Bcl-2 expression in the human cornea.
The purpose of this study was to determine the localization of Bcl-2 protein in the human cornea. Anti-human Bcl-2 monoclonal antibodies (MAbs) against selective Bcl-2 peptide sequences were used to localize Bcl-2 protein immunocytochemically in fresh eye bank donor human corneas (n = 4). Specificity of each MAb was determined by Western blot analysis of pooled protein extracted from human corneal epithelium (n = 3). Expression of Bcl-2 protein in apoptotic surface epithelial cells was detected by co-labeling with TUNEL assay and anti-Bcl-2 antibody staining. Two MAbs specific for amino acids residues (aa) 41-54 within the loop domain of Bcl-2 protein stained nuclei of all corneal epithelial cell layers. MAb specific for aa 61-76, also within the loop domain, produced faint nuclei and nuclear envelope staining. Occasional corneal surface epithelial cells however, consistently lacked anti-Bcl-2 nuclear staining with these three MAbs; concomitant TUNEL assay revealed that all TUNEL positive-surface cells were Bcl-2 negative. In the stroma, keratocytes showed similar but weak anti-Bcl-2 staining. All corneal endothelial cells showed intense nuclear staining with MAbs, with no gradient or absence of staining. In summary, Bcl-2 protein can be localized to the nuclei and nuclear envelope of corneal epithelial cells, keratocytes and endothelial cells with the use of MAbs specific for the loop domain of Bcl-2. TUNEL-labeled surface epithelial cells did not stain with MAbs to Bcl-2, suggesting degradation or epitope masking perhaps by specific phosphorylation of the loop domain during apoptosis. Taken together, these findings suggest that Bcl-2 protein may play a critical role in modulating apoptotic cell desquamation in the human corneal epithelium.
Yamamoto
K
K
The Department of Ophthalmology, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-9057, USA.
Ladage
P M
PM
Ren
D H
DH
Li
L
L
Petroll
W M
WM
Jester
J V
JV
Cavanagh
H D
HD
D000368:Aged; D000704:Analysis of Variance; D017209:Apoptosis / Q000502:physiology; D051017:Apoptosis Regulatory Proteins; D000072224:Bcl-2-Like Protein 11; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D003315:Cornea / Q000378:metabolism*; D003319:Corneal Stroma / Q000378:metabolism; D004728:Endothelium, Corneal / Q000378:metabolism; D019573:Epithelium, Corneal / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D020287:In Situ Nick-End Labeling; D008565:Membrane Proteins; D008875:Middle Aged; D011518:Proto-Oncogene Proteins
D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; D002352:Carrier Proteins; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins