Regulation of keratinocyte proliferation and differentiation by secoiridoid oleacein in monoculture and fibroblast co-culture models

Aberrant differentiation of keratinocytes is closely associated with both pathological skin disorders and non-pathological skin conditions, making the maintenance of normal differentiation process essential for skin integrity and homeostasis. This study investigated the effect of olive-derived secoiridoid oleacein (OC) on keratinocyte proliferation and differentiation in vitro and further validated it in a co-culture model with fibroblasts mimicking a skin-like environment. OC was compared with oleuropein (OP) as a reference compound having similar chemical structure and reported effects on skin barrier formation and wound healing. Notably, OC significantly increased the proliferation makers KRT5 and KRT14 and demonstrated wound healing effect under low-calcium condition, reflecting characteristics of the basal layer. Under high-calcium condition, OC markedly upregulated differentiation markers KRT10, IVL, FLG, and TGM1, along with differentiation characteristics such as cytoplasmic extensions and cell adhesion. Transcriptomic analysis revealed that OP and OC shared a common upstream pathway, Integrin/E-cadherin-Rho-MAPK, at the cytoplasm, while they showed distinct regulatory mechanisms within the nucleus. OP induced differentiation by suppressing stemness genes through epigenetic regulation, whereas OC secured differentiation stability by suppressing proliferative gene ESR1 and activating the DNA damage response from DNA damage or mechanical stress occurring during differentiation. Our study is the first to elucidate the dual regulatory effects of OC on keratinocyte proliferation and differentiation stage-dependently as well as its underlying molecular mechanisms, suggesting that the divergent regulatory mechanisms may be due to their structural differences. These findings highlight OC as a skin protective agent for maintaining skin health and suggest its therapeutic potential for skin disorders related to abnormal differentiation.