SM22α调节AngⅡ诱导的血管生理及病理学效应的机制
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摘要
血管紧张素II（angiotensinII,AngII）是肾素-血管紧张素-醛固酮系统的主要效应因子，是一种多功能的血管活性肽。通过活化多条信号通路，参与心血管系统生理功能的调节，并介导多种心血管疾病发生发展的病理过程。AngII急性刺激可诱导血管收缩，升高血压；而长时间慢性刺激可促进血管平滑肌细胞（vascular smooth muscle cells, VSMCs）增殖、肥大及衰老。
SM22α又被称为transgelin，是一种平滑肌分化标志物，高表达于哺乳动物平滑肌中，被广泛用于平滑肌细胞的鉴定。SM22α的主要功能是与actin结合，促进actin细丝聚合及捆绑。我室以往的研究证实，SM22α通过促进actin纤维聚合成束，进而增强VSMC收缩性；此外，SM22α还参与调节细胞增殖、氧化应激和炎症相关信号转导分子的活性。然而，SM22α是否参与收缩信号分子活性的调节以及分子机制尚不清楚。细胞内SM22α蛋白水平与VSMC的表型状态密切相关，丝裂原和炎性因子可诱导该蛋白表达下调。然而，在多种衰老细胞中，发现SM22α表达上调，该蛋白已成为非肌细胞衰老的新标志物。但是，SM22α是否参与VSMC衰老的调节，尚缺乏深入的研究。本研究主要探讨SM22α在AngII诱导的VSMC收缩及衰老中的作用。
方法：
本研究利用SM22α基因敲除小鼠经背皮下植入AngII微渗透泵复制高血压模型，用智能无创血压计检测小鼠血压，在整体水平探讨病理情况下SM22α与血压的关系；用微血管张力测定仪检测血管环等长张力；用细胞长度测量和胶原胶收缩分析分别检测细胞收缩性；通过敲低SM22α及特异性信号通路阻断剂，确定受其调节的信号途径及靶分子；用RT-PCR、Western blot检测mRNA及蛋白质表达变化；用免疫共沉淀分析和免疫双荧光染色确定蛋白质相互作用；用免疫沉淀分析蛋白质泛素化修饰；用AngII慢性刺激建立VSMC衰老模型，探讨SM22α在AngII诱导的VSMC衰老中的作用。
结果：
1SM22α通过调节ERK1/2活性参与AngII诱导的VSMC收缩
1.1SM22α促进AngⅡ诱导的血管平滑肌收缩
选雄性Sm22α+/+及Sm22α-/-小鼠复制高血压小鼠模型。植泵前，Sm22α+/+小鼠的收缩压和舒张压分别为（118.8±2.2）和（76.4±2.7）mmHg，Sm22α-/-小鼠的收缩压和舒张压分别为（111.7±1.9）和（72.5±2.4）mm Hg，二者的基础血压无显著差异。植泵后，两种基因型小鼠的收缩压和舒张压均随着时间逐步升高，至21天时，Sm22α+/+小鼠的收缩压和舒张压分别为（179.5±2.3）和（130.5±2.8）mm Hg，Sm22α-/-小鼠的收缩压和舒张压分别为（158.3±1.8）和（109.3±3.1）mm Hg。Sm22α-/-小鼠的血压显著低于Sm22α+/+小鼠（P <0.05）。
取小鼠主动脉进行血管环张力测定，绘制主动脉血管环对AngII的浓度-收缩曲线，结果表明，AngII可诱发两种基因型小鼠的主动脉环产生明显的收缩应答。但是，Sm22α-/-小鼠主动脉环的收缩反应性较Sm22α+/+小鼠明显减弱，表现为浓度-收缩曲线明显右移，半数有效浓度（EC50）由5.57nmol/L右移至12.13nmol/L（P <0.05）；收缩峰值降低，在Sm22α+/+小鼠的主动脉环中，AngII引起的收缩峰值为45.5±4.3%，而Sm22α-/-小鼠的主动脉环的收缩峰值为28.4±4.0%(P <0.05）。敲低SM22α后，AngII诱导的VSMC长度变化百分比及胶原胶面积变化百分比均显著降低（P <0.05）。并且Sm22α-/-小鼠VSMC对AngII的收缩性显著降低。
1.2ERK1/2通路介导AngⅡ诱导的血管平滑肌收缩
AngII刺激2min时，ERK1/2的磷酸化水平即有显著升高，5min时达高峰；其下游靶蛋白caldesmon磷酸化时程与ERK1/2基本一致。细胞长度变化百分比于AngII刺激5min后达峰值，时程与ERK1/2磷酸化基本一致。PD98059预孵育2h后，AngII诱导的ERK1/2磷酸化受到显著抑制，伴随caldesmon的磷酸化水平显著降低。用PD98059预处理小鼠主动脉环30min后，AngII诱导的收缩反应显著降低。浓度-收缩曲线明显右移（EC50为8.90nmol/L）；收缩峰值由43.4±4.1%降至32.5±4.2%。在细胞水平，PD98059预处理后，AngII诱导的VSMC长度变化百分比及胶原胶面积变化百分比均显著降低。
1.3SM22α调节AngII诱导的ERK1/2激活
用小干扰RNA敲低内源性SM22α后，观察AngII短时间（5min）刺激，ERK1/2的活化情况。结果表明，AngII刺激后，对照组及SM22α敲低组ERK1/2磷酸化水平均有升高，但SM22α敲低组的上升幅度显著低于对照组（P <0.05）。AngII刺激5min后，原代培养的Sm22α-/-组VSMC的ERK1/2磷酸化水平较Sm22α+/+组显著降低。
2SM22α通过促进MKP3降解调节AngII-ERK1/2通路信号转导
2.1SM22α通过MKP3调节AngII诱导的ERK1/2通路活化
