RNA-destabilizing Factor Tristetraprolin Negatively Regulates NF-kappa B Signaling

Tristetraprolin (TTP) is a CCCH zinc finger-containing protein that destabilizes mRNA by binding to an AU-rich element. Mice deficient in TTP develop a severe inflammatory syndrome mainly because of overproduction of tumor necrosis factor alpha. We report here that TTP also negatively regulates NF-kappa B signaling at the transcriptional corepressor level, by which it may repress inflammatory gene transcription. TTP expression inhibited NF-kappa B-dependent transcription. However, overexpression of TTP did not affect reporter mRNA stability. Instead, TTP functioned as a corepressor of p65/NF-kappa B. In support of this concept, we found that TTP physically interacted with the p65 subunit of NF-kappa B and was also associated with HDAC1, -3, and -7 in vivo. Treatment with histone deacetylase inhibitors or small interfering RNA induced HDAC1 or HDAC3 knockdown completely or partly abolished the inhibitory activity of TTP on NF-kappa B reporter activation. Consistently, chromatin immuno-precipitation showed decreased recruitment of HDAC1 and increased recruitment of CREB-binding protein on the Mcp-1 promoter in TTP(-/-) cells compared with wild-type cells. Moreover, overexpression of TTP blocked CREB-binding protein-induced acetylation of p65/NF-kappa B. Taken together, these data suggest that TTP may also function in vivo as a modulator in suppressing the transcriptional activity of NF-kappa B

Macrophage-Stimulated Cardiac Fibroblast Production of IL-6 Is Essential for TGF β/Smad Activation and Cardiac Fibrosis Induced by Angiotensin II

Interleukin-6 (IL-6) is an important cytokine participating in multiple biologic activities in immune regulation and inflammation. IL-6 has been associated with cardiovascular remodeling. However, the mechanism of IL-6 in hypertensive cardiac fibrosis is still unclear. Angiotensin II (Ang II) infusion in mice increased IL-6 expression in the heart. IL-6 knockout (IL-6-/-) reduced Ang II-induced cardiac fibrosis: 1) Masson trichrome staining showed that Ang II infusion significantly increased fibrotic areas of the wild-type mouse heart, which was greatly suppressed in IL-6-/- mice and 2) immunohistochemistry staining showed decreased expression of α-smooth muscle actin (α-SMA), transforming growth factor β1 (TGF-β1) and collagen I in IL-6-/- mouse heart. The baseline mRNA expression of IL-6 in cardiac fibroblasts was low and was absent in cardiomyocytes or macrophages; however, co-culture of cardiac fibroblasts with macrophages significantly increased IL-6 production and expression of α-SMA and collagen I in fibroblasts. Moreover, TGF-β1 expression and phosphorylation of TGF-β downstream signal Smad3 was stimulated by co-culture of macrophages with cardiac fibroblasts, while IL-6 neutralizing antibody decreased TGF-β1 expression and Smad3 phosphorylation in co-culture of macrophage and fibroblast. Taken together, our results indicate that macrophages stimulate cardiac fibroblasts to produce IL-6, which leads to TGF-β1 production and Smad3 phosphorylation in cardiac fibroblasts and thus stimulates cardiac fibrosis

Cathepsin S Deficiency Results in Abnormal Accumulation of Autophagosomes in Macrophages and Enhances Ang II–Induced Cardiac Inflammation

BACKGROUND: Cathepsin S (Cat S) is overexpressed in human atherosclerotic and aneurysmal tissues and may contributes to degradation of extracellular matrix, especially elastin, in inflammatory diseases. We aimed to define the role of Cat S in cardiac inflammation and fibrosis induced by angiotensin II (Ang II) in mice. METHODS AND RESULTS: Cat S-knockout (Cat S(-/-)) and littermate wild-type (WT) C57BL/6J mice were infused continuously with Ang II (750 ng/kg/min) or saline for 7 days. Cat S(-/-) mice showed severe cardiac fibrosis, including elevated expression of collagen I and α-smooth muscle actin (α-SMA), as compared with WT mice. Moreover, macrophage infiltration and expression of inflammatory cytokines (tumor necrosis factor α, transforming growth factor β and interleukin 1β) were significantly greater in Cat S(-/-) than WT hearts. These Ang II-induced effects in Cat S(-/-) mouse hearts was associated with abnormal accumulation of autophagosomes and reduced clearance of damaged mitochondria, which led to increased levels of reactive oxygen species (ROS) and activation of nuclear factor-kappa B (NF-κB) in macrophages. CONCLUSION: Cat S in lysosomes is essential for mitophagy processing in macrophages, deficiency in Cat S can increase damaged mitochondria and elevate ROS levels and NF-κB activity in hypertensive mice, so it regulates cardiac inflammation and fibrosis

Akt2 Mediates TGF-�1-Induced Epithelial to Mesenchymal Transition by Deactivating GSK3�/Snail Signaling Pathway in Renal Tubular Epithelial Cells

