Interaction Of SM22α With Vascular Inflammation Responses

Inflammation has been considered as an important etiological factor ofcardiovascular diseases, such as atherosclerosis, restenosis. Smooth muscle(SM)22α, an actin-binding protein, is downregulated in atheroscleroticarteries. Loss of SM22α in apolipoprotein E knockout mice led to enlargedatherosclerotic lesions with prominent macrophage infiltration, a sign ofenhanced inflammation. Disruption of SM22α promotes arterial inflammationand chondrogenic conversion of VSMCs through activation of ROS-mediatedNF-κB pathways. Our recent studies revealed that overexpression of SM22αinhibited VSMCs proliferation and neointimal formation induced by ballooninjury via blockade of the Ras-ERK1/2signaling pathway. More recently, wedemonstrated that SM22α, as a PKCδ-regulating and PKCδ-regulated adaptorprotein, modulated vascular oxidative stress in vitro and in vivo throughactivation of PKCδ-p47phoxaxis via itself phosphorylation at Ser181site.Both proliferation and oxidative stress are two major pathological features invascular inflammatory diseases. However, the molecular events that linkSM22α disruption to inflammatory signal transduction pathways are poorlycharacterized. Loss of SM22α resulting in enhanced vascular inflammationprovided the possibility that SM22α may regulate the NF-κB signalingpathway.1Loss of SM22α aggravates vascular inflammation.To verify this finding and to get a more complete picture of SM22α-dependenteffector functions, we first performed transcriptome profiling of aortic tissuesin Sm22α-/-mice and wild type (WT) mice littermates (Sm22α+/+). We foundthat loss of SM22α was accompanied by increased expression ofpro-inflammatory genes.The upstream regulator analysis by IPA showed that NF-κB(RelA) was the major transcription regulator that was activated in Sm22α-/- mice. Using immunohistochemical staining, we next confirmed thatexpression of inflammatory molecules, including MCP-1, VCAM-1, ICAM-1,MMP-2and MMP-9, were significantly increased in carotid neointimalformation of Sm22α-/-mice compared with their Sm22α+/+mice littermates. Wethen looked for evidence of SM22α involvement in vascular inflammation ofhuman, and found that the expression of SM22α significantly decreased in therenal neointima and the carotid artery atherosclerotic plaques, accompanied byincreased expression of these inflammatory molecules, compared with that ofthe normal arteries. These findings suggest a correlation between decreasedSM22α expression and the arterial injury.2TNF-α epigenetically silences SM22α gene via EZH2-mediatedH3K27me32.1TNF-α downregulates SM22α gene expressionTo understand the upstream regulatory mechanism by which the expression ofSM22α is decreased under inflammatory conditions, we first examined theexpression pattern of SM22α following TNF-α stimulation. The resultsshowed that TNF-α reduced both mRNA and protein levels of SM22α.Furthermore, treatment of VSMCs with TNF-α resulted in a decreased SM22αpromoter activity.2.2TNF-α increases histone H3K27trimethylation at SM22α promoterWe quantified histone methylation at the SM22α gene promoter region usingChIP followed by quantitative PCR (ChIP-qPCR) assay. We found that thetrimethylation of histone H3lysine27(H3K27me3) but not lysine9enrichedin the SM22α promoter region upon TNF-α stimulation. To further confirm theepigenetic link between H3K27me3and SM22α transcriptional silencing, thebinding activity of SRF, a key transcriptional regulator for SMC marker genes,was detected using ChIP assay. We showed that recruitment of SRF to theSM22α CArG box chromatin decreased under the same conditions, associatedwith increased H3K27me3and the transcriptional silencing following TNF-αstimulation.2.3EZH2mediates epigenetic silencing of SM22α gene EZH2is the major enzyme for H3K27methylation. We found that TNF-αinduced EZH2acetylation, consistent with increased H3K27me3, suggestingthat the acetylation of EZH2may be an active form. Furthermore,overexpression of EZH2increased TNF-α-induced global H3K27me3levels.ChIP-qPCR data also showed significant