Classical NF-κB activation impairs skeletal muscle oxidative phenotype by reducing IKK-α expression

Background: Loss of quadriceps muscle oxidative phenotype (OXPHEN) is an evident and debilitating feature of chronic obstructive pulmonary disease (COPD). We recently demonstrated involvement of the inflammatory classical NF-κB pathway in inflammation-induced impairments in muscle OXPHEN. The exact underlying mechanisms however are unclear. Interestingly, IκB kinase α (IKK-α: a key kinase in the alternative NF-κB pathway) was recently identified as a novel positive regulator of skeletal muscle OXPHEN. We hypothesised that inflammation-induced classical NF-κB activation contributes to loss of muscle OXPHEN in COPD by reducing IKK-α expression.Methods: Classical NF-κB signalling was activated (molecularly or by tumour necrosis factor α: TNF-α) in cultured myotubes and the impact on muscle OXPHEN and IKK-α levels was investigated. Moreover, the alternative NF-κB pathway was modulated to investigate the impact on muscle OXPHEN in absence or presence of an inflammatory stimulus. As a proof of concept, quadriceps muscle biopsies of COPD patients and healthy controls were analysed for expression levels of IKK-α, OXPHEN markers and TNF-α.Results: IKK-α knock-down in cultured myotubes decreased expression of OXPHEN markers and key OXPHEN regulators. Moreover, classical NF-κB activation (both by TNF-α and IKK-β over-expression) reduced IKK-α levels and IKK-α over-expression prevented TNF-α-induced impairments in muscle OXPHEN. Importantly, muscle IKK-α protein abundance and OXPHEN was reduced in COPD patients compared to controls, which was more pronounced in patients with increased muscle TNF-α mRNA levels.Conclusion: Classical NF-κB activation impairs skeletal muscle OXPHEN by reducing IKK-α expression. TNF-α-induced reductions in muscle IKK-α may accelerate muscle OXPHEN deterioration in COPD.     

Tumor necrosis factor-α receptor type 1, not type 2, mediates its acute responses in the kidney

Acute administration of tumor necrosis factor-α (TNF-α) resulted in decreases in renal blood flow (RBF) and glomerular filtration rate (GFR) but induced diuretic and natriuretic responses in mice. To define the receptor subtypes involved in these renal responses, experiments were conducted to assess the responses to human recombinant TNF-α (0.3 ng·min(-1)·g body wt(-1) iv infusion for 75 min) in gene knockout (KO) mice for TNF-α receptor type 1 (TNFαR1 KO, n = 5) or type 2 (TNFαR2 KO, n = 6), and the results were compared with those obtained in corresponding wild-type [WT (C57BL/6), n = 6] mice. Basal levels of RBF (PAH clearance) and GFR (inulin clearance) were similar in TNFαR1 KO, but were lower in TNFαR2 KO, than WT mice. TNF-α infusion in WT mice decreased RBF and GFR but caused a natriuretic response, as reported previously. In TNFαR1 KO mice, TNF-α infusion failed to cause such vasoconstrictor or natriuretic responses; rather, there was an increase in RBF and a decrease in renal vascular resistance. Similar responses were also observed with infusion of murine recombinant TNF-α in TNFαR1 KO mice (n = 5). However, TNF-α infusion in TNFαR2 KO mice caused changes in renal parameters qualitatively similar to those observed in WT mice. Immunohistochemical analysis in kidney slices from WT mice demonstrated that while both receptor types were generally located in the renal vascular and tubular cells, only TNFαR1 was located in vascular smooth muscle cells. There was an increase in TNFαR1 immunoreactivity in TNFαR2 KO mice, and vice versa, compared with WT mice. Collectively, these functional and immunohistological findings in the present study demonstrate that the activation of TNFαR1, not TNFαR2, is mainly involved in mediating the acute renal vasoconstrictor and natriuretic actions of TNF-α.     

