| BACKGROUND AND OBJECTIVEβ-thalassemia, results from quantitative abnormalities of the adult β-globin chain, is one of the common genetic diseases that cause considerable morbidity and mortality throughout the world. Mutations or deletions of β-globin gene cause low synthesis of β-globin chains, resulting in free α-globin chains deposit on red blood cell surface, which leads to decline of erythrocyte deformability and increase of hematoclasis in spleen. To date there is no safety, effective and easy curing method of β-thalassemia.Induction of fetal hemoglobin (HbF) is a most promising therapeutic for p-thalassemia because reactivation of HbF expression decreases the imbalance of non-a/a-globin chains inβ-thalassemia and ameliorates the clinical symptoms of individuals with the disorder. Hence, research efforts aim to develop different classes of pharmacological agents that reactivate y-globin expression for therapeutic purposes are ongoing and great progress has been made during the last3decades.The drugs seeking to up-regulate HbF include①DNA methyl transferase (DNMT) inhibitors, such as5’-aza-cytidine (5-AZAC),②Histone deacetylase (HDAC) inhibitors, such as trichostatin A(TSA) and apicidin,③short-chain fatty acid and its metabolites, such as butyrate and valproic acid,④cytotoxic agents, such as hydroxyurea (HU),⑤imune modulation drugs, such as thalidomide and pomaliomide,⑥ormones agents, such as progesterone,⑦growth factors, such as erythropoietin (EPO),⑧drugs from the nature world, such as Furocoumarins, Rapamycin and Resveratrol and astragalus polysaccharides. Their effective concentration can be variable from individual to individual and they showed steep dose-response profiles with relatively narrow margins of safety. And it still has a long way to go for clinical use of above agents because of the potential carcinogenicity and their high cost. Therefore, it is important to determine the mechanism of HbF inducers and then develop more effective and safe drugs that are applicable to treat P-thalassemia.For identification of novel HbF inducers, understanding the potential mechanisms that involved in y-globin gene reactivation is the focus of intense investigation. A number of studies have been performed to determine the molecular events that stimulate HbF synthesis and γ-globin gene was thought to be reactived through inhitbitting DNA synthesis (HU) and changing the erythroid cytodynamics (resveratrol). Recently several important signal transduction pathways, epigenetic modifications and transcription factors have been shown to be associated with HbF increase in K562cells and primary human CD34+cells. Recent evidence has emerged demonstrating the important role of Mitogen-activated protein kinases (MAPK) signaling pathways in mediating the effects of drug-induced HbF production. The MAPK family consists of extracellular signal-related kinases (ERK1, ERK2), c-Jun NH2-terminal kinase, and p38MAPKs. Erythroid differentiation in K562cells is augmented by butyrate and HU through of p38activation and ERK MAPK inactivation. A role for p38MAPK signaling has been further confirmed for HbF introduction using ertyhroid progenitors, knockout mice, and K562stable lines. Despite the various pharmacological properties of these compounds, HDAC inhibitors (TSA, MS-275, scriptaid, etc), cototoxic agents and DNMT inhibitors may induce y-globin gene expression via p38MAPK signaling. Our previous study have found that p38phosphorylation was involved in sodium butyrate (NaB) and astragalus polysaccharides-induced y-globin gene expression in K562cells, and the sustained activation ofp38MAPK cell signaling can up-regulate γ-globin gene mRNA levels and increase HbF synthesis.These studies indicate that different pharmacologic agents converge on the p38MAPK pathway to activate γ-globin expression.Our previous study also proved that acetylation of histone H3and H4and phosphorylation of H3were induced by the activatin of p38MAPK cell signaling, and this effect was abolished by SB203580, a specific inhibitor of p38MAPK cell signaling. The results proved that γ-globin gene can be induced by p38MAPK-mediated epigenetic modifications at y-globin gene promoter regions.Through electrophoretic mobility shift assay (EMSA) and super-EMSA assay, we also observed the binding