β联蛋白参与骨形成蛋白9诱导的C3H10T1/2细胞向心肌细胞样细胞分化
|
摘要
目的探讨骨形成蛋白9(BMP9)诱导C3H10T1/2细胞向心肌细胞样细胞分化过程β联蛋白(β-catenin)的作用。方法利用重组腺病毒BMP9(Ad-BMP9)诱导C3H10T1/2细胞分化,通过Western blot法检测Ad-BMP9及不同剂量XAV-939处理后β-catenin的激活情况;在完全抑制β-catenin的情况下,Ad-BMP9诱导1周,通过实时定量PCR法检测心肌细胞增强因子2C(MEF2C)、心肌组织特异性转录因子GATA结合蛋白4(GATA4);Ad-BMP9处理3周后,通过Western blot法及免疫荧光技术检测心肌连接子蛋白43(Cx43)、心肌肌钙蛋白T(c Tn T)的表达。结果在感染效率约50%的情况下,BMP9可过度激活β-catenin,使其磷酸化水平增高;在XAV-939抑制β-catenin后,Ad-BMP9诱导的C3H10T1/2细胞表达的c Tn T、GATA4、Cx43和MEF2C均受到抑制。结论β-catenin能被BMP9激活并参与BMP9诱导的C3H10T1/2细胞向心肌细胞样细胞分化。
Objective To investigate the role of β-catenin in the differentiation of C3H10T1/2 cells into cardiomyocyte-like cells induced by bone morphogenetic protein 9( BMP9). Methods C3H10T1/2 cells were transfected with the recombinant adenovirus expressing BMP9( Ad-BMP9) and differentiated into cardiomyocytes in vitro for up to 21 days. The activated level of β-catenin in the cells after cultivated with Ad-BMP9 and different concentrations of β-catenin specific inhibitor XAV-939 was detected by Western blotting. Real-time quantitative PCR( qRT-PCR) was performed to evaluate the expressions of cardiac specific gene myocyte enhancer factor 2C( MEF2C) and GATA binding protein 4( GATA4) in the cells one week after induced by Ad-BMP9 and different concentrations of XAV-939. Three weeks after the transfection,the expressions of connexin 43( Cx43) and cardiac troponin T( c Tn T) were analyzed by Western blotting,and the location of Cx43 in the cells was observed by immunofluorescence technique. Results While the transfection was successful in 50% of the cells positive for BMP9,β-catenin was excessively activated and their phosphorylation level remarkably increased. After XAV-939 inhibited the activity of β-catenin,the expressions of MEF2 C,GATA4,Cx43,c Tn T in C3H10T1/2 cells induced by BMP9 were significantly suppressed. Conclusion The β-catenin can be activated by BMP9 and the activation of β-catenin plays an important role in the differentiation of C3H10T1/2 cel s into cardiomyocyte-like cel s induced by BMP9.
Objective To investigate the role of β-catenin in the differentiation of C3H10T1/2 cells into cardiomyocyte-like cells induced by bone morphogenetic protein 9( BMP9). Methods C3H10T1/2 cells were transfected with the recombinant adenovirus expressing BMP9( Ad-BMP9) and differentiated into cardiomyocytes in vitro for up to 21 days. The activated level of β-catenin in the cells after cultivated with Ad-BMP9 and different concentrations of β-catenin specific inhibitor XAV-939 was detected by Western blotting. Real-time quantitative PCR( qRT-PCR) was performed to evaluate the expressions of cardiac specific gene myocyte enhancer factor 2C( MEF2C) and GATA binding protein 4( GATA4) in the cells one week after induced by Ad-BMP9 and different concentrations of XAV-939. Three weeks after the transfection,the expressions of connexin 43( Cx43) and cardiac troponin T( c Tn T) were analyzed by Western blotting,and the location of Cx43 in the cells was observed by immunofluorescence technique. Results While the transfection was successful in 50% of the cells positive for BMP9,β-catenin was excessively activated and their phosphorylation level remarkably increased. After XAV-939 inhibited the activity of β-catenin,the expressions of MEF2 C,GATA4,Cx43,c Tn T in C3H10T1/2 cells induced by BMP9 were significantly suppressed. Conclusion The β-catenin can be activated by BMP9 and the activation of β-catenin plays an important role in the differentiation of C3H10T1/2 cel s into cardiomyocyte-like cel s induced by BMP9.
