Extrinsic skin aging is driven by environmental factors, including ultraviolet (UV) radiation and air pollution. While melanocytes serve as key protectors against UV-induced damage, their role in aging, particularly through the process of senescence, remains underexplored. Here, we exposed human neonatal melanocytes and ex vivo skin explants to UV (UVA + UVB), urban particulate matter (UPM), and their combination (UV + UPM) to assess the effects on melanocyte function and skin aging. We demonstrate that combined UV + UPM exposure triggers oxidative stress, mitochondrial and DNA damage, senescence, apoptosis, and modulation of melanogenesis in human neonatal melanocytes. In addition, skin explants subjected to the same treatments showed hallmark features of aging, including epidermal thinning, barrier disruption, fibrosis, and altered pigmentation. These findings highlight that melanocytes respond to environmental stress through multiple interconnected mechanisms, potentially affecting both cell survival and pigmentary function. Our model offers a useful platform to study how environmental stressors affect melanocyte function and skin biology, potentially supporting the development of future strategies targeting pigmentation disorders and environmentally driven skin aging.

Photobiomodulation (PBM) is emerging as a promising non-invasive approach for managing inflammatory skin conditions. However, its precise molecular mechanisms, especially within the green light spectrum, remain elusive. In this study, we investigated the anti-inflammatory mechanisms of 520 nm green light in primary human keratinocytes (KCs) exposed to the contact sensitizer 2,4-dinitrochlorobenzene (DNCB). Our data revealed that green light effectively reduces the mRNA expression of pro-inflammatory cytokines IL-6, IL-8, and TNF-α, comparably to the effect of dexamethasone, a conventional anti-inflammatory agent. As Nuclear factor erythroid-2-related factor 2 (Nrf2) is involved in the red light response, we explored Nrf2′s role in green light anti-inflammatory activity. Green light exposure activated the Nrf2 pathway, leading to Nrf2 increased accumulation in KCs and the induction of Nrf2 target genes, including HO-1 and GCLC. Invalidation of Nrf2 with si-RNA diminished the green light's regulatory effect, indicating Nrf2′s essential role in the green light's anti-inflammatory action. As the Transient Receptor Potential Vanilloid 1 (TRPV1) channel is a potential target for green light, we investigated its role in PBM response. Blocking TRPV1 with capsazepine (CPZ) abolished the anti-inflammatory effect of green light and prevented the upregulation of Nrf2 target genes. This finding highlights TRPV1′s integral role in green light beneficial activity via the activation of the Nrf2 pathway. Overall, our study identifies TRPV1 and Nrf2 as critical players in the green light response, highlighting the versatility of PBM in controlling skin inflammation.

Palmitoylation is a lipid modification involving the attachment of palmitic acid to a cysteine residue, thereby affecting protein function. We investigated the effect of palmitoylation of tyrosinase, the rate-limiting enzyme in melanin synthesis, using a human three-dimensional skin model system and melanocyte culture. The palmitoylation inhibitor, 2-bromopalmitate, increased melanin content and tyrosinase protein levels in melanogenic cells by suppressing tyrosinase degradation. The palmitoylation site was Cysteine500 in the C-terminal cytoplasmic tail of tyrosinase. The nonpalmitoylatable mutant, tyrosinase (C500A), was slowly degraded and less ubiquitinated than wild-type tyrosinase. Screening for the Asp-His-His-Cys (DHHC) family of proteins for tyrosinase palmitoylation suggested that DHHC2, 3, 7, and 15 are involved in tyrosinase palmitoylation. Knockdown of DHHC2, 3, or 15 increased tyrosinase protein levels and melanin content. Determination of their subcellular localization in primary melanocytes revealed that DHHC2, 3, and 15 were localized in the endoplasmic reticulum, Golgi apparatus, and/or melanosomes, whereas only DHHC2 was localized in the melanosomes. Immunoprecipitation showed that DHHC2 and DHHC3 predominantly bind to mature and immature tyrosinase, respectively. Taken together, tyrosinase palmitoylation at Cysteine500 by DHHC2, 3, and/or 15, especially DHHC2 in trans-Golgi apparatus and melanosomes and DHHC3 in the endoplasmic reticulum and cis-Golgi apparatus, regulate melanogenesis by modulating tyrosinase protein levels.