AngII刺激5min后，SM22α敲低组及对照组MEK磷酸化水平均有显著升高，但两组的升高幅度并无显著差异。而在敲低SM22α后，其磷酸酶MKP3的表达明显上调，同时伴有AngII诱导的ERK1/2磷酸化减弱。Sm22α-/-小鼠血管组织中MKP3的表达水平较Sm22α+/+小鼠显著升高。用MKP3抑制剂BC（I10μmol/L）预孵育30min可逆转敲低SM22α对ERK1/2通路活化的抑制作用。
2.2MKP3通过抑制ERK1/2参与AngII诱导的血管平滑肌收缩
BCI预处理后，小鼠主动脉对AngII的收缩应答显著增强，浓度-收缩曲线明显左移（EC50为3.83nmol/L）；收缩峰值由46.1±3.8%升至61.1±5.1%。在细胞水平，BCI (10μmol/L)预孵育2h后，AngII诱导的VSMC长度变化百分比及胶原胶面积变化百分比均显著增加（P <0.05）。与GFP空质粒对照组相比，过表达GFP-MKP3后，AngII诱导的VSMC长度变化百分比及胶原胶面积变化百分比均显著降低。BCI (10μmol/L)预孵育2h后，ERK1/2及caldesmon的磷酸化水平显著升高；而过表达GFP-MKP3融合蛋白后，与转染GFP空质粒的VSMC相比，ERK1/2及caldesmon的磷酸化水平显著降低。
2.3SM22α抑制MKP3与ERK1/2的相互作用
免疫共沉淀结果显示，在未刺激时，MKP3与ERK1/2即有较弱的相互作用；敲低SM22α后，MKP3与ERK1/2的结合量显著增加。与Sm22α+/+小鼠相比，Sm22α-/-小鼠VSMC中二者的相互作用增强。细胞免疫荧光染色分析结果显示，敲低SM22α后，细胞内MKP3荧光强度及其与ERK1/2的共定位均显著增加。
2.4SM22α促进MKP3的泛素-蛋白酶体降解过程
敲低SM22α后，VSMC中MKP3的mRNA水平并无显著改变，并且Sm22α+/+与Sm22α-/-两种基因型小鼠主动脉组织中MKP3的mRNA水平也无显著差异。用放线菌酮阻断新生蛋白质合成，测定蛋白质半寿期。结果表明，MKP3半寿期很短，约为30min左右；90min时，蛋白降解明显。敲低SM22α后，MKP3的半寿期延长至90min以上。用蛋白酶体抑制剂MG132处理细胞，观察对MKP3半寿期的影响。结果显示，MG132（10μmol/L）预处理2h可显著延长MKP3的半寿期。泛素化分析结果显示，敲低SM22α后，MKP3的泛素化水平显著降低。
3SM22α促进AngII诱导的VSMC衰老
3.1SM22α在AngII诱导的VSMC衰老中表达上调
AngII刺激3天后VSMC中SA-β-gal阳性染色细胞百分比为5.4±1.4%，与对照组（6.5±1.6%）相比，无显著差异；刺激5天，SA-β-gal阳性细胞百分比为43.9±5.3%，显著高于对照组（22.1±4.1%，P <0.05），刺激7天和10天，SA-β-gal阳性细胞数大幅度增加，阳性细胞百分比高达78.9±5.2%及92.3±4.4%。AngII刺激7天及10天，p21和p53的表达显著高于对照组（P <0.05），并且表达量随刺激时间延长而升高；相反，PCNA的表达随刺激时间逐渐降低。AngII刺激5天后，SM22α的表达表达显著高于对照组（P <0.05），并随AngII刺激时间延长而增加。
3.2AngII上调SM22α基因转录活性
AngII刺激3天，SM22α的mRNA水平开始升高，与刺激前相比，约升高3.8倍；至10天时达到峰值，约为刺激前的12.1倍，与其蛋白水平变化一致。而AngII刺激不同时间，SM22α的泛素化水平没有显著改变。
3.3SM22α促进AngII诱导的VSMC衰老
在VSMC中敲低SM22α，观察对细胞衰老的影响。敲低SM22α后，AngII诱导的SA-β-gal阳性细胞百分比分别为25.3±4.3%和32.3±4.6%较对照组（79.2±4.2%及89.2±5.2%）显著降低（P <0.05）。并且，衰老标志蛋白p21及p53的表达显著减少，而增殖标志PCNA表达上调（P <0.05）。
结论：
1. SM22α通过调节ERK1/2通路活化参与AngII诱导的血管平滑肌收缩。
2. SM22α通过促进MKP3的泛素-蛋白酶体降解和抑制MKP3与ERK1/2的相互作用，进而调节AngII诱导的ERK1/2通路活化。
3. SM22α促进AngII诱导的VSMC衰老。
4. SM22α是一种新的血管收缩信号转导分子的调节因子，参与调节AngII诱导的血管生理及病理学过程。
AngiotensinII (AngII), is a multifunctional vasoconstrictor peptide,which regulates both the cardiovascular physiology and pathology viatriggering a series of signal transduction cascades. Acute stimulation withAngII regulates vasoconstriction, and modulating blood pressure, whilechronic stimulation promotes hyperplasia, hypertrophy and senescence ofvascular smooth muscle cells (VSMCs).
