Green Tea Polyphenol-Induced Epidermal Keratinocyte Differentiation Is Associated with Coordinated Expression of p57/KIP2 and Caspase 14 Stephen Hsu, Tetsuya Yamamoto, James Borke, Douglas S. Walsh, Baldev Singh, Sushma Rao, Kamatani Takaaki, Nam Nah-Do, Carol Lapp, David Lapp, Emily Foster, Wendy B. Bollag, Jill Lewis, John Wataha, Tokio Osaki, and George Schuster Department of Oral Biology and Maxillofacial Pathology (S.H., J.B., B.S., S.R., N.N.-D., C.L., E.F., J.L., J.W., G.S.), School of Dentistry, Department of Biochemistry and Molecular Biology (D.L.), and Institute of Molecular Medicine and Genetics (W.B.B.), Medical College of Georgia, Augusta, Georgia; Department of Oral Surgery (T.Y., K.T., T.O.), Kochi Medical School, Kochi, Japan; and Dermatology Service (D.S.W.), Eisenhower Army Medical Center, Fort Gordon, Georgia Received August 18, 2004; accepted November 8, 2004 ABSTRACT Epigallocatechin-3-gallate (EGCG), the most abundant poly- phenol in green tea, exerts chemopreventive effects by selec- tively inducing apoptosis in tumor cells. In contrast, EGCG accelerates terminal differentiation in normal human epidermal keratinocytes (NHEK) mediated partially by up-regulation of p57/KIP2, a cyclin-dependent kinase inhibitor that confers growth arrest and differentiation. However, it is unclear if EGCG modulates caspase 14, a unique regulator of epithelial cell terminal differentiation associated with cornification. Here, we examined the effect of EGCG on caspase 14 expression in NHEK and correlated the protein and mRNA expression of p57/KIP2 with those of caspase 14 in either normal keratino- cytes or p57/KIP2-expressing tumor cells (OSC2, an oral squa- mous cell carcinoma cell line). Additionally, paraffin-embedded normal and untreated psoriatic (aberrant keratinization) skin sections from humans were assessed for caspase 14 by im- munohistochemistry. In NHEK, EGCG induced the expression of caspase 14 mRNA and protein levels within a 24-h period. The expression of p57/KIP2 in OSC2 cells was adequate to induce caspase 14 in the absence of EGCG; this induction of caspase 14 was down-regulated by transforming growth fac- tor-1. In human psoriatic skin samples, caspase 14 staining in the upper epidermis was reduced, especially in nuclear areas. These results suggest that, in addition to p57/KIP2, EGCG- induced terminal differentiation of epidermal keratinocytes in- volves up-regulation of caspase 14. Further understanding of how EGCG modulates cellular differentiation may be useful in developing green tea preparations for selected clinical applica- tions. Unique characteristics of green tea polyphenols include their ability to induce growth arrest and apoptosis in tumor cells, especially in epithelial-type cells (Hsu et al., 2002b; Adhami et al., 2003), as well as protecting normal epithelial cells from carcinogens (Mukhtar and Ahmad, 2000; Katiyar, 2003). Among the four major polyphenols present in green tea, (-)-epigallocatechin-3-gallate (EGCG) is the most abun- dant (Miyazawa, 2000). We previously reported that green tea polyphenols, EGCG in particular, activate a pathway for cell differentiation in normal human epidermal keratinocytes (NHEK). Unlike epithelial-derived tumor cells, NHEK undergo an accelerated differentiation that is associated with p57/KIP2 induction when exposed to EGCG (Hsu et al., 2001, 2002a, 2003a). The p57/KIP2 gene product is a p53-independent G1 cyclin/cyclin- dependent kinase inhibitory protein (Lee et al., 1995); the C terminus contains a binding domain for proliferating cell nuclear antigen (Watanabe et al., 1998). Embryonic develop- ment in mice requires p57/KIP2 expression, lack of which causes early postnatal death and growth retardation (Taka- hashi et al., 2000). Conversely, in continuously dividing hu- This study was supported in part by a grant from the Medical College of Georgia Research Institute, a National Cancer Institute Grant R21 CA097258- 01A1, and funding through the Department of Oral Biology and Maxillofacial Pathology, School of Dentistry, Medical College of Georgia (to S.H.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.104.076075. ABBREVIATIONS: EGCG, (-)-epigallocatechin-3-gallate; NHEK, normal human epidermal keratinocytes; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate-buffered saline; RT-PCR, reverse transcription- polymerase chain reaction; TGF-1, transforming growth factor-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluores- cent protein. 0022-3565/05/3123-884 –890$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 312, No. 3 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 76075/1192672 JPET 312:884–890, 2005 Printed in U.S.A. 884 at ASPET Journals on April 10, 2016 jpet.aspetjournals.org Downloaded from