[Efficacy and Safety of Different Dosages of Decitabine in the Treatment of High-Risk Patients with Myelodysplastic Syndrome].

OBJECTIVE: To investigate the efficacy of high-risk myelodysplastic syndrome (MDS) patients treated by different doses of decitabine (DAC) and its safety. METHODS: Thirty patients with high-risk MDS were all treated by demethylation drug DAC. According to the doses of DAC, 30 patients were divided into 10-day regimen [6 mg/(m2·d)×10 d, 15 patients] group and 5-day regimen [15 mg/(m2·d)×5 d, 15 patients] group. The efficacy and adverse events of the patients in the two groups were observed. RESULTS: The patients were followed up to May 2020, in the 10-day regimen group, 10 cases achieved complete remission (CR), 3 cases achieved partial remission (PR), and 2 cases were progressive disease (PD). Four cases died, including 1 case for heart failure, 2 cases for respiratory failure and 1 case for serious infection. In the 5-day regimen group, 11 cases achieved CR, 1 case achieved PR, 3 cases were PD. Five cases died, including 2 cases for heart failure and 3 for serious infection. The CR rate and ORR of the patients in the two groups were 66.67% vs 73.33%, 86.67% vs 80.00%, respectively, which showed no significant differences, and the efficacy also showed no significant difference. After treatment, the levels of WBC, NE, Hb and PLT of the patients in 10-day regimen group were higher than those in 5-day regimen. In the 10-day regimen group, there were 11 cases of pneumonia, 2 cases of bacteremia, 1 case of skin infection and 1 case of urinary tract infection. While in the 5-day regimen group, 13 cases of pneumonia, 5 cases bacteremia, 1 case of skin infection and 3 cases of urinary tract infection. There were 2 cases with mild gastrointestinal response in the 10-day regimen group, and 7 cases with obvious nausea and anorexia in the 5-day regimen group. The symptoms were relieved after the treatment of acid suppression, stomach protection and antiemetic. The liver, kidney and heart function were monitored. One case liver function damage and 2 cases cardiac insufficiency were observed in the 10-day regimen group. Seven cases regimen cardiac insufficiency and 4 cases regimen liver function damage were observed in the 5-day regimen group. CONCLUSION: 10-day regimen and 5-day regimen are equally effective, but 10-day regimen is less myelosuppressive and more safer, which can be applied in clinical.

Accumulation of Smooth Muscle 22α Protein Accelerates Senescence of Vascular Smooth Muscle Cells via Stabilization of p53 In Vitro and In Vivo.

OBJECTIVE: Smooth muscle (SM) 22α, an actin-binding protein, displays an upregulated expression as a marker during cellular senescence. However, the causal relationship between SM22α and senescence is poorly understood. This study aimed to investigate the role of SM22α in angiotensin II (Ang II)-induced senescence of vascular smooth muscle cells (VSMCs). APPROACH AND RESULTS: We prepared a model of VSMC senescence induced by Ang II and found that the expression of SM22α in VSMCs was increased in response to chronic Ang II treatment. Overexpression of SM22α promoted Ang II-induced VSMC senescence, whereas knockdown of SM22α suppressed this process. Moreover, this effect of SM22α was p53 dependent. Increased SM22α protein obstructed ubiquitination and degradation of p53 and subsequently improved its stability. Furthermore, SM22α inhibited phosphorylation of Mdm2 (mouse double minute 2 homolog), an E3 ubiquitin-protein ligase, accompanied by a decreased interaction between Mdm2 and p53. Using LY294002, a PI3K/Akt inhibitor, we found that PI3K/Akt-mediated Mdm2 phosphorylation and activation was inhibited in senescent or SM22α-overexpressed VSMCs, in parallel with decreased p53 ubiquitination. We further found that SM22α inhibited activation of PI3K/Akt/Mdm2 pathway via strengthening actin cytoskeleton. In the in vivo study, we showed that the disruption of SM22α reduced the increase of blood pressure induced by Ang II, associated with decreased VSMC senescence through a mechanism similar to that in VSMCs in vitro. CONCLUSIONS: In conclusion, these findings suggest that the accumulation of SM22α promotes Ang II-induced senescence via the suppression of Mdm2-mediated ubiquitination and degradation of p53 in VSMCs in vitro and in vivo.

