Transcriptional regulation and the role of murine 8S-lipoxygenase in mouse skin carcinogenesis

Abstract

8S-lipoxygenase (8S-LOX), the murine homolog of human 15S-LOX-2, has unique expression features in mouse skin, i.e., it is not expressed at a detectable level in normal skin but is strongly expressed in several stages of skin tumorigenesis, such as 12-O-tetradecanoylphorbol-13-acetate (TPA) treated skin or in skin tumors. In this study, we studied a specific mechanism by which 8SLOX gene expression is regulated by TPA in keratinocytes and the functional role of 8S-LOX during mouse skin carcinogenesis. In the first study, we found TPAinduced 8S-LOX mRNA expression in SSIN primary mouse keratinocytes was the result of increased transcription. Therefore, transcriptional regulation of 8SLOX expression was further studied by cloning its promoter. The cloned 8S-LOX promoter (~ 2 kb) has neither a TATA box nor a CCAAT box, however, it was highly responsive to TPA in SSIN primary keratinocytes. We then identified a Sp1 binding site (-68/-77) as a TPA responsive element (TRE) in the promoter and showed that Sp1, Sp2, and Sp3 proteins bind to the TRE. Binding of these proteins to the TRE was significantly increased by TPA treatment and 8S-LOX transcription was decreased when the binding of these proteins was inhibited. We thus concluded that increased binding of Sp1, Sp2, and Sp3 to the TRE of the 8SLOX promoter is a mechanism by which TPA induces 8S-LOX expression in keratinocytes. In the second study, we found 8S-LOX is closely associated with keratinocyte differentiation and forced expression can inhibit mouse skin tumorigenesis. Targeted C57BL/6J transgenic mice overexpressing the 8S-LOX gene under control of the loricrin promoter exhibited more differentiated epidermal phenotypes as well as reduced papilloma development in a two-stage skin carcinogenesis protocol. Forced expression of the 8S-LOX gene in tumorderived cell lines, MT1/2 (papilloma) and CH72 (carcinoma), also caused increased differentiation and inhibition of cell proliferation in vitro as well as in in vivo xenografts, respectively. Moreover, histochemical analyses showed 8S-LOX expression was strictly confined to the differentiated region of the skin in the course of skin tumorigenesis. Collectively, these findings suggest that 8S-LOX plays a role as a prodifferentiating, an anticarcinogenic, and a tumor suppressing gene in mouse skin.