Background: The epithelial-mesenchymal transition (EMT) induced by growth factors or cytokines, particularly transforming growth factor-β (TGF-β1), plays an important role in kidney tubulointerstitial injury. However, signaling pathways mediating TGF-β1-induced EMT are not precisely known. In this study, we examined the role of Akt2 on EMT. Methods: HK-2 cells were exposed to 10 ng/ml TGF-β1 to establish a model of EMT. The expression of proteins were detected by western blot assay and Immunofluorescence. The levels of genes were tested by RT-PCR. Results: We found that treatment of HK-2 cells, a human proximal tubular cell line, with 10 ng/ml TGF-β1 resulted in activation of phosphatidylinositol 3-kinase (PI3K)/Akt2 signaling as evidenced by increased p-PI3K, Akt2 and p-Akt (Ser 473) expression. Importantly, TGF-β1 treatment decreased zona occludins 1 (ZO-1) and E-cadherin (epithelial markers) expression, increased fibronectin and vimentin (mesenchymal makers) expression, which were prevented by Ly294002 (the inhibitor of PI3K) or small interfering RNA (siAkt2), suggesting that Akt2 mediated TGF-β1-induced EMT. Meanwhile, RNA and protein levels of Snail1, the key inducer of EMT, were significantly elevated in TGF-β1-treated HK-2 cells. TGF-β1 also induced inactivation of glycogen synthase kinase-3β (GSK3β), an endogenous inhibitor of Snail. Knockdown of Akt2 using siRNAs or the PI3K inhibitor Ly294002 inhibited TGF-β1-induced phosphorylation of GSK3β and expression of Snail1. Conclusion: These findings revealed that knockdown of Akt2 antagonized TGF-β1-induced EMT by inhibiting GSK3β/Snail signaling pathway

Mcp-Induced Protein 1 Suppresses Tnfα-Induced Vcam-1 Expression In Human Endothelial Cells

Endothelial inflammation plays a critical role in the development and progression of cardiovascular disease, albeit the mechanisms need to be fully elucidated. We here report that treatment of human umbilical vein endothelial cells (HUVECs) with tumor necrosis factor (TNF) α substantially increased the expression of MCP-induced protein 1 (MCPIP1). Overexpression of MCPIP1 protected ECs against TNFα-induced endothelial activation, as characterized by the attenuation in the expression of the adhesion molecule VCAM-1 and monocyte adherence to ECs. Conversely, small interfering RNA-mediated knock down of MCPIP1 increased the expression of VCAM-1 and monocytic adherence to ECs. These studies identified MCPIP1 as a feedback control of cytokines-induced endothelial inflammation. © 2010 Federation of European Biochemical Societies

RNA-destabilizing Factor Tristetraprolin Negatively Regulates NF-κB Signaling*

Tristetraprolin (TTP) is a CCCH zinc finger-containing protein that destabilizes mRNA by binding to an AU-rich element. Mice deficient in TTP develop a severe inflammatory syndrome mainly because of overproduction of tumor necrosis factor α. We report here that TTP also negatively regulates NF-κB signaling at the transcriptional corepressor level, by which it may repress inflammatory gene transcription. TTP expression inhibited NF-κB-dependent transcription. However, overexpression of TTP did not affect reporter mRNA stability. Instead, TTP functioned as a corepressor of p65/NF-κB. In support of this concept, we found that TTP physically interacted with the p65 subunit of NF-κB and was also associated with HDAC1, -3, and -7 in vivo. Treatment with histone deacetylase inhibitors or small interfering RNA induced HDAC1 or HDAC3 knockdown completely or partly abolished the inhibitory activity of TTP on NF-κB reporter activation. Consistently, chromatin immunoprecipitation showed decreased recruitment of HDAC1 and increased recruitment of CREB-binding protein on the Mcp-1 promoter in TTP−/− cells compared with wild-type cells. Moreover, overexpression of TTP blocked CREB-binding protein-induced acetylation of p65/NF-κB. Taken together, these data suggest that TTP may also function in vivo as a modulator in suppressing the transcriptional activity of NF-κB

Endogenous Sulfur Dioxide Inhibits Vascular Calcification in Association with the TGF-beta/Smad Signaling Pathway

The study was designed to investigate whether endogenous sulfur dioxide (SO2) plays a role in vascular calcification (VC) in rats and its possible mechanisms. In vivo medial vascular calcification was induced in rats by vitamin D3 and nicotine for four weeks. In vitro calcification of cultured A7r5 vascular smooth muscle cells (VSMCs) was induced by calcifying media containing 5 mmol/L CaCl2. Aortic smooth muscle (SM) alpha-actin, runt-related transcription factor 2 (Runx2), transforming growth factor-beta (TGF-beta) and Smad expression was measured. VC rats showed dispersed calcified nodules among the elastic fibers in calcified aorta with increased aortic calcium content and alkaline phosphatase (ALP) activity. SM alpha-actin was markedly decreased, but the osteochondrogenic marker Runx2 concomitantly increased and TGF-beta/Smad signaling was activated, in association with the downregulated SO2/aspartate aminotransferase (AAT) pathway. However, SO2 supplementation successfully ameliorated vascular calcification, and increased SM alpha-actin expression, but inhibited Runx2 and TGF-beta/Smad expression. In calcified A7r5 VSMCs, the endogenous SO2/AAT pathway was significantly downregulated. SO2 treatment reduced the calcium deposits, calcium content, ALP activity and Runx2 expression and downregulated the TGF-beta/Smad pathway in A7r5 cells but increased SM alpha-actin expression. In brief, SO2 significantly ameliorated vascular calcification in association with downregulation of the TGF-beta/Smad pathway.Funding Agencies|National Natural Science Foundation of China [81400311, 31130030]; Major Basic Research Development Program of China [2012CB517806, 2013CB933801]</p