Autophagy drives fibroblast senescence through MTORC2 regulation.

ABSTRACT

Sustained macroautophagy/autophagy favors the differentiation of fibroblasts into myofibroblasts. Cellular senescence, another means of responding to long-term cellular stress, has also been linked to myofibroblast differentiation and fibrosis. Here, we evaluate the relationship between senescence and myofibroblast differentiation in the context of sustained autophagy. We analyzed markers of cell cycle arrest/senescence in fibroblasts in vitro, where autophagy was triggered by serum starvation (SS). Autophagic fibroblasts expressed the senescence biomarkers CDKN1A/p21 and CDKN2A/p16 and exhibited increased senescence-associated GLB1/beta-galactosidase activity. Inhibition of autophagy in serum-starved fibroblasts with 3-methyladenine, LY294002, or ATG7 (autophagy related 7) silencing prevented the expression of senescence-associated markers. Similarly, suppressing MTORC2 activation using rapamycin or by silencing RICTOR also prevented senescence hallmarks. Immunofluorescence microscopy showed that senescence and myofibroblast differentiation were induced in different cells, suggesting mutually exclusive activation of senescence and myofibroblast differentiation. Reactive oxygen species (ROS) are known inducers of senescence and exposing fibroblasts to ROS scavengers decreased ROS production during SS, inhibited autophagy, and significantly reduced the expression of senescence and myofibroblast differentiation markers. ROS scavengers also curbed the AKT1 phosphorylation at Ser473, an MTORC2 target, establishing the importance of ROS in fueling MTORC2 activation. Inhibition of senescence by shRNA to TP53/p53 and shRNA CDKN2A/p16 increased myofibroblast differentiation, suggesting a negative feedback loop of senescence on autophagy-induced myofibroblast differentiation. Collectively, our results identify ROS as central inducers of MTORC2 activation during chronic autophagy, which in turn fuels senescence activation and myofibroblast differentiation in distinct cellular subpopulations. Abbreviations 3-MA 3-methyladenine; ACTA2 actin, alpha 2, smooth muscle, aorta; AKT1 AKT serine/threonine kinase 1; p-AKT1 AKT1 Ser473 phosphorylation; t-AKT1 total AKT serine/threonine kinase 1; ATG4A autophagy related 4A cysteine peptidase; ATG7 autophagy gene 7; C12FDG 5-dodecanoylaminofluorescein Di-ß-D-Galactopyranoside; CDKN1A cyclin dependent kinase inhibitor 1A; CDKN2A cyclin dependent kinase inhibitor 2A; Ctl control; DAPI 4',6-diamidino-2-phenylindole, dilactate; ECM extracellular matrix; GSH L-glutathione reduced; H2O2 hydrogen peroxide; HLF adult human lung fibroblasts; Ho Hoechst 33342 (2'-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2.5'-bi-1H-benzimidazole); HSC hepatic stellate cells; LY LY294002; MAP1LC3B/LC3B microtubule-associated protein 1 light chain 3 beta; MTORC1/2 mechanistic target of rapamycin kinase complex 1/2; N normal growth medium; NAC N-acetyl-L-cysteine; PBS phosphate-buffered saline; PDGFA platelet derived growth factor subunit A; PRKCA/PKCα protein kinase C alpha; PtdIns3K class III phosphatidylinositol 3-kinase; PTEN phosphatase and tensin homolog; R rapamycin; RICTOR RPTOR independent companion of MTOR complex 2; ROS reactive oxygen species; RPTOR regulatory associated protein of MTOR complex 1; SA-GLB1/ß-gal senescence-associated galactosidase beta 1; SGK1 serum/glucocorticoid regulated kinase 1; shRNA short hairpin RNA; siCtl control siRNA; siRNA small interfering RNA; SQSTM1 sequestosome 1; SS serum-free (serum starvation) medium; TP53 tumor protein p53; TUBA tubulin alpha; V vehicle.