The epidermis, a critical barrier protecting the body, is a highly organized tissue where keratinocytes undergo a regulated balance between proliferation and differentiation to maintain its layered structure. A significant issue in skin health, particularly with aging, is the breakdown of the epidermal barrier function and increased susceptibility to damage, with epidermal thinning being a known symptom. Previous studies, including prior work by some of the current authors, have shown that long-term deficiency in vitamin C (VC) leads to epidermal atrophy in mouse models, suggesting a crucial role for this vitamin in maintaining epidermal thickness. However, the specific mechanisms by which VC influences keratinocyte proliferation and differentiation in human skin were not fully understood. Given that VC is a known cofactor for ten-eleven translocation (TET) enzymes involved in DNA demethylation, a key epigenetic process regulating gene expression, researchers hypothesized that VC might regulate epidermal cell dynamics through epigenetic changes, specifically DNA methylation. This research was undertaken to investigate this potential epigenetic mechanism and clarify how VC contributes to epidermal health.
Methods
This study utilized a three-dimensional cultured human epidermal equivalent model constructed from normal human epidermal keratinocytes to recapitulate the structure of the human epidermis. The models were treated with physiological concentrations of vitamin C sodium salt (0.1 mM and 1.0 mM) for 7 or 14 days, with daily medium changes. Experiments also included treatment with a TET inhibitor. The effects of VC were evaluated through histological analysis of epidermal thickness, measurement of cell viability, and assessment of DNA methylation levels (5-mC and 5-hmC) using dot blot and immunofluorescence. Transcriptional profiling was performed using microarray analysis, and whole-genome DNA methylation patterns were assessed using whole-genome bisulfite sequencing (WGBS). An integrated analysis of microarray and WGBS data was conducted to identify genes regulated by VC through DNA demethylation, and the expression of selected genes was validated using qPCR.
Key Findings
•VC promotes epidermal proliferation and thickness: Treatment with vitamin C significantly increased epidermal cell layer (ECL) thickness and cell viability in the human epidermal equivalent model, particularly at 1.0 mM concentration over 14 days. This was accompanied by an increase in Ki-67 positive cells, a marker of proliferation, in the basal layer.
•VC increases global DNA demethylation (5-hmC): Vitamin C treatment led to a drastic increase in 5-hydroxymethylcytosine (5-hmC) levels in a dose-dependent manner after both 7 and 14 days, as confirmed by dot blot and immunofluorescence. Global 5-methylcytosine (5-mC) levels did not significantly change.
•VC’s effects are dependent on TET activity: The increases in 5-hmC levels, ECL thickness, cell viability, and cell proliferation induced by VC were all significantly attenuated by the presence of a TET inhibitor. This indicates that VC promotes keratinocyte proliferation by promoting DNA demethylation via TET enzyme activity.
•VC alters transcriptional profiles and upregulates proliferation genes: Microarray analysis showed that VC treatment significantly changed the overall transcriptional profile, leading to upregulation of genes involved in cell proliferation and DNA replication. Differentiation-related genes were not significantly affected by VC treatment.
•VC induces widespread DNA hypomethylation: Whole-genome bisulfite sequencing revealed that VC treatment resulted in distinct DNA methylation profiles. A large number of differentially methylated regions (DMRs) were identified, with the vast majority (10,138 out of 10,279) being hypomethylated upon VC treatment. This hypomethylation primarily occurred in CpG shore and shelf regions, which are correlated with gene expression.
•Identification of proliferation-related genes regulated by VC-induced demethylation: An integrated analysis identified 81 genes that were both hypomethylated and upregulated in expression in the presence of VC. Twelve of these genes (CAST, CYP1B1, EMP1, EREG, FAM83A, FBXO32, HDAC1, NTN4, RHCG, ROS1, RUNX2, and SOX9) were previously known to be associated with cell proliferation. qPCR validated the increased expression of all twelve genes, supporting the conclusion that VC promotes the expression of proliferation-related genes by increasing their DNA demethylation.
This study provides novel insight into the mechanism by which vitamin C promotes epidermal health. By demonstrating that VC increases epidermal thickness and keratinocyte proliferation via TET-mediated DNA demethylation in a human epidermal equivalent model, it elucidates the underlying mechanisms for previously observed epidermal atrophy in VC deficiency. The identification of twelve specific proliferation-related genes whose expression is upregulated through this epigenetic mechanism highlights key targets influenced by VC. This research indicates that VC-mediated epigenetic modifications could serve as a basis for possible clinical treatment strategies. Given that epidermal thinning is a feature of skin aging and 5-hmC levels decrease in aged skin, VC’s ability to restore 5-hmC and promote proliferation suggests it is a promising molecule for developing treatments for epidermal thinning and potentially rejuvenating aging skin. Future implications include further investigation into the specific functions of the identified proliferation-related genes, such as ROS1, analyzing protein expression levels, and exploring the potential of VC to influence other epigenetic factors or combat aging through these pathways.
