Effects Of P38 MAPK Signaling On The Epigenetic Modification At γ Globin Gene Promoter Region

BACKGROUND AND OBJECTIVE1. Increasing y-globin expression by pharmacological approaches ameliorates the clinical symptoms of patients with p-thalassemiaIt has been known that increase of y-globin expression is clinically beneficial in patients with P-thalassemia. Increasing y-globin expression decreases the imbalance of non-a-/a-globin chains inβ-thalassemia and subsequently promotes fetal hemoglobin (Hb F, a2y2) synthesis, substantially ameliorating the clinical symptoms of patients with this disorder.Pharmacological approaches have been developed which aim to reactivate y-globin gene and subsequently increase Hb F. This effort has led to the development of several pharmacologic agents, as lead compounds capable of inducing y-globin expression and increasing Hb F levels. Unfortunately, a number of factors, such as no drug response, hematopoietic suppression, short half-live in vivo, limit the usefulness of these compounds.2. Mechanisms of inducing y-globin gene expression by drugsIt is important to understand the mechanisms of y-globin gene reactivation for identification of novel Hb F inducors, so far, however, the exact mechanisms is not yet fully understood. Traditionally, most Hb F inducing agents have been viewed as affecting y-globin gene expression by altering local promoter chromatin structure, or by regulating the expression via different cell signaling pathways, or by altering the kinetics of erythroid differentiation.3. The role of p38MAPK in mediating drug-induced y-goblin gene expressionp38 mitogen-activated protein kinases (MAPK) signaling is one of the major MAPK pathways. Evidences indicate that p38MAPK may play an important role in y-globin gene reactivation. Some agents, including short chain fatty acids, histone deacetylase (HDAC) inhibitors, cototoxic agents, and even DNA methyltransferase (DNMT) inhibitors, may induce y-globin gene expression via p38MAPK signaling, although the pharmacological properties of these agents are greatly various. This indicates that p38MAPK signaling may locate in the center of mechanisms of inducing y-globin gene expression by drugs.The mechanisms of p38MAPK-mediated y-globin gene expression are not clear yet, but it has been proved that the activation of cAPM response element binding protein (CREB), activating transcription factor 2 (ATF2), or GATA-1 by p38MAPK is closely associated with y-globin gene expression.4. Our previous studies on regulation of y-globin gene expression via p38MAPK signalingIn our previous studies, (1) We further proved that p38MAPK activation mediated y-globin gene expression in transfacted K562 cells with stable expression of hyperphosphorylated or hypophosporylated p38MAPK. (2) We first revealed that not only CREB and ATF2, but also GATA-1 was closely associated with p38MAPK-mediated y-globin gene expression. (3) The role of GATA-1 in p38MAPK-mediated y-globin gene expression was further comfirmed in the K562 cell models with low level of GATA-1.5. Effects of chromatin epigenetic modifications on y-globin gene expressionGene expression is controlled not only by transcription factors, but also by chromatin remodeling and epigenetic modifications. Chromatin epigenetic modifications play important roles in the "open" and "close" ofβ-like globin genes during their developmental swiths. Some of y-globin gene inductors, such as DNMT inhibitors, HDAC inhibitors, short chain fatty acids, also are the inductors of epigenetic modifications. They can induce y-globin gene expression through DNA demethylation, or histone acetylation.6. Hypothesis:p38MAPK signaling may regulate y-globin gene expression via modulating the epigenetic modifications at y-globin gene promoter regionsFurther studies should be carried out for exploring the mechanisms of regulating y-globin gene expression via p38MAPK signaling. It is clear that activation of CREB by p38MAPK signaling leads to the activation of CREB binding protein CBP and p300. CBP/p300 directly acetylates histone depending on their capability of histone acetyltransferase. In addition, butyrate, a well-known Hb F inducer, induces y-globin gene expression via its property of HDAC inhibitor and activation of p38MAPK. These facts raise question as whether p38MAPK signaling upregulate y-globin expression through epigenetic modifications. So far, very few studies focused on answering this question. We hypothesize that p38MAPK may signal to y-globin gene promoter and histones, cause them modified, in turn, promot y-globin expression.7. The goal and significance of our current studyIn this study, we examined the changes of histone acetylation and/or phosphorylation, DNA methylation at y-globin gene promoter regions under different levels of p38MAPK phosphorylation in butyrate-treated K562 cells and transfected K562 cells with hyperphosphorylation/hypophosphorylation of p38MAPK. The major goal of this study was to investigate the effects of p38MAPK signal on epigenectic modifications at y-globin gene