SQSTM1/p62 and PPARGC1A/PGC-1alpha at the interface of autophagy and vascular senescence

Abstract

ABSTRACT Defective macroautophagy/autophagy and mitochondrial dysfunction are known to stimulate senescence. The mitochondrial regulator PPARGC1A (peroxisome proliferator activated receptor gamma, coactivator 1 alpha) regulates mitochondrial biogenesis, reducing senescence of vascular smooth muscle cells (VSMCs); however, it is unknown whether autophagy mediates PPARGC1A-protective effects on senescence. Using ppargc1a−/- VSMCs, we identified the autophagy receptor SQSTM1/p62 (sequestosome 1) as a major regulator of autophagy and senescence of VSMCs. Abnormal autophagosomes were observed in VSMCs in aortas of ppargc1a−/- mice. ppargc1a−/- VSMCs in culture presented reductions in LC3-II levels; in autophagosome number; and in the expression of SQSTM1 (protein and mRNA), LAMP2 (lysosomal-associated membrane protein 2), CTSD (cathepsin D), and TFRC (transferrin receptor). Reduced SQSTM1 protein expression was also observed in aortas of ppargc1a−/- mice and was upregulated by PPARGC1A overexpression, suggesting that SQSTM1 is a direct target of PPARGC1A. Inhibition of autophagy by 3-MA (3 methyladenine), spautin-1 or Atg5 (autophagy related 5) siRNA stimulated senescence. Rapamycin rescued the effect of Atg5 siRNA in Ppargc1a+/+ , but not in ppargc1a−/- VSMCs, suggesting that other targets of MTOR (mechanistic target of rapamycin kinase), in addition to autophagy, also contribute to senescence. Sqstm1 siRNA increased senescence basally and in response to AGT II (angiotensin II) and zinc overload, two known inducers of senescence. Furthermore, Sqstm1 gene deficiency mimicked the phenotype of Ppargc1a depletion by presenting reduced autophagy and increased senescence in vitro and in vivo. Thus, PPARGC1A upregulates autophagy reducing senescence by a SQSTM1-dependent mechanism. We propose SQSTM1 as a novel target in therapeutic interventions reducing senescence. Abbreviations 3-MA: 3 methyladenine; ACTA2/SM-actin: actin, alpha 2, smooth muscle, aorta; ACTB/β-actin: actin beta; AGT II: angiotensin II; ATG5: autophagy related 5; BECN1: beclin 1; CAT: catalase; CDKN1A: cyclin-dependent kinase inhibitor 1A (P21); Chl: chloroquine; CTSD: cathepsin D; CYCS: cytochrome C, somatic; DHE: dihydroethidium; DPBS: Dulbecco’s phosphate-buffered saline; EL: elastic lamina; EM: extracellular matrix; FDG: fluorescein-di-β-D-galactopyranoside; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; γH2AFX: phosphorylated H2A histone family, member X, H2DCFDA: 2ʹ,7ʹ-dichlorodihydrofluorescein diacetate; LAMP2: lysosomal-associated membrane protein 2; MASMs: mouse vascular smooth muscle cells; MEF: mouse embryonic fibroblast; NBR1: NBR1, autophagy cargo receptor; NFKB/NF-κB: nuclear factor of kappa light polypeptide gene enhancer in B cells; MTOR: mechanistic target of rapamycin kinase; NFE2L2: nuclear factor, erythroid derived 2, like 2; NOX1: NADPH oxidase 1; OPTN: optineurin; PFA: paraformaldehyde; PFU: plaque-forming units; PPARGC1A/PGC-1α: peroxisome proliferator activated receptor, gamma, coactivator 1 alpha; Ptdln3K: phosphatidylinositol 3-kinase; RASMs: rat vascular smooth muscle cells; ROS: reactive oxygen species; SA-GLB1/β-gal: senescence-associated galactosidase, beta 1; SASP: senescence-associated secretory phenotype; SIRT1: sirtuin 1; Spautin 1: specific and potent autophagy inhibitor 1; SQSTM1/p62: sequestosome 1; SOD: superoxide dismutase; TEM: transmission electron microscopy; TFEB: transcription factor EB; TFRC: transferrin receptor; TRP53/p53: transformation related protein 53; TUBG1: tubulin gamma 1; VSMCs: vascular smooth muscle cells; WT: wild type