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p63(TP63) elicits strong trans-activation of the MFG-E8/lactadherin/BA46 gene through interactions between the TA and ΔN isoforms

Abstract

We report here that human MFGE8 encoding milk fat globule-EGF factor 8 protein (MFG-E8), also termed 46 kDa breast epithelial antigen and lactadherin, is transcriptionally activated by p63, or TP63, a p53 (TP53) family protein frequently overexpressed in head-and-neck squamous cell carcinomas, mammary carcinomas and so on. Despite that human MFG-E8 was originally identified as a breast cancer marker, and has recently been reported to provide peptides for cancer immunotherapy, its transcriptional control remains an open question. Observations in immunohistochemical analyses, a tetracycline-induced p63 expression system and keratinocyte cultures suggested a physiological link between p63 and MFGE8. By reporter assays with immediately upstream regions of MFGE8, we determined that the trans-activator (TA) isoforms of p63 activate MFGE8 transcription though a p53/p63 motif at −370, which was confirmed by a chromatin immunoprecipitation experiment. Upon siRNA-mediated p63 silencing in a squamous cell carinoma line, MFG-E8 production decreased to diminish Saos-2 cell adhesion. Interestingly, the ΔN-p63 isoform lacking the TA domain enhanced the MFGE8-activating function of TA-p63, if ΔN-p63 was dominant over TA-p63 as typically observed in undifferentiated keratinocytes and squamous cell carcinomas, implying a self-regulatory mechanism of p63 by the TA:ΔN association. MFG-E8 may provide a novel pathway of epithelial–nonepithelial cell interactions inducible by p63, probably in pathological processes.

Introduction

p63 (TP63), a member of the p53 (TP53) gene family (Osada et al., 1998; Yang et al., 1998), is essential for embryonic epithelial tissue development (Celli et al., 1999; Mills et al., 1999; Yang et al., 1999; Pellegrini et al., 2001; Brunner et al., 2002). It is frequently overexpressed in head-and-neck squamous cell carcinomas and in other cancers derived from epithelial basal cells of breast, lung, cervix, urinary tracts, and so on (Hibi et al., 2000; Quade et al., 2001; DiRenzo et al., 2002; Koga et al., 2003; Massion et al., 2003) as well as in keratinocyte stem cells (Pellegrini et al., 2001).

Reflecting the considerable structural similarity to p53, p63 also trans-activates various genes through p53-binding consensus sequences (p53 motifs) and/or related sites (Osada et al., 1998, 2005b). The p63 gene is transcribed into 5′-terminal RNA isoforms encoding either the trans-activator (TA) domain or the N-terminally truncated (ΔN) domain, each of which is further processed to the α, β and γ variants by 3′-terminal splicing. Among them, TAp63γ (p51A) was first identified as a TA corresponding to tumor suppressor p53. In contrast, the ΔN-p63 isoforms were initially described as dominant-negative type proteins against TA-p63. With the oligomerization domain, ΔN-p63 binds to TA-p63. Recent studies, however, detected positive effects of the ΔN isoforms upon certain promoters (Kurata et al., 2004; Wu et al., 2005; Helton et al., 2006).

Breast epithelial antigen, also termed lactadherin and human milk fat globule protein (HMFG) (GenBank 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 (MFG-E8) of mouse and other mammals (Mather et al., 1993; Hvarregaard et al., 1996). In this report, we refer to the human protein as MFG-E8, and the gene as MFGE8. MFG-E8 is a membrane-associated, heavily glycosylated secretory protein with two distinct types of adhesion sites: the Arg-Gly-Asp triplet arranged to bind to integrin αvβ3 or αvβ5; and the discoidin domains (C1 and C2) that interact with phosphatidylserine located on the inner side of plasma membrane (Taylor et al., 1997; Shi and Gilbert, 2003). Mouse Mfge8 produces MFG-E8-L and MFG-E8-S, in which the former is mouse-specific, and the latter conserved among humans and animals (Watanabe et al., 2005). While the L form containing a Pro/Thr-enriched domain facilitates phagocytosis of apoptotic cells by macrophages (Hanayama et al., 2002, 2004), the S form was reported to support the gamete interaction upon fertilization (Ensslin and Shur, 2003). Very importantly, recent studies determined peptide sequences derived from human MFG-E8 as the potential targets for tumor immunotherapy (Carmon et al., 2002; Liu et al., 2005).

