title: Up-regulation of heat shock protein 70-1 (Hsp70-1) in human limbo-corneal epithelial cells cultivated on amniotic membrane: A proteomic study.
abstract: Transplantation of cultivated human limbo-corneal epithelial (HLE) cells has been recognized as an effective stem cell (SC) therapy for treating corneal epithelial SC deficiency caused by burn or other diseases. With this technique, cryo-preserved human intact amniotic membrane (IAM) has been successfully used as a cell culture substrate and carrier, and is reported to preferentially preserve HLE stem/progenitor cells in vitro. However, little is known about what factors released by HLE cells are involved in the progenitor cell-preserving mechanism. Using proteomic method, we identified 13 proteins over-expressed by HLE cells cultured on IAM, which included heat shock protein 70-1 (Hsp70-1), Hsp-27, glutathione (GSH) S-transferase, annexin A2, galectin-7, and protein S100-A9. Increased Hsp70-1 expression was confirmed by Western blot and real-time PCR. The role of Hsp70-1 in promoting HLE cell survival was demonstrated by increased apoptosis index and increased cleaved poly ADP-ribose polymerase (CPARP) formation in hsp70-1-silenced, but not normal HLE cells after exposure to sublethal UVB irradiation or hydrogen peroxide. To understand the regulatory mechanism of Hsp70-1 expression in HLE cells, the role of transcription factor deltaNp63 (a well-recognized HLE stem cell; SC marker) was studied. We found that over-expression of deltaNp63α by plasmid vector induced a corresponding increase in Hsp70-1 protein production. Likewise, Hsp70-1 expression decreased in HLE cells after addition of deltaNp63α SiRNA. Immunoconfocal microscopy also revealed a paralleled expression of both proteins in corneal specimens. Thus, deltaNp63α-associated Hsp70-1 over-expression may promote HLE progenitor cell survival on IAM, possibly through the cytoprotective and anti-apoptotic effect of Hsp70-1.
authors.affiliation: Limbal Stem Cell Laboratory, Department of Ophthalmology, Chang Gung Memorial Hospital, Taoyuan, Taiwan. davidhkma@yahoo.com
authors.forename: David Hui-Kang
authors.initials: DH
authors.lastname: Ma
authors.forename: Jui-Yang
authors.initials: JY
authors.lastname: Lai
authors.forename: Szu-Tzu
authors.initials: ST
authors.lastname: Yu
authors.forename: Jia-Ye
authors.initials: JY
authors.lastname: Liu
authors.forename: Unique
authors.initials: U
authors.lastname: Yang
authors.forename: Hung-Chi Jesse
authors.initials: HC
authors.lastname: Chen
authors.forename: Lung-Kung
authors.initials: LK
authors.lastname: Yeh
authors.forename: Yi-Ju
authors.initials: YJ
authors.lastname: Ho
authors.forename: Grace
authors.initials: G
authors.lastname: Chang
authors.forename: Su-Fang
authors.initials: SF
authors.lastname: Wang
authors.forename: Jan-Kan
authors.initials: JK
authors.lastname: Chen
authors.forename: Ken-Kuo
authors.initials: KK
authors.lastname: Lin
mesh_terms: D000650:Amnion / Q000166:cytology* / Q000378:metabolism; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D020868:Gene Silencing; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D016850:Limbus Corneae / Q000166:cytology*; D040901:Proteomics / Q000379:methods*; D013234:Stem Cells / Q000166:cytology / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays; D015854:Up-Regulation
chemical_list: D018840:HSP70 Heat-Shock Proteins; C115051:TP63 protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C081884:heat-shock protein 70.1; D006861:Hydrogen Peroxide