promoter. The results of our study may provid novel evidences for the mechanisms of y-globin gene expression induced by drugs, and be greatly helpful for exploring novel drugs for treatment of patients withβ-thalassemia and other hemoglobin disorders.METHOD1. Cell cultureK562 cell line or Hela cell line was cultured in RPMI-1640 medium containing 10%fetal bovine serum, 100U/ml penicillin and 100μg/ml streptomycin at 37℃in a 5% C02 humidified atmosphere.2. Establishment of K562 cell models with different levels of phosphorylated p38MAPK2.1 Sodim butyrate-treated K562 cells K562 cells were treated with sodium butyrate (NaB) at final concentration of 0.5 mM for 48h to induce hyperphosphorylated p38MAPK [K562(NaB)]. In another group of K562 cells, 10μM SB203580, the p38MAPK inhibitor, were used to previously treat cells for 1h before NaB treatment [K562(SB+NaB)]. K562 cells without any treatment were used as blank control (K562).2.2 Transfected K562 cells with stable expression of hyperphosphorylation /hypophosphorylation of p38MAPK The transfected K562 cells with hyperphosphorylation/hypophosphorylation of p38MAPK were constructed in our previous study. In this current study, these cell models were grouped as:(1) Transfected K562 cells with hyperphosphorylation of p38MAPK (K562-MKK3-Glu);(2) Transfected K562 cells with hypophosphorylation of p38MAPK (K562-MKK3-Ala);(3) Transfected K562 cells with vector (K562-vector);(4) SB203580-treated transfected K562 cells with hyperphosphorylation of p38MAPK [k562-MKK3-Glu(SB)];(5) Blank control of K562 cells (K562).3. Characterization of K562 cell models with different levels of phosphorylated p38MAPKRT-PCR was employed to determine the relative levels of p38MAPK mRNA, Gy-and Ay-globin mRNA. Western blot was used to measure the relative levels of total p38MAPK, phosphorylated p38MAPK, and Hb F.4. Analysis of acetylation and/or phosphorylation of histone at y-globin gene promoter regionReal time PCR based-chromatin immunoprecipitation (ChIP) was employed to determine the levels of acetylation of histone H3 and H4 (acH3 and acH4), phosphorylation/acetylation of histone H3 (ph/acH3) at Gy-and Ay-globin gene promoter regions.5. Analysis of DNA methylation at y-globin gene promoterThe DNA methylation at y-globin gene promoter was analyzed using bisculfite-treatment-based DNA sequencing.6. Statistical analysisData are presented as means±SD. Statistical analysis was performed by one-way ANOVA using SPSS 17.0 software. The least significant difference post hoc test (LSD or Dunnett's T3) was used for ANOVA statistics. P values<0.05 are considered statistically significant.RESULTS1. Characterization of different K562 cell models1.1 The levels of p38MAPK mRNA in different K562 cell models Results from RT-PCR analysis showed that there was no significant difference in the levels of p38MAPK mRNA among the various K562 cell models (F=2.420, P=0.081).1.2 The levels of total p38MAPK and phosphorylated p38MAPK in different K562 cell models Among the different K562 cell models, no difference in the total p38MAPK level was found (F=0.302, P<0.926), while the levels of phosphorylated p38MAPK were significantly different (F=8154.574, P=0.000). There was a 4.2-fold or 4.3-fold increase of the levels of phosphorylated p38MAPK in K562 (NaB) or K562-MKK3-Glu cells, respectively, in comparison with the K562 cells. The levels of phosphorylated p38MAPK of K562(SB+NaB), K562-MKK3-Glu(SB), K562-MKK3-Ala, K562-vector, and K562 cells were similar (P>0.05).1.3 The levels of Gy-and Ay-globin mRNA in different K562 cell models The levels of Gy-globin mRNA in K562 (NaB) or K562-MKK3-Glu cells was higher than that in K562 cells (1.4-fold increase, respectively. P=0.000). But the previous treatment of SB203580 reduced the levels of Gy-globin mRNA in these two cells. There was no difference in the levels of Gγ-globin mRNA between K562-MKK3-Ala and K562 cells (P>0.05). The level of Ay-globin mRNA in K562-MKK3-Ala cells was lower than other cell models (P<0.05).1.4 The levels of Hb F in different K562 cell models Expectedly, K562 (NaB) or K562-MKK3-Glu cells had higher Hb F levels than the K562 cells, and there was 3.4-fold increase in the Hb F levels, respectively (P=0.000). SB203580 decreased the levels of Hb F in K562 (NaB) and K562-MKK3-Glu. K562-MKK3-Ala cells, which expressed low level of phosphorylated p38MAPK, had significantly lower level of Hb F than K562 cells (P=0.000).2. Analysis of acH3 and acH4 at Gy-and Ay-globin gene promoter regions2.1 The levels of acH3 or acH4 at Gy-globin gene, Ay-globin gene, or necdin gene promoter regions in each group of K562 cell models. In each group of K562 cell models, hyperacetylation of histone H3 and H4 was found at Gy-and Ay-globin gene promoters, while hypoacetylation of H3 and H4 was detected at necdin gene promoter, the negative control (P<0.05). In Hela cells which never express globin chains, the levels of acH3 or acH4 at Gy-, Ay-globin gene and necdin gene promoter regions were not different (P>0.05).2.2 NaB increases the levels of acH3 and acH4 at Gy-and