Direct involvement of tumor necrosis factor-α in the regulation of glucose uptake in rainbow trout muscle cells

The proinflammatory cytokine TNF-α is known to have a direct action on skeletal muscle in mammals. However, little is known regarding the potential effects of cytokines on nonimmune tissues, particularly in skeletal muscle, in fish. The aim of this study was to investigate the effects of recombinant trout TNF-α (rtTNF-α) on skeletal muscle carbohydrate metabolism in rainbow trout (Oncorhynchus mykiss). We used a primary cell culture of muscle cells from rainbow trout to show that rtTNF-α stimulates glucose uptake in myoblasts and myotubes at concentrations that do not affect the viability of the cells, requiring de novo protein synthesis as shown by the impairment of rtTNF-α-stimulated glucose uptake by cycloheximide. With the use of specific inhibitors, we show that rtTNF-α-stimulated glucose uptake is mediated by the p38MAPK, NF-κB, and JNK pathways. Additionally, we provide evidence that the stimulatory effects of rtTNF-α on glucose uptake in trout skeletal muscle cells may be caused, at least in part, by an increase in the amount of GLUT4 at the plasma membrane. Incubation of trout muscle cells with conditioned medium from LPS-stimulated trout macrophages, enriched in TNF-α, increased glucose uptake. Our results indicate that recombinant, as well as native trout TNF-α, directly stimulates glucose uptake in trout muscle cells and provide evidence, for the first time in nonmammalian vertebrates, for a potential regulatory role of TNF-α in skeletal muscle metabolism.     

Tumor necrosis factor-α stimulation endothelial-to-mesenchymal transition during cardiac fibrosis via endothelin-1 signaling

Cardiac fibrosis is an important pathological change after myocardial infarction (MI). High concentration of tumor necrosis factor-α (TNF-α) contributes to cardiac fibrosis, and TNF-α has been demonstrated to be involved in transforming growth factor-β1-induced endothelial-to-mesenchymal transition (EndMT). However, the role and molecular mechanisms of TNF-α during cardiac fibrosis remain largely unexplored. In this study, we demonstrated that TNF-α and endothelin-1 (ET-1) were upregulated in cardiac fibrosis after MI, and genes associated with EndMT were also upregulated. An in vitro model of EndMT demonstrated that TNF-α promoted EndMT by upregulation of vimentin and α-smooth muscle actin, and which strongly increased ET-1 expression. ET-1 promoted TNF-α-induced expression of gene program through phosphorylation levels of SMAD family member 2, while subsequent inhibition of ET-1 almost abolished the effect of TNF-α during the process of EndMT. In summary, these findings demonstrated that ET-1 is involved in the EndMT induced by TNF-α during cardiac fibrosis.     

Genetically recessive mutant of human monocytic leukemia U937 resistant to tumor necrosis factor-α-induced apoptosis

Tumor necrosis factor-α (TNF-α) is a cytokine that induces apoptosis in various cell systems by binding to the TNF receptor (TNFR). To study TNF-α-induced apoptosis, we isolated and characterized a novel TNF-α-resistant variant, U937/TNF clone UA, from human monocytic leukemia U937 cells. The UA cells resist apoptosis induced by TNF-α and anti-Fas antibody but not by anticancer drugs, such as VP-16 and Ara-C. Somatic cell hybridization between U937 and UA showed that apoptosis resistance to TNF-α in UA was genetically recessive. The hybridization analysis also showed that UA and another recessive mutant clone, UC, belong to different complementation groups in TNF-α-induced apoptosis signaling. In UA cells, TNF-α-induced disruption of mitochondrial membrane potential and CPP32 activation were abrogated. Expression of TNFR, Fas, and Bcl-2 family proteins was not changed in UA cells. These results suggest that the apoptosis resistant UA cells could have a functional defect in apoptosis signaling from the TNFR to mitochondria and interleukin-1β converting enzyme (ICE) family protease activation. UA cells could be used to study signaling linkage between cell death-inducing receptor and mitochondria. J. Cell. Physiol. 174:179–185, 1998. © 1998 Wiley-Liss, Inc.     

Nox2 and Nox4 mediate tumour necrosis factor-α-induced ventricular remodelling in mice

