Cardioprotection afforded by NF-κB ablation is associated with activation of Akt in mice overexpressing TNF-α

2006 ◽  
Vol 290 (2) ◽  
pp. H590-H598 ◽  
Author(s):  
Yoshihiro Higuchi ◽  
Tung O. Chan ◽  
Maria A. Brown ◽  
Jin Zhang ◽  
Brent R. DeGeorge ◽  
...  

When selectively overexpressed in mouse heart, TNF-α effects the development of a cardiomyopathy that closely mimics that seen in human failing hearts. It has been suggested that two intracellular signaling pathways, the Akt protein kinase and the NF-κB transcription factor, mediated TNF-α signaling. The present experiments assessed the effects of TNF-α overexpression on these two target proteins in vivo. We measured cardiac Akt kinase phosphorylation and NF-κB activity in mice overexpressing TNF-α (TNF1.6). Both basal and insulin-stimulated Akt phosphorylation were reduced by almost 70% by TNF-α overexpression. By contrast, NF-κB was robustly activated. These effects were absent when TNF-α receptor 1 (TNFR1) was selectively ablated. Cardiomyocyte-specific overexpression of the dominant-negative inhibitory κB protein transgene and subsequent inhibition of NF-κB activity attenuated the effects of TNF-α on Akt phosphorylation. NF-κB inhibition also significantly improved fractional shortening and diminished ventricular hypertrophy and survival without affecting infiltrative inflammation or cytokine expression. Thus, while overexpression of TNF-α effected a marked Akt inhibition and NF-κB activation in mouse hearts, inhibition of NF-κB offered salutary benefits mediated at least in part through activation of Akt.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3321-3321
Author(s):  
Meixiao Long ◽  
Aaron M. Slaiby ◽  
Adam T. Hagymasi ◽  
Marianne A. Mihalyo ◽  
Alexander C. Lichtler ◽  
...  

Abstract When Th1 effector CD4 cells encounter tolerizing antigen in vivo their capacity to express the effector cytokines IFN-γ and TNF-α is lost more rapidly than non-effector functions such as IL-2 production and proliferation. To localize the relevant intracellular signaling defects, cytokine expression was compared following restimulation with antigen vs agents that bypass TCR-proximal signaling. IFN-γ and TNF-α expression were both partially rescued when TCR-proximal signaling was bypassed, indicating that both TCR-proximal and distal signaling defects impair the expression of these two effector cytokines. In contrast, bypassing TCR-proximal signaling fully rescued IL-2 expression. T-bet, a transcription and chromatin remodeling factor that is required to direct the differentiation of naive CD4 cells into IFN-γ-expressing Th1 effectors, was partially down-modulated in tolerized Th1 effectors. Enforcing T-bet expression during tolerization selectively rescued the ability to express IFN-γ, but not TNF-α. Conversely, expression of a dominant-negative T-bet in Th1 effectors selectively impaired the ability to express IFN-γ, but not TNF-α. Analysis of histone acetylation at the IFN-γ promoter further suggests that down-modulation of T-bet expression during Th1 effector CD4 cell tolerization does not impair IFN γ expression potential through alterations in chromatin structure.


2005 ◽  
Vol 289 (5) ◽  
pp. L883-L889 ◽  
Author(s):  
Weidong Wu ◽  
Robert A. Silbajoris ◽  
Young E. Whang ◽  
Lee M. Graves ◽  
Philip A. Bromberg ◽  
...  