引文
[1]姜辉,王辉山,汪曾炜,等.体外诱导人骨髓间充质干细胞转化为心肌样细胞的基因表达[J].沈阳部队医药,2010,23(1)5-7.
[2]Elnakish M T,Hassan F,Dakhlallah D,et al.Mesenchymal stem cells for cardiac regeneration:translation to bedside reality[J/OL]Stem Cells Int,2012,2012:646038.doi:10.1155/2012/646038Epub 2012 Jun 7.
[3]贺继刚,李洪荣,桂龙升,等.小鼠骨髓间充质干细胞SCA-1+CD45+CD31+亚群向心肌样细胞分化的分子基础[J].临床心血管病杂志,2015,31(12):1332-1335.
[4]Shankar S,Marsh L,Srivastava R K.EGCG inhibits growth o human pancreatic tumors orthotopically implanted in BALB/c nud mice through modulation of FKHRL1/FOXO3a and neuropilin[J]Mol Cell Biochem,2013,372(1/2):83-94.
[5]耿雪静,刘书霞,陈沅.ERK5和JNK参与BMP9诱导的C3H10T1/2细胞向心肌样细胞分化[J].细胞与分子免疫学杂志,2014,30(8):829-832.
[6]Chen L,Wei H,Tan J,et al.Bone morphogenetic protein 9 and 13induce C3H10T1/2 cell differentiation to cardiomyocyte-like cells in vitro[J].Cell Transplant,2015,24(5):909-920.
[7]Carmena A,Gisselbrecht S,Harrison J,et al.Combinatorial signalin codes for the progressive determination of cell fates in the Drosophila embryonic mesoderm[J].Genes Dev,1998,12(24)3910-3922.
[8]Krausova M,Korinek V.Wnt signaling in adult intestinal stem cell and cancer[J].Cell Signal,2014,26(3):570-579.
[9]Kandyba E,Leung Y,Chen Y B,et al.Competitive balance o intrabulge BMP/Wnt signaling reveals a robust gene network rulin stem cell homeostasis and cyclic activation[J].Proc Natl Acad Sci U S A,2013,110(4):1351-1356.
[10]谭金童,陈沅,向平.BMP9对C3H10T1/2干细胞向心肌样细胞分化的影响[J].重庆医科大学学报,2009,34(6):672-676.
[11]Rebuzzini P,Cebral E,Fassina L,et al.Arsenic trioxide alters th differentiation of mouse embryonic stem cell into cardiomyocyte[J/OL].Sci Rep,2015,5:14993.doi:10.1038/srep14993.
[12]Zamilpa R,Navarro M M,Flores I,et al.Stem cell mechanisms durin left ventricular remodeling post-myocardial infarction:Repair and regeneration[J].World J Cardiol,2014,6(7):610-620.
[13]Feng H Z,Chen G,Nan C,et al.Abnormal splicing in the N-termina variable region of cardiac troponin T impairs systolic function of th heart with preserved Frank-Starling compensation[J/OL].Physiol Rep,2014,2(9).pii:e12139.doi:10.14814/phy2.12139.Print 2014 Sep 1.
[14]Shi S,Wu X,Wang X,et al.Differentiation of bone marrow mesenchyma stem cells to cardiomyocyte-like cells is regulated by the combined low dose treatment of transforming growth factor-β1 and 5-azacytidin[J/OL].Stem Cells Int,2016,2016:3816256.doi:10.11552016/3816256.Epub 2015 Nov 30.
[15]Wang L,Liu Z,Yin C,et al.Improved generation of induced cardiomyocyte using a polycistronic construct expressing optimal ratio of Gata4,Mef2and Tbx5[J/OL].J Vis Exp,2015,(105):e53426.doi:10.3791/53426.
[16]Liu S,Geng X,Fang S,et al.Wnt3a combined with bone morphogenetic protein 9 induce C3H10T1/2 cells differentiation into cardiomyocyte-like cells in vitro[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2015,31(4):448-451.
