In two-stage skin chemical carcinogenesis, phorbol ester 12-Otetradecanoylphorbol-13-acetate (TPA) acts as a promoter essential for clonal expansion of the initiated cells carrying the activated ras oncogenes. Although protein kinase C (PKC) isozymes are the main targets of TPA, their role in tumor promotion remains controversial. We previously reported that mice lacking a Ras/Rap effector phospholipase CE (PLCe À/À mice) exhibited marked resistance to tumor formation in the two-stage skin carcinogenesis. PLCe À/À mice also failed to exhibit basal layer cell proliferation and epidermal hyperplasia induced by TPA, suggesting a role of PLCE in tumor promotion. Here, we show that PLCe À/À mice exhibit resistance to TPA-induced skin inflammation as assessed by reduction in edema, granulocyte infiltration, and expression of a proinflammatory cytokine, interleukin-1A (IL-1A). On the other hand, the proliferative potentials of keratinocytes or dermal fibroblasts in culture remain unaffected by the PLCe background, suggesting that the PLCE's role in tumor promotion may be ascribed to augmentation of inflammatory responses. In dermal fibroblast primary culture, TPA can induce activation of the PLCE lipase activity, which leads to the induction of IL-1A expression. Experiments using small interfering RNA-mediated knockdown indicate that this activation is mediated by Rap1, which is activated by a TPA-responsive guanine nucleotide exchange factor RasGRP3. Moreover, TPA-induced activation of Rap1 and PLCE is inhibited by a PKC inhibitor GF109203X, indicating a crucial role of PKC in signaling from TPA to PLCE. These results imply that two TPA targets, RasGRP3 and PKC, are involved in TPA-induced inflammation through PLCE activation, leading to tumor promotion. [Cancer Res 2008;68(1):64-72]

Phospholipase Cε (PLCε) is an effector of Ras/Rap small GTPases. We previously demonstrated that PLCε plays a crucial role in development of phorbor ester-induced skin inflammation, which is intimately involved in the promotion of skin carcinogenesis. In this study, we have examined its role in local skin inflammatory reactions during development of contact hypersensitivity toward a hapten 2,4-dinitrofluorobenzene (DNFB). PLCε+/+ and PLCε−/− mice were sensitized with DNFB, followed by a DNFB challenge on the ears. PLCε−/− mice exhibited substantially attenuated inflammatory reactions compared with PLCε+/+ mice as shown by suppression of ear swelling, neutrophil infiltration, and proinflammatory cytokine production. In contrast, the extent and kinetics of CD4+ T cell infiltration showed no difference depending on the PLCε background. Adoptive transfer of CD4+ T cells from the sensitized mice to naive mice between PLCε+/+ and PLCε−/− backgrounds indicated that PLCε exerts its function in cells other than CD4+ T cells, presumably fibroblasts or keratinocytes of the skin, to augment inflammatory reactions during the elicitation stage of contact hypersensitivity. Moreover, dermal fibroblasts and epidermal keratinocytes cultured from the skin expressed proinflammatory cytokines in a PLCε-dependent manner on stimulation with T cell-derived cytokines such as IL-17, IFN-γ, TNF-α, and IL-4. These results indicate that PLCε plays a crucial role in induction of proinflammatory cytokine expression in fibroblasts and keratinocytes at the challenged sites, where infiltrated CD4+ T cells produce their intrinsic cytokines, thereby augmenting the local inflammatory reactions.

We describe an organotypic model of mouse skin consisting of a stratified sheet of epidermal keratinocytes and dermal fibroblasts within a contracted collagen gel. The model was designed to maintain the polarity of stratified keratinocytes and permit their long-term culture at an air-liquid interface. After air exposure, the thickness of the keratinocyte sheet transiently increased and then decreased to two cell layers at 2 weeks. The two-cell-layer structure is similar to that of the adult mouse epidermis. Cytokeratin 5 was localized in the lowest cell layer in the epithelial sheet, but cytokeratin 1 and loricrin were localized in the outer cell layers, resembling mouse skin. The expressions of interleukin 1 and 1 in the keratinocytes and of keratinocyte growth factor 1 and 2 in the fibroblasts correlated with keratinocyte stratification. The mouse organotypic coculture is useful in studying epithelial cell-mesenchymal cell interactions in vitro.