Photobiomodulation (PBM) is rapidly gaining traction as a valuable tool in dermatology for treating many inflammatory skin conditions using low levels of visible light or near-infrared radiation. However, the physiological regulatory pathways responsible for the anti-inflammatory effect of PBM have not been well defined. Since previous studies showed that nuclear factor-erythroid 2 like 2 (Nrf2) is a master regulator of the skin inflammatory response, we have addressed its role in controlling inflammation by PBM. Primary human keratinocytes (KCs) stimulated with 2,4-dinitrochlorobenzene (DNCB) to mimic pro-inflammatory stress were illuminated with two wavelengths: 660 nm or 520 nm. Both lights significantly reduced the mRNA expression of the DNCB-triggered TNF-α, IL-6, and IL-8 cytokines in KCs, while they enhanced Nrf2 pathway activation. PBM-induced Nrf2 is a key regulator of the inflammatory response in KCs since its absence abolished the regulatory effect of light on cytokines production. Further investigations of the mechanisms contributing to the immunoregulatory effect of PBM in inflamed human skin explants showed that 660 nm light prevented Langerhans cells migration into the dermis, preserving their dendricity, and decreased pro-inflammatory cytokine production compared to the DNCB-treated group. This study is the first to report that the PBM-mediated anti-inflammatory response in KCs is Nrf2-dependent and further support the role of PBM in skin immunomodulation. Therefore, PBM should be considered a promising alternative or complementary therapeutic approach for treating skin-related inflammatory diseases.

