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

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/ß-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-ß-D-galactopyranoside; GAPDH glyceraldehyde-3-phosphate dehydrogenase; γH2AFX phosphorylated H2A histone family, member X, H2DCFDA 2',7'-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/ß-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.