Nucleotide-binding oligomerization domain protein-1 is expressed and involved in the inflammatory response in human sebocytes.

Sebocytes express Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), which participate in the innate immune response of the skin. Although the roles of TLRs and NLR family pyrin domain-containing 3 (NLRP3) in inflammatory responses in sebocytes have been reported, the expression and functions of other NLR members, such as NOD protein-1 and -2 (NOD1 and NOD2, respectively), remain unclear. In this study, we showed that, in sebocytes, the expression of NOD1 is higher than that of NOD2, and that NOD1 is involved in inflammatory responses, such as the secretion of proinflammatory cytokines. A NOD1 agonist, L-alanyl-γ-D-glutamyl-meso-diaminopimelic acid (Tri-DAP) induced the expression and secretion of interleukin-8 (IL-8) and activated the nuclear factor-kappa B and mitogen-activated protein kinase signaling pathways. On the other hand, a NOD2 agonist, muramyl dipeptide, did not. Either inhibition with a NOD1 inhibitor, ML130, or knockdown of NOD1 expression abolished Tri-DAP-induced inflammatory responses, suggesting that NOD1 is involved in the immunogenic signaling system of sebocytes. Furthermore, Tri-DAP and an agonist of TLR2 or TLR4 additively increased IL-8 expression compared with each agonist alone. Our results reveal the role of NOD1 in the inflammatory responses of sebocytes and may provide a novel therapeutic target for sebaceous gland inflammatory diseases, such as acne vulgaris.

Nicotinamide adenine dinucleotide metabolism and arterial stiffness after long-term nicotinamide mononucleotide supplementation: a randomized, double-blind, placebo-controlled trial.

Many animal studies have shown that oral administration of the nicotinamide adenine dinucleotide (NAD+) precursor nicotinamide mononucleotide (NMN) prevents the reduction of NAD+ levels in organs and tissues, helping alleviate aging-related diseases. However, there are very few clinical reports of NMN supplementation in humans. Thus, this study aimed to investigate the influence of a 12-week NMN oral supplementation on biochemical and metabolic health parameters. A 12-week randomized, double-blind, placebo-controlled, parallel-group clinical trial was conducted. A total of 36 healthy middle-aged participants received one capsule of either 125 mg NMN or placebo twice a day. Among the NAD+ metabolites, the levels of nicotinamide in the serum were significantly higher in the NMN intake group than in the placebo group. Pulse wave velocity values indicating arterial stiffness tended to decrease in the NMN intake group. However, no significant difference was found between the two groups. Long-term NMN supplementation at 250 mg/day was well tolerated and did not cause adverse events. NMN safely and effectively elevated NAD+ metabolism in healthy middle-aged adults. Additionally, NMN supplementation showed potential in alleviating arterial stiffness.

Porcine placental extract increase the cellular NAD levels in human epidermal keratinocytes.

Nicotinamide adenine dinucleotide (NAD) is an essential cofactor for numerous enzymes involved in energy metabolism. Because decreasing NAD levels is a common hallmark of the aging process in various tissues and organs, maintaining NAD levels has recently been of interest for the prevention of aging and age-related diseases. Although placental extract (PE) are known to possess several anti-aging effects, the NAD-boosting activity of PE remains unknown. In this study, we found that porcine PE (PPE) significantly increased intracellular NAD levels in normal human epidermal keratinocytes (NHEKs). PPE also attenuated the NAD depletion induced by FK866, an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT). Interestingly, only the fraction containing nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), and nicotinamide (NAM) restored NAD content in NHEKs in the absence of NAMPT activity. These results suggest that PPE increases intracellular NAD by providing NAD precursors such as NMN, NR, and NAM. Finally, we showed that the application of PPE to the stratum corneum of the reconstructed human epidermis significantly ameliorated FK866-induced NAD depletion, suggesting that topical PPE may be helpful for increasing skin NAD levels. This is the first study to report the novel biological activity of PE as an NAD booster in human epidermal cells.

1,25-Dihydroxyvitamin D3 attenuates IL-1ß secretion by suppressing NLRP1 inflammasome activation by upregulating the NRF2-HO-1 pathway in epidermal keratinocytes.

The nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein (NLRP) inflammasome is a key inflammatory signaling pathway activated via a two-step signaling process consisting of priming and activation steps. Several studies have shown that 1,25-dihydroxyvitamin D3 (1,25(OH)2VD3) inhibits the priming step required for NLRP3 inflammasome activation in immune cells. However, as activating the NLRP1 inflammasome in keratinocytes does not necessarily require a priming step, whether 1,25(OH)2VD3 inhibits NLRP1 activation in unprimed keratinocytes is currently unknown. In this study, we showed that 1,25(OH)2VD3 inhibits nigericin-induced NLRP1 inflammasome activation in unprimed keratinocytes. 1,25(OH)2VD3 suppressed nigericin-induced interleukin-1ß (IL-1ß) secretion and caspase-1 activation in human primary keratinocytes. In addition, 1,25(OH)2VD3 significantly inhibited the formation of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) oligomers and specks, but not caspase-1 enzymatic activity, suggesting that 1,25(OH)2VD3 prevents NLRP1-ASC complex assembly in keratinocytes. Vitamin D receptor (VDR)-knockdown abolished the inhibitory effects of 1,25(OH)2VD3 on nigericin-induced ASC oligomerization and IL-1ß secretion, suggesting that 1,25(OH)2VD3 suppresses inflammasome activation via VDR signaling. Furthermore, nigericin induced K+ efflux and cellular reactive oxygen species (ROS) production, and 1,25(OH)2VD3 pretreatment suppressed nigericin-induced ROS production. 1,25(OH)2VD3 increased the expression of both nuclear factor erythroid 2-related factor 2 (NRF2) and heme oxygenase-1 (HO-1), whereas HO-1 inhibition or NRF2 and HO-1 knockdown abrogated the inhibitory effects of 1,25(OH)2VD3 on IL-1ß secretion. Our results indicate that 1,25(OH)2VD3 inhibits nigericin-induced activation step of NLRP1 inflammasome activation in unprimed keratinocytes. Our findings reveal the mechanism underlying the inhibitory effect of 1,25(OH)2VD3, which involves NRF2-HO-1 pathway activation through the VDR, providing further insight into the potential function of 1,25(OH)2VD3 as a therapeutic agent for inflammasome-related skin diseases.

