PGC-1α通过线粒体介导巨噬细胞极化状态的机制研究
|
摘要
目的探讨通过敲低PGC-1α对肿瘤相关巨噬细胞(tumor associated macrophages,TAMs)极化状态的影响及其机制。方法以小鼠腹腔巨噬细胞(mouse peritoneal macrophages,PMs)为研究对象,与4T1细胞共培养24 h建立TAMs;shRNA转染构建瞬时低表达PGC-1α的TAMs;Western blot检测TAMs中PGC-1α、TAMs极化指标、TAMs线粒体功能蛋白的蛋白表达;Real-time PCR(qRT-PCR)检测TAMs的mt DNA水平;流式细胞术检测TAMs极化标志物,ROS水平的变化;ATP测定试剂盒检测TAMs中ATP水平的变化。结果与PMs组比较,TAMs组PGC-1α蛋白表达上调(P <0. 05),M2型分泌细胞因子蛋白(TGF-β1、IL-10)表达升高(P <0. 05),线粒体功能相关蛋白(NRF-1、TFAM)表达升高(P <0. 05),M2型极化标记物(CD206)表达升高(P <0. 05),M1型分泌细胞因子蛋白(TNF-α、IL-1β)表达降低(P <0. 05);sh-PGC-1αTAMs组较TAMs组发生了相反的变化,即PGC-1α蛋白表达降低(P <0. 05),M2型分泌细胞因子蛋白(TGF-β1、IL-10)表达降低(P <0. 05),线粒体功能相关蛋白(NRF-1、TFAM)表达降低(P <0. 05),M2型极化标记物(CD206)表达降低(P <0. 05),M1型分泌细胞因子蛋白(TNF-α、IL-1β)表达升高(P <0. 05),M1型极化标记物(CD86)表达升高(P <0. 05)。结论 PGC-1α参与了乳腺TAMs向M2极化的过程,其机制可能为癌细胞与TAMs相互作用,增强了TAMs中依赖PGC-1α的线粒体功能,促使细胞向M2分化,并产生M2型细胞因子。
Objective To investigate the effect of knocking down peroxisome proliferator-activated receptor gamma coactivator 1-alpha(PGC-1α) on the polarization in tumor-associated macrophages(TAMs).Methods Mouse peritoneal macrophages(PMs) were co-cultured with mouse mammary tumor cell line 4T1 for 24 h to establish TAMs. shRNAs sequence of PGC-1α was synthesized and transfected to construct TAMs with transient low expression of PGC-1α. Western blotting was used to detect the expression of PGC-1α and mitochondrial function-related proteins. Real-time PCR(qRT-PCR) was employed to measure the mt DNA levels,flow cytometry to detect macrophage polarization markers,reactive oxygen species(ROS) kit to test the generation of ROS,and ATP assay kit to study the ATP level in the TAMs. Results Compared with PMs,PGC-1α was up-regulated in TAMs(P < 0. 05),so were the M2-type secreted cytokines(TGF-β1 and IL-10)(P < 0. 05),mitochondrial function-related proteins [nuclear respiratory factor 1(NRF1) and mitochondrial transcription factor A(TFAM)](P < 0. 05),and M2 type polarization marker(CD206)(P <0. 05),but the levels of M1 type secreted cytokines(TNF-α and IL-1β) were decreased(P < 0. 05). shPGC-1α transfection led to opposite changes when compared with TAMs(all P < 0. 05). Conclusion PGC-1α is involved in the polarization of breast TAMs to M2. The mechanism may be due to the interaction between the tumor cells and TAMs,which enhances the PGC-1α-dependent mitochondrial function in TAMs,promotes cell differentiation to M2,and then produces M2 cytokines.
