An ERK5-NRF2 Axis Mediates Senescence-Associated Stemness and Atherosclerosis

Background: ERK5 (extracellular signal-regulated kinase 5) is a dual kinase transcription factor with an N-terminal kinase domain and a C-terminal transcriptional activation domain. Various ERK5 kinase inhibitors have been developed and tested for treating cancer and inflammatory diseases. However, recent findings have raised questions regarding the role of ERK5′s catalytic activity in proliferation and inflammation. This study aims to explore how ERK5 reprograms myeloid cells into a proinflammatory, senescent phenotype, contributing to the development of atherosclerosis.

Methods: An ERK5 S496A (dephosphorylation mimic) knock-in (KI) mouse model was created using CRISPR/Cas9. Atherosclerosis was induced through hypercholesterolemia. Plaque phenotyping in homozygous ERK5 S496A KI and wild-type (WT) mice was analyzed using imaging mass cytometry. Bone marrow-derived macrophages were isolated from hypercholesterolemic mice and characterized through RNA sequencing and various functional in vitro assays, including senescence, mitochondrial reactive oxygen species (ROS), inflammation assays, and metabolic extracellular flux analysis.

Results: We found that atherosclerosis was inhibited in ERK5 S496A KI mice. ERK5 S496 phosphorylation was shown to mediate both the senescence-associated secretory phenotype and senescence-associated stemness by upregulating AHR (aryl hydrocarbon receptor) in plaque and bone marrow-derived macrophages from hypercholesterolemic mice. Additionally, we discovered that ERK5 S496 phosphorylation induces NRF2 (NFE2-related factor 2) SUMOylation at a novel K518 site, inhibiting NRF2 transcriptional activity without affecting ERK5 catalytic function, and mediates the oxidized LDL (low-density lipoprotein)-induced senescence-associated secretory phenotype. Specific ERK5 kinase inhibitors (AX15836 and XMD8-92) were found to inhibit ERK5 S496 phosphorylation, suggesting that this modification contributes to the anti-inflammatory effects of these inhibitors.

Conclusions: Our study uncovers a novel mechanism by which the macrophage ERK5-NRF2 axis drives the development of a unique senescence-associated secretory phenotype and stemness phenotype by upregulating AHR, contributing to atherogenesis. The discovery of the senescence-associated stemness phenotype offers a molecular explanation for the paradox of senescence in proliferative plaques, allowing myeloid cells to bypass senescence-induced cell cycle arrest during atherosclerosis progression.