SM22α, also known as transgelin is one of the widely used markers toidentify SMCs. The basic molecular function of SM22α is to bind actin, and tofacilitate actin filament assembly and cytoskeletal rearrangements. Ourprevious study showed that SM22α enhanced contractility of VSMCs bymodulating the reorganization of actin filaments. SM22α acts as an adapter orscaffold protein to assemble signaling complexes and regulate signaling.However, the roles of SM22α in regulation of VSMC contraction are far fromclear. Recently, increased SM22α is considered to be a marker of cellularsenescence. However, it is unknown whether SM22α is involved in VSMCsenescence.
In this study, we investigated the effect of SM22α on AngII-inducedvasoconstriction using SM22α gene knock out mouse model, measured theVSMC contraction and isometric tension of the aortic rings to clarify the roleof SM22α in VSMC contraction, and used SM22α siRNA and specificsignaling inhibitor to identify the signal transduction pathway involved in thisprocess. We also explored the relationship between SM22α expression andAngII-induced VSMC senescence using the indicators including SA-β-gal,p21and p53as well as the proliferation marker PCNA.
1. SM22α facilitates AngII-induced VSMC contraction throughregulating the activity of ERK1/2
1.1Disruption of SM22α attenuates AngII-induced vasoconstriction
Sm22α+/+and Sm22α-/-mice that exhibit a similar baseline blood pressure(BP) were infused with AngII. The BP of Sm22α+/+mice infused with AngIIshowed steadily rising. However, both SBP and DBP were significantly lowerin Sm22α-/-mice in response to AngII. In response to AngII at indicatedconcentrations, the aortic rings of Sm22α+/+mice displayed strongcontractions with an EC50of5.57nmol/L. However, theconcentration-response curve for aortic rings from Sm22α-/-mice significantlyshifted to the right with an EC50of12.13nmol/L, and the maximal contractionwas significantly reduced. Knockdown of SM22α by specific siRNAsignificantly decreased the percentage of reduction in cell length. Moreover,the contractile response to AngII in the primary VSMCs from Sm22α-/-micewas remarkably reduced compared with that of the cells from the Sm22α+/+mice. Contraction of the gels was significantly attenuated followingknockdown of SM22α. VSMCs derived from Sm22α-/-mice showed a reducedgel contraction upon AngII stimulation.
1.2Loss of SM22α inhibits AngII-induced activation of ERK1/2signaling inVSMCs.
AngII treatment caused a rapid (2min) and significant elevation ofERK1/2phosphorylation that peaked at5min, accompanied by increasedphosphorylation of caldesmon. Maximal cell shortening occurred at5minutesafter AngII stimulation. Pretreatment of the aorta rings with PD98059resultedin a rightward shift of the concentration-response curve with an increasedEC50of8.90nmol/L, suggesting a decrease in contractile response to AngII.The percentage of reduction in cell length and collagen gel contraction of theVSMCs preincubated with PD98059was remarkably decreased comparedwith that of the DMSO controls. The results of western blot showed thatPD98059remarkably reduced the phosphorylation levels of ERK1/2andcaldesmon.
1.3SM22α regulates AngII-induced ERK1/2signaling pathway
Knockdown of SM22α by specific siRNA blocked AngII-inducedphosphorylation of ERK1/2and caldesmon in rat VSMCs. Primary VSMCsfrom Sm22α-/-mice displayed decreased phosphorylation of this two proteinscompared with Sm22α+/+mice.
