Poly-ubiquitinated p62/SQSTM1-mediated hemeoxygenase-1 stabilization plays a critical role in cadmium-induced apoptosis of mouse monocyte Raw264.7 cells.

ABSTRACT

Cadmium (Cd) is a toxic heavy metal that can affect many organs, leading to serious pathological disorders through immune suppression. Here, we investigated the molecular mechanisms underlying the response of monocytes to Cd exposure. Cd treatment of Raw264.7 cells activated antioxidant enzymes, such as hemeoxygenase-1 (HO-1), superoxide dismutase, and catalase. Cd exposure upregulated p53, p53 phosphorylation, p21, and γH2AX phosphorylation. Cd exposure also induced poly ADP-ribose polymerase 1 (PARP-1) cleavage. These findings indicated that Cd induces apoptosis through oxidative stress-mediated DNA damage. Furthermore, upregulation of microtubule-associated protein 1 light chain 3B-II (LC3B-II), an indicator of autophagy, was found to depend on Cd concentration. Accumulation of an autophagy substrate p62/SQSTM1 in monomeric p62 and polyubiquitinated (polyUb)-p62 forms, was suppressed upon N-acetylcysteine treatment Cd-exposed Raw264.7 cells, indicating an impairment of autophagic degradation during oxidative stress. Knockdown of p62 in Raw264.7 cells using small interfering RNA (siRNA) downregulated HO-1 expression and reduced apoptosis. HO-1 knockdown suppressed apoptosis by decreasing the poly-ubiquitination of p62. Treatment with hemin and MG132 enhanced Cd-mediated increases in HO-1 and polyUb-p62 levels, resulting in increased apoptosis, which indicated that Cd-induced HO-1 accumulation is associated with polyUb-p62 formation. p62 and HO-1 interactions were demonstrated by immunofluorescence and immunoprecipitation assays. Additionally, p62 was downregulated in Raw264.7 cells in response to H2O2 and a low level of HO-1 was induced. Cells that were highly sensitive to Cd did not form polyUb-p62, resulting in insufficient HO-1 accumulation. These results suggest that maintenance of HO-1 stability via poly-ubiquitination of p62 in Cd-exposed monocytes promotes apoptosis, which could be involved in immune suppression.