CKII-SIRT1-SM22α loop evokes a self-limited inflammatory response in vascular smooth muscle cells.

AIMS: Sirtuin 1 (SIRT1) inhibits nuclear factor kappa B (NF-κB) activity in response to the inflammatory cytokine tumour necrosis factor alpha (TNF-α). Smooth muscle (SM) 22α is a phosphorylation-regulated suppressor of IKK-IκBα-NF-κB signalling cascades in vascular smooth muscle cells (VSMCs). Sm22α knockout results in increased expression of pro-inflammatory genes in the aortas which are controlled by NF-κB. This study aimed to investigate the relationship between SM22α and SIRT1 in the control of vascular inflammation. METHODS AND RESULTS: The ligation injury model of Sirt1-Tg/Sm22α-/- mice displayed an increased level of the inflammatory molecules in the carotid arteries compared with Sirt1-Tg mice, accompanied with aggravating neointimal hyperplasia. In the in vitro study, on the one hand, we showed that TNF-α induced the epigenetic silencing of SM22α transcription via EZH2-mediated H3K27 methylation in the SM22α promoter region, contributing to inflammatory response. On the other hand, TNF-α simultaneously induced SIRT1 phosphorylation via CKII and thereby protected against inflammation. Phosphorylated SIRT1 interacted with and deacetylated EZH2 and, subsequently, promoted SM22α transcription by inhibiting EZH2 activity. Increased SM22α in turn facilitated the phosphorylation and activation of SIRT1 via recruitment of CKII to SIRT1, which amplified the anti-inflammatory effect of SIRT1. CONCLUSION: Our findings demonstrate that, in response to TNF-α stimulation, CKII-SIRT1-SM22α acts in a loop to reinforce the expression of SM22α, which limits the inflammatory response in VSMCs in vivo and in vitro. The anti-inflammatory effect of SIRT1 may be dependent on SM22α to some extent. Our data point to targeted activation of SIRT1 in VSMCs as a promising therapeutic avenue in preventing cardiovascular diseases.

EZH2-mediated α-actin methylation needs lncRNA TUG1, and promotes the cortex cytoskeleton formation in VSMCs.

Recent studies have revealed that long non-coding RNAs (lncRNAs) participate in vascular homeostasis and pathophysiological conditions development. But still very few literatures elucidate the regulatory mechanism of non-coding RNAs in this biological process. Here we identified lncRNA taurine up-regulated gene 1 (TUG1) in rat vascular smooth muscle cells (VSMCs), and got 4612bp nucleotide sequence. The expression level of TUG1 RNA was increased in synthetic VSMCs by real-time PCR analysis. Meanwhile, the expression of enhancer of zeste homolog 2 (EZH2) (TUG1 binding protein) increased in cytoplasm of VSMCs under the same conditions. Immunofluoresce analysis displayed the colocalization of EZH2 with α-actin in cytoplasm and F-actin in cell edge ruffles. This leads us to hypothesize the existence of cytoplasmic TUG1/EZH2/α-actin complex. Using RNA pull down assay, we found that TUG1 interacted with both EZH2 and α-actin. Disruption of TUG1 abolished the interaction of EZH2 with α-actin, and accelerated depolymerization of F-actin in VSMCs. Based on EZH2 methyltransferase activity and the potential methylation sites in α-actin structure, we revealed that α-actin was lysine-methylated. Furthermore, the methylation of α-actin was inhibited by knockdown of TUG1. In conclusion, these findings partly suggested that EZH2-mediated methylation of α-actin may be dependent on TUG1, and thereby promotes cortex F-actin polymerization in synthetic VSMCs.

