Cullin 3 mitigates nonesterified fatty acid–induced oxidative stress in mammary epithelial cells: Involvement of BCL2/BECN1 and autophagy

Abstract

High nonesterified fatty acid (NEFA) concentrations in cows with clinical ketosis lead to metabolic dysfunction in mammary cells, resulting in oxidative stress. Studies have shown that autophagy is impaired in the mammary glands of ketotic cows, and enhancing autophagy mitigates oxidative stress in these animals. Cullin 3 (CUL3), an E3 ubiquitin ligase, is integral for maintaining cellular homeostasis, particularly regulation of oxidative stress and autophagy. Whether CUL3 is involved in mitigating NEFA-induced oxidative stress is unknown. This study aimed to investigate the protective effects and underlying mechanisms whereby CUL3 mitigates NEFA-induced oxidative stress in mammary epithelial cells. First, mammary gland tissue and blood samples were collected from healthy cows (n = 12, BHB <0.6 mM) and cows with clinical ketosis (n = 12, BHB >3.0 mM). Compared with healthy cows, cows with clinical ketosis had reduced productive performance, decreased CUL3 expression, impaired autophagic activity, and increased oxidative stress status in mammary tissue. In vitro, incubating the immortalized bovine mammary epithelial cell line (MAC-T) with 1.2 mM NEFA downregulated CUL3 expression, impaired autophagy, and increased oxidative stress. Adenovirus-mediated overexpression of CUL3 attenuated NEFA-induced accumulation of peroxides and reactive oxygen species, whereas silencing of CUL3 via small interfering RNA exacerbated these effects. Even when nuclear factor erythroid 2 related factor 2 (NFE2L2) expression was reduced by overexpression of CUL3, there was no worsening of NEFA-induced reductions in mRNA levels of NFE2L2 downstream target genes (NADPH quinone oxidoreductase 1 [NQO1], heme oxygenase-1 [HMOX1], glutamate-cysteine ligase catalytic subunit [GCLC)], and glutamate-cysteine ligase modifier subunit [GCLM]). The reduction in NEFA-induced oxidative stress by CUL3 was diminished upon autophagy related 5 (ATG5) silencing suggesting that CUL3 alleviates NEFA-induced oxidative stress via autophagy. Additionally, CUL3 overexpression aggravated the NEFA-induced decrease in BCL2 apoptosis regulator (BCL2) expression along with alleviating the NEFA-induced decrease in Beclin1 (BECN1) expression. Under NEFA treatment, overexpression of BCL2 partly mitigated the CUL3-induced elevation in BECN1. Overall, oxidative stress and impaired autophagy are characterized in the mammary tissue of cows with clinical ketosis. CUL3 activation, likely through the BCL2-BECN1 pathway, enhances autophagy and mitigates NEFA-induced oxidative stress in MAC-T cells. Thus, targeting CUL3-mediated autophagy could be a promising therapeutic strategy to reduce oxidative stress-induced damage in bovine mammary epithelial cells.