C-terminal cleavage of DeltaNp63alpha is associated with TSA-induced apoptosis in immortalized corneal epithelial cells.
OBJECTIVE
In the central human corneal epithelium, loss of DeltaNp63 occurs in all surface epithelial cells preparing to undergo desquamation, suggesting a potential role for DeltaNp63 isoforms in mediating surface cell apoptotic shedding. In this study, the authors investigated a role for DeltaNp63 isoforms in caspase-mediated apoptosis in a telomerase-immortalized corneal epithelial cell line.


METHODS
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 DeltaNp63 isoforms, DeltaNp63-EGFP expression plasmids were transiently expressed in hTCEpi cells and evaluated by FRAP. Trichostatin-A (TSA; 3.31 muM) was used to induce cell death as measured by caspase activity. Cleavage and loss of endogenous DeltaNp63alpha, DeltaNp63-EGFP expression plasmids, and p53 were assessed after treatment with TSA and siRNA.


RESULTS
Transient expression of DeltaNp63-EGFP alpha and beta isoforms resulted in the formation of a smaller isoform similar in size to DeltaNp63gamma-EGFP. FRAP demonstrated that DeltaNp63alpha-EGFP has greater immobile fraction than beta or gamma. TSA induced caspase-mediated apoptotic pathways; caspase induction was accompanied by a decrease in endogenous DeltaNp63alpha and p53. TSA upregulated DeltaNp63-EGFP plasmid expression; this was accompanied by a selective increase in cleavage of DeltaNp63alpha-EGFP. siRNA knockdown of DeltaNp63alpha correlated with a reduction in p53 independently of TSA.


CONCLUSIONS
DeltaNp63alpha is the dominant active isoform in corneal epithelial cell nuclei. Loss of DeltaNp63alpha occurs during apoptotic signaling by cleavage at the C terminus. The corresponding loss of p53 suggests that a significant relationship appears to exist between these two regulatory proteins.
Robertson
Danielle M
DM
Department of Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390-9057, USA. danielle.robertson@utsouthwestern.edu
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Su-Inn
SI
Cavanagh
H Dwight
HD
D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D018929:Cell Culture Techniques; D019573:Epithelium, Corneal / Q000378:metabolism / Q000473:pathology*; D036681:Fluorescence Recovery After Photobleaching; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D010957:Plasmids; D020033:Protein Isoforms; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators / Q000502:physiology*; D014157:Transcription Factors; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000502:physiology*; D015854:Up-Regulation
D006877:Hydroxamic Acids; D020033:Protein Isoforms; D011500:Protein Synthesis Inhibitors; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C115051:TP63 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C012589:trichostatin A; D053148:Caspase 3