[17]Lei B,Chai W,Wang Z,et al.Highly expressed UNC119 promotes hepatocellular carcinoma cell proliferation through Wnt/β-catenin signaling and predicts a poor prognosis[J].Am J Cancer Res,2015,5(10):3123-3134.
[18]Papathanasiou I,Malizos K N,Tsezou A.Bone morphogenetic protein-2-induced Wnt/beta-catenin signaling pathway activation through enhanced low-density-lipoprotein receptor-related protein 5 catabolic activity contributes to hypertrophy in osteoarthritic chondrocytes[J/OL].Arthritis Res Ther,2012,14(2):R82.doi:10.1186/ar3805.
[19]Tian X H,Hou W J,Yan F,et al.XAV939,a tankyrase 1 inhibitior,promotes cell apoptosis in neuroblastoma cell lines by inhibiting Wnt/β-catenin signaling pathway[J/OL].J Exp Clin Cancer Res,2013,32:100.doi:10.1186/1756-9966-32-100.
[20]Dregalla R C,Zhou J,Idate R R,et al.Regulatory roles of tankyrase 1 at telomeres and in DNA repair:suppression of T-SCE and stabilization of DNA-PKcs[J].Aging(Albany NY),2010,2(10):691-708.
[21]Paige S L,Osugi T,Afanasiev O K,et al.Endogenous Wnt/betacatenin signaling is required for cardiac differentiation in human embryonic stem cells[J/OL].PLoS One,2010,5(6):e11134.doi:10.1371/journal.pone.0011134.
[22]Wang H,Hao J,Hong C C.Cardiac induction of embryonic stem cells by a small molecule inhibitor of Wnt/β-catenin signaling[J].ACS Chem Biol,2011,6(2):192-197.
[23]Minami I,Yamada K,Otsuji T G,et al.A small molecule that promotes cardiac differentiation of human pluripotent stem cells under defined,cytokine-and Xeno-free conditions[J].Cell Rep,2012,2(5):1448-1460.
[24]Bikkavilli R K,Malbon C C.Dishevelled-KSRP complex regulates Wnt signaling through post-transcriptional stabilization ofβ-catenin mRNA[J].J Cell Sci,2010,123(Pt 8):1352-1362.
[25]Wang Q,Kurita H,Carreira V,et al.Ah receptor activation by dioxin disrupts activin,BMP,and WNT signals during the early differentiation of mouse embryonic stem cells and inhibits cardiomyocyte functions[J].Toxicol Sci,2016,149(2):346-357.
[26]Mungrue I N,Zhao P,Yao Y,et al.Abcc6 deficiency causes increased infarct size and apoptosis in a mouse cardiac ischemia-reperfusion model[J].Arterioscler Thromb Vasc Biol,2011,31(12):2806-2812.
[27]Vanhatupa S,Ojansivu M,Autio R,et al.Bone morphogenetic protein-2 induces donor-dependent osteogenic and adipogenic differentiation in human adipose stem cells[J].Stem Cells Transl Med,2015,4(12):1391-1402.
[28]张芬,陈沅,孙文静,等.p38MAPK通路在BMP9诱导C3H10T1/2干细胞向心肌样细胞分化中的作用[J].重庆医科大学学报,2012,37(8):657-660.
[29]Zhang H,Wang J,Deng F,et al.Canonical Wnt signaling acts synergistically on BMP9-induced osteo/odontoblastic differentiation of stem cells of dental apical papilla(SCAPs)[J].Biomaterials,2015,39:145-154.
[30]Liu Y,Liu Y,Zhang R,et al.All-trans retinoic acid modulates bone morphogenic protein 9-induced osteogenesis and adipogenesis of preadipocytes through BMP/Smad and Wnt/β-catenin signaling pathways[J].Int J Biochem Cell Biol,2014,47:47-56.
[31]Kandyba E,Leung Y,Chen Y B,et al.Competitive balance of intrabulge BMP/Wnt signaling reveals a robust gene network ruling stem cell homeostasis and cyclic activation[J].Proc Natl Acad Sci U S A,2013,110(4):1351-1356.