increased H3K27me3and decreasedSRF recruitment at SM22α promoter region in EZH2-overexpressed VSMCs.Similarly, reduced SM22α promoter activities were observed using reportergene assay. Our findings suggest that EZH2may mediate H3K27me3ofSM22α gene promoter under inflammatory conditions.2.4TNF-α-induced acetylation of EZH2is reduced by increasing SIRT1activityWe focused on the roles of SIRT1in EZH2activation induced by TNF-α. Theresult showed that the selective SIRT1agonist SRT1720significantlydecreased TNF-α-induced acetylation of EZH2and histone H3K27me3ofSM22α promoter, accompanied by increasing of SRF binding to the SM22αpromoter and the reporter gene activity.2.5Proinflammation environment in injured carotids of Sirt1-Tg/Sm22α-/-miceTo understand the significances of SIRT1activating SM22α expression ininflammatory conditions, Sm22α-/-mice were crossed with a Sirt1-Tgbackground to generate Sirt1-Tg/Sm22α-/-mice. The expression ofinflammatory molecules in the injured arteries were increased inSirt1-Tg/Sm22α-/-mice compared with Sirt1-Tg/Sm22α+/+mice, suggestingthat SM22α may contributes to the anti-inflammation action of SIRT1associated with vascular diseases. Taken together, these results indicate thatSIRT1-mediated EZH2deacetylation was involved in activation of SM22αgene transcription via reduction of H3K27methylation, and that SIRT1shouldbe critical to maintain the homeostasis of SM22α expression.2.6SM22α facilitates SITR1activation via recruitment of CKII to SIRT1SIRT1facilitated SM22α expression. We then investigated whether SM22αacts as a feedback regulator of SIRT1activity in inflammatory conditions. Wefound that overexpression of SM22α enhanced SIRT1phosphorylation without changes of total SIRT1protein levels upon TNF-α stimulation.Knockdown of SM22α by siRNA resulted in the reduction of SIRT1phosphorylation, which almost completely abolished binding of SIRT1toEZH2. This result led us to identify a potential kinase that phosphorylatesSIRT1. Using a panel of kinase inhibitors, we observed that TNFα-inducedphosphorylation of SIRT1was blocked by the CKII inhibitor heparin, whichwas verified by decreasing endogenous CKII by specific siRNA. Importantly,the association of SIRT1to EZH2was impaired following CKII knockdownor inhibition, suggesting that CKII is an upstream of SIRT1inactivatingEZH2.2.7SM22α is a molecule platform for interaction between CKII and SIRT1We next asked how SM22α facilitates CKII phosphorylating SIRT1. We foundthat knockdown of SM22α suppressed the interaction of CKII with SIRT1,implying that SM22α may act as a scaffold protein to bring together CKII andSIRT1. Indeed, coimmunoprecipitation assays showed SM22α was present inthe same complex with CKII and SIRT1, which was confirmedby co-localization. Taken together, these results suggest that SM22α recruitsCKII to SIRT1to facilitate SIRT1phosphorylation and subsequentlydeacetylation of EZH2, forming positive feedback loop of SM22α expressiveregulation in response to TNF-α.3SM22α inhibits NF-κB and vascular inflammation3.1SM22α inhibits NF-κB nuclear translocation and NF-κB target genesexpression.We showed that overexpression of SM22α decreased TNF-α-inducedexpression of ICAM-1, VCAM-1and iNOS at the mRNA and protein levelsin VSMCs. The knockdown of endogenous SM22α using specific siSM22αsignificantly enhanced TNF-α-induced nuclear translocation of RelA/p65. Thesimilar results were discovered in VSMCs isolated from Sm22α-/-mice.Conversely，overexpression of SM22α decreased it. The results from agaroseoligonucleotide pull-down assay showed that overexpression of SM22αreduced DNA binding activity of NF-κB. The inhibitory effect of SM22α on NF-κB activity was further determined by transiently co-expressing SM22αwith a luciferase reporter driven by a6-tandem-repeat NF-κB element. Theseresults suggest that SM22α inhibits VSMC inflammatory responses throughblockade of NF-κB activation, and may be a novel target foranti-inflammatory intervention.3.2SM22α inhibits TNF-α-induced IκBα phosphorylation and degradationSM22α overexpression markedly reduced TNF-α-induced IκBαphosphorylation