Insulin-independent GLUT4 translocation in proliferative vascular smooth muscle cells involves SM22α.

The insulin-sensitive glucose transporter 4 (GLUT4) is a predominant facilitative glucose transporter in vascular smooth muscle cells (VSMCs) and is significantly upregulated in rabbit neointima. This study investigated the role of GLUT4 in VSMC proliferation, the cellular mechanism underlying PDGF-BB-stimulated GLUT4 translocation, and effects of SM22α, an actin-binding protein, on this process. Chronic treatment of VSMCs with PDGF-BB significantly elevated GLUT4 expression and glucose uptake. PDGF-BB-induced VSMC proliferation was dependent on GLUT4-mediated glucose uptake. Meanwhile, the response of GLUT4 to insulin decreased in PDGF-BB-stimulated VSMCs. PDGF-BB-induced GLUT4 translocation partially rescued glucose utilization in insulin-resistant cells. Immunofluorescence and western blot analysis revealed that PDGF-BB induced GLUT4 translocation in an actin dynamics-dependent manner. SM22α disruption facilitated GLUT4 translocation and glucose uptake by promoting actin dynamics and cortical actin polymerization. Similar results were observed in VSMCs of SM22α -/- mice. The in vivo experiments showed that the glucose level in the neointima induced by ligation was significantly increased in SM22α -/- mice, accompanied by increased neointimal thickness, compared with those in wild-type mice. These findings suggest that GLUT4-mediated glucose uptake is involved in VSMC proliferation, and provide a novel link between SM22α and glucose utilization in PDGF-BB-triggered proliferation. KEY MESSAGES: • GLUT4-mediated glucose uptake is required for the VSMC proliferation. • PDGF-BB-induced GLUT4 translocation partially rescues glucose uptake in insulin resistance. • SM22α disruption enhances PDGF-BB-induced GLUT4 translocation. • Glucose level in injured vascular tissue is positively correlated with neointimal hyperplasia.

SM22α inhibits lamellipodium formation and migration via Ras-Arp2/3 signaling in synthetic VSMCs.

We previously demonstrated that smooth muscle (SM) 22α promotes the migration activity in contractile vascular smooth muscle cells (VSMCs). Based on the varied functions exhibited by SM22α in different VSMC phenotypes, we investigated the effect of SM22α on VSMC migration under pathological conditions. The results demonstrated that SM22α overexpression in synthetic VSMCs inhibited platelet-derived growth factor (PDGF)-BB-induced cell lamellipodium formation and migration, which was different from its action in contractile cells. The results indicated two distinct mechanisms underlying inhibition of lamellipodium formation by SM22α, increased actin dynamic stability and decreased Ras activity via interference with interactions between Ras and guanine nucleotide exchange factor. The former inhibited actin cytoskeleton rearrangement in the cell cortex, while the latter significantly disrupted actin nucleation activation of the Arp2/3 complex. Baicalin, a herb-derived flavonoid compound, inhibited VSMC migration via upregulation of SM22α expression in vitro and in vivo. These data suggest that SM22α regulates lamellipodium formation and cell migration in a phenotype-dependent manner in VSMCs, which may be a new therapeutic target for vascular lesion formation.

TRAF6-Mediated SM22α K21 Ubiquitination Promotes G6PD Activation and NADPH Production, Contributing to GSH Homeostasis and VSMC Survival In Vitro and In Vivo.

RATIONALE: Vascular smooth muscle cell (VSMC) survival under stressful conditions is integral to promoting vascular repair, but facilitates plaque stability during the development of atherosclerosis. The cytoskeleton-associated smooth muscle (SM) 22α protein is involved in the regulation of VSMC phenotypes, whereas the pentose phosphate pathway plays an essential role in cell proliferation through the production of dihydronicotinamide adenine dinucleotide phosphate. OBJECTIVE: To identify the relationship between dihydronicotinamide adenine dinucleotide phosphate production and SM22α activity in the development and progression of vascular diseases. METHODS AND RESULTS: We showed that the expression and activity of glucose-6-phosphate dehydrogenase (G6PD) are promoted in platelet-derived growth factor (PDGF)-BB-induced proliferative VSMCs. PDGF-BB induced G6PD membrane translocation and activation in an SM22α K21 ubiquitination-dependent manner. Specifically, the ubiquitinated SM22α interacted with G6PD and mediated G6PD membrane translocation. Furthermore, we found that tumor necrosis factor receptor-associated factor (TRAF) 6 mediated SM22α K21 ubiquitination in a K63-linked manner on PDGF-BB stimulation. Knockdown of TRAF6 decreased the membrane translocation and activity of G6PD, in parallel with reduced SM22α K21 ubiquitination. Elevated levels of activated G6PD consequent to PDGF-BB induction led to increased dihydronicotinamide adenine dinucleotide phosphate generation through stimulation of the pentose phosphate pathway, which enhanced VSMC viability and reduced apoptosis in vivo and in vitro via glutathione homeostasis. CONCLUSIONS: We provide evidence that TRAF6-induced SM22α ubiquitination maintains VSMC survival through increased G6PD activity and dihydronicotinamide adenine dinucleotide phosphate production. The TRAF6-SM22α-G6PD pathway is a novel mechanism underlying the association between glucose metabolism and VSMC survival, which is beneficial for vascular repair after injury but facilitates atherosclerotic plaque stability.

Aggravated restenosis and atherogenesis in ApoCIII transgenic mice but lack of protection in ApoCIII knockouts: the effect of authentic triglyceride-rich lipoproteins with and without ApoCIII.

AIM: Previously, our group and others have demonstrated a causative relationship between severe hypertriglyceridaemia and atherogenesis in mice. Furthermore, clinical investigations have shown high levels of plasma Apolipoprotein C-III (ApoCIII) associated with hypertriglyceridaemia and even cardiovascular disease. However, it remains unclear whether ApoCIII affects restenosis in vivo, and whether such an effect is mediated by ApoCIII alone, or in combination with hypertriglyceridaemia. We sought to investigate ApoCIII in restenosis and clarify how smooth muscle cells (SMCs) respond to authentic triglyceride-rich lipoproteins (TRLs) with or without ApoCIII (TRLs ± ApoCIII). METHODS AND RESULTS: ApoCIII transgenic (ApoCIIItg) and knockout (ApoCIII-/-) mice underwent endothelial denudation to model restenosis. Here, ApoCIIItg mice displayed severe hypertriglyceridaemia and increased neointimal formation compared with wild-type (WT) or ApoCIII-/- mice. Furthermore, increased proliferating cell nuclear antigen (PCNA)-positive cells, Mac-3, and vascular cell adhesion protein-1 (VCAM-1) expression, and 4-hydroxynonenal (4HNE) production were found in lesion sites. ApoCIIItg and ApoCIII-/- mice were then crossed to low-density lipoprotein receptor-deficient (Ldlr-/-) mice and fed an atherogenic diet. ApoCIIItg/Ldlr-/- mice had significantly increased atherosclerotic lesions. However, there was no statistical difference in restenosis between ApoCIII-/- and WT mice, and in atherosclerosis between ApoCIII/Ldlr double knockout and Ldlr-/- mice. SMCs were then incubated in vitro with authentic TRLs ± ApoCIII isolated from extreme hypertriglyceridaemia glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1-deficient (GPIHBP1-/-) mice crossed with ApoCIIItg or ApoCIII-/- mice. It was shown that TRLs + ApoCIII promoted SMC proliferation, VCAM-1 expression, and reactive oxygen species (ROS) production, and activated the Akt pathway. Scavenging ROS significantly reduced SMC activation caused by TRLs + ApoCIII. CONCLUSIONS: Severe hypertriglyceridaemia resulting from ApoCIII overexpression promotes restenosis and atherosclerosis. Furthermore, we demonstrated that TRLs + ApoCIII promotes SMC proliferation.