2SM22α regulates AngII-ERK1/2signaling cascades by promotingproteasome-mediated MKP3degradation
2.1Depletion of SM22α upregulates MKP3and thereby facilitatesdephosphorylation of ERK1/2.Knockdown of SM22α did not significantly change AngII-inducedphosphorylation of MEK. SM22α silencing resulted in an increasedexpression of MKP3, accompanied by reduction of ERK1/2and caldesmonphosphorylation. Moreover, a consistent increase in MKP3protein levels wasalso observed in the aortas, femoral and carotid arteries of Sm22α-/-micecompared with the Sm22α+/+controls. Inhibition of MKP3activity with theinhibitor BCI rescued the reduced ERK1/2activity resulted from loss ofSM22α.
2.2MKP3negatively regulates AngII-induced vasoconstriction and VSMCcontraction via inactivation of ERK1/2
The maximal contractile response of aortic rings to AngII wassignificantly increased by pretreatment with the MKP3inhibitor BCI, showinga leftward shift of the concentration-response curve of AngII, with the EC50of3.83nmol/L. Preincubation of VSMCs with BCI resulted in a significantlyincrease in the percentage of reduction in cell length and the shrinkage ofcollagen gels in response to AngII. Furthermore, overexpression of MKP3markedly attenuated the contractile response to AngII, displaying a reducedpercentage of reduction in cell length and collagen gel contraction. BCIincreased the phosphorylation of ERK1/2and caldesmon, whereas an oppositeeffect was observed in VSMCs overexpressing MKP3.
2.3SM22α inhibits the interaction of ERK1/2with MKP3.
The results of co-immunoprecipitation showed a weak interaction of MKP3with ERK1/2in resting VSMCs, which did not change upon AngIItreatment. Knockdown of SM22α led to an enhanced interaction of MKP3with ERK1/2. Similar results were observed in VSMCs derived from Sm22α-/-mice. These findings were further confirmed by immunofluorescence staining.The MKP3expression was increased following knockdown of SM22α, andcolocalized with ERK1/2.
2.4Loss of SM22α inhibits the proteasome-mediated MKP3degradation.
MKP3expression at the mRNA level in aortas form Sm22α-/-mice wasnot significantly different from that in the Sm22α+/+controls. Similarly, theMKP3mRNA level was not significantly altered by knockdown of SM22α. Toexamine the effect of knockdown of SM22α on half-life of MKP3protein, weused cycloheximide (CHX) to inhibit the de novo protein synthesis. Theresults showed that MKP3had a short half-life (less than30min), anddecreased dramatically after90min of incubation with CHX. The delayeddecay of MKP3protein was observed following knockdown of SM22α, with alonger half-life of90min. In the presence of MG132(the proteasomeinhibitor), half-life of MKP3was extended to90min. The ubiquitinationassay showed that knockdown of SM22α inhibited the polyubiquitination ofMKP3.
3SM22α promotes AngII-induced VSMC senescence
3.1SM22α is upregulated in AngII-induced VSMC senescence
The SA-β-gal-positive percentage in the VSMCs stimulated with AngIIfor3days was5.4±1.4%, which was not significantly changed comparedwith that in the control group (6.5±1.6%). After stimulation for5days, theSA-β-gal activity was elevated in a time-dependent manner, with theSA-β-gal-positive percentage of43.9±5.3%, significantly increasedcompared with those in the control group (22.1±4.1%, P <0.05). Afterstimulation for7and10days, the SA-β-gal activity was extremely high, witha SA-β-gal-positive percentage of78.9±5.2%and92.3±4.4%respectively.The expression of SM22α was elevated in VSMCs stimulated for7and10days compared with the control cells (P <0.05). The expressions of p21and p53displayed a similar fashion. However, the expression of PCNA wasreduced in the same conditions.
3.2AngII upregulates the transcriptional activity of SM22α gene.
To explore the mechanism by which SM22α protein was upregulated inAngII-induced senescence, the turnover of SM22α was detected. Treatmentwith AngII significantly increased the mRNA level of SM22α, which wasincreased by12.1folds after AngII stimulation for10days. However,ubiquitination of SM22α was not significantly varied by AngII stimulation.
3.3SM22α facilitates AngII-induced senescence in VSMCs.
Knockdown of SM22α using specific siRNA markedly attenuatedAngII-induced SA-β-gal activity. The percentage of SA-β-gal-positive cellswere25.3±4.3%and32.3±4.6%, respectively, after stimulation for7and10days, which was lower than those in the control group (79.2±4.2%and89.2±5.2%, P <0.05). The expression of p21and p53increased under the same
conditions.
Conclusions:
1. SM22α facilitates AngII-induced VSMC contraction through regulatingthe activity of ERK1/2.
2. SM22α regulates AngII-ERK1/2signaling cascades via promotingproteasome-mediated MKP3degradation and inhibiting the interaction ofERK1/2with MKP3.