In addition to the increased expression of MFGE8 in lactating and transformed breast epithelial cells (Peterson et al., 1990), steady-state expression also occurs in various tissues (Kruger et al., 2000; Watanabe et al., 2005). However, little is known about the transcriptional control of MFGE8. This study shows that p63 enhances the MFGE8 promoter and offers an explanation about the MFG-E8 augmentation in carcinomas.

Results

Induction of MFGE8 by p63

We tested whether p63 affected MFGE8 expression in HEK293 cells, using the tetracycline-inducible p63 expression system (Osada et al., 2005b). After induction by tetracyclin, TAp63γ (57 kDa), also termed p51A (Osada et al., 1998; Yang et al., 1998), and TAp63α (85 kDa), or p51B, increased the MFGE8 transcript approximately fivefold and threefold, respectively, as determined by reverse transcription (RT)–PCR (Figure 1a). Neither ΔNp63γ (47 kDa) nor ΔNp63α (75 kDa) altered the level of MFGE8 mRNA. Western blot analyses supported these findings.

Figure 1
figure1

Enhancement of MFGE8 expression by p63. (a) Tetracycline-inducible p63 expression system. Induced cells (+) and control cells (−) were analysed for the p63 and milk fat globule-EGF factor 8 protein (MFG-E8) proteins (upper panels). Positions of the p63 isoforms were indicated. Isoform-specific reverse transcription (RT)–PCR was carried out (lower panels). PCR cycle number is indicated for each experiment. (b) Neonatal human keratinocyte (NHK) culture. Cells before (0) and after (on days −3 and −7) the Ca2+ (1 mM) input were analysed by western blotting and RT–PCR for p63 and MFG-E8. β-Actin and 18S rRNA were tested to ensure equal sample loading.

Both MFGE8 and p63 are endogenously expressed in neonatal human keratinocytes (NHK) (Figure 1b). After these cells were induced to differentiate by increasing the Ca2+ concentration in the medium to 1 mM, p63 had decreased gradually on day 4 and day 7 at the mRNA and protein levels. MFGE8 mRNA diminished concurrently. In contrast, IVL mRNA coding for involucrin, an early differentiation marker absent in the p63-localized basal layer of epidermis (Li et al., 2000), substantially increased.

Presence of p53/p63 motifs in the MFGE8 promoter/enhancer region

The p53 binding consensus sequences comprise a tandem repeat of the half-site motif, RRRC(A/T)(A/T)GYYY, in which R and Y represent purine and pyrimidine, respectively (el-Deiry et al., 1992). p63 uses the same or similar motifs for trans-activation (Osada et al., 1998, 2005b; Yang et al., 1998). The cloned promoter/enhancer region upstream of MFGE8, encompassing −1890 to +50, nucleotide numbers relative to the transcription start site (+1), contained three full-site motifs at positions −1198, −370 and −27, and a single half site at −742, in agreement with the human genome database (Figure 2A). We constructed luciferase gene (luc) expression plasmids, 2K-luc, 1K-luc and S-luc, having nucleotides −1890 to +50, −877 to +50, and −250 to +50, respectively. Trans-activation assays in HEK293 cells indicated that both p53 and TAp63γ enhanced the 2K-luc promoter approximately threefold, and TAp63α did so 1.5-fold (Figures 2Ba and b). Neither ΔNp63γ nor ΔNp63α affected 2K-luc (Figures 2Bc and d), supporting the results of the drug-inducible system (Figure 1a). The 1K-luc plasmid could also respond to TAp63γ, while S-luc failed to do so, localizing the main p63-responsive element between −877 and −249 (Figure 2Ba). p53, however, appeared to enhance the 2K, 1K and S promoters equally, implying importance of the S region (Figure 2Be).

