title: Elevated IGFBP3 levels in diabetic tears: a negative regulator of IGF-1 signaling in the corneal epithelium.
abstract: To determine the ratio of IGFBP3:IGF-1 in normal and diabetic human tears, and in telomerase-immortalized human corneal epithelial cells (hTCEpi) cultured under elevated glucose conditions and to correlate these changes with total and phosphorylated levels of IGF-1R. Tear samples were collected noninvasively from diabetic subjects and non-diabetic controls; corneal sensitivity was assessed using a Cochet-Bonnet Aesthesiometer. Conditioned media were collected following culture of hTCEpi cells in normal (5 mM) and elevated (25 mM) glucose conditions; mannitol was used as an osmotic control. IGFBP3, IGF-1, and phosphorylated IGF-1R levels were assessed by ELISA. IGFBP3 and IGF-1R mRNA were assessed by real-time polymerase chain reaction (PCR). Total and phosphorylated IGF-1R expression in whole cell lysates was assessed by western blot. There was a 2.8-fold increase in IGFBP3 in diabetic tears compared to non-diabetic controls (P=0.006); IGF-1 levels were not significantly altered. No difference in corneal sensitivity was detected between groups. The concentration of IGFBP3 in tears was independent of IGF-1. Consistent with human tear measurements in vivo, IGFBP3 secretion was increased 2.2 fold (P<0.001) following culture of hTCEpi cells under elevated glucose conditions in vitro. Treatment with glucose and the mannitol control reduced IGFBP3 mRNA (P<0.001). Total IGF-1R levels were unchanged. The increase in the IGFBP3:IGF-1 ratio detected in diabetic tears compared to normal controls blocked phosphorylation of the IGF-1R by IGF-1 (P<0.001) when tested in vitro. Taken together, these in vivo and confirmatory in vitro findings suggest that the observed increase in IGFBP3 found in human tears may attenuate IGF-1R signaling in the diabetic cornea. A long-term increase in IGFBP3 may contribute to epithelial compromise and the pathogenesis of ocular surface complications reported in diabetes.
authors.affiliation: Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9057, USA.
authors.forename: Yu-Chieh
authors.initials: YC
authors.lastname: Wu
authors.forename: Benjamin R
authors.initials: BR
authors.lastname: Buckner
authors.forename: Meifang
authors.initials: M
authors.lastname: Zhu
authors.forename: H Dwight
authors.initials: HD
authors.lastname: Cavanagh
authors.forename: Danielle M
authors.initials: DM
authors.lastname: Robertson
mesh_terms: D000328:Adult; D015153:Blotting, Western; D002478:Cells, Cultured; D003316:Corneal Diseases / Q000378:metabolism; D003922:Diabetes Mellitus, Type 1 / Q000378:metabolism*; D003924:Diabetes Mellitus, Type 2 / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000378:metabolism*; D005260:Female; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000235:genetics / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D060888:Real-Time Polymerase Chain Reaction; D017526:Receptor, IGF Type 1 / Q000235:genetics; D013666:Tears / Q000378:metabolism*
chemical_list: C515497:IGFBP3 protein, human; D018972:Insulin-Like Growth Factor Binding Protein 3; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1