18S rRNA. Fold changes were calculated by dividing relative levels of each mRNA derived from TPA-treated cardiocytes by relative mRNA levels measured in cardiocytes not treated with TPA.

Measurements of eIF4E activity
The amount and phosphorylation of eIF4E were determined using isoelectric focusing to resolve phosphorylated and nonphosphorylated isoforms followed by Western blotting using an eIF4E monoclonal antibody (mAb) (20) . Western blotting procedure was done as described before (21) .

The optical densities of the bands were quantified by digital image analysis.

RESULTS

Functional distribution of endogenous c-jun mRNA pools in adult cardiocytes
The purpose of these studies was to determine whether changes in eIF4F activity alter the flux of c-jun mRNA from the mRNP pool into polysomes. Fig. 1 shows the isolation of monosome and polysome fractions on linear sucrose gradients using postmitochondrial supernatants derived from adult cardiocytes. The monosome fractions contain free 40S and 60S ribosome subunits plus mRNPs while the heavier fractions contain polysomes active in translation. In Fig. 1B , the distribution of individual mRNAs along the gradient was determined by quantifying the recovery in each fraction as a percentage of total mRNA. These data show that a large percentage of c-jun mRNA was recovered in the polysome fractions. Conversely, GAPDH mRNA was recovered almost exclusively in the monosome fractions.


Figure 1
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  		Figure 1. Distribution of 18S rRNA, GAPDH mRNA and c-jun mRNA in adult cardiocytes. Cardiocytes were challenged with 200 nM TPA for 1 h, and homogenates were fractionated on linear sucrose gradients. A) Representative absorbance tracings measured at 260 nm. *Indicates that the scale was increased by a factor of 2.5. B) RNA was extracted from individual fractions along the gradient (1.2 ml each). Each RNA species was quantified by real-time RT-PCR and plotted as percentage of total recovery.

To induce expression of c-jun mRNA, cardiocytes were challenged with TPA to activate the gene and generate a "pulse" of transcription. Figure 1 shows that TPA did not change the overall distribution of either c-jun mRNA or GAPDH mRNA between monosome and polysome fractions. Two possible mechanisms could account for the fact that a large percentage of c-jun mRNA is distributed in polysomes, both in nontreated controls and following the TPA-induced increase in c-jun mRNA levels. One is that c-jun mRNA is translated at a relatively high efficiency, such that a large proportion of the total pool is incorporated readily into polysomes. The other is differential stability of c-jun mRNA in monosomes vs. polysomes, that is, the pool of c-jun mRNA not incorporated into polysomes is degraded faster because of destabilizing elements such as AREs in the 3'-UTR.

To examine these possible mechanisms further, translational efficiency of c-jun mRNA in cardiocytes was determined by measuring the flux into polysome fractions after 1 h and 4 h of TPA challenge. By comparing the fold increases of c-jun mRNA in the polysome fractions with the total RNA fraction, flux accounts for rapid and transient changes in c-jun mRNA levels produced in response to TPA. Fig. 2 shows that c-jun mRNA in the polysome fractions increased by 5-fold after 1 h of TPA challenge, while c-jun mRNA levels in the total RNA fraction increased by 10.5-fold. These results indicate that the flux of c-jun mRNA into polysomes was significantly lower at this time point. After 4 h of TPA challenge, c-jun mRNA in polysomes was increased by 2.4-fold, which was not significantly different from the 2.9-fold increase in c-jun mRNA measured in the total RNA fraction. Thus, once the transcriptional pulse of c-jun mRNA was complete, the increase in c-jun mRNA in polysomes closely matched the increase in total c-jun mRNA levels. Figure 2 also shows GAPDH mRNA levels did not increase in the total RNA fraction after 1 or 4 h of TPA challenge. Furthermore, the relative amount of GAPDH mRNA in polysomes was not significantly different at either time point, indicating that flux was unchanged.


Figure 2
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  		Figure 2. Functional distribution of c-jun mRNA in adult cardiocytes. Cardiocytes were challenged with 200 nM TPA for either 1 h or 4 h prior to fractionation of homogenates on linear sucrose gradients. The levels of c-jun mRNA and GAPDH mRNA were measured in the total RNA fraction and in the polysome-bound RNA fractions by real-time RT-PCR. Fold-increases in mRNA levels following TPA challenge were calculated relative to the nontreated controls. Values are the mean ± SE. *P < 0.05 as determined by Student’s t test.