Figure 2
figure2

The promoter/enhancer sequences upstream of MFGE8. (A) Outline of the cloned MFGE8 promoter/enhancer region. The top scheme shows location of the p53-binding consensus sequences. Separate and paired ovals indicate half sites and full sites of the consensus, respectively. Filled boxes indicate two CAT box motifs. Line drawing represents DNA segments, 2K, 1K and S, cloned into the luc vector. The 5′ and 3′ end points are indicated by nucleotide numbers. (B) Trans-activation assays in HEK293 cells. A p63 expression plasmid (pRcCMV-p53, -TAp63γ, -TAp63α or -ΔNp63γ) or control plasmid pRcCMV was co-transfected with a luc expression plasmid (2K-, 1K- or S-luc). Bar graphs indicate relative luciferase activities (-fold activation) in comparison with the activity obtained with pRcCMV (1-fold). Each bar represents the average of triplicates. (C) The p53-binding consensus sequences forming the full-site/half-site motifs within the 1K region (top). Nucleotide sequences at the HS1, HS23 and HS45 sites are aligned, where dotted nucleotides indicate original sequence deviations from the consensus. Altered (T) and preserved (asterisk) sequences in the mutant plasmids, M1, M2, M23 and M45 are indicated. (D) Illustration of the mutant luc plasmids. Open and filled ovals denote the original and mutated motifs. 1K-luc and S-luc are also aligned.

Determination of the main p63-responsive sites in the MFGE8 promoter

We analysed the 1K region, focusing on a half site at −742 (HS1), a full site at −370 (HS2/3) and a full site at −27 (HS4/5) for its responsiveness to p63. Mutant promoter/enhancer plasmids, MI, M2, M1-3, M45 and M1-5, were rendered by introducing a core nucleotide mutation, ‘G to T’, at the seventh position of each half-site decamer in various combinations (Figures 2C and D). In HEK293 cells, the sensitivity of 1K to TAp63γ diminished when HS2 was mutated in the forms of M2-luc, M1-3-luc and M1-5-luc (Figure 3Aa). In contrast, the M1 and M45 mutants retained the full sensitivity to TAp63γ. The TA-p63 proteins including TAp63γ and TAp63α (Figure 3Ab) were thought to activate MFGE8 transcription through HS2/3. However, none of the mutations affected p53 significantly (Figure 3Ae), suggesting involvement of an unidentified site between −250 and +50 or an indirect mechanism.

Figure 3
figure3

Luciferase reporter assay with the wild type and mutated MFGE8 1 K segments. (A) Trans-activation by TAp63γ (a), TAp63α (b), ΔNp63γ (c), ΔNp63α (d) and p53 (e) in HEK293 cells. (B) We tested 1K-luc, M2-luc and M45-luc for activation by TAp63γ (a), TAp63α (b), ΔNp63γ (c) and ΔNp63α (d) in HeLa cells.

Stabilization of TA-p63 by ΔN-p63 in HeLa

In HeLa cells, TAp63γ enhanced 1K-driven luc expression 5.3-fold. Results with the M2 and M45 mutants confirmed HS2/3 as the primary target site of TA-p63 (Figures 3Ba and b). Surprisingly, ΔNp63γ and ΔNp63α activated 1K-luc 12-fold and 25-fold, respectively (Figures 3Bc and d). Apparently, ΔNp63α was fivefold more efficient than TAp63γ in activation of the 1K, M2 and M45 promoters.

To gain an insight into the positive effects of the ΔN isoforms in HeLa, we analysed several epithelial cell lines for p63 expression (Figure 4a). FaDu cells derived from a hypopharyngeal squamous cell carcinoma (Yamaguchi et al., 2000), HSC-1 from a skin squamous cell carcinoma and NHK were ‘p63-positive’: the TA, ΔN, α/β and γ mRNA sequences were detected by RT–PCR, and TAp63α (85 kDa), ΔNp63α (75 kDa), TAp63γ (57 kDa) and ΔNp63γ (47 kDa) by western blotting. HEK293 cells were negative for p63 mRNAs and proteins: the TA and ΔN isoform mRNAs were 4-fold and 32-fold less in HEK293, respectively. Although HeLa cells appeared p63-negative by western blotting, a level of the TA, α/β and γ mRNAs was detectable. The ΔN form mRNA became apparent after four additional PCR cycles. RT–PCR analyses for ITGA3 and MFGE8 (Kurata et al., 2004) suggested a relation between these mRNA levels and the p63 status. The MFGE8 mRNA level in HSC-1 and NHK is lower than that in FaDu, but is certainly (more than threefold) increased in comparison with the level in p63-negative HEK293. Western blotting results were essentially consistent with the RT–PCR analyses. We noted, however, a higher background of MFG-E8 in HeLa cells. In addition to the constitutive, moderate MFGE8 expression due to p63, HeLa has abundant expression of integrin αvβ5 (Smith and Giorgio, 2004), a receptor of MFG-E8, which may have caused longer retention of this protein.

