AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 289: H2543-H2550, 2005. First published August 19, 2005; doi:10.1152/ajpheart.00545.2005
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Phosphorylation and binding of AUF1 to the 3'-untranslated region of cardiomyocyte SERCA2a mRNA

Juliana L. Blum,1 Allen M. Samarel,1,2,3 and Ruben Mestril1,3

1The Cardiovascular Institute and the Molecular Biology Program and the Departments of 2Medicine and 3Physiology, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois

Submitted 23 May 2005 ; accepted in final form 15 August 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental animals and patients with cardiac hypertrophy and heart failure display abnormally slowed myocardial relaxation, which is associated with downregulation of sarco(endo)plasmic reticulum calcium ATPase 2a (SERCA2a), the cardiomyocyte sarcoplasmic reticulum Ca2+ pump. We previously showed that SERCA2a downregulation can be simulated in cultured neonatal rat ventricular myocytes (NRVM) by treatment with the hypertrophic agonist phorbol myristate acetate (PMA) or by overexpression of the novel protein kinase C (PKC) isoenzymes PKC{delta} and PKC{epsilon}. PKC activation, in turn, decreased SERCA2a promoter activity and destabilized the SERCA2a mRNA. Here we demonstrate by using an RSV {beta}-galactosidase reporter system that a 609-nt fragment of the SERCA2a mRNA 3'-untranslated region (UTR), containing five adenylate-uridylate (AU)-rich regions, may be responsible for destabilizing the message following PMA treatment. UV cross-linking analysis demonstrated that several proteins found in the NRVM cell extracts bind to the 609-nt fragment. In addition, protein binding was transiently increased in response to PMA stimulation. 3'-UTR mRNA pull-down assays and Western blot analysis indicated that the AU binding protein AUF1 interacted with the SERCA2a 3'-UTR. AUF1 binding activity was predominantly found in the nuclear fraction, and PMA-induced AUF1 binding was associated with increased threonine phosphorylation of AUF1. These data suggest that the phosphorylation, binding, and location of AUF1 affect the posttranscriptional regulation of the SERCA2a message in NRVM.

neonatal rat ventricular myocytes; adenylate-uridylate-rich regions; heart; signal transduction; protein kinase C

THE SARCO(ENDO)PLASMIC RETICULUM calcium ATPase 2a (SERCA2a) is a 100-kDa member of the P-type family of membrane ATPases. SERCA2a is highly expressed within the sarcoplasmic reticulum membranes of cardiomyocytes, where it catalyzes the translocation of Ca2+ from the cytoplasm into the lumen of the sarcoplasmic reticulum, thereby contributing to muscle relaxation (3). Experimental animals and patients with cardiac hypertrophy and heart failure display abnormally slowed myocardial relaxation, which is associated with downregulation of SERCA2a gene expression (17). Both transcriptional (1, 15, 16, 41, 43, 45) and posttranscriptional (29–31, 39, 40, 46) mechanisms may be involved, although the specific effectors responsible for SERCA2a downregulation during cardiomyocyte hypertrophy and heart failure are only now being identified.

Primary cultures of neonatal rat ventricular myocytes (NRVM) have proven to be useful tools in which to explore the responsible mechanisms regulating SERCA2a gene expression in the heart. For example, previous studies from our laboratory and others have indicated that Ca2+ influx and the novel protein kinase C (PKC) isoenzymes PKC{delta} and PKC{epsilon} are critical factors regulating SERCA2a gene expression in response to neurohormonal and mechanical stimuli (2, 12, 14, 16, 37, 39). In a previous study, we found that treatment of contracting NRVM with phorbol myristate acetate (PMA) induced cellular hypertrophy, which was accompanied by the downregulation of SERCA2a gene expression and prolongation of the intracellular [Ca2+] transient. PMA-induced downregulation of sarcoplasmic reticulum Ca2+ pumps resulted from both a decrease in SERCA2a promoter activity (16) as well as a significant reduction in the half-life of SERCA2 mRNA (39), resulting in both transcriptional and posttranscriptional modulation of SERCA2a gene expression that may be operative in vivo during pathophysiological states.