Cyclooxygenase 2 (COX-2) expression is induced by physiological and inflammatory stimuli. Regulation of COX-2 expression is stimulus and cell type specific. Exposure to Zn2+ has been associated with activation of multiple intracellular signaling pathways as well as the induction of COX-2 expression. This study aims to elucidate the role of intracellular signaling pathways in Zn2+-induced COX-2 expression in human bronchial epithelial cells. Inhibitors of the phosphatidylinositol 3-kinase (PI3K) potently block Zn2+-induced COX-2 mRNA and protein expression. Overexpression of adenoviral constructs encoding dominant-negative Akt kinase downstream of PI3K or wild-type phosphatase and tensin homolog deleted on chromosome 10, an important PI3K phosphatase, suppresses COX-2 mRNA expression induced by Zn2+. Zn2+ exposure induces phosphorylation of the tyrosine kinases, including Src and EGF receptor (EGFR), and the p38 mitogen-activated protein kinase. Blockage of these kinases results in inhibition of Zn2+-induced Akt phosphorylation as well as COX-2 protein expression. Overexpression of dominant negative p38 constructs suppresses Zn2+-induced increase in COX-2 promoter activity. In contrast, the c-Jun NH2-terminal kinase and the extracellular signal-regulated kinases have minimal effect on Akt phosphorylation and COX-2 expression. Inhibition of p38, Src, and EGFR kinases with pharmacological inhibitors markedly reduces Akt phosphorylation induced by Zn2+. However, the PI3K inhibitors do not show inhibitory effects on p38, Src, and EGFR. These data suggest that p38 and EGFR kinase-mediated Akt activation is required for Zn2+-induced COX-2 expression and that the PI3K/Akt signaling pathway plays a central role in this event.


Endocrinology ◽  
2012 ◽  
Vol 153 (9) ◽  
pp. 4502-4510 ◽  
Author(s):  
Isobelle Grant ◽  
Judith E. Cartwright ◽  
Brooke Lumicisi ◽  
Alison E. Wallace ◽  
Guy S. Whitley

Impaired trophoblast invasion is associated with pregnancy disorders such as early pregnancy loss and preeclampsia. There is evidence to suggest that the consumption of caffeine during pregnancy may increase the risk of pregnancy loss; however, little is known about the direct effect of caffeine on normal trophoblast biology. Our objectives were to examine the effect of caffeine on trophoblast migration and motility after stimulation with epidermal growth factor (EGF) and to investigate the intracellular signaling pathways involved in this process. Primary first-trimester extravillous trophoblasts (EVT) and the EVT-derived cell line SGHPL-4 were used to study the effect of caffeine on EGF-stimulated cellular motility using time-lapse microscopy. SGHPL-4 cells were further used to study the effect of caffeine and cAMP on EGF-stimulated invasion of fibrin gels. The influence of caffeine and cAMP on EGF-stimulated intracellular signaling pathways leading to the activation of Akt were investigated by Western blot analysis. Caffeine inhibits both EGF-stimulated primary EVT and SGHPL-4 cell motility. EGF stimulation activates phosphatidylinositol 3-kinase, and Akt and caffeine inhibit this activation. Although cAMP inhibits both motility and invasion, it does not inhibit the activation of Akt, indicating that the effects of caffeine seen in this study are independent of cAMP. Further investigation indicated a role for mammalian target of rapamycin complex 2 (mTORC2) as a target for the inhibitory effect of caffeine. In conclusion, we demonstrate that caffeine inhibits EGF-stimulated trophoblast invasion and motility in vitro and so could adversely influence trophoblast biology in vivo.


Blood ◽  
2003 ◽  
Vol 102 (5) ◽  
pp. 1740-1742 ◽  
Author(s):  
Ingrid Pollet ◽  
Christy J. Opina ◽  
Carla Zimmerman ◽  
Kevin G. Leong ◽  
Fred Wong ◽  
...  

AbstractThe intracellular pathways by which inflammatory mediators transmit their angiogenic signals is not well studied. The effects of a potent inflammatory mediator, bacterial lipopolysaccharide (LPS), are transmitted through Toll-like receptors (TLRs). A major, although not exclusive, LPS/TLR intracellular signaling pathway is routed through TNF (tumor necrosis factor) receptor associated factor 6 (TRAF6). In this report we demonstrate that LPS directly stimulates endothelial sprouting in vitro. By blocking TRAF6 activity using retroviral expression of a dominant-negative TRAF6 in endothelial cells, we show that TRAF6 is absolutely required for the LPS-initiated angiogenic response in vitro and in vivo. Inhibition of either c-Jun N-terminal kinase (JNK) activity or nuclear factor κB (NF-κB) activity, downstream of TRAF6, is sufficient to inhibit LPS-induced endothelial sprouting. In contrast, only inhibition of NF-κB, but not JNK, activity blocks basic fibroblast growth factor (bFGF)–induced angiogenesis. Our findings thus demonstrate a direct endothelial-stimulatory role of LPS in initiating angiogenesis through activation of TRAF6-dependent signaling pathways.


