Oncogene (2008) 27, 308–317 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE p63(TP63) elicits strong trans-activation of the MFG-E8/lactadherin/ BA46 gene through interactions between the TA and DN isoforms T Okuyama1,2,3, S Kurata4,5, Y Tomimori1, N Fukunishi4, S Sato6, M Osada7, K Tsukinoki5, H-F Jin8, A Yamashita8, M Ito8, S Kobayashi2, R-I Hata5, Y Ikawa1,9 and I Katoh1,8 1Ikawa Laboratory, RIKEN, Wako, Japan; 2Department of Molecular Physiology, Kyoritsu University of Pharmacy, Tokyo, Japan; 3Department of Cell Regulation, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan; 4Department of Redox Response Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan; 5Oral Health Science Research Center, Kanagawa Dental College, Yokosuka, Japan; 6Department of Immune Regulation, Tokyo Medical and Dental University, Tokyo, Japan; 7Human Gene Sciences Center, Tokyo Medical and Dental University, Tokyo, Japan; 8Department of Microbiology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Yamanashi, Japan and 9Department of Hematology, Tokyo Medical and Dental University, Tokyo, Japan We report here that human MFGE8 encoding milk fat Introduction globule-EGF factor 8 protein (MFG-E8), also termed 46 kDa breast epithelial antigen and lactadherin, is p63 (TP63), a member of the p53 (TP53) gene family transcriptionally activated by p63, or TP63, a p53 (TP53) (Osada et al., 1998; Yang et al., 1998), is essential for family protein frequently overexpressed in head-and-neck embryonic epithelial tissue development (Celli et al., squamous cell carcinomas, mammary carcinomas and so 1999; Mills et al., 1999; Yang et al., 1999; Pellegrini on. Despite that human MFG-E8 was originally identified et al., 2001; Brunner et al., 2002). It is frequently over- as a breast cancer marker, and has recently been reported expressed in head-and-neck squamous cell carcinomas to provide peptides for cancer immunotherapy, its trans- and in other cancers derived from epithelial basal cells of criptional control remains an open question. Observations breast, lung, cervix, urinary tracts, and so on (Hibi et al., in immunohistochemical analyses, a tetracycline-induced 2000; Quade et al., 2001; DiRenzo et al., 2002; Koga p63 expression system and keratinocyte cultures sug- et al., 2003; Massion et al., 2003) as well as in gested a physiological link between p63 and MFGE8.By keratinocyte stem cells (Pellegrini et al., 2001). reporter assays with immediately upstream regions of Reflecting the considerable structural similarity to MFGE8, we determined that the trans-activator (TA) p53, p63 also trans-activates various genes through isoforms of p63 activate MFGE8 transcription though a p53-binding consensus sequences (p53 motifs) and/or p53/p63 motif at À370, which was confirmed by a related sites (Osada et al., 1998, 2005b). The p63 chromatin immunoprecipitation experiment. Upon siRNA- gene is transcribed into 50-terminal RNA isoforms mediated p63 silencing in a squamous cell carinoma line, encoding either the trans-activator (TA) domain or the MFG-E8 production decreased to diminish Saos-2 cell N-terminally truncated (DN) domain, each of which adhesion. Interestingly, the DN-p63 isoform lacking the is further processed to the a, b and g variants by TA domain enhanced the MFGE8-activating function of 30-terminal splicing. Among them, TAp63g (p51A) was TA-p63, if DN-p63 was dominant over TA-p63 as first identified as a TA corresponding to tumor typically observed in undifferentiated keratinocytes and suppressor p53. In contrast, the DN-p63 isoforms were squamous cell carcinomas, implying a self-regulatory initially described as dominant-negative type proteins mechanism of p63 by the TA:DN association. MFG-E8 against TA-p63. With the oligomerization domain, DN- may provide a novel pathway of epithelial–nonepithelial p63 binds to TA-p63. Recent studies, however, detected cell interactions inducible by p63, probably in pathological positive effects of the DN isoforms upon certain processes. promoters (Kurata et al., 2004; Wu et al., 2005; Helton Oncogene (2008) 27, 308–317; doi:10.1038/sj.onc.1210646; et al., 2006). published online 16 July 2007 Breast epithelial antigen, also termed lactadherin and human milk fat globule protein (HMFG) (GenBank Keywords: p63; MFG-E8; lactadherin; BA46; P53 S56151), was identified as a marker of breast cancers (Larocca et al., 1991; Taylor et al., 1997), and is now known as the human ortholog (GenBank NM_005928, NP_005919) to milk fat globule-EGF factor 8 protein Correspondence: Dr I Katoh, Department of Microbiology, Inter- (MFG-E8) of mouse and other mammals (Mather et al., disciplinary Graduate School of Medicine and Engineering, University 1993; Hvarregaard et al., 1996). In this report, we refer of Yamanashi, Chuo, Yamanashi 409-3898, Japan. to the human protein as MFG-E8, and the gene as E-mail: [email protected] Received 