IL-6 Intracellular signaling in natural and induced senescent cells

Abstract

Senescence is defined as a growth arrest program that preserves physiological cell functions. It has been associated with tumor suppressor mechanisms as well as a resistance mechanism to evade tumor eradication. Interleukin-6 (IL-6) is part of the secretome of senescent cells denominated senescence associated secretory phenotype (SASP). In previous work we demonstrated the relevance of the intracellular IL-6 signaling in vitro and in vivo in a model of senescent pituitary tumor cells. In this work we focus on the characterization of this signaling in a natural and therapy-induced senescent model. In MtT/S cells, a naturally senescent pituitary somatotroph cell line, to discriminate the effect of intracellular action of IL-6 we inhibited secretory pathway using two independent approaches: a) overexpression of a dominant negative form of Rab11 which is a key molecule in anterograde transport, involved in IL-6 associated vesicle exocytosis and b) treatment with brefeldin A (BFA) 100 ng/ml, a drug that blocks intracellular vesicle transport from endoplasmic reticulum to Golgi.In both cases we observed an increase in the senescent biomarkers pRb and p16INK4 (measured by western blot) and the activity of senescence associated β galactosidase (SA-β-Gal) (n=4, p<0.05). To investigate the subcellular localization of the molecules involved, we performed confocal images of MtT/s cells transfected with fluorescent tagged plasmids of IL-6 and IL-6 receptor and observed spatial association of both with structures related to anterograde traffic.We next studied another model of senescence, therapy-induced senescent pulmonary A549 cells treated with doxorubicin (Dox) 132 nM. In these cells stimulation and retention of IL-6 (verified by western blot) with IL-1β 20 ng/ml and BFA also increases the senescent profile according to the biomarkers: increase in p21Cip1 and decrease in pRb.We conclude that IL-6 regulates senescence through a specific intracellular signaling pathway that could represent a general mechanism for tumoral senescent cells.