Anti-EMP2 diabody blocks epithelial membrane protein 2 (EMP2) and FAK mediated collagen gel contraction in ARPE-19 cells.
Epithelial membrane protein 2 (EMP2) regulates collagen gel contraction by the retinal pigment epithelium cell line ARPE-19 by modulating FAK activation. Collagen gel contraction is one in vitro model for an aberrant wound healing response, proliferative vitreoretinopathy (PVR), which occurs as a complication of severe ocular trauma. The purpose of this study is to investigate whether EMP2 specific recombinant diabody decreases activation of FAK and collagen gel contraction in ARPE-19. Anti-EMP2 diabody was recombinantly constructed from a human phage library-derived clone selected for reactivity against an extracellular domain of human EMP2. ARPE-19 cells were exposed to an anti-EMP2 or control diabody, and toxicity, adhesion, and migration were assessed respectively through toluidine blue exclusion, binding to collagen type 1, and a migration assay. Collagen gel contraction was assessed using an in vitro assay. FAK activation was evaluated using Western blot. Exposure to anti-EMP2 diabody, resulted in a 75% reduction in EMP2 protein levels at 4 h. No significant toxicity was observed with anti-EMP2 diabody at levels that maximally reduced EMP2. Anti-EMP2 diabody, but not control diabody, significantly reduced collagen gel contraction (p < 0.001), without changes in adhesion or migration. Concordantly, anti-EMP2 diabody as compared to a control diabody reduced collagen stimulated FAK activation (p = 0.01). Anti-EMP2 diabody decreases EMP2 protein levels, FAK activation, and collagen gel contraction by ARPE-19 cells without an adverse effect on cell survival. Modulation of EMP2 using anti-EMP2 diabody could be a new approach for targeting EMP2 and pathologic consequences associated with EMP2.
Department of Ophthalmology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.
Shawn A
SA
Morales
David G
DG
Telander
Sergey
S
Mareninov
Agnes
A
Nagy
Madhuri
M
Wadehra
Jonathan
J
Braun
Lynn K
LK
Gordon
D019138:Antibodies, Blocking / Q000502:physiology*; D000937:Antigen-Antibody Reactions; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002166:Camptothecin / Q000633:toxicity; D002460:Cell Line; D002465:Cell Movement; D024042:Collagen Type I / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism*; D006801:Humans; D007128:Immunoglobulin Fragments; D008562:Membrane Glycoproteins / Q000276:immunology*; D018613:Microscopy, Confocal; D019151:Peptide Library; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D057127:Single-Chain Antibodies
D019138:Antibodies, Blocking; D024042:Collagen Type I; C486403:EMP2 protein, human; D007128:Immunoglobulin Fragments; D008562:Membrane Glycoproteins; D019151:Peptide Library; D057127:Single-Chain Antibodies; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human; D002166:Camptothecin