2017 ◽  
Vol 312 (5) ◽  
pp. L599-L608 ◽  
Author(s):  
Sherry E. Adesina ◽  
Brandy E. Wade ◽  
Kaiser M. Bijli ◽  
Bum-Yong Kang ◽  
Clintoria R. Williams ◽  
...  

Pulmonary hypertension (PH) is characterized by increased pulmonary vascular resistance, pulmonary vascular remodeling, and increased pulmonary vascular pressures that often result in right ventricular dysfunction, leading to right heart failure. Evidence suggests that reactive oxygen species (ROS) contribute to PH pathogenesis by altering pulmonary vascular cell proliferation and intracellular signaling pathways. However, the role of mitochondrial antioxidants and oxidant-derived stress signaling in the development of hypoxia-induced PH is largely unknown. Therefore, we examined the role of the major mitochondrial redox regulator thioredoxin 2 (Trx2). Levels of Trx2 mRNA and protein were examined in human pulmonary arterial endothelial cells (HPAECs) and smooth muscle cells (HPASMCs) exposed to hypoxia, a common stimulus for PH, for 72 h. Hypoxia decreased Trx2 mRNA and protein levels. In vitro overexpression of Trx2 reduced hypoxia-induced H2O2 production. The effects of increased Trx2 protein level were examined in transgenic mice expressing human Trx2 (TghTrx2) that were exposed to hypoxia (10% O2) for 3 wk. TghTrx2 mice exposed to hypoxia had exacerbated increases in right ventricular systolic pressures, right ventricular hypertrophy, and increased ROS in the lung tissue. Trx2 overexpression did not attenuate hypoxia-induced increases in Trx2 oxidation or Nox4 expression. Expression of a dominant negative C93S Trx2 mutant that mimics Trx2 oxidation exacerbated hypoxia-induced increases in HPASMC H2O2 levels and cell proliferation. In conclusion, Trx2 overexpression failed to attenuate hypoxia-induced HPASMC proliferation in vitro or hypoxia-induced PH in vivo. These findings indicate that strategies to enhance Trx2 expression are unlikely to exert therapeutic effects in PH pathogenesis.


2021 ◽  
Author(s):  
Mary E Law ◽  
Bradley J Davis ◽  
Amanda F Ghilardi ◽  
Elham Yaaghubi ◽  
Zaafir M Dulloo ◽  
...  

Tranexamic Acid (TA) is a clinically used antifibrinolytic that acts as a lysine mimetic to block binding of Plasminogen with Plasminogen activators, preventing conversion of Plasminogen to its proteolytically activated form, Plasmin. Previous studies suggested that TA may exhibit anticancer activity by blockade of extracellular Plasmin formation. Plasmin-mediated cleavage of the CDCP1 protein may increase its oncogenic functions through several downstream pathways. Results presented herein demonstrate that TA blocks Plasmin-mediated excision of the extracellular domain of the oncoprotein CDCP1. In vitro studies indicate that TA reduces the viability of a broad array of human and murine cancer cell lines, and breast tumor growth studies demonstrate that TA reduces cancer growth in vivo. Based on the ability of TA to mimic lysine and arginine, we hypothesized that TA may perturb multiple processes that involve Lys/Arg-rich protein sequences, and that TA may alter intracellular signaling pathways in addition to blocking extracellular Plasmin production. Indeed, TA-mediated suppression of tumor cell viability is associated with multiple biochemical actions, including inhibition of protein synthesis, reduced activating phosphorylation of STAT3 and S6K1, decreased expression of the MYC oncoprotein, and suppression of Lys acetylation. These findings suggest that TA or TA analogs may serve as lead compounds and inspire the production of new classes of anticancer agents that function by mimicking Lys and Arg.


