p51/p63 induces secretory adhesion protein MFG-E8 in epithelial cells

4537 p51/p63, a member of the tumor suppressor p53 family, is essential for skin, limb and craniofacial development, but less important for tumor suppressing function. A high level p51/p63 expression occurs in epidermal stem cells and several types of cancers including squamous cell carcinomas of head and neck, causing transactivation of its target genes: JAG1, REDD1 and ITGA3 as reported to date. The Milk Fat Globule-EGF factor 8 protein (MFG-E8) is a secretory adhesion molecule identified as breast antigen 46 (BA46) or Lactadherin in humans. Mouse MFG-E8 has been found to play critical roles in engulfment of apoptotic cells by macrophages and immature dendritic cells, and in sperm-egg binding for fertilization. MFG-E8 can serve as a linker between cell surface receptor integrin αvβ3/αvβ5 and phospholipid. We detected MFG-E8 expression in the basal and suprabasal cells in human skin tissues as well as in neonatal human keratinocyte cultures, indicating that MFG-E8 expression coincides with p51/p63 expression. Furthermore, both p51/p63 and MFG-E8 are known to be frequently overexpressed in breast cancers. Because p53 (p51/p63) binding consensus sequences existed in the enhancer/promoter region of the human MFGE8 gene, we examined whether p51/p63 has an activity to induce the expression of MFGE8. In the tetracycline-regulated p51/p63 expression system in HEK293 cells, the TA isoforms of p51/p63 enhanced endogenous MFGE8 expression as expected. After cloning an approximatly 2kb region upstream of the MFGE8 coding sequences, we performed a luciferase (luc) reporter assay in HeLa, HEK293 and Saos-2 cells. Activation of the luc gene expression by p53, p51A/TAp63γ and p51B/TAp63α was evident. However, ΔNp51A/ΔNp63γ known as a dominant-negative type isofoms was not found suppressive to the luc expression, and caused a level of the promoter activation in some cell lines. By deletion and mutation analyses, we identified the motifs at -370 and -27 as the target sequence for p51A/TAp63γ and ΔNp51A/ΔNp63γ, respectively. A chromatin immunoprecipitation (ChIP) assay with hemagglutinin (HA)-tagged p51A/TAp63γ supported the results. However, neither of the possible p53 binding sites was not responsive to p53, implying that p53 indirectly affects the MFGE8 enhancer/promoter region. These results suggest that p51/p63 participates in regulation of cell adhesion in the processes of development and tumorgenesis by inducing MFGE8 in epithelial basal cells. (This study was supported by grants-in-aid for Scientific Research on Priority Areas from MEXT Japan.)