Figure 4
figure4

Endogenous p63 expression in HeLa cells and TA-p63 stabilization by transfected ΔN-p63. (a) Cell lines were analysed for p63 and the target gene expression. Numbers in the parentheses indicate amplification cycles in reverse transcription (RT)–PCR. (b) HeLa cells transfected with TAp63γ-, TAp63α-, ΔNp63γ- and ΔNp63α-expression vectors were analysed for p63 and MFGE8 mRNAs (left) and the proteins (right).

When HeLa cells were transfected with the ΔN-α expression vector, amounts of the ΔN-γ and TA-α proteins, in addition to ΔN-α, emerged (Figure 4b, lane 5). A lower level of TA-γ also became detectable. A slight increase in TA-α and ΔN-α also occurred in the ΔN-γ-transfected cells (lane 4). In contrast, neither TA-α nor TA-γ affected other p63 isoforms (lanes 2 and 3). RT–PCR analyses ruled out the possibility that one transfected isoform induced transcription of others. Both the TA and ΔN transfection increased endogenous MFGE8 expression 2.5-fold. The protein amount also increased to the level found in FaDu cells. Thus, we found in HeLa (1) HS2/3 was not only responsible for the trans-activation by TA-p63, but also effective in the enhanced activation by ΔN-p63 and (2) when one ΔN-p63 isoform was introduced by transfection other forms including TA-α and TA-γ accumulated. We therefore speculated that transfected ΔN-p63 caused the MFGE8 promoter activation through endogenously expressed TA-p63 in HeLa cells.

Isoform interactions and binding to the target site

The TA:ΔN combination experiment in HEK293 cells indicated that ΔN-α strongly suppressed TA-γ and TA-α, when the TA:ΔN-α ratio was between 7:1 and 4:4 (Figures 5b and d), supporting the original characterization of ΔN-α as a potent dominant-negative isoform. However, when the ΔN content was increased to 2:6 and 1:7, the luciferase activity normalized to 0.1 μg of the TA plasmid amount (b and d, hatched columns) markedly increased. When the TA:ΔN-γ ratio was between 7:1 and 4:4, ΔN-γ did not affect the TA-γ activity (a, hatched columns), but moderately suppressed TA-α (c, hatched columns). This is consistent with the initial finding that ΔN-γ is a moderate suppressor of TA-p63 (Yang et al., 1998). TA-enhancing effects of ΔN-γ became apparent at 2:6 and 1:7 (a and c). Assays with the TA-α:TA-γ (e) and ΔN-α:ΔN-γ (data not shown) combinations produced luciferase activities proportional to the isoform amounts.

Figure 5
figure5

Enhancement of TA-p63 by dominantly expressed ΔN-p63. 1K-luc trans-activation assays by combined expression of TA-p63 with ΔN-p63. TA:ΔN or TA:TA plasmid ratio employed in each experiment (a–e) is shown at the bottom. Filled columns represent values (RLU) after subtraction of the background count obtained by co-transfection with the pRcCMV vector. Hatched columns indicate luciferase activities corresponding to 0.1 μg of the TA-p63 expression plasmid, provided that the ΔN-p63 isoforms lack an activity to trans-activate this promoter.

When we transfected a suboptimal amount of TAp63α and TAp63γ in HEK293, endogenous MFGE8 transcription was not altered (Figure 6a, lanes 2 and 6). Upon tetracycline-induced, massive ΔNp63α production, however, the transfected cells increased MFGE8 transcription and the protein synthesis, which were accompanied by TA-p63 accumulation (lanes 3, 4, 7 and 8). Without the TA-p63 transfection, chromosomal MFGE8 expression remained unaltered (lanes 9 and 10). Thus, stabilization and activation of TA-p63 concurrently occur by dominantly expressed ΔNp63α.

Figure 6
figure6

Activation of endogenous MFGE8 expression by the p63 isoforms. (a) Cooperation of transiently expressed TA-p63 with tetracycline-induced ΔNp63α in HEK293. TAp63γ and TAp63α are marked with filled and open triangles, respectively, and ΔNp63α by arrows. (b) ChIP experiment detecting an interaction between HA-TAp63γ and the HS2/3 site. The MFGE8 enhancer segment covering HS2/3, ITGA3 intron-1 and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter segment spanning the TATA box were amplified from the immunoprecipitates by PCR.

