| 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 ERK1/21.1Disruption of SM22α attenuates AngII-induced vasoconstrictionSm22α+/+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 pathwayKnockdown 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 MKP3degradation2.1Depletion 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 ERK1/2The 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 senescence3.1SM22α is upregulated in AngII-induced VSMC senescenceThe 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 sameconditions.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... |
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