of GATAbinding protein-1(GATA1)、cAMP responsive element binding protein (CREB) and activating transcription factor2(ATF-2) to specific DNA sequence in y-globin gene promoter regions, suggesting that all of these transcription factors might be involved in p38MAPK-mediated γ-globin gene regulation. Pace et al have reported that y-globin induction by butyrate and trichostatin A (TSA) involves transactivation by transcription factors ATF-2and CREB via p38pathway activated by reactive oxygen species. However, the effect of GATA1on p38MAPK-induced y-globin gene expression was not determined.GATA1is the founding member of GATA family of transcription factors. GATA1is a hematopoietic cell-specific transcription factor that recognizes the consensus sequence (A/T)GATA(A/G). Nearly all erythroid cell-specific genes, including the P-globin gene, contain functionally important GATAl binding sites within their regulatory regions. GATAl is essential for erythroid cell development, as erythroidprecursors in mice deficient for GATAl fail to survive andmature, and the embryos die of anemia. GATAl also can induce terminal erythroid maturation when expressed in a G1E cell line, which lacks GATAl. It was reported that GATAl was able to reverse the γ-globin gene silence effect of Stat3in human K562cells and MEL cells. Then it was found that hypoxia-inducible factor1-mediated human GATA1induction promoted erythroid differentiation under hypoxic conditions.Our previous study revealed that nuclear protein GATAl was able to combine specific DNA sequence of Gγ-globin gene promoter, but whether it was involved in p38MAPK-induced γ-gfobin gene expression remains to be explored.Therefore, we produced various cell models in this study, determined levels of γ-globin mRNA and HbF, and examined the changes of histone H3acetylation and phosphorylation at y-globin gene promoter regions under different levels of GATAl in butyrate-treated K562cells. The results of our study may delineate the role of GATAl in y-globin expression induced by p38MAPK-mediated epigenectic modifications at γ-globin gene promoter regions and shed new light on the mechanisms responsible for the induction of y-globin gene expression by HbF inducers. METHOD1. Cell cultureK562cell line or Hela cell line were cultured in RPMI-1640medium containing10%fetal bovine serum,100U/mL penicillin and100μg/mL streptomycin at37℃in a5%CO2humidified atmosphere.2. Establishment of K562cell models2.1p38hyper-/hypophosphorylationK562cells ①p38hyperphosphorylation K562cells[K562-MKK3(Glu)],②p38hypo-phosphorylation K562cells [K562-MKK3(Ala)]③vector conrrol K562cells (K562-pcDNA3.1),④K562-MKK3(Glu) cells pretreated1h with10μM p38MAPK inhibitor SB203580[K562-MKK3(Glu)+SB],⑤K62cells treated with sodium butyrate (NaB) at final concentration of0.5mM for48h [K562(NaB)],⑥K562(NaB) cells pretreated1h with10μM SB203580[K562-MKK3(Glu)+SB],⑦K562cells,⑧Hela cells.2.2Transfected K562cells with silenced expression of GATA1A pair of64-nt Oligonucleotide was synthesis and then annealed to construct recombinant plasmid pSUPER-shRNA with shRNA expressionvector pSUPER-EGFP. The plasmid was transfected into K562cells to produce cells in which GATA1mRNA expression was knocked down. Cell models were grouped as follows:①transfected K562cells with pSUPER-shRNA Recombinant plasmid (K562-shRNA),②transfected K562cells with pSUPER-EGFP vector (K562-pSUPER),③transfected K562(NaB) cells with pSUPER-shRNA recombinant plasmid [K562(NaB)-shRNA],④transfected K562(NaB) cells with pSUPER-EGFP vector [K562(NaB)-pSUPER],⑤K562cells,⑥K562(NaB) cells.3. Real time RT-PCR was employed to determine the relative levels of GATA1, Aγ-and Gγ-globin mRNA. Western blotting was used to measure the relative levels of GATA1, phospho-GATAl, phospho-p38MAPK and HbF.4. Real time PCR based-chromatin immunoprecipitation (ChIP) was employed to observe the levels of GATA1, acetylation of histone H3(acH3) and phosphorylation/acetylationof histone H3(ph/acH3) at Aγ and Gγ-globin gene promoter regions.5. Statistical analysisData are presented as meansts. Statistical analysis was performed by paired-samples t-test or one-way ANOVA using SPSS13.0software. The least significant difference post hoc test (LSD or Dunnett’s T3) was used for ANOVA statistics. Probability of less than0.05was