[32]Jian J,Sun L,Cheng X,et al.Calycosin-7-O-β-d-glucopyranoside stimulates osteoblast differentiation through regulating the BMP/WNTsignaling pathways[J].Acta Pharm Sin B,2015,5(5):454-460.
[2]Elnakish M T,Hassan F,Dakhlallah D,et al.Mesenchymal stem cells for cardiac regeneration:translation to bedside reality[J/OL]Stem Cells Int,2012,2012:646038.doi:10.1155/2012/646038Epub 2012 Jun 7.
[3]贺继刚,李洪荣,桂龙升,等.小鼠骨髓间充质干细胞SCA-1+CD45+CD31+亚群向心肌样细胞分化的分子基础[J].临床心血管病杂志,2015,31(12):1332-1335.
[4]Shankar S,Marsh L,Srivastava R K.EGCG inhibits growth o human pancreatic tumors orthotopically implanted in BALB/c nud mice through modulation of FKHRL1/FOXO3a and neuropilin[J]Mol Cell Biochem,2013,372(1/2):83-94.
[5]耿雪静,刘书霞,陈沅.ERK5和JNK参与BMP9诱导的C3H10T1/2细胞向心肌样细胞分化[J].细胞与分子免疫学杂志,2014,30(8):829-832.
[6]Chen L,Wei H,Tan J,et al.Bone morphogenetic protein 9 and 13induce C3H10T1/2 cell differentiation to cardiomyocyte-like cells in vitro[J].Cell Transplant,2015,24(5):909-920.
[7]Carmena A,Gisselbrecht S,Harrison J,et al.Combinatorial signalin codes for the progressive determination of cell fates in the Drosophila embryonic mesoderm[J].Genes Dev,1998,12(24)3910-3922.
[8]Krausova M,Korinek V.Wnt signaling in adult intestinal stem cell and cancer[J].Cell Signal,2014,26(3):570-579.
[9]Kandyba E,Leung Y,Chen Y B,et al.Competitive balance o intrabulge BMP/Wnt signaling reveals a robust gene network rulin stem cell homeostasis and cyclic activation[J].Proc Natl Acad Sci U S A,2013,110(4):1351-1356.
[10]谭金童,陈沅,向平.BMP9对C3H10T1/2干细胞向心肌样细胞分化的影响[J].重庆医科大学学报,2009,34(6):672-676.
[11]Rebuzzini P,Cebral E,Fassina L,et al.Arsenic trioxide alters th differentiation of mouse embryonic stem cell into cardiomyocyte[J/OL].Sci Rep,2015,5:14993.doi:10.1038/srep14993.
[12]Zamilpa R,Navarro M M,Flores I,et al.Stem cell mechanisms durin left ventricular remodeling post-myocardial infarction:Repair and regeneration[J].World J Cardiol,2014,6(7):610-620.
[13]Feng H Z,Chen G,Nan C,et al.Abnormal splicing in the N-termina variable region of cardiac troponin T impairs systolic function of th heart with preserved Frank-Starling compensation[J/OL].Physiol Rep,2014,2(9).pii:e12139.doi:10.14814/phy2.12139.Print 2014 Sep 1.
[14]Shi S,Wu X,Wang X,et al.Differentiation of bone marrow mesenchyma stem cells to cardiomyocyte-like cells is regulated by the combined low dose treatment of transforming growth factor-β1 and 5-azacytidin[J/OL].Stem Cells Int,2016,2016:3816256.doi:10.11552016/3816256.Epub 2015 Nov 30.
[15]Wang L,Liu Z,Yin C,et al.Improved generation of induced cardiomyocyte using a polycistronic construct expressing optimal ratio of Gata4,Mef2and Tbx5[J/OL].J Vis Exp,2015,(105):e53426.doi:10.3791/53426.
[16]Liu S,Geng X,Fang S,et al.Wnt3a combined with bone morphogenetic protein 9 induce C3H10T1/2 cells differentiation into cardiomyocyte-like cells in vitro[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2015,31(4):448-451.
[17]Lei B,Chai W,Wang Z,et al.Highly expressed UNC119 promotes hepatocellular carcinoma cell proliferation through Wnt/β-catenin signaling and predicts a poor prognosis[J].Am J Cancer Res,2015,5(10):3123-3134.