24 October 2006; revised 4 June 2007; accepted 5 June 2007; MFGE8. MFG-E8 is a membrane-associated, heavily published online 16 July 2007 glycosylated secretory protein with two distinct types of Transcriptional activation of MFGE8 by p63 T Okuyama et al 309 adhesion sites: the Arg-Gly-Asp triplet arranged to bind a TAp63γ TAp63α Np63γ Np63α to integrin avb3 or avb5; and the discoidin domains (C1 --+ + - + - + tet and C2) that interact with phosphatidylserine located on TAp63α the inner side of plasma membrane (Taylor et al., 1997; ∆Np63α Shi and Gilbert, 2003). Mouse Mfge8 produces MFG- TAp63γ E8-L and MFG-E8-S, in which the former is mouse- ∆Np63γ Western nonspecific specific, and the latter conserved among humans and blot β-actin animals (Watanabe et al., 2005). While the L form containing a Pro/Thr-enriched domain facilitates pha- MFGE8 gocytosis of apoptotic cells by macrophages (Hanayama p21WAF1 (32) et al., 2002, 2004), the S form was reported to support RT-PCR the gamete interaction upon fertilization (Ensslin and MFGE8 (26) Shur, 2003). Very importantly, recent studies deter- 18S rRNA (15) mined peptide sequences derived from human MFG-E8 as the potential targets for tumor immunotherapy b Days after Ca2+ -induction (Carmon et al., 2002; Liu et al., 2005). 0 3 7 In addition to the increased expression of MFGE8 TAp63α in lactating and transformed breast epithelial cells ∆Np63α (Peterson et al., 1990), steady-state expression also TAp63γ occurs in various tissues (Kruger et al., 2000; Watanabe Western ∆Np63γ et al blot ., 2005). However, little is known about the MFG-E8 transcriptional control of MFGE8. This study shows that p63 enhances the MFGE8 promoter and offers an β-actin explanation about the MFG-E8 augmentation in carcinomas. IVL RT-PCR MFGE8 Results 18S rRNA Figure 1 Enhancement of MFGE8 expression by p63. (a) Induction of MFGE8 by p63 Tetracycline-inducible p63 expression system. Induced cells ( þ ) We tested whether p63 affected MFGE8 expression in and control cells (À) were analysed for the p63 and milk fat HEK293 cells, using the tetracycline-inducible p63 globule-EGF factor 8 protein (MFG-E8) proteins (upper panels). expression system (Osada et al., 2005b). After induction Positions of the p63 isoforms were indicated. Isoform-specific reverse transcription (RT)–PCR was carried out (lower panels). by tetracyclin, TAp63g (57 kDa), also termed p51A PCR cycle number is indicated for each experiment. (b) Neonatal (Osada et al., 1998; Yang et al., 1998), and TAp63a human keratinocyte (NHK) culture. Cells before (0) and after (on (85 kDa), or p51B, increased the MFGE8 transcript days À3 and À7) the Ca2 þ (1 mM) input were analysed by western approximately fivefold and threefold, respectively, blotting and RT–PCR for p63 and MFG-E8. b-Actin and 18S as determined by reverse transcription (RT)–PCR rRNA were tested to ensure equal sample loading. (Figure 1a). Neither DNp63g (47 kDa) nor DNp63a (75 kDa) altered the level of MFGE8 mRNA. Western blot analyses supported these findings. promoter/enhancer region upstream of MFGE8, encom- Both MFGE8 and p63 are endogenously expressed in passing À1890 to þ 50, nucleotide numbers relative to neonatal human keratinocytes (NHK) (Figure 1b). After the transcription start site ( þ 1), contained three full-site these cells were induced to differentiate by increasing the motifs at positions À1198, À370 and À27, and a single Ca2 þ concentration in the medium to 1 mM, p63 had half site at À742, in agreement with the human genome decreased gradually on day 4 and day 7 at the mRNA database (Figure 2A). We constructed luciferase gene and protein levels. MFGE8 mRNA diminished concur- (luc) expression plasmids, 2K-luc, 1K-luc and S-luc, rently. In contrast, IVL mRNA coding for involucrin, having nucleotides À1890 to þ 50, À877 to þ 50, and an early differentiation marker absent in the p63- À250 to þ 50, respectively. Trans-activation assays in localized basal layer of epidermis (Li et al., 2000), HEK293 cells indicated that both p53 and TAp63g substantially increased. enhanced the 2K-luc promoter approximately threefold, and TAp63a did so 1.5-fold (Figures 2Ba and b). Presence of p53/p63 motifs in the MFGE8 Neither DNp63g nor DNp63a affected 2K-luc (Figures promoter/enhancer region 2Bc and d), supporting the results of the drug-inducible The p53 binding consensus sequences comprise a system (Figure 1a). The 1K-luc plasmid could also tandem repeat of the half-site motif, RRRC(A/T)(A/ respond to TAp63g, while S-luc failed to do so, T)GYYY, in which R and Y represent purine and localizing the main p63-responsive element between pyrimidine, respectively (el-Deiry et al., 1992). p63 uses À877 and À249 (Figure 2Ba). p53, however, appeared to the same or similar motifs for trans-activation (Osada enhance the 2K, 1K and S promoters equally, implying et al., 1998, 2005b; Yang et al., 1998).