Taken together, the results in Figs. 1 and 2 indicate that the distribution of c-jun mRNA between monosomes and polysomes was not affected by a TPA-induced increase in transcription. The adult cardiocytes did not maintain a large pool of c-jun mRNA in the monosome (mRNP) fractions, which probably reflects stabilization of c-jun mRNA that is incorporated into polysomes. The results in Fig. 2 support this conclusion, since the fold increases in polysome-bound and total c-jun mRNA equilibrated between 1 and 4 h of TPA, while the increase in total c-jun mRNA levels declined from 10.5-fold to 2.9-fold during this same time period.

Modification of eIF4E in adult cardiocytes by adenoviral gene transfer
To modify the activity of eIF4E, cardiocytes were infected with recombinant adenoviruses to overexpress either wild-type (WT) or mutated forms of eIF4E and Mnk1. Cardiocytes were infected at a moi of 4, and the relative levels of expression were determined after 48 h by Western blotting using an antibody (Ab) against the HA epitope tag. Figure 3 A is a representative Western blot, which demonstrates that approximately equal levels of overexpression were achieved for each of the indicated HA-tagged proteins. In Fig. 3B , a phospho-specific Ab to Mnk1 was used to confirm that the KD-Mnk1 mutant was not activated in response to TPA.


Figure 3
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  		Figure 3. Western blots to verify adenoviral-mediated expression and protein overexpression effect in adult cardiocytes. Cardiocytes were infected with the indicated adenoviruses and maintained for 48 h. Western blots were done using an Ab against the HA-epitope tag (A). The sizes of the HA-eIF4E and HA-Mnk1 bands are indicated. The lower blot is a control for loading of cardiocyte protein using the GAPDH Ab. The Western blot in (B) is derived from cardiocytes overexpressing either WT Mnk1 or the KD-Mnk1 mutant using an Ab to phospho-Mnk; (–) nontreated cardiocytes, (+) cardiocytes challenged for 1 h with 200 nM TPA. The phosphorylated protein appearing at ~97 kDa is a nonspecific band. The lower blot shows companion samples probed with anti-Mnk1 Ab.

To demonstrate modifications of eIF4E by adenoviral gene transfer, eIF4E phosphorylation in cardiocyte homogenates was quantified by one-dimensional isoelectric focusing followed by Western blotting with anti-eIF4E Ab (Fig. 4 ). The summary data show that TPA for 1 h increased endogenous eIF4E phosphorylation in ßGal controls from 35 to 67%. In cardiocytes overexpressing 4E-wild-type, the HA tag caused a shift in pI of both phosphorylated and nonphosphorylated isoforms. Similar to endogenous eIF4E, TPA increased phosphorylation of 4E-wild-type increased from 37 to 50%. Overexpression of the 4E-209A mutant blocked phosphorylation of eIF4E below detectable levels. Conversely, overexpression of Mnk1-wild-type increased eIF4E phosphorylation to 66% in nontreated cardiocytes and to 89% following TPA treatment. These data confirmed that Mnk1-wild-type becomes partially activated when expressed in nontreated cardiocytes and that eIF4E kinase activity of Mnk1-wild-type is further induced by TPA, increasing the % eIF4E phosphorylation close to its maximum. Overexpression of KD-Mnk1 reduced endogenous eIF4E phosphorylation to 22% in nontreated cardiocytes and blunted the TPA-induced increase in phosphorylated eIF4E to 48%. Given that activation of KD-Mnk1 was blocked (Fig. 3B ), the remainder of eIF4E phosphorylation was probably due to the Mnk2 isoform, an enzyme that is constitutively activated.


Figure 4
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  		Figure 4. Modification of eIF4E by adenoviral gene transfer. Cardiocytes were infected for 4 h with the indicated adenoviruses at a moi of 4. After a 48 h incubation period, cardiocytes were challenged with TPA for 1 h and compared with nontreated controls. eIF4E was purified from cardiocyte homogenates using 7mGTP-sepharose beads followed by one-dimensional isoelectric focusing to resolve nonphosphorylated and phosphorylated isoforms. The [+] and [–] on the right side of the blot designate the positive and negative poles of the isoelectric focusing gel, respectively. The upper part shows a Western blot probed with anti-eIF4E Ab. The graph below shows summary data of five experiments. The bars are P-eIF4E calculated as a percentage of total eIF4E in nontreated (solid bars) and TPA treated (slashed bars) samples. The values for the 4E-wild-type and 4E-209A samples are derived from the HA-tagged proteins. *P < 0.05 compared with the corresponding nontreated sample. {dagger}P < 0.05 compared with ßGal TPA-treated sample. §P < 0.05 compared with KD-Mnk1 TPA-treated sample. Statistical comparisons were done by ANOVA followed by a Student-Newman-Keuls test. Not determined (ND) = Nondetectable.

