Plastrum testudinis induces γ-globin gene expression through epigenetic histone modifications within the γ-globin gene promoter via activation of the p38 MAPK signaling pathway

Qian,Xinhua; Chen,Jia; Zhao,Danhua; Guo,Lishan; Qian,Xinlai

doi:10.3892/ijmm.2013.1338

Plastrum testudinis induces γ-globin gene expression through epigenetic histone modifications within the γ-globin gene promoter via activation of the p38 MAPK signaling pathway

Authors:
- Xinhua Qian
- Jia Chen
- Danhua Zhao
- Lishan Guo
- Xinlai Qian
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Affiliations: Department of Neonatology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, P.R. China, Pathological Staff Room, Xinxiang Medical University, Xinxiang, Henan 453003, P.R. China
Published online on: April 8, 2013 https://doi.org/10.3892/ijmm.2013.1338
Pages: 1418-1428

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Abstract

The pharmacologically-induced expression of the γ-globin gene, to increase fetal hemoglobin (HbF) production, is a therapeutic strategy used for the treatment of β-thalassemia and sickle cell anemia (SCA). The aim of this study was to investigate the effects of Plastrum testudinis (PT) on differentiation, proliferation, γ-globin gene expression and HbF synthesis in human erythroid cells. For this purpose, we used the K562 human leukemia cell line and human erythroid progenitor cells from normal donors and patients with β-thalassemia cultured using the two-phase liquid culture system. The effects of PT on erythroid differentiation, proliferation, γ-globin gene expression and HbF synthesis, as well as the involvement of epigenetic histone modifications within the γ-globin gene promoter via activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway, were assessed by benzidine staining, trypan-blue dye exclusion, quantitative real-time RT-PCR (qRT-PCR), western blot analysis and chromatin immunoprecipitation (ChIP). PT promoted the erythroid differentiation of K562 cells, and increased γ-globin mRNA accumulation and HbF synthesis without inhibiting cell proliferation in K562 cells and human erythroid progenitors. PT exerted no effect on α- and β-globin gene expression. In human erythroid cells, PT activated the p38 MAPK signaling pathway, and enhanced the acetylation of histone H3 and H4, the phosphorylation of histone H3 within the Gγ- and Aγ-globin gene promoter regions, γ-globin mRNA accumulation and HbF synthesis. These effects were suppressed by pre-treatment with the p38 MAPK inhibitor, SB203580. Epigenetic histone modifications within γ-globin gene promoter regions, via activation of the p38 MAPK signaling pathway, are important for the induction of γ-globin gene expression in human erythroid cells by PT. PT may be a novel potential therapeutic agent for β-hemoglobinopathies, including β-thalassemia and SCA.