A wide variety of genes are regulated posttranscriptionally, including c-myc, c-fos, parathyroid hormone, cox-2, tumor necrosis factor-{alpha}, and a number of cytokines (23, 42, 51). Posttranscriptional regulation is often mediated by cis-acting adenylate-uridylate (AU)-rich elements (AREs) within the 3'-untranslated region (UTR) of the message sequence. Studies have shown that AREs range in size from 50 to 150 nucleotides long and contain either a pentamer sequence (AUUUA) or a nonamer sequence [UUAUUUA(U/A)(U/A)] that is of sufficient length to bind cellular proteins (21, 51–53). Several of these trans-acting, AU binding proteins alter the stability of the message by binding directly to the 3'-UTR mRNA at the ARE sites (28, 30). An Elav-like RNA-binding protein Hu antigen R (HuR) has been identified and characterized as a stabilizing factor, which when bound to the 3'-UTR of posttranscriptionally regulated genes, allows protein levels to remain constant (4, 7, 9, 11, 26, 32, 48). AUF1, another extensively studied AU binding protein, has been characterized as a destabilizing factor that also binds directly to the 3'-UTR of posttranscriptionally regulated genes. When AUF1 is bound to the message, the transcript becomes destabilized and is degraded, leading to a decrease in mRNA levels and a subsequent reduction in protein expression (22, 42, 50).

Both HuR and AUF1 have been shown to bind the same sequences within the 3'-UTR, and under normal conditions both proteins work together by associating directly with the transcript to regulate mRNA levels (13, 35). Examination of the published sequence of the rat SERCA2a mRNA reveals five AU-rich sequences within the 670-nt 3'-UTR. Here we demonstrate that insertion of the SERCA2a 3'-UTR downstream of a heterologous message encoding {beta}-galactosidase ({beta}-Gal) increases its expression levels when transfected into NRVM and confers PMA-responsive downregulation of {beta}-Gal activity. Furthermore, we show that AUF1, but not HuR, binds to the 3'-UTR of SERCA2a. Finally, phorbol myristate acetate (PMA) increases the threonine phosphorylation of AUF1, which appears to increase its nuclear binding activity. These results suggest a novel mechanism of SERCA2a posttranscriptional regulation that may contribute to the pathogenesis of diastolic dysfunction in heart failure.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Neonatal rat ventricular myocyte isolation and culture. NRVMs were isolated from 2-day-old Sprague-Dawley rats and cultured as previously described (18, 33). Myocytes were plated at a density of ~4 million cells per 100-mm culture dish. Primary cultures of NRVM were left in serum-rich medium for 48 h and then changed to serum-free, maintenance medium (24 h) before experiments were started. In some experiments, cells were treated with PMA (200 nM; 5 min-24 h) to activate PKC isoenzymes. Control or treated cells were then washed twice with cold phosphate-buffered saline (PBS) and scraped into 0.5 ml PBS. Cells were collected by centrifugation (2 min at 8,000 rpm) and lysed in solution B (25 mM Tris·HCl, 25 mM NaCl, 0.1 mM EDTA, 1% Triton X-100, and 0.5% deoxycholic acid). Cells were resuspended in 110 µl of solution B and placed on ice for 20 min. The cell mixture was vortexed for 15 s every 5 min of the incubation. The mixture was centrifuged at 15,000 rpm for 15 min, and the supernatant was stored at –20°C. Protein concentration of the cell lysate was determined by Bradford assay (Pierce, Rockford, IL).