Effects of modifying eIF4E on the flux of c-jun mRNA
In the next series of experiments, the effects of modifying eIF4E on translational efficiency were determined by measuring the flux of c-jun mRNA into polysomes (Fig. 5 ). Following infection with either 4E-wild-type, 4E-209A, Mnk1-wild-type, or KD-Mnk1 adenovirus, cardiocytes were maintained for 48 h to allow time for the resultant changes in the amount and/or phosphorylation of eIF4E. Subsequently, cardiocytes were challenged with TPA for 1 h in order to induce maximal expression of c-jun mRNA. Flux was determined by comparing the fold-increases of c-jun mRNA levels in the polysome fractions and the total RNA fraction relative to the nontreated controls. Figure 5A shows that overexpression of 4E-wild-type had no effect on induction of total c-jun mRNA as compared with ßGal nor did it alter the flux of c-jun mRNA into polysomes. Although overexpression of eIF4E increases eIF4F complex formation, it was not sufficient to improve the translational efficiency of c-jun mRNA.


Figure 5
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  		Figure 5. Effects of modifying eIF4F activity on the flux of c-jun mRNA. Cardiocytes were infected for 4 h with the indicated adenoviruses at a moi of 4. A ßGal control group was included for each experimental group. After a 48-h incubation period, cardiocytes were challenged with TPA for 1 h. Relative levels of c-jun mRNA and 18S rRNA were measured by real-time RT-PCR in the total RNA fraction and in the pooled polysome fractions. The c-jun mRNA/18S rRNA ratios are expressed as the fold increase over the nontreated controls. *P < 0.03, ßGal total vs. KD-Mnk1 total as determined by Student’s t test. The numbers of experiments for each adenovirus and its corresponding ßGal control are indicated.

Figure 5B shows the effects of Mnk1-wild-type, which incorporates into existing eIF4F complexes and increases phosphorylation of eIF4E. Mnk1-wild-type did not affect the induction of c-jun mRNA as measured in the total RNA fraction after 1 h of TPA challenge. Despite the fact that 89% of eIF4E was phosphorylated in response to TPA, the flux of c-jun mRNA into polysomes was not increased significantly. These results suggest that a lower percentage eIF4E phosphorylation (67% in ßGal controls) was probably sufficient to enable maximal efficiency of c-jun mRNA translation. Figure 5C demonstrates that the 4E-209A mutant did not affect flux of c-jun mRNA. These data indicate that eIF4E phosphorylation was not required to sustain flux into polysomes. Figure 5D shows the effects of overexpressing KD-Mnk1 to inhibit inducible eIF4E phosphorylation. The level of c-jun mRNA increased by 6.0-fold in the total RNA fraction after 1 h of TPA challenge, which was significantly less than the 9.8-fold increase in c-jun mRNA produced in the ßGal controls. However, cardiocytes overexpressing KD-Mnk1 still exhibited a 6.0-fold increase in c-jun mRNA in the polysome fractions, comparable with the change measured in the ßGal controls. These findings indicate that flux of c-jun mRNA into polysomes was near its maximum and that the remainder of c-jun mRNA was degraded. None of the modifications to eIF4F had an effect on the levels of GAPDH mRNA in the total RNA fraction or in the polysome fractions (data not shown).

Effects of modifying eIF4F activity on the stability of c-jun mRNA
The results in Fig. 5 indicate that flux of c-jun mRNA into polysomes was not affected by modifying either eIF4E levels or eIF4E phosphorylation. Furthermore, as shown in Fig. 6 , none of the modifications altered the distribution of c-jun mRNA as greater than 90% was recovered in polysomes in the presence or absence of TPA. In contrast, a large pool of GAPDH mRNA was maintained in monosomes since 20–35% was recovered in polysomes. Given that KD-Mnk1 significantly reduced the relative levels of c-jun mRNA in the total RNA fraction, we hypothesized that Mnk1 activity regulates stability of c-jun mRNA independent of its role as an eIF4E kinase. To test this hypothesis, cardiocytes overexpressing either ßGal, Mnk1-wild-type or KD-Mnk1 were treated for 1 h with TPA to induce c-jun mRNA expression followed by actinomycin D treatment to block transcription. In Fig. 7 , c-jun mRNA levels in the total RNA fraction are plotted as a function of time after actinomycin D treatment. This assay did not detect any significant changes in the half-life of c-jun mRNA as measured in the total RNA fraction.


