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

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

SPECIFIC AIMS

The activity of eukaryotic initiation factor (elF) 4E (eIF4E) is increased during hypertrophic growth of the adult myocardium. Previous studies have shown that increasing either eIF4E expression or the extent of eIF4E phosphorylation is not sufficient to accelerate the rate of total protein synthesis. The specific aim was to determine whether changes in eIF4E activity have a more selective function during cardiocyte growth, that is regulating the translational efficiency of mRNAs with extensive secondary structure in the 5'-untranslated region (5'-UTR). The mRNA encoding for c-jun is representative of this class because its 5'-UTR is 973 nucleotides long and is predicted to have a high degree of stable secondary structure. Using adenoviral-mediated gene transfer to modify eIF4E activity in adult cardiocytes, the effects on translational efficiency were determined by measuring the movement of c-jun mRNA between monosomes and the translationally active polysome pool, a process referred to as "flux".

PRINCIPAL FINDINGS

1. Translational efficiency and mRNA stability coordinately regulate expression of c-jun mRNA in adult cardiocytes
Following a hypertrophic stimulus, c-jun functions as an early response gene encoding for the activating protein (AP)-1 transcription factor, which in turn regulates transcription of target genes involved in cardiac growth. To induce expression of c-jun mRNA, adult cardiocytes in primary culture were challenged with 0.2 µM 12-O-tetradecanoyl phorbol 13-acetate (TPA) to activate the gene and generate a "pulse" of transcription. Fig. 1 shows that c-jun mRNA in the polysome fractions increased by 5-fold after 1 h of TPA, which by comparison to the 10.5-fold increase in the total RNA fraction, indicated 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 than the corresponding increase measured in the total RNA fraction. Thus, once the transcriptional pulse of c-jun mRNA was completed, the increase in c-jun mRNA in polysomes matched the increase in total c-jun mRNA. Additional studies revealed that a small percentage of c-jun mRNA was recovered in the monosome fractions at 1 and 4 h, which indicates c-jun mRNA was stabilized by incorporation into polysomes. Taken together, c-jun mRNA levels in the adult cardiocyte are determined ultimately by flux into polysomes (translational efficiency) and by stabilization in polysomes.

View larger version (16K): [in this window] [in a new window]   Figure 1. Functional distribution of c-jun mRNA in adult cardiocytes. Cardiocytes were challenged with 200 nM TPA for either 1 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 polymerase chain reaction. 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.

2. Changes in eIF4E expression or the extent of eIF4E phosphorylation do not alter translational efficiency of c-jun mRNA in adult cardiocytes
Changes in eIF4E activity are hypothesized to facilitate translational initiation of mRNAs such as c-jun by relieving secondary structure in the 5'-UTR. The activity of eIF4E was modified by adenoviral gene transfer, and its effects on translational efficiency of c-jun mRNA were determined after 1 h of TPA challenge by measuring its flux into polysomes (Fig. 2 ). These data show that overexpression of eIF4E was not sufficient to increase the flux of c-jun mRNA into polysomes, although prior studies have shown that eIF4F complex formation was increased. The effects of increasing eIF4E phosphorylation were examined following overexpression of the eIF4E kinase Mnk1. Although TPA-induced phosphorylation of eIF4E was enhanced to 89%, there was no effect on the flux of c-jun mRNA into polysomes. Figure 2 further shows that eIF4E phosphorylation was not necessary to maintain translational efficiency since overexpression of the 4E-209A mutant to block phosphorylation on Ser-209 did not alter flux of c-jun mRNA into polysomes.

View larger version (18K): [in this window] [in a new window]   Figure 2. 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. 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 nontreated controls. *P < 0.03 as determined by Student’s t test. The numbers of experiments for each group are indicated.

3. The inducible activity of Mnk1 stabilizes the pool of c-jun mRNA incorporated into polysomes
Overexpression of a kinase-deficient mutant of Mnk1 (KD-Mnk1) was sufficient to block TPA-inducible phosphorylation of eIF4E. Figure 2D shows that the increase in total c-jun mRNA generated in response to TPA was significantly lower than the ßbeta;Gal controls, while flux into polysomes was sustained. Because 90% of the total c-jun mRNA pool was recovered in the polysome fractions, we posited that total c-jun mRNA declined in cardiocytes overexpressing KD-Mnk1 as a function of its half-life in polysomes. To test this possibility, cardiocytes overexpressing either ßbeta;Gal or KD-Mnk1 were challenged for 1 h with TPA, and c-jun mRNA levels were measured in polysome fractions over 30 min of actinomycin D treatment. In ßbeta;Gal controls, c-jun mRNA in polysomes did not change between 0 and 30 min of actinomycin D. These results indicate that polysome-bound c-jun mRNA was relatively stable over this time period. In cardiocytes overexpressing KD-Mnk1, c-jun mRNA in polysomes was decreased significantly after 30 min of actinomycin D (37±3%, P<0.03). These findings point to a novel mechanism whereby Mnk1 activity stabilizes c-jun mRNA incorporated into polysomes, independent of its role as an eIF4E kinase.

CONCLUSIONS AND SIGNIFICANCE

Figure 3 is a schematic to illustrate how expression of c-jun mRNA is regulated in the adult cardiocyte. A hypertrophic stimulus initially activates the gene to generate a "transcriptional pulse" that feeds mRNA into the monosome pool. Subsequently, translational efficiency determines the flux of c-jun mRNA from monosomes into polysomes. After 1 h of TPA challenge, the flux of c-jun mRNA into polysomes is significantly less than predicted on the basis of c-jun mRNA levels measured in the total RNA fraction, indicating that translational efficiency is suboptimal during the transcriptional pulse. The percentage of c-jun mRNA recovered in the monosome fractions remains constant (10%) in both control and TPA-treated cardiocytes. Thus, c-jun mRNA is stabilized by incorporation into polysomes and comprises most of the total pool, while the remainder is degraded. Overexpression of KD-Mnk1 does not disrupt the flux of c-jun mRNA into polysomes, rather it reduces stability of c-jun mRNA in polysomes. Thus, the results indicate that activation of Mnk1 maintains expression of c-jun mRNA by slowing its flux out of polysomes.

View larger version (12K): [in this window] [in a new window]   Figure 3. Schematic illustrating the proposed model for coordinate regulation of c-jun mRNA expression in adult cardiocytes.

The majority of studies on gene expression during cardiac hypertrophy have focused on endpoints that regulate transcription and induce synthesis of specific mRNAs. However, microarray analyses indicate that the actual number of mRNA species that change with respect to abundance is quite small in models of pressure overload hypertrophy. By examining endogenous expression of c-jun mRNA, these studies demonstrate that both translational efficiency and mRNA stability function as additional mechanisms to coordinately regulate mRNA expression in the adult cardiocyte. Furthermore, increases in either eIF4E expression or eIF4E phosphorylation do not improve translational efficiency of c-jun mRNA despite the extensive amount of secondary structure in the 5'-UTR. The activity of Mnk1 stabilizes c-jun mRNA in polysomes independent of its function as an eIF4E kinase. Given that Mnk1 is an inducible kinase that serves as a direct substrate for ERK and P38 MAP kinase family members, this mechanism for polysome stability is controlled by the same signaling pathways that regulate transcription and translation in a wide range of cell types.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-6245fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-6245fjev1 20/12/2133    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Spruill, L. S. Articles by McDermott, P. J. Search for Related Content PubMed PubMed Citation Articles by Spruill, L. S. Articles by McDermott, P. J.