Reactive oxygen species (ROS) and pro-inflammatory cytokines are crucial in ventricular remodelling, such as inflammation-associated myocarditis. We previously reported that tumour necrosis factor-α (TNF-α)-induced ROS in human aortic smooth muscle cells is mediated by NADPH oxidase subunit Nox4. In this study, we investigated whether TNF-α-induced ventricular remodelling was mediated by Nox2 and/or Nox4. An intravenous injection of murine TNF-α was administered to a group of mice and saline injection was administered to controls. Echocardiography was performed on days 1, 7 and 28 post-injection. Ventricular tissue was used to determine gene and protein expression of Nox2, Nox4, ANP, interleukin (IL)-1β, IL-2, IL-6, TNF-α and to measure ROS. Nox2 and Nox4 siRNA were used to determine whether or not Nox2 and Nox4 mediated TNF-α-induced ROS and upregulation of IL-1β and IL-6 in adult human cardiomyocytes. Echocardiography showed a significant increase in left ventricular end-diastolic and left ventricular end-systolic diameters, and a significant decrease in the ejection fraction and fractional shortening in mice 7 and 28 days after TNF-α injection. These two groups of mice showed a significant increase in ventricular ROS, ANP, IL-1β, IL-2, IL-6 and TNF-α proteins. Nox2 and Nox4 mRNA and protein levels were also sequentially increased. ROS was significantly decreased by inhibitors of NADPH oxidase, but not by inhibitors of other ROS production systems. Nox2 and Nox4 siRNA significantly attenuated TNF-α-induced ROS and upregulation of IL-1β and IL-6 in cardiomyocytes. Our study highlights a novel TNF-α-induced chronic ventricular remodelling mechanism mediated by sequential regulation of Nox2 and Nox4 subunits.     

Role of Complement 3 in TNF-α-Induced Mesenchymal Transition of Renal Tubular Epithelial Cells In Vitro

Injured renal tubular epithelial cells (RTECs) have been recently thought to directly contribute to the accumulation of myofibroblasts in renal tubulointerstitial fibrosis through a process of epithelial to mesenchymal transition (EMT). However, the factors inducing RTECs to undergo EMT and the underlying mechanisms need to be further elucidated. This study aimed to determine the EMT-inducing activity of proinflammatory cytokine TNF-α and the role for complement 3 (C3) in this activity in an in vitro model of human RTECs (HK-2 cells). Wild type HK-2 cells were treated with TNF-α, IFN-γ or C3a; C3 siRNA- or control siRNA-carrying HK-2 cells were treated with TNF-α. Changes in the cell morphology and phenotype were assessed by microscopy, RT-PCR, western blotting, and immunostaining. TNF-α effectively induced HK-2 cells to express C3 and to transform into morphologically myofibroblast-like cells that lost E-cadherin (a classical epithelial cell marker) expression but acquired alpha-smooth muscle actin (α-SMA, a classical myofibroblast differentiation marker) expression. C3 siRNA robustly attenuated all the morphologic and phenotypic changes induced by TNF-α but the control siRNA showed no effect. Our preliminary observations suggest that TNF-α may induce EMT in RTECs through inducing C3 expression.     

TNF-α promotes human retinal pigment epithelial (RPE) cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression through activation of Akt/mTORC1 signaling

Tumor necrosis factor-alpha (TNF-α) promotes in vitro retinal pigment epithelial (RPE) cell migration to initiate proliferative vitreoretinopathy (PVR). Here we report that TNF-α promotes human RPE cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression. Inhibition of MMP-9 by its inhibitor or its neutralizing antibody inhibited TNF-α-induced in vitro RPE cell migration. Reversely, exogenously-added active MMP-9 promoted RPE cell migration. Suppression Akt/mTOR complex 1(mTORC1) activation by LY 294002 and rapamycin inhibited TNF-α-mediated MMP-9 expression. To introduce a constitutively active Akt (CA-Akt) in cultured RPE cells increased MMP-9 expression, and to block mTORC1 activation by rapamycin inhibited its effect. RNA interference (RNAi)-mediated silencing of SIN1, a key component of mTOR complex 2 (mTORC2), had no effect on MMP-9 expression or secretion. In conclusion, this study suggest that TNF-α promotes RPE cell migration by inducing MMP-9 expression through activation of Akt/ mTORC1, but not mTORC2 signaling.     

Divergent effects of 17-β-estradiol on human vascular smooth muscle and endothelial cell function diminishes TNF-α-induced neointima formation