Objective To investigate the effect of knocking down peroxisome proliferator-activated receptor gamma coactivator 1-alpha(PGC-1α) on the polarization in tumor-associated macrophages(TAMs).Methods Mouse peritoneal macrophages(PMs) were co-cultured with mouse mammary tumor cell line 4T1 for 24 h to establish TAMs. shRNAs sequence of PGC-1α was synthesized and transfected to construct TAMs with transient low expression of PGC-1α. Western blotting was used to detect the expression of PGC-1α and mitochondrial function-related proteins. Real-time PCR(qRT-PCR) was employed to measure the mt DNA levels,flow cytometry to detect macrophage polarization markers,reactive oxygen species(ROS) kit to test the generation of ROS,and ATP assay kit to study the ATP level in the TAMs. Results Compared with PMs,PGC-1α was up-regulated in TAMs(P < 0. 05),so were the M2-type secreted cytokines(TGF-β1 and IL-10)(P < 0. 05),mitochondrial function-related proteins [nuclear respiratory factor 1(NRF1) and mitochondrial transcription factor A(TFAM)](P < 0. 05),and M2 type polarization marker(CD206)(P <0. 05),but the levels of M1 type secreted cytokines(TNF-α and IL-1β) were decreased(P < 0. 05). shPGC-1α transfection led to opposite changes when compared with TAMs(all P < 0. 05). Conclusion PGC-1α is involved in the polarization of breast TAMs to M2. The mechanism may be due to the interaction between the tumor cells and TAMs,which enhances the PGC-1α-dependent mitochondrial function in TAMs,promotes cell differentiation to M2,and then produces M2 cytokines.
引文
[1] OBEID E,NANDA R,FU Y X,et al. The role of tumorassociated macrophages in breast cancer progression(review)[J]. Int J Oncol,2013,43(1):5-12. DOI:10. 3892/ijo.2013. 1938.
[2] NOY R,POLLARD J W. Tumor-associated macrophages:from mechanisms to therapy[J]. Immunity,2014,41(1):49-61. DOI:10. 1016/j. immuni. 2014. 06. 010.
[3] EVANS-KNOWELL A, LARUE A C, FINDLAY V J.MicroRNAs and their impact on breast cancer,the tumor microenvironment,and disparities[J]. Adv Cancer Res,2017,133:51-76. DOI:10. 1016/bs. acr. 2016. 08. 003.
[4] TANG X,MO C,WANG Y,et al. Anti-tumour strategies aiming to target tumour-associated macrophages[J]. Immunology,2013,138(2):93-104. DOI:10. 1111/imm. 12023.
[5] BELGIOVINE C,BELLO E,LIGUORI M,et al. Lurbinectedin reduces tumour-associated macrophages and the inflammatory tumour microenvironment in preclinical models[J]. Br J Cancer,2017,117(5):628-638. DOI:10. 1038/bjc.2017. 205.
[6] CHANMEE T,ONTONG P,KONNO K,et al. Tumor-associated macrophages as major players in the tumor microenvironment[J]. Cancers(Basel),2014,6(3):1670-1690. DOI:10. 3390/cancers6031670.
[7] KOVALEVA O V,SAMOILOVA D V,SHITOVA M S,et al.Tumor associated macrophages in kidney cancer[J]. Anal Cell Pathol(Amst),2016,2016:9307549. DOI:10. 1155/2016/9307549.
[8] CHAWLA A. Control of macrophage activation and function by PPARs[J]. Circ Res,2010,106(10):1559-1569.DOI:10. 1161/CIRCRESAHA. 110. 216523.
[9] COFFELT S B,TAL A O,SCHOLZ A,et al. Angiopoietin-2regulates gene expression in TIE2-expressing monocytes and augments their inherent proangiogenic functions[J]. Cancer Res,2010,70(13):5270-5280. DOI:10. 1158/0008-5472. CAN-10-0012.
[10]代朦,靳鑫,雷优扬,等.过表达miR-382的肿瘤相关巨噬细胞对三阴性乳腺癌生物学特性的影响[J].第三军医大学学报,2018,40(15):1375-1382. DOI:10. 16016/j.1000-5404. 20180110.DAI M,JIN X,LEI Y Y,et al. Effect of miR-382 overexpressing tumor-associated macrophages on biological properties of triple-negative breast cancer 4T1 cells[J]. J Third Mil Med Univ,2018,40(15):1375-1382. DOI:10. 16016/j.1000-5404. 20180110.
[11] CHIBA S,HISAMATSU T,SUZUKI H,et al. Glycolysis regulates LPS-induced cytokine production in M2 polarized human macrophages[J]. Immunol Lett,2017,183:17-23. DOI:10. 1016/j. imlet. 2017. 01. 012.
