title: Effect of nerve growth factor on the in vitro induction of apoptosis of human conjunctival epithelial cells by hyperosmolar stress.
abstract: OBJECTIVE
  To evaluate the effects of nerve growth factor (NGF), which is activated during inflammatory episodes of ocular diseases, on the apoptotic response in cultured human primary conjunctival epithelial cells (pHCECs).
  
  
  METHODS
  Levels of NGF transcripts and NGF protein in pHCEC grown in medium with normal osmolarity (307 mOsm/L) or hyperosmolar medium (350, 400, and 450 mOsm/L) were determined using RT-PCR or ELISA, respectively. To assess apoptosis, pHCEC were cultured in normal or 400 mOsm/L hyperosmolar medium with neutralizing anti-NGF antibody or recombinant human NGF for 6 hours before analysis by flow cytometry. Levels of Bcl-xL, Bax, phosphorylated JNK, and cleaved caspase-3 were detected using Western blotting. Levels of the inflammatory cytokine IL-6 was analyzed using ELISA.
  
  
  RESULTS
  Hyperosmolar conditions increased NGF levels in cultured pHCECs. Hyperosmolarity and exposure to neutralizing anti-NGF antibody significantly increased the number of apoptotic cells. Addition of recombinant human NGF to 400 mOsm/L medium decreased the number of apoptotic cells, reduced the expression of phosphorylated JNK, Bax, and cleaved caspase-3 and increased the expression of Bcl-xL. Levels of IL-6 were increased by hyperosmotic stress but decreased by exposure to recombinant human NGF.
  
  
  CONCLUSIONS
  Hyperosmolarity induces apoptosis of pHCECs by activating JNK signaling. Increased levels of NGF under hyperosmolar conditions may contribute, at least in part, to the reduced apoptosis of pHCECs and may be beneficial in recovering conjunctival damage due to chronic hyperosmolar stress.
authors: Department of Ophthalmology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea.
authors: Soon-Suk
authors: SS
authors: Kang
authors: Sae-Jin
authors: SJ
authors: Ha
authors: Eun-Soon
authors: ES
authors: Kim
authors: Jin-A
authors: JA
authors: Shin
authors: Jae Yong
authors: JY
authors: Kim
authors: Hungwon
authors: H
authors: Tchah
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002460:Cell Line; D003228:Conjunctiva / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D020932:Nerve Growth Factor / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D009994:Osmolar Concentration; D010766:Phosphorylation; D012313:RNA / Q000235:genetics*; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction
chemical_list: D011994:Recombinant Proteins; D012313:RNA; D020932:Nerve Growth Factor; D048031:JNK Mitogen-Activated Protein Kinases
keywords: apoptosis; human primary conjunctival epithelial cells; hyperosmolarity; nerve growth factor