Transcription factor TCF4 maintains the properties of human corneal epithelial stem cells.
TCF4, a key transcription factor of Wnt signaling system, has been recently found to be essential for maintaining stem cells. However, its signaling pathway is not well elucidated. This study was to explore the functional roles and signaling pathway of TCF4 in maintaining adult stem cell properties using human corneal epithelial stem cells as a model. With immunofluorescent staining and real-time polymerase chain reaction, we observed that TCF4 was exclusively expressed in the basal layer of human limbal epithelium where corneal epithelial stem cells reside. TCF4 was found to be well colocalized with ABCG2 and p63, two recognized epithelial stem/progenitor cell markers. Using in vitro culture models of primary human corneal epithelial cells, we revealed that TCF4 mRNA and protein were upregulated by cells in exponential growth stage, and RNA interference by small interfering RNA-TCF4 (10-50 nM) transfection blocked TCF4 signaling and suppressed cell proliferation as measured by WST-1 assay. TCF4 silence was found to be accompanied by downregulated proliferation-associated factors p63 and survivin, as well as upregulated cyclin-dependent kinase inhibitor 1C (p57). By creating a wound healing model in vitro, we identified upregulation and activation of β-catenin/TCF4 with their protein translocation from cytoplasm to nuclei, as evaluated by reverse transcription-quantitative real-time polymerase chain reaction, immunostaining, and Western blotting. Upregulated p63/survivin and downregulated p57 were further identified to be TCF4 downstream molecules that promote cell migration and proliferation in wound healing process. These findings demonstrate that transcription factor TCF4 plays an important role in determining or maintaining the phenotype and functional properties of human corneal epithelial stem cells.
Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Sun Yat-Sen University, Guangzhou, People's Republic of China.
Rong
R
Lu
Yangluowa
Y
Qu
Jian
J
Ge
Lili
L
Zhang
Zhitao
Z
Su
Stephen C
SC
Pflugfelder
De-Quan
DQ
Li
D000328:Adult; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D050761:Cyclin-Dependent Kinase Inhibitor p57 / Q000378:metabolism; D015536:Down-Regulation / Q000235:genetics; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism*; D019573:Epithelium, Corneal / Q000166:cytology*; D020868:Gene Silencing; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology / Q000378:metabolism; D008875:Middle Aged; D008954:Models, Biological; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism*; D000077022:Survivin; D000073940:Transcription Factor 4; D014157:Transcription Factors / Q000378:metabolism*; D015854:Up-Regulation / Q000235:genetics; D014945:Wound Healing; D055815:Young Adult; D051176:beta Catenin / Q000378:metabolism
C107489:BIRC5 protein, human; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D015415:Biomarkers; C493964:CDKN1C protein, human; D050761:Cyclin-Dependent Kinase Inhibitor p57; D051034:Inhibitor of Apoptosis Proteins; D034741:RNA, Small Interfering; D000077022:Survivin; C532096:TCF4 protein, human; D000073940:Transcription Factor 4; D014157:Transcription Factors; D051176:beta Catenin