Impairment of FOS mRNA induction by a translation inhibitor puromycin in granulocytes from myelodysplastic syndrome patients

Field: Medicine (Health Sciences)

Myelodysplastic syndrome (MDS) is a clonal disorder characterized by ineffective haematopoiesis and dysplasia in multilineages of blood cells. Terminally differentiated cells exhibit quantitative, morphological and functional abnormalities. However, the underlying causes of neutrophil dysfunction in MDS are largely unknown. DNA microarray analysis of peripheral neutrophils has revealed that the expression of FOS and ITGB2 (CD18) are down-regulated in half of MDS patients (Pellagatti et al, 2004). FOS is an immediate-early gene that is rapidly induced in response to chemical and physical stresses (Angel & Karin, 1991) and cytokines, including a neutrophil activator, granulocyte-macrophage colony-stimulating factor (GM-CSF) (McColl et al, 1991). In MDS patients, GM-CSF priming effects on reactive oxygen species production are impaired (Fuhler et al, 2003). We recently identified CSF2RB_v79 (βC79; Shikama et al, 2005), a splice variant of CSF2RB (GM-CSF receptor β chain) that retained intron V, in MDS neutrophils (Shikama et al, 2005). CSF2RB_v79 mRNA, in which the first premature termination codon (PTC) is generated more than 50 nucleotides upstream from the junction of VIII and IX exons, is a target of nonsense-mediated mRNA decay (NMD), an mRNA surveillance system that degrades mRNA harbouring a PTC more than 50 nucleotides upstream from an exon-exon junction (Maquat, 1995). We hypothesized that excessive production of NMD-sensitive mRNAs resulted in their aberrantly low expression, causing cellular dysfunction in MDS. It has been shown that translation inhibitors stop NMD, leading to an increase in NMD-sensitive mRNAs (Noensie & Dietz, 2001). To test our hypothesis, the effects of translation inhibition on ITGB2, FOS and PTC-bearing CSF2RB_v79 in neutrophils from MDS patients were analysed. Twelve MDS patients, including eleven with refractory anaemia (RA) and one with RA with ringed sideroblasts (RARS), and twelve healthy controls provided informed consent in accordance with the Institutional Human Research Committee and Helsinki Declaration. Three patients with RA were receiving treatment at the time of the study, one with erythropoietin, one with prednisolone, and one with red blood cell transfusions. The RARS patient was treated with prednisolone and red blood cell and platelet transfusions. The remaining eight RA patients were under observation without any treatment. The granulocyte fraction was obtained from heparinized peripheral blood by centrifugation through Lymphoprep (Axis-Shield PoC AS, Oslo, Norway) followed by haemolysis. The staining of the fractionated cells with May-Grünwald and Giemsa solutions revealed that more than 93% of the cells were neutrophilic granulocytes. The granulocytes were resuspended in RPMI 1640 medium (Wako Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Sigma Chemical Co., St. Louis, MO, USA) at the concentration of 1x106 cells/ml, and incubated with or without 20 μg/ml puromycin, which maximally inhibited NMD (Shikama et al, 2010), for up to 4·5 h at 37°C in a humidified atmosphere with 5% CO2. The cell viability measured by trypan blue staining was not affected by puromycin in the preliminary experiments (99·7 ± 0·3% at 0 h, 99·5 ± 0·4% without puromycin and 99·3 ± 0·2% with puromycin after 4·5 h, n = 6 including four healthy controls and two patients). Total cellular RNA was extracted from 4 to 5 × 106 cells cultured with or without puromycin at 0-, 1·5-, 3- and 4·5-h time points using RNeasy Plus Mini Kit (Qiagen GmbH, Hilden, Germany), and reverse-transcribed using random primers and Moloney murine leukaemia virus reverse transcriptase (Invitrogen, Carlsbad, USA). The expression levels of CSF2RB_v79, total CSF2RB, ITGB2 and FOS were quantified by real-time polymerase chain reaction using the primers listed in Table I and SYBR Premix Ex Taq (Takara Bio. Inc., Otsu, Japan) in LightCycler (Roche Diagnostics GmbH, Mannheim, Germany). Each value was normalized by that of an internal control ACTB. The increase in normalized values by puromycin treatment was compared between MDS and control groups by the Mann–Whitney test. The protein-coding region in FOS mRNA was sequenced using DYEnamic ET terminator kit (GE Healthcare, Buckinghampshire, UK) in an ABI PRISM 3100 DNA sequencer (Applied Biosystems, Foster City, CA, USA). When granulocytes from eight healthy controls were cultured in the absence of puromycin, mRNA of CSF2RB_v79, total CSF2RB and ITGB2 decreased to 40·6 ± 13·2%, 56·2 ± 15·0% and 57·1 ± 14·1%, respectively, of baseline levels in the first 1·5 h, and no further significant changes were observed at the 4·5-h time point (46·7 ± 23·1%, 48·7 ± 22·0% and 57·7 ± 10·6%, respectively). FOS mRNA levels at 1·5 h were 3·6 ± 0·2% of baseline levels and 0·09 ± 0·5% at 4·5 h. The changes of mRNA levels at each time point in an RA patient and an RARS patient were within the mean value ± standard deviation of controls. The increase in each mRNA following puromycin treatment, which was evaluated by the comparison of mRNA levels between the cells cultured with and without puromycin for the indicated period, is presented in Fig 1. In healthy controls, puromycin up-regulated CSF2RB_v79 mRNA in a time-dependent manner, and the increase at 4·5 h was 3·37 ± 1·39-fold. The time-dependent up-regulation of CSF2RB_v79 mRNA was also observed in MDS with no significant differences compared with controls, suggesting that there was no excessive production of PTC-bearing CSF2RB_v79 in MDS. Total CSF2RB mRNA was not increased more than 2-fold after 4·5 h in both groups. Fold increase of mRNA by puromycin. White and black boxes represent the mean values of healthy controls and patients, respectively. Error bars represent SD. *P < 0·01, **P < 0·05. ITGB2 expression was not significantly changed at 4·5 h in both controls (1·28 ± 0·57-fold) and patients (0·67 ± 0·30-fold), suggesting that NMD-sensitive ITGB2 mRNA is rarely synthesized in both groups. Surprisingly, the increase of FOS mRNA (6·22 ± 1·91-fold after 1·5 h) was more rapid than that of CSF2RB_v79 in controls. In MDS patients, the increase of FOS was significantly smaller compared to controls at each time point (2·56 ± 1·62-fold at 1·5 h, P < 0·01). This up-regulation, however, was not due to cessation of NMD, because no PTC was detected in FOS mRNA isolated after 4·5-h puromycin treatment. The stresses by other translation inhibitors, anisomycin and cycloheximide, were previously shown to induce FOS mRNA in fibroblasts (Rahmsdorf et al, 1987) and osteosarcoma cell lines (Bébien et al, 2003). The present study found that FOS mRNA was also induced in response to translation-inhibiting stress in peripheral granulocytes and that the response was significantly impaired in MDS. This impairment may be relevant to cellular dysfunction in MDS. This study was partially supported by Fukushima Medical Research Fund, Nishinomiya Basic Research Fund, and the Smoking Research Foundation. We thank Dr. Midori Yatabe (Fukushima Medical University, Fukushima, Japan) for English correction. YS was the principal investigator and takes primary responsibility for the paper. XF and TO contributed to laboratory work. TS, HN, KI, and KO recruited the patients. TS, YT and JK contributed to drafting the paper. The authors declare no competing financial interest.