Impairment of HuR-Mediated FOS mRNA Stabilization in Granulocytes From Myelodysplastic Syndrome Patients.

Abstract 2805

Infection is a major cause of death in patients with myelodysplastic syndromes (MDS). Although qualitative and quantitative granulocyte defects have been described, the underlying molecular basis of granulocyte dysfunction in MDS is largely unknown. Recently, we found that FOS mRNA elevation following translation inhibition was significantly smaller in MDS granulocytes than in healthy granulocytes (Shikama et al, Br J Haematol 154, 525–527, 2011). FOS is an immediate early gene that is rapidly expressed in response to various stimuli and cellular stresses, and possesses an AU-rich element (ARE) in its mRNA 3'UTR, to which mRNA stability-regulating proteins, such as HuR, bind. The aim of this study was to clarify the cause of impaired FOS induction following translation inhibition in MDS.

We first confirmed impaired FOS induction in MDS by comparing 17 patients with MDS (refractory anemia) with 17 age-matched healthy controls. Granulocyte fractions, enriched to >90% neutrophils, were cultured with 200 ng/mL of the translation inhibitor emetine at 37 °C for 30 min. FOS mRNA increased 6.2±1.1-fold in controls, vs. 3.5±0.8-fold in MDS (p<0.01). In contrast, when 5 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) or 100 ng/mL lipopolysaccharide (LPS) was added instead of emetine, there were no significant differences in the FOS mRNA elevation between controls and MDS (GM-CSF: 2.4±0.5-fold vs. 2.3±1.3-fold, LPS: 2.2±0.9-fold vs. 2.4±1.1-fold), suggesting that FOS mRNA induction by a translation inhibitor was specifically impaired in MDS granulocytes.

To study the mechanisms of FOS mRNA elevation by emetine, we next examined the involvement of (1) MAPK signaling pathways, (2) FOS transcription and mRNA stabilization, and (3) ARE-binding protein HuR, using healthy granulocytes. Inhibition of MAPK p38, which completely blocked the FOS-upregulating effect of LPS, reduced FOS elevation by emetine to nearly 50%. Neither inhibition of JNK nor ERK altered the effects of emetine. Thus, emetine increases FOS mRNA via MAPK p38-dependent and MAPK-independent mechanisms. Emetine treatment for 30 min increased nascent FOS mRNA 3.5±0.8-fold, which was reduced to 1.1±0.8-fold by MAPK p38 inhibition (p<0.01). When transcription was inhibited, FOS mRNA decayed to 19.2±9.1% of the initial level in 45 min. In the presence of emetine, 78.1±23.5% of FOS mRNA remained at 45 min, and was not affected by p38 inhibition. These data indicate that FOS mRNA elevation by emetine results from both upregulation of transcription via MAPK p38 and p38-independent mRNA stabilization. RNA-protein complex immunoprecipitation showed that 2.5±1.2-fold more FOS mRNA 3'UTR coprecipitated with HuR in emetine-treated cells than in untreated cells (p<0.05). GAPDH mRNA 3'UTR without ARE was not detected regardless of emetine treatment. Although HL60 cells treated with HuR and control siRNA showed similar FOS mRNA decay rates in the absence of emetine, emetine treatment led to a significantly lower FOS mRNA at 45 min in HuR-reduced cells (48.0±12.8%) than in control cells (77.4±13.9%, p<0.05). Thus, emetine stabilizes FOS mRNA via upregulated interaction of HuR with FOS mRNA.

Finally, we investigated which process was impaired in MDS, p38-mediated FOS transcription, or HuR-mediated mRNA stabilization. The increase of FOS transcription in MDS (2.0±0.3-fold) did not significantly differ from that in controls (2.4±0.3-fold). When FOS transcription was blocked by a MAPK p38 inhibitor, FOS mRNA increase by emetine was significantly smaller in MDS (2.9±0.7-fold) than in controls (4.4±1.5-fold, p<0.05), suggesting that the aberrant FOS induction in MDS was not due to impaired FOS transcription. FOS mRNA, which decayed similarly in MDS and control cells in the absence of emetine, remained 41.9±24.6% and 61.0±19.9% in MDS and control granulocytes, respectively, at 45 min in the presence of emetine (p<0.05).

In conclusion, the aberrant FOS mRNA induction by emetine in MDS results from the impairment of HuR-mediated FOS mRNA stabilization. Since nearly 15% of the transcriptome in mammalian cells is targeted by HuR, these results may provide new insights into the functional defects in MDS.