3. SM22α promotes AngII-induced VSMC senescence.
4. SM22α is a novel regulator of vasoconstrictor signaling pathway, and amediator of vascular physiological and pathological processes
SM22α又被称为transgelin，是一种平滑肌分化标志物，高表达于哺乳动物平滑肌中，被广泛用于平滑肌细胞的鉴定。SM22α的主要功能是与actin结合，促进actin细丝聚合及捆绑。我室以往的研究证实，SM22α通过促进actin纤维聚合成束，进而增强VSMC收缩性；此外，SM22α还参与调节细胞增殖、氧化应激和炎症相关信号转导分子的活性。然而，SM22α是否参与收缩信号分子活性的调节以及分子机制尚不清楚。细胞内SM22α蛋白水平与VSMC的表型状态密切相关，丝裂原和炎性因子可诱导该蛋白表达下调。然而，在多种衰老细胞中，发现SM22α表达上调，该蛋白已成为非肌细胞衰老的新标志物。但是，SM22α是否参与VSMC衰老的调节，尚缺乏深入的研究。本研究主要探讨SM22α在AngII诱导的VSMC收缩及衰老中的作用。
方法：
本研究利用SM22α基因敲除小鼠经背皮下植入AngII微渗透泵复制高血压模型，用智能无创血压计检测小鼠血压，在整体水平探讨病理情况下SM22α与血压的关系；用微血管张力测定仪检测血管环等长张力；用细胞长度测量和胶原胶收缩分析分别检测细胞收缩性；通过敲低SM22α及特异性信号通路阻断剂，确定受其调节的信号途径及靶分子；用RT-PCR、Western blot检测mRNA及蛋白质表达变化；用免疫共沉淀分析和免疫双荧光染色确定蛋白质相互作用；用免疫沉淀分析蛋白质泛素化修饰；用AngII慢性刺激建立VSMC衰老模型，探讨SM22α在AngII诱导的VSMC衰老中的作用。
结果：
1SM22α通过调节ERK1/2活性参与AngII诱导的VSMC收缩
1.1SM22α促进AngⅡ诱导的血管平滑肌收缩
选雄性Sm22α+/+及Sm22α-/-小鼠复制高血压小鼠模型。植泵前，Sm22α+/+小鼠的收缩压和舒张压分别为（118.8±2.2）和（76.4±2.7）mmHg，Sm22α-/-小鼠的收缩压和舒张压分别为（111.7±1.9）和（72.5±2.4）mm Hg，二者的基础血压无显著差异。植泵后，两种基因型小鼠的收缩压和舒张压均随着时间逐步升高，至21天时，Sm22α+/+小鼠的收缩压和舒张压分别为（179.5±2.3）和（130.5±2.8）mm Hg，Sm22α-/-小鼠的收缩压和舒张压分别为（158.3±1.8）和（109.3±3.1）mm Hg。Sm22α-/-小鼠的血压显著低于Sm22α+/+小鼠（P <0.05）。
取小鼠主动脉进行血管环张力测定，绘制主动脉血管环对AngII的浓度-收缩曲线，结果表明，AngII可诱发两种基因型小鼠的主动脉环产生明显的收缩应答。但是，Sm22α-/-小鼠主动脉环的收缩反应性较Sm22α+/+小鼠明显减弱，表现为浓度-收缩曲线明显右移，半数有效浓度（EC50）由5.57nmol/L右移至12.13nmol/L（P <0.05）；收缩峰值降低，在Sm22α+/+小鼠的主动脉环中，AngII引起的收缩峰值为45.5±4.3%，而Sm22α-/-小鼠的主动脉环的收缩峰值为28.4±4.0%(P <0.05）。敲低SM22α后，AngII诱导的VSMC长度变化百分比及胶原胶面积变化百分比均显著降低（P <0.05）。并且Sm22α-/-小鼠VSMC对AngII的收缩性显著降低。
1.2ERK1/2通路介导AngⅡ诱导的血管平滑肌收缩
AngII刺激2min时，ERK1/2的磷酸化水平即有显著升高，5min时达高峰；其下游靶蛋白caldesmon磷酸化时程与ERK1/2基本一致。细胞长度变化百分比于AngII刺激5min后达峰值，时程与ERK1/2磷酸化基本一致。PD98059预孵育2h后，AngII诱导的ERK1/2磷酸化受到显著抑制，伴随caldesmon的磷酸化水平显著降低。用PD98059预处理小鼠主动脉环30min后，AngII诱导的收缩反应显著降低。浓度-收缩曲线明显右移（EC50为8.90nmol/L）；收缩峰值由43.4±4.1%降至32.5±4.2%。在细胞水平，PD98059预处理后，AngII诱导的VSMC长度变化百分比及胶原胶面积变化百分比均显著降低。
1.3SM22α调节AngII诱导的ERK1/2激活
用小干扰RNA敲低内源性SM22α后，观察AngII短时间（5min）刺激，ERK1/2的活化情况。结果表明，AngII刺激后，对照组及SM22α敲低组ERK1/2磷酸化水平均有升高，但SM22α敲低组的上升幅度显著低于对照组（P <0.05）。AngII刺激5min后，原代培养的Sm22α-/-组VSMC的ERK1/2磷酸化水平较Sm22α+/+组显著降低。
2SM22α通过促进MKP3降解调节AngII-ERK1/2通路信号转导
2.1SM22α通过MKP3调节AngII诱导的ERK1/2通路活化