Coronary heart disease (CHD) is a condition characterized by increased levels of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α). TNF-α can induce vascular endothelial cell (EC) and smooth muscle cell (SMC) dysfunction, central events in development of neointimal lesions. The reduced incidence of CHD in young women is believed to be due to the protective effects of estradiol (E2). We therefore investigated the effects of TNF-α on human neointima formation and SMC/EC functions and any modulatory effects of E2. Saphenous vein (SV) segments were cultured in the presence of TNF-α (10 ng/ml), E2 (2.5 nM) or both in combination. Neointimal thickening was augmented by incubation with TNF-α, an effect that was abolished by co-culture with E2. TNF-α increased SV-SMC proliferation in a concentration-dependent manner that was optimal at 10 ng/ml (1.5-fold increase), and abolished by E2 at all concentrations studied (1-50 nM). Surprisingly, E2 itself at low concentrations (1 and 5 nM) stimulated SV-SMC proliferation to a level comparable to that of TNF-α alone. SV-EC migration was significantly impaired by TNF-α (42% of control), and co-culture with E2 partially restored the ability of SV-EC to migrate and repair the wound. In contrast, TNF-α increased SV-SMC migration by 1.7-fold, an effect that was completely reversed by co-incubation with E2. Finally, TNF-α potently induced ICAM-1 and VCAM-1 expression in both SV-EC and SV-SMC. However there was no modulation by E2 in either cell-type. In conclusion, TNF-α induced SV neointima formation, increased SMC proliferation and migration, impaired SV-EC migration and increased expression of adhesion molecules. E2 exerted distinct cell-type and function-specific modulation, the mechanisms underlying which are worthy of further detailed study.     

Modulation of reactive oxygen species in skeletal muscle by myostatin is mediated through NF-κB

Abnormal levels of reactive oxygen species (ROS) and inflammatory cytokines have been observed in the skeletal muscle during muscle wasting including sarcopenia. However, the mechanisms that signal ROS production and prolonged maintenance of ROS levels during muscle wasting are not fully understood. Here, we show that myostatin (Mstn) is a pro-oxidant and signals the generation of ROS in muscle cells. Myostatin, a transforming growth factor-β (TGF-β) family member, has been shown to play an important role in skeletal muscle wasting by increasing protein degradation. Our results here show that Mstn induces oxidative stress by producing ROS in skeletal muscle cells through tumor necrosis factor-α (TNF-α) signaling via NF-κB and NADPH oxidase. Aged Mstn null (Mstn(-/-) ) muscles, which display reduced sarcopenia, also show an increased basal antioxidant enzyme (AOE) levels and lower NF-κB levels indicating efficient scavenging of excess ROS. Additionally, our results indicate that both TNF-α and hydrogen peroxide (H(2) O(2) ) are potent inducers of Mstn and require NF-κB signaling for Mstn induction. These results demonstrate that Mstn and TNF-α are components of a feed forward loop in which Mstn triggers the generation of second messenger ROS, mediated by TNF-α and NADPH oxidase, and the elevated TNF-α in turn stimulates Mstn expression. Higher levels of Mstn in turn induce muscle wasting by activating proteasomal-mediated catabolism of intracellular proteins. Thus, we propose that inhibition of ROS induced by Mstn could lead to reduced muscle wasting during sarcopenia.     

Acetyl-l-carnitine inhibits TNF-α-induced insulin resistance via AMPK pathway in rat skeletal muscle cells

In this study, we demonstrated effects of acetyl-l-carnitine (ALC) on insulin resistance induced by tumor necrosis factor-α (TNF-α) in rat L6 cells. TNF-α downregulated insulin-stimulated glucose uptake and increased Serine 307 phosphorylation of insulin receptor substrate-1 (IRS-1). However, the treatment of ALC improved insulin-stimulated glucose uptake via AMP-activated protein kinase (AMPK) activation in a dose-dependent manner. Together, our data suggest that ALC inhibits TNF-α-induced insulin resistance through AMPK pathway in skeletal muscle cells.     

TNF-alpha induces macroautophagy and regulates MHC class II expression in human skeletal muscle cells

Macroautophagy, a homeostatic process that shuttles cytoplasmic constituents into endosomal and lysosomal compartments, has recently been shown to deliver antigens for presentation on major histocompatibility complex (MHC) class II molecules. Skeletal muscle fibers show a high level of constitutive macroautophagy and express MHC class II molecules upon immune activation. We found that tumor necrosis factor-α (TNF-α), a monokine overexpressed in inflammatory myopathies, led to a marked up-regulation of macroautophagy in skeletal myocytes. Furthermore, TNF-α augmented surface expression of MHC class II molecules in interferon-γ (IFN-γ)-treated myoblasts. The synergistic effect of TNF-α and IFN-γ on the induction of MHC class II surface expression was not reflected by higher intracellular human leukocyte antigen (HLA)-DR levels and was reversed by macroautophagy inhibition, suggesting that TNF-α facilitates antigen processing via macroautophagy for more efficient MHC class II loading. Muscle biopsies from patients with sporadic inclusion body myositis, a well defined myopathy with chronic inflammation, showed that over 20% of fibers that contained autophagosomes costained for MHC class II molecules and that more than 40% of double-positive muscle fibers had contact with CD4(+) and CD8(+) immune cells. These findings establish a mechanism through which TNF-α regulates both macroautophagy and MHC class II expression and suggest that macroautophagy-mediated antigen presentation contributes to the immunological environment of the inflamed human skeletal muscle.     