SM22α inhibits vascular inflammation via stabilization of IκBα in vascular smooth muscle cells.

Smooth muscle (SM) 22α, an actin-binding protein, is down-regulated in atherosclerotic arteries. Disruption of SM22α promotes arterial inflammation through activation of reactive oxygen species (ROS)-mediated nuclear factor (NF)-κB pathways. This study aimed to investigate the mechanisms by which SM22α regulates vascular inflammatory response. The ligation injury model of SM22α(-/-) mice displayed up-regulation of inflammatory molecules MCP-1, VCAM-1, and ICAM-1 in the carotid arteries. Similar results were discovered in human atherosclerotic samples. In vitro studies, overexpression of SM22α attenuated TNF-α-induced IκBα phosphorylation and degradation, accompanied by decreased NF-κB activity and reduced inflammatory molecule expression. Using coimmunoprecipitation, we found that SM22α interacted with and stabilized IκBα in quiescent VSMCs. Upon TNF-α stimulation, SM22α was phosphorylated by casein kinase (CK) II at Thr139, leading to dissociation of SM22α from IκBα, followed by IκBα degradation and NF-κB activation. Our findings demonstrate that SM22α is a phosphorylation-regulated suppressor of IKK-IκBα-NF-κB signaling cascades. SM22α may be a novel therapeutic target for human vascular diseases and other inflammatory conditions.

Smooth muscle 22α facilitates angiotensin II-induced signaling and vascular contraction.

UNLABELLED: Smooth muscle 22α (SM22α) is involved in stress fiber formation and enhances contractility in vascular smooth muscle cells (VSMCs). In many cases, SM22α acts as an adapter protein to assemble signaling complexes and regulate signaling, but whether SM22α regulates contractile signaling induced by angiotensin II (AngII) remains unclear. To address this issue, we established a hypertension model of Sm22α(-/-) mice, and demonstrated that hypertension induced by AngII was attenuated in Sm22α(-/-) mice. A decreased vasoconstriction was observed in aortic rings from Sm22α(-/-) mice. Furthermore, loss of SM22α resulted in a reduced contractile response to AngII in VSMCs in vitro. The phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) induced by AngII was impaired following depletion of SM22α, in parallel with a reduced contractility. The decay of ERK1/2 activity was associated with increased expression of mitogen-activated protein kinase phosphatase 3 (MKP3). Inhibition of MKP3 activity rescued ERK1/2 activity. SM22α depletion caused an enhanced interaction of MKP3 with ERK1/2, and a reduced ubiquitination and degradation of MKP3. Knockdown of SM22α extended the half-life of MKP3. In conclusion, SM22α promotes AngII-induced contraction by maintenance of ERK1/2 signaling cascades through facilitating ubiquitination and degradation of MKP3. KEY MESSAGE: The vasoconstriction is attenuated in aortic rings from Sm22α(-/-) mice. MKP3 mediates dephosphorylation of ERK1/2 in AngII-induced VSMC contraction. SM22α inhibits the interaction of ERK1/2 with MKP3. SM22α promotes ubiquitination and degradation of MKP3. SM22α facilitates AngII-induced contraction by maintenance of ERK1/2 signaling.

Phosphorylation of smooth muscle 22α facilitates angiotensin II-induced ROS production via activation of the PKCδ-P47phox axis through release of PKCδ and actin dynamics and is associated with hypertrophy and hyperplasia of vascular smooth muscle cells in vitro and in vivo.