Plasmids. A 2.3-kb fragment containing coding sequences and the entire 3'-UTR of the rat SERCA2a cDNA was kindly provided by Dr. Wolfgang Dillmann, University of California-San Diego. The SERCA2a cDNA was removed by restriction enzyme digestion and was used as a template to amplify 609 nt of the rat SERCA2a 3'-UTR by PCR. Primers designed to anneal to each end of the 3'-UTR sequence were used. The two primers were designed to contain unique restriction enzyme sites, allowing for directional insertion of the SERCA2a 3'-UTR fragment into the 3' polylinker XbaI and BamH1 sites of an rous sarcoma virus long terminal repeat (RSV-LTR)/LacZ reporter construct, kindly provided by Dr. Ed Kislauskis (20). The reporter gene plasmid contained (from 5' to 3') the RSV-LTR, the coding sequence for the bacterial {beta}-Gal gene (lacZ), a polylinker, 609 nt of the rat SERCA2a 3'-UTR, and SV40 polyadenylation signals (MMP-609) (Fig. 1A). The parent plasmid, devoid of the SERCA2a 3'-UTR (RSV-{beta}-Gal), served as a control. A cytomegalovirus (CMV)-luciferase reporter plasmid was also used to control for transfection efficiency and was kindly provided by Dr. Kaie Ojamaa, North Shore University Medical Center, Manhassat, NY. Another plasmid construct for use in UV cross-linking and RNA pulldown assays was generated by cloning the same 609-bp 3'-UTR sequence into the 5' BamHI and 3' XbaI sites of a T3/T7 transcription vector (GIBCO-BRL, Grand Island, NY).



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  		Fig. 1. 3' Untranslated region (UTR) of sarco(endo)plasmic reticulum calcium ATPase 2a (SERCA2a) mRNA confers phorbol myristate acetate (PMA)-responsive downregulation to {beta}-galactosidase ({beta}-Gal). A: 609 bp of the rat SERCA2a 3'-UTR was cloned downstream of the lacZ gene. Black boxes, relative positions of AUUUA sequences within the 3'-UTR. B: parent plasmid rous sarcoma virus-{beta}-Gal (RSV-{beta}-gal) or MMP-609 were transfected, along with a cytomegalovirus (CMV)-luc expression plasmid, into neonatal rat ventricular myocytes (NRVM). Cells were then maintained in control medium or stimulated with PMA (200 nM, 24 h). Data are normalized {beta}-Gal activity in PMA (filled bars) versus control medium (open bars) for the two expression plasmids. Data are means ± SE for n = 4 experiments. *P < 0.05 vs. RSV-{beta}-Gal in control medium. C: ratios of normalized {beta}-Gal activity in PMA per control medium of myocytes transfected with either RSV-{beta}-Gal or MMP-609 for the same experiments are depicted. P < 0.05 vs. RSV-{beta}-Gal.

 
NRVM transfection. NRVM were transfected using the method of Chen and Okayama (6). Briefly, 10 µg of a CMV-luciferase control plasmid and 20 µg of either the parental RSV-LTR/LacZ or the MMP-609 plasmid were dissolved in 0.5 ml of sterile 0.25 M CaCl2, to which 0.5 ml of sterile 2x bicarbonate buffered saline (50 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, pH 6.95, 280 mM NaCl, and 1.5 mM Na2HPO4) was added and incubated at room temperature for 20 min. The mixture was then added dropwise while swirling into a 100-mm dish containing ~2 x 106 cells. Plates were then placed in a 3% CO2 incubator overnight. Cells were then maintained in fresh, serum-free control medium, or treated with PMA (200 nM, 24 h). NRVM were then harvested, and {beta}-Gal and luciferase activities were assayed as previously described (36).

UV cross-linking analysis. Radiolabeled RNA transcripts of the full-length 3'-UTR were generated using a T3/T7 Maxi Script kit (Ambion, Austin, TX). In brief, the T3/T7 plasmid containing the desired transcript fragment was first linearized with HindIII and then transcribed in the presence of T3 polymerase and [{alpha}-32P]UTP (Amersham Biosciences, Piscatway, NJ), according to kit specifications. The radiolabeled transcript was gel purified, as suggested in the protocol, and counted for 32P radioactivity by scintillation spectroscopy (44). UV cross-linking reactions were performed by incubating NRVM cell lysates (35 µg of total protein) with the 32P-labeled, 609-nt, 3'-UTR probe (2 x 105 counts/min) and subjected to UV radiation as previously described (24, 27, 44). The radiolabeled 3'-UTR mRNA probe and cell lysate were then incubated for 30 min at room temperature, followed by the addition of RNase T1 and heparin, and the reactions were incubated for another 10 min at room temperature. Samples were transferred to a 96-well microplate dish and UV irradiated in a GS Genelinker (Bio-Rad, Hercules, CA), ~3 inches from the light source, for 10 min. Next, unbound RNA was digested with RNase A, and remaining RNA-protein complexes were resolved on 12% SDS-acrylamide gels and exposed to film.