Effect of tumor necrosis factor-α on the phosphorylation of tyrosine kinase receptors is associated with dynamic alterations in specific protein-tyrosine phosphatases

Tumor necrosis factor-α (TNF-α) can modulate the signalling capacity of tyrosine kinase receptors; in particular, TNF-α has been shown to mediate the insulin resistance associated with animal models of obesity and noninsulin-dependent diabetes mellitus. In order to determine whether the effects of TNF-α might involve alterations in the expression of specific protein-tyrosine phosphatases (PTPases) that have been implicated in the regulation of growth factor receptor signalling, KRC-7 rat hepatoma cells were treated with TNF-α, and changes in overall tissue PTPase activity and the abundance of three major hepatic PTPases (LAR, PTP1B, and SH-PTP2) were measured in addition to effects of TNF-α on ligand-stimulated autophosphorylation of insulin and epidermal growth factor (EGF) receptors and insulin-stimulated insulin receptor substrate-1 (IRS-1) phosphorylation. TNF-α caused a dose-dependent decrease in insulin-stimulated IRS-1 phosphorylation and EGF-stimulated receptor autophosphorylation to 47–50% of control. Overall PTPase activity in the cytosol fraction did not change with TNF-α treatment, and PTPase activity in the particulate fraction was decreased by 55–66%, demonstrating that increases in total cellular PTPase activity did not account for the observed alterations in receptor signalling. However, immunoblot analysis showed that TNF-α treatment resulted in a 2.5-fold increase in the abundance of SH-PTP2, a 49% decrease in the transmembrane PTPase LAR, and no evident change in the expression of PTP1B. These data suggest that at least part of the TNF-α effect on pathways of reversible tyrosine phosphorylation may be exerted through the dynamic modulation of the expression of specific PTPases. Since SH-PTP2 has been shown to interact directly with both the EGF receptor and IRS-1, increased abundance of this PTPase may mediate the TNF-α effect to inhibit signalling through these proteins. Furthermore, decreased abundance of the LAR PTPase, which has been implicated in the regulation of insulin receptor phosphorylation, may account for the less marked effect of TNF-α on the autophosphorylation state of the insulin receptor while postreceptor actions of insulin are inhibited. J. Cell. Biochem. 64:117–127. © 1997 Wiley-Liss, Inc.     

Activation of Class I histone deacetylases contributes to mitochondrial dysfunction in cardiomyocytes with altered complex activities

Histone deacetylases (HDACs) play vital roles in the pathophysiology of heart failure, which is associated with mitochondrial dysfunction. Tumor necrosis factor-α (TNF-α) contributes to the genesis of heart failure and impairs mitochondria. This study evaluated the role of HDACs in TNF-α-induced mitochondrial dysfunction and investigated their therapeutic potential and underlying mechanisms. We measured mitochondrial oxygen consumption rate (OCR) and ATP production using Seahorse XF24 extracellular flux analyzer and bioluminescent assay in control and TNF-α (10 ng/ml, 24 h)-treated HL-1 cells with or without HDAC inhibition. TNF-α increased Class I and II (but not Class IIa) HDAC activities (assessed by Luminescent) with enhanced expressions of Class I (HDAC1, HDAC2, HDAC3, and HDAC8) but not Class IIb HDAC (HDAC6 and HDAC10) proteins in HL-1 cells. TNF-α induced mitochondrial dysfunction with impaired basal, ATP-linked, and maximal respiration, decreased cellular ATP synthesis, and increased mitochondrial superoxide production (measured by MitoSOX red fluorescence), which were rescued by inhibiting HDACs with MPT0E014 (1 μM, a Class I and IIb inhibitor), or MS-275 (1 μM, a Class I inhibitor). MPT0E014 reduced TNF-α-decreased complex I and II enzyme (but not III or IV) activities (by enzyme activity microplate assays). Our results suggest that Class I HDAC actions contribute to TNF-α-induced mitochondrial dysfunction in cardiomyocytes with altered complex I and II enzyme regulation. HDAC inhibition improves dysfunctional mitochondrial bioenergetics with attenuation of TNF-α-induced oxidative stress, suggesting the therapeutic potential of HDAC inhibition in cardiac dysfunction.