Human telomerase represses ROS-dependent cellular responses to Tumor Necrosis Factor-α without affecting NF-κB activation Marina Mattiussi, Gaëlle Tilman, Sandrine Lenglez, Anabelle Decottignies ⁎ Genetic and Epigenetic Alterations of Genomes research group, de Duve Institute, Faculty of Pharmacy and Biomedical Sciences, Catholic University of Louvain, 75 Avenue Hippocrate, 1200 Brussels, Belgium abstract article info Article history: Received 21 September 2011 Received in revised form 20 October 2011 Accepted 4 November 2011 Available online 13 November 2011 Keywords: Telomerase Reactive oxygen species TNF-α NF-κB In addition to its well-established role in telomere synthesis, telomerase exerts non-canonical functions that may promote cancer and stem cell survival, notably by lowering reactive oxygen species (ROS) levels and acting as transcriptional cofactor in Wnt-β-catenin signaling pathway. We investigated the impact of telomerase on ROS-dependent and -independent cellular responses to Tumor Necrosis Factor-α (TNF-α), a potent inducer of endogenous ROS production and activator of NF-κB signaling pathway. Strikingly, telome- rase overexpression in normal human fibroblasts treated with TNF-α strongly repressed ROS-dependent activation of both ERK1/2 mitogen-activated protein kinases and cell death. Telomerase overexpression also considerably diminished TNF-α-induced transcription of SOD2 Superoxide Dismutase 2 gene by reducing ROS contribution to SOD2 gene induction, both in normal fibroblasts and in cancer cells. Conversely, telome- rase did not impair TNF-α-induced transcription of various ROS-insensitive NF-κB target genes. These data were in apparent contrast with the striking observation that telomerase overexpression induced strong con- stitutive nuclear accumulation of NF-κBp65. Accumulated NF-κBp65, however, lacked Ser-536 activating phosphorylation, was not associated with global constitutive NF-κB activation and did not impair subsequent nuclear translocation of phosphorylated NF-κBp65 in response to TNF-α. Our results demonstrate that human telomerase represses ROS-dependent intracellular signaling and gene induction in response to TNF-α. © 2011 Elsevier Inc. All rights reserved. 1. Introduction Telomerase is a ribonucleoprotein enzyme whose main function resides in telomere repeat synthesis and chromosome end protection. The minimal components of active enzyme include hTERT 2 , the human telomerase reverse transcriptase catalytic subunit, and hTR, the RNA template component. While most normal human somatic cells display a finite lifespan due to the lack of telomerase activity, ectopic expression of hTERT prevents in vitro replicative senescence and extends cellular lifespan [1]. In cancer cells, telomerase is fre- quently up-regulated and ensures cellular immortality and metastasis [2]. In the last decade, accumulating evidences pointed toward the existence of telomere-independent functions of telomerase that may promote survival of cancer cells. These include protection against apoptosis or oxidative stress, enhanced DNA repair activity and modulation of gene expression profiles [3]. A protective role of telomerase against oxidative stress was clearly documented and asso- ciated with reduced intracellular reactive oxygen species (ROS) levels and improved mitochondrial function [4–9]. Additional studies dem- onstrated that telomerase – whether overexpressed or at endogenous levels – can be exported out of the nucleus to mitochondria, thanks to the presence of a mitochondrial targeting sequence at the N-terminus [6,9–14]. Nuclear export of hTERT is exacerbated under oxidative stress conditions, resulting in increased mitochondrial membrane potential, higher levels of overall respiratory chain activity and in- creased activity of cytochrome c oxidase, the rate-limiting enzyme in the mitochondrial electron transport chain [6,7,9,10]. Improved mitochondrial function of telomerase-expressing cells is likely to account for the protective role of telomerase against apoptosis that has been previously reported in numerous studies [4,5,7,9,15,16]. Additional extra-telomeric roles of telomerase include modulation of gene expression through an impact on unrelated cellular mech- anisms like RNA interference [17] or Wnt/β-catenin signaling path- way [18]. Notably, telomerase was identified as a transcriptional modulator of the Wnt/β-catenin signaling pathway through its Cellular Signalling 24 (2012) 708–717 Abbreviations: hTERT, human telomerase reverse transcriptase catalytic subunit; hTR, human telomerase RNA component; ROS, reactive oxygen species; TNF-α, tumor necrosis factor-α; NAC, N-acetyl-L-cysteine; CHX, cycloheximide; ERK1/2, extracellular signal-regulated kinases 1 and 2; SOD2, superoxide dismutase 2; IL-6, interleukin 6; TGF-β, transforming growth factor-β; IKK, IκB kinase; IK-SR, IκB-α “Super-Repressor”; MAPK, mitogen-activated protein kinase; H 2 DCF-DA, 2′,7′-dicholorodihydrofluores- cein diacetate; AACOCF 3 , arachidonyl trifluoromethyl ketone; cPLA2, cytosolic phos- pholipase A2; 5-LOX, 5-lipoxygenase; SV40, T/t antigens of Simian virus 40; ALT, alternative lengthening of telomeres; U0126, inhibitor of MEK1/2 ERK-activating kinases. ⁎ Corresponding author. Tel.: + 32 2 7647574; fax: + 32 2 7647507. E-mail addresses: marina.mattiussi@uclouvain.be (M. Mattiussi), gaelle.tilman@uclouvain.be (G. Tilman), sandrine.lenglez@uclouvain.be (S. Lenglez), anabelle.decottignies@uclouvain.be (A. Decottignies). 0898-6568/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2011.11.004 Contents lists available at SciVerse ScienceDirect Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig