Melatonin Signaling in Epidermal Keratinocytes

Neonatal Human Epidermal Keratinocytes (HEKn, primary cells) were isolated from foreskins collected at the Woman and Infant Hospital (UAB) and cultured on Petri dishes (TPP) in EpiGRO™ Human Epidermal Keratinocyte Complete Culture Media Kit (Millipore Merck KGaA, Darmstadt, Germany) as described previously [12 (link)]. Cells were seeded into 24-well plates, maintained until they reached 70% confluency, and stimulated for 24 h with melatonin, 6(OH)Mel or AFMK at the final concentration of 10⁻⁵ M or with solvent only (control, 0.1% EtOH). HaCaT cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) media containing 5% charcoal stripped FBS followed by treatment with 10⁻⁴ or 10⁻⁵ M melatonin and 10⁻⁵ M 6(OH)Mel and 2-hydroxymelatonin (2(OH)Mel) at 37 °C under 5% CO₂ for 6 and 24 h. We have used similar doses of ligands in our previous studies, which demonstrated the paracrine mechanism of action secondary to production and metabolism of melatonin in the epidermis [12 (link),13 (link),29 (link),30 (link)]. After 6 and 24 h, cells were collected in lysis reagent and RNA isolated using Absolutely RNA Miniprep Kit (Agilent Technologies, Santa Clara, CA, USA).