[18]Papathanasiou I,Malizos K N,Tsezou A.Bone morphogenetic protein-2-induced Wnt/beta-catenin signaling pathway activation through enhanced low-density-lipoprotein receptor-related protein 5 catabolic activity contributes to hypertrophy in osteoarthritic chondrocytes[J/OL].Arthritis Res Ther,2012,14(2):R82.doi:10.1186/ar3805.
[19]Tian X H,Hou W J,Yan F,et al.XAV939,a tankyrase 1 inhibitior,promotes cell apoptosis in neuroblastoma cell lines by inhibiting Wnt/β-catenin signaling pathway[J/OL].J Exp Clin Cancer Res,2013,32:100.doi:10.1186/1756-9966-32-100.
[20]Dregalla R C,Zhou J,Idate R R,et al.Regulatory roles of tankyrase 1 at telomeres and in DNA repair:suppression of T-SCE and stabilization of DNA-PKcs[J].Aging(Albany NY),2010,2(10):691-708.
[21]Paige S L,Osugi T,Afanasiev O K,et al.Endogenous Wnt/betacatenin signaling is required for cardiac differentiation in human embryonic stem cells[J/OL].PLoS One,2010,5(6):e11134.doi:10.1371/journal.pone.0011134.
[22]Wang H,Hao J,Hong C C.Cardiac induction of embryonic stem cells by a small molecule inhibitor of Wnt/β-catenin signaling[J].ACS Chem Biol,2011,6(2):192-197.
[23]Minami I,Yamada K,Otsuji T G,et al.A small molecule that promotes cardiac differentiation of human pluripotent stem cells under defined,cytokine-and Xeno-free conditions[J].Cell Rep,2012,2(5):1448-1460.
[24]Bikkavilli R K,Malbon C C.Dishevelled-KSRP complex regulates Wnt signaling through post-transcriptional stabilization ofβ-catenin mRNA[J].J Cell Sci,2010,123(Pt 8):1352-1362.
[25]Wang Q,Kurita H,Carreira V,et al.Ah receptor activation by dioxin disrupts activin,BMP,and WNT signals during the early differentiation of mouse embryonic stem cells and inhibits cardiomyocyte functions[J].Toxicol Sci,2016,149(2):346-357.
[26]Mungrue I N,Zhao P,Yao Y,et al.Abcc6 deficiency causes increased infarct size and apoptosis in a mouse cardiac ischemia-reperfusion model[J].Arterioscler Thromb Vasc Biol,2011,31(12):2806-2812.
[27]Vanhatupa S,Ojansivu M,Autio R,et al.Bone morphogenetic protein-2 induces donor-dependent osteogenic and adipogenic differentiation in human adipose stem cells[J].Stem Cells Transl Med,2015,4(12):1391-1402.
[28]张芬,陈沅,孙文静,等.p38MAPK通路在BMP9诱导C3H10T1/2干细胞向心肌样细胞分化中的作用[J].重庆医科大学学报,2012,37(8):657-660.
[29]Zhang H,Wang J,Deng F,et al.Canonical Wnt signaling acts synergistically on BMP9-induced osteo/odontoblastic differentiation of stem cells of dental apical papilla(SCAPs)[J].Biomaterials,2015,39:145-154.
[30]Liu Y,Liu Y,Zhang R,et al.All-trans retinoic acid modulates bone morphogenic protein 9-induced osteogenesis and adipogenesis of preadipocytes through BMP/Smad and Wnt/β-catenin signaling pathways[J].Int J Biochem Cell Biol,2014,47:47-56.
[31]Kandyba E,Leung Y,Chen Y B,et al.Competitive balance of intrabulge BMP/Wnt signaling reveals a robust gene network ruling stem cell homeostasis and cyclic activation[J].Proc Natl Acad Sci U S A,2013,110(4):1351-1356.
[32]Jian J,Sun L,Cheng X,et al.Calycosin-7-O-β-d-glucopyranoside stimulates osteoblast differentiation through regulating the BMP/WNTsignaling pathways[J].Acta Pharm Sin B,2015,5(5):454-460.