DHCR24 Eases DNA Damage in Aging Endothelial Cells Through ENKUR/Ca2+ Pathway

Scientists have identified a previously unrecognized molecular pathway that protects aging blood vessel cells from the DNA damage that drives vascular decline, and the discovery could point toward new strategies for preventing age-related cardiovascular disease. The study, published in Aging Cell, centers on DHCR24, an enzyme embedded in the endoplasmic reticulum that has long been known for its role in cholesterol biosynthesis. A research team led by investigators at Tongji Hospital, Huazhong University of Science and Technology, has now shown that DHCR24 also acts as a guardian of genomic integrity in endothelial cells, the thin layer of cells lining blood vessels whose dysfunction is a leading cause of chronic disability and mortality in the elderly. The work reveals that DHCR24 suppresses the DNA damage response that accumulates as endothelial cells age, and that it does so by controlling a calcium-signaling pathway mediated by a protein called ENKUR.

The connection between DNA damage and aging is one of the most intensively studied themes in modern biology, and the new findings place endothelial cells squarely within that framework. As blood vessels age, oxidative stress, chronic inflammation, metabolic imbalance and epigenetic disruption converge to impair endothelial function. DNA damage is increasingly recognized as a key driver of this decline. When DNA strands break or bases are oxidized, the cell mounts a DNA damage response, or DDR, characterized by the recruitment of the kinase ATM to sites of injury and the phosphorylation of histone H2AX at serine 139, producing the well-known γH2AX marker. γH2AX in turn promotes activation of the transducer kinases Chk1 and Chk2, which converge on the p53/p21 axis to arrest the cell cycle and push the cell into senescence, a state of permanent growth arrest that secretes inflammatory signals harmful to surrounding tissue.

To explore how this process unfolds in blood vessels, the researchers used several complementary senescence models. They studied human umbilical vein endothelial cells at early passage, around passage 5, and compared them with late-passage, replicatively senescent cells at passage 13, in which proliferation has nearly ceased. They also isolated pulmonary microvascular endothelial cells from young, 3-month-old mice and naturally aged, 14-month-old mice. Western blotting for γH2AX, immunofluorescent staining for 8-hydroxydeoxyguanosine, a marker of oxidative DNA damage, and analysis of aortic tissue all confirmed that DNA damage accumulates robustly with endothelial aging. Critically, ATM was activated in parallel, and DHCR24 expression fell consistently as the DNA damage response intensified, suggesting that the loss of this enzyme might be permissive for vascular aging.

To forge a direct causal link, the team turned to doxorubicin, a chemotherapy drug well known for inducing DNA damage and cardiac toxicity. Treating endothelial cells with a low dose of 0.05 micromolar doxorubicin for 48 hours, followed by three days of recovery, produced a reproducible model of premature senescence: SIRT1 declined, p16 rose, senescence-associated beta-galactosidase staining increased, proliferation fell, and ATM-mediated DNA damage signaling was engaged. In this model, DHCR24 was again downregulated. Conversely, when the researchers silenced DHCR24 with siRNA in young cells, γH2AX and phosphorylated ATM rose markedly, and the effect was mirrored in pulmonary endothelial cells taken from mice engineered to lack DHCR24 specifically in the vascular endothelium, generated by crossing mice carrying a floxed DHCR24 allele with Tie2-Cre mice. En face staining of aortas from these knockout animals showed strikingly elevated γH2AX along the vessel wall.

Restoring DHCR24, by contrast, proved protective. The team delivered the enzyme into senescent and doxorubicin-treated cells using a lentiviral vector, and the results were unambiguous: overexpression of DHCR24 reduced γH2AX and ATM phosphorylation, lowered phosphorylated p53, decreased senescence-associated beta-galactosidase positivity, restored proliferative capacity measured by EdU incorporation, and reduced the excessive intracellular reactive oxygen species generated by doxorubicin exposure. Flow cytometry revealed that doxorubicin pushed cells into a G2-phase arrest, accompanied by declines in CDK1, Cyclin A2 and Cyclin B1, the kinase and cyclin partners that govern the G2/M transition. DHCR24 overexpression reversed these changes, restoring cell cycle progression. RNA sequencing and KEGG pathway analysis independently reinforced the conclusion that DHCR24’s biological functions cluster around cell cycle regulation.