[12] NIU Z,SHI Q,ZHANG W,et al. Caspase-1 cleaves PPARγfor potentiating the pro-tumor action of TAMs[J].Nat Commun,2017,8(1):766. DOI:10. 1038/s41467-017-00523-6.
[13] LI Y,XU S,LI J,et al. SIRT1 facilitates hepatocellular carcinoma metastasis by promoting PGC-1α-mediated mitochondrial biogenesis[J]. Oncotarget,2016,7(20):29255-29274. DOI:10. 18632/oncotarget. 8711.
[14] WANG C,KAR S,LAI X,et al. Triple negative breast cancer in Asia:an insider’s view[J]. Cancer Treat Rev,2018,62:29-38. DOI:10. 1016/j. ctrv. 2017. 10. 014.
[15] KIM M S,SHIGENAGA J K,MOSER A H,et al. Suppression of estrogen-related receptor alpha and medium-chain acylcoenzyme A dehydrogenase in the acute-phase response[J].J Lipid Res,2005,46(10):2282-2288. DOI:10. 1194/jlr. M500217-JLR200.
[16] BULER M,AATSINKI S M,SKOUMAL R,et al. Energysensing factors coactivator peroxisome proliferator-activated receptorγcoactivator 1-α(PGC-1α)and AMP-activated protein kinase control expression of inflammatory mediators in liver[J]. J Biol Chem,2011,287(3):1847-1860.DOI:10. 1074/jbc. m111. 302356.
[17] PINEDA-TORRA I,GAGE M,DE JUAN A,et al. Isolation,culture,and polarization of murine bone marrow-derived and peritoneal macrophages[J]. Methods Mol Biol,2015,1339:101-109. DOI:10. 1007/978-1-4939-2929-0_6.
[18] ZHANG B,WANG J,GAO J,et al. Alternatively activated RAW264. 7 macrophages enhance tumor lymphangiogenesis in mouse lung adenocarcinoma[J]. J Cell Biochem,2009,107(1):134-143. DOI:10. 1002/jcb. 22110.
[19] LEHMANN B D,JOVANOVIC'B,CHEN X,et al. Refinement of triple-negative breast cancer molecular subtypes:implications for neoadjuvant chemotherapy selection[J].PLo S ONE,2016,11(6):e0157368. DOI:10. 1371/journal. pone. 0157368.
[20] TRIKHA P,SHARMA N,PENA C,et al. E2f3 in tumor macrophages promotes lung metastasis[J]. Oncogene,2016,35(28):3636-3646. DOI:10. 1038/onc. 2015. 429.
[21] FU X F,YAO K,DU X,et al. PGC-1αregulates the cell cycle through ATP and ROS in CH1 cells[J]. J Zhejiang Univ Sci B,2016,17(2):136-146. DOI:10. 1631/jzus.B1500158.
[22] VAZQUEZ F,LIM J H,CHIM H,et al. PGC1αexpression defines a subset of human melanoma tumors with increased mitochondrial capacity and resistance to oxidative stress[J].Cancer Cell,2013,23(3):287-301. DOI:10. 1016/j.ccr. 2012. 11. 020.
[23] LEBLEU V S,O’CONNELL J T,GONZALEZ HERRERA K N,et al. PGC-1αmediates mitochondrial biogenesis and oxidative phosphorylation in cancer cells to promote metastasis[J]. Nat Cell Biol,2014,16(10):992-1003. DOI:10. 1038/ncb3039.
[24] XU H S,LYU S J,XU J H,et al. Effect of lipopolysaccharide on the hemocyte apoptosis of eriocheir sinensis[J]. J Zhejiang Univ Sci B,2015,16(12):971-979. DOI:10.1631/jzus. B1500098.
[25] CAMPBELL C T, KOLESAR J E, KAUFMAN B A.Mitochondrial transcription factor A regulates mitochondrial transcription initiation,DNA packaging,and genome copy number[J]. Biochim Biophys Acta,2012,1819(9/10):921-929. DOI:10. 1016/j. bbagrm. 2012. 03. 002.
[26] IVANOVA M M,RADDE B N,SON J,et al. Estradiol and tamoxifen regulate NRF-1 and mitochondrial function in mouse mammary gland and uterus[J]. J Mol Endocrinol,2013,51(2):233-246. DOI:10. 1530/JME-13-0051.
[27] KRAWCZYK C M,HOLOWKA T,SUN J,et al. Toll-like receptor-induced changes in glycolytic metabolism regulate dendritic cell activation[J]. Blood,2010,115(23):4742-4749. DOI:10. 1182/blood-2009-10-249540.
[28] WANG C,YU X,CAO Q,et al. Characterization of murine macrophages from bone marrow,spleen and peritoneum[J].BMC Immunol,2013,14:6. DOI:10. 1186/1471-2172-14-6.
[2] NOY R,POLLARD J W. Tumor-associated macrophages:from mechanisms to therapy[J]. Immunity,2014,41(1):49-61. DOI:10. 1016/j. immuni. 2014. 06. 010.
[3] EVANS-KNOWELL A, LARUE A C, FINDLAY V J.MicroRNAs and their impact on breast cancer,the tumor microenvironment,and disparities[J]. Adv Cancer Res,2017,133:51-76. DOI:10. 1016/bs. acr. 2016. 08. 003.
[4] TANG X,MO C,WANG Y,et al. Anti-tumour strategies aiming to target tumour-associated macrophages[J]. Immunology,2013,138(2):93-104. DOI:10. 1111/imm. 12023.
[5] BELGIOVINE C,BELLO E,LIGUORI M,et al. Lurbinectedin reduces tumour-associated macrophages and the inflammatory tumour microenvironment in preclinical models[J]. Br J Cancer,2017,117(5):628-638. DOI:10. 1038/bjc.2017. 205.
[6] CHANMEE T,ONTONG P,KONNO K,et al. Tumor-associated macrophages as major players in the tumor microenvironment[J]. Cancers(Basel),2014,6(3):1670-1690. DOI:10. 3390/cancers6031670.
[7] KOVALEVA O V,SAMOILOVA D V,SHITOVA M S,et al.Tumor associated macrophages in kidney cancer[J]. Anal Cell Pathol(Amst),2016,2016:9307549. DOI:10. 1155/2016/9307549.
[8] CHAWLA A. Control of macrophage activation and function by PPARs[J]. Circ Res,2010,106(10):1559-1569.DOI:10. 1161/CIRCRESAHA. 110. 216523.
[9] COFFELT S B,TAL A O,SCHOLZ A,et al. Angiopoietin-2regulates gene expression in TIE2-expressing monocytes and augments their inherent proangiogenic functions[J]. Cancer Res,2010,70(13):5270-5280. DOI:10. 1158/0008-5472. CAN-10-0012.
[10]代朦,靳鑫,雷优扬,等.过表达miR-382的肿瘤相关巨噬细胞对三阴性乳腺癌生物学特性的影响[J].第三军医大学学报,2018,40(15):1375-1382. DOI:10. 16016/j.1000-5404. 20180110.DAI M,JIN X,LEI Y Y,et al. Effect of miR-382 overexpressing tumor-associated macrophages on biological properties of triple-negative breast cancer 4T1 cells[J]. J Third Mil Med Univ,2018,40(15):1375-1382. DOI:10. 16016/j.1000-5404. 20180110.
[11] CHIBA S,HISAMATSU T,SUZUKI H,et al. Glycolysis regulates LPS-induced cytokine production in M2 polarized human macrophages[J]. Immunol Lett,2017,183:17-23. DOI:10. 1016/j. imlet. 2017. 01. 012.
[12] NIU Z,SHI Q,ZHANG W,et al. Caspase-1 cleaves PPARγfor potentiating the pro-tumor action of TAMs[J].Nat Commun,2017,8(1):766. DOI:10. 1038/s41467-017-00523-6.
[13] LI Y,XU S,LI J,et al. SIRT1 facilitates hepatocellular carcinoma metastasis by promoting PGC-1α-mediated mitochondrial biogenesis[J]. Oncotarget,2016,7(20):29255-29274. DOI:10. 18632/oncotarget. 8711.