Ay-globin gene promoter regions via activation of p38MAPK In K562 (NaB) cells, the levels of acH3 and acH4 at Gy-and Ay-globin gene promoters were significantly higher than that in K562 cells, and there was a 3.1-fold or 2.6-fold increase in Gy-acH3 or Gy-acH4 (P<0.05), a 3.7-fold or 3.2-fold increase in Ay-acH3 or Ay-acH4 (P<0.05), respectively, in K562(NaB) cells. The experiment of SB203580 inhibition revealed that the levels of acH3 and acH4 at Gy-and Ay-globin gene promoters in K562(SB+NaB) cells were decreased, in comparison with K562(NaB) cells (48.7% and 40.6%,41.8% and 28.9% levels, respectively).2.3 The levels of acH3 and acH4 at Gy-and Ay-globin gene promoters increase in transfected K562 cells with hyperphosphorylated p38MAPK The K562-MKK3-Glu cells with hyperphosphorylated p38MAPK produced higher levels of acH3 and acH4 at Gy-and Ay-globin gene promoter regions than K562 cells (a 3.1-fold and 2.1-fold,4.2-fold and 3.2-fold increase was observed, respectively. P<0.05). K562-MKK3-Ala cells, the model with hypophosphorylated p38MAPK, did not show difference in the levels of acH3 and acH4 at Gy-and Ay-globin gene promoter in comparison with K562 cells (P>0.05). When K562-MKK3-Glu cells were previous treated with SB203580, the levels of acH3 and acH4 decreased.3. Analysis of ph/acH3 at Gy-and Ay-globin gene promoter regions3.1 Levels of ph/acH3 at Gy-globin gene, Ay-globin gene, or necdin gene promoter regions in each group of K562 cell models. In each group of K562 cell models, high levels of ph/acH3 was found at Gy-and Ay-globin gene promoters, while low levels of ph/acH3 was detected at necdin gene promoter (P<0.05). In Hela cells, in which the globin gene never express, there was no difference in the levels of ph/acH3 at Gy-, Ay-globin gene and necdin gene promoter regions (P>0.05).3.2 NaB increases the levels of ph/acH3 at Gy-and Ay-globin gene promoter regions via activation of p38MAPK In K562(NaB) cells, the levels of ph/acH3 at Gy-and Ay-globin gene promoters were significantly higher than that in K562 cells, and there was a 2.9-and 3.2-fold increase (P<0.05), respectively, in K562(NaB) cells. The results from experiment of SB203580 inhibition showed that the levels of ph/acH3 at Gy-and Ay-globin gene promoters in K562(SB+NaB) cells were decreased, in comparison with K562(NaB) cells (39.5% and 36.8%, respectively).3.3 The levels of ph/acH3 at Gy-and Ay-globin gene promoters increase in transfected K562 cells with hyperphosphorylated p38MAPK The K562-MKK3-Glu cells with hyperphosphorylated p38MAPK produced higher levels of ph/acH3 at Gy-and Ay-globin gene promoter regions than K562 cells (a 2.7-fold and 2.8-fold increase was observed, respectively). K562-MKK3-Ala cells, the model with hypophosphorylated p38MAPK, did not show difference in the levels of ph/acH3 in comparison with K562 cells (P>0.05). When K562-MKK3-Glu cells were previous treated with SB203580, the levels of Gy-and Ay-ph/acH3 decreased.4. The levels of DNA methylation at y-globin gene promoters in different K562 cell modelsThe results from bisculfite-treatment-based DNA sequencing showed that the methylation levels at-350,-256,-162,-53,-50,+6,+17, and+50 CpG sites at y-globin gene promoters in all K562 cell models were very low (ranging from 0% to 2.5%). But in Hela cells, the methylation rates at-256,-53, and-50 CpG sites were 100%, and the total rate at the eight sits of CpG was 37.5%.Conclusions1. Using the NaB-treated K562 cells with hyperphosphorylated p38MAPK and transfected K562 cells with hyperphosphorylation/hypophosphorylation of p38MAPK, we further confirmed that activated p38MAPK signal induces y-globin gene expression and promots Hb F synthesis.2. A real time PCR-based chromatin immunoprecipitation assay is developed to analyze the acetylation and phosphorylation of histone at y-globin gene promoters.3. A bisculfite-based DNA sequencing assay is developed to detect the DNA methylation at y-globin gene promoters.4. This study provides more evidences to confirm that activated p38MAPK signal increases acH3 and acH4 at y-globin gene promoter regions. This effect of p38MAPK on acH3 and acH4 can be inhibited by SB203580. The results indicate p38MAPK may mediate acetylation of histone at y-globin gene promoter regions.5. We first reveales that activated p38MAPK signal increases the level of ph/acH3 at y-globin gene promoter regions. SB203580 inhibits this effect of p38MAPK. The results indicate that p38MAPK may also mediate phosphorylation of histone H3 at y-globin gene promoter regions.6. We first explore the effects of p38MAPK on the DNA methylation at y-globin gene promoters. The results of this experiment disclose that the methylation level at y-globin gene promoter in K562 cells is very low. It indicates that, in K562 cells, p38MAPK signal-mediated y-globin gene expression may not depend on the change of DNA methylation at y-globin gene promoter.7. In summary, the results of our study indicate that p38MAPK signal regulates y-globin gene expression via histone modification at y-globin gene promoter regions.