Enzymatically Hydrolyzed Eggshell Membrane Promotes Keratinocyte Differentiation Associated With PKC and Akt...

Skin aging is characterized by physiological and morphological changes, and nutritional interventions are key strategies to mitigate this process. Eggshell membrane (ESM), a natural byproduct of egg processing, is typically discarded as industrial waste; however, recent studies have revealed its health benefits, including antiobesity and anti-inflammatory effects. We previously reported that enzymatically hydrolyzed ESM (eESM) promotes keratinocyte differentiation in NHEK-Neo cells, although the underlying mechanisms remain unclear. In this study, we show that treatment of NHEK-Neo cells with varying concentrations of eESM upregulated keratinocyte differentiation markers (KRT1, KRT10, IVL, FLG, KRT13) and induced dose-dependent morphological changes. Calcium, a key regulator of keratinocyte differentiation, is present in eESM. Supplementation of calcium significantly increased transcription of early differentiation markers (KRT1, KRT10) but had little effect on late markers (IVL, FLG, KRT13), suggesting that additional bioactive components in eESM contribute to differentiation. Furthermore, eESM enhanced the expression of PKC-related genes (IRF6, GRHL3, OVOL1) and increased phosphorylation of Akt and ERK2. Pharmacological inhibition of PKC and Akt signaling suppressed keratinocyte differentiation. Ultra-high-performance liquid chromatography analysis further suggested that eESM may contain components with a molecular weight of approximately 1000 Da, although further characterization is required. Collectively, these findings indicate that eESM promotes keratinocyte differentiation associated with PKC and Akt signaling pathways, likely through the coordinated action of calcium and other bioactive molecules, and that the observed effects cannot be attributed to calcium alone. eESM may, therefore, represent a potential food-derived functional ingredient for skin health.