We next examined p63 for interaction with the HS2/3 site on chromosome DNA (15q25) by chromatin immunoprecipitation (ChIP). The generally used anti-p63 antibody, 4A4, was nonreactive with p63 unless proteins were fully denatured, and therefore unsuitable for ChIP. We produced hemagglutinin (HA)-tagged TAp63γ in the drug-inducible system (Supplementary Information 2), and analysed DNA sequences co-precipitated with HA-TAp63γ by an anti-HA antibody. PCR analyses indicated that the HS2/3 segment was significantly more abundant in the immunoprecipitate from the HA-TAp63γ-expressing cells (Figure 6b) than in the precipitate from the control cells. The ITGA3 intron-1 segment having the p63 target sites (Kurata et al., 2004) also efficiently co-precipitated with HA-TAp63γ, whereas the TATA box region of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) failed to do so. Control of nonimmune serum did not efficiently precipitate the HS2/3 segment (data not shown).

MFGE8 transcription controlled by p63 in carcinoma cells

FaDu cells were transfected with small interfering RNA (p63si) targeting the DNA-binding domain-encoding sequences of p63. On day 2 and 4 post-transfection, western blotting indicated suppression of the p63 protein level to 20±10% (deviation depending on the experiments) in comparison with the control siRNA (Csi)-transfected cells (Figure 7a). The p63si-transfected cells showed 40% (±10%) decrease in MFGE8 mRNA and the protein on day 4. In the immunofluorescent staining, a 70% (±10%) cell population displayed significant decrease in p63 on day 4. In the parallel culture, the cytoplasmic and membrane label with the anti-MFG-E8 antibody decreased in 50% (±10%) cells.

Figure 7
figure7

p63 silencing in FaDu cells by siRNA. (a) Analyses of the siRNA-transfected cells for p63 and milk fat globule-EGF factor 8 protein (MFG-E8). For immunofluorescent staining, p63si- and Csi-transfected cells were recultured in chambers on the same slide. p63 (Alexa Fluor 488, green, left) and MFG-E8 (Alexa Fluor 594, red, right) were detected with mouse monoclonal antibodies, while control stain was with a rabbit anti-cytochrome c antibody (Alexa Fluor 594, left). Reverse transcription (RT)–PCR and western blot analyses of the whole cell lysates are shown. (b) Saos-2 cell attachment to the plates coated with supernatants of the FaDu cell cultures transfected with Csi and p63si. Assays after blocking with an anti-MFG-E8 antibody (+) and control mouse IgG (−) are shown. Binding efficiency (%, ±s.d.) is indicated in relation to the control experiment (100%) with Csi-transfected cells. (c) Detection of secreted MFG-E8 by western blotting. Untransfected cell lysates (100 or 1 μg) and supernatants (10 μg; Csi, p63si) were analysed in the slots. Secreted forms (42–44 kDa) were indicated by bars.

Using Saos-2 osteosarcoma cells presenting integrins αVβ3 and αVβ5 (Koistinen et al., 1999), receptors of MFG-E8, we tested FaDu culture supernatants for cell-binding activity as described by Taylor et al. (1997). Plastic multi-well plates were coated with concentrated supernatants from serum-free cultures of FaDu transfected with Csi and p63si. In the wells with the Csi-transfected supernatant (12 μg/ml), approximately 40% of the total Saos-2 cell binding was blocked by a monoclonal antibody reactive with native form MFG-E8, corresponding to MFG-E8-dependent Saos-2 cell adhesion (Figure 7b). Total adherent cell count was 35% decreased by p63si-transfection, in which the antibody blocking caused only an 8.3% reduction. Consistent with earlier results (Taylor et al., 1997), western blot analysis of the supernatants detected two broad bands at apparent molecular masses of 42–44 kDa (Figure 7c), indicating secretion of MFG-E8 in poorly glycosylated forms (Oshima et al., 2002; Watanabe et al., 2005). The bands’ intensity was decreased to approximately 40% by the p63 suppression. Thus, we confirmed that p63 influences MFG-E8-mediated cell adhesion in vitro.