2008 ◽  
Vol 294 (6) ◽  
pp. H2879-H2888 ◽  
Author(s):  
Masao Takahashi ◽  
Etsu Suzuki ◽  
Ryo Takeda ◽  
Shigeyoshi Oba ◽  
Hiroaki Nishimatsu ◽  
...  

We examined whether ANG II and TNF-α cooperatively induce vascular inflammation using the expression of monocyte chemoattractant protein (MCP)-1 as a marker of vascular inflammation. ANG II and TNF-α stimulated MCP-1 expression in a synergistic manner in vascular smooth muscle cells. ANG II-induced MCP-1 expression was potently inhibited to a nonstimulated basal level by blockade of the p38-dependent pathway but only partially inhibited by blockade of the NF-κB-dependent pathway. In contrast, TNF-α-induced MCP-1 expression was potently suppressed by blockade of NF-κB activation but only modestly suppressed by blockade of p38 activation. ANG II- and TNF-α-induced activation of NF-κB- and p38-dependent pathways was partially inhibited by pharmacological inhibitors of ROS production. Furthermore, ANG II- and TNF-α-stimulated MCP-1 expression was partially suppressed by ROS inhibitors. We also examined whether endogenous ANG II and TNF-α cooperatively promote vascular inflammation in vivo using a wire injury model of the rat femoral artery. Blockade of both ANG II and TNF-α further suppressed neointimal formation, macrophage infiltration, and MCP-1 expression in an additive manner compared with blockade of ANG II or TNF-α alone. These results suggested that ANG II and TNF-α synergistically stimulate MCP-1 expression via the utilization of distinct intracellular signaling pathways (p38- and NFκB-dependent pathways) and that these pathways are activated in ROS-dependent and -independent manners. These results also suggest that ANG II and TNF-α cooperatively stimulate vascular inflammation in vivo as well as in vitro.


2004 ◽  
Vol 287 (3) ◽  
pp. H1207-H1213 ◽  
Author(s):  
Ines Hasanbasic ◽  
Jessica Cuerquis ◽  
Brian Varnum ◽  
Mark D. Blostein

Gas6 is a γ-carboxylated ligand for the receptor tyrosine kinase Axl. Gas6-Axl interactions can rescue endothelial cells from apoptosis, and this study examined the intracellular signaling mechanisms responsible for this phenomenon. Using flow cytometry, we first confirmed that Gas6 can abrogate apoptosis induced by serum starvation of primary cultures of human umbilical vein endothelial cells (HUVECs). This effect is mediated through phosphorylation of the serine-threonine kinase Akt, with maximal phosphorylation observed after 4 h of treatment with 100 ng/ml Gas6. Inhibition of Akt phosphorylation and abrogation of gas6-mediated survival of HUVECs by wortmannin implicated phosphatidylinositol 3-kinase as the mediator of Akt phosphorylation. Dominant negative Akt constructs largely abrogated the protective effect of Gas6 on HUVECs, underscoring the importance of Akt activation in Gas6-mediated survival. Several downstream regulators of this survival pathway were identified in HUVECs, namely, NF-κB as well as the antiapoptotic and proapoptotic proteins Bcl-2 and caspase 3, respectively. We showed that NF-κB is phosphorylated early after Gas6 treatment as evidenced by doublet formation on Western blotting. As well, the level of Bcl-2 protein increased, supporting the notion that the Bcl-2 antiapoptotic pathway is stimulated. The levels of expression of the caspase 3 activation products p12 and p20 decreased with Gas6 treatment, consistent with a reduction in proapoptotic caspase 3 activation. Taken together, these experiments provide new information about the mechanism underlying Gas6 protection from apoptosis in primary endothelial cell cultures.


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