AngII刺激5min后，SM22α敲低组及对照组MEK磷酸化水平均有显著升高，但两组的升高幅度并无显著差异。而在敲低SM22α后，其磷酸酶MKP3的表达明显上调，同时伴有AngII诱导的ERK1/2磷酸化减弱。Sm22α-/-小鼠血管组织中MKP3的表达水平较Sm22α+/+小鼠显著升高。用MKP3抑制剂BC（I10μmol/L）预孵育30min可逆转敲低SM22α对ERK1/2通路活化的抑制作用。
2.2MKP3通过抑制ERK1/2参与AngII诱导的血管平滑肌收缩
BCI预处理后，小鼠主动脉对AngII的收缩应答显著增强，浓度-收缩曲线明显左移（EC50为3.83nmol/L）；收缩峰值由46.1±3.8%升至61.1±5.1%。在细胞水平，BCI (10μmol/L)预孵育2h后，AngII诱导的VSMC长度变化百分比及胶原胶面积变化百分比均显著增加（P <0.05）。与GFP空质粒对照组相比，过表达GFP-MKP3后，AngII诱导的VSMC长度变化百分比及胶原胶面积变化百分比均显著降低。BCI (10μmol/L)预孵育2h后，ERK1/2及caldesmon的磷酸化水平显著升高；而过表达GFP-MKP3融合蛋白后，与转染GFP空质粒的VSMC相比，ERK1/2及caldesmon的磷酸化水平显著降低。
2.3SM22α抑制MKP3与ERK1/2的相互作用
免疫共沉淀结果显示，在未刺激时，MKP3与ERK1/2即有较弱的相互作用；敲低SM22α后，MKP3与ERK1/2的结合量显著增加。与Sm22α+/+小鼠相比，Sm22α-/-小鼠VSMC中二者的相互作用增强。细胞免疫荧光染色分析结果显示，敲低SM22α后，细胞内MKP3荧光强度及其与ERK1/2的共定位均显著增加。
2.4SM22α促进MKP3的泛素-蛋白酶体降解过程
敲低SM22α后，VSMC中MKP3的mRNA水平并无显著改变，并且Sm22α+/+与Sm22α-/-两种基因型小鼠主动脉组织中MKP3的mRNA水平也无显著差异。用放线菌酮阻断新生蛋白质合成，测定蛋白质半寿期。结果表明，MKP3半寿期很短，约为30min左右；90min时，蛋白降解明显。敲低SM22α后，MKP3的半寿期延长至90min以上。用蛋白酶体抑制剂MG132处理细胞，观察对MKP3半寿期的影响。结果显示，MG132（10μmol/L）预处理2h可显著延长MKP3的半寿期。泛素化分析结果显示，敲低SM22α后，MKP3的泛素化水平显著降低。
3SM22α促进AngII诱导的VSMC衰老
3.1SM22α在AngII诱导的VSMC衰老中表达上调
AngII刺激3天后VSMC中SA-β-gal阳性染色细胞百分比为5.4±1.4%，与对照组（6.5±1.6%）相比，无显著差异；刺激5天，SA-β-gal阳性细胞百分比为43.9±5.3%，显著高于对照组（22.1±4.1%，P <0.05），刺激7天和10天，SA-β-gal阳性细胞数大幅度增加，阳性细胞百分比高达78.9±5.2%及92.3±4.4%。AngII刺激7天及10天，p21和p53的表达显著高于对照组（P <0.05），并且表达量随刺激时间延长而升高；相反，PCNA的表达随刺激时间逐渐降低。AngII刺激5天后，SM22α的表达表达显著高于对照组（P <0.05），并随AngII刺激时间延长而增加。
3.2AngII上调SM22α基因转录活性
AngII刺激3天，SM22α的mRNA水平开始升高，与刺激前相比，约升高3.8倍；至10天时达到峰值，约为刺激前的12.1倍，与其蛋白水平变化一致。而AngII刺激不同时间，SM22α的泛素化水平没有显著改变。
3.3SM22α促进AngII诱导的VSMC衰老
在VSMC中敲低SM22α，观察对细胞衰老的影响。敲低SM22α后，AngII诱导的SA-β-gal阳性细胞百分比分别为25.3±4.3%和32.3±4.6%较对照组（79.2±4.2%及89.2±5.2%）显著降低（P <0.05）。并且，衰老标志蛋白p21及p53的表达显著减少，而增殖标志PCNA表达上调（P <0.05）。
结论：
1. SM22α通过调节ERK1/2通路活化参与AngII诱导的血管平滑肌收缩。
2. SM22α通过促进MKP3的泛素-蛋白酶体降解和抑制MKP3与ERK1/2的相互作用，进而调节AngII诱导的ERK1/2通路活化。
3. SM22α促进AngII诱导的VSMC衰老。
4. SM22α是一种新的血管收缩信号转导分子的调节因子，参与调节AngII诱导的血管生理及病理学过程。
AngiotensinII (AngII), is a multifunctional vasoconstrictor peptide,which regulates both the cardiovascular physiology and pathology viatriggering a series of signal transduction cascades. Acute stimulation withAngII regulates vasoconstriction, and modulating blood pressure, whilechronic stimulation promotes hyperplasia, hypertrophy and senescence ofvascular smooth muscle cells (VSMCs).