[14] WANG C,KAR S,LAI X,et al. Triple negative breast cancer in Asia:an insider’s view[J]. Cancer Treat Rev,2018,62:29-38. DOI:10. 1016/j. ctrv. 2017. 10. 014.
[15] KIM M S,SHIGENAGA J K,MOSER A H,et al. Suppression of estrogen-related receptor alpha and medium-chain acylcoenzyme A dehydrogenase in the acute-phase response[J].J Lipid Res,2005,46(10):2282-2288. DOI:10. 1194/jlr. M500217-JLR200.
[16] BULER M,AATSINKI S M,SKOUMAL R,et al. Energysensing factors coactivator peroxisome proliferator-activated receptorγcoactivator 1-α(PGC-1α)and AMP-activated protein kinase control expression of inflammatory mediators in liver[J]. J Biol Chem,2011,287(3):1847-1860.DOI:10. 1074/jbc. m111. 302356.
[17] PINEDA-TORRA I,GAGE M,DE JUAN A,et al. Isolation,culture,and polarization of murine bone marrow-derived and peritoneal macrophages[J]. Methods Mol Biol,2015,1339:101-109. DOI:10. 1007/978-1-4939-2929-0_6.
[18] ZHANG B,WANG J,GAO J,et al. Alternatively activated RAW264. 7 macrophages enhance tumor lymphangiogenesis in mouse lung adenocarcinoma[J]. J Cell Biochem,2009,107(1):134-143. DOI:10. 1002/jcb. 22110.
[19] LEHMANN B D,JOVANOVIC'B,CHEN X,et al. Refinement of triple-negative breast cancer molecular subtypes:implications for neoadjuvant chemotherapy selection[J].PLo S ONE,2016,11(6):e0157368. DOI:10. 1371/journal. pone. 0157368.
[20] TRIKHA P,SHARMA N,PENA C,et al. E2f3 in tumor macrophages promotes lung metastasis[J]. Oncogene,2016,35(28):3636-3646. DOI:10. 1038/onc. 2015. 429.
[21] FU X F,YAO K,DU X,et al. PGC-1αregulates the cell cycle through ATP and ROS in CH1 cells[J]. J Zhejiang Univ Sci B,2016,17(2):136-146. DOI:10. 1631/jzus.B1500158.
[22] VAZQUEZ F,LIM J H,CHIM H,et al. PGC1αexpression defines a subset of human melanoma tumors with increased mitochondrial capacity and resistance to oxidative stress[J].Cancer Cell,2013,23(3):287-301. DOI:10. 1016/j.ccr. 2012. 11. 020.
[23] LEBLEU V S,O’CONNELL J T,GONZALEZ HERRERA K N,et al. PGC-1αmediates mitochondrial biogenesis and oxidative phosphorylation in cancer cells to promote metastasis[J]. Nat Cell Biol,2014,16(10):992-1003. DOI:10. 1038/ncb3039.
[24] XU H S,LYU S J,XU J H,et al. Effect of lipopolysaccharide on the hemocyte apoptosis of eriocheir sinensis[J]. J Zhejiang Univ Sci B,2015,16(12):971-979. DOI:10.1631/jzus. B1500098.
[25] CAMPBELL C T, KOLESAR J E, KAUFMAN B A.Mitochondrial transcription factor A regulates mitochondrial transcription initiation,DNA packaging,and genome copy number[J]. Biochim Biophys Acta,2012,1819(9/10):921-929. DOI:10. 1016/j. bbagrm. 2012. 03. 002.
[26] IVANOVA M M,RADDE B N,SON J,et al. Estradiol and tamoxifen regulate NRF-1 and mitochondrial function in mouse mammary gland and uterus[J]. J Mol Endocrinol,2013,51(2):233-246. DOI:10. 1530/JME-13-0051.
[27] KRAWCZYK C M,HOLOWKA T,SUN J,et al. Toll-like receptor-induced changes in glycolytic metabolism regulate dendritic cell activation[J]. Blood,2010,115(23):4742-4749. DOI:10. 1182/blood-2009-10-249540.
[28] WANG C,YU X,CAO Q,et al. Characterization of murine macrophages from bone marrow,spleen and peritoneum[J].BMC Immunol,2013,14:6. DOI:10. 1186/1471-2172-14-6.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |