Regulation of limbal keratinocyte proliferation and differentiation by TAp63 and DeltaNp63 transcription factors.
OBJECTIVE
To examine the effects of TAp63 and DeltaNp63 on the proliferation and differentiation of rabbit limbal keratinocytes cultured on human amniotic membrane.


METHODS
Real-time Q-RT-PCR was used to quantify the relative abundance of TAp63 and DeltaNp63 transcripts in limbal, peripheral corneal, and central corneal epithelia. Antisense oligonucleotides were designed specifically to suppress the expression of TAp63 or DeltaNp63 in limbal keratinocytes, and their effects on cell proliferation and differentiation were examined. Immunofluorescence was used to examine the expressions of p63 and keratin-3 and -14.


RESULTS
The expressions of TAp63 and DeltaNp63 transcripts appeared to be site specific. TAp63 was expressed at the highest level in limbus, decreased by approximately 10-fold in peripheral cornea and was undetectable in the central cornea. DeltaNp63 was also expressed at the highest level in limbus, decreased by approximately 35% in peripheral cornea, and was undetectable in the central cornea. Suppression of TAp63 expression inhibited limbal keratinocyte proliferation but promoted differentiation. Suppression of DeltaNp63 expression also inhibited cell proliferation but had no obvious effect on cell differentiation.


CONCLUSIONS
TAp63 and DeltaNp63 affect the proliferation of limbal keratinocytes by a different mechanism. The inhibition by TAp63 antisense oligos appeared to be secondary to the promotion of cell differentiation. In contrast, the inhibition by DeltaNp63 antisense oligos appeared to be independent of cell differentiation.
Wang
Der-Yuan
DY
Department of Physiology, College of Medicine, Chang Gung University Kweishan, No. 259 Wenhua First Road, Kweishan, Taoyuan 333, Taiwan.
Cheng
Chien-Chia
CC
Kao
Ming-Hui
MH
Hsueh
Yi-Jen
YJ
Ma
David H K
DH
Chen
Jan-Kan
JK
D000650:Amnion / Q000166:cytology; D000818:Animals; D001483:Base Sequence; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology*; D002465:Cell Movement; D049109:Cell Proliferation; D004268:DNA-Binding Proteins; D019573:Epithelium, Corneal / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D016147:Genes, Tumor Suppressor / Q000502:physiology*; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D015534:Trans-Activators / Q000037:antagonists & inhibitors / Q000502:physiology*; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins
D004268:DNA-Binding Proteins; D016376:Oligonucleotides, Antisense; D010750:Phosphoproteins; D012333:RNA, Messenger; C115051:TP63 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins