SUMO特异性蛋白酶3通过调控巨噬细胞极化促进磷酸钙诱导的小鼠腹主动脉瘤形成

目的:探讨SUMO特异性蛋白酶3(SENP3)调控巨噬细胞极化在磷酸钙诱导的小鼠腹主动脉瘤(AAA)形成中的作用。方法:(1)分离C57BL/6背景的Senp3flox/flox(野生型,WT)小鼠和Senp3flox/flox;Lyz2-Cre(SENP3单核细胞特异性敲除,即条件性敲除,c KO)小鼠骨髓来源的单核细胞(BMDMs),分别诱导其M1/M2型分化,采用RT-q PCR、Western blot和细胞免疫荧光比较SENP3表达以及M1/M2型巨噬细胞分布差异。(2)8~12周龄雄性WT小鼠和c KO小鼠用改良型磷酸钙诱导14 d构建小鼠AAA模型,比较两组小鼠的成瘤率和生存率;RT-q PCR和Western blot检测SENP3、白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNFα)、IL-6及基质金属蛋白酶9(MMP-9)在两组小鼠AAA组织中的表达差异;二氢乙啶染色检测组织中活性氧簇(ROS)生成差异。(3)为探讨其中机制,通过Western blot和免疫共沉淀验证SENP3与丝裂原活化蛋白激酶激酶7(MKK7)的相互作用;在BMDMs中转染FlagMKK7野生型(Flag-MKK7 WT)和SUMO修饰位点K18突变体(Flag-MKK7 K18R mutant)后诱导BMDMs进行M1极化,用Western blot检测p-JNK和MMP-9表达的差异。结果:(1)SENP3在M1型巨噬细胞中表达显著升高(P<0.01),在M2型巨噬细胞中显著下调(P<0.01);SENP3在M1向M2型巨噬细胞转化过程中表达显著下降(P<0.01),而在M2向M1型巨噬细胞转化时表达显著上调(P<0.01);c KO组BMDMs经M1/M2型诱导后,巨噬细胞M1型少于WT组,而M2型多于WT组;(2)SENP3在AAA组织中表达上调(P<0.05);c KO小鼠磷酸钙诱导后成瘤率显著降低(P<0.01),生存率显著提高(P<0.05),最大腹主动脉外直径显著减小(P<0.01);c KO小鼠AAA组织中IL-1β、IL-6和TNFα表达显著下调(P<0.01),ROS生成减少,MMP-9表达也显著下调(P<0.05)。(3)M1巨噬细胞中,MKK7的SUMO2/3修饰减少,与SENP3的相互作用增强;突变回补实验说明,转染Flag-MKK7 K18R mutant的M1型巨噬细胞中p-JNK和MMP-9表达显著上调(P<0.05)。结论:SENP3通过去SUMO化修饰MKK7,激活MAPK/JNK通路,调控巨噬细胞的极化,上调MMP-9的表达,从而促进AAA形成。

SUMO-specific protease 3 aggravates CaPO4-induced mouse abdominal aortic aneurysm formation by promoting macrophage polarization

AIM:To investigate the role of SUMO-specific protease 3(SENP3)in macrophage polarization and calcium phosphate(Ca PO4)-induced abdominal aortic aneurysm(AAA)formation in mice.METHODS:(1)Bone marrow-derived monocytes(BMDMs)in Senp3flox/flox(wild-type,WT)mice and Senp3flox/flox;Lyz2-Cre(monocyte-specific SENP3 knockout,i.e.conditioned knockout,c KO)mice were isolated and induced for M1 and M2 polarization.The m RNA and protein expression level of SENP3 were detected by RT-q PCR,Western blot and immunocytofluorescence,and the differential distribution of M1/M2 BMDMs from WT and c KO mice was analyzed.(2)Ca PO4was administrated to induce AAA model in 8~12-week-old male WT and c KO mice.The AAA incidence,survival rate and maximal aortic diameter were analyzed between the 2 groups.Aortic aneurysm tissues were collected for pathological analysis,and the expression levels of SENP3,interleukin-1β(IL-1β),tumor necrosis factor-α(TNFα),IL-6 and matrix metalloproteinases-9(MMP-9)were measured by RT-q PCR and Western blot.Dihydroethidium staining in situ in frozen sections was used to analyze the production of reactive oxygen species(ROS).(3)To explore the potential mechanisms,Western blot and coimmunoprecipitation were used to verify the de-SUMO modification of mitogen-activated protein kinase kinase 7(MKK7)induced by SENP3.Besides,BMDMs were transfected with Flag-MKK7 wild type(Flag-MKK7 WT)and SUMO-modified site K18 mutant(Flag-MKK7 K18R mutant),and then M1 polarization of the cells was induced.The protein levels of pJNK and MMP-9 in the 2 groups were determined by Western blot.RESULTS:(1)SENP3 expression was up-regulated in M1 polarized macrophages(P<0.01),but was down-regulated in M2 polarized macrophages(P<0.01).The expression of SENP3 was decreased during the transformation of M1 to M2 in the macrophages(P<0.01),but was significantly up-regulated during the opposite process(P<0.01).Besides,more M1 macrophages and less M2 macrophages after induction were observed in the BMDMs from c KO mice than those from WT mice.(2)SENP3 expression was up-regulated in AAA tissues(P<0.05).The AAA incidence of c KO mice was significantly reduced after Ca PO4induction(P<0.01),the survival rate was significantly improved(P<0.05),and maximal aortic diameter was significantly reduced in c KO group(P<0.01).The levels of IL-1β,IL-6 and TNFα,and the production of ROS were significantly down-regulated(P<0.01),meanwhile MMP-9 expression was also down-regulated in c KO mice(P<0.05).(3)the SUMO2/3 modification of MKK7 was reduced during M1 polarization,and MKK7 interaction with SENP3 was enhanced.Significantly up-regulated protein level of p-JNK and MMP-9 were verified in the M1 macrophages transfected with Flag-MKK7 K18R mutant(P<0.05).CONCLUSION:SENP3 activates the MAPK/JNK pathway via de-SUMOylation of MKK7,regulates the M1/M2polarization of macrophages and promotes the protein level of MMP-9,thus aggravating AAA formation.