considered significant. RESULTS1. Changes of GATA1in p38MAPK-induced y globin gene expression1.1levels of p38pophosphorylation in hyper-/hypophosphorylation p38MAPK K562cellsLevels of p38phosphorylation (p-p38) in K562(NaB) cells and K562-MKK3(Glu) cells were higher than that in K562cells (2.189-fold and2.159-fold increase, respectively, P<0.026). But the previous treatment of SB203580reduced the levels of p-p38to37.764%of that in cells treated with NaB alone and56.461%of that in K562-MKK3(Glu) cells (P<0.030), respectively. Levels of p-p38in K562-MKK3(Ala) cells decreased to42.235%compared with K562cells (P<0.001). There was no significant difference in the levels of p-p38between K562cells and K562-pDNA3.1cells (P=0.997).1.2GATA1mRNA expression in hyper-/hypophosphoiylation p38MAPK K562cellsK562(NaB) cells and K562-MKK3(Glu) cells produced higher levels of GATA1mRNA than that in K562cells (1.565-fold and1.533-fold increase, respectively, P≤0.002). But the previous treatment of SB203580diminished the levels of GATA1mRNA to50.209%of that in cells treated with NaB alone and73.114%of that in K562-MKK3(Glu) cells (P≤0.009), respectively. There was no significant difference in the levels of GATA1mRNA among K562cells, K562-pDNA3.1cells and K562-MKK3(Ala) cells (P≥0.111).1.3GATA1expression and phosphorylation in hyper-/hypophosphorylation p38MAPKK562cellsLevels of GATA1in K562(NaB) cells was1.532-fold increased compared with untreated K562cells (P<0.001). Levels of p-GATA1in K562(NaB) cells and K562-MKK3(Glu) cells were higher than that in K562cells(2.066-fold and1.532-fold increase, respectivelyP<0.001). But the previous treatment of SB203580reduced the levels of p-GATA1to38.802%of that in K562(NaB) cells and30.996%of that in K562-MKK3(Glu) cells (P<0.001), respectively. Levels of GATA1mRNA decreased in K562-MKK3(Ala) cells compared with K562cells(46.632%, P<0.001). There was no significant difference in the levels of GATA1mRNA between K562and K562-pDNA3.1cells (P=0.077).1.4Levels of Ay-and Gy-globin mRNA in hyper-/hypophosphoiylation p38MAPKK562cellsLevels of Ay-mRNA in K562(NaB) cells was3.1-fold increased compared with untreated K562cells (P<0.001). Levels of p-GATAl in K562(NaB) cells and K562-MKK3(Glu) cells were up-regulated in K562cells (3.0-fold increase, P<0.001). But levels of GATA1mRNA decreased to27.1%of that in K562(NaB) cells and27.1%of that in K562-MKK3(Glu) cells (P<0.001) after the pretreatment of SB203580. GATA1mRNA levels decreased in K562-MKK3(Ala) cells compared with K562cells(3.0-fold increase, P<0.001). There was no significant difference in the levels of GATA1mRNA between K562cells and K562-pDNA3.1cells (P=0.057).1.5Levels of HbF mRNA in hyper-/hypophosphorylation p38M APK K562cellsLevels of HbF in K562(NaB) cells and K562-MKK3(Glu) cells were higher than that in K562cells (1.841-fold and1.786-fold increase respectively, P<0.001). But the pretreated of SB203580reduced the levels of HbF to51.515%of that in cells treated with NaB alone and35.142%of that in K562-MKK3(Glu) cells (P<0.001). HbF level decreased to45.144%in K562-MKK3(Ala) cells compared with K562cells(P<0.001). There was no difference in HbF levels between K562cells and K562-pDNA3.1cells (P=0.613). HbF was undetectable in Hela cells.The data revealed that γ-globin gene was induced by the active of p38MAPK cell signaling and the changes of GATA1expression is related to levels of p38phosphorylation.2Influence of GATA1knockdown on p38MAPK-induced y-globin gene expression in K562cells2.1GATA1mRNA levels in GATA1knockdown K562cellsLevels of GATA1mRNA in K562-shRNA cells and K562(NaB)-shRNA cells decreased to47.904%and48.644%(P<0.023) of that in K562-pSUPER cells and K562(NaB)-pSUPER cells, respectively.2.2GATA1and p-GATA1levels in GATA1knockdown K562cellsLevels of GATA1in K562-shRNA cells and K562(NaB)-shRNA cells reduced to58.810%and32.522%(P<0.001) of that in K562-pSUPER cells and K562(NaB)-pSUPER cells, respectively. And when it came to levels of p-GATAl, there were59.792%and49.023%(P<0.023) decrease in K562-shRNA cells and K562(NaB)-shRNA cells copared twith K562-pSUPER cells and K562(NaB)-pSUPER cells, respectively.2.3Aγ and Gγ-mRNA levels in GATA1knockdown K562cellsLevels of GATA1mRNA in K562-shRNA cells was1.137-fold increased than that in K562-pSUPER cells (P=0.009). Levels of GATA1mRNA in K562(NaB)-shRNA cells diminished to50.599%of that in K562(NaB)-pSUPER cells (P<0.001).2.4HbF synthesis in GATA1knockdown K562cellsExpectedly, K562-shRNA cells and K562(NaB)-shRNA cells had lower HbF levels than K562-pSUPER cells and K562(NaB)-shRNA cells (decreased to63.019%and80.332%, respectively, P<0.001). These data suggested that the knockdown of GATA1gene express have the ability to inhibit the phosphorylation of GATA1, y-globin mRNA expression and HbF synthesis.3Role of GATA1in y-globin Gene Expression Induced by p38MAPK-mediated Epigenetic Modifications3.1%Input of amplification fragment at y-globin gene promoter regions3.1.1%Input of GATA1at Ay-globin gene promoter regions%Input of amplification fragment immunoprecipitated by GATA1antibody at Ay-globin gene promoter regions in K562(NaB) cells were significantly higher than that immunoprecipitated by IgG antibody (t=7.282,t=0.018).%Input of amplification fragment immunoprecipitated by GATA1antibody and the IgG antibody did not show difference in K562cells (t=2.567, P=0.124), K562(NaB)+SB cells (t=3.445,P=0.075) and K562-shRNA cells (t=4.193,P=0.052). Hela cells produced lower%Input of amplification fragment immunoprecipitated by GATA1antibody at Aγ-globin gene promoter regions compared with IgG antibody (t=15.741, P=0.004).3.1.2%Input of GATA1at Gy-globin gene promoter regions%Input of amplification fragment immunoprecipitated by GATA1antibody at Gγ-globin gene promoter regions in K562(NaB) cells (t=7.895, P=0.016) and K562cells (/=17.312, P=0.003) were significantly higher than that immunoprecipitated by IgG antibody.%Input of amplification fragment immunoprecipitated by GATA1antibody and the IgG antibody did not show difference in K562(NaB)-shRNA cells (t=2.067, P=0.175) and K562(NaB)+SB cells (t=0.068, P=0.952).%Input of amplification fragment immunoprecipitated by GATA1antibody at Gγ-globin gene promoter regions in Hela cells was lower compared with that by IgG antibody (t=21.840, P=0.002). 3.1.3%Input of ac-H3at Ay-globin gene promoter regions%Input of amplification fragment immunoprecipitated by ac-H3antibody at Aγ-globin gene promoter regions in K562cells (t=15.412, P=0.004) and K562(NaB) cells (t=9.295, P=0.011)were significantly higher than that immunoprecipitated by IgG antibody.%Input of amplification fragment immunoprecipitated by ac-H3antibody at Aγ-globin gene promoter regions in K562(NaB)+SB cells were significantly lower than that immunoprecipitated by IgG antibody (t=12.401, P=0.006).%Input of amplification fragment immunoprecipitated by ac-H3antibody and the IgG antibody did not show difference in K562(NaB)-shRNA cells (t=0.749, P=0.532). Hela cells had a lower%Input of amplification fragment immunoprecipitated by ac-H3antibody at Aγ-globin gene promoter regions compared with IgG antibody (t=6.092, P=0.026).3.1.4%Input of ac-H3at Gy-globin gene promoter regions%Input of amplification fragment immunoprecipitated by ac-H3antibody at Vglobin gene promoter regions in K562(NaB) cells (t=10.320, P=0.009) and K562cells (t=8.474,.P=0.014) were significantly higher than that immunoprecipitated by IgG antibody.%Input of amplification fragment immunoprecipitated by ac-H3antibody and the IgG antibody did not show difference in K562(NaB)-shRNA cells (t-0.478, P=0.680) and K562(NaB)+SB cells0=3.277, P=0.082). Hela cells produced lower%Input of amplification fragment immunoprecipitated by ac-H3antibody at Gγ-globin gene promoter regions compared with IgG antibody (t=31.261, P=0.001).3.1.5%Input of ph/ac-H3at Aγ-globin gene promoter regions%Input of amplification fragment immunoprecipitated by ph/ac-H3antibody at Aγ-globin gene promoter regions in K562(NaB) cells (t=22.589, P=0.002), K562cells (t=6.755, P=0.021), K562(NaB)+SB cells (t=4.778, P=0.041) and K562(NaB)-shRNA cells (t=10.333, P=0.009) were significantly higher than that immunoprecipitated by IgG antibody. There was no significant difference between%Input of amplification fragment immunoprecipitated by ph/ac-H3antibody and IgG antibody at Ay-globin gene promoter regions in Hela cells (t=2.090, P=0.172).3.1.6%Input of ac-H3at Gγ-globin gene promoter regions%Input of amplification fragment immunoprecipitated by ph/ac-H3antibody at Gγ-globin gene promoter regions in K562(NaB) cells (t=23.144, P=0.002), K562cells (t=10.130, P=0.010) and K562(NaB)-shRNA cells (t=15.524, P=0.004) were significantly higher than that immunoprecipitated by IgG antibody.