Ultraviolet (UV) radiation from the sun is widely considered as a major cause of human skin photoaging and skin cancer. Granzyme B (GrB) and perforin (PFN) are two proteins contained in granules and implicated in one of the mechanisms by which cytotoxic lymphocytes and natural killer cells exert their cytotoxicity against virus-infected, alloreactive, or transformed cells. The distribution of GrB and PFN in the skin has received little attention. However, Berthou and co-workers (Berthou, C., Michel, L., Soulie, A., JeanLouis, F., Flageul, B., Dubertret, L., Sigaux, F., Zhang, Y., and Sasportes, M. (1997) J. Immunol. 159, 5293-5300) described that, whereas freshly isolated epidermal cells did not express GrB or PFN, keratinocyte growth to confluence was associated with GrB and PFN mRNA and protein synthesis. In this work, we have investigated the possible role of UV-B on GrB and PFN expression in keratinocytes. We found that UV-B induces GrB and PFN expression in these cells through redox-, epidermal growth factor receptor-, and mitogen-activated protein kinase-dependent signaling. Furthermore, under UV irradiation, keratinocytes acquire a significant cytotoxicity, which is GrB and PFN dependent, toward a variety of cellular targets including transformed T-lymphocytes, melanocytes, and keratinocytes. This phenomenon may have important functional consequences in the regulation of skin inflammatory response and in the emergence of cancer skin.

Evaluation of the photoprotection provided by sunscreens is performed either through the induction of erythema and expressed as the sun protection factor (SPF), or by the UVA-mediated persistent pigment darkening (PPD). None of these two endpoints has a link with skin cancer, the most deleterious consequence of excess exposure to solar UV radiation. We thus set up a complementary approach to evaluate the protection provided by sunscreens to the genome of human skin. This is based on the quantification of the thymine cyclobutane dimer (TT-CPD), the main DNA lesion induced by both UVB and UVA radiations. Irradiations were performed ex vivo on human skin explants and the level of TT-CPD in DNA was determined by HPLC associated with tandem mass spectrometry. The technique was first optimized and validated with three standard sunscreens. The study was then extended to the evaluation of a commercial high SPF sunscreen exhibiting efficient UVA photoprotection. The DNA protecting factor was found to reflect the ratio between UVB and UVA photoprotection, although the absolute values of the genomic protection were, as a general trend, lower than either SPF or PPD. These data show the usefulness of the proposed approach for the evaluation of the genoprotection afforded by sunscreens.

In a previous study, we have described that UVB induces granzyme B (GrB) in human keratinocyte cells, and that confers potent cellular cytotoxicity against various cellular models, including immune cells (Hernandez-Pigeon, H., Jean, C., Charruyer, A., Haure, M. J., Titeux, M., Tonasso, L., Quillet-Mary, A., Baudouin, C., Charveron, M., and Laurent, G. (2006) J. Biol. Chem. 281, 13525-13532). Herein, we have found that, in contrast to UVB, UVA failed to enhance keratinocyte cellular cytotoxicity but was still able to trigger GrB production. We show that GrB is accumulated through a p38 MAPK-dependent transcriptional mechanism stimulated by redox-dependent migration inhibitory factor release. Moreover, GrB purified from UVA-treated cellular extracts was found to degrade fibronectin in vitro. Treatment with antisense oligonucleotide directed against GrB resulted in the inhibition of UVA-induced cell detachment and cell death and facilitated cell migration through fibronectin and vitronectin matrix upon UVA exposure. Altogether, these results suggest another function for GrB in the context of the UV response. Indeed, combined with our previous study, it appears that, whereas this enzyme mediates keratinocyte cellular cytotoxicity following UVB irradiation, GrB supports the capacity of keratinocyte to degrade extracellular matrix components following UVA irradiation. UV-mediated GrB production may thus have important consequences in photoaging and photocarcinogenesis.

Previous studies have established that UV irradiation results in epidermal growth factor receptor (EGFR) activation in keratinocytes. However, the signaling pathways and cellular effects related to this process remain incompletely elucidated. Herein, we describe for the first time that UVA-mediated EGFR activation results in B-catenin tyrosine phosphorylation at the Y654 residue responsible for the dissociation of E-cadherin/ A-catenin/B-catenin complexes. Moreover, UVA induces an EGFR-dependent, but Wnt-independent, B-catenin relocalization from the membrane to the nucleus followed by its association with T-cell factor 4 (TCF4). This newly formed B-catenin/TCF4 complex binds to a specific site on matrix metalloproteinase 1 (MMP1) promoter and governs MMP1 gene and protein expression, as well as cell migration in collagen and gelatin. Altogether, these results suggest that UVA stimulates keratinocyte invasiveness through two coordinated EGFR-dependent processes: loss of cell-to-cell contact due to B-catenin/E-cadherin/A-catenin dissociation and increased cell migration through extracellular matrix component degradation due to B-catenin/TCF4-dependent MMP1 regulation. These events may represent an important step in epidermis repair following UVA injury and their abnormal regulation could contribute to photoaging and photocarcinogenesis. [Cancer Res 2009;69(8):3291-9]

BackgroundGlycation is an aging reaction of naturally occurring sugars with dermal proteins. Type I collagen and elastin are most affected by glycation during intrinsic chronological aging.AimTo study the in vitro and ex vivo assays in human skin cells and explants and the antiaging effects of glycylglycine oleamide (GGO).Materials and methodsThe antiglycation effect of GGO was assessed in a noncellular in vitro study on collagen and, ex vivo, by immunohistochemical staining on human skin explants (elastin network glycation). The ability of GGO to contract fibroblasts was assessed in a functional assay, and its anti-elastase (MMP-12) activity was compared to that of oleic acid alone, glycylglycine (GG) alone, and oleic acid associated with GG.ResultsIn vitro, GGO reduced the glycation of type I collagen. Ex vivo, GGO restored the expression of fibrillin-1 inhibited by glycation. Furthermore, GGO induced a tissue retraction of almost 30%. Moreover, the MMP-12 activity was inhibited by up to 60%.ConclusionUnder the present in vitro and ex vivo conditions, GGO prevents glycation of the major structural proteins of the dermis, helping to reduce the risk of rigidification. By maintaining the elastic function of the skin, GGO may be a promising sparring partner for other topical antiaging agents.