Figure 6
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  		Figure 6. Percent of total c-jun and GAPDH mRNA in polysomes after TPA stimulation. Cardiocytes were infected for 4 h with the indicated adenoviruses, incubated for 48 h, and then treated with TPA for 1 h. After polysome fractionation and RNA extraction, real-time RT-PCR was performed to determine the percentage of total c-jun (A) and GAPDH (B) mRNA that was polysome bound. Error bars = SE, n = 4 experiments.


Figure 7
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  		Figure 7. Effects of Mnk1 activity on the stability of c-jun mRNA. Adult cardiocytes were infected with the indicated adenovirus, incubated for 48 h and treated with TPA for 1 h. Subsequently, cardiocytes were treated with actinomycin D and RNA was extracted at the indicated time points. Values are plotted as the percentage of c-jun mRNA measured at time 0. The extrapolated half-life of c-jun was 102 min in noninfected cardiocytes, 94 min in ßGal infected cardiocytes, 93 min in Mnk1 infected cardiocytes, and 102 min in KD-Mnk1 infected cardiocytes. n = 3 experiments.

The findings of Figs. 5D and 7 seemed contradictory because KD-Mnk1 reduced the relative level of total c-jun mRNA while the half-life of total c-jun mRNA was unchanged. Based on the evidence in Figs. 1 and 2 , we reasoned that changes in half-life derived from measurements of total c-jun mRNA could be difficult to detect since it contains a mixture of monosome and polysome-bound pools with marked differences in stability. Furthermore, because ~90% of the total c-jun mRNA pool was distributed in the more stable polysome fractions, total c-jun mRNA levels would decline as a function of its half-life in polysomes. To test this possibility, stability of TPA-induced c-jun mRNA in polysomes was measured in cardiocytes overexpressing either ßGal or KD-Mnk1. The results in Fig. 8 show the relative amount of polysome-bound c-jun mRNA after 30 min of actinomycin D normalized to time 0. In ßGal controls, c-jun mRNA levels in polysomes were relatively stable over 30 min. In contrast, a significant decrease in the amount of polysome-bound c-jun mRNA occurred over 30 min in cardiocytes overexpressing KD-Mnk1. These data indicate that TPA-inducible activity of Mnk1 does not improve flux of c-jun mRNA into polysomes, rather it functions by stabilizing the pool of polysome-bound c-jun mRNA.


Figure 8
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  		Figure 8. Effects of eIF4F activity on the stability of polysome-bound c-jun mRNA. The stability of c-jun mRNA in polysomes was measured in cardiocytes overexpressing either ßGal or KD-Mnk1 after 1 h of TPA challenge. Cardiocytes were treated with actinomycin D and polysomes isolated after 30 min. The relative amount of polysome-bound c-jun mRNA was normalized to time 0. * P < 0.03 as determined by a Student’s t test, n = 4 experiments.

DISCUSSION

Cardiac hypertrophy develops by accelerating the rate of total protein synthesis to produce a general increase in cardiocyte protein content (3) . In addition to cellular enlargement, the anabolic response is characterized by altered patterns in gene expression that cause specific structural and functional changes at different phases of cardiac hypertrophy (8) . The expression of these mRNAs is regulated by mechanisms that either activate or repress transcription of specific genes associated with cardiac hypertrophy (36) . The studies herein demonstrate that gene expression is regulated by utilizing additional mechanisms that control the flux of a given mRNA between different functional compartments in the adult cardiocyte. The conclusions are based on tracking endogenous expression of c-jun mRNA, a well-characterized marker gene for cardiac hypertrophy that is activated following TPA challenge (37 , 38) . As the level of c-jun mRNA rises following transcriptional activation of the gene, translational efficiency functions as a checkpoint that responds to dynamic changes in the size of the mRNA pool by adjusting the relative proportion in the translationally active polysome-bound compartment. In this manner, the expression of proteins that are needed to trigger an anabolic response is tightly controlled and limited mainly to periods of cardiocyte growth.