The question then became mechanistic: how does a cholesterol-biosynthesis enzyme in the endoplasmic reticulum protect the genome? The researchers suspected calcium might be the link. Calcium is a universal intracellular second messenger, and the endoplasmic reticulum is the cell’s principal calcium reservoir. When ER calcium is depleted, store-operated calcium entry, or SOCE, allows extracellular calcium to flood in and replenish stores, but excessive ER calcium accumulation triggers ER stress, activates the unfolded protein response sensor IRE1, and drives reactive oxygen species overproduction. Calcium released from the ER at mitochondria-associated membranes passes through the voltage-dependent anion channel VDAC and the mitochondrial calcium uniporter MCU into the mitochondrial matrix, where overload disrupts the electron transport chain, depolarizes the mitochondrial membrane potential and further amplifies ROS generation. This ROS, in turn, attacks DNA.

The experimental data fit this framework precisely. In replicatively senescent cells, in doxorubicin-treated cells, and in cells with DHCR24 knocked down, the team measured elevated total cellular calcium using the Fluo4-AM fluorescent probe, elevated ER-releasable calcium revealed by thapsigargin challenge, activated store-operated calcium entry, increased IRE1 phosphorylation, and upregulated VDAC and MCU alongside reduced OPA1, a marker of mitochondrial dynamics. Transmission electron microscopy showed the structural consequences directly: in DHCR24-deficient cells and in the aortas of endothelial-specific knockout mice, mitochondria were shrunken, swollen and had ruptured cristae, while the ER showed vesicle dilatation and degeneration. JC-1 staining confirmed loss of mitochondrial membrane potential, and MitoSOX Red fluorescence documented surging mitochondrial ROS. ATP content, measured by luminescent assay and normalized to protein, fell in parallel.

The most novel discovery emerged from the transcriptome. RNA sequencing of DHCR24-silenced cells identified 384 upregulated genes, among which ENKUR, located on chromosome 10p12.1, stood out. ENKUR encodes enkurin, a protein discovered through yeast two-hybrid screening as an interactor of TRPC calcium channels, where it serves as an adaptor that localizes calcium-signaling machinery to channels and mediates calcium influx. Prior studies had implicated ENKUR in tumor suppression and in calcium homeostasis disorders of myeloproliferative neoplasms, but its role in senescence had not been defined. When the researchers performed double knockdowns of DHCR24 and ENKUR, the calcium overload, IRE1 activation, VDAC and MCU upregulation, γH2AX elevation and ATM phosphorylation caused by DHCR24 loss were all substantially reversed. Knocking down ENKUR alone also alleviated calcium overload in senescent cells.

Notably, the regulation appeared to operate at the level of messenger RNA stability. Using actinomycin D chase assays to block new transcription, the team found that ENKUR mRNA degraded far more slowly in DHCR24-deficient cells than in controls, meaning DHCR24 normally promotes the degradation of ENKUR transcripts. When DHCR24 is lost, ENKUR mRNA persists, enkurin protein accumulates, calcium channels remain engaged, ER calcium overloads, and the cascade of ER stress, mitochondrial dysfunction, ROS production and DNA damage follows. Restoring DHCR24 in doxorubicin-treated cells lowered ENKUR expression and broke this chain. The authors therefore propose a DHCR24–ENKUR–Ca2+ axis as the mechanistic link between ER–mitochondrial stress and endothelial senescence.

The findings carry several caveats that the authors themselves acknowledge. The mechanistic experiments were confined to endothelial models—HUVECs, pulmonary microvascular endothelial cells and endothelial-specific knockout mice—so it remains unknown whether the DHCR24–ENKUR pathway operates in other senescing cell types such as fibroblasts. The team also notes that the effects of DHCR24 overexpression on ER and mitochondrial function in doxorubicin-treated cells were not directly validated with ultrastructural imaging. Nevertheless, the study significantly expands the known portfolio of DHCR24, which earlier work from the same group had shown to delay endothelial senescence by inhibiting ROS generation through the Caveolin-1/ERK signaling axis and by relieving ER stress. With the new findings, DHCR24 emerges as a central node coordinating cholesterol metabolism, calcium homeostasis, mitochondrial health and genomic stability in the aging vasculature.

For the broader field of vascular aging research, the work offers a compelling conceptual advance: calcium signaling, long recognized as a marker of cellular aging, is positioned here as an actionable upstream regulator of the DNA damage response in endothelial cells. Because age-related endothelial dysfunction underlies hypertension, atherosclerosis and heart failure, interventions that stabilize the DHCR24–ENKUR–Ca2+ axis—whether by promoting ENKUR mRNA degradation, buffering ER calcium stores or shielding mitochondria from calcium-driven ROS—could in principle slow the vascular component of aging. Translating that insight into therapies will require much additional work, but the identification of a single, druggable signaling axis connecting organelle stress to genome protection in blood vessels gives researchers a concrete molecular target in the fight against cardiovascular aging.