The Effects of Spermidine on Functional and Transcriptomic Markers in Human Primary Keratinocytes

Recent enthusiasm has surrounded the homeostatic roles that polyamines have in a variety of cell types. Thus, the purpose of this exploratory in vitro study was to determine how spermidine (SPD), a polyamine commonly consumed as a nutritional supplement, affected general markers of cellular health and function in human primary epidermal keratinocytes. Commercial HEKa cells were seeded onto either six-well (transcriptomics and immunoblotting) or 96-well culture plates (viability, ATP, and JC-1 assays) and cultured to ~90+% confluency through complete growth media (CGM) changes every 48 h. Once cells reached this level of growth, treatments included either CGM + phosphate-buffered saline (PBS control, or CTL), CGM + 1 µM SPD, and CGM + 10 µM SPD for either 6 or 24 h depending upon the outcome being assessed. Cellular ATP levels were not significantly affected by 1 µM or 10 µM SPD treatments lasting 24 h. However, cell counts were 9% greater (p = 0.007) when comparing 24 h 10 µM versus CTL treatments indicating increased cell viability. Transcriptomic analyses indicated that 6 h treatments with 10 µM SPD significantly altered 162 transcripts versus non-treated CTL cells (65 up-regulated and 97 down-regulated, p < 0.01). Four pathways were predicted to be enriched based on differential gene expression including protein deubiquitination (GO:0016579), membrane lipid biosynthesis (GO:0046467), DNA metabolic process (GO:0006259), and cell cycle process (GO:0022402). Additionally, the HR gene (essential for keratinocyte hair follicle formation) was significantly up-regulated at the mRNA level with 6 h 10 µM SPD, and immunoblotting confirmed a 96% increase in protein levels with 24 h 10 µM SPD treatments, albeit this did not reach statistical significance (p = 0.102). Pan-keratin protein content was also 60% greater in the 1 µM and 10 µM 24 h treatments than CTL (p ≤ 0.029). Finally, although select markers of mitochondrial content and biogenesis were not significantly altered with 6 h and 24 h treatments, mitochondrial membrane potential (an aspect of mitochondrial function) was 84% greater with 24 h 1 µM versus CTL (p < 0.001). In conclusion, these preliminary screening experiments in unperturbed human keratinocytes suggest that exogenous SPD positively affects various aspects of homeostasis by stimulating transcriptomic and functional alterations (e.g., increased cell viability and enhanced keratinocyte protein levels).