and degradation without change of IKK phosphorylationcompared with VSMCs infected with vehicle Ad-GFP upon TNF-αstimulation. Conversely, knockdown of endogenous SM22α expressionresulted in excessive phosphorylation and degradation of IκBα, decreasing ofIκBα compared with siCon. Similarly, TNF-α-induced IκBα phosphorylationand degradation in VSMCs of Sm22α-/-mice were much higher than that inWT mice.Previous study indicated that IκBα physically interacted with acytoskeleton-associated protein through its signal response domain to regulateNF-κB activation. These let us to hypothesize that SM22α interacts with IκBαto prevent it from phosphorylation and degradation. To test this hypothesis, weperformed cross-coimmunoprecipitation using anti-IκBα and anti-SM22αantibodies, respectively. The results showed that IκBα was associated withSM22α in quiescent VSMCs, and was almost dissociated from SM22αfollowing TNF-α stimulation, which was verified by immunofluorescentstaining. This interaction was also confirmed by coimmunoprecipitationexperiments in HEK293cells. Furthermore, VSMCs of Sm22α-/-mice wereinfected with Ad-GFP-SM22α, to rescue expression of SM22α. The resultsalso showed a marked reduction of the interaction between SM22α and IκBαupon TNF-α stimulation. Collectively, these results indicate that SM22α formsa complex with IκBα under basal conditions, which may be disrupted byTNF-α stimulation.3.3SM22α Thr139phosphorylation by CKII promotes IκBα phosphorylationand degradation through its dissociation from IκBα Based on the previous finding that CKII activity increased after TNF-αtreatment, we first sought to determine whether TNF-α induces SM22αphosphorylation in VSMCs. The results showed that the phosphorylation ofSM22α at Ser and Thr sites increased, while total SM22α levels remainedunchanged during TNF-α short-time stimulation. The pre-incubation withheparin significantly attenuated TNF-α-induced phosphorylation of SM22α atThr but not Ser site. To verify this argument, the specific CKII siRNA or adominant-negative form of CKII (CKIIDN) were transfected into VSMCs todisrupt CKII, respectively. We showed that disruption of CKII abolishedTNF-α-induced Thr phosphorylation of SM22α. In addition, in a rescueexperiment, re-expression of CKII could restore TNF-α-induced SM22αphosphorylation in CKII-knocked down VSMCs. Furthermore, we observedThr phosphorylation of SM22α in the VSMCs of Sm22α-/-mice followingtransduction with SM22α WT, but not the mutants of T139D (to mimicthreonine phosphorylation) or T139A (to inhibit SM22α phosphorylation),suggesting that CKII selectively phosphorylates SM22α Thr139in response toTNF-α stimulation.To determine that CKII-mediated Thr139phosphorylation results indissociation of SM22α from IκBα, we analyzed IκBα binding to SM22α WT,as well as T139D and T139A mutants using coimmunoprecipitation,respectively. SM22α T139D mutant revealed a reduced interaction with IκBα,while SM22α T139A had an increase in binding to IκBα compared withSM22α WT, suggesting that phosphorylation of SM22α is required for IκBαdissociation. To assess whether this dissociation contributes to IκBαphosphorylation and degradation, SM22α WT and these two phosphorylationmutants were transducted into Sm22α-/-mice VSMCs, respectively. SM22αT139A mutant markedly decreased TNF-α-induced IκBα phosphorylation anddegradation compared to SM22α WT or T139D mutant, accompanied bydecreased expression of nuclear RelA/p65. Taken together, these data indicatethat the phosphorylation-mediated dissociation SM22α from IκBα facilitatesIκBα phosphorylation and degradation, and subsequent NF-κB nuclear translocation upon TNF-α stimulation. We identify a novel regulatorymechanism by which the degree of inflammation is controlled by SM22αexpression.CONCLUSION1Loss of SM22α aggravates vascular inflammation.2EZH2-mediated H3K27trimethylation epigenetically silencestranscription of SM22α gene and is abrogated by SIRT1upon TNF-αstimulation.3SM22α suppresses TNF-α-induced NF-κB activation by stabilizing IκBα,and SM22α phosphorylation regulates its suppression of NF-κB activation.