Melanogenesis in melanocytes and the transfer of melanin into the surrounding keratinocytes mainly contribute to skin color and pigmentation. Melanocytes in the skin and hair transfer their melanin to surrounding keratinocytes via a mechanism called melanin transfer. After melanin is internalized into keratinocytes, it forms a supra-nuclear cap, which acts as a 'sunscreen', and protects the genetic material by absorbing and scattering ultraviolet (UV) radiation. Several models for the melanin transfer mechanism have been proposed, and two of them have drawn attention recently [ [1] Wu X. Hammer J.A. Melanosome transfer: it is best to give and receive. Curr. Opin. Cell Biol. 2014; 29: 1-7 Crossref PubMed Scopus (114) Google Scholar ]: the shedding of melanosome-loaded vesicles by melanocytes that are subsequently phagocytosed by keratinocytes [ 2 Ando H. Niki Y. Ito M. Akiyama K. Matsui M.S. Yarosh D.B. Ichihashi M. Melanosomes are transferred from melanocytes to keratinocytes through the processes of packaging, release, uptake, and dispersion. J. Invest Dermatol. 2012; 132: 1222-1229 Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar , 3 Ando H. Niki Y. Yoshida M. Ito M. Akiyama K. Kim J.H. Yoon T.J. Matsui M.S. Yarosh D.B. Ichihashi M. Involvement of pigment globules containing multiple melanosomes in the transfer of melanosomes from melanocytes to keratinocytes. Cell Logist. 2011; 1: 12-20 Crossref PubMed Google Scholar , 4 Wu X.S. Masedunskas A. Weigert R. Copeland N.G. Jenkins N.A. Hammer J.A. Melanoregulin regulates a shedding mechanism that drives melanosome transfer from melanocytes to keratinocytes. Proc. Natl. Acad. Sci. USA. 2012; 109: E2101-E2109 Crossref PubMed Scopus (62) Google Scholar ] and the exocytosis by melanocytes of naked melanin (melanocores), which are then endocytosed by keratinocytes [ 5 Correia M.S. Moreiras H. Pereira F.J.C. Neto M.V. Festas T.C. Tarafder A.K. Ramalho J.S. Seabra M.C. Barral D.C. Melanin Transferred to Keratinocytes Resides in Nondegradative Endocytic Compartments. J. Invest Dermatol. 2018; 138: 637-646 Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar , 6 Tarafder A.K. Bolasco G. Correia M.S. Pereira F.J.C. Iannone L. Hume A.N. Kirkpatrick N. Picardo M. Torrisi M.R. Rodrigues I.P. Ramalho J.S. Futter C.E. Barral D.C. Seabra M.C. Rab11b mediates melanin transfer between donor melanocytes and acceptor keratinocytes via coupled exo/endocytosis. J. Invest Dermatol. 2014; 134: 1056-1066 Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar ]. A recent study using chicken embryonic skin found evidence for the shedding/phagocytosis model [ [7] Tadokoro R. Murai H. Sakai K.I. Okui T. Yokota Y. Takahashi Y. Melanosome transfer to keratinocyte in the chicken embryonic skin is mediated by vesicle release associated with Rho-regulated membrane blebbing. Sci. Rep. 2016; 6: 38277 Crossref PubMed Scopus (23) Google Scholar ]. Despite this, there is still limited evidence of how the melanosome transfer process occurs from melanocytes to keratinocytes, while the signaling pathway involved in melanin synthesis and transport in melanocytes is relatively well characterized. Ando and colleagues used scanning and transmission electron microscopy of human melanocyte/keratinocyte co-cultures to demonstrate the presence of melanosome-rich protrusions or pigment globules (PGs) on the surface of melanocyte dendrites and filopodia [ 2 Ando H. Niki Y. Ito M. Akiyama K. Matsui M.S. Yarosh D.B. Ichihashi M. Melanosomes are transferred from melanocytes to keratinocytes through the processes of packaging, release, uptake, and dispersion. J. Invest Dermatol. 2012; 132: 1222-1229 Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar , 3 Ando H. Niki Y. Yoshida M. Ito M. Akiyama K. Kim J.H. Yoon T.J. Matsui M.S. Yarosh D.B. Ichihashi M. Involvement of pigment globules containing multiple melanosomes in the transfer of melanosomes from melanocytes to keratinocytes. Cell Logist. 2011; 1: 12-20 Crossref PubMed Google Scholar ]. We are focusing on the endocytosis/phagocytosis process, which is one aspect of melanin transfer in the report. Here, we developed a new fluorescent marking technique allowing the dynamic tracking and visualization of the endocytosis/phagocytosis process. Carbocyanine dyes are widely used for visualizing lipid double membranes because of their hydrophilic/hydrophobic characteristics and radiate the strongest fluorescence when they are in membranes[ [8] Lukas J.R. Aigner M. Denk M. Heinzl H. Burian M. Mayr R. Carbocyanine postmortem neuronal tracing. Influence of different parameters on tracing distance and combination with immunocytochemistry. J. Histochem Cytochem. 1998; 46: 901-910 Crossref PubMed Scopus (57) Google Scholar ]. These dyes enter the membrane, diffuse rapidly, stain the entire cell surface, and rarely migrate to adjacent cells. In the present study, we utilized these membrane-staining methods in live-imaging to investigate melanin transfer in a dynamic manner.

Skin structure and function results from a dynamic interplay between dermal and epidermal cell types. Optimizing skin health through an effective and long-lasting skin care regime therefore requires a global approach, encompassing various mechanisms to stimulate this interplay beyond the action scope of a classical topical solution. This study evaluates the impact of a novel home-use device combining a topical serum, light-emitting diodes and massage on the clinical signs of extrinsic skin aging. The innovative principle relies on potentiating the effect of active ingredients contained in the topical serum with visible and near infra-red photons to prevent extracellular matrix degradation and promote its reconstruction. After in vitro and ex vivo investigations, a clinical study assessed the safety and efficacy of a daily treatment with the home-use device for 28 days. A significant increases in skin density and radiance while reducing the wrinkles was obtained with no side effects.

