Ciliary neurotrophic factor released by corneal endothelium surviving oxidative stress ex vivo.
OBJECTIVE
To demonstrate that corneal endothelial (CE) cells that survive oxidative stress in corneas release ciliary neurotrophic factor (CNTF), which does not possess the secretion signal sequence.


METHODS
CNTF and CNTF receptor alpha subunit (CNTFRalpha) in CE cells and cell-conditioned medium (H2O2 and PBS placed in bovine corneal cups for 30 minutes at 37 degrees C) in explant cultures were demonstrated by Western blot (WB) and immunoprecipitation (IP). The number of dead CE cells was determined microscopically with a viability kit, by an observer uninformed of the explants' identities. CNTF and CNTFRalpha synthesis and release by CE cells in 35S-methionine-labeled (0.1 mCi/mL for 8 hours at 37 degrees C) corneal cups were shown by autoradiography and WB. RESULTS. CE cells in fresh bovine eyes expressed a 25-kDa CNTF that was recognized by three different antibodies. CE cells expressed a 61-kDa CNTF-immunoreactive molecule (IM), which disappeared from the CE cells in H2O2-conditioned corneal cups, concomitant with the appearance of the 25-kDa CNTF in the conditioned medium. Corneal cups containing 0, 0.006, 0.012, 0.023, 0.045, 0.09, 0.18, and 0.35 mM H2O2 demonstrated relative levels of CE cell 61-kDa CNTF-IM of 100%, 84%, 77%, 61%, 52%, 39%, 35%, and 35%, respectively, whereas levels of 25-kDa CNTF in the conditioned medium were 23%, 32%, 39%, 63%, 80%, 90%, 100%, and 63%, respectively. CE cells expressed a 53-kDa CNTFRalpha that, along with trace amounts of a 61-kDa CNTFRalpha-IM, appeared concomitantly with the 25-kDa CNTF in the conditioned medium. H2O2 (0-0.56 mM) did not affect the viability of CE cells (15 dead cells per 600 cells). CE cells in 35S-methionine-labeled corneal cups synthesized and released a 35S 61-kDa molecule that was both CNTF- and CNTFRalpha-immunoreactive in an H2O2-dependent manner, whereas 25-kDa CNTF was detected in the (35)S-methionine labeling medium.


CONCLUSIONS
CE cells release autocrine CNTF under sublethal oxidative stress by a mechanism that involves CNTFRalpha and the formation of a 61-kDa CNTF/CNTFRalpha-IM.
Koh
Shay-Whey M
SW
Department of Ophthalmology, University of Maryland at Baltimore, Baltimore, Maryland, USA. skoh@umaryland.edu
D000818:Animals; D001345:Autoradiography; D015153:Blotting, Western; D002417:Cattle; D020934:Ciliary Neurotrophic Factor / Q000378:metabolism*; D017077:Culture Media, Conditioned; D004305:Dose-Response Relationship, Drug; D004728:Endothelium, Corneal / Q000187:drug effects* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D018384:Oxidative Stress; D011233:Precipitin Tests; D020801:Receptor, Ciliary Neurotrophic Factor / Q000378:metabolism
D020934:Ciliary Neurotrophic Factor; D017077:Culture Media, Conditioned; D020801:Receptor, Ciliary Neurotrophic Factor; D006861:Hydrogen Peroxide