SM22α, also known as transgelin is one of the widely used markers toidentify SMCs. The basic molecular function of SM22α is to bind actin, and tofacilitate actin filament assembly and cytoskeletal rearrangements. Ourprevious study showed that SM22α enhanced contractility of VSMCs bymodulating the reorganization of actin filaments. SM22α acts as an adapter orscaffold protein to assemble signaling complexes and regulate signaling.However, the roles of SM22α in regulation of VSMC contraction are far fromclear. Recently, increased SM22α is considered to be a marker of cellularsenescence. However, it is unknown whether SM22α is involved in VSMCsenescence.
In this study, we investigated the effect of SM22α on AngII-inducedvasoconstriction using SM22α gene knock out mouse model, measured theVSMC contraction and isometric tension of the aortic rings to clarify the roleof SM22α in VSMC contraction, and used SM22α siRNA and specificsignaling inhibitor to identify the signal transduction pathway involved in thisprocess. We also explored the relationship between SM22α expression andAngII-induced VSMC senescence using the indicators including SA-β-gal,p21and p53as well as the proliferation marker PCNA.
1. SM22α facilitates AngII-induced VSMC contraction throughregulating the activity of ERK1/2
1.1Disruption of SM22α attenuates AngII-induced vasoconstriction
Sm22α+/+and Sm22α-/-mice that exhibit a similar baseline blood pressure(BP) were infused with AngII. The BP of Sm22α+/+mice infused with AngIIshowed steadily rising. However, both SBP and DBP were significantly lowerin Sm22α-/-mice in response to AngII. In response to AngII at indicatedconcentrations, the aortic rings of Sm22α+/+mice displayed strongcontractions with an EC50of5.57nmol/L. However, theconcentration-response curve for aortic rings from Sm22α-/-mice significantlyshifted to the right with an EC50of12.13nmol/L, and the maximal contractionwas significantly reduced. Knockdown of SM22α by specific siRNAsignificantly decreased the percentage of reduction in cell length. Moreover,the contractile response to AngII in the primary VSMCs from Sm22α-/-micewas remarkably reduced compared with that of the cells from the Sm22α+/+mice. Contraction of the gels was significantly attenuated followingknockdown of SM22α. VSMCs derived from Sm22α-/-mice showed a reducedgel contraction upon AngII stimulation.
1.2Loss of SM22α inhibits AngII-induced activation of ERK1/2signaling inVSMCs.
AngII treatment caused a rapid (2min) and significant elevation ofERK1/2phosphorylation that peaked at5min, accompanied by increasedphosphorylation of caldesmon. Maximal cell shortening occurred at5minutesafter AngII stimulation. Pretreatment of the aorta rings with PD98059resultedin a rightward shift of the concentration-response curve with an increasedEC50of8.90nmol/L, suggesting a decrease in contractile response to AngII.The percentage of reduction in cell length and collagen gel contraction of theVSMCs preincubated with PD98059was remarkably decreased comparedwith that of the DMSO controls. The results of western blot showed thatPD98059remarkably reduced the phosphorylation levels of ERK1/2andcaldesmon.
1.3SM22α regulates AngII-induced ERK1/2signaling pathway
Knockdown of SM22α by specific siRNA blocked AngII-inducedphosphorylation of ERK1/2and caldesmon in rat VSMCs. Primary VSMCsfrom Sm22α-/-mice displayed decreased phosphorylation of this two proteinscompared with Sm22α+/+mice.
