The focus of this proposal is to examine the tissue-specific regulation of a cytochrome P450 family member, CYP1A1, in normal human skin. CYP1A1 is a polycyclic and halogenated aromatic hydrocarbon-inducible monooxygenase. Since more than half of all cancers arise in epithelia having direct contact with the environment, an understanding of the endogenous regulation and expression of CYP1A1 in these tissues is important. Little is known about the tissue-specific regulation of CYPlA1 in normal human stratified squamous epithelia. However, PAHs and HAHs-dependent regulation of CYP1A1 is mediated by a basic helix-loop-helix transcription factor complex containing the aryl hydrocarbon receptor (AhR) and its dimerization partner, AhR nuclear translocator protein (Arnt). The role that AhR and Arnt play in tissue-specific CYP1A1 regulation in skin in not well understood. Furthermore, it is unknown if commitment to differentiation is necessary for CYP1A1 expression in skin or what role, if any, CYP1A1, AhR, and Arnt play in the terminal differentiation program of keratinocytes, the major cell type of stratified squamous epithelia. We have recently found that expression of the CYPlAl gene and enzyme activity is rapidly induced by cellular signaling events initiated by suspension of cultured human keratinocytes in the absence of xenobiotics or other differentiation-inducing conditions, such as high levels of calcium and serum. We suspect that similar cellular signaling events contribute to the induction of CYP1A1 in stratified squamous epithelia in vivo. This novel mechanism of CYP1A1 induction should enhance our understanding of mammalian cytochrome P450 gene regulation in stratified squamous epithelia and potentially aid in the identification of endogenous inducers and substrates for CYP1A1. The results we have generated thus far have led us to hypothesize that changes in cell adhesion are important regulators of CYP1A1 in normal human stratified epithelia.
The specific aims are: 1) Characterize the endogenous expression of CYP1A1 in human keratinocytes, and in AhR+/+ and AhR-/- mouse keratinocytes at different stages of differentiation in vitro and in situ., 2) Investigate the molecular mechanism of CYP1A1 induction in keratinocytes following loss of cell-cell and cell-substratum contact, and 3) Identify specific cell-cell or cell-substratum interaction that, when disrupted, cause rapid induction of keratinocyte CYP1A1 and investigate the role of the AhR/Arnt signaling in adhesion-mediated CYP1A1 induction using AhR-/- mouse keratinocytes.
