C-Terminal Cleavage of (cid:1) Np63 (cid:2) Is Associated with TSA-Induced Apoptosis in Immortalized Corneal Epithelial Cells

P URPOSE . In the central human corneal epithelium, loss of (cid:1) Np63 occurs in all surface epithelial cells preparing to undergo desquamation, suggesting a potential role for (cid:1) Np63 isoforms in mediating surface cell apoptotic shedding. In this study, the authors investigated a role for (cid:1) Np63 isoforms in caspase-mediated apoptosis in a telomerase-immortalized cor- neal epithelial cell line. M ETHODS . For in vitro studies, hTCEpi cells were cultured in KGM-2 serum-free culture media containing 0.15 mM calcium. To assess dynamic protein interactions among individual (cid:1) Np63 isoforms, (cid:1) Np63-EGFP expression plasmids were transiently expressed in hTCEpi cells and evaluated by FRAP. Tri- chostatin-A (TSA; 3.31 (cid:1) M) was used to induce cell death as measured by caspase activity. Cleavage and loss of endogenous (cid:1) Np63 (cid:2) , (cid:1) Np63-EGFP expression plasmids, and p53 were assessed after treatment with TSA and siRNA. R ESULTS . Transient expression of (cid:1) Np63-EGFP (cid:2) and (cid:3) isoforms resulted in the formation of a smaller isoform similar in size to (cid:1) Np63 (cid:4) -EGFP. FRAP demonstrated that (cid:1) Np63 (cid:2) -EGFP has greater immobile fraction than (cid:3) or (cid:4) . TSA induced caspase-mediated apoptotic pathways; caspase induction was accom- panied by a decrease in endogenous (cid:1) Np63 (cid:2) antibiotic-free media for a total of 1 mL; siRNA oligonucleotides were transfected (Oligofectamine; Invitrogen). Cells were collected 24 after transfection. Efﬁcacy of knockdown was evaluated by Western blot analysis. To control for off-targeting effects, HTK cells that did not express (cid:1) Np63 transfected under identical conditions to the hTCEpi cell line and assessed for p53 expression. Live Cell Caspase Assay; Invitrogen) was used to assess caspase-3 activation in hTCEpi cells. Thirty microliters of a 2.4 (cid:2) 10 5 cell/mL suspension of hTCEpi cells was seeded onto glass coverslips and allowed to adhere for 24 hours. Cells were treated with 3.31 (cid:1) M TSA (Sigma) overnight in serum-free KGM2 media. Cells were labeled for caspase-3 according to manufacturer’s instructions. Nuclei were counterstained with a 1:1000 dilution of cell-permeable anthra-quinone (DRAQ5; Axxora, San Diego, CA) and imaged directly under the laser scanning confocal microscope. study. Of more signiﬁcance is the apparent increase in cleavage of ectopically expressed (cid:1) Np63 (cid:2) and (cid:1) Np63 (cid:3) after incubation in TSA. Although cleavage of the (cid:2) variant of (cid:1) N and TA isoforms has been previously reported, in those studies cleav- age was mediated through a caspase cleavage site within the terminal inhibitory domain. 22 Importantly, this domain, though present within the C terminus of (cid:1) Np63 (cid:2) , was not present within the C terminus of (cid:1) Np63 (cid:3) , suggesting the possibility of a second proteolytic cleavage site encoded by both isoforms. Collectively, these results underscore two important ﬁndings in this work. First, cleavage was limited to (cid:1) Np63 (cid:2) and (cid:1) Np63 (cid:3) , producing a smaller isoform equivalent to the un-cleaved (cid:1) Np63 (cid:4) indicating that a proteolytic cleavage site resided within the C terminus of the (cid:2) and (cid:3) isoforms that was not present in the (cid:4) isoform. Second, speciﬁc-TSA–associated cleavage that occurred during caspase-mediated apoptosis ap- peared restricted to (cid:1) Np63 (cid:2) , and the apparent increase in cleavage of (cid:1) Np63 (cid:3) was an indirect result of the increased levels of plasmid expression.