Mechanism And Signification Of SM22α Modulating Vascular Oxidative Stress

Vascular smooth muscle cell (VSMC) proliferation and hypertrophy arepathological basis of vascular remodeling disease, including hypertension andatherosclerosis. The renin-angiotensin system (RAS) plays an essential role inregulating the physiological processes of the cardiovascular system. As theprimary effector molecule of this system, angiotensin (Ang) II has multiplepathophysiological effects on vascular smooth muscle cells (VSMCs),including increase in arterial pressure, induction of VSMC hypertrophy and soon. A significant body of evidence indicates that the reactive oxygen species(ROS) are involved in mediating the signal transduction of Ang II. Multiplerecent reports indicate that the p47^phoxsubunit of NADPH oxidase is the pivotfactor in Ang II-stimulated ROS production in vascular cells. The molecularand cellular actions of Ang II in cardiovascular diseases are almostexclusively mediated by angiotensin II type1receptors (AT1R). The proteinkinase C (PKC), as downstream of AT1R, is shown to be essential in AngII-accelerated ROS production via promoting of phosphorylation andmembrane translocation of p47^phox. PKCδ is one of the main isoforms in ratVSMCs. However, no information is available regarding a role of PKCδ inAng II-mediated ROS production.Despite increasing knowledge about the regulation of oxidative stress, theexact molecular mechanisms of functional protein governing ROS productionin VSMCs have not yet been fully defined. The latest study has revealed thatthe disruption of smooth muscle (SM)22α, an actin-associated protein,induces vascular inflammation through activation of ROS-mediated NF-κBpathways,but the precise mechanism of SM22α in controlling oxidative stressremains to be identified.In the present study, we speculate that dysfunction of SM22α may be involved in Ang II-induced vascular oxidative stress. Then, we investigatedthe effect of Ang II on the expression and function of SM22α in VSMC,which was related to ROS generation, VSMC proliferation and hypertrophy.Using protein interaction analysis technique, the effect of SM22α onPKCδ-p47^phoxaxis activation and the signal regulating mechanism wasexplored. Morever, the model of vascular hypertrophy and neointimalhyperplasia verified the findings in vitro. Our findings not only reveal afundamental biological function for SM22α in VSMC hypertrophy andproliferation, but also define a novel genetic pathway for vascular diseases.1Downregulation of SM22α Increases Oxidative Stress in Ang II-InducedVSMCs1.1Ang II-induced downregulation of SM22α expression increases ROSgeneration in VSMCsWestern blot analysis showed that chronic treatment with Ang II (10-7M)decreased the expression of SM22α in a time-dependent manner. Meantime,ROS production of VSMCs upon Ang II was detected using dihydroethidium(DHE) staining for superoxide and TBA assay for malondialdehyde (MDA).Compared with untreated cells, ROS production was increased with with twopeaks at0.5to1hour and24hours, respectively. Moreover, knockdown ofSM22α enhanced ROS production, while overexpression of SM22α inhibitedit in VSMCs. These findings suggested that downregulation of SM22αexpression increased Ang II-induced oxidative stress.1.2Downregulation of SM22α is required for the activation of p47^phoxandROS production induced by Ang II in VSMCsTo elucidate the role of p47^phoxin the Ang II-induced ROS production, theimmunoprecipitation was performed using anti-phosphor-Ser and anti-p47^phoxantibodies. The phosphorylation of p47^phoxby Ang II was maximal at10minutes, in a manner similar to the change of ROS production. Based onabove establishing a correlation between SM22α and ROS production, wenext investigated the effect of SM22α on p47^phoxactivation. Knockdown ofSM22α significantly enhanced AngII-induced phosphorylation of p47^phoxand ROS generation, while overexpression of SM22α decreased it. These datasuggested that SM22α disruption may be responsible for p47^phoxand ROSproduction in VSMCs.2Phosphorylation of SM22α Facilitates Ang II-Inducted ROS Productionvia Activation of PKCδ-P47phoxAxis through Release of PKCδ and ActinDynamics2.1Phosphorylation of SM22α at Ser181mediates p47^phoxactivation and ROSproduction via promoting interaction of PKCδ with p47^phoxTo confirm the possible roles of SM22α in early oxidative stress, based onthree potential phosphorylation sites in SM22α, immunoprecipitation assayshowed that SM22α phosphorylation was maximal at10to30minutes, anddeclined by60minutes after Ang II stimulation. To determine the correlationbetween phosphorylation of SM22α and p47^phoxactivation, VSMCs wereinfected by SM22α site-mutant adenovirus, Ad-GFP-S181D (substitution ofserine181to aspartic acid to mimic serine phosphorylation) andAd-GFP-S181A (mutation181residue from serine to alanine to inhibitSM22α phosphorylation). Immunoprecipitation results showed thatoverexpression of SM22α and S181A significantly suppressed p47^phoxphosphorylation and membrane translocation induced by Ang II. These dataindicated that phosphorylation of SM22α at Ser181may facilitate p47^phoxactivation.Previous studies found that both SM22α and p47^phoxcould bephosphorylated by PKC in vitro and in vivo. Using co-immunoprecipitationanalysis, we found that in quiescent VSMCs, PKCδ was associated withSM22α, but not p47^phox. However, upon Ang II stimulation for10minutes,PKCδ was dissociated from SM22α, and in turn interacted with