Low-molecular-weight whey proteins promote collagen production in dermal fibroblasts via the TGF-ß receptor/Smad pathway.

Whey proteins (WPs) reportedly enhance cutaneous tissue regeneration in in vivo studies. However, the underlying mechanisms of such regenerative processes are poorly understood. In this study, we show that low-molecular-weight WPs (LMWPs; 1-30 kDa) accelerate the dermal collagen production via the transforming growth factor ß receptor (TßR)/Smad pathway. We showed that LMWPs increased type I and III collagen expression in normal human dermal fibroblasts. Moreover, LMWPs rapidly induced Smad protein phosphorylation and nuclear translocation. Notably, type I TßR/Smad signaling inhibitor treatment or type II TßR siRNA knockdown blocked the LMWP-induced type I collagen expression. To identify the active components, we fractionated LMWPs and identified ß-lactoglobulin and α-lactalbumin as potential TßR/Smad signaling inducers. Our findings unravel novel biological functions of WPs, involving the TßR/Smad-dependent induction of dermal collagen synthesis, highlighting the therapeutic potential of LMWPs in wound healing.

Restoring NAD+ by NAMPT is essential for the SIRT1/p53-mediated survival of UVA- and UVB-irradiated epidermal keratinocytes.

Nicotinamide adenine dinucleotide (NAD+) is a crucial coenzyme in energy production. The imbalance of NAD+ synthesis has been found to trigger age-related diseases, such as metabolic disorders, cancer, and neurodegenerative diseases. Also, UV irradiation induces NAD+ depletion in the skin. In mammals, nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the NAD+ salvage pathway and essential for NAD+ homeostasis. However, but few studies have focused on the role of NAMPT in response to UV irradiation. Here, we show that NAMPT prevents NAD+ depletion in epidermal keratinocytes to protect against the mild-dose UVA and UVB (UVA/B)-induced proliferation defects. We showed that poly(ADP-ribose) polymerase (PARP) inhibitor rescued the NAD+ depletion in UVA/B-irradiated human keratinocytes, confirming that PAPR transiently exhausts cellular NAD+ to repair DNA damage. Notably, the treatment with a NAMPT inhibitor exacerbated the UVA/B-induced loss of energy production and cell viability. Moreover, the NAMPT inhibitor abrogated the sirtuin-1 (SIRT1)-mediated deacetylation of p53 and significantly inhibited the proliferation of UVA/B-irradiated cells, suggesting that the NAMPT-NAD+-SIRT1 axis regulates p53 functions upon UVA/B stress. The supplementation with NAD+ intermediates, nicotinamide mononucleotide and nicotinamide riboside, rescued the UVA/B-induced phenotypes in the absence of NAMPT activity. Therefore, NAD+ homeostasis is likely essential for the protection of keratinocytes from UV stress in mild doses. Since the skin is continuously exposed to UVA/B irradiation, understanding the protective role of NAMPT in UV stress will help prevent and treat skin photoaging.

H(+)/peptide transporter (PEPT2) is expressed in human epidermal keratinocytes and is involved in skin oligopeptide transport.

Peptide transporter 2 (PEPT2) is a member of the proton-coupled oligopeptide transporter family, which mediates the cellular uptake of oligopeptides and peptide-like drugs. Although PEPT2 is expressed in many tissues, its expression in epidermal keratinocytes remains unclear. We investigated PEPT2 expression profile and functional activity in keratinocytes. We confirmed PEPT2 mRNA expression in three keratinocyte lines (normal human epidermal keratinocytes (NHEKs), immortalized keratinocytes, and malignant keratinocytes) by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. In contrast to PEPT1, PEPT2 expression in the three keratinocytes was similar or higher than that in HepG2 cells, used as PEPT2-positive cells. Immunolocalization analysis using human skin showed epidermal PEPT2 localization. We studied keratinocyte transport function by measuring the oligopeptide content using liquid chromatography/tandem mass spectrometry. Glycylsarcosine uptake in NHEKs was pH-dependent, suggesting that keratinocytes could absorb small peptides in the presence of an inward H(+) gradient. We also performed a skin-permeability test of several oligopeptides using skin substitute, suggesting that di- and tripeptides pass actively through the epidermis. In conclusion, PEPT2 is expressed in keratinocytes and involved in skin oligopeptide uptake.