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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.
AuthorsDavid Hui-Kang Ma, Jui-Yang Lai, Szu-Tzu Yu, Jia-Ye Liu, Unique Yang, Hung-Chi Jesse Chen, Lung-Kung Yeh, Yi-Ju Ho, Grace Chang, Su-Fang Wang, Jan-Kan Chen, Ken-Kuo Lin
JournalJournal of cellular physiology (J Cell Physiol) Vol. 227 Issue 5 Pg. 2030-9 (May 2012) ISSN: 1097-4652 [Electronic] United States
PMID21751214 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
CopyrightCopyright © 2011 Wiley Periodicals, Inc.
Chemical References
  • HSP70 Heat-Shock Proteins
  • TP63 protein, human
  • Transcription Factors
  • Tumor Suppressor Proteins
  • heat-shock protein 70.1
  • Hydrogen Peroxide
Topics
  • Amnion (cytology, metabolism)
  • Cell Culture Techniques
  • Cells, Cultured
  • Electrophoresis, Gel, Two-Dimensional
  • Epithelial Cells (cytology, drug effects, physiology, radiation effects)
  • Gene Silencing
  • HSP70 Heat-Shock Proteins (genetics, metabolism)
  • Humans
  • Hydrogen Peroxide (pharmacology)
  • Limbus Corneae (cytology)
  • Proteomics (methods)
  • Stem Cells (cytology, physiology)
  • Transcription Factors (genetics, metabolism)
  • Tumor Suppressor Proteins (genetics, metabolism)
  • Ultraviolet Rays
  • Up-Regulation

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