RATIONALE: We have demonstrated that smooth muscle (SM) 22α inhibits cell proliferation via blocking Ras-ERK1/2 signaling in vascular smooth muscle cells (VSMCs) and in injured arteries. The recent study indicates that SM22α disruption can independently promote arterial inflammation through activation of reactive oxygen species (ROS)-mediated NF-κB pathways. However, the mechanisms by which SM22α controls ROS production have not been characterized. OBJECTIVE: To investigate how SM22α disruption promotes ROS production and to characterize the underlying mechanisms. METHODS AND RESULTS: ROS level was measured by dihydroethidium staining for superoxide and TBA assay for malondialdehyde, respectively. We showed that downregulation and phosphorylation of SM22α were associated with angiotensin (Ang) II-induced increase in ROS production in VSMCs of rats and human. Ang II induced the phosphorylation of SM22α at Serine 181 in an Ang II type 1 receptor-PKCδ pathway-dependent manner. Phosphorylated SM22α activated the protein kinase C (PKC)δ-p47phox axis via 2 distinct pathways: (1) disassociation of PKCδ from SM22α, and in turn binding to p47phox, in the early stage of Ang II stimulation; and (2) acceleration of SM22α degradation through ubiquitin-proteasome, enhancing PKCδ membrane translocation via induction of actin cytoskeletal dynamics in later oxidative stress. Inhibition of SM22α phosphorylation abolished the Ang II-activated PKCδ-p47phox axis and inhibited the hypertrophy and hyperplasia of VSMCs in vitro and in vivo, accompanied with reduction of ROS generation. CONCLUSIONS: These findings indicate that the disruption of SM22α plays pivotal roles in vascular oxidative stress. PKCδ-mediated SM22α phosphorylation is a novel link between actin cytoskeletal remodeling and oxidative stress and may be a potential target for the development of new therapeutics for cardiovascular diseases.

Clinicopathological correlation of Krüppel-like factor 5 and matrix metalloproteinase-9 expression and cartilage degeneration in human osteoarthritis.

The present study was designed to investigate the clinicopathological correlation between the expression of KLF5 and MMP-9, which are associated with extracellular matrix degradation and cartilage degeneration in human knee osteoarthritis (OA). Tibiofemoral joint samples from 20 patients with OA, treated with surgery alone, were divided into two groups: 0=no change (NC, n=17), and severe changes with a higher mean score (≥ 3) (SC, n=29). The latter group contains samples with severe damages in cartilages and subchondral bones at medial tibial plateaux. The expression of the proteins was detected by immunofluorescence and quantitative RT-PCR, respectively. Neurovascular invasion was evaluated by protein gene product (PGP) 9.5 and CD34-positive staining and scanning electron microscopy, respectively. Safranin O staining showed that the sections from the SC group had increased cartilage degeneration. The number of vascular invasions in the SC group (16/29, 55.2%) was higher than that in NC controls (2/17, 11.7%, P<0.05). The expression of KLF5 and MMP-9 increased, and was co-localized in the same cells of SC cartilages. The severity of cartilage degeneration and vascular invasion was associated with upregulation of the two protein expressions and was significantly different between SC and NC samples (P<0.05). Taken together, the expression of KLF5 and MMP-9 may be involved in cartilage degeneration, contributing to human OA.

