Exposure to UVB results in formation of cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts in DNA. These can be quantified by a variety of techniques including alkaline gel electrophoresis, ELISAs, Southwestern blotting, and immunohistochemistry. Damage to DNA results in activation of damage response pathways, as indicated by Western blotting using antibodies specific for p53 and breast cancer-associated gene 1 (BRCA1) phosphorylation. The signal from DNA damage to activation of these response pathways appears to be mediated by FKBP12-rapamycin-associated protein (FRAP), since these phosphorylation events are blocked by rapamycin. UVB-induced DNA damage also leads to induction of immunosuppressive cytokines including tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-10 in skin. Induction of TNF-alpha by UVB is readily detectable in cultured normal human epidermal keratinocytes (NHEKs) using ELISA, while induction of IL-10 is readily detectable in cultured mouse keratinocytes but not in NHEKs. Induction of DNA damage by liposome-encapsulated HindIII results in induction of immunosuppressive responses similar to UVB. Clinical testing shows that liposome-encapsulated T4 endonuclease V or photolyase stimulates repair of CPDs in the skin of human subjects, and prevents UVB-induced immunosuppression. Stimulation of repair and prevention of immunosuppression have been linked to prevention of skin cancer by liposome-encapsulated T4 endonuclease V in repair-deficient xeroderma pigmentosum patients.

. Skin wrinkling and xerosis associated with aging result from decreases in dermal collagen and stratum corneum ceramide content. This study demonstrated that ursolic acid incorporated into liposomes (URA liposomes) increases both the ceramide content of cultured normal human epidermal keratinocytes (NHEK), and the collagen content of cultured normal human dermal fibroblasts. In addition, URA liposomes increased the ceramide content of the skin of human subjects, with increases in hydroxy ceramides occurring after only 3 days of treatment. Both URA liposomes and retinoic acid decreased markers of keratinocyte differentiation (keratin 1, keratin 10 and involucrin) in cultured NHEK. Thus, URA liposomes have effects on keratinocyte differentiation and dermal fibroblast collagen synthesis similar to those of retinoids. However, this study showed that URA liposomes increase ceramides in NHEK, in contrast to the decreases previously shown to be caused by retinoids. URA liposomes have the potential to be used alone or in combination with other agents to restore or maintain skin ceramide and collagen content.

Ultraviolet radiation induces signal transduction at both early (<6 h) and late (>6 h) times after exposure. The inflammatory and immunosuppressive cytokine tumor necrosis factor alpha is induced at late times, and is induced by ultraviolet-induced DNA damage, as defects in DNA repair increase, and enhanced photoproduct repair reduces, tumor necrosis factor alpha expression. Here we show that late tumor necrosis factor alpha gene expression is sensitive to rapamycin, implicating FKBP12-rapamycin-associated protein, a member of the DNA protein kinase family, as a signal transducer of ultraviolet-induced DNA damage. FKBP12-rapamycin-associated protein was localized in the nucleus of keratinocytes and its level was increased following ultraviolet irradiation. Immuno- precipitated FKBP12-rapamycin-associated protein was stimulated by ultraviolet-irradiated DNA to phosphorylate p53 in vitro, and in vivo rapamycin reduced ultraviolet induction of p53 by 20%. Rapamycin further inhibited the ultraviolet-induced phosphorylation of the FKBP12-rapamycin-associated protein downstream target kinase p70S6K. In mice, topical application of rapamycin before ultraviolet exposure protected against suppression of the contact hypersensitivity that is a hallmark of ultraviolet-induced cytokine gene expression. These results demonstrate that the FKBP12-rapamycin-associated DNA protein kinase transduces the signal of ultraviolet-induced DNA damage into production of immunosuppressive cytokines at late times after ultraviolet irradiation.

TNFα is a primary cytokine responsible for inflammatory and immunosuppressive responses in skin. After UV‐B irradiation of cultured human keratinocytes, we found that TNFα was released into the media, as monitored by ELISA, and was bound to cells, as observed by immunofluorescence microscopy. The release of TNFα into cell culture supernatant during the 24 h after UV‐B irradiation was augmented by the addition of IL‐1α to the cells. Further, we found this secretion was unaffected by rapamycin, and therefore independent of FRAP DNA‐protein kinase mediated signal transduction. However, UV‐B also induced expression of membrane‐bound TNFα, and this was dependent on FRAP signaling. In wild type mice, TNFα bound to skin increased immediately after irradiation, declined at 6 h, and then rose again at 12 h before falling by 24 h. This pattern of induction was confirmed by RT‐PCR of TNFα mRNA message in cultured epidermal cells. Induction of membrane‐bound TNFα was also found in c‐fos gene knockout mice deficient in the AP‐1 transcription factor, suggesting that, although AP‐1 containing c‐fos signaling is required for some UV responses, AP‐1 containing c‐fos is not required for this TNFα activation. However, in homozygous p53 knockout mice the basal level of TNFα bound to the epidermis was greatly elevated without UV irradiation. This level declined and remained constant following irradiation. This implies that p53 directly or indirectly represses TNFα gene expression and that modification of p53 mRNA stability or phosphorylation of p53 protein after UV may be responsible for TNFα induction in the membrane. Overexpression of the immunosuppressive cytokine TNFα in this locale may contribute to the carcinogen‐susceptibility of p53 knockout mice.