Novel Role of P62 in the Regulation of Nox4, Ros Levels, Senescence and Atherosclerosis

Introduction: Atherosclerosis is a complex disease caused by gradual build-up of plaque inside the arteries leading to hardening of the arteries. Atherosclerosis is not only the major cause of death in the United State, but also a worldwide healthcare burden. Aging is regarded as an independent and the most important risk factor for atherosclerosis after controlling other known factors. The accumulation of senescent cells in vivo during aging contributes to senescent phenotypes leading to organismal aging and age-related diseases. Autophagy is a tightly regulated catabolic process of cellular self-degradation. SQSTM1/p62, an autophagy adaptor, regulates autophagy by recruiting damaged proteins and organelles. Impaired autophagy in vascular aging causes inefficient removal of damaged and dysfunctional mitochondria in vascular cells leading to increased ROS production and inflammation in the vessel wall and eventually atherosclerosis. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases are major source of ROS in the vasculature. Nox4 plays an important role in maintaining vasculature health, however high levels of Nox4 induce oxidative stress leading to CVD progression. Our preliminary data showed that p62 gene deficiency increased senescence and the expression of Nox4, but not Nox1, in vitro. Therefore, we hypothesized that down-regulation of p62 up-regulates Nox4 expression, which increases the levels of ROS, accelerating vascular senescence and atherosclerosis. Methods: We tested the role of p62 in senescence using p62 knock out (p62-/-) and measuring senescence in aortas of males and females in vivo and in VSMCs in vitro. Senescence was measured using SA- -gal. Protein expression was measured using western blot. To test the role of p62 in atherosclerosis, we generated a ApoE-/-p62-/- animal model and measure plaque accumulation in aortas of males and females in response to high fat diet. Results: Both male and female p62-/- mice showed obese phenotypes and started to increase body weight at 4 and 6 months of age, respectively. SA- -gal activity was increased in aortas of male p62-/- mice before and after the increase in body weight, and was also increased in aortas of 5 to 8 month-old female p62-/- mice. The molecular mechanism involved in p62 deficiency-induced senescence was tested in mouse aortic smooth muscle cells (MASMs). p62-/- MASMs showed increased SA- -gal activity and production of ROS, including hydrogen peroxide, superoxide, and mitochondrial ROS, as well as expression of Nox4, signaling proteins, including phosphorylated Akt, p38MAPK and ERK, and phosphorylated PGC1 that associated with mitochondrial dysfunction. Overexpression of Nox4 by adenovirus upregulated SA- -gal activity in wild type MASM cells, while downregulation of Nox4 by short hairpin RNA (shNox4) reduced SA- -gal activity in p62-/- MASMs, suggesting the Nox4 is required for p62 deficiency-induced senescence. Furthermore, the upregulation of Nox4 by p62 deficiency was mediated by a transcription-dependent mechanism, since inhibition of transcription using actinomycin D reduced Nox4 expression in p62-/- MASMs and mRNA levels of p62 were higher in p62-/-, compared with control cells. Antioxidants including N-acetyl cysteine (NAC) and mitoTEMPO, a mitochondrial ROS scavenger, showed not affect in Nox4 expression in p62+/+ and p62-/- cells, but decreased senescence in both cell types, suggesting that ROS is responsible for p62 deficiency-induce senescence, but not for Nox4 upregulation. Moreover, NAC treatment decreased expression of phosphorylated Akt and PGC1 . Confirming the physiological role of Nox4 in vivo, Nox4 was upregulated in aortas of males at all ages, but not female p62-/- mice. We observed that p62 gene deficiency only increased atherosclerotic plaque on the aortic arch in male ApoE-/-p62-/- mice at 4 months of age, but not in young male animals or in females. Surprisingly, atherosclerotic plaque was reduced in aortas isolated from 1 year old ApoE-/-p62-/- mice for both male (total aortas) and female (total, arch and descending aortas). Conclusions: p62 deficiency upregulates Nox4 transcription increasing Nox4 protein expression, causing increased cytosolic ROS and mitochondrial ROS production via Akt-PGC1 pathway, which leads to vascular senescence in vitro. Senescence could be Nox4 dependent in males, but not in female mice. p62 deficiency-induced senescence is not associated with increased atherosclerosis development or with the onset of obesity. p62 deficiency protects mice from atherosclerosis during natural aging.