The present studies illustrate how mechanisms involving 1) transcription, 2) translation, and 3) mRNA decay are regulated coordinately and contribute to net protein expression of c-jun in the adult cardiocyte. The 10.5-fold induction in c-jun mRNA levels by TPA challenge was produced by transcriptional activation of the gene. The corresponding levels of c-jun mRNA in the polysome-bound fractions were increased by 5-fold, demonstrating that the flux of c-jun mRNA was significantly lower. Finally, expression of c-jun mRNA was transient as the levels declined between 1 and 4 h of TPA treatment. Rapid decay of c-jun mRNA is mediated by a U-rich, non-AUUUA type of ARE situated in the 3'-UTR (39) . In contrast to other types of ARE, this element does not tightly couple the elongation step of translation with ARE-directed decay of c-jun mRNA. Thus, changes in the distribution of c-jun mRNA following TPA challenge reflected its flux into polysomes rather than a decrease in the stability of c-jun mRNA due to ongoing translation in polysomes.

A major determinant of flux into polysomes is translational efficiency, which is controlled at the initiation step by intrinsic features of the mRNA and by the activity of eIFs and associated components (40 , 41) . The 5'-UTR of c-jun mRNA has several intrinsic features that are predicted to reduce translational efficiency: 1) an extensive amount of secondary structure as predicted using m-fold, 2) a primary sequence consisting of 70% G+C bases, and 3) a length of 973 nucleotides (42 , 43) . These studies did not test directly the extent to which the 5'-UTR of c-jun mRNA diminishes its translational efficiency, but analysis of a human full-length mRNA database with a classification and regression tree (CART) model determined that these features are reliable predictors of mRNAs that are weak with respect to translational efficiency (25) . Furthermore, using adenoviral-mediated expression of mRNA reporter constructs in adult cardiocytes, we verified experimentally that increasing the predicted amount of secondary structure in the 5'-UTR caused a corresponding decrease in translational efficiency of the reporter mRNA (28) . Studies are underway to determine whether the 5'-UTR of c-jun mRNA directly affects translational efficiency of a reporter mRNA that is expressed in adult cardiocytes.

Given that individual mRNAs compete for a limited pool of ribosomes in order to undergo initiation, translational efficiency is also determined by the activity of eIFs and associated components (44) . It has been hypothesized that by increasing the availability of eIF4E, the consequent increase in eIF4F complexes disproportionately enhances the ability of weak mRNAs with a highly structured 5'-UTR to be translated (24) . These mRNAs are posited to have a greater dependence on eIF4F activity for the initiation step of translation, possibly because the helicase activity of eIF4A is needed to relieve excessive secondary structure in the 5'-UTR. By comparison, since the 5'-UTR of strong mRNAs has relatively little secondary structure, translation is efficient even when the availability of eIF4E is limiting for eIF4F complex formation. The present studies do not support this hypothesis since eIF4E overexpression had no effect on translational efficiency of c-jun mRNA as measured by its flux into polysomes, although it was demonstrated that eIF4F complexes were increased (21) . Similarly, overexpression of eIF4E in adult cardiocytes did not improve the translational efficiencies of reporter mRNAs with increasing amounts of secondary structure in the 5'-UTR (28) . To the best of our knowledge, there are no data demonstrating that an increase of eIF4E availability directly enhances translation of c-jun mRNA. The mRNA encoding for the closely related family member JunD contains a G-C rich, highly structured 5'-UTR that can repress its translation (45) . But, translational repression of JunD mRNA was not relieved by overexpression of eIF4E in fibroblasts.

In vitro studies have shown that eIF4E phosphorylation causes a marked reduction in cap binding affinity, which has led to the hypothesis that eIF4E phosphorylation exerts a positive effect on protein synthesis by triggering the release of eIF4E from the 5'-cap of mRNA along with other components of the initiation complex (17 , 19) . As a result, translational efficiency could be improved overall by promoting scanning along the 5'-UTR to the start codon and by enabling additional rounds of initiation on the 5'-end to produce larger polysomes. Given that an increase in eIF4E phosphorylation was not sufficient to accelerate the rate of protein synthesis in adult cardiocytes, it is unlikely that eIF4E-controlled release from the 5'-cap would operate as a rate-limiting step in translational initiation (21) . An alternative mechanism was proposed that eIF4E phosphorylation causes complete dissociation of the initiation complex from mRNA, thereby expanding the pool of translational components such as ribosomes and eIFs that are accessible to other mRNAs for initiation. The data are not consistent with this possibility, since overexpression of Mnk1 in cardiocytes did not affect the flux of c-jun mRNA into polysomes despite the fact that eIF4E was 89% phosphorylated following TPA challenge. However, constitutive levels of eIF4E phosphorylation were maintained in the ßGal controls in the presence or absence of TPA, probably due to the activity of Mnk2 (46) . This mechanism, therefore, cannot be ruled out entirely because it is possible that lower levels of eIF4E phosphorylation catalyzed by Mnk2 were sufficient for release of translational components from mRNA.