RNA pulldown assay. Biotinylated SERCA2a 3'-UTR mRNA was used in an RNA pulldown assay adopted from an immunoprecipitation protocol (10). Biotin end-labeling of the 3'-UTR transcript was performed using a biotin 3' End DNA Labeling kit (Pierce, Rockford, IL), according to manufacturer's instructions. Although the kit was designed for DNA, the addition of anti-RNase to the solutions made it compatible for RNA labeling. End labeling was assessed using a slot blot technique. The biotin end-labeled probe ({approx}150 ng) was then incubated (18 h, 4°C with gentle agitation) with NRVM cell lysate (35 µg total protein) in HEPES-Ringer buffer (19). Anti-RNase (1 U) was included in each reaction mixture to protect the 3'-UTR fragments from degradation. Adsorbed proteins bound to the 3'-UTR were recovered by addition of streptavidin beads (15 µg; Sigma, St. Louis, MO). After brief centrifugation, the beads were washed three times with 500 µl HEPES-Ringer solution. After the third wash, the samples were centrifuged for 45 s, the supernatant was discarded, and 20 µl of SDS sample buffer were added. After incubation (95°C; 5 min), recovered proteins were separated by SDS-PAGE and Western blot analysis. Blots were probed with anti-AUF1 rabbit pAb (1:5,000; Upstate Biotechnology, Lake Placid, NY) or anti-HuR mouse mAb (1:1,000; Molecular Probes, Eugene, OR) (10). Primary antibody binding was detected with appropriate horseradish peroxidase-conjugated secondary antibodies and Super Signal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL).

Immunoprecipitation assays. NRVM lysate (35 µg) was immunoprecipitated overnight (at 4°C with shaking) using rabbit anti-AUF1 pAb (1 µg; Upstate Biotechnology, Lake Placid, NY) followed by the addition of protein A-agarose beads (Sigma, St. Louis, MO) for 1 h at 4°C. Reactions were performed using HEPES-Ringer buffer to adjust the final volume to 0.5 ml. Recovered proteins were washed three times with HEPES-Ringer solution, collected, resolved on 12% SDS-acrylamide gels, and electrotransferred to membrane for Western blot analysis. Membranes were probed first for phosphotyrosine and then stripped and sequentially probed for phospho-serine, phospho-threonine, and AUF1 (10).

Subcellular fractionation. NRVM were treated with 200 nM PMA for 30 min and then harvested in cold PBS. Cells were collected by centrifugation, and subcellular fractions were generated using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL) according to the manufacturer's protocol. Resulting nuclear and cytoplasmic fractions were used in RNA pulldown assays as described above. Resulting Western blot analyses were analyzed by using an antibody to AUF1.

