Estrogen Modulates NFκB Signaling by Enhancing IκBα Levels and Blocking p65 Binding at the Promoters of Inflammatory Genes via Estrogen Receptor-β

NFκB signaling is critical for expression of genes involved in the vascular injury response. We have shown that estrogen (17β-estradiol, E2) inhibits expression of these genes in an estrogen receptor (ER)-dependent manner in injured rat carotid arteries and in tumor necrosis factor (TNF)-α treated rat aortic smooth muscle cells (RASMCs). This study tested whether E2 inhibits NFκB signaling in RASMCs and defined the mechanisms.TNF-α treated RASMCs demonstrated rapid degradation of IκBα (10-30 min), followed by dramatic increases in IκBα mRNA and protein synthesis (40-60 min). E2 enhanced TNF-α induced IκBα synthesis without affecting IκBα degradation. Chromatin immunoprecipitation (ChIP) assays revealed that E2 pretreatment both enhanced TNF-α induced binding of NFκB p65 to the IκBα promoter and suppressed TNF-α induced binding of NFκB p65 to and reduced the levels of acetylated histone 3 at promoters of monocyte chemotactic protein (MCP)-1 and cytokine-induced neutrophil chemoattractant (CINC)-2β genes. ChIP analyses also demonstrated that ERβ can be recruited to the promoters of MCP-1 and CINC-2β during co-treatment with TNF-α and E2.These data demonstrate that E2 inhibits inflammation in RASMCs by two distinct mechanisms: promoting new synthesis of IκBα, thus accelerating a negative feedback loop in NFκB signaling, and directly inhibiting binding of NFκB to the promoters of inflammatory genes. This first demonstration of multifaceted modulation of NFκB signaling by E2 may represent a novel mechanism by which E2 protects the vasculature against inflammatory injury

Apocynum Tablet Protects against Cardiac Hypertrophy via Inhibiting AKT and ERK1/2 Phosphorylation after Pressure Overload

Background. Cardiac hypertrophy occurs in many cardiovascular diseases. Apocynum tablet (AT), a traditional Chinese medicine, has been widely used in China to treat patients with hypertension. However, the underlying molecular mechanisms of AT on the hypertension-induced cardiac hypertrophy remain elusive. The current study evaluated the effect and mechanisms of AT on cardiac hypertrophy. Methods. We created a mouse model of cardiac hypertrophy by inducing pressure overload with surgery of transverse aortic constriction (TAC) and then explored the effect of AT on the development of cardiac hypertrophy using 46 mice in 4 study groups (combinations of AT and TAC). In addition, we evaluated the signaling pathway of phosphorylation of ERK1/2, AKT, and protein expression of GATA4 in the cardioprotective effects of AT using Western blot. Results. AT inhibited the phosphorylation of Thr202/Tyr204 sites of ERK1/2, Ser473 site of AKT, and protein expression of GATA4 and significantly inhibited cardiac hypertrophy and cardiac fibrosis at 2 weeks after TAC surgery (P<0.05). Conclusions. We experimentally demonstrated that AT inhibits cardiac hypertrophy via suppressing phosphorylation of ERK1/2 and AKT

O-GlcNAc Modification of NFκB p65 Inhibits TNF-α-Induced Inflammatory Mediator Expression in Rat Aortic Smooth Muscle Cells

BACKGROUND: We have shown that glucosamine (GlcN) or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) treatment augments O-linked-N-acetylglucosamine (O-GlcNAc) protein modification and attenuates inflammatory mediator expression, leukocyte infiltration and neointima formation in balloon injured rat carotid arteries and have identified the arterial smooth muscle cell (SMC) as the target cell in the injury response. NFκB signaling has been shown to mediate the expression of inflammatory genes and neointima formation in injured arteries. Phosphorylation of the p65 subunit of NFκB is required for the transcriptional activation of NFκB. This study tested the hypothesis that GlcN or PUGNAc treatment protects vascular SMCs against tumor necrosis factor (TNF)-α induced inflammatory stress by enhancing O-GlcNAcylation and inhibiting TNF-α induced phosphorylation of NFκB p65, thus inhibiting NFκB signaling. METHODOLOGY/PRINCIPAL FINDINGS: Quiescent rat aortic SMCs were pretreated with GlcN (5 mM), PUGNAc (10(-4) M) or vehicle and then stimulated with TNF-α (10 ng/ml). Both treatments inhibited TNF-α-induced expression of chemokines [cytokine-induced neutrophil chemoattractant (CINC)-2β and monocyte chemotactic protein (MCP)-1] and adhesion molecules [vascular cell adhesion molecule (VCAM)-1 and P-Selectin]. Both treatments inhibited TNF-α induced NFκB p65 activation and promoter activity, increased NFκB p65 O-GlcNAcylation and inhibited NFκB p65 phosphorylation at Serine 536, thus promoting IκBα binding to NFκB p65. CONCLUSIONS: There is a reciprocal relationship between O-GlcNAcylation and phosphorylation of NFκB p65, such that increased NFκB p65 O-GlcNAc modification inhibits TNF-α-Induced expression of inflammatory mediators through inhibition of NFκB p65 signaling. These findings provide a mechanistic basis for our previous observations that GlcN and PUGNAc treatments inhibit inflammation and remodeling induced by acute endoluminal arterial injury

