Impairment of FOS mRNA stabilization following translation arrest in granulocytes from myelodysplastic syndrome patients

Abstract

Although quantitative and qualitative granulocyte defects have been described in myelodysplastic syndromes (MDS), the underlying molecular basis of granulocyte dysfunction in MDS is largely unknown. We recently found that FOS mRNA elevation under translation-inhibiting stimuli was significantly smaller in granulocytes from MDS patients than in healthy individuals. The aim of this study is to clarify the cause of the impaired FOS induction in MDS. We first examined the mechanisms of FOS mRNA elevation using granulocytes from healthy donors cultured with the translation inhibitor emetine. Emetine increased both transcription and mRNA stability of FOS. p38 MAPK inhibition abolished the emetine-induced increase of FOS transcription but did not affect FOS mRNA stabilization. The binding of an AU-rich element (ARE)-binding protein HuR to FOS mRNA containing an ARE in 3'UTR was increased by emetine, and the knockdown of HuR reduced the FOS mRNA stabilizing effect of emetine. We next compared the emetine-induced transcription and mRNA stabilization of FOS between MDS patients and healthy controls. Increased rates of FOS transcription by emetine were similar in MDS and controls. In the absence of emetine, FOS mRNA decayed to nearly 17% of initial levels in 45 min in both groups. In the presence of emetine, however, 76.7±19.8% of FOS mRNA remained after 45 min in healthy controls, versus 37.9±25.5% in MDS (P<0.01). To our knowledge, this is the first report demonstrating attenuation of stress-induced FOS mRNA stabilization in MDS granulocytes.

Figure 1. FOS mRNA elevation by various stimuli in human granulocytes.

(A) Experimental design. After 30-min preincubation, granulocytes were cultured with an indicated FOS inducer for another 30 min. (B) Dose response to emetine. Granulocytes isolated from healthy volunteers were cultured with the indicated concentrations of emetine. Expression levels of FOS mRNA were normalized by those of an internal control β-actin mRNA. The normalized value in the absence of emetine was set as 1.0. Mean ± SD of three to seven experiments are presented. (C)(D)(E) Comparison of FOS mRNA elevation between MDS patients and healthy controls. Granulocytes were cultured with 200 µg/mL emetine (C), 5 ng/mL GM-CSF (D), or 100 ng/mL LPS (E). The fold increase of the ratios of FOS mRNA to β-actin mRNA by a FOS inducer was plotted, and the horizontal bars represent the means. The statistical comparison was performed by the Mann-Whitney test. **P<0.01.

Figure 2. Effects of MAPK inhibitors.

(A) Experimental design. Granulocytes isolated from healthy individuals were cultured in the presence of a MAPK inhibitor for a total of 60 min. After the initial 30 min, a FOS inducer was added. (B)(C)(D) Effects of MAPK inhibition. A p38 MAPK inhibitor SB203580 (B), an ERK inhibitor U0126 (C), or a JNK inhibitor SP600125 (D) was used. The ratio of FOS mRNA to β-actin mRNA in untreated cells was assigned for 1.0. The results shown are mean ± SD of three to seven experiments. The values were statistically compared by ANOVA. *P<0.05, **P<0.01.

Figure 3. Effects of emetine on FOS mRNA synthesis and decay.

(A) Effects of emetine on FOS transcription. Healthy granulocytes were cultured in the presence of 0.5 mM ethynyl uridine (EU) for a total of 60 min with or without a p38 inhibitor SB203580, and after the initial 30 min, emetine was added. The ratio of EU-incorporated nascent FOS mRNA to nascent β-actin mRNA in untreated cells was set as 1.0, and the mean ± SD of thirteen experiments with emetine alone, and eight experiments with emetine and SB203580 were presented. *P<0.05, **P<0.01 by ANOVA. (B) Effects of emetine on FOS mRNA decay. Transcription was inhibited by 25 µg/mL of DRB, and emetine was added to culture medium after the initial 30 min. The cells were harvested for FOS mRNA quantification every 15 min. The mean ± SD of four experiments were shown. *P<0.05 by the Mann-Whitney test. (C) Effects of MAPK p38 inhibition on nascent FOS mRNA decay. Following the incubation of granulocytes with 1.0 mM EU for 30 min, EU was washed out by RPMI 1640 twice, and the cells were treated with emetine in the presence and absence of SB203580 for a further 45 min. The ratio of EU-incorporated nascent FOS mRNA to β-actin mRNA at 0 min was assigned for 100%. The graph shows mean ± SD of six experiments. **P<0.01 by ANOVA.

Figure 4. Involvement of HuR and AUF1 on FOS mRNA stabilization by emetine.

(A) Location of PCR primers for RIP assay and sequence of pull-down assay probe. The sequence of biotinylated transcript for pull-down assay is in the square. The forward and reverse PCR primers used for RIP assay are highlighted in gray. The bold italic letters indicate the binding site for HuR. (B) RIP assay with anti-HuR and anti-AUF1. The whole cell lysates of emetine-treated and untreated granulocytes were subjected to immunoprecipitation of HuR and AUF1. FOS and GAPDH mRNAs contained in the precipitants were quantified by real-time RT-PCR and normalized by precipitated protein amounts. The values in untreated cells, which is shown by white bars, were assigned for 1.0. Black bars present the data from emetine-treated cells. *P<0.05 by paired t test. (C) Pull-down assay with biotinylated RNA. Cytoplasmic (Cyto) and nuclear (Nuc) lysates were incubated with biotinylated transcripts of FOS or GAPDH, and HuR and AUF1 bound to the transcripts were detected by immunoblotting. The left two lanes show the lysates without pull down as positive controls. (D) Effects of HuR knockdown on FOS mRNA stabilization by emetine. Control or HuR-directed siRNAs were introduced to HL60 by electroporation. After 40 hours, HuR protein levels were examined (right panel), and the cells were cultured in the presence of DRB with or without emetine for 45 min for the analysis of the remaining FOS mRNA levels (left panel). Each point presents mean value of four to seven independent experiments. Error bars indicate SD. *P<0.05 by the Mann-Whitney test.

Figure 5. Comparison of FOS transcription and mRNA stabilization by emetine between MDS patients and the healthy controls.

(A) FOS transcription by emetine. Granulocytes were treated as shown in Figure 3A. The increase in the ratios of EU-incorporated nascent FOS to β-actin mRNA was plotted. The bars represent the mean values. (B) FOS mRNA elevation by emetine in the presence of MPAK p38 inhibitor. Granulocytes from seven patients and nine controls were treated as in Figure 2A with 5 µM SB203580. The values in untreated cells were set as 1.0. Gray and white bars indicate MDS and control, respectively. *P<0.05 by ANOVA. (C) Nascent FOS mRNA remaining for 45 min. The decay of the nascent mRNA labeled with EU was compared between MDS and the controls at 12, 25, and 45 min. The mean ± SD of nascent FOS mRNA levels normalized by β-actin were presented. **P<0.01 by the Mann-Whitney test. (D) Comparison of HuR protein expression between MDS and the controls by western blotting. The numbers above the photos indicate the ratio of HuR band intensity to actin band.