To gain insight for the role of mast cell-produced heparin in the regulation of epidermal homeostasis and skin pigmentation, we have investigated the effect of heparin on melanosome uptake and proinflammatory responses in normal human epidermal keratinocytes (NHEKs). We quantified phagocytic activity of NHEKs with uptake of melanosomes or fluorescent microspheres. Heparin exhibited the inhibitory effect on keratinocyte phagocytosis through blocking PI3k/Akt and MEK/ERK signaling pathways. In fact, the heparin-treated NHEKs showed impaired activation of Akt and ERK during phagocytosis, whereas PI3k and MEK inhibitors significantly suppressed melanosome uptake by NHEKs. In addition, the inflammation marker cycloxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2 ) production were induced during phagocytosis, while these effects were downregulated in the presence of heparin. Our observations suggest that heparin may play an antiphagocytic and anti-inflammation role in epidermis of human skin.

Background/purpose: Human skin is constantly exposed to ultraviolet A (UVA), which can generate reactive oxygen species and cause iron release from ferritin, leading to oxidative damage in biomolecules. This is particularly true in post-menopausal skin due to an increase in iron as a result of menopause. As iron is generally released through desquamation, the skin becomes a main portal for the release of excess iron in this age group. In the present study, we examined a strategy for controlling UVA- and iron-induced oxidative stress in skin using a keratinocyte post-menopausal cellular model system.Methods: Keratinocytes that had been cultured under normal or high-iron, low-estrogen conditions were treated with (2-nitrophenyl) ethyl pyridoxal isonicotinoyl hydrazone (2-PNE-PIH). 2-PNE-PIH is a caged-iron chelator that does not normally bind iron but can be activated by UVA radiation to bind iron. Following incubation with 2-PNE-PIH, the cells were exposed to 5 J/cm(2) UVA and then measured for changes in lipid peroxidation and ferritin levels.Results: 2-PNE-PIH protected keratinocytes against UVA-induced lipid peroxidation and ferritin depletion. Further, 2-PNE-PIH was neither cytotoxic nor did it alter iron metabolism.Conclusion: 2-PNE-PIH may be a useful deterrent against UVA-induced oxidative stress in postmenopausal women.

We have developed a technology to incorporate micronized titanium dioxide (TiO_2), together with antioxidants, in particles of a UV-visible transparent polymer gel. These particles are coated with silica to avoid clustering and the size of the micronized TiO_2 reduces the back scattering of white light. gel-trapped TiO_2 minimizes the oxidative stress exerted by UV radiation, increases the photo-stability of some accompanying ingredients, such as avobenzone. The size of the particles is in the micrometre range. This favors their permanence on the top of the stratum corneum . Gel-trapped TiO_2-based sunscreens provide a larger SPF and two-fold larger UVA protection than equal-composition sunscreens that contain larger amounts of untrapped TiO_2.

Background: Differences in structural and functional skin characteristics have been linked with ethnical background. But racial differences in skin have not been thoroughly investigated by objective methods and the data are often contradictory.Objectives: This study was undertaken to compare skin barrier-related parameters of the stratum corneum on African American, Caucasian and East Asian skin by objective measurements.Methods: Baseline values of trans epidermal water loss were collected on the face. Consecutive stratum corneum D-squame (R) tape strippings were collected on the panelist's ventral forearm and face to evaluate skin barrier strength and cohesion. Stratum corneum ceramides, maturation, measured as the transglutaminase-mediated cross-linking of stratum corneum proteins, and stratum corneum trypsin like enzyme activity were measured on the D-squame (R) tape strippings.Results: East Asian and to some extent Caucasian skin was characterized by low maturation and relatively weak skin barrier. African American skin was characterized by low ceramide levels and high protein cohesion in the uppermost layers of the stratum corneum. These data can be interpreted in terms of the high prevalence of xerosis in black skin and increased skin sensitivity in East Asian skin.Conclusion: These results demonstrate that skin properties at the level of the stratum corneum vary considerably among these ethnic groups. This contributes to an improved understanding of physiological differences between these study populations. (C) 2010 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.