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EIF2α-ATF4-CHAC1 Signalling Links ER Stress to Ferroptosis in Human Aortic Smooth Muscle Cells: Mechanistic Insights
Changwen Fang1,2, Xiaolong Du3, Yadan Wang4
1Department of Vascular Surgery, The Second Affiliated Hospital of Anhui Medical University, Hefei, China.
Abstract:
Ferroptosis-a regulated, iron-dependent cell death driven by lipid peroxidation-has been implicated in vascular pathology, yet how endoplasmic reticulum (ER) stress couples to ferroptotic execution in vascular smooth muscle cells (VSMCs) remains unclear. We investigated whether the PERK-EIF2α-ATF4-CHAC1 signalling axis links ER stress to ferroptosis in human aortic SMCs. Human aortic smooth muscle cells (HASMCs) and primary aortic smooth muscle cells (AoSMCs) were exposed to erastin (± ferrostatin-1), with pharmacologic modulation using buthionine sulfoximine (BSO; glutamate-cysteine ligase inhibitor) and salubrinal (selective EIF2α dephosphorylation inhibitor). Viability (CCK-8), ER-stress markers (p-PERK, p-EIF2α, GRP78, ATF4, CHAC1) and ferroptosis effectors (GPX4, ACSL4, ALOX15) were quantified by Western blotting and densitometry. Immunofluorescence assessed GPX4 and p-EIF2α/CHAC1 colocalization. Lipid peroxidation was evaluated by C11-BODIPY 581/591 imaging and malondialdehyde (MDA) content; labile Fe2+ was measured by calcein-AM quenching; apoptosis was examined by TUNEL. Statistics used t-tests or one-way ANOVA. Erastin reduced viability and activated the EIF2α-ATF4-CHAC1 pathway in both HASMCs and AoSMCs, as evidenced by increased p-EIF2α/ATF4/CHAC1 and decreased GPX4, with concomitant upregulation of ACSL4 and ALOX15. GPX4 immunofluorescence declined, while C11-BODIPY and MDA indicated robust lipid peroxidation, and calcein-AM revealed expansion of the labile Fe2+ pool; ferrostatin-1 mitigated these changes. BSO further amplified p-PERK→p-EIF2α → ATF4 → CHAC1 signalling and deepened GPX4 loss, whereas salubrinal sustained EIF2α phosphorylation, augmented ATF4/CHAC1, and further depressed GPX4. Across conditions, effects were consistent in both cell types and reached statistical significance (most p < 0.05 to < 0.01). ER-stress signalling via PERK-EIF2α-ATF4-CHAC1 constitutes a proximal driver of ferroptosis in human aortic SMCs by eroding glutathione tone and disabling GPX4, thereby promoting lipid peroxidation in an iron-rich milieu. Pharmacologic tuning of EIF2α phosphorylation and glutathione biosynthesis modulates ferroptotic susceptibility, nominating EIF2α-ATF4-CHAC1, GPX4/GSH homeostasis and iron handling as actionable nodes to preserve VSMC integrity in oxidizing vascular environments.
Insights
Endoplasmic reticulum (ER) stress triggers ferroptosis in human aortic smooth muscle cells (SMCs) via the PERK-EIF2α-ATF4-CHAC1 pathway, increasing lipid peroxidation and cell death. Modulating this pathway and glutathione levels may protect against vascular disease.
Area of Science:
- Vascular Biology
- Cell Death Mechanisms
- Molecular Signaling
Background:
- Ferroptosis, an iron-dependent cell death, is implicated in vascular pathology.
- The link between endoplasmic reticulum (ER) stress and ferroptosis in vascular smooth muscle cells (VSMCs) is not well understood.
Purpose of the Study:
- To investigate the role of the PERK-EIF2α-ATF4-CHAC1 signaling axis in coupling ER stress to ferroptosis in human aortic SMCs.
- To explore potential therapeutic targets for preserving VSMC integrity.
Main Methods:
- Human aortic smooth muscle cells (HASMCs) and primary aortic smooth muscle cells (AoSMCs) were treated with erastin, buthionine sulfoximine (BSO), or salubrinal.
- Cell viability, ER stress markers, ferroptosis effectors, lipid peroxidation, labile iron, and apoptosis were quantified using Western blotting, immunofluorescence, and biochemical assays.
Main Results:
- Erastin induced ferroptosis by activating the EIF2α-ATF4-CHAC1 pathway, decreasing GPX4, and increasing lipid peroxidation and labile iron.
- BSO amplified ER stress signaling and GPX4 loss, while salubrinal sustained EIF2α phosphorylation and further reduced GPX4.
- These effects were consistent across both cell types and statistically significant.
Conclusions:
- The PERK-EIF2α-ATF4-CHAC1 pathway drives ferroptosis in human aortic SMCs by impairing glutathione homeostasis and GPX4 activity.
- Pharmacological modulation of EIF2α phosphorylation and glutathione biosynthesis can influence ferroptotic susceptibility.
- Targeting this pathway and iron handling may be crucial for maintaining VSMC health in oxidative vascular conditions.
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