HDAC2 phosphorylation-dependent Klf5 deacetylation and RARα acetylation induced by RAR agonist switch the transcription regulatory programs of p21 in VSMCs.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) occurs in hypertension, atherosclerosis and restenosis after angioplasty, leading to pathophysiological vascular remodeling. As an important growth arrest gene, p21 plays critical roles in vascular remodeling. Regulation of p21 expression by retinoic acid receptor (RAR) and its ligand has important implications for control of pathological vascular remodeling. Nevertheless, the mechanism of RAR-mediated p21 expression in VSMCs remains poorly understood. Here, we show that, under basal conditions, RARα forms a complex with histone deacetylase 2 (HDAC2) and Krüppel-like factor 5 (Klf5) at the p21 promoter to inhibit its expression. Upon RARα agonist stimulation, HDAC2 is phosphorylated by CK2α. Phosphorylation of HDAC2, on the one hand, promotes its dissociation from RARα, thus allowing the liganded-RARα to interact with co-activators; on the other hand, it increases its interaction with Klf5, thus leading to deacetylation of Klf5. Deacetylation of Klf5 facilitates its dissociation from the p21 promoter, relieving its repressive effect on the p21 promoter. Interference with HDAC2 phosphorylation by either CK2α knockdown or the use of phosphorylation-deficient mutant of HDAC2 prevents the dissociation of Klf5 from the p21 promoter and impairs RAR agonist-induced p21 activation. Our results reveal a novel mechanism involving a phosphorylation-deacetylation cascade that functions to remove the basal repression complex from the p21 promoter upon RAR agonist treatment, allowing for optimum agonist-induced p21 expression.

Krüppel-like factor 4 interacts with p300 to activate mitofusin 2 gene expression induced by all-trans retinoic acid in VSMCs.

AIM: To elucidate how krüppel-like factor 4 (KLF4) activates mitofusin 2 (mfn-2) expression in all-trans retinoic acid (ATRA)-induced vascular smooth muscle cell (VSMC) differentiation. METHODS: The mfn-2 promoter-reporter constructs and the KLF4 acetylation-deficient or phosphorylation-deficient mutants were constructed. Adenoviral vector of KLF4-mediated overexpression and Western blot analysis were used to determine the effect of KLF4 on mfn-2 expression. The luciferase assay and chromatin immunoprecipitation were used to detect the transactivation of KLF4 on mfn-2 gene expression. Co-immunoprecipitation and GST pull-down assays were used to determine the modification of KLF4 and interaction of KLF4 with p300 in VSMCs. RESULTS: KLF4 mediated ATRA-induced mfn-2 expression in VSMCs. KLF4 bound directly to the mfn-2 promoter and activated its transcription. ATRA increased the interaction of KLF4 with p300 by inducing KLF4 phosphorylation via activation of JNK and p38 MAPK signaling. KLF4 acetylation by p300 increased its activity to transactivate the mfn-2 promoter. CONCLUSION: ATRA induces KLF4 acetylation by p300 and increases the ability of KLF4 to transactivate the mfn-2 promoter in VSMCs.

Angiotensin II stimulates KLF5 phosphorylation and its interaction with c-Jun leading to suppression of p21 expression in vascular smooth muscle cells.

Krüppel-like factor 5 (KLF5) and c-Jun are involved in angiotensin II (Ang II)-induced cell proliferation and play an important role in p21 expression. But the direct and functional implications of KLF5 and c-Jun in regulating p21 expression in vascular smooth muscle cells (VSMCs) are unclear. Here, we show that Ang II upregulated KLF5 and c-Jun expression and inhibited p21 expression in VSMCs, and silencing of KLF5 expression by KLF5-specific small interfering RNA (siRNA) neutralized the inhibitory effects of Ang II on p21 expression. Exposure of VSMCs to Ang II rapidly and strongly stimulated KLF5 phosphorylation, which results in an increase of the interaction of KLF5 with c-Jun. Treating VSMCs with PD98059, the ERK inhibitor, inhibited ERK activation and KLF5 phosphorylation as well as the interaction between KLF5 and c-Jun. Reporter analysis showed that both KLF5 and c-Jun cooperatively repressed the promoter of p21. Furthermore, KLF5 bound to its cis-elements in the p21 promoter, and meanwhile interacted with c-Jun in Ang II-induced VSMCs. These results suggest that Ang II induces KLF5 phosphorylation mediated by the ERK signalling in VSMCs, which in turn stimulates the interaction of KLF5 with c-Jun, subsequently leads to the suppression of p21 expression.