2SM22α regulates AngII-ERK1/2signaling cascades by promotingproteasome-mediated MKP3degradation
2.1Depletion of SM22α upregulates MKP3and thereby facilitatesdephosphorylation of ERK1/2.Knockdown of SM22α did not significantly change AngII-inducedphosphorylation of MEK. SM22α silencing resulted in an increasedexpression of MKP3, accompanied by reduction of ERK1/2and caldesmonphosphorylation. Moreover, a consistent increase in MKP3protein levels wasalso observed in the aortas, femoral and carotid arteries of Sm22α-/-micecompared with the Sm22α+/+controls. Inhibition of MKP3activity with theinhibitor BCI rescued the reduced ERK1/2activity resulted from loss ofSM22α.
2.2MKP3negatively regulates AngII-induced vasoconstriction and VSMCcontraction via inactivation of ERK1/2
The maximal contractile response of aortic rings to AngII wassignificantly increased by pretreatment with the MKP3inhibitor BCI, showinga leftward shift of the concentration-response curve of AngII, with the EC50of3.83nmol/L. Preincubation of VSMCs with BCI resulted in a significantlyincrease in the percentage of reduction in cell length and the shrinkage ofcollagen gels in response to AngII. Furthermore, overexpression of MKP3markedly attenuated the contractile response to AngII, displaying a reducedpercentage of reduction in cell length and collagen gel contraction. BCIincreased the phosphorylation of ERK1/2and caldesmon, whereas an oppositeeffect was observed in VSMCs overexpressing MKP3.
2.3SM22α inhibits the interaction of ERK1/2with MKP3.
The results of co-immunoprecipitation showed a weak interaction of MKP3with ERK1/2in resting VSMCs, which did not change upon AngIItreatment. Knockdown of SM22α led to an enhanced interaction of MKP3with ERK1/2. Similar results were observed in VSMCs derived from Sm22α-/-mice. These findings were further confirmed by immunofluorescence staining.The MKP3expression was increased following knockdown of SM22α, andcolocalized with ERK1/2.
2.4Loss of SM22α inhibits the proteasome-mediated MKP3degradation.
MKP3expression at the mRNA level in aortas form Sm22α-/-mice wasnot significantly different from that in the Sm22α+/+controls. Similarly, theMKP3mRNA level was not significantly altered by knockdown of SM22α. Toexamine the effect of knockdown of SM22α on half-life of MKP3protein, weused cycloheximide (CHX) to inhibit the de novo protein synthesis. Theresults showed that MKP3had a short half-life (less than30min), anddecreased dramatically after90min of incubation with CHX. The delayeddecay of MKP3protein was observed following knockdown of SM22α, with alonger half-life of90min. In the presence of MG132(the proteasomeinhibitor), half-life of MKP3was extended to90min. The ubiquitinationassay showed that knockdown of SM22α inhibited the polyubiquitination ofMKP3.
3SM22α promotes AngII-induced VSMC senescence
3.1SM22α is upregulated in AngII-induced VSMC senescence
The SA-β-gal-positive percentage in the VSMCs stimulated with AngIIfor3days was5.4±1.4%, which was not significantly changed comparedwith that in the control group (6.5±1.6%). After stimulation for5days, theSA-β-gal activity was elevated in a time-dependent manner, with theSA-β-gal-positive percentage of43.9±5.3%, significantly increasedcompared with those in the control group (22.1±4.1%, P <0.05). Afterstimulation for7and10days, the SA-β-gal activity was extremely high, witha SA-β-gal-positive percentage of78.9±5.2%and92.3±4.4%respectively.The expression of SM22α was elevated in VSMCs stimulated for7and10days compared with the control cells (P <0.05). The expressions of p21and p53displayed a similar fashion. However, the expression of PCNA wasreduced in the same conditions.
3.2AngII upregulates the transcriptional activity of SM22α gene.
To explore the mechanism by which SM22α protein was upregulated inAngII-induced senescence, the turnover of SM22α was detected. Treatmentwith AngII significantly increased the mRNA level of SM22α, which wasincreased by12.1folds after AngII stimulation for10days. However,ubiquitination of SM22α was not significantly varied by AngII stimulation.
3.3SM22α facilitates AngII-induced senescence in VSMCs.
Knockdown of SM22α using specific siRNA markedly attenuatedAngII-induced SA-β-gal activity. The percentage of SA-β-gal-positive cellswere25.3±4.3%and32.3±4.6%, respectively, after stimulation for7and10days, which was lower than those in the control group (79.2±4.2%and89.2±5.2%, P <0.05). The expression of p21and p53increased under the same
conditions.
Conclusions:
1. SM22α facilitates AngII-induced VSMC contraction through regulatingthe activity of ERK1/2.
2. SM22α regulates AngII-ERK1/2signaling cascades via promotingproteasome-mediated MKP3degradation and inhibiting the interaction ofERK1/2with MKP3.
3. SM22α promotes AngII-induced VSMC senescence.
4. SM22α is a novel regulator of vasoconstrictor signaling pathway, and amediator of vascular physiological and pathological processes
引文
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