C-terminal cleavage of δNp63α Is associated with TSA-induced apoptosis in immortalized corneal epithelial cells

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Abstract

PURPOSE. In the central human corneal epithelium, loss of ΔNp63 occurs in all surface epithelial cells preparing to undergo desquamation, suggesting a potential role for ΔNp63 isoforms in mediating surface cell apoptotic shedding. In this study, the authors investigated a role for ΔNp63 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 ΔNp63 isoforms, ΔNp63-EGFP expression plasmids were transiently expressed in hTCEpi cells and evaluated by FRAP. Trichostatin-A (TSA; 3.31 μM) was used to induce cell death as measured by caspase activity. Cleavage and loss of endogenous ΔNp63 α ΔNp63-EGFP expression plasmids, and p53 were assessed after treatment with TSA and siRNA. RESULTS. Transient expression of ΔNp63-EGFP α and β isoforms resulted in the formation of a smaller isoform similar in size to ΔNp63 α -EGFP. FRAP demonstrated that ΔNp63 α -EGFP has greater immobile fraction than α or β. TSA induced caspasemediated apoptotic pathways; caspase induction was accompanied by a decrease in endogenous ΔNp63 α and p53. TSA upregulated ΔNp63-EGFP plasmid expression; this was accompanied by a selective increase in cleavage of ΔNp63 α -EGFP. siRNA knockdown of ΔNp63 α correlated with a reduction in p53 independently of TSA. CONCLUSIONS. ΔNp63 α is the dominant active isoform in corneal epithelial cell nuclei. Loss of ΔNp63 α 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.

Original languageEnglish (US)
Pages (from-to)3977-3985
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume51
Issue number8
DOIs
StatePublished - Aug 2010

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Protein Isoforms
Epithelial Cells
Apoptosis
Caspases
Plasmids
Small Interfering RNA
trichostatin A
Corneal Epithelium
Telomerase
Serum-Free Culture Media
Cell Nucleus
Cultured Cells
Proteins
Cell Death
Calcium
Cell Line

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience
  • Medicine(all)

Cite this

@article{5f79036d92ca48f8b7970d4c74fbcc03,
title = "C-terminal cleavage of δNp63α Is associated with TSA-induced apoptosis in immortalized corneal epithelial cells",
abstract = "PURPOSE. In the central human corneal epithelium, loss of ΔNp63 occurs in all surface epithelial cells preparing to undergo desquamation, suggesting a potential role for ΔNp63 isoforms in mediating surface cell apoptotic shedding. In this study, the authors investigated a role for ΔNp63 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 ΔNp63 isoforms, ΔNp63-EGFP expression plasmids were transiently expressed in hTCEpi cells and evaluated by FRAP. Trichostatin-A (TSA; 3.31 μM) was used to induce cell death as measured by caspase activity. Cleavage and loss of endogenous ΔNp63 α ΔNp63-EGFP expression plasmids, and p53 were assessed after treatment with TSA and siRNA. RESULTS. Transient expression of ΔNp63-EGFP α and β isoforms resulted in the formation of a smaller isoform similar in size to ΔNp63 α -EGFP. FRAP demonstrated that ΔNp63 α -EGFP has greater immobile fraction than α or β. TSA induced caspasemediated apoptotic pathways; caspase induction was accompanied by a decrease in endogenous ΔNp63 α and p53. TSA upregulated ΔNp63-EGFP plasmid expression; this was accompanied by a selective increase in cleavage of ΔNp63 α -EGFP. siRNA knockdown of ΔNp63 α correlated with a reduction in p53 independently of TSA. CONCLUSIONS. ΔNp63 α is the dominant active isoform in corneal epithelial cell nuclei. Loss of ΔNp63 α 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.",
author = "Robertson, {Danielle M} and Ho, {Su Inn} and Cavanagh, {Harrison D}",
year = "2010",
month = "8",
doi = "10.1167/iovs.09-4919",
language = "English (US)",
volume = "51",
pages = "3977--3985",
journal = "Investigative Ophthalmology and Visual Science",
issn = "0146-0404",
publisher = "Association for Research in Vision and Ophthalmology Inc.",
number = "8",

}

TY - JOUR

T1 - C-terminal cleavage of δNp63α Is associated with TSA-induced apoptosis in immortalized corneal epithelial cells

AU - Robertson, Danielle M

AU - Ho, Su Inn

AU - Cavanagh, Harrison D

PY - 2010/8

Y1 - 2010/8

N2 - PURPOSE. In the central human corneal epithelium, loss of ΔNp63 occurs in all surface epithelial cells preparing to undergo desquamation, suggesting a potential role for ΔNp63 isoforms in mediating surface cell apoptotic shedding. In this study, the authors investigated a role for ΔNp63 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 ΔNp63 isoforms, ΔNp63-EGFP expression plasmids were transiently expressed in hTCEpi cells and evaluated by FRAP. Trichostatin-A (TSA; 3.31 μM) was used to induce cell death as measured by caspase activity. Cleavage and loss of endogenous ΔNp63 α ΔNp63-EGFP expression plasmids, and p53 were assessed after treatment with TSA and siRNA. RESULTS. Transient expression of ΔNp63-EGFP α and β isoforms resulted in the formation of a smaller isoform similar in size to ΔNp63 α -EGFP. FRAP demonstrated that ΔNp63 α -EGFP has greater immobile fraction than α or β. TSA induced caspasemediated apoptotic pathways; caspase induction was accompanied by a decrease in endogenous ΔNp63 α and p53. TSA upregulated ΔNp63-EGFP plasmid expression; this was accompanied by a selective increase in cleavage of ΔNp63 α -EGFP. siRNA knockdown of ΔNp63 α correlated with a reduction in p53 independently of TSA. CONCLUSIONS. ΔNp63 α is the dominant active isoform in corneal epithelial cell nuclei. Loss of ΔNp63 α 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.

AB - PURPOSE. In the central human corneal epithelium, loss of ΔNp63 occurs in all surface epithelial cells preparing to undergo desquamation, suggesting a potential role for ΔNp63 isoforms in mediating surface cell apoptotic shedding. In this study, the authors investigated a role for ΔNp63 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 ΔNp63 isoforms, ΔNp63-EGFP expression plasmids were transiently expressed in hTCEpi cells and evaluated by FRAP. Trichostatin-A (TSA; 3.31 μM) was used to induce cell death as measured by caspase activity. Cleavage and loss of endogenous ΔNp63 α ΔNp63-EGFP expression plasmids, and p53 were assessed after treatment with TSA and siRNA. RESULTS. Transient expression of ΔNp63-EGFP α and β isoforms resulted in the formation of a smaller isoform similar in size to ΔNp63 α -EGFP. FRAP demonstrated that ΔNp63 α -EGFP has greater immobile fraction than α or β. TSA induced caspasemediated apoptotic pathways; caspase induction was accompanied by a decrease in endogenous ΔNp63 α and p53. TSA upregulated ΔNp63-EGFP plasmid expression; this was accompanied by a selective increase in cleavage of ΔNp63 α -EGFP. siRNA knockdown of ΔNp63 α correlated with a reduction in p53 independently of TSA. CONCLUSIONS. ΔNp63 α is the dominant active isoform in corneal epithelial cell nuclei. Loss of ΔNp63 α 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.

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U2 - 10.1167/iovs.09-4919

DO - 10.1167/iovs.09-4919

M3 - Article

VL - 51

SP - 3977

EP - 3985

JO - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

SN - 0146-0404

IS - 8

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