Discussion

We determined that MFGE8 is a target gene of p63 in epithelial cells. Only the TA isoforms were able to directly enhance the promoter. However, if one ΔN-isoform, ΔNp63α in particular, was overexpressed together with a significantly lower level of the TA isoforms, the TA proteins were stabilized to induce the MFGE8 transcription. The dominant expression of ΔN-p63 in squamous cell carcinomas may contribute to the MFG-E8 abrogation by this mechanism.

HeLa cells constitutively transcribe TA-p63 as efficiently as immunologically p63-positive cells including FaDu, HSC-1 and NHK, but fail to maintain the TA-p63 proteins at a detectable level. The ΔN type transcription seems roughly 20-fold less efficient in HeLa than in other cells. As we reported earlier, TA-p63 undergoes rapid degradation by 26S proteasome due to the TA peptides (Osada et al., 2001; Okada et al., 2002). Furthermore, TA-p63 and ΔN-p63 can form complexes using the oligomerization domain (Yang et al., 1998; Kojima et al., 2001). Certainly, ΔN-α and ΔN-γ were associated with HA-TAp63γ (Supplementary Information 2), implying formation of mixed dimers and tetramers in p63-expressing keratinocytes. This association may serve as a flexible switch between the activation and suppression status depending on the TA:ΔN ratio (Figure 5). A recent study identified Itch, an E3 ubiquitin ligase, expressed in well-differentiated cell layers of human skin, as a factor controlling the p63 stability (Rossi et al., 2006), indicating a major p63 degradation/inactivation mechanism during the keratinocyte differentiation. In contrast, this study dealt with the functional p63 proteins localized to undifferentiated keratinocytes in the basal layer of stratified epithelia and in carcinomas derived from the stage.

MFG-E8-L, the mouse-specific long isoform, having a Pro/Thr-enriched domain, facilitates phagocytosis of apoptotic cells by macrophages as demonstrated by recent studies with MFGE8-deficient mice (Hanayama et al., 2002; Hanayama and Nagata, 2005). However, the other isoform, MFG-E8-S, lacking the phagocytosis-facilitating activity is more ubiquitously, abundantly expressed (Watanabe et al., 2005) and expected to facilitate various types of cell–cell interactions. Importantly, human MFGE8 codes for a protein similar to mouse MFGE8-S, but not an MFG-E8-L-type protein.

For human MFG-E8, an angiogenesis-promoting activity (Silvestre et al., 2005) and an anticoagulant activity (Shi and Gilbert, 2003) have been reported. Supporting the original characterization (Taylor et al., 1997), we have shown that MFG-E8 released from FaDu influences Saos-2 cell adhesion. Either or both of the αVβ3 and αVβ5 integrins are expressed in particular lineages including vascular cells, osteoblastomas, retinal pigmented epithelial cells and fibroblasts, by which attachment, migration and invasion are regulated (Gailit et al., 1997; Koistinen et al., 1999; Hoffmann et al., 2005). MFG-E8 might affect these events during carcinoma tissue growth and/or wound healing, since no substantial defect appeared in the corresponding tissues of MFGE8-deficient mice. As reported recently, p63 induces membrane-anchored molecules including the integrin α3 and β4 subunits, envoplakin, Perp and bullous pemphigoid antigen-1/2 (Kurata et al., 2004; Ihrie et al., 2005; Osada et al., 2005a, 2005b; Carroll et al., 2006) for cell–cell and cell–matrix binding. Soluble protein MFG-E8 may provide a novel mechanism of epithelial–nonepithelial cell interactions around the p63-expressing cells.

p53 seemed to act on MFGE8 indirectly in the reporter assay. In fact, the p53 pathway is frequently inactivated in carcinoma cells including FaDu by p53 mutations or by viral oncogene products. In normal epithelial cells, p53 is silenced by the intrinsic degradation mechanism with MDM2, unless DNA damage or other stress signals are induced. Furthermore, association of p53 with p63 is unlikely (Kojima et al., 2001). Contribution of p53 to the high-level expression of MFGE8 is less probable.

Materials and methods

Cell culture

The Health Science Research Resources Bank (Osaka, Japan) provided us with HeLa, HEK293 and Saos-2 cells. We obtained FaDu from American Type Culture Collection. Primary human neonatal keratinocyte culture was prepared as described by Kurata et al. (2004).