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1	Sankaran VG: Targeted therapeutic strategies for fetal hemoglobin induction. Hematology Am Soc Hematol Educ Program. 2011:459–465. 2011. View Article : Google Scholar : PubMed/NCBI
2	Higgs DR, Engel JD and Stamatoyannopoulos G: Thalassaemia. Lancet. 379:373–383. 2012. View Article : Google Scholar : PubMed/NCBI
3	Frenette PS and Atweh GF: Sickle cell disease: old discoveries, new concepts, and future promise. J Clin Invest. 117:850–858. 2007. View Article : Google Scholar : PubMed/NCBI
4	Lawson SE, Roberts IA, Amrolia P, Dokal I, Szydlo R and Darbyshire PJ: Bone marrow transplantation for beta-thalassaemia major: the UK experience in two paediatric centres. Br J Haematol. 120:289–295. 2003. View Article : Google Scholar : PubMed/NCBI
5	Sadelain M, Lisowski L, Samakoglu S, Rivella S, May C and Riviere I: Progress toward the genetic treatment of the beta-thalassemias. Ann N Y Acad Sci. 1054:78–91. 2005. View Article : Google Scholar : PubMed/NCBI
6	Sadelain M: Recent advances in globin gene transfer for the treatment of beta-thalassemia and sickle cell anemia. Curr Opin Hematol. 13:142–148. 2006. View Article : Google Scholar : PubMed/NCBI
7	Pace BS and Zein S: Understanding mechanisms of gamma-globin gene regulation to develop strategies for pharmacological fetal hemoglobin induction. Dev Dyn. 235:1727–1737. 2006. View Article : Google Scholar : PubMed/NCBI
8	Weatherall DJ and Clegg JB: The Thalassaemia Syndromes. 4th edition. Blackwell Science; Oxford: 2001, View Article : Google Scholar
9	Gambari R and Fibach E: Medicinal chemistry of fetal hemoglobin inducers for treatment of beta-thalassemia. Curr Med Chem. 14:199–212. 2007. View Article : Google Scholar : PubMed/NCBI
10	DeSimone J, Koshy M, Dorn L, et al: Maintenance of elevated fetal hemoglobin levels by decitabine during dose interval treatment of sickle cell anemia. Blood. 99:3905–3908. 2002. View Article : Google Scholar : PubMed/NCBI
11	Bradai M, Abad MT, Pissard S, Lamraoui F, Skopinski L and de Montalembert M: Hydroxyurea can eliminate transfusion requirements in children with severe beta-thalassemia. Blood. 102:1529–1530. 2003. View Article : Google Scholar : PubMed/NCBI
12	Mankidy R, Faller DV, Mabaera R, et al: Short-chain fatty acids induce gamma-globin gene expression by displacement of a HDAC3-NCoR repressor complex. Blood. 108:3179–3186. 2006. View Article : Google Scholar : PubMed/NCBI
13	Johnson J, Hunter R, McElveen R, Qian XH, Baliga BS and Pace BS: Fetal hemoglobin induction by the histone deacetylase inhibitor, scriptaid. Cell Mol Biol (Noisy-le-grand). 51:229–238. 2005.PubMed/NCBI
14	Witt O, Monkemeyer S, Ronndahl G, et al: Induction of fetal hemoglobin expression by the histone deacetylase inhibitor apicidin. Blood. 101:2001–2007. 2003. View Article : Google Scholar : PubMed/NCBI
15	Perrine SP, Castaneda SA, Boosalis MS, White GL, Jones BM and Bohacek R: Induction of fetal globin in beta-thalassemia: Cellular obstacles and molecular progress. Ann N Y Acad Sci. 1054:257–265. 2005. View Article : Google Scholar : PubMed/NCBI
16	Quek L and Thein SL: Molecular therapies in beta-thalassaemia. Br J Haematol. 136:353–365. 2007. View Article : Google Scholar : PubMed/NCBI
17	Fathallah H, Sutton M and Atweh GF: Pharmacological induction of fetal hemoglobin: Why haven’t we been more successful in thalassemia? Ann N Y Acad Sci. 1054:228–237. 2005.
18	Saunthararajah Y, Lavelle D and DeSimone J: DNA hypo-methylating agents and sickle cell disease. Br J Haematol. 126:629–636. 2004. View Article : Google Scholar : PubMed/NCBI
19	Normile D: Asian medicine. The new face of traditional Chinese medicine. Science. 299:188–190. 2003. View Article : Google Scholar : PubMed/NCBI
20	Bianchi N, Zuccato C, Lampronti I, Borgatti M and Gambari R: Fetal hemoglobin inducers from the natural world: A novel approach for identification of drugs for the treatment of {beta}-thalassemia and sickle-cell anemia. Evid Based Complement Alternat Med. 6:141–151. 2009.PubMed/NCBI
21	Fang S, Wu Z, Zhang X, et al: Clinical observation on YiSuiShengXueGranule on treating 156 patients with β-thalassemia major and the molecular mechanism study. Biol Pharm Bull. 30:2084–2087. 2007.PubMed/NCBI
22	Yang M, Qian XH, Zhao DH and Fu SZ: Effects of Astragalus polysaccharide on the erythroid lineage and microarray analysis in K562 cells. J Ethnopharmacol. 127:242–250. 2010. View Article : Google Scholar : PubMed/NCBI
23	Guo ZM, Li HJ and Qian XH: γ-globin synthesis in K562 cells induced with Tortois plastron, Astragali, Salviae miltiorrhizae and Codonopsis pilosulae. J Exp Hematology. 16:520–524. 2008.(In Chinese).
24	Miao XY: Shennong Grass after Drainage (Shen Nong’s Herbal Classic). China Press of Traditional Chinese Medicine; Beijing: 1997
25	State Pharmacopoeia Commission of the People’s Republic of China. Carapax et Plastrum Testudinis. Pharmacopoeia of The People’s Republic China. 1:15–16. 2000.