Data analysis. Results were expressed as means ± SE. Normality was assessed using the Kolmogorov-Smirnov test, and homogeneity of variance was assessed using Levene's test. Data from multiple groups were compared by one-way blocked analysis of variance (ANOVA) followed by Student-Newman-Keuls test. Data from two groups were compared by paired t-test. Differences among means were considered significant at P < 0.05. Data were analyzed using the SigmaStat Statistical Software Package, Ver. 1.0 (Jandel Scientific, San Rafael, CA).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
3'-UTR of SERCA2a mRNA confers PMA-responsive regulation to {beta}-Gal. As a first attempt to identify potential cis-acting sequences within SERCA2a mRNA that may be important in regulating its stability, MMP-609 or RSV-{beta}-Gal (Fig. 1A) were transiently transfected into NRVM (along with the constitutively active luciferase reporter plasmid CMV-luc to control for transfection efficiency), and {beta}-Gal and luciferase activities were measured in cell extracts 48 h later. Insertion of the SERCA2a 3'-UTR into the RSV-{beta}-Gal parent vector increased normalized {beta}-Gal activity approximately fourfold in untreated, control myocytes (0.52 ± 0.05 vs. 0.12 ± 0.02 normalized {beta}-Gal activity units for MMP-609 and RSV-{beta}-Gal vectors, respectively; P < 0.05; Fig. 1B). PMA treatment significantly increased normalized {beta}-Gal activity in myocytes transfected with RSV-{beta}-Gal (0.38 ± 0.07 vs. 0.12 ± 0.02 normalized {beta}-Gal activity units for PMA and untreated cells, respectively; P < 0.05; Fig. 1B). However, PMA failed to significantly increase normalized {beta}-Gal activity in cells transfected with MMP-609 (0.55 ± 0.10 vs. 0.52 ± 0.05 normalized {beta}-Gal activity units for PMA and untreated cells, respectively; Fig. 1B). The ratio of normalized {beta}-Gal activity in PMA and control myocytes is further depicted in Fig. 1C. As is evident from Fig. 1C, treatment of MMP-609-transfected NRVM with PMA markedly reduced normalized {beta}-Gal activity, compared with PMA-treated NRVM transfected with RSV-{beta}-Gal. Taken together, these results suggested that insertion of the SERCA2a 3'-UTR downstream of the {beta}-Gal coding sequence increased the stability of the {beta}-Gal mRNA and conferred PMA-responsive regulation to a this heterologous message. The degree of reporter gene downregulation by PMA was quite similar to the effects of PMA on the stability of the endogenous SERCA2a mRNA transcript (39).

3'-UTR of SERCA2a mRNA binds multiple cardiomyocyte cellular proteins. To determine the protein-binding capability of the 3'-UTR of SERCA2a, UV cross-linking analysis was then performed using the same 609-nt 3'-UTR segment as described above. Furthermore, we examined whether the levels of protein binding to the 3'-UTR were altered in response to activation of PKC isoenzymes with PMA. As seen in Fig. 2A, numerous cellular proteins bound to the SERCA2a 3'-UTR under control culture conditions, and there was an increase the intensity of specific protein bands in the 26- to 50-kDa range following PMA treatment compared with control cells. Changes in protein binding were observed as early as 1 h after PMA treatment, and these changes persisted for up to 8 h. However, examination of cell extracts after 24 h of continuous PMA treatment revealed no major differences in protein binding compared with untreated cells (data not shown). Figure 2B is a protein stain of the same gel used for autoradiography, indicating equal loading of samples. These results suggest that as soon as 1 h after PKC activation of NRVM by PMA, protein binding to the 3'-UTR of SERCA2a was enhanced. However, this alteration in binding activity returned to control levels by 24 h posttreatment.



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  		Fig. 2. UV cross-linking analysis of the 3'-UTR of SERCA2a mRNA. NRVM were maintained under control (Con) conditions or treated with PMA (200 nM; 1, 4, or 8 h). Cells were harvested, and extracted proteins (35 µg) were mixed with radiolabeled, 609-nt SERCA2a 3'-UTR (2 x 105 counts/min of [32P]UTP-incorporated probe). Reaction mixtures were UV irradiated for 10 min, and the cross-linked protein-RNA complexes were resolved by SDS-PAGE. The gels were fixed, stained for protein (B), dried, and autoradiographed (A). Position of molecular weight markers is indicated to the left of each image. Results depicted are representative of 3 separate experiments.