%Input of amplification fragment immunoprecipitated by ph/ac-H3antibody and the IgG antibody did not show difference in K562(NaB)+SB cells (t=4.263,P=0.051). Hela cells had a tower%Input of immunoprecipitated by ph/ac-H3antibody at Gγ-globin gene promoter regions compared with IgG antibody (t=6.465, P=0.023).3.2Changes of GATA1and epigenetic modifications at γ-globin gene promoter regions in NaB-induced γ-globin gene expression3.2.1NaB increased the levels of GATA1at Ay-and Gy-globin gene promoter regions in K562cellsHyperacetylation of histone H3was found at Aγ-and Gγ-globin gene promoter regions in K562(NaB) cells (1.740-fold and1.721-fold increase, respectively, P<0.004) compared with K562cells. Levels of Ay-GATAland Gy-GATA1in K562(NaB)+SB cells decreased to26.064%and33.823%respectively of that in K562(NaB) cells after1h pretreatment of SB (P≤0.001).3.2.2NaB increased the levels of ac-H3at Aγ-and Gy-globin gene promoter regions in K562cellsIn K562(NaB) cells, the levels of ac-H3at Aγ-and Gγ-globin gene promoter regions were significantly higher than that in K562cells, and there were3.049-fold and4.960-fold increase respectively in Aγ-acH3and Gγ-acH3levels (P<0.017) in K562(NaB) cells. The experiment of SB203580inhibition revealed that the levels of ac-H3at Aγ-and Gγ-globin gene promoter regions in K562(NaB)+SB cells were decreased, compared with K562(NaB) cells (to2.248%and0.584%, respectively, P≤0.011).3.2.3NaB increased the levels of ph/ac-H3at Aγ-and Gγ-globin gene promoter regions in K562cellsK562(NaB) cells produced higher levels of ph/ac-H3at Aγ-and Gγ-globin gene promoter regions than K562cells (8.639-fold and19.45-fold increase were observed respectively, P<0.004). The experiment of SB203580inhibition revealed that the levels of ph/acH3at Aγ-and Gγ-globin gene promoter regions in K562(NaB)+SB cells were decreased, compared with K562(NaB) cells (decreased to12.324%and6.433%respectively, P<0.004).3.3Influence of GATA1knockdown to epigenetic modifications mediated by NaB-induced p38MAPK hyperphosphorylated3.3.1The levels of GATA1was inhibited by GATA1shRNAat Aγ-and Gγ-globin gene promoter regionsWe found that levels of GATA1at Aγ-and Gγ-globin gene promoter regions in K562(NaB)-shRNA cells were significantly lower than that in K562(NaB) cells (reduced to17.846%and18.457%, respectively, P<0.001).3.3.2The levels of ac-H3was inhibited by GATA1shRNA at Aγ and Gγ-globin gene promoter regionsCompared with K562(NaB) cells, levels of ac-H3at Aγ-and Gγ-globin gene promoter regions in K562(NaB)-shRNA cells cut down to14.845%and11.213%, respectively (P<0.008). 3.3.3The levels of ph/ac-H3was inhibited by GATA1shRNA at Aγ-and Gγ-globin gene promoter regionsK562(NaB)-shRNA cells with hyperphosphorylated p38MAPK produced fower levels of ph/ac-H3at Aγ-and Gγ-globin gene promoter regions than K562(NaB) cells (decreased to18.100%and9.584%, respectively, P≤0.003).The results revealed the important role of GATA1in γ-globin gene expression induced by p38MAPK-mediated epigenetic modifications at γ-globin gene promopter regions.Conclusions1. We found that the expression of GATA1-mRNA, p-GATA1, Aγ-and Gγ-globin mRNA and HbF were up-regulated in hyperphosphorylation p38MAPK K562cells and down-regulated in hypophosphorylation p38MAPK K562cells, and the increase efforts were abolished by SB203580.The data futher suggests that the changes of GATA1has close relation with the levels ofp38phosphorylation.2. We first described the knockdown of GATA1gene may inhibit the phosphorylation of GATA1, γ-globin mRNA expression and HbF synthesis in NaB-induced K562cells with p38hyperphosphorylation. The results reveal the important role of GATA1in p38MAPK-induced y-globin gene expression.3. We first described the decrease of GATA1, p-GATA1, ac-H3and ph/ac-H3at γ-globgene promopter regions and Aγ/Gγ-globin mRNA expression and HbF synthesis in mediated by GATA1shRNA in NaB-induced p38hyperphosphorylation K562cells. The data implies the important role of GATA1in γ-globin gene expression induced by p38MAPK-mediated epigenetic modifications atγ-globin gene promopter regions. |
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