The results show that overexpression of either KD-Mnk1 or 4E-209A decreased the percentage of phosphorylated eIF4E. While KD-Mnk1 reduced relative levels of c-jun mRNA in the total RNA fraction, 4E-209A did not cause any effect. These data indicate that Mnk1 activity enhances the stability of c-jun mRNA independent of eIF4E phosphorylation. Overexpression of KD-Mnk1 increased decay of c-jun mRNA as measured in the polysome fractions without a corresponding change in the decay rate of total c-jun mRNA. It seems counterintuitive that increasing the decay rate of polysome-bound c-jun mRNA would diminish the relative levels of c-jun mRNA in the total RNA fraction, but not in the polysome fractions. Our interpretation is that a large percentage of the c-jun mRNA pool is preserved in the polysome fractions because it is more stable than c-jun mRNA in monosomes (mRNPs). This concept is supported by the observation that relative levels of c-jun mRNA in the total RNA fraction and the polysome fractions were equivalent after 4 h of TPA challenge, a time when the transcriptional pulse was essentially complete. The increase in decay rate doesn’t involve the initiation step directly since the flux of c-jun mRNA into polysomes was not affected by overexpression of KD-Mnk1.

Like other proto-oncogenes, the stability of c-jun mRNA is regulated by an ARE-mediated mechanism of decay in the 3'-UTR (47) . These cis-acting elements are targets for large family of ARE binding proteins that can either stabilize or destabilize the mRNA, probably by controlling the access and/or activity of enzymes involved in deadenylation-dependent decay. It is unlikely that KD-Mnk1 increased the decay rate of c-jun mRNA in polysomes by modifying its translational activity for two main reasons: 1) there was no change in the flux of c-jun mRNA into polysomes, which indicated that translational initiation was sustained; and 2) the destabilizing function of the ARE in c-jun mRNA is not strongly coupled to translational elongation (39) . Rather, the destabilizing effect of KD-Mnk1 on c-jun mRNA indicates that eIF4E is not the only effector for Mnk1. A recent study by Buxade et al. demonstrated that the ARE binding protein hnRNP A1 functions as a substrate for Mnk1 (48) . Phosphorylation of hnRNP A1 by activated Mnk1 decreased its ability to bind to the 3'-UTR of tumor necrosis factor-{alpha} mRNA (TNF-{alpha}), thus preventing the destabilization and subsequent degradation of TNF-{alpha} mRNA. Thus, the expression of specific mRNAs could be regulated by Mnk1-dependent activity of RNA binding proteins.

In summary, these studies demonstrate how gene expression in the adult cardiocyte is determined by mechanisms that coordinately control the distribution of mRNA between the monosome- and polysome-bound pools. By utilizing TPA to activate transcription and rapidly expand the total pool of c-jun mRNA, these data show that translational efficiency limited the flux of c-jun mRNA into polysomes. Furthermore, flux into polysomes was not affected by increasing the amount of eIF4E or by changing the extent of eIF4E phosphorylation, even though the 5'-UTR of c-jun mRNA is predicted to have a high degree of secondary structure. Flux appears to have an indirect role in regulating the expression of c-jun mRNA by its stabilization in the polysome fractions. Consequently, the majority of c-jun mRNA was recovered in polysomes. This pool of polysome-bound c-jun mRNA was stabilized by Mnk1 activity independent of eIF4E phosphorylation. Mnk1 is primarily an inducible enzyme that is direct substrate for ERK and P38 MAP kinase family members (34 , 46) . Thus, this mechanism for polysome stability is controlled by the same hypertrophic signaling pathways that regulate transcription and translation. Further understanding of this mechanism will depend on identifying the putative Mnk1 substrate that regulates stability of c-jun mRNA in polysomes and on determining whether it is involved in regulating the expression of other ARE containing mRNAs that are expressed during hypertrophic growth.

ACKNOWLEDGMENTS

We thank Daisy Dominick, Mary Barnes, and Christina DeRienzo for their excellent technical assistance. This work was supported by National Institutes of Health Grant PO1 HL-48788 and the Research Service of the Department of Veterans Affairs.

Received for publication April 5, 2006. Accepted for publication May 22, 2006.

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