 
AUF1 binds to the 3'-UTR of SERCA2a. To determine whether the ARE binding proteins AUF1 and/or HuR bind to the 3'-UTR of the SERCA2a mRNA, a biotin-labeled, 609-nt probe was used to "pull down" proteins capable of binding to the 3'-UTR in control cells and cells stimulated with PMA. Recovered proteins were then analyzed by SDS-PAGE and Western blot analysis with antibodies specific for AUF1 and HuR. As seen in Fig. 3A, several polypeptides in NRVM extracts cross-reacted with the AUF1 antibody, corresponding to the multiple splice variants of AUF1 previously identified in human tissues (47). Furthermore, the largest AUF1 iosoform (~48 kDa) appeared to preferentially bind to the biotin-labeled, 3'-UTR probe under control conditions. The binding of this isoform appeared to increase following PMA stimulation, with a peak at 30 min and decreasing thereafter. However, examination of cell extracts after 24 h of continuous PMA treatment revealed no major differences in AUF1 binding compared with untreated cells (data not shown).



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  		Fig. 3. RNA pulldown assay to identify proteins binding to the 3'-UTR of SERCA2a. NRVM were maintained in Con medium or stimulated with PMA (200 nM, 5–120 min). Cell lysates (35 µg of total protein) were added to biotin-labeled SERCA2a 3'-UTR (~150 ng), and the bound proteins were separated by SDS-PAGE and Western blot (WB) analysis. Blots were probed with anti-AUF1 IgG (A) or anti-HuR IgG (B). Total cell lysate protein (140 µg) was also separated as a positive control. Position of molecular weight markers is indicated to the left of each blot.

 
HuR has previously been identified as another AU-rich 3'-UTR binding protein in several cell types. However, we were unable to detect HuR bound to the 3'-UTR of SERCA2a under control conditions or after short-term (5–120 min) PMA stimulation (Fig. 3B). Nevertheless, analysis of NRVM cell lysates confirmed the presence of HuR within cardiomyocytes.

AUF1 is serine and threonine phosphorylated in NRVM. We next examined whether activation of PKC isoenzymes with PMA affected the level of phosphorylation of AUF1 in total cell extracts of NRVM. As seen in Fig. 4A, tyrosine phosphorylation of AUF1 was not detected under basal conditions or in response to PMA. In contrast, the 48-kDa AUF1 isoform was found to be serine phosphorylated under basal conditions. PMA treatment did not acutely increase the level of serine phosphorylation, and in each experiment, the amount of serine phosphorylation appeared to decrease below basal levels after 60 min of exposure to the drug (Fig. 4B). Furthermore, all four of the AUF1 isoforms were threonine phosphorylated under basal conditions, and the level of threonine phosphorylation of AUF1 transiently increased in response to PMA (Fig. 4C). Western blot analyses with the AUF1 antibody confirmed equal loading of the gels (Fig. 4D).



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  		Fig. 4. Serine and threonine phosphorylation of adenylate-uridylate-rich element binding factor 1 (AUF1) in NRVM in response to PMA. NRVM were maintained in Con medium or stimulated with PMA (200 nM, 5–120 min). Cell lysates (35 µg of total protein) were immunoprecipitated with anti-AUF1 pAb (1 µg). Recovered proteins were separated by SDS-PAGE and WB analysis with anti-phosphotyrosine (pTyr, A), anti-phosphoserine (pSer; B), anti-phosphothreonine (pThr; C), or anti-AUF1 antibodies (D). Position of molecular weight markers is indicated to the left of each blot. Results depicted are representative of 3 separate experiments.

 
We also examined whether AUF1 bound to the 3'-UTR of SERCA2a mRNA was phosphorylated under basal conditions or in response to PMA. Western blot analyses were generated as in Fig. 3A and probed with phosphotyrosine, phosphoserine, phosphothreonine, and AUF1 antibodies. Although AUF1 was detected under these conditions, we were unable to detect any phosphorylation of the proteins using this technique (data not shown).

AUF1 interaction with the 3'-UTR of SERCA2a occurs predominantly within the nuclear fraction of NRVM. Nuclear and cytoplasmic fractions were then separated, and equal amounts of total protein were used to determine the subcellular distribution of AUF1 in NRVM. As seen in Fig. 5A, most of the AUF1 was found within the nuclear fraction of the cells. All four of the AUF1 isoforms were detected in the nuclear extracts, and their distribution was not substantially altered in response to PMA treatment. Similar subcellular fractions were then analyzed in the RNA pulldown assay. As seen in Fig. 5B, the majority of the AUF1 binding activity was present in the nuclear fraction in both untreated and PMA-stimulated cells. Levels of AUF1 binding to the 3'-UTR of SERCA2a mRNA increased in both fractions in response to PMA, with a statistically significant increase observed in the nuclear fraction. The quantitative analysis of three RNA pulldown assays is depicted in Fig. 5C.



