Nicotinamide Metabolism Modulates the Proliferation/Differentiation Balance and Senescence of Human Primary Keratinocytes

Abstract

Nicotinamide (NAM) is the main precursor of nicotinamide adenine dinucleotide (NAD þ), a coenzyme essential for DNA repair, glycolysis, and oxidative phosphorylation. NAM has anti-aging activity on human skin, but the underlying mechanisms of action are unclear. Using 3-dimensional organotypic skin models, we show that NAM inhibits differentiation of the upper epidermal layers and maintains proliferation in the basal layer. In 2-dimensional culture, NAM reduces the expression of early and late epidermal differentiation markers and increases the proliferative capacity of human primary keratinocytes. This effect is characterized by elevated clonogenicity and an increased proportion of human primary keratinocyte stem cell (holoclones) compared to controls. By contrast, preventing the conversion of NAM to NAD þ using FK866 leads to premature human primary keratinocyte differentiation and senescence, together with a dramatic drop in glycolysis and cellular adenosine triphosphate levels while oxidative phosphorylation is moderately affected. All these effects are rescued by addition of NAM, known to compete with FK866, which suggests that conversion to NAD þ is part of the mechanistic response. These data provide insights into the control of differentiation, proliferation, and senescence by NAM and NAD þ in skin. They may lead to new therapeutic advances for skin conditions characterized by dysregulated epidermal homeostasis and premature skin aging, such as photoaging.

Niacinamide mitigates SASP‐related inflammation induced by environmental stressors in human epidermal keratinocytes and skin

“…Human Primary Keratinocytes (HPKs) were obtained from healthy human skin samples (6-year-old male donor, foreskin) from de-identified surplus surgical waste with written informed patient consent and ethical clearance. HPKs were isolated as previously described [21]. For maintenance, HPKs were cultured on lethally irradiated murine 3T3-J2 feeder cells in cFAD medium (3:1 DMEM/ Ham's F-12) supplemented with 10% foetal calf serum (FCS), 1% penicillin/streptomycin and 10 ng mL À1 epidermal growth factor as previously described [21].…”

“…Components of SASP factors secreted by senescent cells include the cytokines IL-1a, IL-6 and IL-8 [20]. We had previously shown that Nam can reduce senescence induced by NAD+ metabolism modulation in keratinocytes [21]. Thus, we asked whether Nam can mitigate stress-induced inflammation in skin in vitro and in vivo models.…”

Nicotinamide (aka Niacinamide) Mitigates SASP-Related Inflammation Induced by Environmental Stressors in Human Epidermal Keratinocytes

“…Human Primary Keratinocytes (HPKs) were obtained from healthy human skin samples (6-year old male donor, foreskin) from de-identified surplus surgical waste with written informed patient consent and ethical clearance. HPKs were isolated as previously described [22]. For maintenance, HPKs were cultured on lethally irradiated murine 3T3-J2 feeder cells in cFAD medium (3:1 DMEM/Ham's F-12) supplemented with 10% fetal calf serum (FCS), 1% penicillin/streptomycin and 10 ng/ml epidermal growth factor as previously described [22].…”

“…HPKs were isolated as previously described [22]. For maintenance, HPKs were cultured on lethally irradiated murine 3T3-J2 feeder cells in cFAD medium (3:1 DMEM/Ham's F-12) supplemented with 10% fetal calf serum (FCS), 1% penicillin/streptomycin and 10 ng/ml epidermal growth factor as previously described [22]. The medium was replaced every 2-3 days.…”

“…Under these conditions Nam inhibited the induced IL-8 levels to the control treated levels. To more closely mimic in vivo oxygen conditions in the epidermis, we compared keratinocytes grown under 5% or 20% 02 conditions [22]. Interestingly, keratinocytes grown at 5% 02 had higher levels of basal and UV-induced PGE2 compared to cells grown at 20% 02.…”