Background:Small ubiquitin-like modifiers(SUMO)ylation is a dynamic and reversible post-translational modification playing pivotal roles in the regulation of cancer,diabetes,heart failure,and neurological diseases.How...Background:Small ubiquitin-like modifiers(SUMO)ylation is a dynamic and reversible post-translational modification playing pivotal roles in the regulation of cancer,diabetes,heart failure,and neurological diseases.However,whether SUMO inhibitors also have anti-hypertension effect remains yet to be explored.Methods:Blood pressure was monitored in spontaneously hypertensive rats(SHR)after Tannic acid(TA)administration for 4 weeks.The contents of nitric oxide(NO)and endothelin-1(ET-1)in the serum of SHR were measured.Isolated endothelium-intact mesenteric artery rings were used to study relaxation effect of SUMO inhibitors.ERK5 SUMOylation was determined using co-immunoprecipitation(co-IP)and immunofluorescence(IF).NO levels were analyzed by IF.The expression levels of KLF2 and p-eNOS were semi-quantified by Western blot analysis.The transcriptional activity of eNOS promotor was assayed using ChIP-PCR.Results:Three SUMO inhibitors all reduced the phenylephrine(PE)-induced contraction of mesenteric artery rings in a concentration-dependent manner.Co-IP revealed that ponatinib promoted ERK5 SUMOylation,which was nulled following pre-treatment with the SUMO inhibitors.IF displayed that TA increased ERK5 accumulation and its co-localization with SUMO-1 in the nucleus.ChIP-PCR unveiled TA-induced enhancement of KLF2-dependent eNOS promoter activity and upregulation of eNOS/NO expression in HUVECs.In vivo,TA significantly lowered the blood pressure and improved the vascular reactivity by activating the KLF2/eNOS/NO pathway.Additionally,the level of NO was elevated along with decreased ET-1 levels in the serum of SHR.Conclusions:SUMO inhibitors inhibit ERK5 SUMOylation to promote KLF2-eNOS/NO signaling,indicating their therapeutic potential for the treatment of hypertension.展开更多
目的构建KLF2过表达BGC823和KLF2敲减MGC803胃癌稳转细胞株。方法克隆人源KLF2基因,连接到Age I/Age I酶切后GV358载体上,构建GV358-KLF2重组质粒,转染293T细胞,包装慢病毒;构建KLF2shRNA,连接到Bam HI和EcoRI双酶切后的PHBLV-U6-ZSGree...目的构建KLF2过表达BGC823和KLF2敲减MGC803胃癌稳转细胞株。方法克隆人源KLF2基因,连接到Age I/Age I酶切后GV358载体上,构建GV358-KLF2重组质粒,转染293T细胞,包装慢病毒;构建KLF2shRNA,连接到Bam HI和EcoRI双酶切后的PHBLV-U6-ZSGreen-Puro载体上,经鉴定后转染293T细胞。采用RT-PCR及Western印迹法检测稳转细胞株中KLF2的表达。结果利用慢病毒介导,重组质粒G V358-K L F2和K L F2-shRN A转导入B G C823细胞、M G C803细胞并稳定表达。RT-PC R和W estern印迹法结果均显示过表达稳转株KLF2表达量明显升高(P<0.05),敲减稳转株KLF2表达量明显下降(P<0.05)。结论成功构建了K L F2过表达B G C823细胞株和K L F2敲减M G C803细胞株,为后续K L F2功能实验奠定了基础。展开更多
基金supported by the National Natural Science Foundation of China(82330011,U21A20339)the CAMS Innovation Fund for Medical Sciences(CIFMS,2020-I2M-5-003)+2 种基金the National Natural Science Foundation of China(No.82370302,31871175)Natural Science Foundation of Heilongjiang Province of China(No.YQ2019H003)College of Pharmacy,Harbin Medical University Excellent Young Talents Funding(No.2020-YQ-01).
文摘Background:Small ubiquitin-like modifiers(SUMO)ylation is a dynamic and reversible post-translational modification playing pivotal roles in the regulation of cancer,diabetes,heart failure,and neurological diseases.However,whether SUMO inhibitors also have anti-hypertension effect remains yet to be explored.Methods:Blood pressure was monitored in spontaneously hypertensive rats(SHR)after Tannic acid(TA)administration for 4 weeks.The contents of nitric oxide(NO)and endothelin-1(ET-1)in the serum of SHR were measured.Isolated endothelium-intact mesenteric artery rings were used to study relaxation effect of SUMO inhibitors.ERK5 SUMOylation was determined using co-immunoprecipitation(co-IP)and immunofluorescence(IF).NO levels were analyzed by IF.The expression levels of KLF2 and p-eNOS were semi-quantified by Western blot analysis.The transcriptional activity of eNOS promotor was assayed using ChIP-PCR.Results:Three SUMO inhibitors all reduced the phenylephrine(PE)-induced contraction of mesenteric artery rings in a concentration-dependent manner.Co-IP revealed that ponatinib promoted ERK5 SUMOylation,which was nulled following pre-treatment with the SUMO inhibitors.IF displayed that TA increased ERK5 accumulation and its co-localization with SUMO-1 in the nucleus.ChIP-PCR unveiled TA-induced enhancement of KLF2-dependent eNOS promoter activity and upregulation of eNOS/NO expression in HUVECs.In vivo,TA significantly lowered the blood pressure and improved the vascular reactivity by activating the KLF2/eNOS/NO pathway.Additionally,the level of NO was elevated along with decreased ET-1 levels in the serum of SHR.Conclusions:SUMO inhibitors inhibit ERK5 SUMOylation to promote KLF2-eNOS/NO signaling,indicating their therapeutic potential for the treatment of hypertension.
文摘目的构建KLF2过表达BGC823和KLF2敲减MGC803胃癌稳转细胞株。方法克隆人源KLF2基因,连接到Age I/Age I酶切后GV358载体上,构建GV358-KLF2重组质粒,转染293T细胞,包装慢病毒;构建KLF2shRNA,连接到Bam HI和EcoRI双酶切后的PHBLV-U6-ZSGreen-Puro载体上,经鉴定后转染293T细胞。采用RT-PCR及Western印迹法检测稳转细胞株中KLF2的表达。结果利用慢病毒介导,重组质粒G V358-K L F2和K L F2-shRN A转导入B G C823细胞、M G C803细胞并稳定表达。RT-PC R和W estern印迹法结果均显示过表达稳转株KLF2表达量明显升高(P<0.05),敲减稳转株KLF2表达量明显下降(P<0.05)。结论成功构建了K L F2过表达B G C823细胞株和K L F2敲减M G C803细胞株,为后续K L F2功能实验奠定了基础。