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  		Fig. 5. AUF1 interaction with the 3'-UTR of SERCA2a occurs predominantly within the nuclear fraction of NRVM. NRVM were maintained in Con medium or treated with PMA (200 nM, 30 min). Cells were harvested and separated into nuclear and cytosolic fractions. A: total protein from each subcellular fraction (20 µg) was separated by SDS-PAGE and WB analysis with anti-AUF1 IgG. B: an RNA pulldown assay was performed using 35 µg of total protein from each fraction and 150 ng of biotinylated SERCA2a 3'-UTR probe. Proteins bound to the probe were recovered and separated by SDS-PAGE and WB analysis with anti-AUF1 IgG. Position of molecular weight markers is indicated to the left of each blot. C: quantitative analysis of 3 RNA pulldown assays is depicted. Data are means ± SE.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The ARE-mediated, posttranscriptional stabilization/destabilization of several short-lived mRNA transcripts have been shown to play a crucial role in regulating the intracellular content of variety of transcription factors and cyokines. In this report, we have begun to examine the mRNA binding proteins and signal transduction pathways responsible for the posttranscriptional regulation of SERCA2a, a message with a relatively long half-life. Interestingly, we provide evidence to suggest that both groups of mRNAs share common regulatory mechanisms.

In our previous study, we demonstrated by actinomycin D-stability assay that SERCA2a mRNA half-life was ~67 h in control NRVM maintained in serum-free culture. PKC activation with PMA decreased SERCA2a mRNA half-life to ~28 h (39). This relatively modest reduction in mRNA stability was, however, in part responsible for PMA-induced decreases in SERCA2a mRNA and protein levels and for prolongation of the intracellular [Ca2+] transient. Misquitta et al. (31) have previously suggested that SERCA2a expression is at least partly regulated posttranscriptionally. The cardiac and stomach smooth muscle isoforms of SERCA2 (SERCA2a and SERCA2b, respectively) are products of the same gene but differ in their 3'-UTRs as a result of alternative splicing. Nuclei isolated from both tissues demonstrated similar rates of SERCA2 transcription as measured by nuclear run on, but mRNA and protein levels of the two transcripts were markedly different (cardiac > stomach), suggesting an important element of posttranscriptional control. This was confirmed by direct measurement of SERCA2a and SERCA2b half-lives in cultured cells derived from both tissues. Our results extend their findings and indicate that the 3'-UTR of SERCA2a mRNA can prolong the half-life of a heterologous mRNA. Addition of the 3'-UTR of SERCA2a downstream of the lacZ gene stabilized {beta}-Gal production under basal conditions. However, after PMA stimulation, {beta}-Gal production was significantly reduced. These results suggest that the 3'-UTR functions to stabilize the SERCA2a message under control conditions, but following activation of a PKC-dependent signaling pathway, the 3'-UTR then destabilizes the message.

UV cross-linking analysis demonstrated that several proteins bind to the 3'-UTR of SERCA2a, and AUF1 was identified as one of the RNA binding proteins in a novel, RNA pulldown assay. Interestingly, AUF1 in cell extracts derived from both control and PMA-stimulated cardiomyocytes was capable of binding to a synthetic RNA fragment, suggesting that AUF1 functions to maintain a continual turnover of SERCA2a message under basal conditions. However, PMA stimulation, which activates all of the conventional and novel PKC isoenzymes in cardiomyocytes (8, 38), increased the level of AUF1 binding to the 3'-UTR. Increased AUF1 binding correlated with decreased SERCA2 mRNA stability and reduced transcript levels in cultured NRVM (16, 37, 39). Additional studies will be required, however, to ascertain whether AUF1 binding to SERCA2 mRNA contributes to the overall downregulation of SERCA2a gene expression reported in hypertrophied and failing cardiomyocytes in vivo (17, 25).

Of note, we found that the PMA-induced increase in AUF1 binding activity was transient, which correlates well with the transient activation, followed by downregulation of PKC isoenzymes by PMA (38). Indeed, we previously demonstrated that prolonged exposure to PMA substantially reduced levels of PKC{alpha}, PKC{delta}, and PKC{epsilon} but that continuous PKC activation was not necessary to reduce SERCA2 mRNA levels in NRVM. In fact, the stimulus for SERCA2 mRNA downregulation was generated within 30 min of PMA exposure, but the reduction in SERCA2 mRNA took ~12 h to become manifested (39). These results are consistent with a mechanism in which only transient activation of PKCs are required to initiate a cascade of events that ultimately leads to reduced SERCA2 gene expression (37). Furthermore, our results indicate that AUF1 is phosphorylated at serine and threonine (but not tyrosine) residues in control NRVM, and threonine phosphorylation transiently increased in response to PKC activation with PMA. Of note, Wilson et al. (49) have recently shown that the p40AUF1 isoform of AUF1 undergoes reversible phosphorylation following treatment of a human monocyte cell line with PMA. Thus there appears to be a relationship between the phosphorylation of AUF1 and its increased binding activity to the 3'-UTR of SERCA2a. We were unable to detect whether the AUF1 bound to the 3'-UTR was serine or threonine phosphorylated. This was likely due to the very small amount of protein bound to the 3'-UTR and the lack of sensitivity of the antibodies to detect phosphorylation by Western blot analysis. Prolonging exposure time and using extracts from larger number of cells also did not demonstrate either serine or threonine phosphorylation of the AUF1 bound to the 3'-UTR. At present we cannot explain this negative result, but it is conceivable that AUF1 must be dephosphorylated before binding to the 3'-UTR. PKC-dependent AUF1 phosphorylation could then regulate its availability for mRNA binding via reversible dissociation from another intracellular protein. Proof of this speculation, however, will require additional experiments, including more sensitive methods to detect AUF1 phosphorylation state.

AUF1 binding activity to the 3'-UTR of SERCA2a was concentrated in the nuclear rather than the cytoplasmic fraction, suggesting that the decay of SERCA2a mRNA begins within the nucleus and continues during transport to the cytoplasm. Our findings extend those of Misquitta et al. (31), who showed that cardiomyocyte nuclear extracts produced a faster rate of SERCA2a mRNA decay than cytoplasmic extracts. These authors proposed that nuclear decay is an initial step in the control of SERCA2a mRNA abundance and that this control is maintained or modulated in the cytoplasm. Recent studies focusing on the posttranscriptional deadenylation, decapping, and degradation of mRNAs by an exosome protein complex similar to that found in yeast suggest additional potential target proteins that may mediate PKC-dependent SERCA2 mRNA degradation (5, 32, 34). A clear understanding of the molecular mechanisms and pathways by which SERCA2a gene expression is regulated may be useful in preventing the untoward effects of SERCA2a downregulation during cardiac hypertrophy and heart failure.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
These studies were supported by National Heart, Lung, and Blood Institute RO1 Grants HL-63711 (to A. M. Samarel) and HL-61339 (to R. Mestril), an American Heart Association National Center Grant-in-Aid (to R. Mestril), and a grant to the Cardiovascular Institute from the Ralph and Marian Falk Trust for Medical Research. J. L. Blum was a recipient of an Eli Lilly Cardiovascular Research Predoctoral Fellowship during the time these studies were performed.


   ACKNOWLEDGMENTS
 
The authors thank Tina Valdez for excellent technical assistance and Drs. Gary Brewer and Henry Furneaux for provision of valuable reagents.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. M. Samarel, The Cardiovascular Institute, Loyola Univ. Medical Center, Bldg 110, Rm. 5222, 2160 South First Ave., Maywood, IL 60153 (email: asamare{at}lumc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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