RT–PCR

Total RNA was isolated with RNAwiz (Ambion, Austin, TX, USA) and subjected to semiquantitative RT–PCR with the Platinum Taq One-step RT–PCR kit (Invitrogen, Carlsbad, CA, USA). Primer sequences are shown in Supplementary Information 3.

Western blotting

We used anti-MFG-E8 (HMFG1, clone EDM45, Lab Vision, Fremont, CA, USA at a dilution of 1:1000), anti-p63 (4A4, Santa Cruz Biotechnology Inc., Fremont, CA, USA 1:1000), anti-β-actin (A5441, Sigma-Aldrich, St Louis, MO, USA, 1:5000), anti-integrin α3 (Biogenesis, Poole, UK, 1:1000) antibodies. EDM45 IgG is specifically, strongly reactive with the major form of 46 kDa MFG-E8 identified by an anti-MFG-E8 monoclonal antibody (MAB2767, R&D Systems, Minneapolis, MN, USA). Alexa Fluor 680 (Molecular Probes, Invitrogen) and IRDye 800CW were used for quantitation using the Odyssey Infrared Imaging System (LI-COL/Aloka).

Cloning of the promoter/enhancer region of MFGE8

Genomic DNA was from peripheral blood cells of a healthy donor (YT) with informed consents. The 2K (−1890 to +50), 1K (−877 to +50) and S (−250 to +50) regions were amplified by PCR with Ex Taq (Takara Bio, Kyoto, Japan), cloned into pGEM-T Easy (Promega, Madison, WI, USA), and sequenced.

Transfection and luciferase assay

Recipient cells were plated 24 h before transfection at a density of 0.4 × 105 cells in each well on 24-well plates. Typically, a pRcCMV-based, p63 isoform expression vector (0.1 μg for each well) was co-transfected with a luc-expression vector (0.1 μg) with Effectene (Qiagen, Hilden, Germany). At 40 (HeLa) or 48 h (HEK293) posttransfection, we measured luciferase activity using the Steady-Glo Luciferase Assay System (Promega). We used the GeneTailor Site-Directed Mutagenesis System (Invitrogen) to render the ‘G to T’ mutations and the SiLentFect Lipid Reagent (Bio-Rad, Hercules, CA, USA) for siRNA transfection.

ChIP

Tetracycline-induced, HA-tagged TAp63γ-expressing HEK293 cells and uninduced cells were crosslinked with formaldehyde and subjected to enzymatic chromatin DNA fragmentation (ChIP-I Enzymatic Shearing Kit, Active Motif, Carlsbad, CA, USA). HA-p63 and associated materials were immunoprecipitated with an anti-HA monoclonal antibody (Santa Cruz, 7392 X).

Saos-2 binding assay

FaDu cells transfected with Csi and p63si were expanded on day 2. Cells at 80% confluency on day 4 were washed and fed with serum-free KGM-2 medium supplemented with transferrin and insulin (Lonza). The supernatants, at 24 h, were clarified and concentrated with centrifugal filter units (Vivaspin 500, Sartorius, Goettingen, Germany). Binding assay was performed in wells on 24-well plates as described by Taylor et al. (1997). An anti-MFG-E8 monoclonal antibody (2 μg/ml, Chemicon, Temecula, CA, USA, CBL421) was used for blocking.

Accession codes

Accessions

GenBank/EMBL/DDBJ

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Acknowledgements

This work was supported by Grant-in Aids from MEXT Japan, for Scientific Research on Priority Areas (YI) and for High-Tech Research Center Project (RH) and by Strategic Research Fund from University of Yamanashi (IK). We thank N Shirato, Photography Department, Tokyo Medical and Dental University, for data file making.

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Correspondence to I Katoh.

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Supplementary Information accompanies the paper on the Oncogene web site (http://www.nature.com/onc).

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Okuyama, T., Kurata, S., Tomimori, Y. et al. p63(TP63) elicits strong trans-activation of the MFG-E8/lactadherin/BA46 gene through interactions between the TA and ΔN isoforms. Oncogene 27, 308–317 (2008). https://doi.org/10.1038/sj.onc.1210646

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Keywords

  • p63
  • MFG-E8
  • lactadherin
  • BA46
  • P53

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