Structural and Functional Basis of JAMM Deubiquitinating Enzymes in Disease

Deubiquitinating enzymes (DUBs) are a group of proteases that are important for maintaining cell homeostasis by regulating the balance between ubiquitination and deubiquitination. As the only known metalloproteinase family of DUBs, JAB1/MPN/Mov34 metalloenzymes (JAMMs) are specifically associated with tumorigenesis and immunological and inflammatory diseases at multiple levels. The far smaller numbers and distinct catalytic mechanism of JAMMs render them attractive drug targets. Currently, several JAMM inhibitors have been successfully developed and have shown promising therapeutic efficacy. To gain greater insight into JAMMs, in this review, we focus on several key proteins in this family, including AMSH, AMSH-LP, BRCC36, Rpn11, and CSN5, and emphatically discuss their structural basis, diverse functions, catalytic mechanism, and current reported inhibitors targeting JAMMs. These advances set the stage for the exploitation of JAMMs as a target for the treatment of various diseases

Smad3-mSin3A-HDAC1 Complex is Required for TGF-β1-Induced Transcriptional Inhibition of PPAR&#x03B3; in Mouse Cardiac Fibroblasts

Background: We have recently demonstrated that activated transforming growth factor-β (TGF-β) signaling suppresses myocardial peroxisome proliferator-activated receptor &#x03B3; (PPAR&#x03B3;) expression in the pressure overloaded heart. In this study, we aim to further define the molecular mechanisms that underlie TGF-β-induced PPAR&#x03B3; transcriptional inhibition. Methods: Adult mouse cardiac fibroblasts were isolated and cultured. PPAR&#x03B3; promoter activity was measured by the dual-Luciferase reporter assay. Interactions between transcription factors and the target gene were identified. Results: In cultured cardiac fibroblasts transfected with a plasmid containing a human PPAR&#x03B3; promoter, co-transfection of Smad3 and Smad4, but not Smad2, plasmids significantly enhanced TGF-β1-induced inhibition of PPAR&#x03B3; promoter activity. Promoter deletion analysis and site-directed mutagenesis assays defined two Smad binding elements on the promoter of the PPAR&#x03B3; gene. Utilizing chromatin immunoprecipitation analysis and DNA-affinity precipitation methods, we demonstrated that the transcriptional regulatory complex consisting of Smad3, mSin3A and HDAC1 bound to the promoter of the PPAR&#x03B3; gene in cardiac fibroblasts in response to TGF-β1 stimulation. Either silencing endogenous mSin3A expression by Lentivirus-mediated transduction of mSin3A shRNA or pretreatment with the specific HDAC1 inhibitor MS-275 effectively attenuated TGF-β-induced transcriptional suppression of PPAR&#x03B3;. Conclusion: These results suggest that TGF-β1-induced inhibition of PPAR&#x03B3; transcription depends on formation of a functional transcriptional regulatory complex that includes Smad3, mSin3A and HDAC1 at the PPAR&#x03B3; promoter

cGMP inhibits TGF-beta signaling by sequestering Smad3 with cytosolic beta2-tubulin in pulmonary artery smooth muscle cells

Atrial natriuretic peptide (ANP) and TGF-β play counterregulatory roles in pulmonary vascular adaptation to chronic hypoxia. We have demonstrated that ANP-cyclic GMP (cGMP)-protein kinase G (PKG) signaling inhibits TGF-β signaling by blocking TGF-β-induced nuclear translocation of mothers against decapentaplegic homolog (Smad)3 in pulmonary artery smooth muscle cells (PASMC). The current study tested the novel hypothesis that activation of the ANP-cGMP-PKG pathway limits TGF-β-induced Smad3 nuclear translocation by enhancing Smad3 binding to cytosolic anchoring proteins in isolated pulmonary artery smooth muscle cells. Cells were pretreated with vehicle or cGMP and then exposed to TGF-β1 treatment. Cytosolic fractions were isolated and immunoprecipitated with a selective anti-Smad3 antibody. Differential proteomic analysis of the cytosolic Smad3-interacting proteins by two-dimensional differential in-gel electrophoresis and mass spectroscopy followed by coimmunoprecipitation and immunostaining demonstrated that Smad3 was bound to β2-tubulin in a TGF-β1/cGMP-dependent manner: binding of Smad3 to β2-tubulin was decreased by TGF-β1 and increased by cGMP treatment. A site-directed mutagenesis study demonstrated that mutating Smad3 at Thr388, but not Ser309, two potential sites of PKG-induced hyperphosphorylation, inhibited cGMP-induced Smad3 binding to β2-tubulin. Further, luciferase reporter analysis showed that muation of T388 in Smad3 abolished the inhibitory effect of cGMP on TGF-β1-induced plasminogen activator inhibitor-1 (PAI-1) transcription. In addition, disruption of β2-tubulin with the microtubule depolymerizers nocodazole and colchicine promoted Smad3 dissociation from β2-tubulin, increased both TGF-β1-induced Smad3 nuclear translocation and PAI-1 mRNA expression, and abolished the inhibitory effects of cGMP on these processes. In contrast, the microtubule stabilizers paclitaxel and epothilone B increased cytosolic Smad3 binding to β2-tubulin and enhanced the inhibitory effect of cGMP on Smad3 nuclear translocation and PAI-1 expression in response to TGF-β1. These provocative findings suggest that sequestering Smad3 by β2-tubulin in cytosol is a key mechanism by which ANP-cGMP-PKG signaling interferes with downstream signaling from TGF-β and thus protects against pulmonary arterial remodeling in response to hypoxia stress