26	Chen DF, Zeng HP, Du SH, et al: Extracts from Plastrum testudinis promote proliferation of rat bone-marrow-derived mesenchymal stem cells. Cell Prolif. 40:196–212. 2007. View Article : Google Scholar : PubMed/NCBI
27	Xie XM, Li XC, Zhong YS, Huang CH and Chen DF: Study of antioxidant activities of Plastrum Testudinis in vitro. China Pharmacy. 17:1368–1370. 2006.
28	Chen DF, Li H and Du SH: Effects of plastrum testudinis on differentiation of transplanted mesenchymal stem cells into neurons in the injured spinal cord of rat. Chinese J of Neuroanatomy. 22:233–237. 2006.
29	Fathallah H, Weinberg RS, Galperin Y, Sutton M and Atweh GF: Role of epigenetic modifications in normal globin gene regulation and butyrate-mediated induction of fetal hemoglobin. Blood. 110:3391–3397. 2007. View Article : Google Scholar : PubMed/NCBI
30	Guo ZM, Li HJ and Qian XH: Accumulation of Gγ-globin mRNA and induction of HbF in K562 cells induced by serum contained Huangqi. Pharm J Chin PLA. 24:15–19. 2008.
31	Fibach E, Manor D, Oppenheim A and Rachmilewitz EA: Proliferation and maturation of human erythroid progenitors in liquid culture. Blood. 73:100–103. 1989.PubMed/NCBI
32	Fibach E: Cell culture and animal models to screen for promising fetal hemoglobin-stimulating compounds. Semin Hematol. 38:374–381. 2001. View Article : Google Scholar : PubMed/NCBI
33	Wu QQ, Qian XH and Xu MJ: Effect of low-dose hydroxyurea with sodium butyrate on globin gene expression in human erythroid progenitor cells. Nan Fang Yi Ke Da Xue Xue Bao. 29:2073–2076. 20812009.(In Chinese).
34	Lam LT, Ronchini C, Norton J, Capobianco AJ and Bresnick EH: Suppression of erythroid but not megakaryocytic differentiation of human K562 erythroleukemic cells by notch-1. J Biol Chem. 275:19676–19684. 2000. View Article : Google Scholar : PubMed/NCBI
35	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods. 25:402–408. 2001.
36	Im H, Grass JA, Johnson KD, Boyer ME, Wu J and Bresnick EH: Measurement of protein-DNA interactions in vivo by chromatin immunoprecipitation. Methods Mol Biol. 284:129–146. 2004.PubMed/NCBI
37	Pace BS, Qian XH, Sangerman J, et al: p38 MAP kinase activation mediates gamma-globin gene induction in erythroid progenitors. Exp Hematol. 31:1089–1096. 2003. View Article : Google Scholar : PubMed/NCBI
38	Mukhopadhyay A, Deplancke B, Walhout AJ and Tissenbaum HA: Chromatin immunoprecipitation (ChIP) coupled to detection by quantitative real-time PCR to study transcription factor binding to DNA in Caenorhabditis elegans. Nat Protoc. 3:698–709. 2008. View Article : Google Scholar : PubMed/NCBI
39	Haring M, Offermann S, Danker T, Horst I, Peterhansel C and Stam M: Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization. Plant Methods. 3:112007. View Article : Google Scholar : PubMed/NCBI
40	Rutherford TR, Clegg JB and Weatherall DJ: K562 human leukaemic cells synthesise embryonic haemoglobin in response to haemin. Nature. 280:164–165. 1979. View Article : Google Scholar : PubMed/NCBI
41	Bianchi N, Ongaro F, Chiarabelli C, et al: Induction of erythroid differentiation of human K562 cells by cisplatin analogs. Biochem Pharmacol. 60:31–40. 2000. View Article : Google Scholar : PubMed/NCBI
42	Mabaera R, West RJ, Conine SJ, et al: A cell stress signaling model of fetal hemoglobin induction: what doesn’t kill red blood cells may make them stronger. Exp Hematol. 36:1057–1072. 2008.PubMed/NCBI
43	Kiefer CM, Hou C, Little JA and Dean A: Epigenetics of beta-globin gene regulation. Mutat Res. 647:68–76. 2008. View Article : Google Scholar : PubMed/NCBI
44	Thein SL, Menzel S, Lathrop M and Garner C: Control of fetal hemoglobin: new insights emerging from genomics and clinical implications. Hum Mol Genet. 18:R216–R223. 2009. View Article : Google Scholar : PubMed/NCBI
45	Bauer DE and Orkin SH: Update on fetal hemoglobin gene regulation in hemoglobinopathies. Curr Opin Pediatr. 23:1–8. 2011. View Article : Google Scholar : PubMed/NCBI
46	Witt O, Sand K and Pekrun A: Butyrate-induced erythroid differentiation of human K562 leukemia cells involves inhibition of ERK and activation of p38 MAP kinase pathways. Blood. 95:2391–2396. 2000.PubMed/NCBI
47	Kouzarides T: Chromatin modifications and their function. Cell. 128:693–705. 2007. View Article : Google Scholar : PubMed/NCBI
48	Bernstein BE, Meissner A and Lander ES: The mammalian epigenome. Cell. 128:669–681. 2007. View Article : Google Scholar
49	Turner BM: Defining an epigenetic code. Nat Cell Biol. 9:2–6. 2007. View Article : Google Scholar
50	Wozniak RJ and Bresnick EH: Epigenetic control of complex loci during erythropoiesis. Curr Top Dev Biol. 82:55–83. 2008. View Article : Google Scholar : PubMed/NCBI
51	Fathallah H, Portnoy G and Atweh GF: Epigenetic analysis of the human alpha- and beta-globin gene clusters. Blood Cells Mol Dis. 40:166–173. 2008. View Article : Google Scholar : PubMed/NCBI
52	Clayton AL and Mahadevan LC: MAP kinase-mediated phosphoacetylation of histone H3 and inducible gene regulation. FEBS Lett. 546:51–58. 2003. View Article : Google Scholar : PubMed/NCBI