p47^phox, with aconcomitant increase in SM22α phosphorylation and p47^phoxactivation.Immunofluorescent staining also showed the similar result. To verify theinvolvement of SM22α phosphorylation in direct activation of PKCδ-p47^phoxaxis, low endogenous SM22α expressing cell, the293A cells were transfectedwith GFP-S181D, GFP-S181A and WT constructs respectively. PKCδ was markedly associated with S181A in resting conditions, In contrast, aftertreatment with Ang II for10minutes, there was an increased interactionbetween PKCδ and p47^phoxin S181D-expressed cells. The glutathioneS-transferase (GST) pull-down assay showed that PKCδ in VSMC whole celllysates could be pulled down by GST-S181A fusion proteins rather thanGST-S181D. These results strongly supported the speculation thatphosphorylation of SM22α at Ser181triggered its dissociation from PKCδ,and subsequently facilitated the interaction of PKCδ with p47^phox.2.2AT1R-PKCδ signaling pathway mediates Ang II-induced phosphorylationof SM22αTo investigate the possible signal pathway of SM22α phosphorylation,AT1R antagonist valsartan, PKC agonist phorbol myristate acetate (PMA),PKC broad inhibitor Staurosporine, PKCδ selective inhibitor Rottlerin,siPKCδ, and PKCα and β selective inhibitor G6976were used. Western blotand immunoprecipitation analysis indicated that AT1R-PKCδ pathwaymediated Ang II-induced phosphorylation of SM22α in VSMCs.2.3Downregulation of SM22α may be responsible for the long-term effects ofAng II on oxidative stress via increasing actin dynamics in VSMCsIn eukaryocyte, protein ubiquitination generally is dependent on itsphosphorylation, then whether SM22α phosphorylation is contribute to itsubiquitination. Firstly, immunoprecipitates and Western blot showed Ang IIindeed induced SM22α ubiquitination. Furthermore, PKCδ selective inhibitorRottlerin and overexpression of S181A inhibited Ang II-induced SM22αubiquitination. We showed that S181D, but not S181A, had an increase effecton ubiquitylation in the293A cells transfected by plasmids. These resultssuggest that Ang II-induced phosphorylation facilitates SM22α degradationvia ubiquitin-proteasome pathway.SM22α plays critical role in regulating and stabilizing the actincytoskeleton, so we investigated the relationships between downregulation ofSM22α mediating actin dynamics and ROS production. VSMCs were treatedwith Ang II for0,12,24and48hours, and the distribution of actin in the cytosolic soluble (Sol, G-actin) and cytoskeletal fractions (Csk, F-actin) wasextracted respectively. Western blot showed the ratio of Csk/Sol-actin wasdecreased with prolonging the time of stimulation. Using cytochalasin B (CB)to destroy actin cytoskeleton, immunoprecipitates showed the interaction ofPKCδ with p47^phoxand activation of p47^phoxwere increased. In contrast, theactin-stabilizing toxin jasplakinolide (JPK) inhibited them. Moreover, aboveresults had shown the interaction between PKCδ and p47^phoxwas obviouslyincreased after SM22α downregulation, parallel with the decreasedCsk/Sol-actin ratio. These data suggested that depolymerization of actincytoskeleton enhances oxidative stress via increasing interaction of PKCδwith p47^phox. Taken together, Ang II-induced actin dynamics may be requiredfor long-term effects of oxidative stress via degradation of SM22α byubiquitin-proteasome pathway.3Phosphorylation of SM22α Is Involved in both Hypertrophy andHyperplasia of VSMCs via Increased ROS Generation in Vitro and inVivo3.1Overexpression of SM22α and S181A mutant decreased Ang II-inducedVSMC hypertrophy and peolieration in vitroTo investigate the role of SM22α phosphorylation mediating oxidativestress in hypertrophy and proliferation of VSMCs in vitro, BrdU incorporationand total protein amount per cell analysis were used. The BrdU incorporationand the total protein amount per cell increased following Ang II stimulationfor24and72hours, respectively, which was decreased by overexpression ofSM22α and S181A mutant.3.2Phosphorylation of SM22α participates in both hypertrophy andhyperplasia of VSMCs via increased ROS generation in VivoWe directly examined the effects of SM22α phosphorylation on vascularhypertrophy and blood pressures under basal conditions and after treatmentwith Ang II, following infection of the adenovirus constructs expressing wildtype and mutants of SM22α. Systolic blood pressure (SBP) was significantlyincreased in rats receiving Ang II plus the adenovirus constructs, compared with vehicle. The medial thickness and medial cross-sectional area (CSA) ofthe carotid arteries were increased after Ang II infusion plus GFP and S181Doverexpression, respectively, in consistent with increasing of ROS generation.Using the balloon injury model of rat carotid arteries, we also found that thephosphoralation of SM22α enhanced ROS generation and neointimalhyperplasia induced by injury.Taken together, these results suggest that SM22α may play key roles incellular hypertrophy and hyperplasia of VSMCs via modulating oxidativestress in vitro and in vivo. Thus, SM22α Ser181site may be an effectivemolecular target for prevention of hypertension and restenosis afterangioplasty.CONCLUSION1Downregulation of SM22α increases oxidative stress in Ang II-inducedVSMCs.2Phosphorylation of SM22α facilitates Ang II-inducted ROS production viaactivation of PKCδ-p47^phoxaxis through release of PKCδ and actindynamics.3Phosphorylation of SM22α is involved in both hypertrophy andhyperplasia of VSMCs via increased ROS generation in vitro and in vivo.